Rana temporaria) may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca2+ buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca2+-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction.Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO). In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins–parvalbumins–present in the cutaneous mucus of the common frog ( Many predators have evolved highly specialized sensory systems to detect and locate their prey Rana temporaria) Lumbricus terrestris) Rana temporariaThamnophis marcianus, Thamnophis sirtalis and Nerodia fasciata) that are representative of the two major clades of new world natricine snakes. They share similar dietary habits, principally fish and amphibians Lithobates catesbeianus and Pimephales promelas) and two fish species  that are not part of the natural diet of these snakes 2) to establish the phylogenetic extent of this chemical based prey-predator relationship and to 3) to investigate the physiological significance of presence of parvalbumins in cutaneous secretions of fish and amphibians.Snakes have the most highly developed vomeronasal organ of all vertebrates, and vomeronasal chemoreception is known to be the dominant sensory mode used by most snakes to detect and locate prey Lithobates catesbeianus, Pimephales promelas and Osmerus eperlanus) were submitted to unambiguous snake bioassays  skin sections also showed specific α parvalbumin immunoreactivity localized in the epidermal mucous cells (Ambystoma mexicanum (data not shown), suggesting that these proteins may be widely present among vertebrates with mucus-covered skin, such as fish and amphibians.The α and β parvalbumins, proteins found in the cutaneous mucus of the frog sbeianus , consistus cells . MoreoveLithobates catesbeianus, Osmerus eperlanus and Pimephales promelas crude extracts are necessary and sufficient to elicit prey attack. The parvalbumin threshold for eliciting prey-attack in natricine snakes was evaluated by testing different quantities of purified Osmerus eperlanus parvalbumins on Nerodia fasciata. Although an individual variability in the responses was observed in our bioassays, the threshold for eliciting lure attack was in the order of tens µg of for four out of five snakes. The lowest parvalbumin detection threshold recorded was on the order of µg  and several fish genera from both fresh and salt water environments . Moreover parvalbumin-like proteins were also found in the cutaneous mucus of an urodele amphibian, Ambystoma mexicanum (data not shown), suggesting that these proteins may be widely present among vertebrates with mucus-covered skin such as fishes and amphibians.In a recent study Thamnophis marcianus, Thamnophis sirtalis and Nerodia fasciata). Moreover, of all the proteins isolated from crude extracts, only parvalbumins showed a chemo-attractive activity. This finding should be interpreted with caution as the denaturing conditions of our protein extraction procedure, semi-preparative SDS-PAGE, are known to disturb protein tertiary structure and thus protein function. Parvalbumins have a relatively high tolerance to denaturation and thus still interact with calcium and thus keep their chemo-attractive properties. As the majority of proteins are altered by these conditions, other putative phagostimulting proteins may have lost their activity during this procedure. Moreover, it seems unlikely parvalbumins are the only chemical cues used by thamnophiine snakes to locate and recognize prey. Other chemical cues are certainly involved in prey recognition and it is possible that our study, focused on the protein skin secretome, did not detect all compounds present in the cutaneous mucus of prey. However, our results do suggest that parvalbumins play a critical role in the chemical prey recognition mechanism of new world natricine snakes. In addition, two old world natricine snakes (Natrix natrix and Natrix maura) respond to proteinous chemo-attractants from frog skin Boiga irregularis, strongly respond to mammalian blood serum fraction known to contain parvalbumins All the parvalbumins and parvalbumin-like proteins identified were found to be chemoattractive for the three thamnophiine snakes tested . Pimephales promelas and Osmerus eperlanus specimens were purchased from a commercial dealer. Nine Thamnophis marcianus specimens were kindly provided by Prof. G. Toubeau and by the Natural History Museum of Tournai (Belgium). Two Thamnophis sirtalis and seven Nerodia fasciata specimens were purchased from licensed dealers. All snakes were adults and maintained in the laboratory prior to the beginning of the experiments. The snakes were housed individually in plastic cages (40 cm×25 cm×20 cm) in a common holding room kept on a 12∶12 hr light/dark cycle. Room temperatures ranged from 23 to 27°C. Snakes were fed twice a week on a laboratory-controlled diet composed of fish (Osmerus eperlanus), frogs (Limnonectes macrodon) and mice (Mus musculus). Water was available ad libitum. Prior to tests, snakes were deprived of food for one week. All experiments were approved by the animal ethics committee of the University of Mons. Maintenance and care of animals was in compliance with guidelines of the animal care and use committee.Juvenile specimens of Lithobates catesbeianus, Pimephales promelas and from frozen Osmerus eperlanus. Crude extracts were obtained from aqueous washes of the prey according to the procedure described by Leroy et al Pimephales promelas and Osmerus eperlanus) to one hour (Lithobates catesbeianus). After filtration, Dithioerythritol (DTE: 50 mM) and PMSF (2 mM) were added to the washing solutions. Filtered extracts were concentrated by lyophilization and dissolved in 7 mM β-mercaptoethanol (β-MSH). Extracts were then dialyzed three times against 7 mM β-MSH for two hours at 4°C. Dialysed extracts were lyophilized again and stored at −20°C.Cutaneous mucus extracts were prepared from living Chemoattractive proteins of cutaneous mucus extract were isolated using semipreparative SDS-PAGE as described by Leroy et al. Rana temporariaLithobates catesbeianus, Pimephales promelas and Tanichthys albonubes was performed using anti-α parvalbumin antibodies. Tanichthys albonubes, a fish that does not figure in the normal diet of natricine snakes, was used instead of Osmerus eperlanus due to the impossibility of obtaining skin sections from frozen specimens. The paraffin embedded sections were cleared and the sections were blocked with 3% H2O2 in TBS for 10 min., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 0.5% casein for 10 min. at room temperature (RT) and incubated firstly with 1/400 antibody for 1 hr at RT, then with biotinylated Goat Anti-Rabbit (Prosan)) at 1/50 for 30 min. at RT, and finally with Strep-ABC complex  at 1/100 for 30 min. at RT. The sections were developed with AEC substrate kit  at RT for 20 min., counterstained with Luxol Blue and after drying were mounted with DAKO aqueous mount .The Ph14 antibodies were obtained from the purified mucus α parvalbumin of The specificity of the different primary antibodies was monitored by pre-incubation with its antigen 10×in excess. For each skin section, no labelling was observed without primary antibodies or in the presence of pre-absorbed primary antibodies.Excised Protein bands were subjected to in-gel trypsin digestion and analyzed using LC-MS/MS with an HCT ultra-plus ion trap instrument (Bruker) Rana temporaria) control tests were carried out. The sample and control solutions were presented in a randomly selected order. In order to determine if the chemoattractivity is due to proteins, protein extracts were treated with proteinase K before being submitted to snake bioassay. The protein extracts were incubated in presence of proteinase K (solubilized in Tris-HCl buffer 50 mM pH 7.5) during 1 hour at 55°C. The action of the proteolytic enzyme was analyzed using SDS-PAGE.The chemoattractivity of crude mucus extracts and purified proteins was assessed using a standard “all-or-none” snake bioassay Nerodia fasciata bioassays were filmed at 75 frames per second in a glass aquarium (35×17.5×24.5 cm) using four synchronized high speed cameras  mounted on tripods . Illumination during filming came from two cold light spots .E. coli (20 mL of culture in each 50 ml flask) was cultivated at 28°C and 150 rpm in minimum medium 284 containing 0.2% glucose (27). Alpha parvalbumin (0.5 mM) was added to test its potential anti-bacterial activity. Cell growth was monitored by measuring the value of OD600 using a Beckman DU-640 spectrophotometer (Beckman).Video S1Nerodia fasciata attacking a parvalbumin-coated lure after lure-directed tongue flicks. The video was recorded at 75 fps.The movie shows a positive bioassay with the snake (MOV)Click here for additional data file.


Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth.Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels.These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth. Understanding the cellular dynamics involved in microvascular network growth is critical for future development of cell-specific therapies targeted at manipulating the microcirculation during tumor growth, diabetic retinopathy, myocardial infarction and other pathological conditions. Microvascular network growth in adult tissues is largely attributed to angiogenesis defined as the growth of new vessels from pre-existing vessels. Angiogenesis is commonly associated with two modes: capillary sprouting and intussusception . CapillaWe recently identified vascular islands, defined as endothelial cell segments disconnected from neighboring microvascular networks . While vThe objective of this study was to determine if vascular islands are involved in angiogenesis in the adult rat mesentery. The mesentery was selected because it allows for visualization of entire microvascular networks. We show that vascular islands are multi-cellular and associated with cell proliferation during angiogenesis. The number of vascular islands present in a network decreases during later stages of network growth. Our observations implicate vascular island proliferation and incorporation as an alternative mode of growth during the initial stages of angiogenesis in adult microvascular networks.All experimental protocols were approved by Tulane University’s Institutional Animal Care and Use Committee. Similar to a previously established protocol ,11, a siMesenteric windows, defined as the thin translucent connective tissues between the mesenteric arterial/venous vessels feeding the small intestine, were labeled for BRDU + PECAM. Rats were anesthetized with intramuscular injections of ketamine (80 mg/kg bw) and xylazine (8 mg/kg bw). The peritoneal cavities were then injected with BRDU . After a two-hour period, the rats were euthanized via intra-cardiac injection of Beuthanasia . 8–10 mesenteric windows were carefully dissected starting from the ileum and immediately placed in 10 mM Phosphate Buffered Saline . Next, the windows were whole mounted on positively charged slides, fixed in 100% methanol for 30 minutes at −20°C, and labeled according to the following BRDU and PECAM protocol: 1) 6 N HCl for 1 hour at 37°C; 2) 1:100 monoclonal mouse anti-bromodeoxyuridine (BRDU)  in antibody buffer solution (PBS + 0.1% saponin + 2% BSA) with 5% NGS overnight at 4°C; 3) 1:100 goat anti-mouse CY2 Fab fragments  in antibody buffer solution for 1 hour at room temperature; 1:200 biotinylated mouse monoclonal anti-CD31 (PECAM) in antibody buffer solution at 4°C overnight; 4) 1:500 Strep-CY3 in antibody buffer solution for 1 hour at room temperature. Following each labeling step, tissues were washed every 10 minutes for 30 minutes with PBS containing 0.1% saponin. A subset of BRDU + PECAM labeled tissues was also labeled with DAPI  for 10 minutes at room temperature to confirm cell nuclei.202 in PBS; 2) 20 minute in 2.5% normal horse blocking serum ; 3) 1 hour incubation at room temperature with 1:1000 rabbit anti-mouse Type IV collagen Ab  or 1:100 rabbit polyclonal Neuron-glia antigen 2 ; 4) 1 hour incubation at room temperature with horse anti-rabbit secondary agent ; 5) 5–10 minute incubation at room temperature with Vector Nova Red (Vector Laboratories) substrate. All labeling steps were followed by washes with PBS + 0.1% saponin, except after the Nova Red developing step, which was followed by 5-minute incubation in distilled water. For subsequent PECAM labeling, tissues were stained following a previous protocol [Additional mesenteric windows from unstimulated rats were harvested, fixed with 4% paraformaldehyde for 1 hour at 4°C, and labeled for PECAM, Collagen IV (basement membrane marker), PECAM + Collagen IV, or PECAM + NG2 (pericyte marker) using colorimetric methods. PECAM colorimetric labeling has been previously described . Collageprotocol  with thePositive immunostaining was confirmed by comparing labeling patterns to expected cell morphologies and to the appropriate controls: unstained, secondary antibody alone, or IgG plus secondary antibody labeled tissues.Images were digitally captured with one of the following systems: an inverted microscope  coupled with a PixelFly camera  and a Olympus 4x dry, 10x dry, 20x oil or 60x oil objective; a digital camera (FUJIFILM FinePix S1 Pro) mounted on an inverted microscope (Olympus IX70) with a Cooke 5x dry, Olympus 20x dry, or Olympus 60x oil immersion objective; or on an inverted microscope (Leica DM IRE2) with a Leica SP2 AOB confocal microscopy system with a 20x dry or 63x oil objective.Two vascularized tissues were randomly selected from each rat per experimental group. Thus, a total of 8 tissues were analyzed per experimental group: unstimulated , 3 days post-stimulation , and 10 days post-stimulation . Network montages, generated by overlaying 4x images, were used to measure vascular area per tissue area. Vascular area was defined as the cumulative area circumscribed around every microvascular network in a tissue. Vascular length density was measured for two randomly chosen representative 4x fields of view per tissue . Vascular length density was calculated by the sum of vessel segment lengths in a field of view divided by the corresponding circumscribed vascular area in that field. Vascular area and vascular length metrics were based on measurements of PECAM positive microvessels along intact networks and did not include vascular island segments.For the unstimulated and day 3 groups, the total number of vascular islands, the number of vascular islands with at least one proliferating cell, the branch points per vascular island, the number of blind-ended capillary sprouts, and the number of capillary sprouts with proliferating cells were measured. Vascular islands were defined as blood endothelial cell segments disconnected from the neighboring blood microvascular network based on PECAM labeling. Disconnections were confirmed by focusing throughout the full tissue thickness, which is approximately 20 – 40 μm ,15. BRDUIn additional unstimulated animals, a bolus of 2.5 mL FITC-albumin  was injected via the femoral vein in adult male Wistar rats . ImmediaMeasurements are presented as means +/− SEM. Microvascular network growth metrics were compared across the 10 day time course using a one-way ANOVA or one-way ANOVA on Ranks followed by a Student-Newman-Keuls pairwise comparison test. Capillary sprout proliferation was compared between unstimulated and day 3 groups using a Student’s t-test. The vascular island proliferation and branch point metrics were compared between unstimulated and day 3 groups using a Mann–Whitney Rank Sum Test. All statistical comparisons were made using SigmaStat . p < 0.05 was regarded as statistically significant.In unstimulated microvascular networks, PECAM labeling served to identify endothelial cells along all hierarchies of microvascular networks down to the capillary level. PECAM-positive labeling also revealed numerous vascular islands. Vascular islands had both blood and lymphatic vessel morphologies Figure . LymphatSimilar to previous reports from our laboratory , compounDuring the initial stages of microvascular growth, BRDU-positive cells were commonly observed along vascular islands Figure . On day The primary findings from this study are that 1) during the initial stages of angiogenesis vascular islands undergo proliferation comparable to capillary sprouting and 2) during later stages of angiogenesis vascular islands are no longer present, in line with the hypothesis that these segments are able to incorporate into growing microvascular networks. In addition, vascular islands are associated with vascular pericytes, which have been attributed to play a role in capillary growth and stabilization .The mesentery was selected for this study because it allows for observation of intact microvascular networks (as compared to tissue cross-sections) with a resolution down to the single cell level. Use of the mesentery to investigate cellular dynamics during angiogenesis has served to identify endothelial cell phenotypic changes along capillary sprouts ,19, the The origin of the endothelial cells along vascular islands is currently unknown emphasizing the need for future lineage studies. Potential sources could be attributed to the migration from existing vessels, a resident population of endothelial precursor cells, or vascular regression. Support for regression is provided by the observation of increased vascular islands during vascular pruning. In rat juvenile retinas hyperoxia was shown to increase the number of disconnected vascular segments. These segments were associated with endothelial cell apoptosis . In contSince proliferation along vascular islands was assessed with a single BRDU pulse, only the cells in S-phase at the time of tissue harvesting were captured. At day 3, the percentage of vascular islands with at least one proliferating cell was 33%. This value most likely underestimates the cellular proliferation associated with the vascular islands given that cells along the vascular islands are presumably in different stages of the cell cycle. Capillary sprouting is generally associated with endothelial cell proliferation. During peak capillary sprouting, the percentage of sprouts with a BRDU-positive cell was comparable to the percentage of vascular islands with a BRDU-positive cell. Thus, cells along vascular islands undergo proliferation similar to cells along capillary sprouts.Previous dye injection experiments in the rat mesentery  confirmeA limitation of the current study is that individual vascular islands were not tracked over the time course of angiogenesis in the same tissue. This type of time lapse investigation is required to confirm the fate of vascular islands, to determine whether vascular islands increase their length, or whether the number of endothelial cells increases along a vascular island. In our current study, the average length of a vascular island was not different between the unstimulated and 3 days post stimulation scenario (data not shown). This lack of difference can be attributed to a high variability in both vascular island length and cell number. In spite of this heterogeneity, we do report that vascular islands form new branch points during angiogenesis. The increase in cellular extensions indicates that the vascular islands are dynamic and capable of changing their structure.The increase in cellular extensions during angiogenesis combined with the increase in cell proliferation and the drastic reduction in number results provide strong support that vascular islands are able to incorporate into growing microvascular networks. However, how these vascular islands contribute to network growth remains unclear. Future experiments will be required to determine whether vascular islands actively direct the growth of nearby vessels or whether vascular islands represent a significant cell source for new vessels in an expanding network.in vitro are able to develop intact networks after implantation [The ability of vascular islands to connect to a surrounding vascular network is supported by the endothelial cell dynamics observed during embryonic vasculogenesis. Progenitor cells aggregate and elongate into chord like formations subsequently producing vascular islands composed of endothelial cells . Over tiantation ,28. ThesThe involvement of vascular islands in angiogenesis is in line with a similar growth mechanism seen in lymphangiogenesis. Lymphatic vascular islands, identified as lymphatic endothelial cell segments disconnected from the surrounding network have been associated with proliferation, migration, and recruitment of cells during lymphangiogenesis ,9,29. ThVascular islands, defined as endothelial cell segments disconnected from nearby microvascular network, are associated with cellular proliferation and increased branching during angiogenesis in the adult rat mesentery. In addition, the number of vascular islands decreases as microvascular area and length density increase. While future studies are required in other tissues and during other angiogenic scenarios, our results implicate a new mode for adult microvascular network growth involving vascular island proliferation and incorporation. The next step in confirming that vascular islands contribute to network growth requires a longitudinal study in the same tissue that allows for comparing the location of a vascular island before and after angiogenesis.PECAM: Platelet endothelial cell adhesion molecule; PBS: Phosphate buffered saline; BRDU: Bromodeoxyuridine; BSA: Bovine serum albumin; NG2: Neuron-glia antigen 2.The authors declare that they have no competing interests.MRK-G and ERW were responsible for tissue harvesting, immunohistochemistry, imaging, data collection, statistical analysis, and writing of the manuscript. PCS, MY, and RSS performed 48–80 injections and contributed to the tissue harvesting and immunohistochemistry. GWS-S contributed to the interpretation of the data and the writing of the manuscript. WBS contributed immunohistochemical reagents that led to the initial observation of the vascular islands and to the interpretation of the data. WLM conceived the study, and contributed to imaging, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.


