Salmonella, Shigella, Yersinia, and enteropathogenic Escherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins.The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogens  The demia (CML) . Soon afia (CML) . The disia (CML) , 6. Thesia (CML)  and cellCrkI and CrkII are alternatively spliced products from a gene locus located on human chromosome 17p13.3 , 12. Crk BCR-ABL fusion gene [ E. coli.There are two different CrkL K.O. mouse models. One was used to develop a transgenic mouse bearing theion gene . Unexpecion gene  that cauion gene . Importaion gene  that, muCrk proteins are prototypical adaptors  consisting of Src homology 2 and Src homology 3  domains. They possess one N-terminal SH2 domain, followed by one SH3 domain in the case of CrkI. However, CrkII and CrkL have two SH3 domains named according to whether they are more N- or C-terminally located, nSH3 and cSH3, respectively . The preThe SH2 domain is an approximately 100 amino acid module with intrinsic folding capacity. Structural studies performed with the Src kinase determined that SH2 domains have two binding surfaces and consequently bind simultaneously to a phosphorylated tyrosine (pTyr) and to motifs containing specific residues located C-terminal to the pTyr (residues +1 to +5). The first pocket contains highly conserved arginine and histidine residues whose mutations abrogate phosphate binding . The secA closer look into the structure of the SH2 domain of CrkI and CrkII shows that, compared with CrkL, they possess an extra stretch of 17 amino acids that contains a proline-rich region  and class II (PXXPX+) . The nSHCrkII and CrkL each contain a cSH3 defined by its inability to bind to classic polyproline type II (PPII) motifs (reviewed in ). NucleaThe nSH3 and cSH3 domains are bound by an approximately 50-residue-long linker (spacer) region that contains the so-termed regulatory tyrosines, Y221 in CrkII and Y207 in CrkL. It was proposed early on that the phosphorylated Y221 of CrkII could bind to its own SH2 domain . Later, The CrkI and CrkII structure were studied using small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy  was proposed to be another regulatory element. In the cis conformation the nSH3 binding surface would be blocked by P238 and other residues from the cSH3 acting as a “reversible lid”  oncogene, comprises Abl and Arg . This family of nonreceptor tyrosine kinases has a modular structure, which includes one SH2 and one SH3 domain. They have a complex regulation and they play essential roles in regulating the actin-cytoskeleton, as indicated by the presence of an actin-binding domain in their C-terminus .The functional relationships between Crk and Abl are bidirectional (reviewed in ). The reCML and some adult acute lymphoblastic leukemia (ALL) patients frequently present the so called Bcr-Abl fusion protein, generated by a reciprocal chromosomal translocation ) that originates from the “Philadelphia chromosome” . The chiAn elegant recent NMR spectroscopy study has uncovered a different global organization for CrkII and CrkL that would explain the predilection of the Bcr-Abl fusion protein for CrkL over CrkII , 43. CrkAlthough Crk adaptor proteins can enter the nucleus, the mechanisms governing their nuclear import/export are not well characterized. These proteins seem not to have a cognate nuclear localization signal (NLS); therefore, it is thought that they are imported through their interaction with other proteins that contain an NLS . On the CrkL nuclear export is mediated by the interaction with CrmI/exportin . The cSHThe current paradigm in the signaling of Crk adaptors is that interactions through the nSH3 are constitutive, while binding to the SH2 domain is primarily inducible. In other words, their SH2 domain senses pathway activation by upstream tyrosine kinases. However, phosphotyrosine signaling is now envisioned as more dynamic , with coCrkII itself and some of its key partners are focal adhesion (FA) proteins. FAs are multiprotein complexes containing plasma membrane-associated integrins that link the extracellular matrix and the actin cytoskeleton . TwFA formation and dynamics are challenging subjects to investigate because many different molecular assemblies of proteins are formed that are difficult to dissect. FAs are enriched in kinases, such as focal adhesion kinase (FAK) and Src family kinases (SFK). FAK is recruited to the cytoplasmic domain of integrin receptors. In one of the most studied cell adhesion pathways, a FAK-Src complex is formed that promotes paxillin and p130Cas phosphorylation at FAs. The adaptor protein p130Cas contains binding-sites not only for Crk, as mentioned above, but also for FAK, Nck, and Src.Many proteins found at FAs participate in the formation of other “adhesive structures” such as the so-called “phagocytic cup.” Ig-opsonized pathogens are phagocytosed by professional phagocytes, mainly macrophages and neutrophils, as well as by M cells in the intestine, in a process that requires remodeling of the actin cytoskeleton through the action of the small GTPase Rac. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Using Crk mutants and Crk siRNA, it was proposed that CrkII is required for DOCK180 recruitment, Rac activation, and pathogen engulfment . The pos Salmonella and Shigella). On the contrary, Yersinia uses “the zipper mechanism.”Numerous reports indicate that Crk may contribute to bacterial pathogenesis in a variety of ways, including helping bacteria to enter host cells or serving as “targets of bacterial toxins that disrupt essential cellular functions” . On one Salmonella enterica serovar Typhimurium targets intestinal epithelial cells that are normally nonphagocytic. Despite this, Salmonella is able to induce its phagocytic uptake after bacterial attachment to the intestinal epithelium [ Salmonella T3SS effectors directly target and manipulate host signaling pathways involved in actin filament assembly and cytoskeletal rearrangement leading to bacterial entry [ Salmonella invasion utilizes several pathways that converge on Crk: on one side the bacteria manipulate the Fak-p130Cas-Crk axis and on the other side the Abl/Arg-Crk signaling pathway.ithelium . Invasioithelium . Salmoneal entry , 72. Sal Salmonella induces the assembly of FA-like complexes that lack integrins but require the recruitment of FAK and p130Cas [ Salmonella-induced cytoskeletal rearrangement. Thus, p130Cas−/− cells complemented with p130Cas lacking the Crk-binding domain were impaired for invasion as compared with p130Cas+/+ cells. These data suggest that FAK, p130Cas, and Crk work in concert to regulate Salmonella invasion [To stimulate phagocytic uptake by host cells, p130Cas . FAK appinvasion . Salmonella is the manipulation of Abl kinases to promote lamellipodia formation through activation of the WASP family verprolin homologous protein (WAVE) complex [Another important strategy used by complex , also ca complex ). Their  complex , 77. Salmonella internalization where it colocalized with F-actin. Salmonella infection led to increased phosphorylation of CrkII, while CrkII phosphorylation deficient variants block Salmonella entry. Furthermore, Salmonella infection led to increased phosphorylation of the Abelson-interacting protein (Abi1) [ Salmonella invasion. Because these pathways have largely been studied independently of each other, it will be interesting to determine if they are indeed interconnected.The role of Abl/Arg kinases and Crk phosphorylation has been studied by Casanova's group . Abl is n (Abi1) , a compon (Abi1) . Thus, i Salmonella, Shigella flexneri enters intestinal epithelial cells, which are not inherently phagocytic, and causes bacillary dysentery in humans. Shigella T3SS effectors interact with host proteins to induce dramatic rearrangements of the host actin cytoskeleton to form actin-rich extensions, similar to lamellipodia, which engulf the bacterium [ Salmonella, which replicates and survives inside a special compartment , Shigella remains in the cell cytoplasm [Likeacterium , 79. Howytoplasm . Thus, aShigella stimulates the tyrosine phosphorylation of some host cell proteins, including Crk adaptors. Abl- and Arg-deficient MEFs have a dramatic decrease in intracellular bacteria after Shigella infection, as compared with WT cells. Similar to Salmonella infection, treating cells with Imatinib (which was developed to inhibit the kinase activity of the BCR-Abl fusion protein in Philadelphia-positive CMLs) reduced Shigella invasion compared with untreated cells, indicating that efficient Shigella infection requires Abl and Arg kinase activity [ Shigella uptake promotes phosphorylation of Crk, indicating that the Abl-Crk module participates in Shigella invasion of host cells. In addition, a Crk-phosphorylation deficient mutant (CrkII-Y221F) showed a significant decrease in Rac and Cdc42 activation suggesting that the activation of these GTPases is at least in part mediated by Crk phosphorylation [activity . It was rylation . Shigella entry and is tyrosine-phosphorylated in a Src kinase-dependent manner [ Shigella invasion, Src phosphorylates cortactin and then phosphocortactin localizes to the site of Shigella invasion. Furthermore, it was proposed that phosphocortactin association with Crk promotes Shigella entry [ Shigella, cortactin expression downregulation by RNA interference does not inhibit Salmonella invasion, although the protein is recruited to the sites where the bacteria invade [Cortactin, a type II NPF that activates the actin related protein (Arp) 2/3 complex and regulates the neural Wiskott-Aldrich syndrome protein (N-WASP) activity , is recrt manner . Bougnèrla entry . Contrara invade . Shigella invasion by inhibiting Abl/Arg tyrosine kinases, which results in Crk phosphorylation downregulation [ Salmonella, the WAVE complex has been well studied, relatively little data are available for Shigella, while the opposite is true for cortactin. Therefore, potential future studies could examine the roles of these respective proteins to determine if they are indeed important to Shigella and Salmonella invasion, respectively.Unc119 was initially identified as an adaptor protein that activates specific SFK members, such as Lyn . Interesgulation . While fYersinia pseudotuberculosis and Y. enterocolitica are enteric pathogens that cause infections which are usually self-limiting, in contrast to Y. pestis, the causative agent of bubonic plague (reviewed in [ Yersinia species deliver virulence proteins (Yop effectors) directly into the host cell via the T3SS [ Yersinia spp. utilize a β1-integrin-mediated zippering mechanism for bacterial uptake in an actin-dependent process. Uptake is mediated by the interaction between invasin, a bacterial transmembrane protein, and β1-integrins on the host cell surface. The invasin-integrin association initiates a signaling cascade, characterized by tyrosine phosphorylation, leading to activation of the Rho GTPases Rac1 and Cdc42, possibly to promote the activation of N-WASP and the Arp2/3 complex [iewed in ). Yersinthe T3SS . Yersini complex  for the  Y. pseudotuberculosis uptake [ Yersinia invasion of epithelial cells. Furthermore, p130Cas-Crk complex formation was induced in response to infection, which was coupled to Rac1 activation. However, the existence of a Fak-dependent and p130Cas-Crk independent pathway for Yersinia uptake has also been proposed [Crk-p130Cas signaling was implicated ins uptake . Overexpproposed .