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Mammals produce volatile odours that convey different types of societal information. In Homo sapiens, this is now recognised as body odour, a key chemical component of which is the sulphurous thioalcohol, 3-methyl-3-sulfanylhexan-1-ol (3M3SH). Volatile 3M3SH is produced in the underarm as a result of specific microbial activity, which act on the odourless dipeptide-containing malodour precursor molecule, S-Cys-Gly-3M3SH, secreted in the axilla (underarm) during colonisation. The mechanism by which these bacteria recognise S-Cys-Gly-3M3SH and produce body odour is still poorly understood. Here we report the structural and biochemical basis of bacterial transport of S-Cys-Gly-3M3SH by Staphylococcus hominis, which is converted to the sulphurous thioalcohol component 3M3SH in the bacterial cytoplasm, before being released into the environment. Knowledge of the molecular basis of precursor transport, essential for body odour formation, provides a novel opportunity to design specific inhibitors of malodour production in humans.https://doi.org/10.7554/eLife.34995.001
Human body odour contains a number of chemicals, but the most pungent and recognisable are thioalcohols. These molecules are created through a series of chemical reactions that start with an odourless precursor, a compound produced in glands located in our armpits. Then, a type of bacteria called Staphylococcus hominis takes in these molecules and transforms them into smelly thioalcohols. The precise details of how the bacteria do this are not clear.
Now, Minhas, Bawdon et al. show how S. hominis uses a transport protein in its membrane to bring the odourless precursor inside. In the experiments, tools such as X-ray crystallography captured snapshots of this transporter as it was moving the compound into the bacteria. This helped to understand how the bacteria recognize these precursors, as well as the exact structure of these molecules and of their transporters.
The experiments also reveal that bacteria which do not create odour can also ingest the precursors through this same process. This suggests that the odour production is a unique process that happens once these molecules are inside S. hominis.
The findings imply that humans and their body odour-producing bacteria likely evolved together. In other mammals, the bacterial production of bodily smells is linked to the release of pheromones, which are chemicals involved in communication and in selecting sexual partners. It is not clear whether this is also true for humans. Ultimately, learning more about how S. hominis converts precursor molecules into thioalcohols could lead to new ways of nipping body odours in the bud.https://doi.org/10.7554/eLife.34995.002
In Homo sapiens the skin of the underarm (axilla) provides a unique niche for bacteria. Through the secretions of various glands, that open onto the skin or into hair follicles, this environment is nutrient-rich and hosts a unique microbial community (Taylor et al., 2003). Esterified fatty acids and other lipids are secreted by the sebaceous gland, while the eccrine glands produce a dilute salt solution with lactate and other organic solutes being secreted (Bovell, 2015). A third exocrine gland, the apocrine, which is more specialized and found only in the axilla, areolae, genitalia and external auditory meatus (ear canal) (Collins, 1989), starts secreting an odourless lipid-rich viscous secretion with the onset of puberty (Figure 1A). The lipid rich secretion is not involved in thermoregulation like the eccrine gland and in mammals has a likely role in scent generation (Collins, 1989).
