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Pseudomonas aeruginosa is an opportunistic pathogen, which usually presents multiple antibiotic resistance. Host-directed therapy involves modulating the host defense system and the interplay between innate and adaptive immunity is a new strategy for designing anti-infection drugs. Memantine (MEM), a drug used to treat Alzheimer's disease, has a good inhibitory effect on neonatal mice with Escherichia coli-associated bacteremia and meningitis; however, the inhibitory effect and mechanisms of MEM against P. aeruginosa infection remain unclear. Here, we investigated whether MEM could inhibit P. aeruginosa infection and explored the potential mechanisms. MEM significantly promoted the bactericidal effect of neutrophils against P. aeruginosa and its drug-resistant strain. The combination index of MEM and amikacin (AMK) was

The present study analyzed the relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures (VTs) and cryoprotectant agent concentrations (CPAs). Cumulus oocyte complexes were randomly divided into five groups: control, vitrified in liquid nitrogen (LN; -196 °C) with 5.6 M CPAs (LN 5.6 M), LN with 6.6 M CPAs (LN 6.6 M), liquid helium (LHe; -269 °C) with 5.6 M CPAs (LHe 5.6 M), and LHe with 6.6 M CPAs (LHe 6.6 M). After vitrification and warming, oocytes of vitrified and control groups were subjected to in vitro maturation (IVM), in vitro fertilization and in vitro culture. The blastocyst rate in LHe 5.6 M group was the highest among the four vitrified groups (13.7% vs. 9.4%, 1.3%, and 8.4%; P 0.05). The mRNA expression level of 8 apoptotic- and 12 mitochondria-related genes were detected through qRT-PCR after IVM. Lower VT (LHe, -269 °C) positively affected the mRNA expression levels of apoptotic genes (BAD, BID, BTK, TP53, and TP53I3) and mitochondrial genes (COX6B1, DERA, FIS1, NDUFA1, NDUFA4, PRDX2, SLC25A5, TFB1M, and UQCRB), and reduced oxidative stress from freezing. Decreased CPAs (5.6 M) positively affected mRNA expression levels of apoptotic genes (BAD, BCL2A1, BID, and CASP3) in LHe vitrification but negatively affected apoptotic genes (BAD, BAX, BID, BTK, and BCL2A1) in LN vitrification. In conclusion, decreased VTs and CPAs in LHe vitrification may increase the blastocyst rate by changing the mRNA expression levels of these apoptotic and mitochondrial genes for the vitrified oocytes.

Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.

The present study aimed to investigate the effect of vitrification temperature (VT) and cryoprotective agent concentrations (CPAs) on the mRNA transcriptome of bovine mature oocytes after vitrifying at immature stage. Cumulus oocyte complexes (COCs) were randomly divided into the following five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; -269 °C) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; -196 °C) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). We performed two experiments in this study. In experiment 1, after vitrification and thawing, oocytes of vitrified and control groups were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The rates of normal morphology, maturation, cleavage, and blastocyst formation in LHe 5.6 M were higher than those in LN 5.6 M (P 0.05). The rates of normal morphology and cleavage in LHe 6.6 M were higher than those in LN 6.6 M (P 0.05). However, the maturation and blastocyst rates were similar (P 0.05) between LHe 6.6 M and LN 6.6 M. The blastocyst rate of 13.31% in LHe 5.6 M was the highest among all vitrified groups (P 0.05). In experiment 2, the mRNA transcriptome of each sample was analyzed by Smart-Seq4, and the differentially expressed genes (DEGs) were detected by edgeR (P ≤ 0.05; fold-change ≥ 2). A total of 505 DEGs (342 upregulated and 163 downregulated genes) were detected in LHe 5.6 M; 609 DEGs (493 upregulated and 116 downregulated genes) were detected in LHe 6.6 M; 218 DEGs (101 upregulated and 117 downregulated genes) were determined in LN 5.6 M; and 221 DEGs (104 upregulated and 117 downregulated genes) were detected in LN 6.6 M. LHe vitrification affected the mRNA transcriptome of bovine mature oocytes after vitrifying at immature stage mainly by upregulating gene expression. Decreased CPAs (5.6 M) reduced the effect of vitrification on mRNA transcriptome when LHe vitrification was used. Among the DEGs closely related to bovine oocytes, the genes possibly related to VT were ND2, MPV17L2, PIF1, LPIN1, IMP3, BRD1, DCTN3, DERA, ATP7B, NEK5, HVCN1, and MARK2. The gene that may be associated with CPAs is CC2D2A. Genes that may be affected by VT and CPAs included PGK1, SLC7A3, FITM2, NPM3, ISCU, CWC15, and PSAP.