β1-integrin receptors with high affinity, YadA is a Y. pseudotuberculosis outer membrane adhesin that binds integrins indirectly through the extracellular matrix (ECM). Hudson et al. characterized the mechanisms by which invasin and YadA promote adherence and phagocytic signaling events in macrophages and studied how ECM proteins differentially influence their ability to bind integrins. At low ECM concentrations, invasin binds directly and with high-affinity to β1-integrin to induce integrin clustering and to stimulate integrin-dependent phagocytosis, through FAK-p130Cas-Rac1 signaling. However, at high ECM concentrations, indirect YadA binding to β1-integrin predominates, which also promotes adherence to and entry into host cells [ Yersinia infection is likely influenced not only by the expression levels of invasin and YadA during infection, but also by the extracellular environment at the infection site.In contrast to invasin, which binds directly to st cells . Togethe Salmonella and Shigella, and in the zipper mechanism of invasion for Yersinia. This may not be surprising because Crk is implicated in cytoskeletal remodeling via Rac GTPase and possibly WAVE regulation, whereas it is also involved in FA remodeling through integrin signaling.It is noteworthy that Crk is implicated in the triggering mechanism for Escherichia coli  adhere to intestinal epithelial cells and deliver proteins into the host via the T3SS, resulting in microvilli effacement and intimate attachment to cells, forming the so-called attaching and effacing (A/E) lesions. Because most of the data obtained to date regarding Crk signaling has been from studies of EPEC, rather than EHEC, we will primarily focus on EPEC in this review. It will be interesting in the future to determine to what extent these data are translatable to EHEC.Enteropathogenic and enterohemorrhagicAmong the effector proteins delivered is the translocated intimin receptor (Tir), which is inserted into the host cell plasma membrane where it binds intimin, located on the outer membrane of EPEC . The TirEPEC Tir is phosphorylated on residue Y474  redundanThe current paradigm establishes that the major pathway to actin polymerization in typical EPEC infections takes place through phosphorylation of Tir Y474, and subsequent formation of a Tir-Nck-N-WASP complex to promote actin polymerization by the Arp2/3 complex . This pr Citrobacter rodentium (TirCR Y471 and Y451) was used [ in vitro versus in vivo contradictory results might be explained at least in part by our recent unexpected findings indicating that Nck adaptors possibly have a subsidiary function in activating N-WASP during pedestal formation by EPEC . We found decreased levels of translocated Tir within Nck1/2-deficient MEFs that were corroborated in HeLa cells with down-regulated expression of Nck by siRNA.However, infection studies with human intestinal tissue using an EPEC Tir Y454F,Y474F variant also lead to actin nucleation and pedestal formation . Likewiswas used . These i−/− fibroblasts, detachment could not be detected [In the early 1960s it was reported that EPEC induces epithelial cell shedding in the intestine  which madetected . Howeverdetected . Shigella late T3SS effector OspE has been reported [β1-integrin tails in FAs that mediates the communication between cells and the ECM. Future work will undoubtedly address whether the anticell lifting effect mediated by OspE binding to ILK, demonstrated for Shigella, applies to the OspE orthologs found in EPEC, EHEC, and Salmonella [ Shigella OspE, and through EspM, seem to regulate RhoA GTPase activity to stabilize FAs [Related to that, an increase in FAK dephosphorylation by thereported . OspE inlmonella , 114. Aslize FAs . Since CUsing immunofluorescence staining, Goosney et al. localized other FA components besides FAK, including p130Cas, vinculin, and CrkII . CrkII lThese and other unanswered questions prompted us to investigate the role of Crk adaptors in pedestal formation by EPEC . UnexpecκB signaling pathway. This is the case for Tir, whose interaction in the cytoplasm with the TNF-alpha receptor-associated factor (TRAF) adaptor proteins induces their degradation by, at the moment, an undefined mechanism [Several EPEC and EHEC T3SS effectors have been found to inhibit the innate immune response of intestinal epithelial cells by altering the activity of various components of the proinflammatory NF-echanism . EPEC Tiechanism . While Techanism , this inκB in the nucleus to increase the affinity of the NF-κB complex for a subset of gene promoters [κB-kinase-β (IKKβ) phosphorylation of RPS3 S209, preventing RPS3 nuclear translocation [κB activity in infected cells [κB activation, yet neither RPS3 nor IKKβ appeared to be the target for NleH1 kinase activity [ in vitro kinase array, CrkL was identified as an NleH1 kinase substrate and was also found to interact with IKKβ [κB activity, suggesting that CrkL may act as an adaptor protein, possibly by recruiting NleH1 to the IKKβ-RPS3 complex to prevent RPS3 phosphorylation and subsequently inhibit NF-κB activation [Several years ago it was discovered that the ribosomal protein S3 (RPS3) interacts with the p65 subunit of NF-romoters . The nonlocation  and thused cells . Wan et activity . Using aith IKKβ . Downregtivation . It is c Salmonella and Shigella infection of cells, an observation that appears promising regarding its potential use in animal models. Evolving knowledge on microRNAs  could b in vitro studies [ in vitro and in vivo models, as previously mentioned, will dictate careful reassessment. The subject gets even more complicated because, as discussed above, on one side, CrkII and CrkL block Tir signaling to achieve pedestal formation, which could be explained by the recent finding that Crk adaptors function in a heterocomplex, as reported to occur during podocyte morphogenesis [κB, which could imply a different mechanism of action and a direct role of Crk adaptors in promoting innate immune responses. Clearly, more research is needed to gain further insight into how Crk adaptors participate in the host response to Enterobacteriacea infections.On the contrary, according to studies , chemicaogenesis . TherefoMuch has been learned about the role of Crk adaptors in host-pathogen interactions . However


There is increasing recognition of the importance for local biodiversity of post-mining sites, many of which lie near communities that have suffered significant social and economic deprivation as the result of mine closures. However, no studies to date have actively used the knowledge of local communities to relate the history and treatment of post-mining sites to their current ecological status. We report a study of two post-mining sites in the Yorkshire coalfield of the UK in which the local community were involved in developing site histories and assessing plant and invertebrate species composition. Site histories developed using participatory GIS revealed that the sites had a mixture of areas of spontaneous succession and technical reclamation, and identified that both planned management interventions and informal activities influenced habitat heterogeneity and ecological diversity. Two groups of informal activity were identified as being of particular importance. Firstly, there has been active protection by the community of flower-rich habitats of conservation value  and distinctive plant species (e.g. orchids) which has also provided important foraging resources for butterfly and bumblebee species. Secondly, disturbance by activities such as use of motorbikes, informal camping, and cutting of trees and shrubs for fuel, as well as planned management interventions such as spreading of brick rubble, has provided habitat for plant species of open waste ground and locally uncommon invertebrate species which require patches of bare ground. This study demonstrates the importance of informal, and often unrecorded, activities by the local community in providing diverse habitats and increased biodiversity within a post-mining site, and shows that active engagement with the local community and use of local knowledge can enhance ecological interpretation of such sites and provide a stronger basis for successful future management. Extraction of coal and other minerals often involves the dumping of spoil or the stripping of surface soil. After mining has ceased, many sites receive technical reclamation, typically consisting of a cover of fertile topsoil, and sowing with productive grass/herb species or tree planting. The alternative approach is spontaneous succession, which involves no direct planting or sowing but aims only to control alien or invasive species: the typical successional communities on mine spoil are then influenced by factors such as the shallow soil depth, poor soil nutrient status and soil contamination. The rate of vegetation establishment may be slow on the infertile site conditions which remain with spontaneous succession, but the spatial heterogeneity in soil conditions can ultimately lead to a more diverse flora e.g. , 2). Unu. Unu2]).However, the influence of local residents on the plant and insect communities that become established on post-mining sites, and their role in maintenance of a high ecological value, has rarely been considered. Furthermore, these communities, who have often suffered significant social and economic deprivation as a result of mine closures, may have important local knowledge about the history and development of a site after mining ceased. The importance of informal local knowledge to understanding the ecology and management of urban ecosystems has been established in a number of studies. For example, Tsuchiya and colleagues  showed tHere we describe a novel study at two post-mining sites, which involved working with local residents with the specific aims of (a) establishing restoration histories for the sites and (b) assessing links between the formal management and informal community activities and the richness of the plant and invertebrate communities now present. Our approach is a hybrid of citizen science and participatory action research (see  for a tyThe research was part of the OPAL (Open Air Laboratories) program, which aimed to engage citizens in collecting novel ecological data (e.g. ) and to The two sites, Upton and Fitzwilliam, are situated south-east of the city of Wakefield. Both sites mined coal mainly from the Upper and Middle Coal Measures of the Carboniferous period. At Fitzwilliam the site is on sandstone and waste siltstones: mudstones and sandstones formed the spoil heaps that completely covered the site. The Fitzwilliam pithead was closed in 1967, but a drift mine was opened on part of the site in 1977, with final site closure in 1987. While part of the Upton site is on siltstones, mudstones and sandstones, it also has an area of Permian Magnesian Limestone. Unlike Fitzwilliam, most pit spoil from Upton was transported away from the colliery site. A disused railway cutting forms the southern boundary of the site. The Upton site closed in 1964, although clay extraction continued for the on-site brickworks. The UK Index of Multiple Deprivation shows that Upton is in the 40% most deprived areas in England, and Fitzwilliam is in the bottom 20% of areas in England, and that for both areas, deprivation levels got worse over the period 2004–2010 .A number of methods were used to recruit participants in order to maximize their numbers. Initially a press release appealing for volunteers to monitor wildlife on the sites and provide historical information was sent to the local newspapers. Posters were placed in local libraries, shops and post offices, and contact was made with local schools and colleges. In addition, conversations with local residents while the researchers were visiting the sites led to some individuals taking part in the study, as well as providing useful anecdotal information on site history.Participatory GIS (PGIS)  workshopFor the PGIS workshops, a series of size A0 topographic maps dating back over the last 40 years were provided in order to aid recollection. In addition, participants brought a selection of photographic and historical material to the workshops. At the beginning of the workshop, participants were asked a series of questions in order to create a historical timeline of what had happened at the site and when. This timeline was then used to generate maps indicating where management and development changes had occurred and the impacts these changes had on the diversity of flora, fauna, habitats and community use. This process was iterative as mapping sparked remembrance of new activities and events that were added to the timeline. Participants’ conversations were recorded to capture the details of these discussions allowing further annotation of the timeline. The workshops provided