17ß-Trenbolone activates androgen receptor, upregulates transforming growth factor beta/bone morphogenetic protein and Wnt signaling pathways, and induces masculinization of caudal and anal fins in female guppies (Poecilia reticulata).

Sexually mature female guppies (Poecilia reticulata) were exposed to environmentally relevant concentrations (20, 200, and 2000 ng/L) of 17ß-trenbolone for four weeks. As evidenced by the increased caudal fin index and anal fins developing into gonopodium-like structures, exposed females displayed masculinized secondary sexual characteristics. Differential gene expression and subsequent pathway analysis of mRNA sequencing data revealed that the transcription of transforming growth factor beta/bone morphogenetic protein signaling pathway and Wnt signaling pathway were upregulated following 17ß-trenbolone exposure. Enzyme-linked immunosorbent assays showed that the bone morphogenetic protein 7 protein content was elevated after 17ß-trenbolone exposure. Finally, real-time PCR revealed that 17ß-trenbolone treatment significantly increased androgen receptor mRNA levels, and molecular docking showed potent interaction between 17ß-trenbolone and guppy androgen receptor. Furthermore, 17ß-trenbolone-induced masculinization of caudal and anal fins in female guppies, concomitant to the upregulated expression of differentially expressed genes involved in the above-mentioned two signaling pathways, was significantly inhibited by flutamide (androgen receptor antagonist). These findings demonstrated that 17ß-trenbolone masculinized fins of female guppies by activating the androgen receptor. This study revealed that 17ß-trenbolone could upregulate signaling pathways related to fin growth and differentiation, and eventually cause caudal and anal fin masculinization in female guppies.

Quantitative Relation between Ionic Diffusivity and Ionic Association in Ionic Liquid-Water Mixtures.

Molecular dynamic simulations of aqueous mixtures of imidazolium ionic liquids (ILs) were performed to elucidate the dependence of the ionic diffusivity on the microscopic structures changed by water. Two distinct regimes of the average ionic diffusivity (Dave) were identified with the increased water concentrations: the jam regime with slowly increased Dave and the exponential regime with rapidly increased Dave, which are found to be directly correlated to the ionic association. Further analysis leads to two general relationships independent of IL species between Dave and the degree of ionic association: (i) a consistent linear relationship between Dave and the inverse of ion-pair lifetimes (1/τIP) in the two regimes and (ii) an exponential relationship between normalized diffusivities (D̃ave) and short-ranged interactions between cations and anions (Eions), with different interdependent strengths in the two regimes. These findings revealed and quantified the direct correlation between dynamic properties and ionic association in IL-water mixtures.

[Transcriptional analysis of grape in response to weak light stress].

Grape (Vitis vinifera L.) in production is frequently exposed to inadequate light, which significantly affects its agronomic traits via inhibiting their physiological, metabolic and developmental processes. To explore the mechanism how the grape plants respond to the weak light stress, we used 'Yinhong' grape and examined their physiology-biochemistry characteristics and transcriptional profile under different levels of weak light stress. The results showed that grape seedlings upon low intensity shading treatments were not significantly affected. As the shading stress intensity was strengthened, the epidermis cells, palisade tissue, and spongy tissue in the leaves were thinner, the intercellular space between the palisade tissue and spongy tissue was larger compared with that of the control, and the activities of superoxide dismutase, catalase and peroxidase were decreased gradually. Additionally, the soluble protein content increased and the free proline content decreased gradually. Compared with the control, significant changes in plant photosynthetic characteristics and physiology-biochemistry characteristics were observed under high intensity of shading (80%). RNA-seq data showed that the differentially expressed genes between CK and T2, CK and T4, T2 and T4 were 13 913, 13 293 and 14 943, respectively. Most of the enrichment pathways were closely related with the plant's response to stress. Several signaling pathways in response to stress-resistance, e.g. JA/MYC2 pathway and MAPK signal pathway, were activated under weak light stress. The expression level of a variety of genes related to antioxidation (such as polyphenol oxidase and thioredoxin), photosynthesis (such as phytochrome) was altered under weak light stress, indicating that 'Yinhong' grape may activate the antioxidation related pathways to cope with reactive oxygen species (ROS). In addition, it may activate the expression of photosynthetic pigment and light reaction structural protein to maintain the photosynthesis activity. This research may help better understand the relevant physiological response mechanism and facilitate cultivation of grape seedlings under weak light.

mRNA-miRNA sequencing reveals mechanisms of 2,2'-dipyridyl disulfide-induced thyroid disruption in Japanese flounder (Paralichthys olivaceus).