Cellular senescence is a program of irreversible cell cycle arrest that cells undergo in response to a variety of intrinsic and extrinsic stimuli including progressive shortening of telomeres, changes in telomeric structure or other forms of genotoxic and non-genotoxic stress. The role of nuclear factor-κB in cellular senescence is controversial, as it has been associated with both proliferation and tumour progression, and also with growth arrest and ageing. This research perspective focuses on the evidence for a functional relationship between NF-κB and senescence, and how disruption of the NF-κB pathway can lead to its bypass. CIP1, which inhibits cyclinE-CDK2 complexes from phosphorylating the pRB family of proteins. This leads to activation of the pRB tumour suppressor pathway in which the hypophosphorylated pRB family of proteins complex with E2F heterodimers, thereby preventing cell cycle progression [CIP1 can also arrest cell cycle progression by inhibiting cyclinA/B-CDK1 and cyclinD/CDK4 complexes [INK4A upon non-genotoxic stress, which inhibits cyclinD-CDK4/6 complexes from phosphorylating the pRB family of proteins [Somatic cells usually undergo a limited number of divisions termed the Hayflick limit . When thgression . P21CIP1omplexes . The pRBSenescence can dampen the repair and regenerative ability of a tissue due to an increased population of growth arrested cells and a diminished stem cell population; and thereby play a hand in tissue ageing. It can also act as a tumour suppressor mechanism by preventing the proliferation of potentially cancerous cells. Bypass of the cell cycle arrest can lead to a limitless replicative potential, a key step in malignant transformation . BlagoskThe role of nuclear factor (NF)-κB in senescence is controversial, as it is divisively associated with proliferation and tumour progression, and contrastingly with growth arrest and ageing. The NF-κB family of transcription factors are ubiquitously expressed and regulate the response to cellular and environmental stress . In the The non-canonical pathway Figure  depends Persistent inflammation is an important underlying condition that aids tumour development and is characterised by the activation of NF-κB signalling. The NF-κB family of proteins have been implicated in tumour development due to their sequence similarity to the viral oncogene v-Rel , 18. MutINK4A expression, both of which are reduced upon NF-κB inhibition [In parallel to its role in tumour development, chronic inflammation also plays an important role in the ageing process. Increased expression of inflammatory markers and an associated increase in NF-κB DNA binding activity have been demonstrated in cells from older donors over cells from younger donors . This mahibition . Moreovehibition .Recently, Rovillain et al.  used a nAlthough it is not known how NF-κB signalling is activated in senescence, a pathway linking genotoxic stress to activation of NF-κB Figure  in mouseInsulin/IGF1 signalling can promote NF-κB signalling and potentiate ageing and age-related degeneration by activation of the phosphoinositide 3-kinase (PI-3K) /AKT pathway (Figure CR may also increase expression of SIRT1 Figure , a nicotFOXM1 is a highly conserved forkhead transcription factor that regulates the expression of numerous regulators of cell cycle progression, particularly in the G2 phase . FOXM1 iSenescent cells secrete growth factors, proteases, and inflammatory cytokines as part of the SASP -60. DespIn this perspective, we have reviewed the recent findings about the role of NF-κB signalling in ageing and senescence, and weighed up the contradictory evidence. Previous studies had proposed NF-κB signalling as a proliferative message, as well as others conversely implicating NF-κB signalling in growth arrest. With such controversy, many questions remain to be answered. Our latest results show that silencing of NF-κB allowed cells to bypass senescence, and that activation of NF-κB signalling leads to upregulation of genes associated with SASP  and supp


TCERG1L. Overall, we conclude that DNMT3B is involved in a wave of de novo methylation during reprogramming, a portion of which contributes to the unique hiPSC methylation signature. These 82 CpG methylation sites may be useful as biomarkers to distinguish between hiPSCs and hESCs.Whether human induced pluripotent stem cells (hiPSCs) are epigenetically identical to human embryonic stem cells (hESCs) has been debated in the stem cell field. In this study, we analyzed DNA methylation patterns in a large number of hiPSCs (n = 114) and hESCs (n = 155), and identified a panel of 82 CpG methylation sites that can distinguish hiPSCs from hESCs with high accuracy. We show that 12 out of the 82 CpG sites were subject to hypermethylation in part by DNMT3B. Notably, DNMT3B contributes directly to aberrant hypermethylation and silencing of the signature gene,  •Eighty-two DNA methylation sites distinguish hiPSCs and hESCs with high accuracy•Twelve aberrantly hypermethylated signature CG sites are due in part to DNMT3B•DNMT3B also contributes to previously reported aberrant hypermethylation in hiPSCs Huang et al. identify a panel of 82 CpG methylation sites that can robustly distinguish hiPSCs from hESCs with high accuracy. Part of the signature consists of aberrantly hypermethylated CpG sites, which are partially abrogated in the absence of DNMT3B. In addition, DNMT3B-mutant hiPSCs do not exhibit other aberrant hypermethylation signatures as previously reported. DNA cytosine methylation is a major epigenetic factor that contributes to regulating important biological processes such as genomic imprinting, X inactivation, and gene regulation. DNA methylation is established and maintained by a family of DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, and deficiency in any Dnmt enzyme leads to embryonic death in mice. Additionally, aberrant DNA methylation is associated with human diseases such as cancer, immunodeficiency, and neurological disorders . CollectDNA methylation is also important for both stem cell differentiation and cellular reprogramming. In differentiation, dynamic DNA methylation changes are critical for lineage specification as a wave of de novo methylation takes place to silence pluripotency genes and establish tissue-specific methylation patterns . During Induced pluripotent stem cells (iPSCs) have the characteristics of human embryonic stem cells (hESCs), and many studies have investigated the similarities between iPSCs and hESCs, including genome stabilities, transcriptome  and histTo address this issue, we systemically compared the methylation profiles of a large number of human iPSCs (hiPSCs) and hESCs from multiple labs to revisit the question of whether hiPSCs have a unique CpG methylation signature. We identified a panel of 82 CpGs that can distinguish hiPSCs from hESCs with high accuracy. Remarkably, 12 signature CpG sites tended to be hypermethylated compared with both somatic cells and hESCs, suggesting that these methylation signatures are not a form of residual somatic epigenetic memory. Hypermethylation of these 12 sites is partially disrupted in DNMT3B-deficient hiPSCs, consistent with the function of DNMT3B for de novo methylation during reprogramming. Together, our results demonstrate a robust hiPSC molecular signature that is partially a consequence of DNMT3B-mediated de novo methylation during reprogramming.In previous reports, investigators have debated whether hiPSCs exhibit a unique CpG methylation profile due to either residual somatic cell memory or aberrant methylation in select domains when compared with hESCs. In this study, we investigated this topic by analyzing CpG methylation in a large number of pluripotent cell samples (n = 269) from multiple labs, thus increasing the power of the statistical analyses. Using Illumina Infinium BeadChip assays, we first examined the methylation profiles of 25 cell lines, including five hESCs, five parental somatic cells, and 15 lines of hiPSCs covering hiPSCs generated by both vector-containing and vector-free methods. Globally, hierarchical clustering analysis demonstrated that hiPSCs are highly similar to hESCs, but distinctively different from somatic cells A. To ideUltimately, a robust signature should be able to accurately discriminate between hiPSCs and hESCs in independent data sets regardless of the laboratory of origin or the quantitation method used. Although hierarchal clustering is one way to visualize how multiple samples are grouped, we turned to more robust and quantitative classification methods. To provide an unbiased estimate of predictive accuracy for cell type, we used a leave-one-out analysis in which the support vector machine (SVM) learning model was fit on all but one sample and its prediction was related to the truly observed cell type of the left-out sample see . Using oWe found that our panel of CG signature sites was able to accurately discriminate between hiPSCs and hESCs despite the varying sample sizes among different studies. For example, we analyzed an independently derived Infinium 27k BeadChip data set that profiled DNA methylation in 20 hiPSCs and 11 hESCs , and shoRemarkably, our DNA methylation signature was also able to accurately discriminate between hiPSCs and hESCs regardless of the quantitation platform or technique used. In a data set compiled using the Infinium 450k BeadChip system, we examined DNA methylation in 29 hiPSCs and 18 hESCs . InteresMEG3, PEG3, ZIM2, and other imprinted loci  or not expressed in hiPSCs  were also uniquely hypermethylated in hiPSCs, but hypomethylated in both somatic cells and a portion of hESCs B. RemarkBecause Dnmt3B is more dramatically upregulated in hiPSCs when compared with the levels of Dnmt3A and Dnmt1 , we hypoTCERG1L was consistently identified as an aberrantly hypermethylated gene in two previous studies and confirmed in our current study .Up to now, it was not clear whether hiPSCs have distinct transcriptomes and methylomes when compared with hESCs. Although one initial study reported the presence of iPSC-specific gene expression in a small number of iPSCs , severalBecause iPSCs exhibit a significant increase in genome-wide methylation when compared with parental somatic cells, we suspected that de novo methylation plays an important role in establishing a unique iPSC methylation signature. By comparing methylation patterns in mutant ICF-iPSCs, we indeed found some altered methylation signatures, suggesting that DNMT3B contributes to de novo methylation during reprogramming. In particular, we identified five signature CpGs (out of the 82 CpG signature sites) that undergo DNMT3B-mediated de novo methylation. This conclusion was also extended to hypermethylation signatures identified by others .Our methylation signature is different from what was previously identified by either microarray or high-throughput sequencing analysis. Earlier studies suffered primarily from limited sample sizes due to the costly approach required to measure genome-wide DNA methylation levels on a comprehensive scale. Several previous studies using RRBS attempted to verify reported signatures in the literature and found a lack of reproducibility , arguingOCT4, SOX2, KLF4, and c-MYC or OCT4, NANOG, KLF4, and LIN-28. This study of hESCs and hiPSCs was approved by the UCLA Embryonic Stem Cell Research Oversight Committee. We used the HumanMethylation27 DNA Analysis BeadChip from Illumina to interrogate 26,837 CpG sites over 14,152 genes. Full experimental procedures and data analysis are available in the Briefly, hiPSCs were generated from IMR90, CCD-1097SK, BJ1, and NPC cells derived from 11-week-old fetal brain using retroviral expression of