information about when and where reclamation of the sites took place, as most formal records were either destroyed or lost; indeed, at the Upton site, local residents had maps and reclamation information that would normally have been held by the local authority.RAP-GIS involved approaching site users with an aerial photograph of the site, and asking them the following questions: which areas of the site do you use and why, which areas of the site do you not use and why, and where have you seen wildlife. They were asked to draw these areas onto the aerial photograph and notes were taken about any species they mentioned. This process provided an overview of the types of information held by a cross-section of users about the amenity value of the sites in relation to land cover, habitats and their associated biodiversity potential.Plant surveys using quadrats were conducted by the researchers in the grassland areas in summer 2010 (permission granted by Wakefield District Metropolitan Council). Raw data can be found in A species list of invertebrates was created for each site. Raw data can be found in Volunteers were asked to walk specified routes (transects) on a weekly basis to assess the presence and number of selected butterfly and bumblebee species, and record their observations on a form which was adapted from the Butterfly Monitoring Scheme  and the The timeline generated from the PGIS workshop at Upton is shown in The pit was closed in 1964, although some clay extraction continued after this date. No active remediation of the site took place, with natural regeneration of the spoil to form a heath and scrub matrix. Local residents noted that this provided excellent bird habitat over the following two decades. Participants highlighted that some coal remained un-extracted and plans were developed in the late 1970s to exploit the remaining resources by open-cast methods, and to quarry the limestone on the eastern half of the site. However a successful campaign by the local community during the 1980s stopped this, as they recognized and valued the biodiversity which had naturally regenerated on the site since the mine closed, and there were also concerns about the extra heavy goods vehicle traffic that the site would create in the village. Once the mine and quarry plans were abandoned, the local council developed plans for landscaping and reclamation. However, members of the local community who were keen wildlife observers protected valued species and habitats from what they perceived as destructive reclamation techniques, and negotiated a plan that preserved some of the most ecologically significant features of the site. For example, participants were aware of a variety of orchids establishing themselves by the 1980s, and described an orchid transplantation scheme that took them from areas of the site due for reclamation, to areas of the site that had naturally regenerated and would be protected. It is important to note that throughout the disputes over use and reclamation of the site, there was no organized external pressure group at work, and opposition was driven by the interests of long-term local residents. Reclamation measures that did take place included coalmine buildings being demolished and crushed for hardcore and the resulting aggregates spread over the west of the site. As part of the reclamation process in the early 1990s, a fishing pond was established; the Angler’s group formed around this have actively engaged young people with the aim of reducing anti-social activity on the site. Members of this group have also been involved in planting of ornamental cyclamen species, spindle and apple trees. Finally, in the late 2000s when a new drainage scheme was installed, local residents were involved in identifying ways to reduce its impact on biodiversity.All the surveyed areas of the Upton site showed fits to NVC habitat classes of high conservation value  or that were unexpected at such as site, including calcareous dune grassland, maritime cliff grassland and vegetation of open habitats. The timeline and map developed at the Fitzwilliam PGIS workshop, using the same format as for Upton, is shown in Rhinanthus minor. As for Upton, both historical planned management (such as vegetation control for the benefit of the anglers and to maintain public footpaths) and informal community activities were reflected in the habitat classification. However, at Fitzwilliam, areas of conservation value were associated with poorly planned reclamation and anti-social activities, rather than active community protection of areas of spontaneous succession. The outcome here was ‘wasteland’ which was left to become enriched by natural processes. The only surveyed site of low ecological value, F6, was in an area known to have been actively reclaimed for the planned golf course in the early 1990s. Areas of old spoil with little topsoil addition (site F1) and of brick rubble (site F3), from site reclamation in the 1990s, were both associated with open habitats of waste ground, reflecting the slow rate of succession on these substrates, as at Upton. The affinities with maritime cliff communities at F2 were associated particularly with the use of the area as a BMX track providing long-term disturbance. At sites F4 and F5 the disturbances that led to plant communities of high conservation value were associated with the traveller camp, and with coal digging and tree removal respectively.Five of the six surveyed areas showed fits to NVC habitat classes of high conservation value or that were unexpected at such a site, including many of those identified at Upton. The community surveys at Fitzwilliam added further positive indicator species, for example, the presence of orchids and the hemi-parasite rich flower resource is well represented by the invertebrate fauna at both sites, but was not accounted for by records prior to this study. The Lepidoptera and Hymenoptera groups contribute most towards this code. This code reflects and confirms that these sites, in the areas identified on Figs unshaded early successional mosaic, which is related to the specific assemblage type open short sward, all increased with addition of species identified in this study. The majority of species recorded for this particular habitat code were in areas that had most recently been disturbed, or were recorded as having plant communities of open habitats  and butterfly species (e.g. Polyommatus icarus) that were identified in significant numbers in the volunteer surveys [The additional invertebrate species identified in collaboration with volunteers in this study highlighted the importance of two habitats, rich flower resource and early successional mosaic, in providing a rich invertebrate fauna with unusual or rare species. The former also relates to the lowland grassland communities of conservation importance that were identified in the plant survey. Specific positive indicators of grassland quality may have value as food resources for the Lepidoptera and Hymenoptera species that contributed most to the value of this habitat. For example,  quality , is also surveys , 26. The surveys  also idePoecilus versicolor), the pill woodlouse (Cylisticus convexus) the mottled grasshopper (Myrmeleotettix maculatus), and the yellow meadow ant (Lasius flavus).The participatory mapping exercises also revealed that use by local residents of different areas of the site stopped or decelerated successional processes and encouraged more of the valued open mosaic habitat for invertebrates, as well as plant communities characteristic of habitats of low nutrient status with significant amounts of bare ground. These activities included tree cutting for fuel during the miner’s strike, use of motorbikes and BMX bikes on the site, and use for informal camping, as well as external planned management, such as the creation of new ponds to help downstream drainage at Upton in 2009. Regular disturbance on urban brownfield sites has been recognised as contributing to high plant and invertebrate species richness , 27. ManThe Millennium Ecosystem Assessment  recognisOur research demonstrated that our understanding of the ecology of brownfield sites may require the gathering of detailed informal knowledge of the interactions between the site and the local community over time. This is because the current ecological value of such sites is strongly influenced by time since abandonment, and informal management activities such as those described here can have a significant impact over that time period. Many brownfield sites will have had complex histories since their abandonment, which, as for our sites, are undocumented or only partially documented, and interpretation of current ecological patterns and spatial diversity may depend on such information. Despite the fact that the conservation value of brownfield sites in general, and post-mining sites in particular, is increasingly recognised, urban and industrial areas have traditionally been neglected in ecological survey programmes. For example, in the UK, Open Mosaic Habitat on Previously Developed Land was only identified in 2007 as a new Priority Habitat within the National Biodiversity Action Plan , and theAlthough engagement of communities in ecological surveys has become more widespread, to our knowledge no study has adopted the approach described here, where active engagement with the local community provided an enhanced understanding of how site history has influenced the ecology of different areas. Many features of our findings will be unique to these sites or to the Yorkshire coalfield region, and using local history as a ‘hook’ for engagement may have been particularly effective here because of the emotive cultural impact of the large scale closure of mines, and accompanying loss of thousands of jobs in the Yorkshire region. Nevertheless, the approach that we have taken, and in particular the use of PGIS and RAP-GIS methods to stimulate discussion and focus field investigations, may be useful to inform future practice in community engagement with the ecology of brownfield sites or of other areas that have experienced rapid land use change which has not been well documented.S1 Document(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167 S.pombe Cdc2 (CDK1) can override the cell cycle control by Wee1 kinase . To. Tocdc2.extracts . We alsoextracts .cdc2.T14A cells. This identified Cdc2-Hf as a strong candidate for carrying the T14 and the Y15 modifications as the intensity of the F species significantly declined in the cdc2.T14A mutant. The second Y15 modified form, Cdc2-He, was not affected in the mutant strain , the anti-Y15P antibody identified only the two most negative species E and F . This imt strain . Unfortu mitosis  and thercdc13-HA strain to find out which Cdc2 forms associate with it. This assay indicates T167 phosphorylation as this modification is required for the stable formation of the Cdc2-cyclin B complex [CDC2-cyclin B complex [Since we could not test the T167 phosphorylation directly, we decided to immunoprecipitate cyclin B (Cdc13) from an affinity tagged  complex . As show complex , Cdc13 aS.pombe [chk1-HA strain [chk1-HA pull down material contained only the two unphosphorylated forms Cdc2-Ua and Cdc2-Ub, but none of the phosphorylated species. Activation of Chk1 by the DNA damage checkpoint in the presence of CPT was evident from the two phosphorylated forms of the kinase which migrate closer to the alkaline end of the strip as reported previously [We reported recently two Cdc2 protein complexes in S.pombe . A largeA strain . The celeviously . This unS.pombe CDK1 (Cdc2) kinase is restricted to the three amino acids T14, Y15 and T161Hs (T167Sp) [S.pombe cells [The phosphorylation of human and (T167Sp) , 38. Earbe cells . How thecdc2.T14A mutant (Sp (T161Hs) is not essential for cyclin B binding [S.pombe Cdc2-cyclin B complex for its roles in S phase.Our first key finding is the observation that only two Cdc2 forms are modified at tyrosine 15 (Y15). This identifies Cdc2-He and Cdc2-Hf as the target of Wee1 and Mik1 kinase in cell cycle regulation Figs  and 4C. A mutant . It is tA mutant  strongly binding , it sign binding . Further binding , and rea binding . It seem binding , the samcdc2.T14A mutant  appears on the Phos-Tag gel   . This fo(Cdc2-M) . Its abumutation . Taken tw mutant  and thatw mutant . One pos and the response to DNA damage (Cdc2-Ua + Cdc2-Ub) . Further