This study was conducted to evaluate the thyroid-disrupting effects of 2,2'-dipyridyl disulfide using Japanese flounder (Paralichthys olivaceus) as an animal model and to reveal the underlying mechanisms from the perspective of miRNA-mRNA interactions. The results indicated that 2,2'-dipyridyl disulfide exposure decelerated the metamorphic progress of P. olivaceus, suggesting its thyroid-disrupting property as an antagonist. Furthermore, radioimmunoassays, thyroid histological observation, real-time polymerase chain reaction, and mRNA sequencing showed that 2,2'-dipyridyl disulfide exposure exerted its thyroid-disrupting effects on larval and juvenile P. olivaceus by targeting multiple processes and pathways involved in the thyroid system, including peripheral metabolism of thyroid hormones, the thyroid hormone synthesis pathway, and the thyroid hormone/thyroid hormone receptor signaling pathway. In particular, global upregulation of the gene expression of three deiodinases caused decreases in thyroid hormone levels after 2,2'-dipyridyl disulfide exposure that are believed to be responsible for the inhibition of metamorphosis in P. olivaceus. Finally, miRNA sequencing suggested that several evolutionarily conserved miRNAs play important roles in the mechanism of 2,2'-dipyridyl disulfide-induced thyroid disruption. Specifically, overexpression of pny-miR-723a and pny-miR-216a resulted in upregulation of deiodinase 1 mRNA levels in the 2,2'-dipyridyl disulfide exposure group. This study provides the first evidence that 2,2'-dipyridyl disulfide has thyroid-disrupting properties and is also the first study remarking on the roles of miRNA-mRNA interactions in the action mechanisms of thyroid disruptors.

Polystyrene microplastics increase estrogenic effects of 17α-ethynylestradiol on male marine medaka (Oryzias melastigma).

Microplastics (MPs) and endocrine disrupting chemicals are ubiquitous pollutants in marine environments, but their combined ecological risk is unclear. This study exposed male marine medaka (Oryzias melastigma) to 10 ng/L 17α-ethynylestradiol (EE2) alone or EE2 plus 2, 20, and 200 µg/L polystyrene MPs for 28 days to investigate the impacts of MPs on the reproductive disruption of EE2. The results showed that 10 ng/L EE2 alone did not affect biometric parameters, while co-exposure to EE2 and 20, 200 µg/L MPs suppressed the growth and decreased gonadosomatic and hepatosomatic indices. Compared to EE2 alone, EE2 plus MPs exposure significantly increased plasma 17ß-estradiol (E2) levels in a dose-dependent manner, and co-exposure to EE2 and 20, 200 µg/L MPs significantly increased the ratios of E2/testosterone (T). Moreover, EE2 plus MPs exposure elevated the transcription levels of estrogen biomarker genes vitellogenin and choriogenin, and estrogen receptor (ERα and ERß). Morphological analysis also showed that co-exposure to EE2 and MPs induced more severe damage to the testes and livers, indicating that MPs increased the toxicity of EE2. The actual EE2 concentrations in the solution increased with the exposure concentrations of MPs, suggesting that MPs changed the fate and behavior of EE2 in the seawater. These findings demonstrate that MPs could increase the estrogenic effects of EE2 on marine fish, suggesting that the combined health risk of MPs and endocrine disrupting chemicals on marine organisms should be paid great attention.

Mechanistic revealing of reproductive behavior impairment in male guppy (Poecilia reticulata) induced by environmentally realistic 2,2'-dithiobis-pyridine exposure.

