Spectrochemical determination of effects on rat liver of binary exposure to benzo[a]pyrene and 2,2',4,4'-tetrabromodiphenyl ether.

Benzo[a]pyrene (B[a]P) and polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants. The effects in organisms of exposures to binary mixtures of such contaminants remain obscure. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy is a label-free, non-destructive analytical technique allowing spectrochemical analysis of macromolecular components, and alterations thereof, within tissue samples. Herein, we employed ATR-FTIR spectroscopy to identify biomolecular changes in rat liver post-exposure to B[a]P and BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) congener mixtures. Our results demonstrate that significant separation occurs between spectra of tissue samples derived from control versus exposure categories (accuracy = 87%; sensitivity = 95%; specificity = 79%). Additionally, there is significant spectral separation between exposed categories (accuracy = 91%; sensitivity = 98%; specificity = 90%). Segregation between control and all exposure categories were primarily associated with wavenumbers ranging from 1600 to 1700 cm-1 . B[a]P and BDE-47 alone, or in combination, induces liver damage in female rats. However, it is suggested that binary exposure apparently attenuates the toxic effects in rat liver of the individual contaminants. This is supported by morphological observations of liver tissue architecture on hematoxylin and eosin (H&E)-stained liver sections. Such observations highlight the difficulties in predicting the endpoint effects in target tissues of exposures to mixtures of environmental contaminants.

Intrauterine exposure to 2,3',4,4',5-pentachlorobiphenyl alters spermatogenesis and testicular DNA methylation levels in F1 male mice.

Polychlorinated biphenyls (PCBs) are synthetic biphenyl compounds with high toxicity. There are a total of 209 homologs, among which 2,3',4,4',5-pentachlorobiphenyl (PCB118) is one of the dioxin-like PCBs. PCB118 can accumulate in pregnant mice, leading to fetus directly exposure during development. The stage of migration of mouse primordial germ cells ranges from 8.5 to 13.5 days of pregnancy, which is the stage undergoing a genome-wide DNA demethylation process. In this study, the mice were exposed to 20 µg/kg/day and 100 µg/kg/day PCB118 from 8.5 to 13.5 days of pregnancy. During the embryo stage at 18.5 days (E18.5 days), the expression level of DNA methyltransferase 1 (Dnmt1) was reduced in the testes, and the DNA methylation level in mouse testes were also decreased. We found that the seminiferous tubules showed vacuolization and that the sperm deformity rate increased in the treated groups compared with the control group in 7-week-old mice. Because exposure to PCB118 during pregnancy causes damage to the reproductive system of male offspring mice, attention should be devoted to the toxicity transmission of persistent environmental pollutants such as PCBs.

Effects of Polychlorinated Biphenyls on Animal Reproductive Systems and Epigenetic Modifications.

Polychlorinated biphenyls (PCBs) are a group of highly toxic endocrine-disrupting chemicals comprising 209 homologs. PCBs are extensively found in the environment and can induce typical estrogenic and profound, long-lasting effects on animals. In this article, the introduction of PCB residues into the environment and the pathways of PCB enrichment in animals are described. PCBs are widely deposited and eventually accumulate in human tissues and body fluids through biomagnification. PCBs can significantly decrease animal fertility and interfere with endocrine processes, leading to the development of various diseases and even cancer. The effects of PCBs on the reproductive systems of animals can also be passed to their offspring, indicating that PCBs may affect the epigenetic modification process. There is currently no treatment to effectively inhibit the toxicity of PCBs in organisms; therefore, the severity of PCB toxicity needs to be widely recognized.

Deletion of BAF250a affects oocyte epigenetic modifications and embryonic development.

