Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation.Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks.Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily , neuregulin 1, retinoid X receptor, and nerve growth factor family.This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation. Female teleost fishes show remarkable diversity in reproductive strategies. Some reproductive strategies include continuous and semi-synchronous spawning, sex reversal, and synchronous or simultaneous hermaphroditism. Fish that are fractional spawners develop eggs rapidly for fertilization over relatively short time scales (days to weeks) while synchronous spawning fish develop their eggs gradually over an entire breeding cycle (months). Despite the wide diversity in reproductive strategies, there are characteristic morphological and physiological changes that occur as the oocytes grow and mature. In general, active nuclear transcription and DNA recombination drives meiotic divisions of oogonia during primary growth phases of development. The primary oocyte stage is characterized by the formation of the follicle including the granulosa cells, which surround the oocyte, the basal lamina, produced by the granulosa layer and the theca cells including blood vessels. Also, one can discern the beginning of formation of oocyte microvilli, extending towards the granulosa layer, followed by extensions of microvilli from the granulosa layer towards the oocyte. During this phase, meiosis is arrested at the diplotene stage of prophase I and the oocyte is characterized by intensive mRNA transcription Micropterus salmoides) are widely distributed throughout the southern continental USA. LMB have significant economic value because they are highly prized in the sport-fishing industry, in addition to being ecologically important as apex predators in their freshwater environments. LMB are semi-synchronous reproducers and many populations in central Florida typically spawn during mid to late spring (end of March-early April) when water temperatures are approximately 75°F. Floridian LMB sampled in mid-late spring have higher gonadosomatic indices (GSI) when compared to individuals sampled in other seasons, and in the summer months Florida LMB are sexually recrudescent, exhibiting little ovarian development Transcriptomics-based studies in the teleostean ovary have provided valuable insight into the molecular events leading to ovulation. In many cases, the transcriptional response can be associated with the physiological and morphological changes that are occurring in the ovary. Gene expression studies have been performed in teleost fishes with different reproductive strategies, including both fractional and seasonal spawners The present study uses a transcriptomics-based approach and bioinformatics to characterize the molecular events in the LMB ovary throughout a complete breeding season. Microarray analysis was conducted for eight distinct histological stages in wild female LMB that included ovulated eggs and ovaries that contained atretic oocytes. We point out here that this study was conducted in the whole ovary and not in individual oocytes. The ovarian stages used to categorize individuals reflect the emerging and dominant oocyte stage within the ovary in relation to the total volume of the ovary. LMB offer the opportunity to characterize gene expression patterns at specific points in the reproductive process, since a cohort of maturing eggs go through the process in a synchronous manner. The present study contributes to an increased mechanistic understanding of the gene networks that are activated and inhibited in the teleostean ovary during oocyte development and identifies potential biomarkers of atresia that could be utilized in both aquaculture and ecotoxicology as predictors of disrupted reproductive capabilities in wild populations of fish.Adult female and male LMB were collected by electroshocking from October 2005 to April 2007 at Welaka, FL USA , located approximately 20 miles south of Palatka, FL on the St. John’s River in an area that is considered to be relatively free from the influence of industrial effluent and agricultural runoff Approximately 10 males and 10 females were collected during each sampling period. At the time of capture, 3–5 ml of blood was drawn from the caudal vein using a heparinized vacutainer and stored on ice for Vtg and steroid measurements. Body weight (g), body total length , and gonad weight (±0.01 g) were recorded. Gonadosomatic index (GSI) was calculated for all individuals as [gonad weight/absolute body weight] X 100%. Multiple tissues were harvested at time of dissection for different studies and included brain, liver, ovary or testes, pituitary, and kidney. Tissues were frozen in liquid nitrogen in the field and stored in the laboratory at −80°C until processed for microarray analysis. Ovulated eggs (clear and gold in color), when present in the tissues, were collected directly from an excised ovary by cutting, along the longitudinal plane and flash frozen in liquid nitrogen. All microarrays were performed on ovary tissue except for a set performed on ovulated eggs (OV).Ovaries were fixed in buffered formalin, 1∶20 dilution  and then embedded in plastic and cut into 4 µm sections. Staining of the ovary was done with standard hematoxylin and eosin. Female LMB ovaries were categorized into eight ovarian stages based on histology following the categories described by Grier et al. Concentrations of plasma Vtg were determined by Enzyme-Linked Immunosorbent Assay (ELISA) using monoclonal antibody, 3G2 (HL1393) as previously described 3H]estradiol or [3H]testosterone  prior to extraction. Sex steroid levels were determined by ELISA  as previously described 2 and T was 20 pg/mL and 25 pg/mL, respectively. Intraday coefficient of variation was less than 5% across all standards, ranging from 0 to 4.5% as measured for each assay.Plasma steroids were evaluated after extracting plasma samples with n-butylchloride (Acros Organics) as previously described −4) were used for pathway analyses.Custom LMB oligonucleotide microarrays were designed by the Denslow laboratory Gene expression analysis was performed with four biological replicates for each ovarian stage (N = 32 microarrays). Total RNA for microarray analysis was extracted from LMB ovary samples using RNA STAT-60 reagent . RNA purity and quantity were measured using the NanoDrop ND-1000  and RNA integrity was evaluated using the Agilent 2100 BioAnalyzer (RNA 6000 Nanochip) and had average RNA integrity values ± SD of 8.9±0.48; RINs>8). Microarray hybridizations were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol using Cyanine 3 (Cy3). Microarrays were scanned at 5 µm at both 10 and 100 PMT (Agilent G2505 B Microarray Scanner), as previously described Method 1: ANOVA using JMP® Genomics v4.0 software. Raw intensity data for each microarray was normalized using LOESS normalization with a smoothing factor of 0.2 or by Quantile normalization and the two methods were in close agreement in identifying differentially expressed transcripts. The two normalization procedures identified >90% of the same transcripts as differentially expressed and a regression of fold-change estimates between the two normalization methods for transcript fold change was R2>0.95. One microarray slide in the atresia group had a low global intensity signal relative to all other slides and was removed from all downstream analyses. Differentially expressed transcripts from one stage to the next were identified using a one-way analysis of variance (ANOVA) followed by an FDR (5%) post hoc test for multiple comparisons.Raw expression data were analyzed by two different methods. Method 2: Significance analysis of microarrays (SAM). To identify genes which may change as a result of a linear sequential development we performed a SAM Hierarchical clustering was performed in JMP Genomics, using the Fast Ward algorithm. For some analyses, data sets for primary growth stages (PG and CA), vitellogenesis (eVtg and lVtg), and maturation (eOM and lOM) were combined to strengthen the power of the bioinformatics analysis. OV and AT remained separate. Functional enrichment was conducted in JMP Genomics and an adaptive false discovery rate was applied with 10,000 bootstrap iterations.−4). For GSEA, genes were permutated 400 times using the Kolmogorov-Smirnov classic approach as an enrichment algorithm. To broaden the analysis, all pathways were expanded to include cell processes and functional classes (enrichment p<0.05). GSEA was conducted on curated pathways in Pathway Studios. A sub-network enrichment analysis (SNEA) was performed to determine which gene expression networks were differentially activated/inhibited during follicular development.PathwayStudio™ 7.1  was used for GSEA. LMB genes were annotated with human homologs using Entrez Gene IDs for pathway enrichment  growth hormone receptor 1(ghr-1), luteinizing hormone receptor (lhr), and follicle-stimulating hormone receptor (fshr) were developed because of their key roles in oocyte development. Transcripts for mPR-α and ghr were present on the microarray. Total RNA used for real-time PCR was DNase treated with DNAse TURBO  as per manufacturer’s protocol. cDNA synthesis using Superscript-II was performed using 3 µg of RNA . Detailed methods for cloning and sequencing LMB genes have been previously described 8 to 1×101 copy number. Standard curves showed between 90–110% efficiency and were linear at R2>0.990. Two minus RT controls from separate cDNA sample pools and four no template controls (water) were run on each plate to ensure genomic DNA contamination was negligible and not a factor after DNase treatment. Melt curves were also generated for all genes on every real-time PCR plate to verify a single product/dissociation curve.Standard curves were used to obtain copy numbers of each gene and ranged from 1×1018S rRNA expression by month and by stage (p<0.001) was noted in ovarian samples. Trends in expression of conventional housekeeping genes by reproductive stage in ovarian tissue from aquatic species have been previously reported in the literature 18S rRNA data were normalized using the method utilized in the most recent publication on LMB ovarian gene expression 18S rRNA values were then used to normalize the gene of interest (GOI) as a simple ratio of GOI input amount/standardized 18S rRNA for each individual sample.Real-time PCR analysis of gene expression was performed using 1X iQ SYBR Green Supermix  as