Podophyllotoxin (PPT) is a lignan extracted from podophyllum genera and it shows potent antitumor activity since it could effectively inhibit the assembly of microtubule in tumor cells. However, the effects of podophyllotoxin exposure on porcine oocyte quality is still unclear. In present study we tried to examine whether podophyllotoxin exposure was toxic to porcine oocyte maturation. Our results showed that podophyllotoxin exposure inhibited porcine oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on porcine oocytes was dose-depended. Moreover, the meiotic spindle formation was disturbed and the chromosomes were misaligned in the podophyllotoxin-treated porcine oocytes. However, there was no different expression for p-MAPK and ace-tubulin between the control and podophyllotoxin treatment group. In addition, after 0.01 μM podophyllotoxin treatment, the intracellular reactive oxygen species (ROS) levels and the Annexin-V signal at MI stage significantly increased compared to the control group, indicating the occurrence of oxidative stress and early apoptosis. Taken together, our results suggested that the toxic effects of podophyllotoxin exposure on porcine oocyte maturation might be through its effects on spindle formation and the induction of oxidative stress-mediated early apoptosis.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l -carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

SummaryKaempferol (KAE) is one of the most common dietary flavonols possessing biological activities such as anticancer, anti-inflammatory and antioxidant effects. Although previous studies have reported the biological activity of KAE on a variety of cells, it is not clear whether KAE plays a similar role in oocyte and embryo in vitro culture systems. This study investigated the effect of KAE addition to in vitro maturation on the antioxidant capacity of embryos in porcine oocytes after parthenogenetic activation. The effects of kaempferol on oocyte quality in porcine oocytes were studied based on the expression of related genes, reactive oxygen species, glutathione and mitochondrial membrane potential as criteria. The rate of blastocyst formation was significantly higher in oocytes treated with 0.1 µm KAE than in control oocytes. The mRNA level of the apoptosis-related gene Caspase-3 was significantly lower in the blastocysts derived from KAE-treated oocytes than in the control group and the mRNA expression of the embryo development-related genes COX2 and SOX2 was significantly increased in the KAE-treated group compared with that in the control group. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased after KAE treatment. Mitochondrial membrane potential (ΔΨm) was increased and the activity of Caspase-3 was significantly decreased in the KAE-treated group compared with that in the control group. Taken together, these results suggested that KAE is beneficial for the improvement of embryo development by inhibiting oxidative stress in porcine oocytes.

L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm−1 (phenylalanine) and 1529 cm−1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells.

The developmental potential and the changes in cytoskeleton structures in immature oocytes of cattle resulting from liquid helium (LHe) vitrification was evaluated in this study. Immature oocytes were randomly divided into three groups: fresh oocytes (negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In Experiment 1, the proportions for normal morphology, maturation, cleavage, and blastocyst were greater in the LHe group than in the LN group (88.3% compared with 79.1%, 51.7% compared with 43.3%, 42.6% compared with 33.0%, and 11.0% compared with 4.7%, respectively; P

// Yu Wu 1 , Na Zhang 1 , Ying-Hua Li 2 , Liang Zhao 3 , Mo Yang 1 , Yimei Jin 1 , Yong-Nan Xu 2 and Hongyan Guo 1 1 Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, P.R. China 2 College of Agriculture, Yanbian University, Yanji 133002, P.R. China 3 Department of Obstetrics and Gynecology, Beijing Jishuitan Hospital, Beijing 100035, P.R. China Correspondence to: Hongyan Guo, email: bysyghy@163.com Yong-Nan Xu, email: xuyongnan@ybu.edu.cn Keywords: citrinin, oocyte, cytoskeleton, apoptosis, oxidative stress Received: November 30, 2016 Accepted: January 11, 2017 Published: February 28, 2017 ABSTRACT Citrinin is one of the mycotoxins and has been shown to have various toxic effects in animals and humans. Although previous study showed the toxic effects of citrinin on the female reproductive system, especially on oocyte maturation, however, the causes or mechanism of citrinin on oocyte quality is unclear. In present study we deeply investigated this topic. We found thatcitrinin toxin exposure inhibited mouse oocyte maturation and early embryo development. Further investigation showed that the actin distribution in oocytes and embryos was disrupted, and the reduced expression of actin nucleator ARP2 expression in the oocyte cortex further confirmed this. We also found that meiotic spindle morphology was abnormal after citrinin treatment. These results indicated that citrinin toxin exposure could disrupt cytoskeleton dynamics to affect oocyte maturation and early embryo development. We also examined the ROS level and early apoptosis marker Annexin signals, and the results showed that both levels increased, indicating that citrinin induced oxidative stress and further resulted in oocyte early apoptosis. Taken together, our results indicated that citrinin toxin exposure could reduce mouse oocyte maturation and early embryo development capability by affecting cytoskeletal dynamics, which may be due to the oxidative stress induced early apoptosis.