BRG1-associated factor 250a (BAF250a) is a component of the SWI/SNF adenosine triphosphate-dependent chromatin remodeling complex, which has been shown to control chromatin structure and transcription. BAF250a was reported to be a key component of the gene regulatory machinery in embryonic stem cells controlling self-renewal, differentiation, and cell lineage decisions. Here we constructed Baf250aF/F ;Gdf9-cre (Baf250aCKO ) mice to specifically delete BAF250a in oocytes to investigate the role of maternal BAF250a in female germ cells and embryo development. Our results showed that BAF250a deletion did not affect folliculogenesis, ovulation, and fertilization, but it caused late embryonic death. RNA sequencing analysis showed that the expression of genes involved in cell proliferation and differentiation, tissue morphogenesis, histone modification, and nucleosome remodeling were perturbed in Baf250aCKO MII oocytes. We showed that covalent histone modifications such as H3K27me3 and H3K27ac were also significantly affected in oocytes, which may reduce oocyte quality and lead to birth defects. In addition, the DNA methylation level of Igf2r, Snrpn, and Peg3 differentially methylated regions was decreased in Baf250aCKO oocytes. Quantitative real-time polymerase chain reaction analysis showed that the relative messenger RNA (mRNA) expression levels of Igf2r and Snrpn were significantly increased. The mRNA expression level of Dnmt1, Dnmt3a, Dnmt3l, and Uhrf1 was decreased, and the protein expression in these genes was also reduced, which might be the cause for impaired imprinting establishment. In conclusion, our results demonstrate that BAF250a plays an important role in oocyte transcription regulation, epigenetic modifications, and embryo development.

Effects of 2,3',4,4',5-pentachlorobiphenyl exposure during pregnancy on DNA methylation in the testis of offspring in the mouse.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants, and the widespread use of PCBs has had adverse effects on human and animal health. This study experiment explored the effects of 2,3',4,4',5-pentachlorobiphenyl (PCB118) on the mammalian reproductive system. PCB118 was administered to pregnant mice from 7.5 to 12.5 days of gestation; F1 mice were obtained and the reproductive system of F1 male mice was examined. PCB118 damaged the reproductive system in male F1 mice, as evidenced by negative effects on the testicular organ coefficient (testes weight/bodyweight), a decrease in the diameter of seminiferous tubules and a significant reduction in the anogenital distance in 35-day-old F1 mice. In addition, methylation levels of genomic DNA were reduced, with reductions in the expression of the DNA methyltransferases DNMT1, DNMT3A and DNMT3B, as well as that of the epigenetic regulatory factor ubiquitin like with PHD and ring finger domains 1 (Uhrf1). Together, the results of this study provide compelling evidence that exposure of pregnant mice to PCB118 during primordial germ cell migration in the fetus affects the reproductive system of the offspring and decreases global methylation levels in the testis.

Effects of intrauterine exposure to 2,3',4,4',5-pentachlorobiphenyl on the reproductive system and sperm epigenetic imprinting of male offspring.

Polychlorinated biphenyls (PCBs) are a class of persistent organic environmental pollutants with a total of 209 homologs. The homolog 2,3',4,4',5-pentachlorobiphenyl (PCB118) is one of the most important dioxin-like PCBs and is highly toxic. PCB118 can accumulate in human tissues, serum and breast milk, which leads to direct exposure of the fetus during development. In the present study, pregnant mice were exposed to 0, 20 and 100 µg/kg/day of PCB118 during the stage of fetal primordial germ cell migration. Compared with the control group, we found morphological alterations of the seminiferous tubules and a higher sperm deformity rate in the male offspring in the treatment groups. Furthermore, the methylation patterns in the treatment groups of the imprinted genes H19 and Gtl2 in the sperm were altered in the male offspring. We also characterized the disturbance of the expression levels of DNA methyltransferase 1 (Dnmt1), Dnmt3a, Dnmt3b, Dnmt3l, and Uhrf1. The results indicated that intrauterine exposure to low doses of PCB118 could significantly damage the reproductive health of the male offspring. Therefore, attention should be paid to the adverse effects of PCB118 exposure during pregnancy on the reproductive system of male offspring.

Effects of 2,3',4,4'5-pentachlorobiphenyl exposure during pregnancy on epigenetic imprinting and maturation of offspring's oocytes in mice.

