Research on the preparation process of the cytarabine/daunorubicin dual-encapsulation liposome and its physicochemical properties and performances in vitro/vivo.

As the only Food and Drug Administration (FDA)-approved dual-encapsulation liposome injection for treating Acute myeloid leukemia (AML), CPX-351 outperforms the standard chemotherapy treatment "DA 7 + 3″ in terms of clinical effectiveness. Although research on dual-loaded liposomes has increased in recent years, little attention has been paid to their preparation, which can affect their quality, efficacy, and safety. This study explored various preparation processes to create the cytarabine/daunorubicin co-loaded liposome (the Cyt/Daun liposome) and eventually settled on two methods: the sequential loading approach, thin film hydration-extrusion-copper ion gradient, and the simultaneous encapsulation technique, copper ion gradient-concentration gradient. Different preparation methods resulted in different particle sizes and encapsulation efficiencies; the two aforementioned preparation processes generated dual-loaded liposomes with comparable physicochemical properties. The sequential encapsulation technique was selected for the subsequent research owing to its higher encapsulation efficiency prior to purification; the prepared Cyt/Daun liposomes had small and uniform particle size (108.6 ± 1.02 nm, Polydispersity index (PDI) 0.139 ± 0.01), negative charge (-(60.2 ± 1.15) mV), high drug encapsulation efficiency (Cyt 88.2 ± 0.24 %, Duan 94.2 ± 0.45 %) and good plasma stability. To improve its storage stability, the Cyt/Daun liposome was lyophilized (-40 °C for 4 h, maintained for 130 min, and dried for 1200 min) using sucrose-raffinose (mass ratio 7:3; glycolipid ratio 4:1, w/w) as a lyoprotectant. The lyophilized liposomes were purple cakes, redissolved rapidly with insignificant alterations in particle size and encapsulation efficiency, and possessed well storage stability. The pharmacokinetic and tissue distribution studies demonstrated that the Cyt/Daun liposome could achieve long circulation and maintain synergic proportions of drugs within 24 h, increasing the accumulation of drugs at tumor sites. Furthermore, the in vitro/in vivo pharmacodynamic studies confirmed its good anti-tumor activity and safety.

The effects of intermolecular interactions on the stability and in vitro drug release of daunorubicin/cytarabine co-loaded liposome.

Various studies were performed on the intermolecular interactions of daunorubicin (DNR) and cytarabine (Ara-C) co-loaded liposome to predict and elucidate its stability and in vitro drug release behavior. Langmuir monolayer and spectroscopy studies showed interactions between its components. The Langmuir monolayer study and blank liposomes stability study illustrated that interactions between lipids could affect their stability, and the DSPC/DSPG/Chol (7/2/1, mol%) mixed system tended to be thermodynamically and physicochemically stable. The interactions between daunorubicin and copper ions were then investigated by ultraviolet-visible (UV-vis) electronic absorption spectroscopy and circular dichroism (CD) spectroscopy, which revealed that the DNR-Cu complex was composed of daunorubicin and copper ions at a molar ratio of 1:1 or 1:2, and its solubility was related to the acidity of the solution. In vitro release experiment of liposomes with different copper gluconate contents illustrated that the interactions between drugs and copper ions were conducive to the retention and synergetic release of drugs. The stability and release studies of the DSPC/DSPG/Chol (7/2/1, mol%) co-loaded liposome illustrated that it had good storage and plasma stability, and the release behaviors of drugs were pH-related, i.e., drugs could be released faster under acidic condition. These studies indicated that intermolecular interactions could affect the stability and release behavior of the liposome, and a certain ratio of components could be conducive to its stability and synergistic release of drugs.

Impact of Ticagrelor vs. Clopidogrel in Patients With Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention After Risk Stratification With the CHA2DS2-VASc Score.