previously described . Sample sizes for real-time PCR assays were as follows; PN (n = 9), CA (n = 8), eVtg (n = 8), lVtg (n = 3), OM (n = 12) and AT (n = 2). Total number of animals used in the stage specific analysis was n = 42. During analysis of qPCR data, significant variation in 10 transformation. One-way analysis of variance (ANOVA) followed by a Dunnett’s T3 post-hoc test for multiple comparisons were conducted on data that met assumptions of normality and homogeneity. Wilcoxon/Kruskal-Wallis Tests (Rank Sums) were used if data did not meet assumptions. Differences were considered statistically significant when p<0.05. All analyses were performed in JMP Genomics (V4.0).All physiological endpoints and gene expression patterns were assessed for normality using a Shapiro-Wilk W test and homogeneity of variance using a Levene’s test. Data were tested for normality after logThe fact that not all of the oocytes mature exactly at the same time in LMB ovary results in partial overlap in molecular signatures between closely related stages (e.g. early maturation versus late maturation). But, the stages were incremental, with each new stage characterized by visual changes in the overall appearance of the histological section.2 were not normally distributed and were evaluated with Wilcoxon/Kruskal-Wallis. There was significant variation in GSI, Vtg, E2, and T across stages , reaching a peak at lOM and OV. Plasma Vtg ranged in individual females from below detection (PG) to 2.91 (mg/ml) (OV)  peaking during lVtg. Plasma Vtg remained elevated and constant in later stages of oocyte growth (OM) and dramatically decreased in animals exhibiting AT.Log transformation of T had a normal distribution and was evaluated with ANOVA while GSI, Vtg, and Es stages . GSI rans stages  and signml) (OV)  and sign2 ranged in individual females from 3.54 (pg/ml) (PG) to 10695.89 (pg/ml) (OV)  and followed closely the changes in GSI, peaking at late secondary growth phases and remaining relatively constant until AT. T ranged in individual females from 83.34 (pg/ml) (AT) to 52235.71 (pg/ml) (lOM) , peaking during late secondary growth phases and remaining relatively constant until AT. Plasma T in LMB are consistent with other reports in laboratory and wild bass 2.Eml) (OV) . E2 in fl) (lOM) . T in feThe cluster analysis for all transcripts on the LMB oligonucleotide microarray demonstrated that gene expression patterns for each sequential ovarian phase was most similar to the prior stage , suggest−4, and fold change>±1.5, the number of differentially expressed probes for each stage were as follows; PN to CA (n = 0), CA to eVtg (n = 0), eVtg to lVtg (n = 9), lVtg to eOM (n = 351), eOM to lOM (n = 0), lOM to OV (n = 94) and lOM to AT (n = 168). Therefore, gene expression changes were more pronounced with major transitions in ovary morphology.The marked differences in gene expression clustering were also reflected in the number of transcripts that were differentially expressed from stage to stage. Using the criteria of FDR adjusted p-values (5%), NR e-value<10When closely related stages were grouped together to increase power of the analysis and using the same criteria as above, the following numbers of genes were differentially expressed; Primary growth (PN+CA) to secondary growth (eVtg+lVtg) (n = 645), secondary growth to maturation (eOM+lOM) (n = 397), and maturation to ovulation (OV) (n = 552) and maturation to atresia (AT) (n = 2070). A complete list of these genes is provided in SAM identified 1020 transcripts that increased with oocyte development and 1444 transcripts that decreased with abundance in the trajectory towards ovulation (PN -> CA -> eVtg -> lVtg -> eOM -> lOM -> OV), whereas 62 genes were significantly increased and 599 genes decreased in progression towards AT (PN -> CA -> eVtg -> lVtg -> eOM -> lOM -> AT). All significant genes are plotted on separate heat maps showing ovulation  and atreWithin the genes that were identified by the SAM algorithm, gene titles were searched for ‘transcription’ or ‘receptor’ (FDR corrected at 5%). There were 17 genes that fit this search that increased in abundance  and 10 gBiological processes significantly over-represented (over) or under-represented (under) in each ovarian stage are listed in Some noteworthy examples of biological processes associated with the transition from PG to SG were glycolysis, ion transport, and chloride transport. For the transition from SG to OM, lipoprotein metabolic processes, electron transport, chloride transport, ubiquinone biosynthetic process, and aromatic compound metabolic processes were affected and for the transition from OM to OV, lipid transport, chloride transport, and cellular iron ion homeostasis were affected. Some noteworthy examples of biological processes affected with atresia were blood coagulation, lipid transport, protein import into mitochondrial inner membrane, notch signaling pathway, and cell proliferation.Gene set enrichment results for ovarian stages are shown in SNEA identified a number of expression networks that were involved in the different stages of follicular development . Some exWe provide some examples of the SNEA analysis. Expression targets of luteinizing hormone, a hormone well known to be involved in the final stages of ovulation, are low during vitellogenesis but increased over 150% at OV . This an10 transformed and data were determined to be normally distributed. Data were grouped according to month  and stage . mPR-alpha showed higher expression during the primary growth stages (PN and CA) compared to OM. There were no significant variations in ghr1 expression based on either month  or stage .Gene expression data were logto month  and ovarto month  There walhr by month  and by stage . There was also significant variation in fshr mRNA levels by month , while differences in fshr expression between ovarian stages were not significant. As reported in other teleost fishes, LMB fshr showed a different pattern of expression compared to lhr and was significantly higher prior to OV while lhr mRNA levels were higher during OV.There was significant variation in the ovarian mRNA steady state abundance of mPR-alpha and ghr1 were corroborated by both real-time and microarray data in this study, and each approach revealed that there were higher levels of the transcripts at earlier stages of ovary development. The transcripts lhr and fshr were not present on the microarray but were examined with real-time PCR. Transcript expression patterns for genes such as star and esrba have also been previously described in the LMB ovary star is increased in expression towards oocyte maturation and that esrba is also increased in expression at OV and at AT.The expression patterns of Pimephales promelas) Danio rerio) Gene expression patterns of many genes in the maturing LMB ovary  corroborate other reports in fish; rainbow trout cytochrome b and c, NADH dehydrogenase subunit 5). Other regulated genes included cytochrome P450 1A, cholecystokinin, low density lipoprotein receptor (ldlr), and apolipoproteins while some notable transcripts with lower expression during vitellogenesis compared to primary growth stages included follistatin and growth hormone receptor 1.Oocytes in the PN stage begin to accumulate oil droplets, which marks early transition into pre-vitellogenesis. This transition is predominantly dependent upon water temperature and photoperiod; for LMB populations located in Florida, this begins in early to mid-Autumn. Secondary growth is characterized by the active accumulation of plasma Vtg as well as other nutrients important for growth of the oocytes. Vitellogenesis is marked by active receptor-mediated endocytosis of Vtg and other very low density lipoproteins. Related to these events, notable transcripts with higher expression in secondary growth phases included genes involved in mitochondrial function  mRNA was most highly expressed during PN and CA, just before the VTGR protein is required on the plasma membrane for VTG endocytosis into oocytes preproapolipoprotein A-I, apolipoprotein A-IV4 (apoA-IV4) and apoe, but the expression was higher for some (7 to 8-fold) during the secondary growth phase. Apolipoproteins function in the transport of cholesterol, the precursor for all steroid hormones. Other studies have also seen a similar increases in apolipoprotein mRNA. For example, in coho salmon, apoe is significantly higher during early secondary growth stages compared to early growth stages In the LMB ovary, the significant increase in star, apolipoproteins, glutamate decarboxylase 2, claudin 19, steroidogenic factor 1 (sf1), cytochrome p450 3A68, growth hormone receptor 1 (ghr1), aromatase (cyp19a), ldlr, neurogranin, selenoprotein P, and fibrinogens. Transcripts showing a lower abundance at ovulation included protocadherin b, progranulin, aquaporin 1, cathepsin D, cyclin G2, and serine proteinase inhibitor. Many of the LMB transcripts identified here have been previously identified as differentially expressed in specific ovarian stages star transcript levels were significantly and positively correlated with plasma E2 levels in LMB during various stages of the female reproductive cycle, peaking during maturation and significantly decreasing post-maturation in atretic eggs. In previous studies, we have measured LMB star mRNA during late vitellogenesis and maturation and these data correspond to the microarray data Ovulation involves the rupture of the cell wall and oocyte expulsion. It has been reported that inflammatory responses are associated with ovulation as well as protease activity, vasodilation, and leukocyte activation esrba, lldr, progestin and adipoQ receptor family member VII mRNA levels decreased towards ovulation. These data are consistent with previous reports that esrba mRNA in the LMB ovary decreases throughout the stages of LMB ovarian development towards ovulation SAM identified a number of transcription factors and receptor subunits that showed significant temporal changes during follicular development that may be important for final ovulation. Transcripts for gabrb2, gabrb3, gabrg2) and peroxisome proliferator-activated receptor gamma (pparg) were significantly increased in abundance. Glutamate decarboxylase 67 (gad1), the enzyme that decarboxylates glutamate to form GABA, was also differentially expressed in early transitions of the ovary and was expressed at higher levels during earlier stages of development. GABAA receptors are ionic Cl- channels that are composed of five subunits and the subunit