Although (PS)2, the primary degradation product of emerging antifouling biocides metal pyrithiones (MePTs), can disrupt the reproductive behavior of fish at an environmentally relevant ng/L level, the underlying mechanism is still largely unknown. This study exposed sexually mature male guppy (Poecilia reticulata) to 20, 200, and 2000 ng/L (PS)2 to explore the compromised effect of (PS)2 on reproductive behavior through a realistic competing scenario. The results showed that (PS)2 suppressed male guppies' sexual interest to stimulus females, reduced their competitive behavior frequencies toward rival males, and decreased their mating time and frequency. (PS)2 exposure did not affect male guppies' secondary sexual characteristics or induce estrogenic activity. Whole-brain transcriptome sequencing identified 1070 differentially expressed genes (DEGs) with 872 up-regulated genes, which were functionally enriched into Gene Ontology terms pertaining to extracellular matrix (ECM) and extracellular region. KEGG enrichment for the DEGs uncovered that the activations of ECM-receptor interaction and focal adhesion pathways could be the underlying molecular mechanism implicated in the (PS)2 induced reproductive behavior impairment. This work would deliver a substantial contribution to the understanding of the ecological safety of MePTs biocides.

Brightened body coloration in female guppies (Poecilia reticulata) serves as an in vivo biomarker for environmental androgens: The example of 17ß-trenbolone.

In vivo testing systems for environmental androgens are scarce. The aim of this study was to evaluate the potential of male-specific brightened body coloration in female guppies (Poecilia reticulata) to serve as an in vivo biomarker of environmental androgens using 17ß-trenbolone as an example. The high bioaccumulation of 17ß-trenbolone in the skin of female guppies suggests that it is a potential target tissue of environmental androgens. The coloration index, pigment cell ultrastructure, pigment levels, sexual attractiveness, and reproductive capability of female guppies were analyzed following 28 days of exposure to 20 ng/L, 200 ng/L, and 2000 ng/L 17ß-trenbolone. Increases in the coloration index caused by 17ß-trenbolone exposure were attributable to increased pteridine and melanin levels. Decreases in the sexual attractiveness, number of offspring, and survival rate of offspring suggested that the changes in body coloration translated into adverse outcomes. Finally, mRNA sequencing indicated that 17ß-trenbolone increased pteridine levels by activating genomic effects of androgen receptor on xanthine dehydrogenase and increased melanin levels by exerting non-genomic effects targeting microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 1 that were mediated by mitogen-activated protein kinase and calcium signaling pathways. We have derived a robust adverse outcome pathway of environmental androgens, and our findings suggest that indicators at different biological levels related to brightened body coloration in female guppies can serve as less-invasive or noninvasive in vivo biomarkers of short-term exposure to environmental androgens.

Combined exposure to environmentally relevant copper and 2,2'-dithiobis-pyridine induces significant reproductive toxicity in male guppy (Poecilia reticulata).

Metal pyrithiones (MePTs), the most widely used biocides in antifouling paints (AFs) coated on the hulls, are usually used in combination with Cu-containing substances. In the aquatic environment, 2,2'-dithiobis-pyridine ((PS)2), the main degradation product of MePTs, and Cu usually coexist. However, their combined impacts on aquatic organisms are unclear. This study exposed male guppy (Poecilia reticulata) to an environmentally realistic concentration of Cu (10 µg/L) alone or Cu (10 µg/L) combined with 20, 200, and 2000 ng/L (PS)2 to explore their combined reproductive toxicity. The results showed that co-exposure to Cu and (PS)2 increased Cu accumulation in the fish body in a dose-dependent manner and induced obvious spermatozoon apoptosis and necrosis, which was mediated by the peroxidation and caspase activation. Compared to Cu alone, co-exposure to Cu and 200, 2000 ng/L (PS)2 significantly decreased the testosterone level and collapsed spermatogenesis, and depressed male's sexual interest and mating behavior were observed in three co-exposure groups. Moreover, co-exposure to Cu and (PS)2 increased the disturbance on cyp19a and cyp19b transcription and suppressed the "display" reproductive behavior. Eventually, co-exposure to Cu and (PS)2 caused male reproductive failure. Therefore, the concurrence of Cu and (PS)2 induced significant reproductive toxicity in male guppies and would threaten the sustainability of fish populations. Considering the extensive usage of MePTs products in the AFs, their ecological risk warrants more evaluation.

