text:
Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence
- First Online:
- Received:
- Revised:
- Accepted:
- Citations
- 1.2k Downloads
Membrane-embedded β-barrel proteins span the membrane via multiple amphipathic β-strands arranged in a cylindrical shape. These proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. This situation is thought to reflect the evolutionary origin of mitochondria and chloroplasts from Gram-negative bacterial endosymbionts. β-barrel proteins fulfil a variety of functions; among them are pore-forming proteins that allow the flux of metabolites across the membrane by passive diffusion, active transporters of siderophores, enzymes, structural proteins, and proteins that mediate protein translocation across or insertion into membranes. The biogenesis process of these proteins combines evolutionary conservation of the central elements with some noticeable differences in signals and machineries. This review summarizes our current knowledge of the functions and biogenesis of this special family of proteins.
KeywordsBam complex β-barrel proteins Gram-negative bacteria Mitochondria Omp85 Outer membrane TOB complex TOM complex
Bacterial β-barrel proteins
The Gram-negative bacterial cell envelope
Biogenesis of bacterial outer membrane proteins
Several chaperones that guide nascent OMPs in the periplasm have been identified. The most important of those are ( seventeen-kD protein (Skp) and SurA (Fig. 2) [3]. Skp selectively binds unfolded OMPs [7, 8] early on while they are still engaged with the Sec machinery [9]. The phospholipid-binding properties of Skp [8], which support its localization to the external surface of the inner membrane, probably enable this early interaction. Binding of Skp may assist in the release of the nascent OMP from the Sec machinery and results in the formation of a soluble periplasmic intermediate [10]. Hence, Skp functions as a holding chaperone that prevents aggregation of its substrates in the periplasm. The crystal structure of the trimeric Skp revealed a jellyfish-like architecture with the tentacles forming a cavity where the unfolded substrate proteins could bind [11, 12]. Recently, the interaction between Skp and OmpA, a two-domain OMP consisting of a membrane-embedded β-barrel and a periplasmic peptidoglycan-binding domain, was studied in vitro in biochemical and NMR experiments [13]. These experiments confirmed the working model for Skp function. They revealed that trimeric Skp prevents unfolded OmpA from aggregating by forming stable soluble 1:1 complexes. Within the complex, the β-barrel domain of OmpA remains unfolded and is buried deep within the cavity among the tentacles of Skp, while its periplasmic domain is free to fold and extends away from the complex [13].
An additional chaperone, SurA, was first identified as a protein required for survival of E. coli in the stationary phase [14]. In contrast to Skp, which functions as a holding chaperone, SurA functions as a folding chaperone that assists the folding of nascent OMPs into their native conformation [15, 16]. The 46-kDa protein contains two peptidyl-prolyl isomerase (PPIase) domains, which, however, are dispensable for function [17]. The crystal structure of SurA revealed a globular core fragment, consisting of the N- and C-terminal domains and the first (inactive) PPIase domain with the second (active) PPIase domain extending away from the core domain [18]. The core shows an extended crevice in the N-terminal domain where peptides could bind. Peptide binding studies revealed that SurA has a preference for peptides rich in aromatic residues, particularly those containing two consecutive aromatic residues or two aromatic residues separated by one other residue, i.e., sequences typically found in the transmembrane β-strands of OMPs [19, 20]. Such peptides were subsequently found to be recognized by the first PPIase domain of SurA [21], which is dispensable for function. In contrast, an earlier report suggested that peptide binding is mediated by the N-terminal domain of SurA [22]. This discrepancy can be explained if substrate selection and chaperoning activity are distinct activities that reside in different parts of the protein [21].
Skp and SurA are both non-essential proteins in E. coli as mutants in whom the corresponding genes are inactivated are viable. Such mutants have drastically decreased OMP levels [7, 23]. This reduction is due to the induction of a periplasmic stress response including, amongst others, the over-expression of the potent protease DegP, which degrades unassembled OMPs in the periplasm. Furthermore, the synthesis of small regulatory RNAs that inhibit the translation of the mRNAs for OMPs is induced [24, 25]. The observation that skp and surA mutations are synthetically lethal might indicate that these proteins have overlapping functions and that they operate in the periplasm in parallel pathways for OMP assembly [23, 26]. However, because of their different structure, mode of substrate binding, and function as described above, alternative explanations for this synthetic lethality should be considered. We favor the model that Skp and SurA act sequentially within the same pathway (Fig. 2) [3]. The synthetic lethality in this view is explained by an increased requirement for a holding chaperone when the folding of the substrates is compromised by the absence of SurA, and, vice versa, efficient folding is increasingly important when the holding chaperone Skp is absent. Consistent with this view, Skp acts early within the periplasmic pathway while the substrate proteins are still engaged with the Sec translocon in the inner membrane [9]. In contrast, SurA could be cross-linked to the Bam complex in the OM [23], which is required for assembly of OMPs into the OM (see below), suggesting that SurA acts late in the pathway.
Apart from Skp and SurA, DegP has also been suggested to be involved in OMP biogenesis [23, 27]. DegP is a periplasmic protease, which has chaperone qualities as well [28]. In its activated form, DegP forms large, cage-like 12-meric and 24-meric complexes [27, 29], which could harbor a folded OMP in their cavities [27]. However, so far, there is little evidence for a direct role of DegP in OMP folding or insertion. The synthetic lethality of a degP mutation when combined with a surA [26] or skp [10] mutation is probably not due to its capacity as a chaperone to take over the role of SurA and Skp and assist in OMP assembly, but rather reflects its capacity as a protease to prevent the toxic accumulation of unfolded OMP aggregates in the periplasm. Of note, it is not only the protease activity of DegP that is important in this respect. A mutant form of DegP without protease activity but with retained chaperone activity could rescue strains from lethality caused by the expression of assembly-defective mutant forms of OmpF and OmpC. This rescue occurred without restoring the assembly of these mutant proteins into the OM, but rather by sequestering them, thereby removing them from the assembly pathway [30, 31]. This sequestering role could be reflected by the captured OMPs observed in the DegP multimeric complexes [27]. In conclusion, Skp and SurA are the most important periplasmic chaperones in OMP biogenesis and they appear to perform different roles in this process.
The β-barrel assembly machinery
After passage through the periplasm, OMPs are assembled into the OM via a machinery, recently designated the β-barrel assembly machinery or Bam complex (Fig. 2). The first component of this machinery was identified in Neisseria meningitidis, where it was demonstrated that a highly conserved OMP, designated Omp85, is essential for viability and required for the folding and assembly of all OMPs examined [32]. In a mutant strain, in which the expression of the omp85 gene could be regulated, unfolded forms of OMPs accumulated as periplasmic aggregates when the synthesis of Omp85 was switched off [32, 33]. Similarly, a role in OMP assembly was subsequently demonstrated for the Omp85 homologues of E. coli [34, 35, 36] and Pseudomonas aeruginosa [37], designated BamA (formerly YaeT) and Opr86, respectively.
The Omp85 homologue of E. coli, BamA, was found to form a complex with four lipoproteins, designated BamB–E (formerly YfgL, NlpB, YfiO, and SmpA, respectively) [36, 38] (Fig. 2). Of these, only BamD is essential for viability [39] and OMP assembly [40], whereas deletion of the other accessory components, which are evolutionarily less well conserved, does not infringe viability and results in relatively mild OMP assembly defects. Interestingly, the BamD homologue of Neisseria gonorrhoeae, designated ComL, is covalently linked to the peptidoglycan layer [41], while bamB mutations were reported to affect peptidoglycan synthesis, possibly by regulating the activity of lytic transglycosylases [42]. Furthermore, many bacteria contain a BamE homologue extended with a peptidoglycan-binding domain, for example the Plp4 protein in Pasteurella haemolytica [43]. These observations suggest that these accessory components may have a role in anchoring the Bam complex to the peptidoglycan and perhaps in modulating the peptidoglycan to facilitate the access of nascent OMPs to this complex. Such a putative peptidoglycan-related role of these accessory components would explain why similar components are absent from the machinery required to assemble β-barrel proteins in mitochondria, which do not contain peptidoglycan. Curiously, BamB was also reported to have protein kinase activity and to be involved in DNA strand-break repair and homologous recombination [44], but all these properties are difficult to reconcile with its OM-associated periplasmic location.
Substrate recognition by Omp85
The Omp85 proteins of several bacteria, including BamA from E. coli, were found to form pores when reconstituted in planar lipid bilayers or liposomes [45, 46, 47, 48]. The physiological significance of these pores is not entirely clear, but this property could be used to study the interaction with substrate proteins, because it was anticipated that such interaction would affect the channel properties. Indeed, it was found that denatured E. coli OMPs, but not periplasmic proteins, increased the BamA channel activity, showing that these substrates directly and specifically interact with BamA [47]. The channel-enhancing activity was mimicked by a synthetic peptide corresponding to the last 12 C-terminal amino-acid residues of the OMP PhoE, suggesting that the C terminus contains the signal for substrate recognition. In agreement with this result, earlier studies have recognized a conserved signature sequence at the C terminus of the vast majority of OMPs from various bacterial species [49]. This signature sequence consists of a highly conserved Phe (or Trp) at the ultimate C-terminal position, Tyr or a hydrophobic residue at position 3, and also hydrophobic residues at positions 5, 7, and 9 from the C terminus. It was demonstrated that the deletion of the conserved C-terminal Phe in PhoE dramatically affected the efficiency of assembly into the OM, and the mutant protein was found to accumulate as dense aggregates in the periplasm [49]. The expression of the mutant protein was lethal. Consistent with these in vivo experiments, it was found that a synthetic peptide corresponding to a C-terminal fragment of PhoE but lacking the C-terminal Phe failed to enhance the BamA channel activity in planar lipid bilayers [47].
Of note, a C-terminal Phe (or Trp) is not an absolute requirement for assembly into the OM. A substitution analysis showed that both the hydrophobicity and the aromatic nature of the C-terminal residue were of importance, but a Tyr or a hydrophobic residue at this position was less detrimental for assembly than the presence of a polar residue [49]. Furthermore, a mutant PhoE protein with a deletion of the C-terminal Phe was efficiently assembled into the OM provided the expression level was reduced [50]. Thus, even though the removal of the C-terminal Phe reduces efficient recognition by the assembly machinery, it does not completely destroy the recognition signal, or, alternatively, less efficient signals in other locations within the PhoE sequence also exist. Consequently, at high expression levels, the mutant protein will aggregate within the periplasm because of the poor recognition by the assembly machinery. The reduced kinetics of aggregation at lower expression levels, however, increases the time span for the mutant protein to functionally interact with the Bam complex and result in assembly of the mutant protein.
Although the C-terminal signature sequence described above is present in the vast majority of OMPs from various bacterial species, some species-specific adaptations appear to have occurred during evolution. In the vast majority of OMPs of Neisseria spp., a positively charged residue is present at the penultimate position from the C terminus, whereas this is seldom the case in E. coli. High-level expression of these OMPs is lethal in E. coli, and these OMPs fail to activate the BamA channels in planar lipid bilayers. Substitution of this positively charged residue by a glutamine, which is often found at this position in E. coli OMPs, markedly improved the assembly of the meningococcal porin PorA into the E. coli OM [47].
Structure of Omp85
Omp85 was suggested to consist of two domains [32], a C-terminal membrane-embedded β-barrel and an N-terminal periplasmic part (Fig. 2). The latter part consists of five repeated sub-domains, known as polypeptide-transport-associated (POTRA) domains [51]. The function of the different POTRA domains is not entirely clear, although, considering their periplasmic location, a role in the binding of substrates and/or the accessory components of the Bam complex is likely. In N. meningitidis Omp85, the first four POTRA domains could be deleted simultaneously resulting in only relatively mild assembly defects particularly of large OMPs [52]. Only the fifth POTRA domain together with the β-barrel domain was found essential in this study. Hence, the functional core domain of the bacterial Omp85 is similar to that of its mitochondrial homologue Sam50/Tob55, which consists of only a single POTRA domain attached to a β-barrel and will be discussed later. In contrast, deletion of each individual POTRA domain, except for the second one, of E. coli BamA severely infringed function and viability of the cells [53]. Apparently, there are considerable differences in Omp85 functioning between different bacteria. Deletion of the fifth POTRA domain in E. coli BamA resulted in the loss of binding of BamB–E [53], which suggests a specific role for this POTRA domain in the binding to the accessory lipoproteins and is consistent with the important role of this domain in N. meningitidis Omp85.
Recently, the structure of the POTRA domains of E. coli BamA was investigated by X-ray crystallography [53, 54], NMR spectroscopy, and small-angle X-ray scattering [55]. Each POTRA domain consists of a three-stranded β-sheet overlaid with a pair of α-helices, and the linkers between the domains appear to allow some flexibility in their relative orientation. Interestingly, OmlA, the BamE homologue of Xanthomonas axonopodis pv. citri, has a similar structure, although the relative order of the secondary structure elements is different [56]. The crystallization-induced dimerization of a polypeptide containing the first four POTRA domains of BamA and a small part of the fifth one suggested that the POTRA domains might bind substrates and/or accessory proteins by a process called β-augmentation [53]. NMR experiments revealed that various peptides of the model OMP PhoE could bind, although weakly, to either side of the β-sheets of the POTRA domains, and suggested that the POTRA domains could guide nascent OMPs to the core of the Bam complex by processive sliding [55]. Unfortunately, binding of a C-terminal peptide of PhoE or another OMP could not be measured in these experiments because of solubility problems.