composition of the GABAA receptor determines the mechanistic properties of the receptor. The role of GABA in the control of reproduction in teleost fishes is well established from a neuroendocrine viewpoint Interestingly, during the progression of primary growth stages to ovulation, gamma-aminobutyric acid (GABA) receptor subunits  were also decreased, perhaps reflecting a lowered requirement for energy stores.Prior to ovulation, oocytes not needed undergo atresia, which is characterized by hyperplasia of the oocyte, breakdown of cell structures, and reabsorption of the zona pellucida. Interestingly, during this transition, our data indicates that there is preponderance of down-regulated transcripts, similar to data reported for the Senagalese sole ovulatory protein-2 as important for ovulation and it was significantly depressed in LMB at atresia (75-fold). Another intriguing transcript was nkcc corresponding to the NKCC protein which transports Na+, K+ and Cl- into cells and plays a role in maintenance of ionic balance and intracellular pH regulation in human Sertoli cells glutathione peroxidase 3 isoform 1, adenine nucleotide translocator, cathepsin B precursor, and cell division cycle 42, among others. Their roles in atresia need to be investigated.Garczynski and Goetz Many cell-signaling cascades involved in immune response were differentially affected during oocyte maturation. Our data suggest that cell signaling cascades that involve T and B-cell activation, mast cells, and NK cell activation are increased during the early stages of follicular development and are subsequently decreased during AT. Mast cells are located in most tissues and play a key role in the inflammatory process. Mast cells secrete biogenic amines such as histamine and serotonin in response to allergens and cellular injury; these cells also play an important role in the rat ovary during sexual maturation An objective of this study was to gain additional insight into the processes that might determine whether an oocyte follows a path towards OV or AT. If energy stores are inadequate, an individual will reduce the number of eggs (via AT) to reabsorb the nutrients. Pathways such as arachidonic acid metabolism and sphingolipid metabolism were involved in OV, while reactive oxygen species metabolism, FSHR -> FOXO1A signaling, TGF signaling, and cholinergic signaling were associated with AT. In addition, oxidative phosphorylation and pathways such as actin cytoskeleton regulation and gap junction regulation were decreased at AT, reflecting changes in energy requirements for atretic oocytes and the breakdown of the germinal vesicle. Although the majority of affected pathways at OV and AT were different, there were some examples of common signalling pathways between OV and AT, such as T-cell pathways and translation control. Interestingly, the fibronectin receptor -> catenin (cadherin-associated protein), beta 1 signalling cascade was down-regulated at both ovulation and atresia while at early growth stages, this pathway is up-regulated and we hypothesize that this pathway plays a role in ooctye maturation in the ovary.Some other interesting gene expression networks determined to be significantly associated with OM and AT included targets of nerve growth factors (NGF) (increased nearly 110% at ovulation) and expression targets of signal transducers and activators of transcription (STAT5) signaling (decreased 140% during AT). NGF is a protein that stimulates the growth and survival of sympathetic nerve cells. In mammalian ovaries, NGF can be found in the developing oocyte and contributes to the growth and function of sympathetic neurons projecting to the ovaries follistatin (fst) and inhibin B (inhb) on the other hand were significantly decreased during AT (∼130%). A primary role for these cytokine signaling cascades in oocyte maturation in vertebrates are well supported in mammalian studies fst and igf1 and these genes were increased in expression in ovulated eggs.Gene networks that require more discussion include those controlled by activin/inhibin. Both activin and inhibin are cytokines that are found in the CNS as well as in peripheral tissues. Interestingly, in the ovary, the activin network was significantly higher in expression in earlier stages of oocyte development and decreased towards maturation, but then dramatically increased in ovulated eggs (∼100%). Networks involving mPR-alpha mRNA was higher in abundance in secondary growth stages (vitellogenesis) compared to maturation. Progestins are steroidal hormones that include progesterone and 17alpha, 20beta-dihydroxy-4-pregnen-3-one  that function in regulating gametogenesis. Progestin membrane receptors have been extensively characterized in Atlantic croaker (Micropogonias undulatus) Ictalurus punctatus) mPR-alpha is highest immediately preceding the peak in GSI and lhr. In contrast, mPR-beta has a more constitutive expression in catfish throughout the year and mPR-gamma peaks earlier in follicular development and declines in mRNA levels before spawning. In the zebrafish ovary, smaller oocyte sizes showed lower mRNA expression of mPR-alpha compared to later stages of development and growth Carassius auratus), mPR-alpha mRNA was constitutively expressed during oogenesis and was highest in expression (size of 876–1000 µm) before the maximum size of the oocyte mPR-alpha mRNA expression mirrored more closely the seasonal expression patterns of the catfish mPR-gamma. The LMB sequence we obtained is highly homologous to characterized alpha isoforms and the degree in amino acid sequence identity between the α and the β and γ isoforms in catfish is 49% and 30% respectively, thus we are confident that the mPR-alpha isoforms is the isoform being quantified in LMB. Taken together, these studies suggest that there may be differences in mPR isoform expression in teleosts and seasonal expression patterns of all three isoforms need to be further characterized in fishes.Microarray and real-time PCR analysis suggested that LMB ghr mRNA as higher during primary growth stages than vitellogenic oocytes. This corroborates other studies that show higher levels of ghr mRNA early in oocyte development. Using in situ hybridization to detect ghr in the ovary of tilapia (Oreochromis mossambicus), Kajimura et al. (2004) observed intense staining for ghr mRNA in the cytoplasm and nucleus of immature oocytes in the PN stage which suggests an early role for ghr during early maturation. Other studies with salmon ghr1 and ghr2 mRNA expression were highest at sexual maturation compared to regressed stages of reproductive development in tilapia ghr1 isoform was investigated and LMB ghr2 should be investigated to determine whether the two isoforms correspond in expression patterns.Microarray analysis and real-time PCR identified LMB lhr and fshr are comparable to the seasonal patterns observed in other fish, for example female European seabass fshr mRNA and the identification of gonadotropin-signaling pathway activation through FSHR signaling by the transcriptomics data in primary growth stages. Of interest, gene networks associated with LH signaling were significantly affected throughout LMB ovarian development. The analysis does not suggest that LH is responsible for these changes, only that downstream targets of LH signaling are increased. This is at a time when the receptor is not yet expressed or is very low, thus the expression network may not yet be responsive to LH and may be regulated by other hormonal factors. Not surprisingly, there was a significant increase in LH expression targets in the ovary at maturation (120%), which corresponds to increased lhr mRNA expression associated with ovulation.LMB gonadotropin receptor expression was investigated only by real-time PCR experiments since the probes for the two receptors were not present on the microarray. The expression of LMB Molecular based studies are needed to better characterize natural variation in ovarian transcriptomics in wild populations of fish. This comprehensive transcriptomics analysis identified genes that are differentially throughout oocyte maturation and atresia. Many environmental pollutants and abiotic factors  induce atresia in fish. Therefore, it is important to identify the signaling cascades that correspond to atresia to better understand how these molecular changes relate to gonad morphology. These data suggest that there are distinct transcriptomics fingerprints for LMB ovary stages, and this study provides novel mechanistic insight into molecular signaling cascades underlying oocyte maturation. For example, there may be a role for gene target signaling related to LH in early stages of oocyte growth. This has not been studied to date. Also transcriptomics data suggest that activin/inhibin have a prominent role in regulating DNA replication, steroid metabolism, and lipid transport during final maturation and atresia. Lastly, there appears to be a role for neurotransmitters , the immune system , cytokines (e.g. activins) and signaling via prostaglandins and arachidonic acid in oocyte development that require further study.File S1mPR-alpha (B) ghr1 (C) lhr and (D) fshr mRNA. Expression is reported as mean absolute copy number of the transcript ± SEM. Sample sizes/month is n = 4 except for the month of June (n = 3). PG pn (n = 9), PG ca (n = 8), SG eVtg (n = 8), SG lVtg (n = 3), OM (n = 12), and AT (n = 2). Total number of animals used in the stage specific analysis was n = 42. Different letters indicate statistical differences among groups (p<0.05).Figure S1: Hierarchical clustering of the transcriptome of each LMB ovarian stage. Clustering was based on all transcriptomics data from the 15 K microarray to investigate overall patterns in gene expression. Primary and secondary growth stages clustered separately as did ovulation and atresia. Figure S2: Heat maps of receptors and transcription factors that showed significant (A) increases with ovulation (B) decreases at ovulation, and (C) and decreases in mRNA abundance with atresia after a time course analysis (SAM). There were no significant transcripts that were induced following atresia, suggesting many processes that are receptor mediated are down regulated with reabsorption of the oocytes. Figure S3: Seasonal dependent expression of (A) (PDF)Click here for additional data file.File S2(PDF)Click here for additional data file.File S3(XLSX)Click here for additional data file.File S4(XLS)Click here for additional data file.File S5(DOCX)Click here for additional data file.