Young swimmers are particularly susceptible to the onset of oral diseases.  To evaluate the oral health status in young competitive and non-competitive swimmers, involving an assessment of salivary cariogenic bacteria and secretory IgA (S-IgA) concentration. Before training sessions (T1), 54 competitive and 69 non-competitive swimmers had the following parameters assessed: decayed, missing, and filled teeth (DMFT), Plaque Index (PlI), and Gingival Index (GI). At T1 and after training sessions (T2), stimulated saliva was collected and microbiological and immunological analyses were performed.S. mutans was harbored by 18.6% of competitive and the 32.2% of non-competitive swimmers. S. sobrinus was detected in 22.03% of competitive and 91.6% of non-competitive swimmers (p<0.05). S. sanguinis was found only in the saliva of competitive swimmers. The average S-IgA of competitive swimmers decreased significantly at T2 (p<0.05). The pool water had a daily average pH of 7.22. Competitive swimmers trained 2.02±0.09 hours 5 times a week, while non-competitive swimmers trained 2.03±0.18 hours a week. A total of 14.7% of competitive swimmers suffered dental trauma related to sports. Only 11.76% of the competitive swimmers took a daily dose of fluoride, against 32.65% of non-competitive swimmers (p=0.029). Neither group followed an established diet or presented statistically significant differences in terms of nutritional supplement drink and chocolate intake. There were statistically significant differences in terms of oral hygiene. No significant difference in clinical indexes  was present.  Microbial markers, immune status and sporting characteristics are important for establishing guidelines for management of training load in order to minimize physical stress and the risk of oral infection. Dental diseases harm quality of life and have a negative impact on self-esteem, eating ability, and health, causing pain, anxiety, and impaired social functioning,,,,,Many different agents protect the tooth surface against developing caries. Secretory IgA (S-IgA) constitutes the main immunoglobulin isotype found in saliva and is considered the first line of host defense against pathogens which colonize or invade the oral cavity. As part of innate immunity, S-IgA is considered the best indicator of mucosal immunity and acts by preventing microbial pathogens from entering the body via mucosal surfaces, which are mainly responsible for infections of the upper respiratory tractThe purpose of the present study was to evaluate the oral health of hard and soft tissues and the prevalence of caries in young selected competitive and non-competitive swimmers, by assessing salivary cariogenic bacteria and concentration of salivary IgA, both before and after training sessions. An additional objective was to monitor the water pH of the swimming pool during the day to assess its potential role in erosion.The study population was composed of 123 subjects: 54 competitive swimmers (test group) and 69 non-competitive swimmers (control group), 58 females and 65 males. There was no difference between test and control group with respect to age . The swiThe following inclusion criteria were considered:Absence of systemic diseases that could affect the immune response or that could condition bacterial colonization.No use of antibiotics for three months prior to the beginning of the study.No orthodontic therapy.Dental care not in progress.A self-administered questionnaire was used to obtain data concerning hours and frequency of weekly training, complete pathological history, history of hard and soft tissues of the oral cavity, family history, oral hygiene practices, fluoride intake, and eating habits .& Löe Plaque Index (PlI) and the Löe & Silness Gingival Index (GI) were used to evaluate oral hygiene and periodontal status, respectively.Before training sessions (T1), each patient was clinically evaluated. The number of decayed, missing, and filled teeth (DMFT) was recorded to assess the prevalence of caries according to World Health Organization (WHO) criteria. The Silness In addition, an oral examination of intraoral mucosa was performed and the presence/absence of bad habits, malocclusion, and parafunctional habits was assessed.Before (T1) and after training sessions (T2), saliva was collected with paraffin-chewing stimulation. The saliva samples were immediately placed on ice and transferred to the laboratories for processing. An aliquot of 500 μl from each sample was transferred to a microcentrifuge tube and used for microbiological analysis. The remainder of the sample was placed in an eppendorf tube, clarified by centrifugation at 10,000 Xg for 15 min at 4°C and frozen at -20°C for the titration of salivary IgA.Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis) in each saliva specimen was analysed by PCR. S. mutans ATCC31383, S. sobrinus ATCC27607, and S. sanguinis 49297 were used as control species. The sequences of species-specific sets of the primers used in this study are listed in The presence of three oral streptococcal species (Negative controls (no DNA) and positive controls with DNA coming from pure bacterial cultures were tested. Amplification was performed in 100 μl reaction, containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl (1 x PCR Buffer), 1.5 mM MgCl, 200 μM of each nucleotide, 30 ml of each primer, 2.5 U Hot Start Taq DNA Polymerase  and 5 μl of DNA Template. The reaction mixture was denatured at 95°C for 3 min, followed by 30 cycles of denaturation at 98°C for 30 sec, annealing at 70°C for 30 sec, and extension at 72°C for 1 min. The reactions were conducted in a thermocycler iCyclar System . Amplification of ubiquitous primers was conducted in the same way. Electrophoretic bands were visualized and photographed with a UV rays transilluminator  after staining for 30’ with ethidium bromide (1 μg/ml). The sizes of the PCR products were estimated from the electrophoretic migration of products relative to a 100-base-ladder marker .The titration of salivary IgA was performed by using LC-examination Partigen immunodiffusion .Samples of swimming pool water (10 ml) were also collected in test tubes at regular intervals throughout the day and sent to the laboratory for pH evaluation. pH values were analyzed by a pHmeter .The Wilkoxon matched-pairs signed-rank test was used for the statistical analysis. All p-values lower than 0.05 were considered significant.The demographic and clinical characteristics of the studied populations are presented in Data concerning the dietary habits showed that neither group followed an established diet and that their nutritional supplement drink and chocolate intakes were significantly different (p=0.000).Statistically significant differences in oral habits between the two groups were observed. The most frequently reported by the study population were nail biting, sleep bruxism, and atypical swallowing.The analyzed clinical indexes  did not present statistically significant differences . It is nS. mutans in their saliva. S. sobrinus was present in 22.03% of competitive swimmers and 91.6% of non-competitive swimmers, with a statistically significant difference between the two groups. On the other hand, S.sanguinis was determined only in the saliva sample of the competitive swimmers. No difference was noted between pre- and post-training samples.As shown in Competitive swimmers showed a statistically significant decrease of the average value in the titration of S-IgA . The aveThe waters samples from the pools had a daily average pH value of 7.22 and both swimming pools followed hygiene recommendations, which included a double daily pH register, daily assessment of antimicrobial concentration in the water, quantity of purified and renewed water, and water and environmental temperatures.,,,The exercise involved in routine sport practice exposes athletes to the onset of numerous diseases. A number of factors, including the levels of competition and of exposure, can affect the higher or lower incidence. For this reason the present study considered two groups of swimmers attending the same environment (same pools) and same years of practice (4.17±0.27), but with different training loads. Results from this study indicated that athletes engaged in swimming at a competitive level, with more hours of training in pool water, had a higher incidence of dental stains and trauma. Dental injuries associated with various types of sports have often been described in literatureThe incidence of dental stains was higher in competitive swimmers compared to non-competitive ones. Although the difference was not statistically significant, it can be caused, as claimed by Escartin, et al.Competitive swimmers had a low prevalence of parafunctional behaviors. The most frequent oral habits reported by non-competitive swimmers were nail biting, sleep bruxism, and atypical swallowing, with an incidence similar to those reported in young adolescents in the literature,,Data concerning the dietary habits showed that in both groups, because of the age of the sample, no one followed a specific diet for training or competitions. Intake of nutritional supplement drinks by competitive swimmers was significantly different from non-competitive swimmers. Dental erosion is a further potential problem associated with sports drinks,vs 1%).Some authors believe that if the pH of the water in the pool decreases below that of saliva, erosions on the dental enamel may occur,,,,,In this study, after training, there was a statistically significant decrease in the concentration of salivary IgA levels of selected swimmers. S-IgA average values before and after training were significantly higher in competitive athletes than in non-competitive swimmers. The results of this study confirmed the majority of studies in that acute bouts of exercise reduced S-IgA levels in a variety of endurance sports, including swimmingS. mutans and S. sobrinus in their saliva, respectively. On the other hand, these bacteria were detected in 32.2% and 91.6% of non-competitive swimmers. Moreover, S. sanguinis was determined only in the saliva sample of the competitive swimmers. Mutans streptococci (S. mutans and S. sobrinus) are closely associated with the development of dental caries in humans. S. sanguinis was significantly associated with dental health, and the results of this study confirmed that S. sanguinis has been reported to be in relatively high numbers when the numbers of S. mutans are low, as reported in competitive swimmers. Moreover, in competitive swimmers the presence of high numbers of S. sanguinis was associated with low caries risk, as suggested by Seow, et al.The microbiological analysis showed that only 18.36% and 22.03% of competitive swimmers harbored S. sanguinis, associated with low caries risk, as confirmed by lower values of active caries. Several studies,Therefore, the results suggested that the time of training was the period characterized by the least intensive salivary functions and physiologic responses as a decrease in the salivary level of IgA. Swimming at competitive levels determined higher S-IgA average values, both before and after training, than for non-competitive swimmers. A low number of competitive swimmers harbored cariogenic microorganisms in their saliva; on the contrary, they showed a higher frequency of “protective” bacteria as This study confirms the usefulness of using microbial risk markers, including mutans streptococci. Non-microbial caries risk factors included visible plaque, child age, race, salivary and pool water pH, and eating habits. Furthermore, mucosal immune status and sport characteristics  are considered useful for establishing guidelines for nutritional support and management of training load. In this way, it would be possible to minimize physical stress and subsequently the risk for oral infection.