In humans, the link between the axillary apocrine gland, underarm bacteria and body odour (BO), was first recognised over 50 years ago (Shelley et al., 1953) and early culture-based studies on this relationship found the axillary microbiota to be dominated by Staphylococcus, Corynebacterium and Propionibacterium species, with the Corynebacteria in particular correlating with malodour intensity (Taylor et al., 2003; Leyden et al., 1981). More recent culture-independent meta-taxonomic studies have confirmed that the axilla is dominated by these genera, although the presence of additional taxa not previously indicated by culture-based methods is also evident, notably Gram-positive anaerobic cocci (GPAC) belonging to the Anaerococcus and Peptoniphilus genera (Costello et al., 2009; Egert et al., 2011; Troccaz et al., 2015). Various molecules produced in the axilla have been linked to malodour, including 16-androstene steroids (Gower et al., 1994) and short-chain (C2-C5) volatile fatty acids (VFAs) produced by axillary bacteria via several metabolic routes (James et al., 2004). However, 16-androstenes are now believed to be only minor contributors to axillary malodour (James et al., 2013), and it is accepted that the VFAs most heavily involved in BO are a series of structurally-unusual medium-chain (C6-C10) acids originating from N-acylglutamine precursors present in apocrine sweat (Natsch et al., 2003; Natsch et al., 2006). A third class of molecule, the thioalcohols (also called sulfanylalkanols), have recently been identified as important components of human body odour and a number of these have been detected within the volatiles released from the underarm (Troccaz et al., 2004; Natsch et al., 2004; Hasegawa et al., 2004). They are particularly pungent and can be detected in pg.l−1 quantities, an order of magnitude lower than many other volatile chemicals emanating from the skin (Natsch et al., 2004). The most abundant and pungent of these thioalcohols is 3-methyl-3-sulfanylhexan-1-ol (3M3SH) (Figure 1B), which is a primary causal molecule of axillary malodour and subsequent BO (Troccaz et al., 2004; Hasegawa et al., 2004).
Thioalcohols are known to be released as a peptide-conjugated precursor from the apocrine gland, which secretes directly into the hair follicle (Figure 1A). The biosynthetic origin of the thioalcohol precursors is currently poorly understood, but they are thought to be secreted into the axilla via the ABC transporter ABCC11 as odourless glutathione conjugates (Baumann et al., 2014a). The glutathione conjugated form (SG-3M3SH) has never been detected in axillary sweat; however, the L-cysteinylglycine dipeptide conjugated form, S-[1-(2-hydroxyethyl)−1-methylbutyl]-L-cysteinylglycine (S-Cys-Gly-3M3SH) (Figure 1B), is readily detectable (Starkenmann et al., 2005) and recently a γ-glutamyl transferase (GGT1) has been demonstrated to localise to the apocrine sweat gland and convert SG-3M3SH to S-Cys-Gly-3M3SH (Baumann et al., 2014a) (Figure 1C). We and others have demonstrated that S-Cys-Gly-3M3SH can be taken up and metabolised by a highly limited range of staphylococcal species present in the axilla, namely Staphylococcus hominis, Staphylococcus haemolyticus and Staphylocooccus lugdunensis, to liberate 3M3SH, that subsequently exits the cell through an undefined mechanism (Troccaz et al., 2004; Starkenmann et al., 2005; Bawdon et al., 2015). A strong correlation between S. hominis and body odour production has recently been established using culture-independent taxonomic studies of the axillary microbiota (Troccaz et al., 2015), confirming the biological significance of this low-abundance species within the axilla in BO production in humans. However, the mechanism by which the S-Cys-Gly-3M3SH precursor molecule is taken up into S. hominis and related thioalcohol producers is currently unknown, hampering efforts to identify novel mechanisms to disrupt this process and control odour production in humans.
Here we identify the biochemical and structural basis for the recognition and specific transport of the malodour precursor peptide, S-Cys-Gly-3M3SH, into S. hominis. We first show that S-Cys-Gly-3M3SH is actively taken up by S. hominis through a specific secondary active transporter, STAH0001_1446 (SH1446), a member of the proton coupled oligopeptide transporter (POT) family (Hagting et al., 1994; Paulsen and Skurray, 1994). Through a combination of structural, biochemical and cell based assays we reveal how this peptide transporter is able to recognise a thioalcohol-conjugated peptide and further, show that transport is coupled to the inwardly directed proton electrochemical gradient. Our results suggest that SH1446 is a clear target for inhibitor design to prevent malodour production, thus providing further molecular insights into the role of the skin microbiota in human scent generation.