Adaptation of life-history traits and trade-offs in marine medaka (Oryzias melastigma) after whole life-cycle exposure to polystyrene microplastics.

Microplastics are ubiquitous in marine environments and may cause unexpected ecological effects. This study adopted a whole life-cycle exposure to illuminate the impact of polystyrene microplastics on life-history strategies of marine medaka (Oryzias melastigma), including the hatching of embryos, growth and reproduction of F0 generation, and embryonic and larval development of F1 offspring. Microplastics accumulated on the eggshell and reduced embryonic hatching rate and larval body length and weight. Similarly, 150 days of microplastic exposure decreased body mass and gonadosomatic index of adult fish, but accelerated sexual maturity of female fish, showing a trade-off between growth and reproduction. Microplastic exposure also caused obvious histopathological damages to gonads and decreased egg productions and fertilization rates. Moreover, parental microplastic exposure induced elevated heartbeats, premature hatching, and slow growth in F1 offspring. Anti-oxidative stress response, sex hormone disruption, and disturbed transcription of steroidogenic genes in the reproductive axis could partially explain the reproduction impairment and transgenerational trade-offs. Furthermore, transcriptome analysis revealed that the steroid hormone biosynthesis and cytochrome P450 pathways in the testes of male fish were significantly affected after 20 µg/L microplastic exposure. These findings suggest that microplastic pollution may be an emerging threat to the sustainability of marine fish population.

[Progress on reconstruction of proximal femur in the hemiarthroplasty for intertrochanteric fracture with distal fixated long stem].

OBJECTIVE: To summarize the application of distal fixated long stem in the treatment of intertrochanteric fracture (ITF) in the sequence of proximal femoral reconstruction, fixation materials, and other details after operation, in order to improve doctor's attention to the reconstruction of the proximal femur and reduce complications. METHODS: The related literature about the application of distal fixated long stem in the treatment of ITF was extensively reviewed, summarized, and analyzed. RESULTS: The sequence of reconstruction is divided into fracture reconstruction priority and prosthesis reconstruction priority. The former is mainly to provide an anatomical reference for the placement of joint prostheses, the latter is mainly to provide support for fracture fixation. The distal fixated cement long stem and cementless long stem have their own characteristics, and materials of reconstruction are used in combination. There is no uniform standard for the sequence and materials of reconstruction. CONCLUSION: Although the stability of the distal fixated long stem depends on the distal femur, the accurate reconstruction of the proximal femur is still worthy of attention.

New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin.

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17ß-ethinylestradiol (EE2) was measured using these three methods. Exposure to 10 and 50 ng/L EE2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments.

Tripterine up-regulates miR-223 to alleviate lipopolysaccharide-induced damage in murine chondrogenic ATDC5 cells.

Tripterine, also known as celastrol, is a main natural ingredient in Tripterygium wilfordii. Tripterine has a variety of pharmacological functions, and the therapeutic potential of tripterine in many kinds of inflammation-linked diseases has been revealed. However, the function of tripterine on osteoarthritis still remains unclear. The objective of this study was to study the function of tripterine (TPR) on lipopolysaccharide (LPS)-injured chondrocyte. ATDC5 cells were treated with tripterine after LPS stimulation and then cell survival, the release of pro-inflammatory cytokines, and the expression of chondrogenic differentiation-associated proteins were assessed by performing CCK-8, flow cytometry, reverse transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Moreover, the expression of miR-223 and core factors in PI3K/AKT and nuclear factor kappa B (NF-κB) signaling was tested by RT-qPCR/Western blot. LPS stimulation significantly reduced ATDC5 cells viability, induced apoptosis, and increased the release of interleukin (IL)-6 and tumor necrosis factor (TNF)-α. Tripterine protected ATDC5 cells against LPS-induced chondrocyte loss and the release of IL-6 and TNF-α. miR-223 was down-regulated by LPS, while was up-regulated by tripterine. The protective actions of tripterine were eliminated when miR-223 was silenced. Besides, tripterine inhibited hypertrophic differentiation induced by LPS, and the inhibitory effects of tripterine on hypertrophic differentiation could be abolished when miR-223 was silenced. Furthermore, tripterine activated PI3K/AKT pathway and deactivated NF-κB pathway. And the regulatory effects of tripterine on these two pathways were abolished by miR-223 silence. This study revealed that tripterine protected ATDC5 cells against LPS-induced cell damage possibly via up-regulation of miR-223 and modulation of NF-κB and PI3K/AKT pathways.