Backgrounds: The clinical benefit of ticagrelor vs. clopidogrel in unselected patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) remains controversial in the real world. This study was aimed to investigate the impact of ticagrelor vs. clopidogrel in subjects with ACS without atrial fibrillation or flutter (AF) after PCI based on risk stratification using the CHA2DS2-VASc score. Methods: In 2016-2019, patients who underwent PCI with at least one stent implanted in the General Hospital of Northern Theater Command were classified as low- or high-risk groups according to the CHA2DS2-VASc score. Incidences of 12-month ischemia [cardiac death, myocardial infarction (MI), or stroke], all-cause death, Bleeding Academic Research Consortium (BARC) 2,3,5 bleeding, BARC 3,5 bleeding, and net adverse clinical events (NACEs) (all-cause death, MI, stroke, or BARC 3, 5 bleeding) with aspirin plus different P2Y12 inhibitors (clopidogrel or ticagrelor) were appraised among different risk groups. Propensity score matching (PSM) and Cox multivariate analysis were used to balance the groups. Results: A total of consecutive 17,037 patients with ACS were enrolled. The optimal cut-off value of the CHA2DS2-VASc score for ischemic events by the Youden test was 3 points. Among patients with high risk (CHA2DS2-VASc ≥ 3, n = 6,151), ticagrelor was associated with slightly lower risks of ischemic events (2.29% vs. 3.54%, P = 0.02) and stroke (0.39% vs. 1.08%, P = 0.01) without excessive risk of BARC 3, 5 bleeding events (2.16% vs. 2.11%, P = 0.92) compared to clopidogrel within 12 months after PCI. For patients with low risk (CHA2DS2-VASc < 3, n = 10,886), a statistically significant difference was seen in the incidence of overall 12-month BARC 2, 3, 5 bleeding events by P2Y12 receptor inhibitor (4.00% vs. 3.26%) with a similar incidence of the ischemic events (1.40% vs. 1.52%). Results in the PSM cohort and the adjustment with Cox multivariate analysis were consistent with the main outcomes. Conclusion: Higher CHA2DS2-VASc scores were associated with a higher incidence of 1-year ischemic events for the patients with ACS after PCI. Compared with clopidogrel, ticagrelor was associated with lower ischemic events within 12 months after PCI without excessive risk of bleeding in high-risk patients but shows poor safety with excess bleeding in low-risk patients.

N enrichment affects the arbuscular mycorrhizal fungi-mediated relationship between a C4 grass and a legume.

Arbuscular mycorrhizal fungi (AMF) regulate soil nutrient cycling, directly supplying a host plant with nitrogen (N). AMF can also affect the outcome of interspecific interactions, but a mechanistic understanding of how soil N availability affects AMF-mediated interspecific relationships is currently lacking. We selected one dominant (Bothriochloa ischaemum; C4 grass) and one subordinate (Lespedeza davurica; legume) species in a natural grassland climax community to investigate the mechanism by which AMF influence interspecific interaction (mixed and monoculture) under three levels of N addition (0, low, and high N addition). Under the non-N addition treatment, AMF preferentially supplied N to the roots of B. ischaemum at the expense of N uptake by L. davurica, resulting in inhibited AMF benefits for L. davurica shoot growth. Under the low N addition treatment, interspecific interaction via AMF promoted L. davurica growth. Compared to the non-N addition treatment, N addition largely mitigated the effects, both positive (for B. ischaemum) and negative (for L. davurica), of AMF-mediated interspecific interaction on plant N uptake via AMF. When soil N availability severely limited plant growth, preferential N supply to the C4 grass by AMF was important for maintaining the abundance of the dominant species. When the N limitation for plant growth was alleviated by N addition, the interaction between AMF and soil microorganisms improved nutrient availability for the legume by stimulating activity of the enzyme responsible for soil organic matter mineralization, which is important for maintaining the abundance of the subordinate species. These data could influence strategies for maintaining biodiversity.

Mechanistic understanding of interspecific interaction between a C4 grass and a C3 legume via arbuscular mycorrhizal fungi, as influenced by soil phosphorus availability using a 13 C and 15 N dual-labelled organic patch.