This study aimed to investigate the developmental potential of and the ultrastructural changes and gene expression differences resulting from liquid helium (LHe; -269 °C) vitrification in immature bovine oocytes. Immature oocytes were randomly divided into three groups: fresh oocytes (control, negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In experiment 1, the rates of normal morphology, maturation, cleavage, and blastocyst in the LHe group were higher than those in the LN group (87.1% vs. 80.5%, 51% vs. 48%, 41.7% vs. 36.8%, and 13% vs. 8.5%, respectively; P 0.05), and the rates of development in the control group (100%, 73.2%, 62%, and 39.8%) were higher than those in the treated groups (P 0.05). In experiment 2, oocytes displayed various degrees of injury at the ultrastructural level after vitrification, but more severe degeneration was observed in the LN group, such as formation of several lipid droplets, swelling of mitochondria, and absence of cortical granules. Compared with the LN group, fewer lipid droplets, relatively intact mitochondria, and clustered cortical granules were distributed in the cytoplasm of oocytes in the LHe group. In experiment 3, the mRNA expression levels of p53, CDC20, Eg5, and Npm2 were investigated by real-time quantitative polymerase chain reaction. Expression levels of the kinesin Eg5 and the apoptotic gene p53 expression levels were higher in the LN group compared with the control and LHe groups (P 0.05). CDC20 and Npm2 expression did not differ significantly between the LN and LHe groups (P0.05), the CDC20 expression in the LN and LHe groups were lower than control group (P0.05), the Npm2 expression in LHe group was lower than control group (P0.05), but there was no significant difference between the LN and control groups (P0.05). In conclusion, LHe vitrification decreased the negative effect of cryoinjury on the ultrastructure of some organelles and the expression of some related genes, thereby improving the viability of immature bovine oocytes compared to LN vitrification.

The Arp2/3 complex, which nucleates actin filaments, comprises a stable assembly of seven-protein subunits including two actin-related proteins (Arp2 and Arp3). Previous work showed that Arp2/3 binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. In the present study, we show that the Arp2/3 complex is critical for cytokinesis during early embryonic development in porcine parthenotes. The Arp2/3 complex is concentrated at the cortex of each cell at the 1-, 2-, and 4-cell stages, and at the periphery at the morula stage. The amount of Arp2/3 significantly decreased at the blastocyst stage in parthenogenetically activated porcine embryos. Inhibition of the Arp2/3 complex in the pig embryos by the Arp2/3-specific inhibitor CK666 resulted in abnormal cell division, a decrease in developmental rate and total cell numbers, and an increase in the ratio of trophectoderm cell number to inner cell mass number in blastocyst-stage embryos. In addition, 4-cell stage e...

The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P0.05) although the corresponding rates in both treated groups decreased compared with the control group (100%, 75.0%, 64.9%, and 40.8%; P0.05). Normal calves were obtained after the transfer of blastocysts derived from LHe- and LN2-vitrified oocytes. The effects of the different vitrification solutions (EDS30, EDS35, EDS40, EDS45, and EDS50) in LHe vitrification for bovine immature oocytes vitrification were examined. No difference was found in the rates of morphologically normal oocytes among the EDS30 (87.9%), EDS35 (90.1%), EDS40 (89.4%), and EDS45 (87.2%) groups (P0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P0.05). No significant differences were observed in the cleavage and blastocyst rates between the EDS35 (49.0% and 12.1%) and EDS40 (41.7% and 10.2%) groups. However, the cleavage and blastocyst rates in the EDS35 group were higher (P0.05) than those of the EDS30 (36.2% and 6.8%), EDS45 (35.9% and 5.8%), and EDS50 (16.6% and 2.2%) groups. In conclusion, LHe can be used as a cryogenic liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study.

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression.