Sarda sarda, Bloch 1793) belongs to the important marine fish species with a wide geographical distribution covering the Atlantic Ocean, the Mediterranean and its bordering seas. Aquaculture practices for this species are still in their infancies and scientific studies are seldom undertaken, mainly because of difficulties in sampling. Thus for small tuna species like the Atlantic bonito only little is known about its biology and regarding the molecular background even less information is available. In the production of marine fish it is known that the most critical period is the larval stages, as high growth rates as well as significant developmental changes take place. In this study we have investigated the transcriptome of the Atlantic bonito of five larvae stages applying Illumina sequencing technology. For non-model species like aquaculture species, transcriptome analysis of RNA samples from individuals using Illumina sequencing technology is technically efficient and cost effective. In the present study a total number of 169,326,711 paired-end reads with a read length of 100 base pairs were generated resulting in a reference transcriptome of 68,220 contigs with an average length of 2054 base pairs. For differential expression analyses single end reads were obtained from different developmental stages and mapped to the constructed reference transcriptome. Differential expression analyses revealed in total 18,657 differentially expressed transcripts and were assigned to five distinguished groups. Each of the five clusters shows stage specific gene expression. We present for the first time in the Atlantic bonito an extensive RNA-Seq based characterization of its transcriptome as well as significant information on differential expression among five developmental larvae stages. The generated transcripts, including SNP and microsatellite information for candidate molecular markers and gene expression information will be a valuable resource for future genetic and molecular studies.The Atlantic bonito ( Sarda sarda, Bloch 1793), belonging to the family of the Scombridae is a mackerel-like epipelagic teleost fish species. Among the four allopatric species, Sarda australis, Sarda chiliensis, Sarda orientalis and S. sarda, only S. sarda inhabits the Atlantic and Mediterranean waters. Overall the Atlantic bonito has a wide geographical distribution covering the Atlantic Ocean, the Mediterranean and its bordering seas S. chiliensis (Cuvier) has been published Sparus aurata) by GS454 FLX sequencing Ictalurus punctatus) S. aurataDicentrarchus labrax) Thunnus thynnus) in total 11, 453 sequences are published comprising 10,175 expressed sequence tags (ESTs) and 1278 Nucleotide . Regarding the Atlantic bonito even less molecular information is available. Only 469 sequences are published at the NCBI nr database and none in the NCBI EST database and no Next Generation Sequencing (NGS) project has been published so far .The Atlantic bonito  guiding principles for more ethical use of animals in testing, first described by Russell and Burch in 1959. These principles are now followed in many testing establishments worldwide prior to initiation of experiments. The larvae were anaesthetized using 100–200 mg/L MS222  depending on fish size. Afterwards samples were immersed in the RNA later  were transferred to an 80°C ultra-low freezer until preparation of RNA. All experiments were carried out within the specific European RTD program of Framework Program 7, Theme 2-Food, Agriculture, Fisheries and Biotechnology (Project SELFDOTT) which is supervised by the European commission and follows the European Union Directive for the for the protection of animals used for experimental and other scientific purposes.Larval rearing was performed at HCMR and IEO following semi-intensive methodologies In total three larvae (whole larvae) of five different developmental stages  were pooled, collected in RNAlater  and stored at −80°C for transcriptome analysis. Sampling points and maturation are illustrated in Whole larvae of each stage were submitted to RNA extraction. Disruption of the samples was performed in liquid nitrogen using mortar and pestle. After adding lysate buffer, the lysate was homogenized by passing it through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe for 5 times. RNA was subsequently extracted using the RNA extraction Kit II of Machinery Nagel , according to the manufacturers’ instructions. RNA concentrations were determined using NanoDrop ND-1000 spectrophotometer  and the quality was assessed by electrophoresis on a 1% ethidium bromide agarose gel as well as by the A260/280 ratio and by Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis chip.http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and low quality reads were removed with Trimmomatic software Samples for RNA sequencing (RNA-Seq) were prepared by Cornell University Core Laboratories Center using standard methods and sequenced over two lanes of Illumina HiSeq vs 2000. In one lane, extracted RNA from mixed developmental stages were sequenced as paired-end, 100 bp reads. On the second lane, each of the five developmental stages were tagged and sequenced as 100 bp, single-end reads. Reads from each stage were distinguished through the use of mulitplex identifier (MID) tags. Sequence quality was assessed using FastQC  and Msat identification tool Sequences were clustered using the default parameters of the CD-HIT-EST tool p<0.05 and a minimum of 2 fold differences in their expression between different stages were assigned as differentially expressed.Transcripts expression profiles were assessed for each developmental stage using the single read sequences retrieved from each stage by mapping the reads against the reconstructed assembly using bowtie −6 and 10−10 respectively. Annotations and GO terms were assigned using Blast2Go software Assembled transcripts were submitted against the non-redundant protein database (nr) as well as the non-redundant nucleotide database (nr/nt) using the standalone BLAST tools (version 2.2.25) Transcripts assigned to be differentially expressed were submitted to K-means clustering method. K means clustering is the most appropriate clustering method when more than 200 data points are analysed Venn diagram was constructed using all differentially expressed genes using as reference stage Bon0. To find enrichment in gene ontology (GO) terms, enrichment analysis (Fisher’s Exact Test) tool in Blast2GO software was used with term filter value p<0.05, term filter mode “False Discovery Rate (FDR)” and two-tailed test. Reference data set were all mapped genes onto the constructed reference transcriptome and test data set used in the present analysis were all transcripts found to be differentially expressed only at stage 30 when comparing all stages under study to stage 0.In order to infer the module networks the LeMoNe algorithm was used. LeMoNe uses ensemble based probabilistic optimization techniques to identify clusters of co-expressed transcripts as well as their regulators. In this study the normalized log2 values of all stages were used as input of transcript expression. Annotated transcription factors were used as potential regulators. LeMoNe assigned the corresponding regulators in each module characterized by a particular weight. The visualization of the regulatory networks was performed with Cytoscape v. 2.7.0, where transcripts being differentially expressed only between stage Bon0 and Bon5 were filtered out in the present study.Homologue nucleotide sequences of comp49187_c0_seq1 and comp6057_c0_seq1 with the blastx match myozenin-2-like  were useRNA Seq was carried out on RNA extracted from 5 different developmental stages of the Atlantic bonito. A total number of 169,326,711 paired-end reads with a read length of 100 bp were generated. After filtering 162,109,074 clean reads were obtained for assembly analysis .it database (http://www.fish-it.org). This set was used as reference transcriptome for differential expression assessment as well as for the enrichment analysis. Putative single nucleotide polymorphisms (SNPs) and microsatellites were identified and summarized in The assembly returned 68,220 contigs with an average length of 2054 bp  and is sBLASTX and BLASTN searches were performed for annotation of the Atlantic bonito transcriptome assembly. Out of 68,220 contigs obtained after assembly, 43,312 (∼63%) showed a significant match against the protein database (nr) and 46,224 (∼ 68%) had a positive match against the nucleotide database (nr/nt) of GenBank. Blast2GO analysis of the different annotations related to biological process revealed that the majority of transcripts are categorized to nine gene ontology (GO) terms: Cellular process (19%), metabolic process (14%), biological regulation (12%), response to stimulus (9%), multicellular organismal process (8%), signaling (7%) developmental process (7%), localization (7%) and cellular component organization of biogenesis (6%). Blast2GO analysis of the different annotations related to molecular function classified the majority of transcripts to the binding (45%) and catalytic activity (26%) categories .p value <0.05, and minimum of 2 fold difference) in one of the pair-wise comparison of the stages. Those transcripts found to be differentially expressed at least in one of the stages, were subjected to K-means clustering having the largest F-values when the cluster number was set to five (data not shown) (Single end reads were assessed from each stage separately and mapped onto the constructed reference transcriptome . On avert shown) . Clustert shown) . The colStage specific transcripts using stage Bon0 as reference stage  are illustrated by a Venn diagram in de novo assembly of short reads without a reference transcriptome were still difficult. However using Illumina technology of sequencing paired-end reads facilitates the assembly and using a combined assembly and mapping strategy results in a reasonable and accurate reference transcriptome which is also strongly supported by the obtained results of sequence average length and N50 length. Transcripts expression profiles were assessed by high throughput single end sequence (SE) data from each developmental stage which was mapped against the reconstructed assembly using bowtie de novo transcriptome assembly PE reads are of importance, for assessing differentially expressed genes the sequencing depth is significant Oreochromis niloticus]” of the two transcripts comp49187_c0_seq1 and comp6057_c0_seq1. Here homolog sequences for seven fish species were retrieved from NCBI databases followed by phylogenetic tree construction  is not Mitf has been identified but does not show significant differential expression within the five stages. In contrast to Mitf the role of Otx in eye formation is conserved between zebrafish and other vertebrates indicating it importance in development. Besides transcripts which show differential expression between stages also transcripts were found exclusively in only one of the stages. A common step for microarray analysis is cluster analysis of differentially expressed genes. Cluster analysis permits to classify samples of entities into a small number of mutually exclusive groups based on the similarities among the entries. In the present study the obtained groups were confirmed by discriminant analysis in which up to 95% were successfully grouped in the predicted clusters. Four of the five clusters show exclusive up-regulation in one of the developmental stages, i.e. cluster 2 stage Bon 30, cluster 3 stage Bon 0, cluster 4 stage Bon 20 and cluster 5 stage Bon 5. Cluster 1 comprises a small set of genes showing up-regulation exclusively in stage Bon 10 . TranscrS. sarda). We demonstrate that transcriptome analysis as well as assessment of differential expression of RNA samples using Illumina sequencing technology is technically efficient and with low cost. A total of 68,220 contigs have been constructed out of a total number of 169,326,711 100 bp paired-end reads. Differential expression between five important developmental stages has been assessed and stage specific genes were isolated. It has been shown that in earlier stages transcripts like homoebox genes are up-regulated whereas in the later stage transcripts important for muscle development are found to be higher expressed. The generated transcripts, microsatellite and SNP information for candidate molecular markers as well as gene expression information will be valuable information for future genetic and molecular studies in the Atlantic bonito and closely related species pinpointing also to the importance of studying the 3’UTR as well as paralogues during development.The present study is the first report of a transcriptome study in the Atlantic bonito Click here for additional data file.Table S1Transcripts of each of the five generated cluster including their annotations.(XLSX)Click here for additional data file.Table S2http://bioinformatics.bc.edu/marthlab/FreeBayes).Transcripts screened for sites with possible single nucleotide polymorphic (SNP) variation as well as patterns of simple sequence repeats (SSR) using freebayes Click here for additional data file.Table S3Transcripts involved in muscle development. Here all major sarcomeric proteins are shown found after enrichment analysis using enrichment analysis (Fisher’s Exact Test) tool in Blast2GO software. Enrichment analysis was performed with the set of genes found uniquely in Atlantic bonito stage 30 dph and using all transcripts obtained after assembly of mixed developmental stages as reference test.(XLSX)Click here for additional data file.Table S4Summary of all transcripts found in the present study with a relevant role in the post-embryonic development. As described by Yufera et al. (XLSX)Click here for additional data file.


Due to the known potential for fretting and corrosion at modular junctions in orthopaedic implants, this retrospective study evaluated radiographic and clinical outcomes of 85 primary TKA patients implanted with modular stemmed tibial components and followed up for an average of 82 months. There was low incidence of tibial radiolucent lines, excellent functional outcomes, and no complications associated with stem modularity. The findings were comparable to the historical control study involving 107 TKA with a nonmodular tibial stem design. When using surface cemented tibial components combined with a constrained polyethylene bearing, modular stems appear to be a viable option for primary TKA when adequate fixation and rotational stability are maintained.  Central stems on tibial components in total knee arthroplasty (TKA) can exist in many different lengths  and shapes . Whether provided in monoblock or modular form, central stems are thought to aid component fixation by transferring load-bearing stresses to stronger distal bone and by resisting component tilting and liftoff –7. Some Surgeons often restrict modular stem use to revision knee arthroplasty or to primary TKA cases with gross malalignment or osseous defects , 13. DurThe aim of this study is to evaluate clinical outcomes at the 2-to-11-year follow-up interval for a consecutive series of primary TKA patients implanted using a single prosthesis design having a cemented tibial baseplate with five pegs and a short modular stem attached via a Morse taper and a conforming polyethylene insert. The rationale for selecting this prosthesis design for primary TKA was to provide anteroposterior stability at the femoral-tibial articulation, rotational stability from the pegs at the baseplate-bone interface, and varus-valgus stability from the stem to prevent baseplate liftoff. Because this study is focused on primary TKA, we identified a historical control study inclusive of a comparable TKA design with similar tibial component features, except for the stem modularity . The nulBetween May 2001 and October 2002, 121 consecutive knees in 119 patients underwent primary TKA by a single surgeon and coauthor Thomas Pace. While all data points were entered prospectively concurrent with each patient's pre- and postoperative clinical assessment, the results were retrospectively reviewed in this institutional review board-approved study. Only patients who achieved a minimum of 2 years of follow-up were evaluated, resulting in exclusion of 24 patients (25 TKA). Two patients (2 TKA) were lost to follow-up and 9 patients (9 TKA) died before the 2-year follow-up interval. The final cohort consisted of 85 TKA in 84 patients with 2 to 11 years of follow-up. There were 60 females and 24 males, with an average age at index surgery of 66 ± 11 (31–86) years.All patients were implanted with the same prosthesis design  that incorporates a modular stem attached to the tibial baseplate . The tibMedical records were retrospectively reviewed to assess clinical and radiographic outcomes at last the follow-up. The surgical approach and posterior cruciate ligament treatment (retention or sacrificed) were recorded, as well as preoperative and postoperative Knee Society Scores  and postThis level III therapeutic study, including patients treated with a primary TKA having a modular tibial stem, was compared to a historical control group of patients treated with a primary TKA having a nonmodular tibial stem, as reported by Hofmann et al. . This prHofmann et al.  reportedIndication for TKA for all patients was osteoarthritis. Patients with other indications  received a different TKA design and were not included. The posterior cruciate ligament (PCL) was sacrificed in all 85 TKA, with anteroposterior stability provided through the use of a polyethylene insert incorporating an anterior-constrained articular geometry. The patella was unresurfaced in all but one knee. The medial parapatellar approach was used in 83 (97.6%) of the cases, whereas the remaining 2 (2.4%) were exposed using the subvastus approach. The subvastus approach was selected on a case by case basis with the premise that it may offer some benefit by allowing a quicker recovery and less postoperative pain. It was performed on patients if the distal thigh circumference was small enough that it could practically be done; however, patients with a larger distal thigh circumference received the medial parapatellar approach.The average follow-up was 82 ± 38 (24–133) months, including 56 TKA with a minimum of 5-year follow-up. Average preoperative Knee Society Scores were 78.6 ± 4.6 (70–87) and improved to 99.2 ± 2.0 (90–100) postoperatively. Postoperative flexion averaged 118.5° ± 5.4° (95°–128°).No radiographs demonstrated osteolytic lesions around the tibial component. Radiolucent lines adjacent to 2 TKA were noted upon initial radiographic analysis. In one TKA, the radiolucent line was less than 2 mm in thickness, asymptomatic, and not associated with prosthesis failure. In the other TKA, a 2 mm lucent line was noted and further investigated. The patient had indicated mild patellofemoral pain (not requiring medication), most likely related to the unresurfaced patella and is not considered to be associated with failure of the tibial component.None of the knees included in the current study