Familial aggregation of ischemic stroke derives from shared genetic and environmental factors. We present a meta-analysis of genome-wide association scans (GWAS) from 3 cohorts to identify the contribution of common variants to ischemic stroke risk.−5) were incorporated into a multivariate risk profile model.This study involved 1464 ischemic stroke cases and 1932 controls. Cases were genotyped using the Illumina 610 or 660 genotyping arrays; controls, with Illumina HumanHap 550Kv1 or 550Kv3 genotyping arrays. Imputation was performed with the 1000 Genomes European ancestry haplotypes (August 2010 release) as a reference. A total of 5,156,597 single-nucleotide polymorphisms (SNPs) were incorporated into the fixed effects meta-analysis. All SNPs associated with ischemic stroke (P<1×10−8). Secondary analysis identified a significant cumulative effect for age at onset of stroke , as well as a strong effect showing increased risk across samples with a high propensity for stroke among samples with enriched counts of suggestive risk alleles (P<5×10−6). Risk profile scores based only on genomic information offered little incremental prediction.No SNP reached genome-wide significance for ischemic stroke  information may be required to improve clinical risk profiling. Ischemic stroke is known to aggregate in families. The observed familial aggregation is believed to be the result of shared genetic and environmental factors. The true extent of familial aggregation remains unknown, as many individuals who would develop ischemic stroke in later ages may succumb to other morbid conditions, thereby reducing the estimated familial risk. Nonetheless, there are consistent and compelling data that suggest genetic factors play a major role in risk of ischemic stroke.e.g., CADASIL and Fabry disease) have been associated with stroke risk. Additionally, genome-wide approaches have identified several additional loci associated with stroke risk. The well-documented chromosome 9p21 locus associated with myocardial infarction also has been shown to be a risk factor for large vessel (atherosclerotic) ischemic stroke PITX2 gene that is associated with atrial fibrillation is also associated with cardioembolic stroke ZFHX3 gene on chromosome 16q22 associate with both atrial fibrillation and cardioembolic stroke Ischemic stroke is clinically heterogeneous, with multiple etiologic pathways contributing to risk. There is increasing evidence from genetic studies supporting the hypothesized heterogeneity of ischemic stroke This study involves collaboration among three clinical stroke cohorts comprising two independent datasets that have been subjected to genome-wide association scans. Imputation to greater than 5 million SNPs was performed to permit meta-analysis of association with ischemic stroke and secondary analyses of SNP associations with presumed stroke etiology (subtype) and age at stroke onset. No SNP exhibited genome-wide significant levels of association with ischemic stroke. Risk profiling to identify possible genetic factors associated with ischemic stroke phenotypes identified a potential genetic contribution to ischemic stroke etiology.All subjects provided written informed consent to participate in stroke genetics research. SWISS and ISGS protocols are approved by the Mayo Clinic Institutional Review Board, Rochester, MN and the BRAINS protocol is approved by the Ethics Committee of Imperial College London & Hammersmith Hospital.The Ischemic Stroke Genetic Study (ISGS) is a multicenter inception cohort study The Siblings with Ischemic Stroke Study (SWISS) is a multicenter affected sibling pair study Concordant siblings had their diagnosis of ischemic stroke confirmed by review of medical records by a central vascular neurology committee. Concordant siblings had the same eligibility criteria as probands. Subtype diagnoses were assigned to the index strokes of probands and concordant siblings according to TOAST criteria Readily available US controls were utilized, including stroke-free participants from the Baltimore Longitudinal Study of Aging and the National Institute of Neurological Diseases and Stroke neurologically normal control series taken from the Coriell Cell Repositories. All controls had been previously genotyped and described in detail elsewhere BRAINS is an ongoing, multicenter, in-hospital study which recruits consenting acute stroke patients into a highly characterized biobank −4 in controls and P<1×10−7 in cases, nonrandom missingness per haplotype P<1×10−5 and missingness in cases compared to controls (from chi-squared test) P<1×10−5.Both the ISGS/SWISS and the BRAINS genotyping datasets underwent identical quality control procedures. Each case series was genotyped using the Illumina 610 or 660 genotyping arrays, while control series used in the ISGS/SWISS dataset were genotyped using the Illumina HumanHap 550Kv1 or 550Kv3 genotyping arrays. The BRAINS dataset utilized controls genotyped on either the Illumina 610 or 660 genotyping arrays. Genotypes were called using Illumina GenomeStudio software, with all alleles called on the forward strands based on default cluster files provided by Illumina. In addition, all A/T and G/C SNPs were removed prior to merging case and control sample sets, SNPs with discordant minor alleles on the same strand across chips were removed prior to merging datasets as well. Preliminary exclusion criteria per sample included genome-wide SNP call rates <95% and discordance between self-reported gender and sex determined from X chromosome heterozygosity. After merging with control datasets, SNPs were excluded if genotyping success rate <95%, minor allele frequency (MAF) <0.01, Hardy-Weinberg equilibrium (HWE) P<1×10Stroke cases and controls were merged with a subset of samples from HapMap 3  and underwent multidimensional scaling analyses to verify European ancestry for the case-control series. Individuals having estimated principal component vector 1 (PC1) and 2 (PC2) values greater than 3 standard deviations from the combined CEU/TSI means for each vector were excluded as outliers. Evidence of cryptic relatedness was examined using pairwise identical by descent (IBD) estimates. Samples were excluded if they shared greater than a 0.125 proportion of alleles (pi_hat >0.125). After samples were excluded, SNP-based quality control was repeated prior to imputation, with all SNPs passing quality control entering the imputation phase. Basic quality control of genotyped SNP data was carried out using PLINKv1.07 The SNPs passing quality control for the ISGS/SWISS and BRAINS datasets were imputed separately using a two-stage procedure implemented in Markov Chain based haplotyper  e.g., ischemic stroke) or linear regression for continuous phenotypes  as implemented in MACH2QTL All dataset-specific GWAS summary statistics were generated using logistic regression as implemented in MACH2DAT for binomial phenotypes , where ßjj is the parameter estimate for the jth SNP with fixed-effects P<1×10−5 in the ISGS/SWISS dataset, and N is the number of risk alleles at the jth SNP . The effect estimates from ISGS/SWISS data for the chosen SNPs were then applied to BRAINS data. Risk profile associations for each quintile was quantified using the lowest quintile of risk per population as a reference group in logistic regression models, adjusted for estimates of population substructure  in the BRAINS dataset for all binomial (ischemic stroke) phenotypes. Risk profile associations in the BRAINS dataset for the age at onset phenotype were estimated using linear regression and adjusted for population substructure. Overall risk trends (unstratified models) were evaluated for each population using identical covariates in multivariate regression models.The SNPs chosen for the models were based on meta-analysis of both ISGS/SWISS and BRAINS data. All SNPs with fixed-effects P<1×10In the risk profile analysis, age-at-onset was treated as a continuous measure. For this analysis, the quintile groups were based on the distribution of alleles per sample, which is indicative of earlier onset stroke. Therefore the first quintile would be participants possessing the lowest number of alleles associated with early onset of first stroke as the reference population for all analyses, with the fifth quintile as per  being pa−8 cut-off for significance. The small effect size often associated with common variants is likely the reason for this study failing to identify any genome-wide significant SNP associations. Using a similar modeling scenario for the risk profile analysis, based on the entire BRAINS dataset, we were optimistically powered to detect cumulative risk effects at P<0.01 significance level based on effect sizes at an odds ratio of 1.64 as per the lowest estimate for overall stroke in Due to the comparatively small size of this meta-analysis, post-hoc power calculations were carried out. Based on the realistic target of alleles with beta coefficients of roughly 0.3 (odds ratio ∼1.35) in our total series of samples, this study was at 70% power for effect alleles at a frequency of 0.45, 66% power for effect alleles at a frequency of 0.30, and 28% power for effect alleles at a frequency of 0.15, using a P<5×10−8)  exhibited genome-wide significant association with ischemic stroke or for any phenotypes analyzed in this study (P<5×10−8) . Nonetheanalysis . The imp−5) after Bonferroni correction for 5 tests and the risk profile accounts for ∼4% of the variation in the phenotype. This association persists after adjustment for stroke subtypes (1.71×10−5). The surprisingly strong trends for associations with ischemic stroke and presumed etiology suggest genetic effects that should be detected by larger meta-analyses. There are markedly significant risk increases across more common stroke subtypes , suggesting a strong skewing of effect towards genetic variants with a high propensity for late onset stroke. When adding additional covariates of stroke subtype into the age at onset model, the overall trend is still highly. The risk profile scorings provide additional suggestive evidence of genetic components in the etiology of stroke. Nevertheless, all area under the curve (AUC) estimates were below 0.7, suggesting little incremental clinical utility of SNP genotype information at this stage, assuming AUC >0.8 is often the criterion for clinical utility.The risk profile analyses demonstrate significant trends of cumulative genetic effects associated with risk of ischemic stroke and presumed etiology (TOAST subtypes). The association between risk profile SNPs and ischemic stroke age at onset is significant . In the SWISS data, a significant correlation has previously been reported between age at onset for probands with ischemic stroke and age at onset in their ischemic stroke-affected siblings We identified a significant difference that survived correction for multiple testing between the highest quintile and lowest quintile of allele dosages contributing to age at onset of stroke (compared with the lowest quintile), this revealed a 14.78  year difference in age at onset between these two groups  is consistent with the known association between family history of stroke and large vessel and small vessel ischemic stroke Table S1Single nucleotide polymorphisms with the strongest associations for srtoke.(DOC)Click here for additional data file.Table S2This table shows the association between previously identified stroke loci and related stroke phenotypes in this study .(DOC)Click here for additional data file.