BACKGROUND AND PURPOSE: Nodular goiter is one of the most common benign lesions in thyroid nodule. The main treatment of the disease is still the traditional surgical resection, however there are many problems such as general anesthesia, surgical scar, postoperative thyroid or parathyroid function abnormalities, and high nodules recurrence rate in residual gland. The purpose of this study was to compare the efficacy, safety, and cost-effectiveness of 2 treatment methods, surgery and radiofrequency ablation, for the treatment of benign thyroid nodules. MATERIALS AND METHODS: From May 2012 to September 2013, 200 patients with nodular goiters who underwent surgery (group A) and 200 patients treated by radiofrequency ablation (group B) were enrolled in this study. Inclusion criteria were the following: 1) cosmetic problem, 2) nodule-related symptoms, 3) hyperfunctioning nodules related to thyrotoxicosis, and 4) refusal of surgery (for group B). An internally cooled radiofrequency ablation system and an 18-ga internally cooled electrode were used. We compared the 2 groups in terms of efficacy, safety, and cost-effectiveness during a 1-year follow-up. RESULTS: After radiofrequency ablation, the nodule volume decreased significantly from 5.4 to 0.4 mL (P = .002) at the 12-month follow-up. The incidence of complications was significantly higher from surgery than from radiofrequency ablation (6.0% versus 1.0%, P = .002). Hypothyroidism was detected in 71.5% of patients after surgery but in none following radiofrequency ablation. The rate of residual nodules (11.9% versus 2.9%, P = .004) and hospitalization days was significantly greater after surgery (6.6 versus 2.1 days, P < .001), but the cost difference was not significant. CONCLUSIONS: Surgical resection and radiofrequency ablation are both effective treatments of nodular goiter. Compared with surgery, the advantages of radiofrequency ablation include fewer complications, preservation of thyroid function, and fewer hospitalization days. Therefore, radiofrequency ablation should be considered a first-line treatment for benign thyroid nodules.

Objectives We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. Methods Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. Results Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. Significance PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

ObjectivesMammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo.MethodsInducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51–like kinase 1 (ULK1) siRNA to determine mTOR signalling.ResultsmTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage.ConclusionsThis study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

Mitochondria are major powerhouses in all eukaryotic cells, producing ATP through oxidative phosphorylation and the citric acid cycle. An increase in ATP production is required during oocyte maturation, fertilization, and early embryo development in mammals [1, 2]. Previous studies have also reported an association between mitochondrial DNA (mtDNA) copy number and oocyte quality during maturation [1, 3]. For example, mtDNA copy number is an indicator of fertilization potential and oocyte maturation, and oocytes with a low mtDNA copy number have a significantly lower developmental potency [4, 5]. mtDNA copy number also increases during the in vitro maturation of porcine oocytes and after the treatment of oocytes with follicular fluids or EGF (epidermal growth factor), which likely affects the developmental potential of oocytes [6]. Mitochondrial membrane potential (Δφm) is also critical for the production of ATP. During oocyte maturation, there is a significant increase in mitochondrial Δφm [7], and in the absence of an increase, the developmental potential of oocytes decreases [8, 9]. In addition, a high mitochondrial Δφm in mouse and human oocytes and early preimplantation stage embryos is associated with ionic and metabolic regulation [10]. To date, few maternal genes in mammalian oocytes have been characterized. Among these maternal transcripts, C-mos, Cyclin B1, Cdc2 (cell division cycle 2), Gdf9 (growth differentiation factor 9), and Bmp15 (bone morphogenetic protein 15) are well-studied genes considered to be markers of female germ cells. One of the essential regulators of meiosis resumption is formed by Cyclin B1 and Cdc2 kinase [11]. It has been reported that the dynamic change in levels of cyclin B1 is mainly controlled by cytoplasmic polyadenylation during mouse [12] and bovine [13] oocyte maturation. GDF9 and BMP15 belong to the transforming growth factor-β (TGF-β) superfamily, which contains many members with important roles in regulating fertility [14]. GDF9 and BMP15 were recently identified as oocyte-secreted factors involved in folliculogenesis and oocyte maturation, as well as in cooperative regulation of granulosa cells [15]. Recently Ge et al. [16] reported a connection between mouse oocyte quality and both mitochondrial metabolic activity and DNA copy number, specifically with spindle formation, chromosomal alignment, and embryo development. However, the underlying molecular mechanism has not been addressed. In vitro maturation of pig oocytes is useful in the study of the molecular mechanisms that underlie meiosis and fertilization as well as in the production of cloned and transgenic embryos and pigs [17,18,19]. While in vitro culture conditions and/or micromanipulations such as enucleation and injection of DNA or sperm can affect mitochondrial activity in oocytes from several species, this information is not available for porcine oocytes. To determine the effects of mitochondrial metabolic activity and mtDNA copy number on oocyte maturation and developmental competence, we treated immature porcine oocytes with FCCP, which inhibited mitochondrial oxidative phosphorylation, and ddC (2’3-dideoxycytidine), which depleted mtDNA. The effects of these two inhibitors on oocyte dynamics were assessed by determining the mitochondrial Δφm, mtDNA copy number, ATP concentration, target mRNA expression and poly(A) tail length of maternal genes. P34cdc2 kinase activity and mitogen-activated protein kinase (MAPK) phosphorylation were also investigated following FCCP and ddC treatment. To the best of our knowledge, this is the first report to address the relationship among mitochondrial Δφm and copy number, the in vitro maturation of porcine oocytes, and the developmental competence of embryos.