To elucidate how thiol-conjugated dipeptides are transported by bacteria, we first examined the model organism Escherichia coli, which is able to recognise and metabolise the dipeptide version of the non-physiological malodour precursor S-benzyl-L-cysteine, namely, S-benzyl-L-cysteinylglycine (James et al., 2013). Resting cells of E. coli were indeed able to remove S-benzyl-L-cysteinylglycine from solution and break it down to release the thiol active product, benzyl mercaptan (Figure 2—figure supplement 1A,B). Following transport into the cell, the dipeptide must be cleaved to release the benzyl mercaptan, most likely through a cysteine-S-conjugate β-lyase-type activity, for which a number have been described in E. coli, including MetC and MalY (Dwivedi et al., 1982; Zdych et al., 1995; Awano et al., 2003). Interestingly, TnaA which is a tryptophanase known to have L-cysteine desulfhydrase activity (Awano et al., 2005) is also capable of releasing benzyl mercaptan.
Individual disruption of metC and malY did not decrease the amount of benzyl mercaptan produced by E. coli, while disruption of tnaA led to an almost complete loss of biotransformation (Figure 2—figure supplement 1C). This suggests that E. coli takes up the S-benzyl-L-cysteinylglycine, whereupon a host dipeptidase cleaves the glycine residue prior to cleavage of the S-benzyl-cysteine by TnaA to release the benzyl mercaptan.
To discover the genetic basis of the transport and metabolism of these thiol-conjugated dipeptides, we exploited the knock out (KO) library of single gene disruptions in E. coli K-12 (Baba et al., 2006). We hypothesised that the dipeptide moiety might direct the molecule through a peptide transporter. In E. coli the main peptide systems are relatively well characterised, the two main transport systems belong to the ATP binding cassette (ABC) family and the proton dependent oligopeptide transporter (POT) family (Figure 2B). The E. coli Opp and Dpp ABC transporters have been extensively studied in E. coli and the closely related bacterium Salmonella typhimurium (Smith et al., 1999; Klepsch et al., 2011; Abouhamad et al., 1991; Goodell and Higgins, 1987; Tame et al., 1995) and an E. coli BW25113 strain with non-functional Opp and Dpp system (ΔDB1), was used to assess the role of these transporters in the production of benzyl mercaptan. Surprisingly, benzyl mercaptan production was similar to the wild-type strain, suggesting that neither ABC transporter was important for Cys-Gly-conjugate transport (Figure 2A). Having ruled out transport via the ABC peptide transporters, we then broadened our search to include additional peptide transporters that have been described in E. coli. These were the four members of the proton dependent oligopeptide transporter, or POT family, encoded by dtpA-D, that have poorly defined physiological roles during growth on peptides (Harder et al., 2008; Prabhala et al., 2014; Casagrande et al., 2009), the CstA protein, orthologues of which have been demonstrated to be peptide transporters (Garai et al., 2016) and the Sap system, which is implicated in uptake of cationic antimicrobial peptides (Groisman et al., 1992). Most of these strains had similar levels of benzyl mercaptan production to the wild-type, with the exception of dtpB which had an overall 50% drop in thiol production (Figure 2A). These data suggest that transporters belonging to the POT family are most likely responsible for Cys-Gly-conjugate transport, revealing a new biological role for bacterial POT transporters in the uptake of Cys-Gly-(S) conjugates.
Having established that E. coli has the ability to transport and cleave a Cys-Gly-(S) conjugated substrate, we reasoned that overexpression of a POT transporter might confer the ability to recognise the physiological malodour precursor S-Cys-Gly-3M3SH. The four dtp genes were cloned into a pBAD vector commonly used for overexpression of membrane transporters (Mulligan et al., 2009; Mulligan et al., 2012) and transformed into the wild-type E. coli background. The ability of the resulting strains to convert S-Cys-Gly-3M3SH to 3M3SH was subsequently measured (Bawdon et al., 2015). Consistent with the reduction in benzyl mercaptan production reported in Figure 2A, the dtpB gene alone was able to confer a malodour production (BO+) phenotype to E. coli, which was not seen with any of the other dtp genes (Figure 2C). The same pattern was observed with S-benzyl-Cys-Gly as a substrate (Figure 2—figure supplement 2B), suggesting that DtpB has a unique ability to transport these unusual conjugated peptides.