Arbuscular mycorrhizal fungi (AMF) can improve plant nutrient acquisition, either by directly supplying nutrients to plants or by promoting soil organic matter mineralization, thereby affecting interspecific plant relationships in natural communities. We examined the mechanism by which the addition of P affects interspecific interactions between a C4 grass (Bothriochloa ischaemum, a dominant species in natural grasslands) and a C3 legume (Lespedeza davurica, a subordinate species in natural grasslands) via AMF and plant growth, by continuous 13 C and 15 N labelling, combined with soil enzyme analyses. The results of 15 N labelling revealed that P addition affected the shoot uptake of N via AMF by B. ischaemum and L. davurica differently. Specifically, the addition of P significantly increased the shoot uptake of N via AMF by B. ischaemum but significantly decreased that by L. davurica. Interspecific plant interactions via AMF significantly facilitated the plant N uptake via AMF by B. ischaemum but significantly inhibited that by L. davurica under P-limited soil conditions, whereas the opposite effect was observed in the case of excess P. This was consistent with the impact of interspecific plant interaction via AMF on arbuscular mycorrhizal (AM) benefit for plant growth. Our data indicate that the capability of plant N uptake via AMF is an important mechanism that influences interspecific relationships between C4 grasses and C3 legumes. Moreover, the effect of AMF on the activities of the soil enzymes responsible for N and P mineralization substantially contributed to the consequence of interspecific plant interaction via AMF for plant growth.

Impact of 6- versus 12-month dual antiplatelet therapy on clinical prognosis in patients with high bleeding risk: Insights from the 4-year results of the I LOVE IT 2 study.

OBJECTIVES: To explore the impact of 6- versus 12-month dual antiplatelet therapy (DAPT) on the clinical prognosis of high bleeding risk (HBR) patients. BACKGROUND: The optimal DAPT duration after percutaneous coronary intervention (PCI) in HBR patients is unclear. METHODS: This study is a post hoc analysis of the 4-year clinical follow-up results of the I LOVE IT 2 study. Prevalence and prognosis of HBR patients were explored, and clinical outcomes of HBR patients who underwent 6- versus 12-month DAPT were compared. The primary outcome was Bleeding Academic Research Consortium (BARC) type 3 or 5 bleeding. The secondary outcomes were BARC type 2-5 bleeding and net clinical adverse events (NACE), defined as a composite of all-cause death, myocardial infarction (MI), ischemia-driven revascularization, stroke, stent thrombosis, or any bleeding events. RESULTS: HBR occurred in 440 of 2,737 patients (16.0%). HBR patients were associated with a higher risk of BARC type 3 or 5 bleeding (2.95 vs. 1.52%, p = .03), NACE (31.82 vs. 25.99%, p = .01), all-cause death (5.68 vs. 3.13%, p = .008) and stroke (9.09 vs. 3.83%, p < .001) than non-HBR patients at 4 years. There were no significant differences in BARC type 3 or 5 bleeding (3.07 vs. 2.76%, p = 1.00) or NACE rate (31.9 vs. 33.8%, p = .72) between patients who underwent 6- and 12-month DAPT. CONCLUSIONS: HBR patients are at a higher risk of long-term bleeding and ischemic events than non-HBR patients. The safety and efficacy of 6- and 12-month DAPT were comparable in HBR patients.

Effects of natural vegetation restoration on dissolved organic matter (DOM) biodegradability and its temperature sensitivity.