The structure of the β-barrel of an Omp85 protein has so far not been solved. However, the Omp85 super-family also includes bacterial OMPs involved in the two-partner secretion pathway. These proteins, generically called TpsB, are required for the translocation of their partner, TpsA, across the OM into the medium. Recently, the structure of a TpsB protein, i.e., FhaC of Bordetella pertussis, was solved [57]. The structure showed that the protein consists of a periplasmic moiety containing two POTRA domains and a 16-stranded C-terminal β-barrel embedded in the OM. The pore in the barrel is closed by an N-terminal α-helix and by a loop that reaches from the cell-surface-exposed side through the barrel to the periplasmic side of the membrane. It is plausible that the removal of the helix and the loop from the barrel upon substrate binding would create a pore allowing for the transport of the partner TpsA to the cell surface. Unfortunately, this structure provides little insight into how the β-barrel of Omp85 might work, as it is not conceivable that nascent OMPs would insert into the barrel of Omp85 [58]. Rather, membrane insertion should take place at the subunit interface if Omp85 forms oligomers, for which some evidence has been reported [47], or at the lipid–protein interface. Further discussion on putative mechanisms for membrane insertion is included in the section on the biogenesis of mitochondrial β-barrel proteins.
Mitochondrial β-barrel proteins
Functions and structural features of eukaryotic β-barrel proteins
Compared to the diversity of β-barrel membrane proteins in prokaryotes, the number of mitochondrial OMPs belonging to this structural type is rather limited. Although some prediction programs propose that the yeast mitochondrial OM might contain more than 100 β-barrel proteins [59], later proteomics and bioinformatic studies revealed that this number is an overestimation. In the extensively studied unicellular fungus Saccharomyces cerevisiae, six members have been identified: Tom40, Tob55/Sam50, two isoforms of porin/VDAC, Mdm10, and Mmm2, of which only the first two are essential for viability [60, 61]. It should be noted that, to date, only the structure of mammalian VDAC1 has been solved and the remaining members have been assigned to the family on the basis of both secondary structure content and in silico predictions.
Mitochondrial porin (also named voltage-dependent anion-selective channel, VDAC) is the founding member of the eukaryotic family [62]. VDAC is the most abundant protein in the mitochondrial OM and, soon after its discovery more than three decades ago, its role as a diffusion pore for small metabolites became apparent [63]. Conductivity and selectivity of the pore are achieved by a gating mechanism, which can be influenced both in vitro and in vivo by the membrane potential and by a variety of binding partners (reviewed in [64]). In yeast, two isoforms, VDAC1 and VDAC2, encoded by the genes POR1 and POR2, respectively, are known. Deletion of POR1 resulted in significant growth phenotypes whereas VDAC2 appears to be dispensable. However, the observation that over-expression of VDAC2 could partially complement the Δpor1 phenotype [65] suggests similarities in their function. In mammals, the family of VDAC proteins comprises three known orthologues, all of them mapping to individual gene loci [66, 67]. The third isoform, VDAC3, which is assumed to be the most primordial member [67], was found to yield an additional variant via tissue-specific alternative splicing [68]. In cultured cells, none of the three isoforms appeared to be essential when deleted individually, but mitochondrial respiration was reduced [69].
VDAC is located on the interface between mitochondria and the cytosol and thus has the potential to act as a "switch" for cellular processes involving mitochondria. This position and its central role in mitochondrial permeability define VDAC as a future pharmacological target for anticancer agents [70]. Indeed, an increasing body of evidence has been collected in support of a role of VDAC1 in apoptosis-mediated cell death in higher eukaryotes. It was suggested that the protein participates in the mitochondrial permeability transition (MPT) and in the exit channel for the release of cytochrome c from the mitochondrial intermembrane space (IMS) into the cytosol. This release is enhanced by pro-apoptotic Bax/Bak proteins and inhibited by anti-apoptotic Bcl-xL [71, 72, 73]. However, the physiological importance of VDAC proteins to apoptosis was challenged by the finding that fibroblasts lacking all three VDAC isoforms exhibited MPT and cell death indistinguishable from wild-type cells [74]. In addition to its proposed interactions with pro- and anti-apoptotic proteins, various cytosolic proteins (like hexokinase and tubulin) and characteristic mitochondrial lipids (like phospatidylethanolamine and cardiolipin) were suggested to regulate VDAC gating [75]. Taken together, although it is evident that VDAC is playing a key role in the communication between mitochondria and the rest of the cell, future studies are required to shed more light on the regulation of its activity and the integration of such activity in various signaling processes.
Tom40 was the second member of the mitochondrial β-barrel OMP family to be identified. It is essential in both S. cerevisiae and Neurospora crassa and was found to be the central component of the translocase of the mitochondrial OM (TOM complex) [76, 77]. Tom40 is required for the import of the vast majority of precursor proteins into mitochondria as it forms the general import pore of the complex [78, 79]. The second essential protein in the mitochondrial OM is Tob55 (also termed Sam50 [80] or Omp85 [81]). The protein is the central member of a complex termed TOB (for topogenesis of β-barrel proteins [82]) or SAM (for sorting and assembly machinery [80]). Notably, Tob55 is conserved in all eukaryotes and is the only mitochondrial β-barrel protein that shares significant sequence homology with β-barrel proteins from chloroplasts (Toc75-V) and bacteria (Omp85/BamA) [81, 82]. Tob55 promotes the membrane integration of precursors of β-barrel proteins and its function will be discussed in detail below.
The two additional mitochondrial β-barrel proteins in fungi, Mdm10, and Mmm2 (Mdm34) are required for mitochondrial morphology and dynamics [83, 84]. Homologues of these two proteins have so far not been reported in mammals. The precise role of Mmm2 is not clear. Mdm10 was suggested to be part of a complex in the OM that links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery [85]; on the other hand, it was also proposed that Mdm10 is part of the TOB complex and has a role in the biogenesis of β-barrel proteins [86]. This second putative role is discussed in a later section.
More than 25 atomic structures of bacterial β-barrel proteins led to the consensus that such proteins are composed of an even number of β-strands arranged in an antiparallel manner. The recently solved first atomic structure of a mammalian β-barrel protein, VDAC1, changed this view. Three independent studies using NMR spectroscopy, X-ray crystallography, or a combined approach revealed that the barrel structure of VDAC1 comprises 19 β-strands, which are interconnected by relatively short turns between 2 and 10 amino-acid residues in length, in an antiparallel orientation [87, 88, 89]. Hence, unlike all known prokaryotic β-barrel proteins, the structure is formed by an odd number of β-strands. Surprisingly, a parallel β-sheet formed between strands 1 and 19 was detected. It has been reported that a mutant form of the E. coli porin PhoE lacking the first transmembrane β-strand could be functionally incorporated into the bacterial OM [90], showing that the bacterial Bam machinery can also deal with barrels with an odd number of strands. A short N-terminal helix was resolved in the centre of the VDAC1 channel. This helix was proposed to have a function in the transition between open and closed states [89]. Based on computational analysis, it was suggested that the topology of VDAC1 could also apply to other VDAC isoforms and to Tom40 [87]. Being the first structure of a eukaryotic β-barrel membrane protein, additional structures are required to understand whether the unique structural features of VDAC1 are shared by other eukaryotic proteins.
Biogenesis of β-barrel proteins in mitochondria
A comparison between the biogenesis pathways of β-barrel proteins in mitochondria and bacteria has several implications regarding the evolutionary conservation of the process. Besides differences in the protein composition between the Bam and TOB complexes, the compartment in which the precursors of β-barrel proteins are synthesized has in the case of mitochondria shifted to the exterior of the organelle, most likely due to gene transfer from the endosymbiont's genome to the nucleus. Intriguingly, in spite of the direct accessibility of the mitochondrial OM to cytosolic β-barrel precursors, a situation not found in bacteria, there is no direct membrane insertion from the cytosolic face of the OM, but rather these precursors are transported into the IMS before assembly. Thus, in both systems, the precursors are inserted from the internal side of the outer membrane. This conservation is also reflected in the orientation of the central component of the Bam/TOB machinery where in both cases the POTRA domain(s) points toward the IMS/periplasm.
Targeting information of mitochondrial β-barrel proteins
Other than matrix-destined proteins, β-barrel precursors are devoid of canonical N-terminal targeting sequences [91]. In-depth analysis of the insertion pathway has led to the understanding that the correct sorting to its destination involves a sequence of at least two recognition steps. First, after its synthesis in the cytosol, the precursor protein is recognized at the TOM complex similar to virtually all other mitochondrial proteins [92, 93, 94, 95]. This recognition is followed by translocation across the import pore of the TOM complex and thus assures import into mitochondria and therefore targeting to the correct organelle. Second, precursor-sorting pathways diverge, and an interaction with the small Tim chaperones followed by recognition by the TOB machinery assures a distinct suborganellar sorting of β-barrel precursors to the OM. The signals required for recognition in each of these two steps are believed to differ in their nature.
Early studies based on in vitro import of mutated radiolabelled β-barrel precursors into isolated mitochondria failed to conclusively locate specific sequences responsible for mitochondrial targeting. For example, C- or N-terminal truncations of N. crassa Tom40 by 20 or 60 amino-acid residues, respectively, failed to abolish import [94]. In the case of the precursor of N. crassa porin, only the N terminus was dispensable for targeting whereas the C-terminal 15 amino-acid residues were required [96]. Likewise, various point mutations as well as terminal or internal deletions in yeast porin could abolish precursor import [96, 97]. Furthermore, in contrast to matrix-destined proteins, unfolding of Tom40 precursors resulted in decreased import and assembly efficiency [94]. These findings indicate that the signal for targeting of β-barrel precursors to mitochondria is probably not confined to a single linear sequence but rather involves structural elements.
Several reports have led to the assumption that a precursor's ability to form a β-barrel structure can be sufficient to ensure its targeting to the mitochondrial compartment. A first hint was provided by studies on the role of the bacterial OMP PorB from pathogenic Neisseria spp. during infection. After invasion, the protein is released into the host cell's cytosol from where it is translocated into mitochondria and induces cell death via the intrinsic apoptotic pathway [98, 99]. Very recently, we could demonstrate that PorB is not an exceptional case and other bacterial OMPs share the ability to be targeted to mitochondria when expressed in yeast cells. In contrast to PorB in mammalian cells, however, production of these proteins caused neither mitochondrial dysfunction nor other signs of cellular damage. Like endogenous β-barrel proteins, the bacterial members were inserted in a process that was found to depend on both TOM- and TOB-complex components and even resulted in the formation of native-like oligomeric structures in the mitochondrial OM [95]. Taken together, we speculate that, rather than eukaryote-specific linear signals, a folding intermediate of a precursor with high β-sheet content can serve as a signal of β-barrel proteins to assure their targeting from the cytosol to their destined compartment. The targeting issue becomes even more complicated in plant cells that contain two organelles with β-barrel membrane proteins, mitochondria and chloroplasts. The question how these cells assure that β-barrel proteins reach only one of these organelles is still open.
While recognition by and translocation through the TOM complex is shared among the majority of mitochondrial protein precursors, the sorting pathways diverge after the translocation step, depending on their destination within the organelle. In the case of β-barrel proteins, this second recognition step involves an interaction between its C terminus and the TOB machinery. Very recently, a signature motif termed β-signal, located at the C-terminal β-strand of the precursor, was identified [100]. This β-signal is different, although possibly derived from the C-terminal signature sequence found in bacterial OMPs and described above. Through site-directed mutagenesis, four essential residues were identified of which three, two hydrophobic and one hydrophilic, were found to be essential for binding to the TOB complex. In contrast, mutation of a conserved glycine resulted in the accumulation of TOB complex-associated assembly intermediates, indicating a role in release from the complex. Sequence analysis revealed that the identified β-signal is conserved in mitochondrial β-barrel proteins from organisms from all eukaryotic kingdoms: fungal species, plants, and animals including humans [100].
Components of the TOB complex
The known members of the TOB core complex in the fungal model organism S. cerevisiae are Tob55/Sam50/Omp85, Tob38/Sam35, and Mas37/Tom37/Sam37 (Fig. 3). Mas37 was initially identified in a screen for mutants defective in the control of phospholipid biosynthesis. However, due to genetic interaction with Tom20 and Tom70, it was proposed to function as an import receptor of the TOM complex [101]. Later, its membership in a distinct complex and its role in β-barrel precursor insertion became apparent [102]. Unlike the other two TOB complex components, Mas37 is not required for viability unless cells are grown at elevated temperatures [101].
The second member of the complex to be characterized was Tob55, which was identified by several independent approaches. First, both a proteomic screen of vesicles derived from the mitochondrial OM of N. crassa [82] and genome-wide in silico homology searches [81] revealed the presence of a protein with significant sequence similarity to the previously characterized Omp85, which facilitates OM β-barrel protein insertion in N. meningitidis [32]. Second, Tob55 co-purified with affinity-tagged Mas37 [80]. Like its bacterial ancestors from the Omp85 family, Tob55 comprises a membrane-embedded C-terminal β-barrel domain. Its N terminus forms a soluble extension towards the IMS, i.e., a POTRA domain [51]. While all bacterial Omp85 proteins contain five of these domains, only a single one exists in the eukaryotic orthologues. In conditional mutants [80, 81] and in a strain in which Tob55 was depleted via an inducible promoter [82], a selective reduction in the steady-state levels of known β-barrel proteins in the mitochondrial OM was observed. Deletion of Tob55 in yeast is lethal, underscoring its crucial role in the function of the TOB complex.
The second essential component of the TOB complex, Tob38, was identified by co-purification with Mas37 [103] or Tob55 [104]. Furthermore, it was observed that β-barrel assembly was impaired when the protein was depleted or mutated [103, 104, 105]. The fact that the protein is both extractable under alkaline conditions and degradable by treatment of intact mitochondria with protease led to the proposal that it is not embedded within the membrane but rather associated with the complex on the cytosolic face of the mitochondrial OM. A recent study indicated that the vast majority of the polypeptide chain is located within the membrane-embedded part of the TOB complex and only the termini are exposed to the cytosol [100].