Patients with secondary pneumothoraces generally have serious comorbid disease and poor pulmonary function and require that treatment be individualized. Though interventional treatments such as tube thoracostomy and pleurodesis are to be selected first, some cases become obstinate. Even though VATS treatment is attempted ultimately, sometimes it turned out to be inadequate for stapling or simple suturing. We present here our new VATS technique to control refractory air leak by direct infusion of fibrin glue (FG) via double lumen catheter and additional insertion of pieces of PGA (polyglycolic acid) sheet into ruptured bulla alternately.1) VATS is introduced with general anesthesia, 2) detect the responsible focus, 3) confirm that stapling is not good for the lesion, 4) ensure surgical margin to afford loop tie, 5) enlarge the air-leak hole or make a new hole to introduce a catheter, 6) FG infusion via a double lumen catheter, 7) a piece of PGA sheet insertion by endoscopic forceps, (repeat 6 and 7 a couple of times), 8) tie the catheter insertion site, 9) pleurodesis is added.Since Oct. 2011 this operation was performed at our institution 9 times for 8 patients with uncontrolled secondary spontaneous pneumothoraces. Their underlying conditions were COPD in 5, interstitial pneumonia (IP) in 2 and non-tuberculous mycobacterium (NTM) in 1. The NTM case required ECMO support during the operation due to poor pulmonary function. An IP case received this operation twice metachronously for another lesion. Air-leak could be perfectly controlled after this procedure in all cases. Chest tube could be withdrawn within 5 days after surgery and no serious morbidity was occurred.This layered FG and PGA insertion technique into ruptured bulla, named “mille-feuille therapy”, encourages us to perform the VATS for the patients with refractory pneumothoraces. This seems feasible and improves patients quality of life.


Breast cancer mortality is directly linked to metastatic spread. The metastatic cell must exhibit a complex phenotype that includes the capacity to escape from the primary tumour mass, invade the surrounding normal tissue, and penetrate into the circulation before proliferating in the parenchyma of distant organs to produce a metastasis. In the normal breast, cellular structures change cyclically in response to ovarian hormones leading to regulated cell proliferation and apoptosis. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. Their primary function is degradation of proteins in the extracellular matrix to allow ductal progression through the basement membrane. A complex balance between matrix metalloproteinases and their inhibitors regulate these changes. These proteinases interact with cytokines, growth factors, and tumour necrosis factors to stimulate branching morphologies in normal breast tissues. In breast cancer this process is disrupted facilitating tumour progression and metastasis and inhibiting apoptosis increasing the life of the metastatic cells. This paper highlights the role of matrix metalloproteinases in cell progression through the breast stroma and reviews the complex relationships between the different proteinases and their inhibitors in relation to breast cancer cells as they metastasise. Cancer mortality most often results directly from metastatic spread to distant organs –3. ApproBreast carcinomas spread in several ways. They can move directly into the skin and muscle, via lymphatics and other lymph nodes and via the blood stream to lungs, bone, liver, and brain . The preWhatever the route of metastasis, the tumour cells must first migrate through the local tissues. Noninvasive breast cancers remain within the basement membrane of the terminal duct lobule. Invasive breast cancers involve the dissemination of cancer cells outside the basement membrane of the duct and the lobules into the surrounding adjacent normal breast tissue stroma . ChangesThe extracellular matrix (ECM) serves as a medium for cell-cell interactions and can directly signal cells through cell surface ECM receptors such as integrins. In addition, many growth factors and signalling molecules are stored in the ECM . Cell-ceThe matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases . To dateIn the normal breast, cellular structures change cyclically in response to ovarian hormones. Cell proliferation, apoptosis, invasion, and differentiation are integral processes that are precisely regulated but become dysregulated in pathologies such as cancer. ECM remodelling is a prerequisite to ductal progression and MMP activity facilitates this progression by removing or breeching the basement membrane and stromal matrix . MMPs caThese MMPs, including MMPs 1, 5, and 13, catalyse the degradation of fibrillar forms of collagen, types I, II, and III . Elevateβ (TGF-β) and is activated through a proteolytic cascade involving other MMPs. It exhibits a broad spectrum of activity against the ECM. It is released from stromal fibroblasts and facilitates tumour growth through regulating the activity or availability of growth factors sequestered as inactive molecules in the ECM. In breast cancer it is implicated in an uncontrolled degradative process occurring during tumour progression [Collagenase-3 (MMP 13) degrades helices of fibrillar collagens with preferential activity on type II collagen . It is igression . Within gression .α/β fibronectin receptors such as the α4β1 surface receptor that mediates cell-matrix and cell-cell interactions by attachment to laminin. MMP 1 was found to upregulate α5β1 receptors which lead to a reduced number of α4β1 fibronectin receptors relative to mammary epithelial cells [α chains. The denatured α chains are susceptible to hydrolysis by the gelatinases (MMPs 2 and 9) [Attachment to laminin is a key event in the process of local and vascular invasion. In order for vascular invasion to occur, laminin receptors have to be upregulated. They are normally upregulated by cytokines, inflammatory agents, and ECM proteins such as fibronectin . Fibroneal cells . MMP 1 c2 and 9) . This ca2 and 9) .β signalling was shown to decrease growth of the primary tumour but promote metastasis. Inhibition of TGF-β signalling has been shown to decrease metastasis of mammary tumours by impairing invasion, migration, and cellular survival and may prove to be a potential antimetastatic therapy in the future [The gelatinases, for example, MMPs 2 and 9, are type IV collagenases that degrade gelatine (denatured collagen) and types IV, V, VII, IX, and X collagen. Type IV collagen is particularly abundant in basement membranes . In a ste future .β, hepatocyte growth factor (HGF), epidermal growth factor (EGF), and tumour necrosis factor α (TNFα) have been shown to influence MMP 9 induction by fibroblasts in breast cancer cells [A complex variety of cytokines and growth factors such as TGF-er cells . Cell coer cells .Stromelysins, for example, MMPs 3, 7, 10, and 11, have broad substrate specificity, catalysing degradation of many different substances. These include proteoglycans, noncollagenous proteins such as laminin and fibronectin . MMP 3 iThis group of MMPs possesses a membrane-spanning domain and has been shown to catalyse the activation of progelatinase-A which degrades a variety of ECM substances and functions as a fibrinolytic enzyme . The mosα3β1 and α6β1 integrins at the cell membrane leading to attachment to laminin and subsequent local invasion of tissues [Extracellular matrix metalloproteinase inducer (EMMPRIN) is one of the molecules involved in the regulation of several MMPs including MMPs 1, 2, and 3  and MT1- tissues .Tissue inhibitors of metalloproteinases (TIMPs) inhibit protease activity by forming high affinity noncovalent complexes with active MMPs. Four inhibitors have been described; TIMPs 1, 2, 3, and 4 . TIMPs aHigh levels of macrophage infiltrate in breast tumours correlate with a poor prognosis and hypoxic tumours have a higher invasive capacity and poorer prognosis than well-oxygenated tumours . MacrophEpithelial-mesenchymal transition (EMT) is a development process in which epithelial cells take on the characteristics of invasive mesenchymal cells . EvidencThe Breast Cancer Health Disparities study evaluated genetic variation in MMPs 1, 2, 3, and 9 and breast cancer risk. MMP 1 and MMP 2 were associated with breast cancer overall and were associated with oestrogen receptor positive/progesterone positive cancers and with oestrogen positive/progesterone receptor negative tumours. MMP 3 and MMP 9 were associated with oestrogen and progesterone receptor negative tumours. These findings suggest that genetic variation in MMP genes influences breast cancer development and survival .The aim of current research is to identify future treatments for breast cancers by understanding the mechanisms involved in metastatic spread in order to identify potential drug therapies. Unfortunately, to date, research has had to consider specific cancer cell lines in isolation. Breast cancer is not one specific entity, but, by the very nature of the cellular changes, neoplasms are complex and multifactorial with neoplasms displaying characteristic genetic instability with divergent properties even within a single tumour. This will mean resistance to single focus drug therapy. Identifying a rate-limiting step in the complex mechanism to target drug therapy has not yet been possible mainly due to the cellular toxicity of synthetic MMP inhibitors caused by complex interactions with other tissues throughout the body. MMPs may also prove to be useful for prognostic and predictive markers of metastatic disease, enabling doctors to identify high-risk cancers and tailoring therapy accordingly.Future research may one day locate the rate limiting steps that breast cancer cells need to adopt a malignant phenotype. Targeting these mechanisms, so that healthy tissues are not disrupted, could mean that tumour cells would remain constrained within the local tissues. As tumour invasion is the most important diagnostic criteria for malignancies, then by definition the tumour would no longer be malignant. As breast cancer is the largest cause of deaths in women aged 35–55, this would be a ground breaking achievement that would significantly reduce mortality and morbidity associated with breast cancer.