2,2'-Dithiobis-pyridine induced reproductive toxicity in male guppy (Poecilia reticulata).

Metal pyrithiones (MePTs) are frequently used antifouling biocides in marine coatings. Their main degradation product, 2,2'-dithiobis-pyridine ((PS)2), has been widely detected in seawater and may pose potential ecological risks. In the present study, sexually mature guppies (Poecilia reticulata) were exposed to (PS)2 at concentrations of 0, 20, 200, and 2000 ng/L for 28 days to investigate its reproductive toxicity. The results showed that (PS)2 significantly reduced testosterone (T) levels, spermatogenic cyst number and sperm motility, impeded spermatogenic cell differentiation in male guppies and delayed embryo development in females. These results indicated that (PS)2 could cause reproductive toxicity in guppies. We also examined mRNA expression of indices involved in the hypothalamic-pituitary-gonadal axis and reproductive behaviors. We found that 200 and 2000 ng/L (PS)2 decreased T synthesis by downregulating 17ßHSD and CYP17 mRNA levels, and upregulating the mRNA level of CYP19a1a, which converted T to 17ß-estradiol. (PS)2 also upregulated GnRH1, FSHß, LHß, and LHR mRNA levels, a positive feedback regulation due to the decrease of T levels in male guppies. Furthermore, (PS)2 significantly decreased CYP19a1b mRNA levels in all three exposure groups and thus reduced the display frequency of male guppies. This study was the first to report that (PS)2 could induce reproductive toxicity, which would provide a basis for future assessment of its ecological risk.

Oxidative damage induced by copper in testis of the red swamp crayfish Procambarus clarkii and its underlying mechanisms.

Copper (Cu) is one of the most widespread environmental pollutants and is known to exert multiple toxic effects including reproductive toxicity. In this study, we investigated the toxic effect of Cu on reproduction of the red swamp crayfish (Procambarus clarkii), an economic crustacean species, by exposing adult male crayfish to 0.03 and 3.00 mg/L Cu2+ for 7 days. The results showed that Cu2+ exposure induced oxidative stress accompanied by elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels in testes, and resulted in decreased sperm quality and abnormal testicular structures with apoptotic germ cells and vacuolisation in Sertoli cells. To reveal the molecular mechanism of Cu2+-induced oxidative damage in crayfish testes, we sequenced, assembled and annotated the transcriptome for crayfish testes, using the Illumina sequencing approach. After the 3.00 mg/L Cu2+ treatment, 6745 genes with differentially expressed profile were identified, of which many genes were involved in cellular response to ROS based on Gene Ontology enrichment analysis. Further, KEGG analysis demonstrated that genes with up-regulated expression levels significantly enriched in mitochondria oxidative phosphorylation pathway, suggesting disturbed mitochondrial electron transport chain was probably a main source of Cu2+-induced ROS production in testes. This study represented the first use of transcriptome to investigate the toxic effect of Cu2+ on male crayfish reproduction, and the pathways identified underlying Cu2+ toxicity at molecular level provide a novel insight into the reproductive toxicity of Cu in crustaceans.

MiR-25 promotes proliferation, differentiation and migration of osteoblasts by up-regulating Rac1 expression.