Rationale and Objectives Previous research has determined that carotid abnormalities (CAs) are partly located >3 cm above the carotid bifurcation. However, identifying CAs occurring in this location using high-frequency linear probes is difficult. The aim of this study was to explore the efficacy of a combination of high-frequency and low-frequency probes in duplex ultrasonography for identifying morphologic features of the internal carotid artery (ICA). Materials and Methods A total of 1055 ICAs from 532 patients were analyzed. CAs were classified prospectively according to the criteria of Wain et al, Weibel and Fields, and Metz et al. The diagnostic rates of high-frequency ultrasonography alone and a combination of high-frequency and low-frequency ultrasonography were compared. The distances from the carotid bifurcation to the initial point of the CA and to the mandibular angle were also measured. Results High-frequency ultrasonography detected 23.2% of CAs, and the combination detected 32.2% of CAs (P 2.5 and >3 cm above the carotid bifurcation, respectively. The distance between the carotid bifurcation and the mandibular angle was Conclusions Combining high-frequency and low-frequency probes is better than high-frequency probes alone to assess morphologic features of the ICA in duplex ultrasonography. Low-frequency convex probes should be added to traditional ultrasonography for the evaluation of morphologic features of the ICA before carotid endarterectomy, carotid artery stenting, and CA angioplasty.

Objective To observe the clinical effects of the warm-dredging needling method for cerebral infarction sequela.

Study question What is the function of Spindlin 1 (Spin1) in metaphase II stage oocytes in pigs? Summary answer Depletion of Spin1 induces spontaneous oocyte activation and overexpression of Spin1 causes multinuclear formation through induction of DNA damage in porcine oocytes. What is known already Little is known about the function of Spin1 in oocytes and embryos. In mouse oocytes, Spin1 is specifically expressed during gametogenesis and is essential for meiotic resumption. In somatic cells, Spin1 promotes cancer cell proliferation and activates WNT/T-cell factor signaling. Study design size, duration After knockdown (KD) or overexpression of Spin1 in porcine MII-stage oocytes, MII maintenance was checked following additional culture for 24 h. Investigated parthenotes were cultured up to the four cell (72 h) or blastocyst (7 days) stages. Participants/materials, setting, methods Spin1 was knocked down in porcine oocytes and embryos via microinjection of pig Spin1-targeting siRNA. For Spin1 overexpression, porcine Spin1-eGFP cRNA was generated. Additionally, for rescue experiments, cRNA encoding siRNA-resistant mouse Spin1 was added to the pig Spin1-targeting siRNA. For the overexpression and rescue experiments, microinjection and culture were performed using the same methods as the KD experiments. Main results and the role of chance KD of Spin1 in MII-stage porcine oocytes reduced metaphase-promoting factor and mitogen-activated protein kinase activities, resulting in spontaneous pronuclear formation without calcium activation. However, the DNA damage response was triggered by Spin1 overexpression, generating the checkpoint protein γH2A.X. Furthermore, Spin1 overexpression blocked metaphase-anaphase transition and led to multinucleation in oocytes and embryos. Large scale data None. Limitations, reasons for caution This study is based on in vitro investigations with abnormal expression levels of Spin1. This may or may not accurately reflect the situation in vivo. Wider implications of the findings Spin1 is essential to maintain MII arrest, but a high level of Spin1 induces DNA damage in oocytes and embryos. Thus, a system to accurately regulate Spin1 expression operates in porcine MII-stage oocytes and embryos. Study funding and competing interest(s) This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01057629). The authors declare no competing financial interests.

Background The unfolded protein response (UPR) is activated following an endoplasmic reticulum (ER) stress. The aim of this study was to investigate the global expression of UPR genes in human OA chondrocytes in induced (I)-UPR conditions, and to explore the regulation and role of the UPR genes in homeostatic (H)-UPR conditions in human normal and OA chondrocytes. Methods Gene expression was determined by PCR array and qPCR. Protein production in cartilage was determined by immunohistochemistry, gene silencing by specific siRNAs, and gene regulation by treating chondrocytes with cytokines and growth factors associated with cartilage pathobiology. Results Several UPR genes, among them ERN1, PERK, and CREB3L2 were downregulated in OA compared to normal chondrocytes at both the mRNA and protein levels, but the ER stress response triggered by thapsigargin or tunicamycin treatment was similar in normal and OA chondrocytes. The activation of ER stress sensors (phosphorylated PERK, cleavage of ATF6B, and the spliced mRNA forms of XBP1) was not significantly increased in OA chondrocytes/cartilage. PDGF-BB and IL-6 significantly downregulated the expression of ERN1, PERK, and CREB3L2, but not that of ATF6B. Silencing experiments done under conditions of no ER stress (physiological conditions) revealed that decreasing ERN1 expression led to decreased COL2a1, MMP-13, ADAMTS4 and ADAMTS5 expression, while decreasing CREB3L2 and ATF6B led to decreased ADAMTS5 and ADAMTS4 expression, respectively. Importantly, the downregulation of PERK expression increased COL1a1 and suppressed COL2a1 expression. Conclusions Although the level of ER stress is not significantly increased in OA chondrocytes, these cells respond strongly to an acute ER stress despite the decreased expression of ERN1, PERK, and CREB3L2. Emerging findings revealed for the first time that these genes play a role in cartilage biology in conditions where an acute ER stress response is not triggered and OA is not characterized by an overall basal activation of the ER stress response. Importantly, these findings identify PERK as a potential target for new OA treatment avenues. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1070-6) contains supplementary material, which is available to authorized users.