A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban  or enoxaparin . The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR).Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2.These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function. Venous thromboembolism is a significant potential complication following orthopaedic surgery and an important cause of morbidity and mortality in adults. In the absence of thromboprophylaxis, the rate of deep vein thrombosis following major lower extremity orthopaedic surgery is between 40-60%, while the risk of developing fatal pulmonary embolism is between 1-2% ,4. AlthoRivaroxaban or Xarelto™ (Bayer Schering Pharma AG) is an anti-thrombotic drug that was granted marketing approval by the European Commission  in 2008 for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery . A direcin vitro. The effect of each drug was evaluated in terms of its effect on osteoblast viability, function and gene expression.Rivaroxaban therapy is recommended for 5 weeks post hip replacement surgery and 2 weeks post knee replacement surgery . In lighN = 1, 78 year old, Caucasian female). Osteoblast cells were characterised by the supplier as staining positive for alkaline phosphatase and osteocalcin. Cells were routinely cultured in Osteoblast Growth Media  at 37°C in a humidified atmosphere of 95% air and 5% CO2. Growth media contained 10% fetal calf serum, 125 μg/ml sodium phosphate and 50 μg/ml ascorbic acid. Cells were seeded into 6-well plates at a density of 1 × 105 cells  or into 96-well plates at a density of 5 × 104 cells per well (cell viability assay). Cells were then cultured to approximately 80% confluence prior to treatment.A human osteoblast cell line (primary cells) was purchased from PromoCell . Osteoblasts were isolated from bone biopsies (cancellous bone) obtained from the femoral condyles of the knee joint of a patient undergoing knee surgery  in aliquots containing 10 mg of rivaroxaban salt. Rivaroxaban was dissolved in dimethyl sulphoxide  as demonstrated in previous in vitro studies with this drug [in vivo therapeutic range (0.02 - 0.05 μg/ml) [Osteoblast cells were cultured in the presence of enoxaparin  at concentrations of 1, 10 and 100 μg/ml. Cells cultured in the presence of osteoblast growth medium alone served as experimental controls. Rivaroxaban (BAY 59-7939) was obtained directly from Bayerhis drug . Final r5 μg/ml) . Cells c® AQueous One Solution Cell Proliferation Assay  as per manufacturer's instructions and previous literature [4 cells/well and cultured at 37°C in a humidified incubator containing 5% CO2. Once approximately 80% confluent, cells were treated for 72 hours with varying concentrations of enoxaparin or rivaroxaban and their respective controls. A 20 μl aliquot of the CellTitre 96® Aqueous One Solution reagent was added to each culture well and after 4 hours the absorbance was recorded at 490 nm. The cell viability of treatment cultures was reported as a percentage of control cultures. All assays were carried out in triplicate.Cell viability was assessed using the CellTiter 96terature . It is a5 cells/well. Following 7 days of treatment with enoxaparin or rivaroxaban and their respective controls, cell lysates were collected by CelLytic M  and the extracted proteins frozen at -80°C. The alkaline phosphatase activity of cell lysates was determined using a p-nitrophenylphosphate (pNPP) based assay [Alkaline phosphatase activity was measured as a marker of osteoblast function. Cells were seeded into 6-well plates at a density of 1 × 10ed assay . A 5 μl free™ kit . RNA was reverse transcribed to complementary DNA (cDNA) using the Enhanced Avian Reverse Transcriptase kit . cDNA served as template for real-time PCR, which was conducted using the QIAGEN QuantiTect SYBR Green PCR kit . Using gene specific primer pairs, the mRNA expression of the runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2) and Osteocalcin (OC) gene products were measured using absolute quantification and were reported as a function of crossing time (Ct), the cycle number at which PCR amplification becomes linear. mRNA expression was normalised to control and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression resulting in mean fold change values. Assays were carried out in triplicate (3 independent cDNA preparations extracted from 3 independent cultures) , according to the manufacturer's instructions. Contaminating genomic DNA was removed from RNA samples using a DNA-s) Table  Table 2.P < 0.05.Data are given as a mean ± standard deviation. Data were analysed using Student's t-test to determine a significant difference between sample means. Differences were considered significant if ® AQueous One Solution Cell Proliferation Assay , cells treated with enoxaparin were found to have a similar viability when compared to control cells , and in cultures treated with a range of rivaroxaban concentrations, including the therapeutic levels Figure  and 4. ALong term administration of heparin and LMWH has been associated with a negative effect on bone and an increased risk of developing osteoporosis ,4. XarelClinical trials comparing the efficacy and side effects of enoxaparin and rivaroxaban therapy on the outcome of thrombosis following joint replacement surgery favour the use of rivaroxaban for the prevention of thrombotic events post-arthroplasty  . This, cIn this study, neither enoxaparin nor rivaroxaban treatment caused a reduction in osteoblast cell viability indicating that both drugs do not show cytotoxic effects to osteoblasts studied. Our findings in relation to enoxaparin are in accordance with those of other studies, which have shown that enoxaparin does not have a cytotoxic effect on osteoblast viability . SimilarOur findings of reduced alkaline phosphatase activity and osteocalcin expression in the absence of reduced cell viability suggested that enoxaparin and rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function rather than a decrease in osteoblast proliferation. Therefore, to determine if rivaroxaban and enoxaparin influence osteoblast function through changes in osteoblast signalling, the expression of the transcription factor, Runx2, and the pro-osteogenic growth factor, BMP-2 were investigated. The exposure of human osteoblasts to enoxaparin and rivaroxaban resulted in a significant reduction of both Runx2 and BMP-2 expression in this study. Runx2 is the main transcription factor responsible for the development and maintenance of the osteoblast phenotype  and targThese findings suggest that enoxaparin and rivaroxaban may affect osteoblast function by reducing BMP-2 induced bone formation. Since the exposure of human osteoblasts to both drugs did not result in a cytotoxic affect in this study, a downregulation of BMP-2 signalling may explain the reduction in osteoblast functionality. Runx2 regulates the expression of several major extracellular matrix genes expressed by osteoblasts including alkaline phosphatase and osteocalcin ,30. Thusin vitro [Bone metabolism is a continuous remodelling process involving both bone formation by osteoblasts and bone resorption by osteoclasts. Heparin has been found to negatively affect bone formation by promoting the activation of osteoclasts and decreasing bone volume in rats, by inducing bone resorption in rat osteoclasts in vitro ,33 and ein vitro . While tin vivo. Given that these agents are typically promoted for use following hip and knee arthroplasty, and that many modern hip and knee implants are 'uncemented' and depend on effective bone ingrowth for fixation, these findings are of potential clinical importance.In conclusion, rivaroxaban and enoxaparin treatment led to a reduction in alkaline phosphatase activity and a reduction in BMP-2, osteocalcin and Runx2 mRNA expression, indicating that treatment with both drugs leads to a general negative effect on osteoblast activity. Due to the increased duration of therapy (as compared to enoxaparin), rivaroxaban could potentially be more detrimental than enoxaparin in terms of its effects on osteoblast function. While the findings of this study indicate that rivaroxaban negatively affects osteoblast function, further clinical investigations are required to elucidate whether these effects of rivaroxaban therapy translate into demonstrable effects on bone homeostasis The authors declare that they have no competing interests.GNS conceived this study, carried out all experiment work and drafted the manuscript. PMW participated in the co-ordination of the study and the drafting of the manuscript. KJM participated in the design and coordination of this study, and participated in drafting the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/247/prepub


Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse  Sfswap mutants interact genetically with a mutation in Jagged1, which encodes a Notch ligand. We show that expression of some genes involved in Notch signaling is disrupted in Sfswap mutant mice. Our work is one of the first studies to show that a putative splicing factor has specific effects on Notch signaling pathway members and on inner ear development.The organ of Corti is a sensory structure in the cochlea that mediates our sense of hearing. It consists of one row of inner hair cells and three rows of outer hair cells, together with an array of neighboring supporting cells. The precise arrangement of these different cell types is regulated very tightly by a number of signaling pathways during embryonic development, and mutations in genes that regulate this pattern often lead to deafness. We have generated a mouse mutant containing a lentiviral insertion in a gene encoding a putative RNA splicing factor called Sfswap. Homozygous mutant mice have hearing and balance defects, and have an abnormal arrangement of hair cells in their cochlea. These defects are consistent with defects in the Notch signaling pathway. We show that  Notch1, Jag2 or Delta1Hes and Hey family of downstream Notch effectors also cause an increase in hair cell numbers at the expense of supporting cells, with mutations of multiple Hes/Hey family members causing progressively more severe phenotypes The organ of Corti is an excellent system to study mechanisms of cell patterning due to its highly organized array of sensory cells. It contains one row of inner hair cells, three rows of outer hair cells and several classes of specialized supporting cells, including pillar and Deiters' cells. The signals responsible for this intricate and fine-grained cellular pattern are beginning to be understood, and include the Notch signaling pathway. The Notch1 receptor is expressed in supporting cells, while the Notch ligands Jagged2 (Jag2), Delta1 and Delta3 are expressed in hair cells after they differentiate from prosensory precursors Jag1) is expressed in all sensory organs of the inner ear prior to the onset of hair cell differentiation Jag1 is expressed broadly at first, and then becomes excluded from the prosensory domain and restricted to Kölliker's organ by E13.5 Jag1 is down-regulated from Kölliker's organ and is expressed with Notch1 in supporting cells Jag1 in the developing inner ear leads to a severely disrupted organ of Corti Jag1 conditional mutant cochlea, whereas two rows of inner hair cells but no outer hair cells are observed in the apical region of the cochlea Jag1 mutant heterozygotes generated by ENU mutagenesis show a milder phenotype; they lack some cells in the third row of outer hair cells and display ectopic inner hair cells The Notch ligand, Jagged1  protein that is a putative splicing factor. RS-domain containing proteins are known to regulate many aspects of RNA processing, including splicing, transcript elongation, transcript stability, nuclear export and miRNA cleavage as well as genome stability,  lentiviruses can be used for efficient insertional mutagenesis in transgenic mice, we used a tyrosinase-expressing lentiviral vector to infect pre-implantation albino (FVB/N) mouse embryos by subzonal injection. Tyrosinase expression rescues albinism and provides a visible, dosage-sensitive, reporter for different integration sites. Transgenic founder (F0) mice were bred to establish families with single lentiviral integration sites and the mice were then inbred and assayed for evidence of insertional mutations. In one family (OVE2267B), homozygous micSfswap, 115 bases 5′ of exon 5. The mutation was labeled Tg(Tyr)2267BOveSfswap (MGI ID: 5287267), and will be referred to throughout this study as TgSfswap or Tg. The tyrosinase minigene allows for identification of genotype by coat color when transgenics are maintained in an albino background Tg mice are more darkly pigmented than heterozygotes and non-transgenic mice are albino. We back-crossed TgSfswap mice onto the FVB/N background for more than 10 generations. Tyrosinase expression and the inner ear phenotypes in homozygous mutants correlated 100% with the insertion in Sfswap as assayed by PCR. Additionally, Southern blots showed a single lentiviral integration site (data not shown). Northern blot analysis of Sfswap revealed that the wild-type Sfswap transcript was significantly reduced in Tg/TgSfswap mice . This RNA is most likely unspliced or incompletely spliced Sfswap mRNA, suggesting a hypomorphic allele. Reverse transcriptase PCR (RT-PCR), followed by sequencing, reveals that some of the Sfswap mRNA produced in Tg/TgSfswap mice includes sequences from the lentiviral insert and exclusion of exons surrounding the insert (data not shown). These abnormal RNAs are not found in mice heterozygous for the transgene, suggesting that TgSfswap is likely to be a recessive allele. Previous studies have demonstrated that Sfswap can regulate the splicing of its own transcript Sfswap transcript is aberrantly spliced in homozygotes but not heterozygotes.We conducted a random insertional mutagenesis study using a tyrosinase-tagged lentiviral vector  to infec/Tg mice . In its Tg/TgSfswap mutants in our screen because they displayed a significant circling behavior , all indicative of vestibular defects Tg/TgSfswap mutants on the FVB/N background are about 23% smaller than wild-type (WT) littermates at 8 weeks , and this size difference is detectable at birth . Tg/Tg animals mate at very low frequency in the FVB background, but when crossed to a C57Bl/6 background, the circling behavior ceases and homozygous mice are able to mate more successfully.We initially identified behavior . In a 30termates . Since cSfswap mutants also have hearing defects we measured auditory-evoked brainstem responses (ABR) and found that Tg/Tg mice have an average 18 dB increase in threshold to elicit an auditory response, indicating a moderate degree of hearing loss  to identify if the hearing loss is at the level of hair cells. Tg/Tg mutants display an average 15 dB increase in DPOAE thresholds . We stained surface preparations of newborn Tg/Tg cochleas with fluorescently-labeled phalloidin to detect actin in stereocilia and found regions of the mutant cochlea are missing the third row of outer hair cells and regions that have ectopic inner hair cells , respectively Bmp4 expression, which corresponds to the presumptive horizontal and anterior canal cristae, is reduced in mutants at E10.5. The posterior spot, which will develop into the posterior crista, is small or indiscernible at this age . These similarities lead us to hypothesize that Sfswap and Jag1 function in the same genetic pathway. To test this, we crossed Jag1 knockout mice  maintained on a C57BL/6 background, with our Tg/+Sfswap mutants raised in an FVB/N background to obtain Tg/+; Jag1+/−Sfswap F1 progeny. These were then crossed again to Tg/+Sfswap mice on an FVB/N background to generate Tg/TgSfswap; Jag1+/− mutants. Most of these compound mutants (10/14) exhibited semicircular canal truncations, but neither Tg/TgSfswap nor +/−Jag1 mutants have canal truncations on this genetic background , and Sfswap and found no differences in splicing in any gene except for Sfswap  hearing loss. The mutants exhibit a partial loss of the third row of outer hair cells and show ectopic inner hair cells, together with some missing pillar cells. All organs in the vestibular system are smaller than their wild-type counterparts, particularly the utricle and saccule, which are less than 50% of the size of wild-type organs. Our data are consistent with Sfswap regulating aspects of Jagged1 signaling. First, the cochlear phenotype of Tg/TgSfswap mice is strikingly similar to that seen in Jag1 heterozygous point mutants Tg/TgSfswap mice have otocyst patterning defects similar to those seen in Jag1 mutants Sfswap mutant allele exacerbates the phenotype of Jag1 heterozygous mice. We discuss these phenotypes in more detail below.Using a lentivirus-based insertional mutagenesis strategy, we have identified the putative splicing factor Sfswap as an essential gene for inner ear development. Tg/TgSfswap mutants have reduced levels of the early sensory markers Bmp4, Lfng, and Hey1 and significantly smaller sensory organs. Jag1 conditional mutants similarly have severely reduced vestibular structures Jag1 mutants also show changes in expression of sensory markers such as Bmp4, Sox2, Lfng and Hey1 at E10.25 Bmp4 conditional or hypomorphic mutants Bmp4 expression in our mutants may be due to defects in Jag1 signaling and leads to the smaller vestibular structures and reduced eminentia cruciata of the anterior crista that we observe in Tg/TgSfswap mutants.Tg/TgSfswap mutants has only been observed previously in heterozygous knockout or point mutants of Jag1Tg/TgSfswap mutants, although if there are small changes in the prosensory domain, they would be difficult to discern using the markers and techniques currently available. Ectopic rows of inner hair cells can be seen in Jag1 heterozygotes and in apical regions of the Jag1 conditional homozygote cochlea Tg/TgSfswap mutants, we