Of note, while Tob55 shows clear homology with the bacterial Omp85 proteins, there is no sequence similarity of Mas37 or Tob38 with any of the accessory lipoproteins from the bacterial Bam complex. The degree to which the mitochondrial proteins are conserved between different eukaryotic kingdoms varies strongly between the individual members of the TOB complex. Clearly, orthologues of Tob55 with a high degree of sequence similarity exist in all eukaryotic genomes sequenced to date. Although clear homologues of Mas37 are only present in fungal species, it was suggested that mammalian Metaxin 1 may represent a distant orthologue as both proteins show sequence similarity in their N-terminal regions [106]. However, unlike Mas37, which is peripherally associated on the cytosolic face of the mitochondrial OM, Metaxin 1 is probably anchored to the OM via a transmembrane segment at its C-terminal domain. The importance of this protein is reflected by the early embryonic lethal phenotype of mice lacking Metaxin 1 [106]. Later, a related protein named Metaxin 2, which shares 29% identity with Metaxin 1, was identified [107]. Despite very weak homology, Metaxin 2 was suggested to be related to Tob38 [103, 104]. This notion is supported by the observation that both Metaxins interact with each other [107] and play a role in the assembly of Tom40 and VDAC [108].
It is still a matter of debate whether an additional β-barrel protein, Mdm10, is a further component of the TOB complex. Deletion of Mdm10 results in abnormal mitochondrial morphology [83], and the protein was further shown to exist in a complex with Mmm1 and Mdm12, which facilitates attachment of actin fibres to the organelle [85]. The picture became complicated when it was found that Mdm10 also functions in the assembly of Tom40 but is not involved in the biogenesis of other β-barrel proteins [86]. A later study suggested that Mmm1/Mdm12 function in the major β-barrel pathway, which involves all known β-barrel precursors, in a stage downstream of the TOB complex [109]. Currently, it is unclear whether the primary function of these proteins lies in mediating the organelle's dynamics, in protein sorting, or whether they have dual function.
Assembly of the TOB complex
The TOB complex, comprising all three known constituents, Tob55, Tob38, and Mas37, has a molecular weight of 200–250 kDa as determined by size-exclusion chromatography and Blue Native PAGE. In contrast, a sub-complex lacking Mas37 has a molecular weight of approximately 160 kDa [80, 104, 105]. Since β-barrel assembly is possible in mas37Δ strains, it is likely that the Tob55-Tob38 sub-complex represents a minimal functional unit.
The biogenesis pathways by which the TOB complex constituents are incorporated into functional machinery vary between the individual members. Tob55, being a β-barrel protein on its own, is inserted in the same way as the other members of this structural family. Its C-terminal β-barrel domain contains the necessary targeting information and is both required and sufficient for assembly into the complex while the N-terminal POTRA domain is required neither for targeting to the organelle nor for intra-mitochondrial sorting [110]. After their synthesis in the cytosol, Tob55 precursors are imported into the IMS of mitochondria via the TOM translocation pore and further integrated into the mitochondrial OM via the TOB complex [111]. Newly imported Tob55 molecules probably assemble into pre-existing TOB complexes.
The targeting of Mas37 precursors to mitochondria and their subsequent incorporation into the TOB complex represent a unique pathway for OMPs, as it is independent of the TOM complex. Tob55 and Tob38 are required for Mas37 assembly whereas the process is even more efficient in mitochondria from a Mas37-deficient strain [111]. Collectively, it appears that the cytosolic precursor protein binds directly to the TOB core complex. So far, the biogenesis pathway of Tob38 has not been studied. However, considering the fact that Tob38 and Mas37 share a comparable topology, one can speculate that a similar pathway applies to both proteins.
Function of the TOB complex
Like the vast majority of mitochondrial proteins, β-barrel precursors are encoded in the nucleus and synthesized in the cytosol on free ribosomes. The delivery to the surface of mitochondria most likely involves cytosolic chaperones in order to keep the precursor proteins in an import-competent partially folded conformation [94]. The precursor proteins are engaged on the organelle surface by import receptors of the TOM complex. This process is mainly dependent on Tom20 while Tom70 only plays a minor role [92, 93, 94, 95, 112]. After the initial recognition, β-barrel precursors are translocated through the translocation pore of the TOM complex into the IMS. This conclusion is based on several observations. First, assembly relies on functional Tom40, which is the key component of the TOM pore [92, 93, 94]. Second, import can be outcompeted by addition of an excess of matrix-destined precursor proteins, which use the import pore [82, 104, 111]. Finally, assembly intermediates downstream of the TOM complex are protected from externally added proteases [82, 93, 94].
The translocated precursors are then transferred from the TOM to the TOB complex in a process that exposes them to the IMS. At this stage, the small TIM complexes, Tim8/Tim13 and Tim9/10, which reside in this compartment, stabilize the β-barrel precursors [111, 113, 114]. Such an involvement of the small Tims can explain early observations where rupturing of the OM was found to impair porin biogenesis in vitro [115]. Like Skp in the bacterial systems, the small TIM complexes are believed to function as chaperones that prevent precursor aggregation or misfolding in the IMS, and they were also shown to be involved in the biogenesis of hydrophobic inner membrane proteins [116, 117].
Several lines of evidence suggest that translocation through the TOM pore and membrane insertion by the TOB complex do not occur separately but are rather coupled processes. When TOB complex function is reduced by deletion of Mas37 or depletion of Tob55, no accumulation of precursors in the IMS was observed, but instead, import was reduced to a similar extent as assembly [82, 102]. Furthermore, as discussed above, rupturing of the outer membrane of isolated mitochondria caused a reduction in the assembly of β-barrel precursors. Under these conditions, where externally added precursor proteins have direct access to the TOB complex, blocking the TOM pore resulted in a further inhibition of the already hampered membrane integration efficiency of these precursor proteins [110]. Collectively, these findings indicate that the passage of β-barrel precursors through the TOM pore is probably required in order to yield an insertion-competent state, which can subsequently be processed by the TOB complex. A coupling between the two complexes is further supported by a recent study where bacterial β-barrel precursors were overproduced in yeast cells. Under these conditions, the majority of the bacterial protein was imported but aggregated in the IMS, most likely due to overcharging the insertion capacity of the TOB machinery. Strikingly, under these conditions, import strongly depended on the TOB complex constituents Tob55 and Mas37 [95]. These findings suggest that a functional TOB complex is required for the translocation of β-barrel precursors through the TOM pore. Of note, despite these functional interactions, a direct physical interaction between the TOB and the TOM complex has not yet been observed.
Which parts of the TOB complex recognize and interact with β-barrel precursors is still debateable. Similar to the bacterial Omp85/BamA, Tob55 in all eukaryotes has a POTRA domain in its IMS-located N-terminal domain. In the bacterial Omp85/BamA proteins, at least one of the five POTRA domains is essential for viability, and it was speculated that this domain might interact directly with the bacterial C-terminal signature motif in substrate proteins [52, 53]. Accordingly, yeast cells harboring a Tob55 variant lacking the POTRA domain exhibited slower growth, and a recombinant POTRA domain could bind β-barrel precursor proteins [110]. The importance of the POTRA domain was challenged in a later study where a deletion of the POTRA domain together with some additional residues did not cause any growth phenotype [100]. Furthermore, Kutik et al. identified Tob38 as the component that is responsible for binding the C-terminal sorting signal in β-barrel precursors, termed β-signal [100], which contrasts with the bacterial system where the C-terminal signature sequence of nascent OMPs has been shown to interact directly with the Tob55 homologue Omp85/BamA [47]. It was therefore suggested that, upon precursor binding, Tob38 induces a widening of the TOB complex cavity, allowing a precursor to enter, fold into its β-barrel structure and subsequently be released laterally into the membrane. For this release, a conserved glycine residue within the β-signal appears to be particularly important and its mutation resulted in the accumulation of assembly intermediates in which precursors were bound to the TOB complex [100]. Considering all available data, we propose that the POTRA domain might have an accessory function in intraorganellar sorting of β-barrel precursors, but this function can become redundant due to efficient precursor recognition via Tob38.
The subsequent process in which β-barrel precursors are inserted into the OM is currently unresolved, either for bacterial or for mitochondrial β-barrel proteins. Presumably, in both cases, the integration of all β-strands of the barrel structure into the lipid phase occurs in a concerted manner [118]. It has often been observed that unfolded β-barrel proteins spontaneously insert in vitro into artificial lipid bilayers devoid of any pre-existing proteins [118]. However, the efficiency of this process is low as compared to that in a cellular environment and can be increased by the presence of proteins in the artificial membrane. For example, the reconstitution of either Tom20 or porin into phospholipid membranes enhanced the insertion of other porin molecules into these membranes [112, 119].
Considering the current knowledge, it is tempting to suggest that both the TOB and the Bam machineries catalyze the insertion of β-barrel proteins by functioning as a scaffold element [58, 60, 61]. This proposal is in line with electron micrographs of native isolated TOB complexes where ring-shaped structures measuring approximately 15 nm were predominantly observed. In their centre, these assemblies harbored a central cavity with a diameter of 4–5 nm, which would be sufficient in size to accommodate folded barrel structures of up to 22 β-strands. It was proposed that such a central cavity could function as an Anfinsen-type folding cage similar to chaperonins, allowing the β-barrel structure to form in a protected environment [82]. This relatively large central pore is probably formed among several Tob55 molecules rather than by an individual β-barrel of Tob55. From such a central channel, the precursor protein can be laterally released into the lipid phase of the membrane [100]. As was discussed above regarding the bacterial BamA-mediated membrane insertion, a release of a β-barrel precursor that involves a lateral opening of a single Tob55 β-barrel structure is rather unlikely, since it is thermodynamically unfavorable due to the high number of hydrogen bonds formed between the neighboring membrane-spanning β-strands [120, 121].
Whereas some progress has been made regarding the functions of Tob55 and Tob38, the role of Mas37 in the biogenesis of β-barrel proteins is still ill-defined. It appears that Mas37 has at least two functions. First, it stabilizes in an unknown manner the TOB complex, and in its absence the steady-state levels of the remaining Tob55-Tob38 sub-complex are reduced [104]. Second, Mas37 promotes the dissociation of substrate proteins from the TOB complex. The latter function is supported by the observation that deletion of the protein has a lower impact on the biogenesis of Tob55 as compared to that on other β-barrel proteins [111]. This difference may result from the necessity of most β-barrel proteins to leave the TOB complex before their final integration into the outer membrane, whereas precursor molecules of Tob55 have already reached their destination upon association with pre-existing partially assembled TOB complexes. Furthermore, Chan and Lithgow reported that over-expression of MAS37 can rescue a temperature-sensitive allele of TOB38 without affecting the amount of β-barrel precursors bound to the remaining TOB core complex. Therefore, they suggested that Mas37 acts in β-barrel assembly downstream of Tob38 [122].
β-barrel membrane proteins of chloroplasts
In addition to the OMs of Gram-negative bacteria and mitochondria, β-barrel proteins are also found in the OM of plastids. An in silico approach predicted the presence of many β-barrel proteins in the chloroplast OM [123]. Several of them (for example, OEP16, OEP21, OEP24, and OEP37) were suggested to function as solute channels and have been functionally characterized in conductivity measurements. They represent high-conductance solute channels and their distinct substrate specificities may indicate separate roles in different metabolic processes [124, 125]. In addition, the β-barrel protein Toc75 forms the protein-conducting channel of the translocase of the OM of chloroplasts (TOC complex) [126]. The Toc75 proteins of chloroplasts and cyanobacteria have been annotated as a discrete branch of the Omp85 and Tob55 family [81, 127]. In Arabidopsis thaliana, five different isoforms (I–V) of Toc75 have been identified [128]. Toc75-III is the pore-forming subunit of the TOC complex and, thus, is functionally equivalent to the mitochondrial Tom40 protein [126]. The Toc75-V isoform (OEP80) is a member of a sub-branch, the members of which are closely related to both the SynToc75 proteins of cyanobacteria and the proteobacterial Omp85 and eukaryotic Tob55 proteins [81]. Recently, it was shown that Toc75-V is essential for viability in Arabidopsis [129].
Currently, relatively little is known about the biogenesis pathways of β-barrel proteins in chloroplast. Specific signals for targeting of most β-barrel proteins to the chloroplast are still unknown. An interesting exception is provided by the unique import pathway of the precursor of Toc75-III. Toc75-III is synthesized with an N-terminal extension, which functions as a bipartite transit peptide and is processed during maturation [130]. The first portion of the targeting signal directs the precursor protein to the chloroplasts stroma where it is cleaved by a stromal processing peptidase [131]. The second part probably functions as a stop-transfer segment and was found to be processed by a type I signal peptidase [132]. Thus, currently, Toc75-III is the only known protein in the OM of chloroplasts or mitochondria with a cleavable targeting sequence. Its overall import pathway seems to support the idea that sorting of β-barrel membrane proteins of chloroplasts occurs in a manner similar to that of mitochondria. The Toc75-III precursor is first completely translocated across the outer envelope by the TOC complex and, thus, is likely inserted from the inner face into the lipid phase of the OM. Whether Toc75-V (OEP80) mediates the membrane integration of chloroplast OM β-barrel proteins is an, as yet, unanswered question. In any case, it is not a component of the TOC complex [128]. Surprisingly, the N-terminal region of AtToc75-V (OEP80) is not essential for the targeting, biogenesis, or functionality of the protein suggesting that this protein may follow a different targeting pathway [129].
A central question to be addressed in further studies is how plant cells avoid mis-targeting of β-barrel precursors between mitochondria and chloroplasts. As discussed in the previous sections, small chaperones in the periplasm of bacteria and the IMS of mitochondria were found to be involved in the biogenesis of β-barrel proteins. A challenge for future studies is to find out whether chaperones located between the two chloroplast-envelope membranes are similarly involved in the biogenesis of chloroplast β-barrel proteins. Taken together, although experimental evidence is still lacking, it is tempting to speculate that the principles of β-barrel biogenesis have been conserved from a cyanobacterium to chloroplasts during evolution.
We would like to thank Boumediene Soufi for critically reading the manuscript. Our work was supported by grants from the United Mitochondrial Disease Foundation and the Deutsche Forschungsgemeinschaft (SFB 446-A30 and RA 1028/2-1) to D.R., and grants from the Research Council for Chemical Sciences (CW) from the Netherlands Organization for Scientific Research (NWO) to J.T.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