The molecular basis for S-Cys-Gly-3M3H transport into malodour producing staphylococci is unknown, and the genetic intractability of these poorly studied coagulase negative Staphylococcal species makes it difficult to study using genetic approaches. We therefore used an alternative approach exploiting our discovery that expression of a transporter in trans could produce a BO+ phenotype to E. coli. Given our discovery that E. coli POT family peptide transporters can transport S-Cys-Gly-3M3SH, we examined the genome of S. hominis SK1119, as S. hominis are the most abundant malodour producing Staphylococcal species in the underarm microbiota. Unlike E. coli, S. hominis contains only a single POT family member DtpT (SH1446), orthologues of which were seen in all sequenced staphylococci (Figure 2—figure supplement 3A). The POT peptide systems remain largely uncharacterised in staphyoloccci, although in the well-studied pathogen S. aureus, the single POT transporter encoded by dtpT is the sole route for dipeptide uptake in this species (Hiron et al., 2007). We also identified a second system, encoded by STAHO0001_0415 (SH0415), which is annotated as an MFS transporter and only present in S. hominis and S. haemolyticus, two known thioalcohol producers, and which is genetically linked to genes involved in peptide and amino acid metabolism (Figure 2—figure supplement 3B). Both transporters were cloned into pBADcLIC2005 and transformed into E. coli for functional analysis. When levels of 3M3SH production were measured in these strains, uptake was only enhanced by the presence of SH1446, with SH0415 having little effect on 3M3SH activity (Figure 2D). In contrast, with S-benzyl-cysteinlyglycine as a substrate, there was no increase in production of benzyl mercaptan with SH1446 but a significant increase was observed with SH0415 (Figure 2—figure supplement 2B). Together these data strongly support the conclusion that the S-Cys-Gly-3M3SH transporter in S. hominis is the POT transporter STAH0001_1446 (SH1446).
To further demonstrate the importance of SH1446 in the physiological transport of S-Cys-Gly-3M3SH we used a biochemical approach to examine whether peptides could inhibit 3M3SH production by competing with S-Cys-Gly-3M3SH uptake. Firstly, we used our E. coli system where 3M3SH production is seen when SH1446 is expressed in trans and we observed clear inhibition of 3M3SH production with both di- and tri-Ala peptides (Figure 2—figure supplement 4) but not with L-Ala alone nor tetra-Ala. The purified TnaA enzyme that cleaves Cys-3M3SH was not inhibited by any of these peptides (Figure 2—figure supplement 5) , supporting the hypothesis that the whole cell inhibition is due to inhibition of peptide transport. Similarly, we measured the ability of the same peptides to inhibit 3M3SH production directly in S. hominis (Figure 2—figure supplement 4) and observed a comparable profile. It should be noted that a significant affect was observed for 25 mM tetra-Ala in S. hominis wild-type, however this affect is due to the acidic pH that was required to dissolve the peptide which appears to inhibit metabolism in S. hominis but not E. coli.
POT transporters belong to the Major Facilitator Superfamily (MFS) and use an inwardly directed proton electrochemical gradient to drive the concentrative uptake of di- and tri-peptides across the cell membrane for metabolic assimilation (Daniel and Kottra, 2004). To establish SH1446 as the protein responsible for S-Cys-Gly-3M3SH recognition and transport, the gene was cloned and overexpressed in E. coli for functional and structural studies. The purified protein, hereafter referred to as PepTSh (Peptide Transporter Staphylococcus hominis) was purified and reconstituted into liposomes. Transport was studied using a previously employed pyranine based reporter assay, which measures transport via changes to the internal pH of the liposomes (Parker et al., 2014) (Figure 3A). Acidification of the lumen of the liposome was observed in the presence of a membrane potential (ΔΨ, negative inside), and addition of external di- or tri-peptide substrate, demonstrating that PepTSh is a proton coupled peptide transporter. Importantly we also observed acidification in the presence of the S-Cys-Gly-3M3SH precursor peptide, confirming our earlier identification of PepTSh as the transporter for this molecule in S. hominis.