Polychlorinated biphenyls (PCBs) are a class of organic pollutants that have been widely found in the environment. The chemical 2,3',4,4'5-pentachlorobiphenyl (PCB118) is an important dioxin-like PCB compound with strong toxicity. PCB118 can accumulate in adipose tissue, serum and milk in mammals, and it is highly enriched in the follicular fluid. In this study, pregnant mice were exposed to 0, 20 and 100 µg/kg/day of PCB118 during pregnancy at the fetal primordial germ cell migration stage. The methylation patterns of the imprinted genes H19, Snrpn, Peg3 and Igf2r as well as the expression levels of Dnmt1, 3a, 3b and 3l, Uhrf1, Tet2 and Tet3 in fully grown germinal vesicle oocytes were measured in offspring. The rates of in vitro maturation, in vitro fertilization, oocyte spindle and chromosomal abnormalities were also calculated. The results showed that prenatal exposure to PCB118 altered the DNA methylation status of differentially methylated regions in some imprinted genes, and the expression levels of Dnmt1, 3a, and 3l, Uhrf1 and Tet3 were also changed. In addition, PCB118 disturbed the maturation process of progeny mouse oocytes in a dose-dependent manner. Therefore, attention should be paid to the potential impacts of PCB118-contaminated dietary intake during pregnancy on the offspring's reproductive health.

Activated proline-rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte.

Proline-rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p-PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p-PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p-PYK2 colocalizes with γ-tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p-PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.

Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice.

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development.

Exposure to Aroclor-1254 impairs spindle assembly during mouse oocyte maturation.

Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally-related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor-1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 µg mL-1 Aroclor-1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor-1254-treated oocytes. Further studies showed that the normal localization of γ-tubulin and Aurora-A kinase was interfered and α-tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor-1254. The length of spindle from 10 µg mL-1 Aroclor-1254-treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor-1254-treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor-1254. These results will provide the important reference for the prevention of reproductive disorders caused by PCBs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1652-1662, 2016.

[Exploration of influencing factors of price of herbal based on VAR model].

Based on vector auto-regression (VAR) model, this paper takes advantage of Granger causality test, variance decomposition and impulse response analysis techniques to carry out a comprehensive study of the factors influencing the price of Chinese herbal, including herbal cultivation costs, acreage, natural disasters, the residents' needs and inflation. The study found that there is Granger causality relationship between inflation and herbal prices, cultivation costs and herbal prices. And in the total variance analysis of Chinese herbal and medicine price index, the largest contribution to it is from its own fluctuations, followed by the cultivation costs and inflation.

A naphthalimide-based and Golgi-targetable fluorescence probe for quantifying hypochlorous acid.

The Golgi apparatus (GA) is a vital organelle in biological systems and excess reactive oxygen species (ROS) is produced during stress in the Golgi apparatus. Hypochlorous acid (HOCl) is a significant reactive oxygen species and has strong oxidative and antibacterial activity, but excessive secretion of hypochlorous acid can affect Golgi structure or function abnormally, it will lead to a series of diseases including Alzheimer's disease, neurodegenerative diseases, autoimmune diseases, and Parkinson's disease. In present work, a novel fluorescent probe for Golgi localization utilizing naphthalimide derivatives was constructed to detect hypochlorous acid. The fluorescent probe used a derivatived 1,8-naphthalimide as the emitting fluorescence group, phenylsulfonamide as the localization group and dimethylthiocarbamate as the sensing unit. When HOCl was absent, the intramolecular charge transfer (ICT) process of the developed probe was hindered and the probe exhibited a weak fluorescence. When HOCl was present, the ICT process occurred and the probe showed strong green fluorescence. When the HOCl concentration was altered from 5.0 × 10-7 to 1.0 × 10-5 mol·L-1, the fluorescence intensity of the probe well linearly correlated with the HOCl concentration. The detection limit of 5.7 × 10-8 mol·L-1 was obtained for HOCl. The HOCl fluorescent probe possessed a rapid reaction time, a high selectivity and a broad working pH scope. In addition, the probe possessed good biocompatibility and had been magnificently employed to image Golgi HOCl in Hela cells. These characteristics of the probe demonstrated its ability to be used for sensing endogenous and exogenous hypochlorous acids within the Golgi apparatus of living cells.

2,3',4,4',5-Pentachlorobiphenyl induces mitochondria-dependent apoptosis mediated by AhR/Cyp1a1 in mouse germ cells.