required revision surgery. No infections were recorded in this series of patients. One knee experienced dehiscence two weeks following surgery and was treated with secondary wound closure and healed uneventfully. One knee required closed, manual manipulation following implantation because the average flexion was less than 90° at the 10–12 week follow-up visit. The overall survivorship was 100% at an average of 82 months with no pending failures.The historical control study by Hofmann et al.  includedAverage follow-up for the control group study was 95 (range 63–155) months. Postoperative Knee Society Scores averaged 195 (range 162–200), which improved from the preoperative scores that averaged 122 (range 94–152). Postoperative flexion averaged 116°.Radiographic review of the historical control group revealed no osteolytic lesions in any TKA. However, three TKA had nonprogressive radiolucent lines adjacent to the tibial baseplate, which all were asymptomatic and not associated with prosthesis failure. Two other TKA required revision surgery but none of the tibial components were revised for loosening. One revision consisted of a polyethylene exchange due to PCL insufficiency and the other required a femoral component removal due to pain and possible loosening at 4 years following index surgery. There were no infections reported. The overall survivorship was 98% at an average of 95 months.The recorded positive clinical outcomes, including lack of radiolucent lines and osteolysis in these primary TKA with surface-cemented tibial components and short modular stems, indicate that fretting and corrosion were not prominent features of the modular junction at the 2-to-11- year follow-up interval. The lack of revisions, few complications, low incidence of tibial radiolucent lines, and excellent functional outcomes were comparable to the historical control study involving patients implanted with pegged tibial baseplates having a nonmodular central stem . The earThere are several factors that likely contributed to our results. First, tibial stems were used during primary TKA in the absence of large tibial bone defects. In such circumstances, stemmed components endure lower load magnitudes and decreased stresses, resulting in greater baseplate stability than the same components implanted in tibias with bone defects . In the The surgical technique of the senior surgeon used in these PCL-resected primary TKA included routine selection of a conforming polyethylene insert with anterior constraint rather than a cam and post-PCL-substituting (PS) design and implantation of a modular stem tibial baseplate to augment tibial baseplate. Several clinical studies support this surgical technique. Hofmann et al.  report sIt was not the intent of this study to compare complication rates for modular tibial stems used in primary and revision TKA, as considerable differences exist in those different clinical scenarios . AlthougThere are some limitations to this study. The average follow-up time of approximately seven years may not be sufficient to fully capture potential adverse events. However, particulate wear-induced osteolysis can present radiographically within the first three years . TherefoIn these primary TKA patients implanted with surface cemented tibial components having a short modular stem and constrained polyethylene bearing, there were no complications associated with stem modularity. The absence of progressive radiolucent lines is consistent with adequate fixation, including rotational stability provided by the pegs and sufficient varus-valgus stability provided by the modular stem. These findings do not support the null hypothesis that these modular TKA experience greater radiographic evidence of negative tissue reactions and higher revision rates compared to patients receiving TKA without stem modularity. At this 2–11-(average 6.8) year follow-up interval, the option for attaching modular stems appears to be a viable design feature for primary TKA. However, since only one modular stem TKA design was evaluated after used with uniform surgical techniques during routine primary TKA, the authors caution that these findings may not be applicable when generalized to other designs with different modular stems and articular conformity used under different clinical scenarios.


Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform E. grandis (including the clone of which the genome was recently sequenced – Brasuz1) as well as a single E. grandis x urophylla hybrid were established in the laboratory. Light intensity was studied as a means to reduce browning and promote callus growth and shoot regeneration. Gibberelic acid (GA3) concentrations in the micropropagation medium (Murashige and Skoog’s (MS) basal medium containing 0.05 mg/L benzylaminopurine – BAP) were studied to help produce long internodes to aid transformation in vitro. To mitigate phenolic production we used several different antioxidants including ascorbic acid, PVP and PVPP. Shoot regeneration rates were studied by testing a concentration range of cytokinin . We tested different Agrobacterium transformation protocol enhancements including the use of acetorsyringone and two different co-cultivation techniques (whole explant immersion versus pipetting of Agrobacterium suspension onto cut leaf edges). We also tested the suitability of kanamycin and hygromycin as selectable markers during transformation.Five different clones of Light conditions during shoot regeneration were critical to the rate of organ regeneration, with more than 50% of explants producing shoots in the presence of reduced or indirect light, compared to approximately 30% incubated under normal light conditions. In addition, shoot differentiation occurred earlier (10-14% at 40 days) under reduced or indirect light compared to normal light  for shoot regeneration from stem and leaf explants of the E. grandis x urophylla containing different cytokinin combinations, we found that the highest rates of shoot regeneration (11.8%) occurred when using stem explants incubated on SIM containing 1 mg/L NAA (nicotinamide) and 10 mg/L zeatin. Of the five tested E. grandis clones, we found that the highest rates of shoot regeneration (52%) were from leaf explants of clone p207 in the presence of SIM containing 3 µM TDZ and 0.1 µM NAA. Neither the use of two different acetosyringone concentrations (250 and 750 µM) during co-cultivation, nor either of the two co-cultivation techniques (immersion and pipetting) had a significant effect on transient gene expression measured shortly after cocultivation. Testing of different kanamycin and hygromycin concentrations (“kill curve”), revealed that 30 mg/L kanamycin or 5 mg/L hygromycin can be used for the selection of putative transgenics after transformation , and quality  as well as finely-tuned phytohormone concentrations, will play critical roles in enhancing the probability of the successful generation of stable transgenic lines.There is significant variability in the performance and response of different clones of


Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric  forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage.The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O II) heme-iron and molecular oxygen provides the essence of aerobic metabolism, this molecular function is also vulnerable to detrimental effects by auto-oxidation due to electron transfer from the heme-iron to oxygen: FeII-O2→FeIII+O·2→H2O2III, met-) RH as well as active oxygen species, are hazardous to the vascular system, as they can induce pathological events, such as atherosclerosis. The ferric forms of the RH bear peroxidase-like activity leading to the unstable, potentially damaging ferryl (FeIV) state II+FeIV→2FeIII) which is longer-lived and hence its likelihood to provide damage are high Hemoglobin (Hb) and myoglobin (Mb) are collectively known as the respiratory hemoproteins (RH) based on their function to deliver molecular oxygen to the body tissues. While the link between ferrous , which binds to ferrous as well as to ferric heme-iron, and carbon monoxide (CO), which binds with extremely high affinity exclusively to ferrous heme-iron in vivo conditions peroxides in low concentration can assist the reduction in presence of CO.In recent years the essential role of CO, the product of heme catabolism by the enzyme Heme Oxygenase  has been clarified in vitro on a molecular level, whether the potential dangerous oxidative activities of acellular RH and peroxides can be attenuated by the HO product, CO. To mimic in vivo plasma conditions, reactions were carried out in an anaerobic environment using ferric RH and peroxides at their concentration levels reported under a variety of pathological circumstances. It was found that in the presence of CO damaging peroxides are consumed while ferric RH are transformed into their nonhazardous carboxy-forms.The present study was designed to analyze III) and H2O2. To mimic in vivo conditions of restricted free oxygen  the reactions were carried out under oxygen-free conditions in a CO atmosphere which resulted in a CO concentration of 1 mM in water phase 2O2 were used at a concentration range of few micromolars throughout the study. Control reactions were carried out under neutral gases, argon or nitrogen, without CO or in a CO atmosphere in which no peroxides were added. The data in 2O2 practically no changes occurred in the spectra pattern and only minor changes in the optical density (OD) were detected (The first part of the study was designed to test whether CO can affect the interaction between metHb (Fe detected . Similar2O2 as discussed in the literature 2O2 (line b) spectral changes, due to the reduction of metHb under CO atmosphere lacking a reducing agent, have already been described, however they are extremely slow (t1/2 of ∼50 days) The spectral changes are easier noticeable when presented as difference spectra depicted in 2O2-induced alterations were completed within two hours. The kinetic patterns of the CO lacking controls of both hemoproteins exhibited an initially fast absorption increase, followed by a decreasing phase (IV) formation followed by its auto-reduction to ferric (FeIII) forms 2S) two hours after reaction initiation did not result in any formation of the typical