Biodegradation of dissolved organic matter (DOM) plays a key role in regulating both production of greenhouse gases and accumulation and stabilisation of soil organic matter (SOM). However, the mechanisms by which natural vegetation restoration affects the extent, rate, and temperature sensitivity of DOM biodegradation are poorly understood. Elucidating these mechanisms is important for SOM management, especially in light of future climate warming scenarios. In this study, a laboratory DOM solution incubation experiment was conducted to comprehensively investigate the effects of temperature and natural vegetation restoration spanning a period of 160 y on DOM biodegradation in the Loess Plateau, China. The results indicated that dissolved organic C (DOC) biodegradation significantly decreased with vegetation restoration after an incubation period of 60 d. Further, biodegradation of dissolved organic N (DON) and dissolved organic P (DOP) significantly decreased after farmland abandonment. Specifically, the lowest values were observed in pioneer (Populus davidiana) and mingled (Populus davidiana and Quercus liaotungensis) forests. Generally, an increase in temperature significantly promoted the biodegradation of DOC, DON, and DOP by enhancing the microbial utilisation efficiencies of recalcitrant humic substrates (i.e., low-molecular-weight humic materials). Our results suggest that DOM biodegradability and its temperature sensitivity were regulated by DOM substrate quality (i.e, recalcitrant humic materials), and microbial properties (i.e., gram-negative bacterial and fungal PLFA, enzyme activities). Additionally, our results suggest that climax forest communities (Quercus liaotungensis) played a vital role in reducing DOC and DOP losses. This could be attributed to the low Q10 of the DOC and DOP biodegradation rates.

Forest management practices of Pinus tabulaeformis plantations alter soil organic carbon stability by adjusting microbial characteristics on the Loess Plateau of China.

Sustainable management practices can enhance the capacity and potential for soil carbon (C) sequestration, significantly contributing towards mitigating regional climate change. Here, we investigated how the microbial characteristics of a Pinus tabulaeformis plantation responded to different management practices to identify the role of microbial characteristics in influencing the stability of soil organic carbon (SOC). We chose a Pinus tabulaeformis plantation on the Loess Plateau where forest management practices had been conducted since 1999. Five forest management practices were implemented: two at the forest level (P. tabulaeformis with and without ground litter), and three using different vegetation restoration approaches after clear-cutting (P. tabulaeformis seedlings, abandoned grassland, and natural shrub regeneration). Microbial biomass, soil respiration, microbial community structure, microbial metabolic function, and soil oxidizable organic carbon (OC) fractions were evaluated. Forest management practices changed SOC stability by adjusting the microbial characteristics (e.g. soil microbial community diversity and microbial metabolic function diversity). The result of path analysis was that the direct path coefficient of microbial biomass on soil oxidizable OC fractions was the largest, which was 1.499. Path analysis and redundancy analysis showed that microbial biomass had the largest direct influence on soil oxidizable OC fractions. Compared with other forest management practices, natural shrub regeneration increased the nonlabile carbon fraction by increasing soil microbial characteristics, and contributed the most towards stabilizing SOC, which enhanced the stability of the soil ecosystem on the plateau. In conclusion, microbial biomass was the biggest influence factor of SOC stability. In contrast, the stability of SOC may be most stable in the area of natural shrub regeneration.

Plant-microbial feedback in secondary succession of semiarid grasslands.

Plant-soil feedback (PSF) is an important driver of plant community dynamics. The role of plant species in PSF has been emphasized for secondary succession processes; however, microbial responses to PSF and the underlying mechanisms responsible for their effects on plant succession remain poorly understood, particularly in semiarid grassland ecosystems. Here, we conducted a greenhouse experiment using soil collected from early-, mid-, and late-successional plant communities to measure net pairwise PSF for species grown under monoculture. Soils conditioned by pre-successional species had a positive feedback effect on subsequent plant species, whereas soil conditioned by subsequent plant species had a negative feedback effect on pre-successional species. The feedback effect of plants from different successional stages on soil bacterial and fungal communities was mainly positive. However, the bacterial genera in the soil conditioned by early- and mid-successional species and fungal classes in the soil conditioned by early- successional species had a negative feedback effect on late-successional species. Thus, the effects of soil fungal and bacterial communities on species in other successional stages varied with taxonomic level. Our results provide insight into the manner in which soil microbial communities influence PSF responses during secondary succession processes.