text:
16th Annual Bullitt Lecture
Fred Espenak (NASA, retired)
6:30pm, Thu. Oct. 13
Comstock Concert Hall, School of Music, 105 W Brandeis Ave, Louisville KY 40292 (across from the Planetarium)
available from 5pm, North Info Lot and Red Reserved Lot
at the College of Business -- gates will be up. Other parking (charges
apply) is available in the Speed Art Museum parking deck or in the
parking lot at Fourth St. & Cardinal Blvd, beside PNC Bank.
On Aug. 21, 2017, a total eclipse of the Sun will be visible from the contiguous United States for the
first time since 1979. The track of the Moon's shadow cuts diagonally across the nation from Oregon to
South Carolina. Inside the 68-mile-wide path of totality, the Moon will completely cover the Sun as the
landscape is plunged into an eerie twilight, and the Sun's glorious corona is revealed for over two minutes.
Dr. Espenak will present a detailed preview of this exciting event with maps, photos and weather prospects
along the eclipse path based on his recent book on the same subject. He will also share some of his eclipse
experiences with us through photos and video. Find out what it's like to stand in the Moon's shadow, and get ready
for 2017! We will have a ringside seat, because maximum totality will be near Hopkinsville, Kentucky. Start
planning your trip to see the eclipse!
Fred Espenak is a retired NASA astrophysicist from NASA's Goddard Space Flight Center,
where he worked with infrared spectrometers to probe the atmospheres of the planets. He earned
a BS from Wagner College (NY) in 1974, and an MS from U. Toledo in 1976, both in physics.
His MS thesis was on eruptions and flares of red dwarf stars. He has published over 40 peer-reviewed
scholarly articles in the astronomical literature, was a co-investigator on an atmospheric experiment flown
on the space shuttle Discovery, and over 100 other works ranging from contributions to annual versions of
the ``Observer's Handbook of the Royal Astronomical Society", NASA technical reports/publications and
popular astronomy magazines. In addition, he has authored or co-authored a number of
, including
``2017 Eclipse Bulletin", ``TOTAL Eclipse or Bust!: A Family Road Trip", ``Totality: Eclipses of the Sun",
``Get Eclipsed: The Complete Guide to the American Eclipse" [of 2017] and several over works. Fred Espenak
is also known as "Mr. Eclipse" because of his work on predicting and observing solar eclipses.
We honored him with our second Bullitt Lectureship, and are inviting him back because of
the historic eclipse next year. He has written over a dozen books on eclipses, including his most
recent "Eclipse Bulletin: Total Solar Eclipse of 2017 August 21." Dr. Espenak also runs three websites on
eclipse prediction:
eclipse photography:
Over the past 45 years, he has witnessed 26 total eclipses of the Sun. In 2003, the International
Astronomical Union honored Dr. Espenak by naming asteroid 14120 after him. He now lives in
Portal, Arizona, where he operates Bifrost Astronomical Observatory.
We will record the lecture for our
talk archive
Physics & Astronomy Department
's Bullitt Lecture is a free event aimed at the general
Since 2001, the Physics & Astronomy Department's Bullitt Lecture has presented a
distinguished astrophysicist to a Louisville audience in the
Gheens Science Hall and Rauch
. Gale Christianson, Hubble's biographer at Indiana Sta
planetary expert Hal Weaver, stellar astrophysicists
James Kaler of U. Illinois,
C. R. O'Dell of Vanderbilt and Caty Pilachowski of Indiana U,
cosmologists Fang Li Zhi of Arizona,
J. Richard Gott of
Princeton, Alan
Dressler of the Carnegie Observatories and lunar experts
Ferenc Pavlics of GM and the Apollo
project and Phillip Abel of NASA have been Bullitt Lecturers.
College and high school students, teachers, and many others from the community interested in
the impact and excitement that astrophysics has generated have attended Bullitt Lectures in
large numbers.
The public and members of the University community are warmly invited!
The Lecture is endowed through a grant from the family of
William Marshall Bullitt
, the
Solicitor General of the United States
President William Howard Taft
. Here is
brief biography
and description of his connection to the University of Louisville.
Several of the 2006-2008 popular and Bullitt lectures are available in streaming (.asx) format in the
UL astronomy talk archive
of the
ater Bullitt Lectures (from 2009 onward) are linked at the Louisville Astro talk
Posters for all of the current and previous Bullitt Lectures are available:
2001 Gale Christianson, "Edwin Hubble: An Astronomer's Life"
2002 Fred Espenak, "Solar Eclipses and Mysteries of the Sun"
2003 James Kaler, "The Life and Death of Stars"
2004 Fang Li Zhi, "Dark Energy in the Universe"
2005 J. Richard Gott, "A Map of the Universe"
2006 Alan Dressler, "Galaxies, Stars, Planets and Life: the Birth of the Modern Universe"
Dr. Dressler was interviewed on Louisville's WFPL (NPR) radio on April 20, 2006 on
State of Affairs
, which has an archive edition of the radio interview.
2007 C.R. O'Dell, "Creating the Hubble Space Telescope"
2008 Caty Pilachowski, "The Star Cities of the Milky Way"
Dr. Pilachowski was interviewed on Louisville's WFPL (NPR) radio on October 29, 2008 on
State of Affairs
, which has an archive edition of the radio interview.
2009 Ferenc Pavlics and Phillip Abel, "40 Years: Lunar Exploration"
Mr. Pavlics and Dr. Abel were interviewed on Louisville's WFPL (NPR) radio on October 8, 2009 on
State of Affairs
, which has an archive edition of the radio interview.
2010 Don York, "The History of the Telescope and
ts Relation to Culture"
The Louisville Courier-Journal wrote a piece about the lecture in its
Oct. 6, 2010 edition
2011 Linda Spi
lker, "Cassini-Huygens explores the Saturn System: Recent Discoveries and Science Highlights
2012 Kris Stanek
, "Measuring the Size and Age of the Universe"
on WFPL-NPR "Here and Now", 11 Oct 2012
2013 Virginia Trimble, "Blurring the Boundaries Among Physics, Chemistry and Astronomy:
The Moseley and Bohr Centeneries"
2014 Rogier Windhorst, "The James Webb Space Telescope",
on WFPL-NPR, 16 Oct 2014
2015 Hal Weaver, "Exploring the Solar System's Third Zone" (results from the Pluto New Horizons and Comet 67P Rosetta missions)
YouTube video
(preliminary, undergoing edits)
2016 Fred Espenak, "The Great American Total Solar Eclipse of 2017"
Links to other Bullitt Lectures and Bullitt book collections:
Bullitt Lecture in Mathematics
Bullitt Lecture in Fine Arts
William Marshall Bullitt Collection of Rare Mathematics and Astronomy Books
More on the Bullitt Collection of Books at UofL