Growth factors and transcription factors are well known to regulate pluripotent stem cells, but less is known about translational control in stem cells. Here, we use embryonic stem cells (ESCs) to investigate a connection between ESC growth factors and eIF2α-mediated translational control . We find abundant phosphorylated P-eIF2α (P-eIF2α) in both pluripotent mouse and human ESCs, but little P-eIF2α in ESCs triggered to differentiate. We show that the growth factors LIF (leukemia inhibitory factor) and BMP4 (bone morphogenic protein 4) both maintain P-eIF2α in mESCs, but use distinct mechanisms: LIF inhibits an eIF2α phosphatase whereas BMP4 activates an eIF2α kinase. The mRNAs encoding the pluripotency factors Nanog and c-Myc possess uORFs while Oct4 mRNA does not. We find that salubrinal, a chemical that increases eIF2α phosphorylation, promotes Nanog and c-Myc expression, but not Oct4 expression. These experiments connect ESC growth factors to eIF2α phosphorylation and suggest a chemical substitute for LIF to enhance Nanog and c-Myc expression. Best established are the networks of epigenetic chromatin modifications and transcriptional regulation , butipotency , and humipotency . Also, mipotency . In addiipotency –8.A different mechanism of translational control uses upstream open-reading frames (uORFs) to inhibit translation of the coding sequence (CDS) in the same mRNA . Remarkae.g. nutrient deprivation, hypoxia and viral infection) [Constitutive Repressor of eIF2α Phosphorylation) [The enzymes regulating eIF2α phosphorylation include both kinases and phosphatases. The eIF2α kinases, four from the same family, act in response to cellular stresses (fection) –20. The fection)  or CReP ylation)  and oppoylation) . Relevanylation) . The actylation) . Howeverylation) .Here, we investigate a role for eIF2α phosphorylation in ESCs and probe the connection between growth factors and eIF2α. We report that both human and mouse ESCs contain abundant P-eIF2α and that P-eIF2α decreases dramatically upon differentiation. We then show that the growth factors LIF (leukemia inhibitory factor) and BMP4 (bone morphogenic protein 4) act synergistically to maintain P-eIF2α: LIF downregulates the CReP eIF2α phosphatase, and BMP4 activates the eIF2α kinase, Protein Kinase regulated by RNA (PKR). Finally, we show that chemical induction of P-eIF2α, using salubrinal, maintains expression of Nanog and c-Myc proteins in mouse ESCs cultured without LIF. Together, our results connect ESC growth factors to the control of eIF2α phosphorylation in ESCs and identify a chemical tool that promotes ESC maintenance.2 on gelatin-coated tissue culture plates. Medium was exchanged daily, and cells were passaged with Accutase  every 3 days at a 1:8 split ratio.Mouse embryonic stem cells  were propagated in ESGRO complete medium supplemented with GSK3β inhibitor  at 37°C in 5% COFor experiments involving removal of cytokines, ESGRO basal medium  with GSK3β inhibitor was supplemented either with recombinant BMP4  or recombinant LIF . Note that in all cases, GSK3β inhibitor was included with ESCs. All protein and RNA samples were collected 24 h after medium exchange. Salubrinal  was resuspended in DMSO at a stock concentration of 1 mM. Salubrinal was then added to ESGRO basal medium supplemented with GSK3β inhibitor and BMP4 at a final concentration of 10 nM and 100 nM. In all cases, DMSO was added in the same amount as a control. Samples were collected 24 h after medium exchange with the exception of the samples for prolonged salubrinal treatment: medium with 100 nM salubrinal was exchanged daily for four days with a split on day 3. There was no difference in doubling time or apoptosis observed in cells grown with salubrinal or in ESGRO complete medium.The human ES cell line H1 was cultured in E8 medium as previously described . MesendoAnti-eIF2α, P-eIF2α (Ser51), eIF4G, P-eIF4G (Ser1108), eIF4E, P-eIF4E (Ser209) and Oct4 antibodies were obtained from Cell Signaling Technology . Anti-mNanog was obtained from R&D Systems . Anti-hNANOG and pPKR (Thr446) were obtained from Millipore , and anti-Actin (mouse C4) and c-Myc  were obtained from Sigma . Anti-PKR and Nck1/2 were obtained from Santa Cruz Biotechnology . Anti-CReP was obtained from ProteinTech .Mouse and human ESCs were collected using trypsin-EDTA to detach the cells from tissue culture plates. Cells were then washed twice with PBS and resuspended in a minimal volume of SDS-PAGE loading dye. Dilutions  for 30 min at room temperature. Samples were then washed twice with ice-cold PBSTw, and ice-cold methanol was added. Samples were then stored at -20°C for at least 15 min. Samples were washed 3 times with ice-cold PBSTw and blocked for 30 min at room temperature in PBSTw + 0.5% BSA. Samples were then incubated with primary antibody (as indicated in figures) at a 1:100 dilution in PBSTw + 0.5% BSA for three h at room temperature. Samples were washed 3 times with ice-cold PBSTw + 0.5% BSA for 15 min each at room temperature. Samples were then incubated with DAPI and the appropriate secondary antibody (at 1:500 dilution) conjugated to a fluorophore for two h at room temperature. Samples were washed 3 times with ice-cold PBSTw + 0.5% BSA for 15 min each at room temperature. Samples were then imaged on a Leica SP8 confocal microscope. M phase cells were quantitated by counting cells with clear metaphase plates or chromosomes in anaphase. Three biological replicates were quantitated separately.Mouse ESCs were cultured as described but were grown in chamber slides. Prior to fixation, cells were washed in PBS twice and were fixed with 3% paraformaldehyde dissolved in 100 mM KMouse ESCs were cultured as described, and total RNA was extracted with Trizol Reagent according to manufacturer’s instructions . cDNA synthesis was performed with oligo-dT primers and 5 μg total RNA using Superscript III reverse transcriptase again according to manufacturer’s instruction . qPCR was then performed on an Applied Biosystems 7500 Fast Real-Time PCR System . The Taqman assay probe sets used were mouse Nanog (Mm016117761_g1), c-Myc (Mm00487804_m1), Oct4 (Mm03053917_g1) and human eEF1A . In each case, data were normalized to the internal control, eEF1A. Technical replicates were performed in each case, but reported standard deviations are for independent, biological replicates (done three times).Translation initiation is regulated by phosphorylation of multiple initiation factors, which were analyzed earlier . HoweverWe also asked how P-eIF2α and P-eIF4G respond to changes in LIF concentration. To this end, we cultured mESCs with increasing LIF concentrations for 24 hours (note that routine mESC culture uses 10 ng/mL LIF) and then assayed P-eIF2α and P-eIF4G as well as total eIF2α and eIF4G proteins. As LIF increased, P-eIF2α and P-eIF4G increased . We alsoHuman and mouse ESCs differ in growth factor requirements (reviewed in ). We theAbundant P-eIF2α depends on LIF . We postWe also asked how CReP abundance responds to changes in LIF concentration. Mouse ESCs were cultured with increasing LIF concentrations for 24 h  and CReP protein was monitored by western blotting. Increased LIF correlated with decreased CReP protein . Nck1/2 Mouse ESC pluripotency is maintained by BMP4 in addition to LIF , 38. We P-eIF2α can promote CDS expression from mRNAs harboring uORFs see . Nanog aTo test whether LIF affects Nanog and c-Myc protein expression via its control of P-eIF2α, we turned to a chemical agent that induces eIF2α phosphorylation. The drug salubrinal inhibits CReP with high specificity and also increases P-eIF2α levels . We cultWe next explored whether salubrinal treatment might prolong mESC pluripotency. Normally LIF withdrawal rapidly arrests cell divisions in mESCs , so we fThe major finding of this work is that growth factors regulate translation initiation in ESCs, as summarized in LIF and BMP4 can maintain mESC pluripotency , and ourBut open questions remain. For one, how does LIF reduce CReP expression? In mESCs, LIF activates Jak1 via the LIF receptor , which aHuman ESCs are typically maintained with distinct growth factors from mouse ESCs. Routine hESC culture uses FGF2 and TGF-β , and we in vitro [Prior to implantation and oxygenation from the maternal blood supply, embryos live in a hypoxic environment . In factin vitro . Most rein vitro , along win vitro ), we sug