Purpose To assess the benefits of using a low-frequency convex probe and detecting ophthalmic artery blood flow for the differential diagnosis of occlusion from severe stenosis of the internal carotid artery (ICA). Methods A total of 103 patients were enrolled and had three sequential examinations by an experienced sonographer. First, the proximal ICA was examined with a 5.0–7.5-MHz linear probe. Second, the distal ICA was examined with a 3.5–5.0-MHz convex probe. Third, the ophthalmic artery (OA) was examined with a 5.0–7.5-MHz linear probe. Three parallel sets of diagnoses were made based on results from (1) high-frequency ultrasonography; (2) combined use of high- and low-frequency ultrasonography; and (3) combined use of high- and low-frequency ultrasonography along with OAs examination findings. Efficiencies of the three diagnoses were compared by receiver operating characteristic curves with digital subtraction angiography as the gold standard. Results The second diagnostic approach yielded the highest sensitivity (96.6%) and specificity (94.5%). OA blood flow was reversed in most, but not all, cases of ICA occlusion (27/29). Areas under the receiver operating characteristic curves of the three diagnoses were significantly different (p < 0.05). Conclusions The diagnostic performance of combined high- and low-frequency ultrasonography is greater than that of high-frequency ultrasonography alone. Although OA examination does not increase the diagnostic performance, it provides helpful data for the assessment of hemodynamics and collateral circulation. © 2013 Wiley Periodicals, Inc. J Clin Ultrasound 41:408–414, 2013

Clinically diagnosed cases of Babesia gibsoni infection in dogs were evaluated on the basis of polymerase chain reaction (PCR) results and nucleotide sequences of the 18S rRNA gene. Twenty-nine of 117 submitted dogs were PCR positive. The breed of dogs infected with B. gibsoni varied; however, pit bull terriers were the breed most often infected. The infection rate was higher in densely populated regions and in male dogs. Young dogs (age < 3 years) were more sensitive to B. gibsoni infection, and 6 out of the 29 infected dogs had a history of tick exposure. The clinical signs observed during physical examination were related to canine babesiosis, and many dogs showed symptoms similar to those associated with anemia. The results of hematologic analysis revealed severe hemolytic anemia and thrombocytopenia in the infected dogs. However, the blood smears of 29 infected dogs showed very low levels of parasitemia. Nucleotide sequencing of the PCR products revealed that the 18S rRNA gene sequences of B. gibsoni in South Korea were very close to those reported in Spain, Japan, and the United States.

The development of polarized hippocampal neurons with a single axon and multiple dendrites depends on the activity of phosphoinositide 3-kinase (PI3K) and the GTPase Rap1B. Here we show that PI3K regulates axon specification and elongation through the GTPase Rheb and its target mammalian target of rapamycin (mTOR). Overexpression of Rheb induces the formation of multiple axons, whereas its suppression by RNA interference blocks axon specification. mTOR is a central regulator of translation that phosphorylates eIF4E-binding proteins like 4E-BP1. Axon formation was suppressed by inhibition of mTOR and expression of mTOR-insensitive 4E-BP1 mutants. Inhibition of PI3K or mTOR reduced the level of Rap1B, which acts downstream of Rheb and mTOR. The ubiquitin E3 ligase Smurf2 mediates the restriction of Rap1B by initiating its degradation. Suppression of Smruf2 by RNA interference is able to compensate the loss of Rheb. These results indicate that the mTOR pathway is required to counteract the Smurf2-initiated degradation of Rap1B during the establishment of neuronal polarity.

Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.