Contrastive Studies of Cytarabine/Daunorubicin Dual-Loaded Liposomes Prepared by pH Gradient and Cu2+ Gradient Method.

Conventional combination chemotherapy often leads to unsatisfactory clinical outcomes due to the different distribution characteristics in vivo and the superimposed systemic toxicity of the drug cocktail. Co-encapsulated nano preparations have been gradually developed in recent years. In this work, cytarabine (Ara-C)/daunorubicin (DNR) liposomes were prepared by the pH gradient (ADL-pH) and Cu2+ gradient (ADL-Cu) methods. Ara-C did not show significant release from either ADL-Cu or ADL-pH in vitro during 168 h, which related to its logPoct. Different drug-loading patterns showed different release characteristics of DNR due to the different existence forms, ADL-pH contains the citrate form, while in ADL-Cu, there is the Cu2+ complex. To evaluate the release behavior, daunorubicin liposome (DL) and daunorubicin-Cu2+ complex (DNR-Cu) were prepared. The addition of EDTA in the release medium significantly increased the release rate of DNR from DL-Cu, while lower pH accelerated DNR release from both DL-pH and DL-Cu. The PK confirmed that ADL-Cu and ADL-pH could prolong the drug circulation time, and ADL-Cu had a mean retention time 1.5 times that of ADL-pH. Furthermore, both liposomes allowed the two drugs to maintain a relatively constant plasma concentration ratio for a prolonged time. Cytotoxicity assays showed that Ara-C/DNR with a molar ratio of 5:1 and 3:1 exhibited an excellent synergistic effect, which was more obvious at 5:1. In vitro antitumor results revealed that ADL-Cu exhibited more cytotoxicity than ADL-pH. All factors tested in this work suggest the considerable potential of ADL-Cu and ADL-pH for anticancer treatment.

[Effects of Management Measures on Soil Water-soluble Carbon and Nitrogen and Their Three-Dimensional Fluorescence Characteristics of Pinus tabulaeformis Plantations on Loess Plateau].

The Pinus tabulaeformis plantation on the Loess Plateau was selected as the research site to study the changes of soil water-soluble carbon and nitrogen content and components of soil dissolved organic matter (SDOM) under different management measures (litter removal, young Pinus tabulaeformis forest, shrubs, grassland). The uncleared Pinus tabulaeformis plantation was used as the control. Results show that the soil water-soluble organic carbon content of shrubs and young Pinus tabulaeformis forests was significantly higher than that in other management measures. The content of soil water-soluble organic carbon decreased with an increase in soil depth, whereas soil water-soluble organic carbon/soil organic carbon (WSOC/SOC) increased. The change of soil water-soluble nitrogen content corresponded to that of water-soluble carbon. The content of soil water-soluble nitrogen content in the young Pinus tabulaeformis forest was higher than that of other management measures, and generally decreased with an increase in depth. Different management measures have significant effects on SDOM components. Based on three-dimensional fluorescence spectroscopy combined with parallel factor (EEM-PARAFAC) analysis, four components of SDOM are identified, which are mainly divided into categories, humus, protein-like, and soluble microbial metabolites in three major categories, among which the main component of the SDOM is the rich acid humus, and the highest content in the soil of the young forest of Pinus tabulaeformis is rich acid. The results suggested that management measures had certain effects on soil water-soluble carbon and nitrogen content as well as SDOM components, especially in young Pinus tabulaeformis forests and shrubs. The young Pinus tabulaeformis forest and shrubs increased the content of soil water-soluble organic carbon and water-soluble nitrogen, enhanced their ability to migrate and transform in the soil, and changed the structural composition of soil organic matter, hence enhancing the degree of humification and further improving soil quality.

Influence of slope aspect on the macro- and micronutrients in Artemisia sacrorum on the Loess Plateau in China.