Various approaches to calling single-nucleotide variants (SNVs) or insertion-or-deletion (indel) mutations have been developed based on next-generation sequencing (NGS). However, most of them are dedicated to a particular type of mutation, e.g. germline SNVs in normal cells, somatic SNVs in cancer/tumor cells, or indels only. In the literature, efficient and integrated callers for both germline and somatic SNVs/indels have not yet been extensively investigated.We present SNVSniffer, an efficient and integrated caller identifying both germline and somatic SNVs/indels from NGS data. In this algorithm, we propose the use of Bayesian probabilistic models to identify SNVs and investigate a multiple ungapped alignment approach to call indels. For germline variant calling, we model allele counts per site to follow a multinomial conditional distribution. For somatic variant calling, we rely on paired tumor-normal pairs from identical individuals and introduce a hybrid subtraction and joint sample analysis approach by modeling tumor-normal allele counts per site to follow a joint multinomial conditional distribution. A comprehensive performance evaluation has been conducted using a diversity of variant calling benchmarks. For germline variant calling, SNVSniffer demonstrates highly competitive accuracy with superior speed in comparison with the state-of-the-art FaSD, GATK and SAMtools. For somatic variant calling, our algorithm achieves comparable or even better accuracy, at fast speed, than the leading VarScan2, SomaticSniper, JointSNVMix2 and MuTect.http://snvsniffer.sourceforge.net.SNVSniffers demonstrates the feasibility to develop integrated solutions to fast and efficient identification of germline and somatic variants. Nonetheless, accurate discovery of genetic variations is critical yet challenging, and still requires substantially more research efforts being devoted. SNVSniffer and synthetic samples are publicly available at  In , it is bIn this paper, we propose a lightweight indel candidate identification approach by assuming alignments are correct. In this case, realignment of reads around indels becomes an important procedure before applying our approach to call indels. In our approach, we assume that if a site is an indel candidate, there is at least one indel operation on this site relative to the reference. After finding such a site, we conduct two statistical examinations using Fisher’s exact test in order to remove bias caused by sequencing and alignment in practice. The first test computes if the number of variant alleles is statistically significant compared to the number of references bases at this site under a certain mutation rate. If significant, this site will not be considered as an indel candidate; otherwise, we continue to the second test. The second test computes if the number of indel operations is statistically significant compared to the number of references bases at this site. If significant, this site is deemed as an indel candidate and otherwise, we discard this site. In practice, this lightweight approach not only leads to fast speed, but also demonstrates highly competitive indel calling accuracy compared to the leading SAMtools and GATK through our limited evaluations  will be observed from the alignment map and each sequence represents the inserted or deleted consecutive bases relative to the reference. Moreover, these sequences may contain different consecutive bases, due to reasons such as sequencing errors and alignment bias. In practice, this is usually the case based on our observations from real data. In this regard, we have investigated a MUA approach with the intention to find the most significant consensus representation of a set of sequences of varied lengths possibly.Given an indel mutation candidate, a set of sequences lmin and lmax to denote the minimum and maximum sequence length in S, respectively, and Si,j,l to denote the substring of Si that starts at position j and contains l characters. Our MUA approach can be described as follows. Given a specific l (lmin≤l≤lmax), any l-length substring in S is deemed as a seed. Starting from a seed Si,j,l, we align it by not allowing gaps to every sequence Su (0≤u<|S|) and subsequently single out the substring Su,v,l with the largest alignment score for each Su. This set of highest-scoring substrings forms a MUA and is subsequently used to calculate a position-specific weight matrix (PSWM) of size |Σ|×l. Having got the PSWM, we compute a likelihood score for the MUA and then use this score to select the best consensus representation of S. Algorithm 1 shows the pseudocode of our MUA approach.Our MUA approach is inspired by the starting point search procedure of the MEME motif discovery algorithm , 35. DefΣ. For the convenience of discussion, we assume that characters in Σ have already been encoded to distinct integers in the range [ 0,|Σ|). For a PSWM W, its element Wi,j (0≤i<|Σ| and 0≤j<l) is defined as Given a MUA, its PSWM is computed by associating it with a background frequency specification for each character in pi,j denotes the site-specific frequency of character i at column j and is computed by dividing the number of occurrences of character i at column j by |S|. qi represents the background frequency of character i and is computed as In this equation, l columns, we can draw a consensus sequence S′ from the alignment by first computing a PSWM using Eq. , the likelihood score for S′ is calculated as Given a MUA of length sing Eq.  and thenl) to have higher significance.In this case, given an indel mutation candidate, we single out the MUA with the largest likelihood score and then draw a consensus sequence to represent the inserted or deleted consecutive bases. It should be noted that if two MUAs have an identical likelihood score, we consider the one with the more columns  from those present in the matched normal (germline polymorphisms).k≤K) denote the genotypes from the normal and tumor, A and B denote the reference base γ and the non-reference variant (≠γ) in the diploid genotypes, respectively. Moreover, the SNVs are reported in the well-established VCF format [In our algorithm, we have adopted a hybrid approach benefiting from the combination of an independent subtraction analysis and a joint sample analysis, with genotype inference as the core. Based on the genotypes inferred for both samples, SNVs detected in the tumor are classified into four mutation types: Somatic, LOH (loss of heterozygosity), Germline, and Unknown. Table F format .Table 8i) call the genotype ii) if Our subtraction analysis first calls mutations from the normal and tumor samples separately using the aforementioned Bayesian probabilistic models and then contrasts the results like a simple subtraction. This approach can provide reasonable predictions if there exists little noise and variant alleles have large enough frequencies (e.g. exceeding the expected) to be detected. In SNVSniffer, given a site, the subtraction analysis works as follows :  if the new ii) if the new iii) if both iv) classify the variant and report the result in VCF.Having calculated the most likely genotypes sing Eq. , our posFor indel calling, our algorithm separately call mutations from the normal and tumor samples. At a given site, our algorithm will make an attempt to call a somatic indel if only one of the two samples is deemed as an indel candidate at the site. The consensus sequence of a somatic indel is determined using the MUA approach mentioned above.