According to many studies, miRNAs are involved in the control of bone cell differentiation and function. Hence, an understanding of the pathways regulated by miRNAs involved in skeletal function is essential for the development of miRNA-based therapeutic strategies for bone diseases. In this study we evaluated the role of miR-25 in osteoblast differentiation by examining the expressions of key osteoblast differentiation markers like Runx2 and Ocn and also evaluated the effects of miR-25-Rac1 axis on PI3K/AKT and JNK pathways. MC3T3-E1 osteoprogenitors were treated with osteogenic differentiation media which was refreshed every 48?h after the initial differentiation treatment and were then quantified for total miRNA content. The viability, migration as well as Runx2 and Ocn expressions in cells transfected with miR-25 mimic, miR-25 inhibitor, and si-Rac1 were evaluated by CCK-8 assay, wound migration assay, qRT-PCR and Western blot. Finally, the effects of miR-25-Rac1 axis on PI3K/AKT and JNK pathways were studied. MiR-25 was found to significantly enhance cell viability and migration and up-regulate the expressions of Runx2 and Ocn. MiR-25 was also found to enhance the expression levels of Rac1, which contributed to the effects of miR-25 on osteoblastic cell lines. MiR-25 activated PI3K/AKT and JNK pathways possibly by up-regulation of Rac1. Enhanced expression of miR-25 led to the promotion of cell viability and migration, as well as up-regulation of Runx2 and Ocn markers by enhancing Rac1 expression.

Aspirin inhibits the proliferation and migration of gastric cancer cells in p53-knockout mice.

The aim of the present study was to investigate the effect of aspirin on the cell proliferation and migration of gastric cancer cells in p53-knockout mice. Twenty p53-/- male mice aged 6 to 7 weeks, with an average weight of 20±3 g were used. The model of gastric cancer was established by the implantation of a mouse forestomach carcinoma cell line, subcutaneously, at the back of the neck, and then the mice were randomly divided into two groups after establishment of the model (control group, n=10; experimental group, n=10). Aspirin (250 mg/kg) was added to the food in the experimental group one day before model establishment, until the end of the experiment. Mice in the control group were given regular food without aspirin. All mice were sacrificed 3 months afterwards, and gastric cancer tissues were harvested. MTT assay was used to detect the proliferation of the tumor cells. Tumor cell number was also observed. Migration ability was detected by scratch assay, and E-cadherin protein expression was evaluated by immunofluorescence. The results revealed that the proliferation ability of tumor cells in the experimental group was lower than that in the control group. In addition, cell numbers were significantly decreased and the migration ability was diminished. The expression of E-cadherin was also increased in the experimental group, and the differences were statistically significant (P<0.05). In conclusion, aspirin inhibited the cell proliferation and migration of gastric cancer cells in mice.

Effect of low intensity pulsed ultrasound on expression of TIMP-2 in serum and expression of mmp-13 in articular cartilage of rabbits with knee osteoarthritis.

OBJECTIVE: To study the effect of low intensity pulsed ultrasound (LIPUS) on the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the serum and expression of matrix metallopeptidase 13 (MMP-13) in the articular cartilage cells of rabbits with knee osteoarthritis (OA). METHODS: Inner patellar ligament defect method was used to establish the model of knee OA. Four weeks after the modeling, the arterial blood was drawn from the ear of each rabbit, while ELISA was employed to detect the expression of TIMP-2 in the serum. The chondrocytes were separated from animals in each group and then cultured in vitro. All rabbits were divided into control group, OA model group and OA + LIPUS group. Cells in the control and OA groups were not treated, while cells in the OA + LIPUS group were treated with LIPUS (40 mW/cm(2), 1 time/day). Cells were collected 7 d later and the RNA and total protein were extracted respectively. Real-time PCR and Western blotting were employed to analyze the expression of MMP-13 in chondrocytes at the mRNA and protein level, respectively. RESULTS: The success rate of establishment of OA model was 83%. The results of ELISA showed that the content of TIMP-2 in the serum of animals with OA was 22.3%, lower than the one in the control group (P < 0.05). Compared with the normal control group, the expression of TIMP-2 in the OA model group was significantly increased, while the expression of MMP-13 was significantly increased (P < 0.05). After the stimulation of LIPUS, the expression of TIMP-2 and MMP-13 was close to the one in the normal control group. CONCLUSIONS: The inner patellar ligament defect method is a mature method to establish the rabbit OA model, with high success rate. The expression of serum TIMP-2 in the OA model group is significantly decreased. LIPUS can up-regulate TIMP-2 and down-regulate MMP-13.