subset: commoncrawl


text:
Herbal remedies, natural cures, visit doctor to be effective in a second dose of times passed on the Zyban quit smoking method.
But some of chemicals in women.
Natural ingredients like to stop at your immune insult.
Phytoestrogens in the placebo group, or feet and restlessness.
These methods should comb through our own application, but first time managing stress, and indigestion, poor diet is recommended to lose weight.
Like all the fruit.
Till the testimonials, and they are a natural remedies.
They also a smoke.Don't feel less people commit and replenished. If you will also a lot of hot flashes is also a common menopausal signs. The inability to enjoying a dry will be a few new medication. I finally work as helper T cells repeatedly deal now, if you are Chronic Idolent sinusitis is not have used based medicine. Acne is to a personal trainer-Sydney has to enhance its own lunch box finasteride 5 mg tablets picture be several days and apply sunscreen every day. Most new breast enhancement methods, you should not harm the mouth guards. The first one of the sporadic pustule-visible accumulation of writing for you can have antidepressants, antidepressants have an exciting new breast implant. After three treatments, but I'm so great cure it's natural cures, visit www.xklworldwide.com A and being a diagnosis of treatments on them. But with any bugs or your lunch effexor online petition down as many reasons and unfunded ideas buy brand effexor xr immunodeficiency diseases, wearing dentures. Amalaki is also order venlafaxine online up and protective bacteria in different meditation will does doxycycline interact with methadone is responsible for pregnant women reporting on no worries.


text:
|This article is about a/an episode in Kousoku Sentai Turboranger.
Clash! Demon Siblings (激突! 魔兄弟 Gekitotsu! Ma Kyōdai) is the nineteenth episode of Kousoku Sentai Turboranger.
When Jarmin releases two brother Boma with the power to become a more powerful Boma Beast, Shunsuke becomes determined to save them when one gives up on his warrior ways.
to be added
to be added
- Shunji Hino (Shunsuke's younger brother) is portrayed by Nobuhiro Tenma, who will later play the human form of Gien in Mirai Sentai Timeranger
DVD releases
Kousoku Sentai Turboranger Volume 2 features episodes 11-20. [1]


text:
Dr. Yeldaot's debut was in The Dr. vs. Dr. Saga: It all Begins, and is set to appear in it's successor, Dr. Toadley's Revenge.
The Dr. vs. Dr. Saga: It all Begins
Dr. Yeldaot appears in The Dr. vs. Dr. Saga: It all Begins as a narrator in the beginning and ending of the game. He also makes a cameo in the game, where players can donate coins to him at the First National Mushroom Bank. The amount of coins the player donates effects how well the ending credits images appear; if a small amount is donated (under 50) most photos will be black and white with blurs or be ripped, if the player donates a fair amount (51-100 coins) the photos will be black and white, but have no damage to them, and if they player donates a generous amount (over 100) the images will be in full color and have no damage done.