We stopped omalizumab treatment after four years. Recurrences of urticaria symptoms were found to be higher in patients with chronic urticaria than recurrences of asthmatic symptoms in severe persistent asthma patients. For the very first time, we used omalizumab in symptomatic therapy of recurrent laryngeal oedema and urticaria attacks in a patient with postoperative pulmonary carcinoid tumor for eight months. During the four years of follow-up, no recurrence was noted in pulmonary carcinoid tumor. Control PET CT results revealed normal findings. After omalizumab treatment, laryngeal oedema and urticaria symptoms were decreased. The most common adverse reaction from omalizumab is injection site induration, injection site itching, injection site pain, and bruising but the package insert contains warnings regarding parasitic infections. While there are no reports of fatal anaphylaxis as a result of omalizumab, some cases have been serious and potentially life-threatening. Therefore, the FDA requires that people receiving omalizumab be monitored in the physician's office for a period of time after their injections.Omalizumab depletes free IgE in the blood and interstitial space and inhibits IgE binding to Fc In addition to asthma, omalizumab has been investigated in various other conditions including chronic urticaria (CU), perennial and seasonal allergic rhinitis (AR), pruritic bullous pemphigoid, latex allergy, peanut allergy, idiopathic anaphylaxis, hyper-IgE syndrome, chronic rhinosinusitis, interstitial cystitis, aspirin sensitivity, mastocytosis, eosinophilic gastroenteritis, and atopic dermatitis. Most patients with chronic urticaria have an autoimmune cause: some patients produce IgE autoantibodies against autoantigens, such as thyroperoxidase or double stranded DNA, whereas other patients make IgG autoantibodies against FcεRI, IgE, or both, which might chronically activate mast cells and basophils. In the remainder of patients with CSU, the nature of the abnormalities has not yet been identified. Accumulating evidence has shown that IgE, by binding to FcεRI on mast cells without FcεRI cross-linking, can promote the proliferation and survival of mast cells and thus maintain and expand the pool of mast cells. IgE and FcεRI engagement can also decrease the release threshold of mast cells and increase their sensitivity to various stimuli through either FcεRI or other receptors for the degranulation process [Omalizumab, a humanized mAb that binds to the CH3 domain, near the binding site for the high-affinity type-I IgE Fc receptors of human IgE, can neutralize free IgE and inhibit the IgE allergic pathway without sensitizing mast cell and basophils. Omalizumab is a humanized recombinant anti-IgE monoclonal antibody approved for therapeutic use both in adults and in children aged 6–12 years with severe allergic asthma. The coexistence of severe asthma refractory to the conventional pharmacological approach and sensitization to at least one perennial allergen represent the current indications for omalizumab prescription. Its efficacy and safety as an add-on therapy is sustained by several data coming from both clinical trials and real life experiences  and show process , 12. The process –18.εRI on basophils, mast cells, and dendritic cells. Omalizumab cannot bind to IgE that is already bound to FcεRI and does not have a direct effect on FcεRI levels. However, the depletion of free IgE results in the downregulation of FcεRI on cells bearing the receptor, making those cells insensitive to the stimulation by incoming allergens [In a patient with an allergic disease caused by type I hypersensitivity toward specific external antigens, omalizumab induces multifactorial therapeutic effects. Omalizumab depletes free IgE in the blood and interstitial space and inhibits IgE binding to Fcllergens –19.Omalizumab has been approved in over 120 countries for treating patients with SPA. These pharmaceutical developments have validated the IgE pathway as an effective therapeutic target for treating IgE mediated allergic disease .Tumor necrosis factor related apoptosis inducing ligand (TRAIL: Apo-2L) is used as a marker for apoptosis. TRAIL (Apo-2L) is a transmembrane (type II) glycoprotein belonging to the TNF superfamily. The extracellular domain of TRAIL is homologous to that of other family members and shows a homotrimeric subunit structure. Like TNF and FasL, sApo-2L also exists physiologically in a biologically active soluble homotrimeric form. An increase in eosinophil levels has been reported in allergic asthma and is thought to reflect an increase in peripheral blood eosinophil survival promoted by Apo-2L.εRI receptor expression on key cells in the inflammatory cascade. The consequences of these processes are the inhibition of the release of inflammatory mediators from mast cells and diminished recruitment of inflammatory cells, especially eosinophils, into the airways [In our previous study we showed that soluble Apo-2L levels in patients with severe persistent allergic asthma decreased after anti-IgE treatment using omalizumab. These results suggested that sApo-2L may act as a soluble effector molecule and that the decrease in levels after omalizumab treatment may allow us to use this marker to monitor clinical improvement. Combination therapy with omalizumab and specific subcutaneous immunotherapy (SCIT) in patients with severe persistent asthma also suggest that omalizumab is an effective therapy in such individuals. Omalizumab reduces serum IgE levels and Fc airways , 23.Allergen specific immunotherapy (SIT) has the advantage of being the only causal treatment of allergic controlled asthma and rhinitis but is fraught with the dangers of severe systemic or local side effects and anaphylaxis. Omalizumab can possibly overcome these limitations by binding exclusively to circulating IgE molecules and reducing the levels of circulating IgE regardless of allergen specificity by binding to the constant region of circulating IgE molecule. This prevents free IgE from interacting with the high- and low-affinity IgE receptors (Fc′′RI and Fc′′RII) on mast cells, basophils, macrophages, dendritic cells, B lymphocytes and subsequently leads to a decrease in the release of the mediators of the IgE mediated allergic response, namely, cytokines, histamines, and leukotrienes , 24, 25.P = 0.04) [P = 0.017). More patients were able to reach the target maintenance SIT dose (P = 0.004) in the omalizumab group compared to placebo [The first clinical trial looking for the clinical effects of a combined therapy of SIT and omalizumab was performed in grass and birch pollen allergic children and adolescents in Germany. Kuehr and colleagues recruited 221 children and adolescents to evaluate the efficacy and safety of omalizumab with SIT on birch pollen induced allergic rhinitis (AR) . SIT plu = 0.04) . Another placebo , 30, sug placebo , 32. In vivo experiments on finding the cytokines involved in acute-phase protein response showed that there were three major cytokines: interleukin-1-beta, interleukin-6, and TNF-alpha [Ceruloplasmin (CP) is a copper-containing alpha-2-glycoprotein with a molecular weight of approximately 132 kDa. Ceruloplasmin is essential for iron homeostasis, is involved in angiogenesis, and under different conditions can act as either a pro- or antioxidant. The known functions of ceruloplasmin oxidase activity (COA) include copper transportation, iron metabolism, antioxidant defense and involvement in angiogenesis, and coagulation. It was previously reported that synthesis of CP was stimulated by interleukin-1 in normal and copper deficient rat models concluding that CP was dependent on oxidase activity . MoreoveNF-alpha , 34.2 values are lower in the exacerbation period than in the stable period. Blood counts and respiratory function tests were reported not to change between exacerbation and stable periods in patients with COPD. Thus episodes of BA or COPD might be associated with elevated levels of oxidative stress.An imbalance between oxidative stress and antioxidative capacity may play an important role in the development and progression of bronchial asthma (BA) and chronic obstructive pulmonary disease (COPD). The systemic oxidant-antioxidant status changes during exacerbation versus stable periods in patients with BA and COPD. During an exacerbation period of BA, despite the decreases in glutathione peroxidase (GSH-Px), glutathione reductase (GRd) and melatonin levels, malondialdehyde (MDA) and catalase (CAT) levels, and the white blood cell count, the percentage of eosinophils is significantly higher than in the stable period. MDA and superoxide dismutase (SOD) values are higher in the exacerbation period than in the stable period although GSH-Px, GRd, melatonin, pH, and pOA decrease in NO during omalizumab therapy was also previously described by Barber and Cousins . DownregAlternatively, the development of atopy may also be a direct effect of elevated homocysteine or some of its metabolites, which appears to exert a number of diverse effects on immune function. In addition, total homocysteine (Hcy) has been shown to increase in response to immune activation and cell proliferation during a nonallergic Th1-type immune response. Although much less is known about the health effects of sustained postload homocysteine concentrations, there is evidence that it has negative effects on platelet aggregation and endothelial function. A number of studies have indicated that homocysteine may contribute to the development and progression of atherosclerosis, a risk factor for cardiovascular diseases. However, the mechanisms by which Hcy can induce vascular dysfunction are not fully understood –41.Vitamin-D (25(OH)D) has effects on the innate and adaptive immune system. 25(OH)D levels are associated with poor asthma control, reduced pulmonary function, increased medication intake, and exacerbations. Little is known about 25(OH)D in adult asthma patients or its association with asthma severity , 43. MorBullous pemphigoid (BP) is an acquired, autoimmune, bullous disease that is characterized by autoantibodies against the 230-kDa bullous pemphigoid antigen within basal keratinocytes and the 180-kDa type XVII collagen within the basement membrane zone lying between the epidermis and dermis. In addition to skin blisters, patients with BP often experience pruritus and erythematous urticaria-like skin lesions.CD200 (OX-2) is a novel immune-effective molecule, both cell membrane-bound and also existing in a soluble form in serum , act as a proinflamatory through its receptor , 14, 22.β, and OX-2 and had an increased CXCL8, activated pC (APC), antithrombin III (AIII), protein S (pS), and protein C (pC) levels [More interestingly, as in some of our cases we have observed, one with severe persistent asthma (SPA) patient who had protein C/S deficiency history, multiple massive pulmonary embolus, and systemic subacute thrombosis determined in vena saphena parva and in left vena perforantes cruris underwent omalizumab treatment. After a long term (20 months) treatment with omalizumab, he had a decreased fractional exhaled nitric oxide concentrations (FENO), d-dimer (DD), sTRAIL, proinflammatory IL-1) levels . In this) levels .Severe persistent asthma is associated with a procoagulant state in the bronchoalveolar space and is further aggravated by impaired local activities of the anticoagulant pC/S, AIII system, and fibrinolysis, as demonstrated by massive fibrin depositions in the alveoli of a SPA who died from a SPA attack who did not respond to treatment. Recent reports revealed that patients with CU also show signs of thrombin generation and activation of the TF pathway of the coagulation system. DD, a fibrin degradation product formed during the lysis of a thrombus, is also detected in high levels in patients with active CU –51. Afte3, the omalizumab treatment was suspended for 4 weeks until the platelet count rose up to 100,000/mm3   after the 22nd dose of the drug was given. When the platelet count fell down to 55,000/mm00/mm3   is a heterogeneous group of immune disorders. It is characterized by very high concentrations of the serum antibody IgE. Clinically eczema-like rash, cold staphylococcal infection, severe lung infection are seen. An IgE level greater than 2,000 IU/mL is often considered diagnostic, except patients younger than 6 months of age. Extrinsic pathway of coagulation is activated in response to high level of circulatory IgE. Abnormal neutrophil chemotaxis due to decreased production of interferon gamma by T lymphocytes is thought to cause the disease. Both autosomal dominant and recessive inheritance have been described , 55. Mutectively . These cectively , 59. in vivo immunomodulatory activity on T cell responses [Eosinophilic gastroenteritis (EGE) is characterized by patchy or diffuse eosinophilic infiltration of any part of gastrointestinal (GI) tract , 61. Eosesponses .