Chronic lung disease (CLD) of prematurity remains a significant cause of morbidity among premature infants. It is a multifactorial disorder and characterized by an early increased number of neutrophils and alveolar macrophages, with later architectural epithelial and endothelial cell damage. Recently, apoptosis of type 2 pneumocytes in the lung of preterm neonates with acute and chronic lung disease has been examined and apoptosis of mesenchymal cells was detected in the chronic stage of bronchopulmonary dysplasia. Infection and inflammatory responses in the lungs play important roles. However, the contribution of Ureaplasma urealyticum to the development of CLD is debated. We found that U. urealyticum induced apoptosis in human type II lung epithelial cells (A549 cell line) and macrophages (derived from human monocytic cell line THP-1) by measuring the outer leaflets translocation of phosphatidylserine (flow cytometry analysis and fluorescence microscopy assessment), DNA fragmentation analysis, cell morphology changes such as diminution in cell volume, increased cytoplasmic staining, and nuclear pyknosis (hematoxylin and eosin staining) and viable counting (trypan blue exclusion). Anti-TNF-α monoclonal antibody partially protected the macrophages from undergoing apoptosis after infection with U. urealyticum. Our findings imply that U. urealyticum might be involved in impairing lung structure and host immune response during the development of CLD.

The study aims to investigate the relationship between nuclear factor (nuclear factor kappa B (NF-κB)) viability and lactacystin-mediated cell apoptosis in gastric cancer cells. Two gastric cancer cell lines (MKN28 and SGC7901) were treated with lactacystin-a proteasome inhibitor for 24 h. The cell viability, toxicity, and death were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. DNA binding viability of NF-κB and caspase-3 viability were analyzed by ELISA; the expression of p65 NF-κB nuclear protein was detected by immunocytochemistry and Western blot. Lactacystin reduced DNA binding viability of NF-κB (t = 3.0,P = 0.013) and the NF-κB viability (compared to the 5, 10 μmol/L MKN28 cell (p53 mutant) line, P < 0.001) and the expression of p65 NF-κB nuclear protein decreased parallelled to concentrations of lactacystin in MKN28 cell line, while without obvious effects on NF-κB viability in SGC7901 cell line (P = 0.381), while the viability of caspase-3 increased also along with the raising of lactacystin concentrations (compared to control, 5 μmol/L: SGC7901 cell line P = 0.029, MKN28 cell line P < 0.001; 10 μmol/L: SGC7901 cell line, P < 0.001, MKN28 cell line, P < 0.001). It was concluded that lactacystin had diversified killing effects on gastric cancer cells. The mechanism may be related to induce the apoptosis by downregulation of nuclear factor kappa B viability. There may be additional cell survival/death pathway in SGC7901 gastric cancer cells.

Staphylococcus aureus bacteraemia (SAB) originating from local infections can lead to severe secondary infections such as endocarditis. The protective effect of antibodies against secondary infections was studied in a rat model, where a local joint infection leads to bacteraemia and endocarditis on damaged aortic valves. In this study, immunizations with a truncated D2-domain of the S. aureus fibronectin-binding protein displayed on a cow-pea mosaic virus (CPMV-D) carrier induced protection against endocarditis (P < 0.05). Opsonization of S. aureus with antibodies raised against CPMV-D stimulated both neutrophil activity and macrophage phagocytosis in vitro. Furthermore, intravenous administration of these antibodies protected mice from weight loss due to SAB.

Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance ofUreaplasma urealyticumin the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability ofU. urealyticumto stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor κB (NF-κB) in vitro was characterized. The effect of NO on the growth ofU. urealyticumwas also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found thatU. urealyticumantigen (≥4 × 107color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P< 0.05). This effect was further enhanced by gamma interferon (100 IU/ml;P< 0.05) but was attenuated by budesonide and dexamethasone (10−4to 10−6M) (P< 0.05). The mRNA and protein levels of iNOS were also induced in response toU. urealyticumand inhibited by steroids.U. urealyticumantigen triggered NF-κB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced byU. urealyticumcaused a sixfold reduction of its own growth after infection for 10 h. Our findings imply thatU. urealyticummay be an important factor in the development of CLD. The host defense response againstU. urealyticuminfection may also be influenced by NO. The down-regulatory effect of steroids on NF-κB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.

Curli organelles are expressed by commensal Escherichia coli K12 and by Salmonella typhimurium at temperatures

Catechol-O-methyltransferase inhibitors have been newly introduced as adjunct drugs to the levodopa/dopa decarboxylase inhibitor therapy in Parkinson's disease. When given alone, catechol-O-methyltransferase inhibitors seem to affect behaviour. We wanted to determine whether the concentrations of free amine would be increased by catechol-O-methyltransferase inhibition with tolcapone and underpin the positive behavioural effects. To this end, dopamine and noradrenaline levels were analyzed in the microdialysis perfusion fluid collected from several brain regions in chloral hydrate anaesthetized rats. We also analyzed the turnover rate of catecholamines in the brain after single doses of tolcapone and entacapone using the alpha-methyl-p-tyrosine method. On their own, tolcapone (at 10 or 30 mg/kg) did not elevate dopamine or noradrenaline levels in any brain region studied although the formation of catechol-O-methyltransferase-dependent metabolites was strongly reduced. Neither tolcapone nor entacapone (at 30 mg/kg) affected the turnover rate of catecholamines. It seems that catechol-O-methyltransferase inhibitors do not alter behaviour by elevating extracellular levels of free catecholamines levels but other explanations are needed.