A novel FBN2 mutation in a Chinese family with congenital contractural arachnodactyly.

Congenital contractural arachnodactyly (CCA, OMIM: 121050) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS, OMIM: 154700), including contractures, arachnodactyly, dolichostenomelia, scoliosis, crumpled ears and pectus deformities but excluding the ocular and cardiovascular complications that characterize MFS. These two similar syndromes result from mutations in two genes belonging to the fibrillin family, FBN1 and FBN2, respectively. We successfully identified a novel FBN2 mutation (C1406R) in a Chinese family with CCA for over five generations. This mutation was detected in the patients of this family but not in the seven unaffected family members or 100 normal individuals. SIFT and PolyPhen analyses suggested that the mutation was pathogenic. We identified a missense mutation in the calcium binding-epidermal growth factor (cbEGF)-like domain. Our study extends the mutation spectrum of CCA and confirms a relationship between mutations in the FBN2 gene and the clinical findings of CCA.

Two novel mutations in myosin binding protein C slow causing distal arthrogryposis type 2 in two large Han Chinese families may suggest important functional role of immunoglobulin domain C2.

Distal arthrogryposes (DAs) are a group of disorders that mainly involve the distal parts of the limbs and at least ten different DAs have been described to date. DAs are mostly described as autosomal dominant disorders with variable expressivity and incomplete penetrance, but recently autosomal recessive pattern was reported in distal arthrogryposis type 5D. Mutations in the contractile genes are found in about 50% of all DA patients. Of these genes, mutations in the gene encoding myosin binding protein C slow MYBPC1 were recently identified in two families with distal arthrogryposis type 1B. Here, we described two large Chinese families with autosomal dominant distal arthrogryposis type 2(DA2) with incomplete penetrance and variable expressivity. Some unique overextension contractures of the lower limbs and some distinctive facial features were present in our DA2 pedigrees. We performed follow-up DNA sequencing after linkage mapping and first identified two novel MYBPC1 mutations (c.1075G>A [p.E359K] and c.956C>T [p.P319L]) responsible for these Chinese DA2 families of which one introduced by germline mosacism. Each mutation was found to cosegregate with the DA2 phenotype in each family but not in population controls. Both substitutions occur within C2 immunoglobulin domain, which together with C1 and the M motif constitute the binding site for the S2 subfragment of myosin. Our results expand the phenotypic spectrum of MYBPC1-related arthrogryposis multiplex congenita (AMC). We also proposed the possible molecular mechanisms that may underlie the pathogenesis of DA2 myopathy associated with these two substitutions in MYBPC1.

[Effects of light intensity on associated enzyme activity and gene expression during callus formation of Vitis vinifera].

We analyzed the best light intensity for callus induction and maintenance in Vitis vinifera and explored the mechanism of grape callus browning. Tender stem segments of grape cultivar "gold finger" were used to study the effects of different light intensities (0, 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 4 000 Lx) on the induction rate, browning rate and associated enzyme activity and gene expression during Vitis vinifera callus formation. The callus induction rate under 0, 500, 1 000 and 1 500 Lx was more than 92%, significantly higher than in other treatments (P < 0.05). A lower browning rate and better callus growth were also observed during subculture under 1 000 and 1 500 Lx treatments. We found that chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid contents were correlated with the browning rate of callus, among which chlorogenic acid content was positively correlated with the browning rate (P < 0.05). Peroxidase (POD) and polyphenol oxidase (PPO) activities were negatively correlated with the browning rate of callus (P < 0.01). The POD, PPO and phenylalanine ammonialyase (PAL) expression levels were positively correlated with the browning rate at P < 0.05 or P < 0.01. An appropriate light intensity for the tissue culture of Vitis vinifera was 1 000-1 500 Lx, higher or lower light intensities significantly impaired normal callus growth.