εRI on circulating basophils and mast cells, it seems to lower the activity potentials of basophils and mast cells, thereby reducing the potential reactivity of these cells [Mastocytosis is a heterogeneous disorder that results from clonal mast cell proliferation (myeloproliferative neoplasm), characterized by excessive mast cell accumulation in skin (cutaneous form) or multiple tissues, with or without skin involvement (systemic form) , 68. Incse cells , 69. Conse cells .The historic triad of nasal polyposis, asthma, and intolerance to aspirin and related chemicals, recently designated as Samter's syndrome, is an inflammatory condition of unknown pathogenesis. Many patients with Samter's syndrome also have a marked eosinophilia of both bronchial and nasal secretions as well as the circulating blood. Approximately 10% of the patients have urticaria-angioedema, alone or in combination with respiratory inflammation. As with all allergic diseases, the cornerstone of treatment is environmental control with avoidance of respiratory irritants, aspirin, and aspirin-like medications. Management of upper airway disease requires careful prescription of medication supplemented by judicious selection of surgery. Omalizumab demonstrated clinical efficacy in the treatment of nasal polyps with comorbid asthma, supporting the importance and functionality of local IgE formation in the airways , but in Severe refractory atopic dermatitis is a chronic, debilitating condition that is associated with elevated serum IgE levels. The mechanisms of omalizumab in the treatment of atopic dermatitis (AD) need further research in lowering serum IgE subjects. Several case reports investigating anti-IgE therapy in patients with AD found symptomatic improvement with omalizumab . RecentlMetz et al.  assessedThe activation of mast cells and their release of inflammatory mediators are regarded as the ‘‘final common pathway” for a myriad of proinflammatory factors, including those involved in the various types of urticaria , 83.The clinical response of urticaria to H1-antihistamines and the finding of increased concentrations of histamine in skin tissue fluid underscore the role of histamine derived from dermal mast cells as a major mediator of urticaria. Although highly unlikely, it cannot be excluded that in some cases of urticaria, the primary abnormality lies in the mast cells themselves. If this were the case, it would be likely that the condition would be systemic rather than confined to the skin. Therefore it is more likely that skin mast cells in patients with urticaria are not intrinsically abnormal but become increasingly sensitive or ‘‘unstable” or activated as the result of certain abnormal factors present in their surroundings. Although there are many nonimmunologic factors that might influence mast cell function in the skin, such as components of the complement system and neuropeptides, particularly those related to stress, because this review is primarily concerned with the mechanisms by which omalizumab might be effective, nonimmunologic factors will not be considered in detail –89.εRI-bound IgE by antigen or anti-IgE antibodies. This initiates the aggregation of FcεRI, leading to tyrosine kinase activation and subsequent mast cell activation for secretion. However, in 2001, it was suggested independently by 2 groups that monomeric IgE in the absence of antigen can have multiple effects in murine mast cells, including differentiation, proliferation, survival, and mediator and cytokine generation. These effects, which involve the binding of IgE to FcεRI and the aggregation of FcεRI, occur without the mast cells undergoing degranulation. The finding that monomeric IgE can augment mast cell activity has been confirmed by studies using various techniques. In a transcriptome analysis of 8793 genes, sensitization of mast cells with monoclonal IgE alone, without FcεRI cross-linking, was found to upregulate 58 genes more than 2-fold compared with their levels in unsensitized mast cells. These genes included those for cytokines, such as IL-1b, IL-6, and colony stimulating factor 1; chemokines, such as CXCL8, CCL4, and CCL7; and cytokine and chemokine receptors. The genes for various immune regulators, adhesion molecules, antiapoptosis proteins, and cytoskeletal elements, such as RAS protein activator like 1 and fibronectin leucine-rich transmembrane protein 2, were also upregulated [In conventional thinking the involvement of IgE in mast cell activation requires the cross-linking of Fcegulated –91.εRI, the depletion of mast cell-bound IgE by omalizumab and the subsequent downregulation of FcεRI on mast cells and basophils would lead to their decreased state of hyperexcitability. Third, in those patients with IgE autoantibodies against autoallergens, the inhibition of IgE binding to FcεRI by omalizumab and the downregulation of FcεRI would represent a central mechanism of omalizumab [First, omalizumab sequesters monomeric IgE to reduce its priming effect on mast cells. This would be particularly relevant if HC IgE is involved in the pathogenesis of urticaria. Second, in patients with IgG autoantibodies against IgE or Fcalizumab . Other salizumab .We stopped omalizumab treatment after 4 years. Recurrences of urticaria symptoms were found to be higher in patients with chronic urticaria than recurrences of asthmatic symptoms in severe persistent asthma patients. This might be due to transient effect of omalizumab on extrinsic coagulation pathway in the endothelial cells.There have been concerns about the cardiovascular safety in patients initiating omalizumab therapy, because of the most recent study that analyzed the association between omalizumab and arterial thrombotic events –81, 93. The clinical experience during the patient follow-up of omalizumab-treated severe persistent allergic asthma patients with type-2 diabetes mellitus is introduced. Omalizumab is generally considered safe. The most common adverse reaction from omalizumab is injection site pain and bruising but the package insert contains warnings regarding malignancies, geohelminth infections, and a “black box” warning about anaphylaxis. While there are no reports of fatal anaphylaxis as a result of Xolair, some cases have been serious and potentially life-threatening. Therefore, the FDA requires that people receiving Xolair be monitored in the physician's office for a period of time after their injections. Also, it is not yet known what the potential long term effects of Xolair use may have on people who are prone to getting cancer (such as the elderly). While it would appear that Xolair has potentially severe side effects, it must be remembered that anaphylaxis and cancer formation occurred only in a very small number of patients. Moreover, our knowledge about omalizumab use for asthma and other allergic diseases has improved to such an extent that we now better understand the treatment influence on systemic levels of oxidative stress markers, the interaction of oxidant and antioxidant balance, and apoptotic and inflammatory markers. We showed that omalizumab therapy increases blood glucose levels in allergic asthma patients with diabetes mellitus. Although we do not know the exact mechanism behind this relationship, it might be related with vial containing (145 mg sucrose) of omalizumab. Patients with diabetes mellitus should be informed that such a need of insulin dose should be increased due to the possible effect of omalizumab on blood glucose level. In these two patients half of the recommended dosage was given and blood glucose levels were controlled .The most common adverse reaction from omalizumab is injection site induration, injection site itching, injection site pain, and bruising but the package insert contains warnings regarding parasitic infections. While there are no reports of fatal anaphylaxis as a result of omalizumab, some cases have been serious and potentially life-threatening. Therefore, the FDA requires that people receiving omalizumab be monitored in the physician's office for a period of time after their injections .A female patient with severe persistent allergic asthma, aged 58, is introduced. Laboratory tests are as follows: anti-nuclear antibody, and viral hepatitis markers such as HBsAg, HBsAb, anti-HCV HIV, thyroid antibodies were negative, autologous serum skin test was positive. Liver, thyroid, and renal function tests, serum IgG, IgA, and IgM levels were within normal ranges. Skin prick tests (SPTs) were highly positive for mite and mold. The specific IgE levels were correlated with the SPTs. Total IgE level was 480 IU/L. Body temperature was 38.5°C.The patient had not previously reported drug induced anaphylaxis. On omalizumab (on the 23rd dose) therapy, this patient had laryngeal oedema during a complicated common cold infection with bacterial sinusitis after omalizumab injection.Systemic steroids (500 mg prednisolone), ketotifen fumarate (1X1), antibiotic and antihistamines  were given. Oral antihistaminics and mast cell stabilizing agents were used for treatment afterwards. We did not stop omalizumab therapy. We do not suggest omalizumab during the first five days of complicated viral infections such as common cold and bacterial sinus infections .I had a male patients with food allergy with pulmonary carcinoid tumor, aged 23. Autologous serum skin test was positive in patients. Anti-nuclear antibody and viral hepatitis markers such as HBsAg, HBsAb, anti HCV HIV, thyroid antibodies were negative in patients. Liver, thyroid, and renal function tests, serum IgG, IgA, and IgM levels were within normal ranges. Skin prick tests (SPTs) were highly positive for kiwi, tomato, fish, and orange. The specific IgE levels were correlated with the SPTs. Total IgE level was 960 IU/L . A mass was defined on lower lobe of left lung on computerized tomography (PET CT SUVmax: 6). The patient was operated. In postoperative period, he had recurrent laryngeal oedema and urticaria attacks. Omalizumab treatment planned because of the patient was resistant to antihistaminics and steroids. For the very first time, we used omalizumab in symptomatic therapy of recurrent laryngeal oedema and urticaria attacks in a patient with postoperative pulmonary carcinoid tumor for eight months. During the four years of follow-up, no recurrence was noted in carcinoid tumor. Control PET CT and CT results revealed normal findings. After omalizumab treatment, laryngeal oedema and urticaria symptoms were decreased. Oral antihistaminics and mast cell stabilizing drugs were used for treatment afterwards. Oral steroid was given only once .Omalizumab has the potential to be an additional and solitary treatment option in patients with allergic bronchopulmonary aspergillosis and cystic fibrosis. Early onset treatment may be beneficial and patients with early stage of lung disease seem to benefit more . Pooled To sum up what I would like to express as a conclusion is that omalizumab in patients with severe persistent asthma is an effective therapy for asthma and comorbid conditions  just like I have mentioned above. The mechanism of action of omalizumab in the treatment of asthma is believed to be multifactorial and includes effects mediated through altered production of redox metabolites, oxidative markers related mi RNA, TRAIL related mi RNA, and regulation of production of known inflammatory proteins.