also observe an extra row of inner hair cells in the apical region of the cochlea. Since Jag1 is expressed at the boundary of the greater epithelial ridge and the prosensory domain where the first inner hair cells form, it is possible that a partial loss of Jag1-Notch signaling leads to the inappropriate formation of extra inner hair cells in Jag1 and Tg/TgSfswap mutants.The unique combination of loss of outer hair cells and ectopic inner hair cells that we observe in the cochleas of Sfswap and Jag1, we currently have no evidence for a direct interaction. It is possible that Jag1 mRNA splicing or stability is regulated by Sfswap. However, we did not detect significant differences in the levels or splicing pattern of Jag1 transcripts, and we have not detected significant differences in Jag1 protein expression in the cochlea by immunofluorescence. Since the cochlear phenotype of Jag1 mutants can vary according to both the type of mutation (point mutation versus null) and the genetic background on which mutants are maintained Tg/TgSfswap mice are causing small changes in Jag1 expression below the limits of detection that are sufficient to affect cochlear patterning. Alternatively, it is possible that Sfswap is regulating the splicing or stability of modifiers of the Jag1 locus. We screened a variety of Notch pathway genes for possible expression changes in Sfswap mutant cochleas. Several genes showed significant changes by RT-PCR including the mouse neuralized homologue Neurl1A, the Notch transcriptional co-activator MamlD1, and Numb  mouse. Mutation at this locus results in degeneration of inner hair cells after E17.5 Srrm4 suggests this factor regulates splicing of genes involved in synaptic transmission and the secretory pathway. We have now identified a second SR-like protein, Sfswap, which has distinct and highly specific effects on patterning of the inner ear. Our work provides the first evidence that a putative splicing factor is necessary for establishing the size of sensory organs in the ear and for proper patterning of mechanosensory hair cells in the organ of Corti.Many genes undergo complex splicing patterns in the inner ear. These include several genes that are involved in ion transport including Sfswap transgenic mice by lentiviral insertional mutagenesis with a tyrosinase minigene is given in A detailed account of the generation of For RT-PCR, RNA was isolated from E15.5 inner ears using the Ambion PureLink RNA mini kit (12183018A) according to the manufacturer's instructions. cDNA was generated using the Superscript III First Strand system (Invitrogen 18080-051) according to manufacturer's instructions. RT-PCRs for exon splicing events were performed using exon tiling primers  and S2. in situ hybridizations and Northern blots were obtained from Brigid Hogan (Bmp4),Gerry Weinmaster (Jag1), Elias Pavlopoulos (Neurl1A), Yutaka Hata . These include Lfng: F-GTTCCGCTCTGTCCATTGC R- GGATCCTAATACGACTCACTATAGGGAGCCCACTATGGGCGACTTTC and Hey1: F-AGACCTTGGGGGACAGAGAT and R-GGATCCTAATACGACTCACTATAGGGAGAACGGTGAAATCCGTGAGAC.Some For cryosections, heads were fixed in 4% paraformaldehyde overnight at 4 degrees. Heads were then washed briefly in PBS, immersed in 30% sucrose overnight, equilibrated in OCT, then embedded in OCT. Sections were taken between 8 and 14 µm. For P0 and older whole mounts, heads were fixed overnight in 4% paraformaldehyde. Cochleas, cristae, and maculae were then dissected out and processed. For immunohistochemistry, some tissue was first boiled for 10 minutes in 10 mM citric acid for antigen retrieval. All samples were washed 3 times in PBS, then 30 minutes in blocking buffer (PBS with 10% goat serum and 0.1% Triton-X or Tween-20). Tissue was then stained overnight in primary antibody diluted in blocking buffer. Tissue was then washed 3 times in PBS then incubated for 2 hours with the appropriate secondary antibody at 1∶400 dilution in blocking buffer. In tissues where Topro3  or phalloidin  were used, they were added to the secondary antibody cocktail. Tissue was then washed 3 times in PBS and mounted in either Vectashield (Vector Labs) or Prolong Gold+Antifade (Invitrogen).Kip1 (Neomarker), p75 (Advanced Targeting Systems), Alexa 488 anti mouse and rabbit (Invitrogen), Cy5 anti mouse and rabbit (Jackson Immunolabs). In situ hybridization was performed as previously described Antibodies used: Myo6 (Proteus), Prox1 (Millipore Bioscience Research Reagents), Parv (Sigma), Jag1 (Santa Cruz Biotechnology), p27Images were taken using a Zeiss LSM 510 confocal microscope, a Zeiss Axiophot microscope, or a Zeiss dissecting scope. Images were processed using Axiovision software or ImageJ, then further processed in Adobe Photoshop. Inner and outer hair cells were counted in P0 cochleas per 200 µm using Axiovision software. Cochlea lengths were measured at P0 using Axiovision software. Vestibular areas were measured on P0 vestibular flat mounts using ImageJ.Paint fills were performed as previously described Tg/Tg mice fail to present a threshold level startle response, so percent pre-pulse inhibition could not be calculated and only startle responses are reported. ABR and DPOAE were performed as previously described Open field activity was measured using the Versamax System for automated activity recording. Mice were acclimated for at least 30 minutes to the testing room illuminated to 400 lux light and with 60 dB white noise before testing. The open field is a 40×40×30 cm Plexiglas arena. Tests were performed for 30 minutes during which time horizontal and vertical activity were measured via beam breaks. Chambers were cleaned with alcohol before and after each run to minimize interfering odors. Pre-pulse inhibition and startle responses were measured using a San Diego Instruments system. Mice were acclimated for at least 30 minutes in a separate nearby room before testing. Mice were placed in cylindrical restraint tube in a sound-attenuating chamber to minimize movement of the mouse and interfering noise. Mice were acclimated in the testing chamber to 70 dB white noise for 5 minutes before test pulses were delivered. Mice were then presented with 6 rounds of 8 test types in a pseudorandom order with 10–20 seconds between trials. These consist of no stimulus, startle at 120 dB for 40 ms, pre-pulse trials  and pre-pulse inhibition trials of each sound 100 ms prior to a 120 dB startle. Responses were recorded every 1 ms for 65 ms following the stimulus. Significance was measured using students T-Test or ANOVA with repeated measures where appropriate using SPSS software. Graphs were generated using Prism.All animal experiments in this study were carried out in accordance with the Institutional Animal Care and Use Committee at Baylor College of Medicine.Figure S1Tg/TgSfswap ABR traces are reduced compared to WT. Representative ABR traces are shown for WT and Tg/TgSfswap mice. Tg/Tg traces are qualitatively normal but have reduced peak-to-peak amplitude.(TIF)Click here for additional data file.Figure S2Tg/TgSfswap mutants show no distinctive changes in prosensory or nonsensory cochlear markers at E13.5. : Sections from E13.5 Tg/TgSfswap and wild-type mice co-stained with antibodies to p27kip1 (green) and Sox2 (red) to reveal the prosensory domain and Jag1 (red) to show its expression in the adjacent Kölliker's organ. No significant expression differences are seen in the mutant cochleas. (C): In situ hybridization of E13.5 Tg/TgSfswap and wild-type mice with the prosensory marker Hey2 and the outer sulcus marker Bmp4. No significant differences are seen in the size or intensity of either domain.(TIF)Click here for additional data file.Figure S3Tg/TgSfswap mutants show no difference in hair cell mechanotransduction. SfswapTg/Tg and wild-type pups were injected with AM1-43 at postnatal day 1 and examined at P2. Flat mount preparations of the vestibular organs (A) and cochlea (B) showed no significant differences in AM1-43 uptake.(TIF)Click here for additional data file.Figure S4Tg/TgSfswap mutants do not have splicing defects in Jag1, Neurl1A, Mamld1, or Numb. Northern blots were performed to examine splicing in putative Sfswap targets using brain RNA. No splicing differences were found in Jag1, Neurl1A, Mamld1, or Numb.(TIF)Click here for additional data file.Table S1Notch1, Delta1 and Jagged1 exons.List of RT-PCR primers for (PDF)Click here for additional data file.Table S2List of RT-PCR primers for Notch pathway targets and modifiers.(PDF)Click here for additional data file.Text S1Sfswap insertional mutant mice, together with a description of Northern blot methods.This section contains a detailed account of the generation of (PDF)Click here for additional data file.Video S1Tg/TgSfswap mutants exhibit circling and head bobbing behavior. Tg/TgSfswap mutants examined in their home cage exhibit significant circling behavior. Between circling bouts, Tg/TgSfswap mice show head bobbing behavior.(MOV)Click here for additional data file.