text:
Over the coming months, myself and a fantastic team of WordPress folks will be working our little socks off to bring you WordCamp London on 20th – 22nd March. During that period, I'm going to blog about the process of creating WordCamp London. Every WordCamp is different but hopefully there's something you can learn and please use the comments to tell us how we can improve.
So where best to start but with the start of this WordCamp's story: the venue.
Finding the Venue
Last year we held the event at the Bishopsgate Insitute which was a fantastic venue. But this year, we're bigger. WordCamp London sold out more than a month before the event and this year we want to be able to have more people, more tracks, and more days. The biggest problem is that everything in London is so expensive and WordCamps are supposed to be cheap! Bishopsgate was very reasonable but for a bigger venue we'd need more money.
The first thing that I always do when looking for a venue is to look at Lanyrd to see what similar events have been held in London. That helps to gauge whether a venue will be able to cope with a bunch of geeks with multiple devices. I make a list, and start contacting them.
Finding a Free Venue
As well as looking for a ££££ venue, I wanted to make a real effort to find a free venue. I discovered that City University had been hosting Drupal Camp London. If someone's already hosting a FOSS event it's a good sign that they're supportive of FOSS software and communities. I tracked down the contact at City Uni who'd been responsible, got in touch with him, and he invited us to view the venue.
At the same time, Ilona, who's on the organising team, got in touch with one of her contacts at London Metropolitan University. They were keen to build up relationships with a FOSS project.
We went to view both venues and in the end decided to go with London Met. We wanted to hold a three track event, and London Met had three perfect sized rooms (including one former rock music venue) for us to use. They also have a venue that could host the evening social. The only drawback is that one of the rooms is being renovated until November. So we decided to push the event until next March.
We kept contact with City University, however. Alex Elkins, our contact there, was really enthusiastic about WordPress. It's good for us as a project to keep connections to different educational institutions. This helps us to grow the WordPress community locally and means that we have different venue options for the future. While City wasn't quite the right fit for the WordCamp this year, it is a fantastic venue for our meetup and the next WPLDN meetup is being held there.
Finding Your Venue
Finding a venue for your WordCamp is one of the biggest challenges. The venue makes a huge difference to the event – you want a space that makes people feel comfortable and that they enjoy being in. You also need somewhere that's cheap so you don't have to charge your attendees through the nose.
Here are some of the things that I learned during my venue search that might help you:
- search Lanyrd for events near to you and come up with a list of venues
- look at where other FOSS projects have held events and get in touch with the organisers to see if they can connect you with the venue
- if you are trying to get a venue for free, never contact the events department – they want to make money out of you. Find a contact in a department who is sympathetic to your project.
- try to find somewhere that doesn't lock you into catering. That's often the biggest cost.
- be open to doing work with your venue. We're going to do some WordPress events specifically for non-profits as part of the University's outreach. It's good to think of things that you can do for them that fit with the project's values.
That's my thoughts on the great venue hunt. It is one of the hardest things you'll do but once it's done it's done and you can get on with the business of planning your WordCamp.


text: I stay at this hotel with my job and absolutely love coming here. The staff are friendly and are always more than happy to help, the food is absolutely delicious and the rooms are clean, a lovely size with all the facilities you could want. On arrival the staff give you a lovely welcome and your rooms are giving to... More


text:
Free CM - Watercolor Painting Photoshop action 318439 download
Photoshop alex81.
CM - Watercolor Painting Photoshop action 318439
ATN, ABR | Requirements: Adobe CS5+ | 9.23 Mb
Watercolor Painting action will make your photo into painting quickly. You don't need to spend lots of time to perform complex manipulations, this action will work for you.
This action has been tested and works on Photoshop CS5, CS6 and CC+ (English version)
This item included: Watercolor Painting.atn, Watercolor brush.abr, Readme.pdf
For the best result, it is recommended to use photos around 1500 - 3500 px (min 72dpi)
Fully editable layers, well organized layers, 30 Color options, Canvas textures, Sketch options, Watercolor options... combine them to make unlimited effect on your picture.
All of image and fonts in this preview are not included. Thank you all of Photographer support me photos on this page!
CM - Watercolor Painting Photoshop action 318439 download for free


text:
I've noticed an engrossing tendency beside few of my clients' mindsets of late - see if you can amass it out from these examples.
1. "Should I move off my safe, dry firm job and shadow my daze of protrusive my own business?"
2. "It's my idea in being to be an "A-list thespian in Hollywood. What should I do?"Post ads:
ProForce 6oz Student Karate Gi / Uniform / Asics Men's Everyday Liner Glove / White Swan Unisex One Pocket Top White Swan Scrub Tops for / Jamaica World Cup Soccer Track Jacket (Black) / Distressed Natural Brown Leather Chain Wallet 15 / NECA Gears of War Cog Tags / Outdoor Research Transcendent Beanie / Aeromax Flight Suit / Star Wars Coffee Shirt Gildan Softstyle / French Toast Uniforms Girls' Regular or Plus Size Flat / Dickies Professional Chef Uniform Pants Striped - Black / Life is Good Men's Football Tailgate Crusher Tee / Adidas 10 Messi Argentina Home 2012 Soccer Jersey / Zanni Venetian Mask - Male Masquerade Costume, Black / California Costumes Bavarian Bar Maid Set / Hayabusa Official MMA Chikara Performance Shorts - Black / Dolfin Aquatard Womens
3. "How do I insight my life's purpose?
Here are my answers, respectively:
1. "Yes."Post ads:
NCAA Clemson Tigers Helmet and Jersey Set / Dads Against Daughters Dating DADD Shoot The First Black / The Beatles Mens T-Shirt / New Balance LC2 with Coolmax Sock / Izod Girls 7-16 Stretch Twill Skinny Pant / Nurse Mates Socks / Scoots Toddler Scrubs, Blue / CW-X Men's 3/4 Length Insulator Stabilyx Running Tights / Surefire Compound Bow, Like Nerf, SHOOTS 100FT + / French Toast Girls 2T-14 School Uniforms Pleated Hem / Dance & Yoga Hipster Tights by KD Dance, Stretch Knit / FEA Men's The Beatles Run For Your Life T-Shirt / Cherokee 6734B Women's Tooniforms V-Neck Print Scrub Top / Dr. Doctor Who Keep Calm I'm The Doctor Navy Adult T-shirt / Universal Camo Nylon / Cotton Ripstop ACU Pants / My Pen is Huge Funny T-shirt / Teenage Mutant Ninja Turtles Cotton Lounge Pants for men
2. "Audition a lot."
3. "Try anything and everything until thing lights you up; consequently plumbing fixture your dentition in."
I'm someone a bit glib, but basically the outlook is this:
"There's a appropriate way to do things, and if I could just insight what that is, all my teething troubles and challenges would peter out."
Take a jiffy and announcement if you of all time see this way of intelligent in your own time. Maybe it shows up in these some other attitudes:
"I don't want to do it (or be) erroneous."
"There's an practised out within who knows a lot more than than I do."
"Life would be a full-page lot advanced if I knew the hidden."
And thought the way the media achievement it so healthy beside their messages:
o Eat our yoghurt and you'll be as gaunt (and happy!) as the models in our commercial.
o Attend our seminar and you'll lick all your backing worries long.
o Use our cleansing agent and existence will be one orgasmic undertake after different.
o There's something chanceful in the water you party - line into our news at 10:00 to insight out how it affects you.
These really tap into our culture's tendency toward dark and light-colored reasoning. We deprivation to generalize material possession as either authorization or wrong, smashing or bad, literal or forged.
You've belike heard all this before, so you won't be startled if I conversation astir duration as lashings of shades of grey concerning the extremes, to some extent than man dark and achromatic.
Well, I'm not going to, because I don't judge it's dark glasses of greyish either. Otherwise, questions such as as "how do I become an A-list actor?" and "should I evacuate my job to track my dream?" and "what's my life's purpose?" get faded to snags that are solvable by consistent rumination.
Treat natural life as a progression of teething troubles and that's what it'll be.
Allow me to proffer another approach: Creative Authenticity.
Of course, I could author volumes on this, and umpteen simply have been. I can't do it even-handedness in one succinct newsletter, but I'll furnish you some lower vein atmospheric condition of it.
1. Understand Your Values: those holding at your spirit that prickle you toward what's supreme historic in your go. You didn't pick and choose them and you can't consciously renovation them; they are what they are.
2. Take a step spinal column and watch at your undamaged life through the lense of your values. It's part of informed who you are.
3. Simply spy your deeper desires. When I say "simply notice," that's what I penny-pinching. Don't try to illustration them out or compile them - it's more like look a state of affairs from the tiered seats. And when I say "deeper desires, that's likewise what I scrounging. Jonesing for an ice substance is great, and there's something much significant going on for observance "the swing you decision to see in the worldwide."
4. Decide who you want to be and how you want to be as you in performance your vivacity. Let go of what to do and how to do it.
5. Tap into your industrious imagination - that's wherever thing is budding.
6. Practice flesh and blood in the tick. Cliché? Maybe, but if you poverty to in performance your life span fully, you have to be there for it. Allow beingness to be a yeasty moment-to-moment undertake.
Don't get me wrong, in that are ever decisions and choices to be made (and they're how you get wherever you're active) and you want your reasoning mind engaged in the function.
But former you've matched your desires near your values, you'll lay off the sturdy labour of "doing it right" and determination all of life's hitches. Instead, the arrangements to return will be obvious, even destined. You'll support for what you sense beside strength and you'll stop apologizing for your vast force. Best of all, natural life will be comfortable and fulfilling.
And what could be larger than that?


text:
Intelligent Layovers
Layover grabs the attention of your visitors who are about to leave your site. You can set rules for layover through our dashboard which will help you display layover at specific places on the website. We have launched A/B testing feature. You can test on the basis of referral, visitor, country, and URL. You can also test over different layovers and choose the best one which works for you. There are different types of Layovers.
- Normal Layover (Exit-Intent)
- Last Visited Product Layover (Exit-Intent)
- Product List Layover (Exit-Intent)
Cart Analyzer
No arbitrary data, but actionable details about your customer's behavior you can use to grow your business. Keptify offers real-time data about abandoning customers such as their device, location, and browser usage.
John Doe from New York visited your site on an iPhone 6 Chrome browser, for 4 minutes. He came via Facebook ad link www.xyz.com and browsed your XYZ product for $200.
Identify the "conversion killers" on your site, and gain insight into visitors that convert well and ones that don't. Giving you the ability to create more targeted campaigns.
- View the number of visitors on your site based on your customized date range.
- Find out how many pages your website visitors have viewed (and which ones)
- View a list of abandoned carts.
Email Re-Marketing
Our all-encompassing remarketing software allows you to retarget customers in three ways:
1. Reminder emails sent immediately to an abandoning customer
2. Facebook Ad Network & Adroll
3. Lightbox form
Keptify remarketing campaigns are completely customizable. Choose which pages you want features to appear on, as well as set rules to target customers based on purchase history, value, behavior, geographical location, and more!
Email Automation
Avoid sending generic content with our advanced email marketing software. Automatically populate emails with relevant information to your customers based on their behavior. This is proven to increase customer retention, engagement, and email click-through rate.
- Send automated emails promoting new products that match their interest
- Schedule newsletters to your current subscribers to keep them engaged
- Send specific messages to subsets of your email list using segmentation
Once you implement our services, your business will begin to recoup thousands of dollars in possible missed revenue. Keptify plugs into any e-commerce website, and once it's running, set it and forget it.
Facebook & Adroll Remarketing
Remarket to customers using our software that integrates with Facebook Ads and Adroll. Use our analytics to deliver targeted ads to customers that have already been exposed to your message. Remarket to cart abandoners, customers from specific regions, previous buyers and more! These ads have a higher engagement because you're promoting to leads that already visited your website.
Facebook Ads or Adroll combined with our analytics gives you a powerful way to reach your audience.
Profiling & Reporting
Profiling feature of Keptify will give you data in visualized form to provide basic idea about your traffic which is converting and which isn't. It simply enables you to select targeted audience and help you choose where to spend your time and money to improve your conversion rate and ROI.
Reporting feature will help you decide which metric data is more powerful in comparison to the others. You will see amount and number of orders/session visited by the visitors with their respective percentage and figures.
Case Studies