Slope aspect is an important topographic factor for a micro-ecosystem environment that may affect macro- and micronutrients in plants and soil. The south-, northwest-, and north-facing slopes were selected to investigate the influence of slope aspect on the concentrations, storage, and allocation of macro- and micronutrients in Artemisia sacrorum on the Loess Plateau in China. The concentrations of available manganese (Mn) in both rhizosphere and non-rhizosphere soils reached their maximum on the north-facing slope. The concentrations of available iron (Fe) in rhizosphere soil and available copper (Cu) in non-rhizosphere soil reached their maximum on the south-facing slope. Slope aspect significantly affected the total concentrations of potassium (K), calcium (Ca), magnesium (Mg), Cu, and Mn in rhizosphere and non-rhizosphere soils, and all of these elements reached their maximum on the northwest-facing slope. Slope aspect significantly influenced the concentrations of aboveground K, Ca, and Mg, sodium (Na), Mn, and belowground K in A. sacrorum, and the concentrations of aboveground K, Ca, Mg, and Na and belowground Mn, Na, Fe, Ca, and Mg in weed. Most elements in A. sacrorum and the weeds reached their maximum on the south-facing slope. Slope aspect significantly changed the aboveground-to-belowground concentration ratios of K, Ca, and Na in A. sacrorum and weed. Slope aspect significantly affected the storage of macro- and micronutrients in A. sacrorum and weed but not the storage in the plants of the entire plot. Slope aspect predominantly affected the storage allocation of macro- and micronutrients in A. sacrorum but not those in weed. Slope aspect is an important topographic factor that affects the macro- and micronutrients in plants and soil in micro-ecosystem environments.

Rehabilitation time has greater influences on soil mechanical composition and erodibility than does rehabilitation land type in the hilly-gully region of the Loess Plateau, China.

BACKGROUND: The major landscape in the hilly-gully region of the Loess Plateau is greatly affected by vegetation rehabilitation on abandoned cropland. Although many studies have shown that the rehabilitation have greatly improved soil conditions and protected them from erosion, these effectiveness were not always in consensus possibly due to the land type of vegetation or to the rehabilitation time. To close this gap, we conducted a long term experiment as follows. METHODS: In this study, we analysed four land types of vegetation rehabilitation (shrub land, woodland, naturally revegetated grassland, and orchard land) with different rehabilitation times and investigated the mechanical composition and erodibility of the soil. Areas of slope croplandand natural forest were selected as controls. RESULTS: The results showed that soil depth, rehabilitation time and rehabilitation land type had strong impacts on soil mechanical composition, micro-aggregation and erodibility. Following rehabilitation, naturally revegetated grassland and shrub land had lower fractal dimensions of particle size distribution (fractal dimensions of PSD), fractal dimensions of micro-aggregation, and erodibility (K factor) than did cropland. Compared to the positive effects of rehabilitation mainly happened in the topsoil layer at other rehabilitation land type, that of woodland happened in the deeper soil layer. Besides, the indispensable rehabilitation time for the significant improvement of soil condition was shorter at naturally revegetated grassland than that at shrub land and woodland. DISCUSSION: Although rehabilitation time was more influential than was rehabilitation land type or soil depth, the differences among the rehabilitation land types showed that naturally revegetated grassland with native plants is the most time-saving rehabilitation vegetation for the Loess Plateau in the conversion from slope cropland. The success of rehabilitation in this forestry practice was mainly contributed by the suited species of rehabilitation land type to the local climate and soil. Based on the differences of rehabilitation effectiveness resulting from land type, we should be cautious to choose land types for the rehabilitation of soil conditions in the Loess Plateau.

Effects of long-term fertilisation on aggregates and dynamics of soil organic carbon in a semi-arid agro-ecosystem in China.