To understand the recognition of S-Cys-Gly-3M3SH compared to natural peptides, we determined IC50 values using competition with 3H-di-alanine. The IC50 value for tri-alanine, di-alanine and Cys-Gly were 23.7 ± 3.4, 72.2 ± 9.7 and 115 ± 22.2 μM, respectively (Figure 3B), indicating that PepTSh is able to recognise tri-Ala more favourably than di-Ala. However, for S-Cys-Gly-3M3SH the IC50 value was 362 ± 21.8 μM, three-fold higher than the Cys-Gly peptide. Our data therefore show that while PepTSh can transport the modified peptide, the addition of the thioalcohol group on the cysteine side chain has substantially impaired the recognition and transport versus natural peptides.
To gain further insight into the mechanism of substrate binding we successfully obtained a high-resolution co-crystal structure of PepTSh at 2.5 Å resolution, bound to S-Cys-Gly-3M3SH using the in meso crystallisation method (Figure 4A, Table 1 and Figure 4—figure supplement 1). PepTSh adopts the canonical MFS fold with helices H1-H6 forming the N-terminal bundle and H7-H12 the C-terminal bundle. PepTSh was crystallised in an inward open state, observed previously for other members of the POT family (Newstead, 2015), wherein the extracellular gate, consisting of helices H1-2 and H7-8 from the N- and C-terminal bundles respectively, is closed and the intracellular gate, consisting of helices H4-5 and H10-11 is open. Similar to other bacterial POT family members, PepTSh contains two additional helices inserted between the N- and C-terminal bundles, termed HA and HB (Figure 4B and Figure 4—figure supplement 2). Unlike previous POT structures however, PepTSh also contains a well-structured β-hairpin motif between helices H7 and H8 on the extracellular side of the membrane, extending out in line with the lipid head groups. Interestingly we observed a number of monoolein lipid molecules that cluster around both the β- hairpin and the cavity created between helices HA and HB with the transporter domain. The role of helices HA and HB in prokaryotic POT family transporters still remains unclear. However, the observation of lipid molecules bound between these helices and the transport domain suggests a possible role in stabilising these transporters in the membrane, which may be an adaption specific to prokaryotic members of the POT family (Newstead et al., 2011).
Following extensive screening and co-crystallisation we observed clear mFo-DFc difference electron density for the S-Cys-Gly-3M3SH ligand in the peptide binding site (Figure 4C and Figure 4—figure supplement 3). The Cys-Gly part of the precursor is positioned such that the carboxy termini of the peptide faces towards the intracellular side of the binding site. The terminal carboxyl group makes two hydrogen bond interactions, one with Tyr41 on H1 and a second interaction to a water molecule (W107), which links the carboxyl group with Asn347 on H8. The carbonyl group of the CG peptide also makes a hydrogen bond to this water molecule, creating a well-coordinated interaction network between the peptide and Asn347 (Figure 4D). The terminal amino group makes two further polar interactions, with a water molecule that sits next to Tyr41 and also to the carbonyl group of Gln344, which sits further up on H8. In contrast, the thioalcohol moiety points towards the extracellular gate, with the sulphur atom sitting close to Gln310 on H7, whereas the alcohol group extends towards helix H8 making interactions with backbone carbonyl and amide groups of Phe343 and Asn347, respectively. The acyl group extends into a large pocket nestled between helices H7 and H10 (Figure 4E). This long pocket (~10 Å) extends from the central peptide binding site into the C-terminal bundle of the transporter and is both hydrophobic and polar in character.
Previously, we showed that a related POT family transporter, PepTSt, is able to bind peptides in different orientations in the binding site, with a di-peptide adopting a horizontal position and a tri-peptide a vertical one (Lyons et al., 2014) and suggested this flexibility may be responsible for the accomodation of larger peptide ligands (Newstead, 2017). Here we see that the S-Cys-Gly-3M3SH peptide adopts a more vertical orientation, similar to the tri-alanine position in PepTSt. However, there are significant differences in the interactions. A structural overlay of the S-Cys-Gly-3M3SH peptide shows that it sits much further up in the binding site compared to the natural peptides captured in PepTSt (Figure 4—figure supplement 4A & B), most likely as a result of the requirement to accommodate the thioalcohol group in the extended pocket. This has a knock-on effect with respect to the position of the amino and carboxy termini. In PepTSt, the amino terminal group of the di-peptide directly interacts with helix H8 and H10, whereas the tri-peptide makes a much weaker interaction through a carbonyl group. In contrast, in PepTSh this interaction is achieved through a water mediated hydrogen bond (Figure 4D).