ferryl product (data not shown) 2O2 used were equal or lower then those of Hb.Time dependent spectral changes of reactions including Hb or Mb  at wavelng phase , lines c2O2 concentration dependent.The results demonstrated in 2O2 can be effective as a ferric Hb reducing agent in the presence of lower CO concentrations. Free oxygen was also avoided in this experiment by using a neutral gas (nitrogen) as a complement. As slower reaction rates are expected by lowering one of reactant concentrations, the reaction mixtures were incubated overnight (ON). The experiments up to this stage were performed in solutions equilibrated with an atmosphere containing CO alone which, as stated, yield about 1 mM of the gas in the liquid phase. Such a concentration is not expected to be formed in the plasma even following long-term activated HO-1. Therefore the following experiment tested whether HTo analyze a possible interaction of hydrophobic peroxides and metHb, oxidized LDL (oxLDL) formed by copper were used initially in vivoHaving demonstrated that the copper oxidized LDL model can replace hydrophilic peroxides as reducing agents for metHb in presence of CO, it was of importance to study the activity of “naturally” oxidized aged LDL which can form 2O2 as ferric heme-iron reducing agents and trap the RH in their carboxy, inactive forms.Once the increased oxidability of aged LDL was demonstrated, it was further employed as a source of hydrophobic peroxides to determine whether they are able to act as reducers of ferric RH in presence of CO. Experimental mixtures containing ferric Hb or Mb and aged LDL were incubated under CO atmosphere. LDL lacking mixtures were used as controls. The difference spectra of time zero and ON incubation are depicted in 2O2 and CO resulted in transformation of ferric RH into their carboxy forms →RH(FeII)+½O2+H2O (very slow)HII)+CO→CORH(FeII) (fast)RH and Hb in vascular pathology is well established It is important to note that other electron donors can affect ferric RH in a similar manner. For example, in presence of CO the reduction of ferric to ferrous Mb occurs in a mutant protein where a correctly positioned cysteine residue is responsible for electron transfer to the heme-iron in vitro data could be found from in vivo total hemoglobin evaluation following abdominal surgery Epidemiological studies evaluating metHb and COHb levels usually refer to total blood containing mostly encapsulated Hb. Since cell-free Hb is a minor part of total Hb, changes in this fraction are usually not observed. Interestingly, support for our It should be noted that diminished oxidability of RH by CO using any electron source, even a weak one like peroxides, probably represents a much broader phenomena than that shown in the current study for the RH and peroxides. Horseradish Peroxidase (HRP) is a classic heme-enzyme with a ferric ground state is also reduced to ferrous iron thereby binding CO with high affinity An additional aspect related to the consequences of the present study involves iron chelators. These molecules, originally designed to strongly bind hazardous free iron and eliminate it from plasma, were recently demonstrated to function as antioxidants as well. By binding to ferric heme-iron, the chelators prevent also oxidative modifications in vitro study specifically used both hemoproteins and peroxides concentration range to mimic in vivo plasma conditions which ensue during acute or chronic vascular hemolysis. At this state of affairs, RH peroxidase-like activity, namely heme iron redox-shuttle (FeIII↔FeIV), is limited and damage may result mostly from unstable hemin RH met-forms As stated, the current III→FeII) is analogous to the on going course of action provided to circulating Hb captured within red cells. Howether at more extreme pathological conditions the unstable hemin in ferric RH may be trapped by hemopexin The current study data indicate that metRH can be reduced to the more stable ferrous states with aid of endogenously HO-produced CO. The latter process  and potassium bromide (KBr) was from Merck, Darmstadt, Germany. PD-10 desalting columns were purchased from GE Healthcare UK Ltd. CM-52 and DE-52 celluloses were obtained from Whatman International, Maidstone, England. Ferricyanide was from Agan Chemical Manufacturers Ltd., Israel. Gases: Carbon monoxide (CO), at least 99.5%, was supplied by Gordon Gas and Chemicals Ltd., Israel; Nitrogen  and Argon  were supplied by Israel Oxygen Centre.Myoglobin (equine), Hemin (from bovine red cells), Albumin , ethylene diamine tetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich, Israel. Hydrogen peroxide  of healthy donors by sequential ultracentrifugation as described 4 (five fold molar protein concentration) for 2 hours in 37°C and immediately passed through two PD-10 columns.Freshly isolated LDL was oxidized by copper according to the literature with some modifications Freshly isolated LDL was sterilized by passing through 0.2 µM filters and further incubated for at least 10 weeks in 4°C XL or thermostated GBC UV/VIS 920 spectrophotometer.Spectral characteristics and time dependent changes spectral in reaction mixtures were monitored by single or repetitive (every 135 sec) scanning of light absorbance spectra at UV and/or visible wavelength range using a Contron – UVIKONAll reactions were carried out in PBS pH 7.4 at 37°C. The appropriate gas atmosphere was reached as follows: the desired gas was bubbled into the media solutions in rubber septa sealed spectrophotometric cuvettes. Protein containing stock solutions were equilibrated a priori by the desired or nitrogen as neutral gas and were further diluted (by injection) into cuvettes up to 5% of volume. Under such conditions the gases concentration in the liquid phase reaches about 1 mM 2O2 - 30 µM (1∶ 250 dilution of stock), LDL - 100 µg protein/ml.The following reactants were used at constant concentrations: hemoproteins - 10 µM, H


Cholestatic liver diseases encompass a wide spectrum of disorders with different causes, resulting in impaired bile flow and accumulation of bile acids and other potentially hepatotoxic cholephils. The understanding of the molecular mechanisms of bile formation and cholestasis has recently improved significantly through new insights into nuclear receptor (patho)biology. Nuclear receptors are ligand-activated transcription factors, which act as central players in the regulation of genes responsible for elimination and detoxification of biliary constituents accumulating in cholestasis. They also control other pathophysiologic processes such as inflammation, fibrogenesis, and carcinogenesis involved in the pathogenesis and disease progression of cholestasis liver diseases. By inhiThe role of FGF19 in cholestasis is yet to be elucidated. Although FGF19 is not expressed in hepatocytes and systemic FGF19 under physiologic conditions originate from the intestine, its hepatocellular expression is highly induced in cholestasis.Apart from repression of BA synthesis, FXR is able to induce alternative basolateral BA transport through organic solute transporter α/β (OSTα/β)Biliary BAs are normally present in the form of mixed micelles together with phospholipids and cholesterol. Importantly, hepatic FXR promotes bile secretion not only through regulation of BA export but also via induction of canalicular phopholipid floppase MDR3 (Mdr2 in rodents)R)-hydroxycholesterol and androsterone has been identified as endogenous FXR activators.In addition to BAs as principal endogenous FXR ligands, an intermediate product of BA synthesis oxysterol 22 prolonged duration of cholestasis may induce adaptive, secondary changes in transporter expression self-protective mechanisms of hepatocytes against retaining cholephils. For example, in PBC patients, repression of BA uptake systems  together with induction of basolateral efflux systems  support the elimination of retained BAs from the liver as cholestasis progresses with advanced disease.An increasing body of evidence suggests that BA and FXR signaling regulates liver cell growth. Mice lacking FXR as well as mice lacking its downstream target SHP develop hepatocellular cancer (HCC).In the last several years, various BA-derived or non-BA-based FXR activators have been developed as potential therapeutics against cholestasis. The protective effects of FXR were demonstrated in several animal models. A non-BA synthetic FXR agonist GW4064 and BA-derived 6α-ethyl derivative of chenodeoxycholic acid  have beneficial effects in mouse models of chemically induced liver injury (α-naphthylisothiocyanate (ANIT) and estradiol-induced) or in bile duct-ligation (BDL).Recently 3 BA-based therapeutic compounds were compared in Mdr2 (mouse ortholog of human phospholipid export pump MDR3) knockout mice, a model of bile duct injury and biliary fibrosis associated with the toxic bile composition caused by absent biliary phospholipidsIn addition to hepatocytes, cholangiocytes also play an important role in bile formation. Importantly, FXR is also expressed in human biliary epithelium, where it may play a critical role in ductular bile generation by alkalinization and fluidization through secretory mechanisms known to be predominantly regulated by complex neuro-endocrine as well as local mechanisms.In addition, CDCA (a potent endogenous FXR ligand) is able to induce expression of cathelecidin, the major anti-microbial peptide known to counteract the LPS, in human cholangiocytes, suggesting that BAs/FXR might play an important role in sterility of the biliary tree and protection against bile duct inflammation.Although many cholestatic liver diseases progress to liver fibrosis and finally cirrhosis, the question of whether FXR affects the fibrogenesis still remains unclear. Interestingly, FXR was also shown to have direct anti-fibrotic effects in hepatic stellate cells (HSCs) via activation of SHP.Collectively, FXR activation by endogenous or synthetic agonists represents an efficient mechanism to counteract cholestasis by a synchronized network of hepatoprotective mechanisms: (1) reducing intrahepatic BA load via repression of BA synthesis and an increase in BA export ; (2) changing bile composition at the hepatocellular level (by increasing relative phospholipid and bicarbonate secretion), ultimately resulting