Polychlorinated biphenyls (PCBs) are environmental organic pollutants widely used in industry that can bioaccumulate and affect the reproductive systems of male animals of different species. 2,3',4,4',5-Pentachlorobiphenyl (PCB118) is a representative of the 209 toxic PCB congeners. In this study, male mice were exposed to PCB118 at 0, 50, and 500 µg/kg/day for 35 days beginning 3-4 weeks after birth. The results of the study showed that PCB118 exposure during puberty reduced testicular quality, caused tissue damage, decreased sperm motility and sperm count, and increased malformation and testicular cell apoptosis in mice. Moreover, PCB118 increased the oxidative stress levels in sperm and testicular tissue and the expression of aryl hydrocarbon receptor (AhR) and Cyp1a1 and siginificantly decreased the level of nuclear factor-erythroid 2-related factor 2 (Nrf2). The results indicate that PCB118 can activate the AhR/Cyp1a1 pathway and inhibit Nrf2 expression to aggravate testicular oxidative stress and induce cell apoptosis, resulting in testicular and sperm quality damage.

[Curative effect of integrative medical therapy for systematic lupus erythematosus].

OBJECTIVE: To observe the curative effect of integrative medicine for treatment of systematic lupu erythematosus (SLE). METHODS: Totally 110 cases of SLE were randomized into two groups, 50 in the control group and 60 in the treated group, both were treated with prednisone, but to the treated group, integrative medica treatment was given additionally according to syndrome differentiation. The course for both groups was 6 months. Clinical symptoms, immunological indexes, peripheral blood figure, erythrocyte sedimentation rate (ESR), levels of C-reactive protein (CRP) and creatinine (Cr) in blood, and 24 h urinary total protein (u-TP/24 h) were observed before and after treatment. RESULTS: Of the 60 patients in the treated group, the treatment on 29 was evaluated as clinical remission, 18 as remarkably effective, 9 as effective, and 4 as ineffective, the total effective rate being 93.33% (56/60), while that in the control group was clinical remission in 11, remarkably effective in 10, effective in 19, ineffective in 10 respectively, and the total effective rate 80% (40/50), the difference on total ef fective rate between the two groups was significant (chi2 = 4.36, P <0.05). Besides, the improvement in the treated group was superior to that in the control group in terms of clinical symptoms such as fever, arthralgia and baldness (P <0.05, P <0.01); the negative reversion rate of anti-nuclear antibody (ANA, 51.3% vs 28.1%), antidouble-stranded DNA (ds-DNA, 53.6% vs 26.1%), anti- ribonucleoprotein (RNP, 63.2% vs 29.4%) and circulating immnue complexes (CIC, 63.2% vs 29.4%, all P <0.05); lowering of immunoglobulin (for IgG, P < 0.05; IgA, P <0.01; IgM, P <0.01); as well as bettering in peripheral blood figure, ESR, CRP, Cr and u-TP/24 h (P <0.05 or P <0.01). CONCLUSION: Integrative medical treatment is obviously effective for SLE patients.

Toxic effects of polychlorinated biphenyls (Aroclor 1254) on human sperm motility.

Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent a considerable risk to reproductive toxicity in exposed human populations. Although some experimental studies have suggested an association between the levels of PCBs and semen quality, the direct effects of PCBs on human sperm parameters remain largely unexplored. To this aim, a short-term in vitro incubation experiment that better imitated the putative exposure of sperm to Aroclor 1254 (a commercial PCB mixture) in male reproduction tissue was conducted. Human sperm were incubated with various concentrations (0, 1, 5, or 25 mg l-1) of Aroclor 1254 for different amounts of time (3 and 6 h) in vitro. Sperm motility parameters were analyzed with computer-assisted sperm analysis (CASA). The proportion of sperm with high mitochondrial membrane potential (ΔΨm) and the levels of intracellular reactive oxygen species (ROS) were detected to explore the probable cause of sperm impairment. Human sperm exposed to continuous Aroclor 1254 exhibited: (i) reduced sperm motility and kinematic parameters, (ii) a proportion of sperm with high ΔΨm that decreased in a dose-dependent manner (P < 0.05), and (iii) increased levels of ROS compared with controls (P < 0.05). In conclusion, Aroclor 1254 can decrease sperm motility, which may culminate in increased ROS and general mitochondrial dysfunction, thus affecting the fertilization potential of sperm. Our findings suggest a broader understanding of the effect of Aroclor 1254 on human sperm.