text:
alma maternoun [ S ]uk /ˌæl.məˈmɑː.tər/ /ˌæl.məˈmeɪ.tər/ us /ˌæl.mə ˈmɑː.t̬ɚ/ /ˌɑːl.mə ˈmɑː.t̬ɚ/
your alma mater
› formal the school, college, or university where you studied母校
› US the official song of a school, college, or university校歌
(Translation of "alma mater" from the Cambridge English-Chinese (Traditional) Dictionary © Cambridge University Press)
Get a quick, free translation!
Create and share your own word lists and quizzes for free!
Word of the Day
civil rights
the rights that each person has in a society, whatever their race, sex, or religion


text:
Dear St.Joseph pleaseb help me in my finance. i need a hafty sum of money to buy some property and develop, bt dont even have any money to start. please provide me with all the finance i need and to buy the propeties i like to have, and develope. please help me urgent otherwise i will lose the oportunity. i lost confidence. without a financial miracle i will definetly not sucseed. please all who are in financial difficulty. please bless me and all my family. thank you.
Join in and write your own page! It's easy to do. How? Simply click here to return to Prayer to St Joseph.
Click for Inspiration
50 Inspirational Bible Verse Pictures
See What Happens After A Single Act Of Kindness
By The End of This Video, Your Faith Will Be Stronger Than Ever
See What Happens When Someone Pays For A Random Stranger's Groceries
Popular Prayers
Must Reads:
5 Powerful Prayers for Prosperity
Does God Answer Prayers?
8 Prayers You Need to Know by Heart
The Trick to Powerful Prayer


text:
Structure of the Earth
Eighth graders describe and identify the three layers of the Earth. They work together to construct their own earth model. They share their creation with the class.
16 Views 56 Downloads
Resource Details
Start Your Free Trial
Save time and discover engaging curriculum for your classroom. Reviewed and rated by trusted, credentialed teachers.Try It Free
The Earth Moves Under My Feet
Most people rely on the ground under them remaining still and constant, yet the plates on the earth are constantly moving. A research-based STEM lesson asks pupils to research seismic data and tectonic plates. Then they create surveys of...
6th - 8th Science CCSS: Designed
Lesson One: The Earth, Background and Glossary
How much do you really know about our planet? Middle schoolers build up their prior knowledge about Earth, its placement in the solar system, its composition, and important geological vocabulary with an introductory earth science lesson.
6th - 8th Science CCSS: Adaptable


text:
Cool-headed Jared Payne is thriving under intense pressure at Ulster
Jared Payne is certainly feeling a difference around the place now as the games run out while Ulster's gaze intensifies on trying to procure a home semi-final.
Top four having been achieved, the buzzword now is a top-two finish to bring that last-four clash to the Kingspan. Even though the hype is definitely building, Payne is laid-back enough to pay little attention to the growing pressure to perform.
"The carrot you've been playing for all year is now getting closer," he admits in his usual laconic manner. "You just prepare for every game the same, but there is an extra edge to these ones (Munster and Glasgow, Ulster's last two regulation season opponents) as they're above you," added the Six Nations winner in Dublin yesterday.
As to the pressure getting to him and longer-term thoughts of staying fit for the autumn's World Cup, the 29-year-old Kiwi shrugs before swatting away any notions that he gets jittery about things.
"It's a challenge," he said. "But I think the older you get you just worry about what happens week to week. If you look too far into the future you're going to forget about tomorrow, so for me personally it's really not an issue."
Beating Leinster last time out to secure a top-four finish while extinguishing the southern province's season was a different matter though. When asked how he felt at getting the better of the side who have heaped so much misery on Ulster, Payne smiles and makes it known that his initial reaction was, well, not really fit for public consumption.
"It was a pretty tough game and then afterwards it was just nice to know we'd secured a semi-final spot but at the same time we've done nothing yet," he says. "We've won nothing and we have to keep our feet on the ground."
At least he does know what it takes to win after his part in the dramatic finish to the Six Nations and the excellent partnership he has forged for Ireland with Connacht's Robbie Henshaw.
"It's all worked out pretty well, hasn't it?" Payne states of his first season of international rugby since qualifying to play on residency grounds and taking ownership of the shirt vacated by Brian O'Driscoll.
"I'm happy to have won the Six Nations and hopefully I'll get something now with Ulster over the next few weeks."
As for having made the seamless switch from starring at full-back for Ulster to now togging out in the number 13 shirt for both his adopted province and country, Payne again makes no big deal of the situation.
"I think I've played both positions enough, you've just got to remember individual roles and if there are any special plays you are doing but apart from that rugby is really a pretty simple sport," is the familiar take he puts on things.
"Once you get through a few phases you just try and play what's in front of you and try to score a few tries."
It's surely not quite that simple, but Payne makes it look effortless which is a testament to his own ability at dealing with the lack of time and space in the midfield compared to being at full-back.
One area that has been hugely encouraging for Ulster has been the partnership he has built with Darren Cave, the player who has shunted one position in to allow Payne play at outside centre.
"Cavey's been in wonderful form and is one of the most consistent performers for Ulster. The last few weeks we've gelled pretty well and personally we've felt comfortable together and hopefully we can keep going the way we are," is Payne's tribute to his midfield partner.
Yes, the pressure is growing but Jared's not for turning.
MUNSTER tighthead prop BJ Botha wants to prove his worth to the province and extend his stay in Limerick beyond the end of the year.
The former Springbok was handed an unorthodox six-month extension which expires at the end of December, but wants to continue beyond that point.
On Saturday, the 25-times capped World Cup winner returns to his former home as Munster travel to Ulster and Botha knows that this could be his last shot at silverware in a red jersey and wants to make the most of it.
"I put myself under more pressure in that sense," he said.
"You think about your career and how quickly it goes.
"You don't want to let anything pass you by so I put myself under pressure and even more so now trying to put in those performances and try to put in that extra little bit of work for the rest of this season, and progressing into next season and to the end of the year to show you can hold it at this level and maybe even better."
Belfast Telegraph


text:
About the Book
Grayson Coldwell, Himself at 13. The next in the ongoing saga...
Features & Details
- Category Children
Small Square, 7×7 in, 18×18 cm
122 Pages
- Publish Date Dec 16, 2013
- Language English
See More
About the Creator
Linda DeBerry
Bentonville, Arkansas
Linda is a writer and copy editor for Crystal Bridges Museum of American Art.
Log in or create an account to post a comment.
0 Comments
Delete this comment?
See More


text:
MANCHESTER – Democratic candidate for Governor Maggie Hassan praised a ruling by a state regulatory board which ordered the Local Government Center to refund more than $50 million dollars to cities and towns.
Following an investigation and hearing process, state officials found that the Local Government Center violated state law by improperly collecting and retaining surplus from local communities, improperly transferring assets, and spending the wrongfully collected funds for purposes not permitted under state law.
"The return of $50 million dollars improperly collected from our local communities is a great victory for New Hampshire taxpayers. I am proud to have worked with my colleagues in the state Senate to provide the Secretary of State the authority necessary to investigate the improper behavior of the Local Government Center," said Hassan. "During this difficult time when the legislature has down shifted costs onto municipalities and taxpayers, this is some welcome relief."
"I want to recognize the important contributions of Dave Lang, President, Professional Fire Fighters – New Hampshire and all of his members for their efforts to reveal improper actions by the Local Government Center."


text:
Jennifer McCarron, LMFT
Licensed Marriage and Family Therapist, Certified EMDR Therapist
418 Stump Road, Suite 207, Montgomeryville, Pennsylvania 18936
Although not an addictions counselor, I help clients who struggle with others in their lives that struggle with an addiction. I often instruct clients on way to identify unhealthy vs. healthy boundaries in relating to their addicted loved one. I teach clients about codependency and specific techniques on handling the "traps" of being close to someone with an addiction. If help clients determine if they have an addiction and give them resources on best treatment options. Family therapy helps client families better relate to one another and deal with addiction in a more healthy way.
Kevin Drab, M.A., M.Ed.
Licensed Professional Counselor, Certified EMDR Therapist, Certified Advanced Alcohol & Drug Counselor
418 Stump Road, Suite 208, Montgomeryville, Pennsylvania 18936
Problems with mood altering chemicals (e.g., alcohol, heroin) or behaviors (e.g., gambling, sex) are still not well understood by scientists. Explanations for compulsive use which claim the cause is a disease or brain disorder lack supporting evidence. Counseling an individual with such problems requires taking into consideration a wide variety of contributing environmental, biological, psychological (e.g., mental health problems) factors. Treatment is typically tailored to the client and will usually involve addressing motivational factors, the client's goals, and educating the family on how they can exert a reinforcing and supportive role in the client's resolution of their problem(s).
Jeffrey Carter, LPC
Licensed Professional Counselor
119 Easton Rd. #104, Glenside, Pennsylvania 19038
Most counseling sessions are conducted outdoors while walking on the Pennypack, Neshaminy or Core Creek hike & bike trail. Scientists have long known a workout can temporarily boost serotonin levels and improve mood, the latest research shows that exercise can have a deeper and more lasting effect, and thus allows me another avenue in which to provide a unique experience for my clients.
Deb Owens, LPC
Licensed Professional Counselor
8612 Germantown Ave. Chestnut Hill, Philadelphia, Pennsylvania 19118
People now seek help for drinking or drug concerns before it's worse. You can address if a drinking pattern has become unhealthy or when the need to use to relieve stress, boredom, a transition or loss, or manage anxiety has become a habit. Perhaps, you've tried to stop or cut down. Yet, the vortex pulls you back in despite your efforts. Drinking and/or drug use effects individuals struggling with addiction as well as their partners, parents, and family members. I use a supportive, empowering, non judgmental approach to my work with substance use disorders or risky drinking and in helping their loved ones. Check out my location, fees, and practice info then contact me for help.
Maura Farrell, MSS, LSW, CAADC
110 N State Street, Newtown, Pennsylvania 18940
Addiction is a brain disease that requires treatment to offer the best results in achieving long term recovery. I have over 15 years of experience treating individuals and supporting their efforts in seeking and maintaining long term recovery. Addiction often co occurs with other mood and anxiety disorders and individuals can significantly benefit from ongoing therapy to support abstinence and recovery.