BACKGROUND: Long-term fertilisation has a large influence on soil physical and chemical properties in agro-ecosystems. The effects on the distribution of aggregates, however, are not fully understood. We determined the dynamic change of the distribution of aggregates and soil organic carbon (SOC) content over time in a long-term field experiment established in 1998 on the Loess Plateau of China and illustrated the relationship between them. METHODS: We determined SOC content and the distribution of aggregates in nine fertiliser treatments: manure (M); nitrogen (N); phosphorus (P); M and N; M, N, and P; M and P; N and P; bare land; and an unfertilised control. These parameters were then used for a path analysis and to analyse the fractal dimension (Dv). RESULTS: The organic fertiliser increased SOC content. The proportions of 0.1-0.25 mm microaggregates and 0.25-0.5 mm macroaggregates were higher and the proportion of the 0.01-0.05 mm size class of the silt + clay fraction was lower in the treatments receiving organic fertiliser (M, MN, MNP, and MP) than that in the control, indicating that the addition of organic fertiliser promoted aggregation. The distribution of aggregates characterised by their fractal dimension (Dv ), however, did not differ among the treatments. DISCUSSION: Dv was strongly correlated with the proportion of the <0.002 mm size class of the silt + clay fraction that did not differ significantly among the treatments. The change in the distribution of aggregates was strongly correlated with SOC content, which could produce organic polymer binding agents to increase the proportion of larger particles. Long-term application of organic fertiliser is thus necessary for the improvement and maintenance of soil quality in semi-arid agricultural land when residues are removed.

The Effects of Nitrogen Addition on the Uptake and Allocation of Macro- and Micronutrients in Bothriochloa ischaemum on Loess Plateau in China.

The effects of nitrogen (N) addition on the macro- and micronutrient concentrations, storage, and allocation of Bothriochloa ischaemum (L.) Keng, a native forage plant on the Loess Plateau in China remain unclear. We studied the effects of N addition at 0 (CK), 2.5 (N1), 5.0 (N2), and 10.0 (N3) g N m-2 y-1. N addition significantly decreased the available copper (Cu), zinc (Zn), and total Cu concentration, but significantly increased the available iron concentration in the soil. Cu, manganese (Mn), and sodium (Na) concentrations in aboveground tissues and potassium (K), magnesium, and Zn concentrations in belowground tissues significantly increased with N addition. Calcium (Ca) concentrations in belowground tissues decreased significantly. The ratios of above- to belowground Ca, Cu, Zn, and Mn significantly increased with N addition. The maximum ratios appeared at N2 for Cu, Zn, and Mn. The aboveground, belowground, and total biomass storage of studied nutrients significantly changed with N addition, and most attained maximum values under N2 treatment. The storage ratios of above- to belowground Cu, Zn, Mn, and Na attained maximum values at N2. We conclude that N addition significantly, but differentially influence the macro- and micronutrient concentrations and storage in B. ischaemum. B. ischaemum allocated and accumulated increased macro- and micronutrients to its aboveground tissues and exhibited high total storage when the amount of N addition reached 5 g N m-2 y-1.

[Histologic characteristics of in vitro constructed skin basement membrane].

OBJECTIVE: To observe the histological characteristics of constructed basement membrane in tissue-engineered skin. METHODS: Forskins from circumcision in normal children were obtained with informed consent of the parents, and then the epidermal keratinocytes (KC) and dermal fibroblasts (Fb) were isolated with trypsin and collagenase D digestion in sequence. Tissue engineered skin with composite chitosan was maintained in a submerged state for 3 days, and then at the air-liquid interface. The tissue-engineered skins were fixed in neutral formalin and then embedded in paraffin after culture for 7, 10 and 15 days, respectively for immunohistological examination of the basement membrane component,including the condition of collagen type IV (COL-IV), collagen type VII (COL-VII), and laminin (LN). RESULTS: HE staining showed that the keratinocytes formed a fine stratified squamous epithelium with the presence of basal, spinous, granular and corneous cell layers, and there was various amount of cells in flat and fusiform shape in each layer. It was found that a regular red staining strip situated at the dermal epidermal junction. Positive staining of collagen IV, collagen VII as well as LN was observed by immunohistological examination. CONCLUSION: The results suggest that the composite chitosan tissue engineered skin has a good prospect for clinical use because it presents a perfect reconstruction of basement membrane.