in a less toxic bile protecting hepatocytes and cholangiocytes; (3) impacting on ductular bicarbonate secretion (via induction of VPAC-1); (4) mediating direct anti-inflammatory effects in hepatocytes (via inhibition of NF-κB and STAT3) and non-parenchymal liver cells; (5) impacting on the gut-liver axis .Because targeted FXR activation has been recognized as a promising therapeutic option for patients with cholestasis, FXR agonists have already entered the clinical trials. Specifically, combination therapy of ursodeoxycholic acid (UDCA) with the INT-747 in phase II clinical trials in PBC patients not responding to UDCA showed substantial reduction of biochemical parameters of liver damage and cholestasis, such as ALT and ALP, after short-term and long-term administration.The primary function of PXR and CAR is to regulate genes responsible for the detoxification and elimination of a broad spectrum of potentially toxic endogenous and exogenous compounds.Various enzymes involved in phase I  and phase II  detoxification as well as many drug transporters are target genes of PXR and CAR, converting their substrates into more hydrophilic and therefore less toxic and easier cleared compounds see .98,100,1Altered function of PXR and CAR is involved in both pathogenesis and adaptation to cholestatic liver disease. Genetic variants of PXR are associated with increased susceptibility for ICP, as well as with lower neonatal weight and Apgar score in South American populations.In patients with obstructive cholestasis, a pronounced increase in PXR and CAR expression is observed, followed by an increase in their target genes (MRP3 and MRP4),Because of their central role in BA detoxification and transport, PXR and CAR represent attractive targets for drug therapy of cholestasis. Ligands for both receptors have already been used to treat cholestasis and pruritus, long before their mode of action has been fully understood. As such, rifampicin is a classic ligand for PXR and not only is effectively used to treat pruritus but also improves liver function tests in PBC, compatible with a direct anti-cholestatic effect.Phenobarbital was also given to patients long before the identification of CAR as its molecular target.In a rodent model, pharmacologic stimulation of PXR counteracted LCA-induced liver toxicity by induction of Cyp3a11 (CYP3A4 in human) and SULT2A1, both involved in BA detoxification.Apart from its anti-cholestatic effects, PXR also has anti-fibrotic and anti-inflammatory properties that may be beneficial in complex cholestatic liver diseases such as PSC and PBC. PXR stimulation in human HSC inhibits their transdifferentiation to fibrogenic myofibroblasts, inhibits expression of the major profibrogenic cytokine TGF-1β, and markedly slows proliferation.Finally, PXR is essential for liver regeneration because mice lacking PXR have impaired hepatocyte proliferation.Collectively, pharmacologic stimulation of PXR and CAR in chronic cholestatic liver disease may improve the disease course via at least 4 potential beneficial mechanisms: (1) repression of BA synthesis and increase in BA and bilirubin detoxification and elimination pathways, which will enhance the ability of the liver to reduce levels of toxic cholephils; (2) suppression of inflammation and fibrosis; (3) promotion of hepatocellular regeneration; and (4) amelioration of pruritus. However, it must be emphasized that both PXR and CAR ligands are potentially hepatotoxic and carcinogenic; therefore, novel compounds targeting PXR and CAR with fewer side effects need to be developed.The main function of VDR is to mediate the effects of its natural ligand calcitriol  on calcium homeostasis, but VDR also regulates cell proliferation and differentiation and has immunomodulatory as well as anti-microbial functions.Multiple polymorphisms in the coding sequence and promoter region of VDR may alter the immune response and specific VDR variants are associated with several immune-mediated liver diseases. As such, VDR polymorphisms are associated with susceptibility and clinical appearance of PBCBecause impaired absorption of fat-soluble vitamins is a hallmark of cholestasis and severe liver dysfunction, low serum vitamin D levels are commonly observed in patients with cholestasis and may alter VDR activity with consequences beyond bone metabolism. Low 1,25-VitD3 levels impair fetal outcome (inversely correlating with meconium staining) in patients with ICP.Accumulation of LCA during cholestasis decreases the effects of vitamin D on human osteoblasts, acting as a competitive ligand for VDRAccording to the predominance of VDR in non-parenchymal liver cells, activation of VDR in the liver has mainly anti-inflammatory and anti-fibrotic effects that may be beneficial in chronic cholestatic liver disease (such as PBC and PSC). “Classical” targeting of VDR through vitamin D substitution improves bone density in patients where cholestasis leads to chronic vitamin D deficiency and increased rates of osteoporosis. The anti-fibrotic potential of VDR stimulation was confirmed by reduced fibrosis in a rat model of liver fibrosis.PPARα, PPARγ, and PPARδ are 3 structurally homologous receptors and are activated by endogenous fatty acids and their derivatives to control important metabolic pathways in lipid and energy homeostasis.Besides its role in the regulation of fatty acid metabolism, PPARα is involved in BA homeostasis. Fibrates, which are PPARα activators, induce the expression of phase II enzymes SULT2A1, UGT2B4, and UGT1A3 as well as ASBT, BA uptake transporter, in cholangiocytes and enterocytes.In contrast to PPARα, a direct role for PPARγ in the regulation of BA metabolism has not yet been reported, probably because of a low expression pattern in hepatocytes. Targeting PPARγ is of particular interest for inflammatory cholestasis because of its crucial role in attenuation of inflammation-mediated transporter and enzyme changes. In the LPS model of inflammatory cholestasis treatment with glitazones, as synthetic PPARγ ligands and accepted anti-diabetic drugs, attenuated repression of NTCP, BSEP, and Cyp3a11, without affecting cytokine levels via inhibition of RXRα, export from the nucleus.PPARγ is involved in inhibition of inflammation and production of pro-inflammatory cytokines. Because bile duct destruction in PBC is Th1 cytokine mediated, it may not be surprising that PPARγ expression, which is high in normal bile ducts, is reduced in damaged bile ducts and may be associated with the Th1-predominant milieu and favor the development of chronic cholangitis in PBC.The effects of PPARα on biliary phospholipid secretion, BA metabolism, and synthesis make PPARα an interesting therapeutic target in the treatment of cholestasis. One of the key rationales for a beneficial role of fibrates in cholangiopathies may be upregulation of MDR3Clinically the beneficial effects of PPAR ligands in cholestasis were recognized for more than a decade and multiple pilot studies have evaluated their therapeutic effectiveness in patients with PBC. More than a dozen uncontrolled pilot trials using bezafibrate and fenofibrate showed beneficial effects on biochemical parameters and in part also on histologic findings in patients with PBC.Moreover, PPARα ligands may also be beneficial in patients with chronic hepatic graft-versus-host disease of the liver. A combination of UDCA and bezafibrate therapy in this patient population significantly improved liver biochemistry after 1 month of treatment.Other hypolipidemic drugs, such as inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (statins), are indirect activators of PPAR, also have pleiotropic anti-inflammatory effects,In addition to PPARα, PPARγ activation may also be effective in cholestatic diseases, in particular by ameliorating fibrosis and inflammation, thus limiting disease progression. The inhibitory effects of PPARγ ligands on collagen synthesis in HSCsGlucocorticoids are natural ligands of GR. GR is expressed in most human cells and plays a role in numerous metabolic pathways including carbohydrate and protein homeostasis, mediates negative feedback on the hypothalamic–pituitary–adrenal axis, and has strong anti-inflammatory and immunosuppressive effects.Activation of GR by glucocorticoids is widely used to treat inflammatory and autoimmune diseasesAlthough (combination) therapy with steroids may be clinically beneficial, their use is limited by classic side effects including bone loss,norursodeoxycholic acid (norUDCA) showed beneficial effects in the Mdr2 knockout mouse model of biliary fibrosis.norUDCA were associated with induction of phase I and phase II detoxification enzymes with simultaneous induction of basolateral efflux systems, resulting in alternative renal BA excretion.norUDCA induced induction of bicarbonate-rich bile flow. However, similar to its parent drug UDCA, no NR has been identified as a potential target for norUDCA and generation of bicarbonate-rich bile flow by norUDCA is thought to be mediated by the cholehepatic shunting of the compound.norUDCA, a characteristic pattern of induction of CAR-regulated genes was observed in the gene expression array study, suggesting CAR involvement in the anti-cholestatic effect of this compound.norUDCA has profound beneficial effects on lipoprotein composition, and hepatic lipid metabolism.norUDCA a very attractive therapeutic candidate for cholestatic and metabolic liver diseases.UDCA is currently used as a therapeutic standard in cholestasis and has multiple beneficial mechanisms,NRs control several important hepatic functions involved in the pathophysiology of cholestatic liver disease such as BA homeostasis and enterohepatic circulation of BAs as well as hepatic inflammation and fibrosis. Novel concepts on NR (patho)physiology have successfully been integrated in the understanding of the development of cholestasis. At present, many drugs used as standard treatments for cholestasis act via NRs and stimulation of their target genes. A revolution of expanding use of NR targeting in the therapy for cholestatic diseases is being witnessed. The translation of expanding knowledge on NRs should result in optimizing the current standard therapy with careful selection of patients’ subgroups benefiting from such novel NR-directed approaches.