Immunogenicity and safety of an inactivated quadrivalent influenza vaccine candidate versus inactivated trivalent influenza vaccines in participants >/=3 years of age: a double-blind, randomized, parallel-controlled phase III clinical trial in China.

BACKGROUND: Viruses from two antigenically distinct influenza B strains have co-circulated since the mid-1980s, yet inactivated trivalent influenza vaccines (TIVs) with either the Victoria or Yamagata lineage could only provide limited protection from influenza B strain. Quadrivalent influenza vaccine (QIV) including both influenza B lineages can improve protection against circulating influenza B viruses. METHODS: Participants >/ = 3 years of age were recruited, stratified by age, and then randomly allocated at a ratio of 2:1:1 to receive one-injection of the experimental QIV, TIV-Victoria (Vic) or TIV-Yamagata (Yam). The primary objective of this study was to demonstrate that the hemagglutination-inhibition (HI) antibodies induced by the QIV candidate are not inferior to the licensed TIVs. RESULTS: First, 3661 participants received the inoculation. The QIV was found to be non-inferior to TIVs in terms of the geometric mean titers (GMTs) and seroconversion rates (SCRs) of the HI antibodies against shared strains 28 days after completion of inoculation, and was superior to the TIVs against the alternate B strain, which is absent from the TIVs. The occurrences of adverse events (AEs) post-vaccination were similar across the treatment groups. CONCLUSION: The experimental QIV showed good immunogenicity and an acceptable safety profile.

Different fates of donor mitochondrial DNA in bovine-rabbit and cloned bovine-rabbit reconstructed embryos during preimplantation development.

The functions of mitochondria depend on precise interaction between nuclear and cytoplasmic genomes. Non-balance of mtDNA has been reported in most nuclear transfer embryos and offspring. The reason of the degradation of donor mtDNA is still not clear. To further investigate the mechanism, in this study, we designed an experiment as follows. Two fibroblast cell lines sharing same nuclear genome but different mitochondria genome backgrounds, namely cells from ear tissues of cloned bovine and its donor, were choose as donor cells and introduced into enucleated rabbit oocytes. Similar developmental potential was observed in cloned bovine-rabbit (clone group) and bovine-rabbit (non-clone group) embryos. Real-time PCR assay showed that, in non-clone group, bovine mtDNA decreased during the development of reconstructed embryo, and that a sharp decrease was detected at the blastocyst stage. In clone group, bovine mtDNA decreased slightly, and the abrupt reduction of donor mtDNA did not occur during preimplantation development. In addition, an obvious increase in rabbit mtDNA was observed in both groups at the blastocyst stage. Our results demonstrate that: 1) the fates of donor mtDNAs in bovine-rabbit and cloned bovine-rabbit reconstructed embryos were different; and 2) recipient mtDNAs replicate at blastocyst regardless of the difference of donor cells.

Dynamic changes in NuMA and microtubules in monkey-rabbit nuclear transfer embryos.

Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.

Prediction and analysis model of temperature and its application to a natural ventilation multi-span plastic greenhouse equipped with insect-proof screen.

The natural ventilation widely used in greenhouses has advantages of saving energy and reducing expense. In order to provide information for climate control of greenhouse, a model was developed to predict the variation of air temperature in the naturally ventilated greenhouse equipped with insect-proof screen. Roof ventilation and combined roof and sidewall ventilation were considered in the model. This model was validated against the results of experiments conducted in the greenhouse when the wind was parallel to the gutters. The model parameters were determined by the least squares method. In the used model, effects of wind speed and window opening height on the air temperature variation were analyzed. Comparison between two types of ventilation showed that there existed a necessary ventilation rate which results in air temperature decrease in natural ventilation under special climatic conditions. In our experiments when wind speed was less than 3.2 ms(-1), wind had a more gradual effect on greenhouse temperature for roof ventilation, compared with combined roof and sidewall ventilation, which had greater air temperature decrease than roof ventilation only.

The DNA methylation events in normal and cloned rabbit embryos.

To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite sequence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and precociously de novo methylated, while the satellite sequence maintained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellite sequences may have aberrant methylation patterns in cloned embryos.