Background c-Jun NH2-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression. Methods Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH2-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy. Results In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy. Conclusions These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression.

Two catechol O-methyltransferase inhibitors, peripherally acting entacapone and also centrally acting tolcapone, were tested regarding their capacity to influence learning and memory in adult intact rats. Tolcapone was also studied in rats treated with scopolamine, in adult rats lesioned in the nuclei basalis magnocellularis, and in aged rats. Spatial working memory performance (radial-arm maze) of intact rats was facilitated following pretraining i.p. administration of tolcapone (10 mg/kg). Entacapone was ineffective at doses of 10 and 30 mg/kg. Senescent poor performers improved their accomplishment in the spatial memory task (linear-arm maze) under the influence of tolcapone. Scopolamine (1 mg/kg) impaired working memory performance. Bilateral lesions in the nucleus basalis magnocellularis reduced choline acetyltransferase activity in the frontal cortex by 26% and retarded the learning rate of spatial place task. Tolcapone was not able to counteract the performance deficits in these models. It is concluded that tolcapone can either slightly improve or impair the memory functions depending on task specific elements and performance factors.

PURPOSE Whether carotid abnormalities (CAs) are congenital or acquired, and whether they are risk factors for cerebrovascular diseases or medical curiosities, is still under debate. The aim of this study was to identify the independent risk factors for CAs. METHODS In this study, subjects (aged 3 days to 102 years) were divided into nine age groups, divided by 10-year intervals. The observed CAs were prospectively classified, according to the nature of the abnormality. Analyses of the factors associated with CAs were performed by univariate and multivariate logistic regression models. RESUITS: Of the 1,838 internal carotid arteries studied, 552 had CA (30%). There were peaks in the prevalence of CA in the 0-9-year-old group and in the above 80 years group; the lowest rate was found among the subjects in the 20-39-year-old group. The significant predictors of CA were age (OR 1.986, in the60-69-year-old group; OR 2.283, in the 70-79-year-old group; and OR 3.566, in the > 80-year-old group vs the 40-49-year-old group reference group), female gender (OR 1.799), hypertension (OR 1.52), and carotid artery atherosclerosis (OR3.176). CAs in youngersubjects were also associated with age (OR 9.894 in the 0-9-year-old group; and OR 3.294 in the 10-19-year-old group vs the 30-39-year-old group reference group). CONCLUSIONS Both congenital and acquired factors may be involved in CAs. In subjects> or = 40years old, CAs seem to be more associated with acquired factors, including ageing, gender, hypertension, and atherosclerosis.

Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (GWL) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that GWL is required for meiotic maturation in porcine oocytes. GWL expression increases from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with progression of the meiotic cell cycle. GWL is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown. Depletion of GWL inhibited or delayed meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of GWL overcame meiotic arrest and initiated progression to MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of GWL progressed to pronuclear formation. Taken together, our data demonstrate that GWL is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

The reaction between methyl (R)-2-(2-chlorophenyl)-2-hydroxyacetate and 3-nitrobenzenesulfonyl chloride gave the title compound, C15H12ClNO7S, which is a promising intermediate for the synthesis of Clopidrogel, an antiplatelet drug used in the prevention of strokes and heart attacks. In the crystal, molecules are linked through C—H...O interactions, and there is also a short Cl...O contact present [Cl...O = 3.018 (2) Å].

Pancreatic ductal adenocarcinoma (PDAC) is amongst the deadliest of human cancers, due to its late diagnosis as well as its intense resistance to currently available therapeutics. To identify mechanisms as to why PDAC are refractory to DNA damaging cytoxic chemotherapy and radiation, we performed a global interrogation of the DNA damage response of PDAC. We find that PDAC cells generally harbor high levels of spontaneous DNA damage. Inhibition of Non-Homologous End Joining (NHEJ) repair either pharmacologically or by RNAi resulted in a further accumulation of DNA damage, inhibition of growth, and ultimately apoptosis even in the absence of exogenous DNA damaging agents. In response to radiation, PDAC cells rely on the NHEJ pathway to rapidly repair DNA double strand breaks. Mechanistically, when NHEJ is inhibited there is a compensatory increase in Homologous Recombination (HR). Despite this upregulation of HR, DNA damage persists and cells are significantly more sensitive to radiation. Together, these findings support the incorporation of NHEJ inhibition into PDAC therapeutic approaches, either alone, or in combination with DNA damaging therapies such as radiation.

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.