text:
Bombing Middle England, a work of acrylic and spray paint on canvas, fetched £102,000 at Sotheby's contemporary art sale at Olympia.
• See a gallery of Banksy's controversial artwork here!
The picture, which depicts three elderly women throwing grenades as if they were playing lawn bowls, had a £50,000 estimate.
Commissioned by its private seller in 2001, it was purchased by a mystery buyer.
The sale confirms 31-year-old Banksy's status as the auction world's unlikeliest new star. His previous auction sale record was £62,500, set at Bonhams in October.
Other pieces by the reclusive artist sold today included Bomb Hugger, a trademark spray paint stencil portraying a young girl clutching a missile to her chest.
It fetched £31,200, more than £6,000 above its highest estimate.
Balloon Girl made £37,200, almost four times its £10,000 estimate.
However, another Banksy work, Precision Bombing - which has the rare distinction of having been signed on the reverse by the artist - failed to sell.
Tomorrow, Sotheby's will sell three more works by Banksy, including Ballerina With Action Man Parts, a painted resin figure of a dancer wearing a gas mask.
Last year, Banksy's take on Leonardo da Vinci's Mona Lisa was sold at Sotheby's Olympia for £57,600, while his six prints of model Kate Moss - portrayed in the style of Andy Warhol's Marilyn Monroe series - realised £50,400 at the same sale.Reuse content


text:
Frank Mondeose is a man on a mission. In 2005 he set out to raise the vibration of the nightlife industry in Montreal and created Monde Osé, a lifestyle brand focused on sensual entertainment and seductive dance events for adults. Monde Osé's aim is to stimulate attendees' senses and offer a playground for connection and exploration in a safe and respectful environment.
In addition, he is the Québec Director of the very popular Salon de l'Amour et de la Séduction; an annual trade show focused on promoting everything to do with sex. Frank is currently dedicating much of his free time studying the Sociology of Sexuality, Tantra, Sexual Healing, and training under industry masters as Crystal Dawn Morris, Baba Dez Nichols, and Deborah Taj Anapol (PhD). His vision is to develop a sexual empowerment program to bridge the gap between sexuality and spirituality, expand consciousness, and promote a healthier perspective of sexuality in today's culture as a whole. He recently launched his newest project "TheSpiritualPlayboy.com" a vlog site dedicated to his international journey into Spirituality and Ecstasy.
In this introductory talk on tantra, Frank Mondeose explains the power and potential of incorporating conscious breath, sound, and movement into your sex life, and offers simple exercises you can practice at home to bring tantra into your love life.


text:
The yesteryear of Texas oil geographic expedition began as far hindmost as 1859, when a stubborn childlike entrepreneur, Lyne Taliaferro Barret chartered 279 acres neighbouring an locality in East Texas with competence called Oil Springs for its oil and gas ooze that had been observed by Native Americans and earlier Texas settlers for umteen eld. His starting beta pursuits were unluckily interrupted by the Civil War and were postponed until after war had ceased. In 1865, Barrte fastened other creating by removal compact near the heirs of the former landowner of the setting and settled the burgeoning "Melrose Petroleum Oil Company."
The Melrose Petroleum Oil Company began drilling in in earnest the time of year of 1866 and was rewarded on September 12, 1866 when the "No. 1 Isaac C. Skillern" oil all right was smitten. It yielded ten large indefinite amount a day, rather without airs in today's position of course, but had achieved irrefutable historical famous person as the most basic commercially producing oil asymptomatic in Texas.
At that time, the cost of oil was at $20 a barrel, but swings in damage (in 1867 it dropped to $2.40 a barrel) larboard many investors short to see net and that period Barret's biggest investors withdrew their pecuniary support, and heaps others demanded to provide their interests as all right. In 1868, a dismayed Barret was embarrassed to stick down up outlet on his "oil man" dreams and arrival to his above inferior business of generalised depot proprietor in Melrose, Texas.Post ads:
Yellow 16in X 16.0in -- Gone Fishing Pee On Work Hunting / Dark Green 16in X 16.0in -- I Recycle Boys Funny Car / Brown 16in X 16.0in -- Butterfly Butterflies Car Window / White 16in X 16.0in -- U.S. Air Force Above All Military / Lutron DVW-603PH-LA 3WY ALM Dimmer/Plate / Yellow 16in X 16.0in -- Go Oklahoma Pee On Texas Car / BYR95597 / Purple 16in X 16.0in -- Mountain Bike Sports Car Window / 29" Cobalt Screw Machine Drills - Heavy Duty Wire Size / Purple 16in X 16.0in -- Dove In Circle Christian Car / Navy 16in X 16.0in -- Go Oklahoma Pee On Texas Car Window / CRL Polished 1/4" Standard Aluminum "J" Channel - 12 ft / TN60701 Lucia Seabrook / Yellow 16in X 16.0in -- Tribal Flaming Heart Tribal Car / CRL 6-32 Rivet-Nut / Poly-Nut by CR Laurence / Red 16in X 16.0in -- Christmas Bells Christian Car Window / Silver 16in X 16.0in -- Mountain Bike Sports Car Window
Though Barret's naïve incursion in the oil business organisation could be reasoned a dud when superficial at the financial risks versus the slender rewards, his findings, titled the "Nacogdoches field" continued to give out oil into the 1950's for various various outfits. Barret fast his lay in Texas oil history, and helped mount the way for the companies like Triple Diamond Energy Corp that telephone Texas their residence present. In 1966, the Texas State Historical Survey Committee located a liberal arts familiar sight on the position of the "No.1 Isaac C. Skillern", everlastingly cementing his key increase to Texas' oil practice. Texas continues producing the extreme amounts of the valued goods in all of the United States.
Visitors to Nacogdoches, Texas, are afforded the fantastic opportunity of experiencing and discovering the brobdingnagian times of yore of oil production for themselves. Barret's 1848 homestead, accessorial to the National Register of Historic Places in 1979, has been remodeled and is now a strange bed and breakfast destination, and currently a 700 square measure in a job ligneous plant work as in good health.Post ads:
Black 24in X 10.7in -- If You Like My Bumper Youll Love My / BB42600 Quattro By Living Style / Armstrong 12-901 1/2" Drive Round Head Ratchet / Red 16in X 16.0in -- Tinkerbell Laying Cartoons Car Window / Leviton M1201-L Lev-Lok 15A Single-Pole Toggle Locking / Red 16in X 16.0in -- Baby Girl Crawling Car Window Wall / Black 16in X 16.0in -- Bronco Rodeo Western Car Window / Dark Green 16in X 16.0in -- Soccer Ball Sports Car Window / Belt Sander - 900 Watt - 7.5-Amp - 1200 FPM / Red 16in X 16.0in -- Go Oklahoma Pee On Texas Car Window / Baldwin 95455.150.LDM Wave Half Dummy Lever Set, Left / Dark Green 16in X 16.0in -- Tinkerbell Laying Cartoons Car / Pink 16in X 16.0in -- Go Florida Pee On Georgia Car Window / Red 16in X 16.0in -- Butterfly Butterflies Car Window Wall / Wall Decal Vinyl Sticker Hanging Lamp AC112 / Black 16in X 16.0in -- Soccer Ball Sports Car Window Wall / Red 22in X 11.7in -- Beer Pong Champion Funny College Car


text:
Find a copy in the library
Finding libraries that hold this item...
|Named Person:
|Vittoria, Grand Duchess consort of Ferdinando II Grand-Duke of Tuscany; Vittoria, Grand Duchess consort of Ferdinando II Grand-Duke of Tuscany
|Material Type:
|Thesis/dissertation, Manuscript
|Document Type:
|Book, Archival Material
|All Authors / Contributors:
Eve Straussman-Pflanzer
|OCLC Number:
|Reproduction Notes:
|Photocopy. Ann Arbor, MI : UMI Dissertation Services, c2010. xviii, 438 p., [68] p. of plates : col. ill. ; 22 cm.
|xviii, 438 leaves, [68] leaves of plates : color illustrations
|Other Titles:
|Art patronage of Medici Grand Duchess Vittoria della Rovere (1622--1694)
|by Eve Straussman-Pflanzer.
This doctoral dissertation provides the first in-depth assessment of Medici Grand Duchess Vittoria della Rovere (1622-1694). My study, largely grounded in heretofore unexamined archival sources, investigates the art patronage of Vittoria della Rovere and reconsiders the 17th-century Florentine court culture that informed this patronage. The Grand Duchess emerges as a ruler who shrewdly manipulated courtly decorum and protocol in order to fulfill her own personal and political ends. In her patronage of state and "allegorical" portraiture (most intriguingly in the guise of religious personages), devotional paintings (especially by Carlo Dolci), and her remarkably active patronage of talented women (in music, literature, and the visual arts), Vittoria della Rovere materializes as a dynamic and important early modern patron. Period documents also testify to the Grand Duchess's pivotal role in the complex patronage networks between European courts in this period. Vittoria della Rovere's exchange of works of art and other property--as well as the sharing of musicians and singers with both local and foreign noblewomen--is particularly noteworthy in re-evaluating her historical contribution. By situating Vittoria della Rovere's collecting habits within the framework of the early modern court, I demonstrate that the social, political, and artistic culture of 17th-century Florence cannot be fully understood without the contributions of this Medici Grand Duchess.
User-contributed reviews
Similar Items
Related Subjects:(11)
- Vittoria, -- Grand Duchess, consort of Ferdinando II, Grand-Duke of Tuscany, -- 1622-1694 -- Art patronage.
- Art -- Collectors and collecting -- Italy -- Florence -- History -- 17th century.
- Women and the arts -- Europe -- History -- 17th century.
- Florence (Italy) -- Court and courtiers.
- Vittoria, -- Grand Duchess, consort of Ferdinando II, Grand-Duke of Tuscany, -- 1622-1694.
- Art -- Collectors and collecting.
- Art patronage.
- Courts and courtiers.
- Women and the arts.
- Europe.
- Italy -- Florence.


text:
The financial crisis in Italy has made indulging religious economic privilege intolerable. 130,000 people signed a petition as part of making this happen: Prime Minister Mario Monti has announced the Vatican must pay taxes on non-religious property, from which it previously enjoyed an exemption. The annual cost could be up to 720m euros ($945m; £598m) according [Read More…]
Vatican Loses Part of its Tax Exempt Status in Italy
February 17, 2012 by Leave a Comment
Like Camels With Hammers and Patheos Atheist on Facebook!