The role of water in mediating interactions between peptides and their binding sites has been previously observed in the ABC transporter binding domains (Tame et al., 1995), albeit for side chain independent binding. A similar mechanism may be employed by PepTSh to facilitate transport of modified peptides by replacing direct interactions between the peptide and the transporter when these are sterically restricted, as we observe with S-Cys-Gly-3M3SH. The interaction of the amino termini of peptides with both the mammalian and bacterial POT family transporters has been identified as facilitating high affinity binding (Weitz et al., 2007; Meredith et al., 2000). The reduced IC50 value for the modified peptide in PepTSh (Figure 3B), may therefore be a combination of steric restraints resulting in the accommodation of the extended side chain group, and the subsequent knock on effects on the position of the interactions between the amino and carboxy groups with conserved side chains in the binding site.
S-Cys-Gly-3M3SH is the first human derived peptide with a modified side chain to be captured in a POT family transporter, and contains biochemical features similar to physiological peptides (Cys-Gly), but also incorporates the non-peptide thioalcohol group (3M3SH). We therefore wished to explore whether S-Cys-Gly-3M3SH is recognised using a similar mechanism to physiological peptides. Several mutations in the binding site that have previously been shown to play an important role in peptide recognition within the bacterial POT family were therefore generated (Newstead, 2017). Tyrosine 41 forms part of a conserved E33xxERFxYY41 motif on TM1 (Solcan et al., 2012), which plays a role in proton coupling and influences peptide uptake in several POT family members (Doki et al., 2013; Ernst et al., 2009; Guettou et al., 2014; Lyons et al., 2014). In PepTSh Tyr41 hydrogen bonds with the carboxyl terminus of the terminal glycine amino acid in S-Cys-Gly-3M3SH (Figure 4C,D). Mutating Tyr41 to phenylalanine or alanine severely reduced transport of di-alanine compared to WT protein (Figure 5A). A similar negative effect on transport was observed in the related POT family transporter PepTSt, from Streptococcus thermophilus (Solcan et al., 2012), suggesting a common role for the conserved tyrosine in peptide recognition within the POT family. We then used the same variants in our whole cell assay for 3M3SH production. Interestingly, while both Tyr41Phe and Tyr41Ala variants had reduced activity, at about 50% of the wild-type protein (Figure 5B), the phenotype was not as severe as that observed in the liposome based assay. We interpret this result to indicate that recognition of S-Cys-Gly-3M3SH is less dependent on this residue than di-Ala.
We next wanted to test the role of other residues we observed sitting close to the S-Cys-Gly-3M3SH ligand. In particular, we were interested in the contribution made by the Asn347 and Glu418, which coordinate a water molecule (W107) linking both the carboxyl and carbonyl groups of the ligand to the binding site (Figure 4B). The equivalent residues in homologous POT family transporters have been implicated in both peptide recognition (Lyons et al., 2014) and gating (Solcan et al., 2012), and in the case of Glu418 may facilitate the coupling between peptide recognition, proton movement and transport (Parker et al., 2017). Although a Glu418Ala variant had no activity in our assays, consistent with its proposed role in coordinating the intracellular gate (Fowler et al., 2015), we observed reduced uptake of di-Ala in the Asn347Ala variant (Figure 5A). Interestingly, this reduction was far more severe in the in vivo assay (Figure 5B), indicating that this interaction is more important for transport of the S-Cys-Gly-3M3SH peptide than di-Ala. This may reflect the requirement of Asn347 to coordinate the water molecule linking the peptide to the intracellular gate side chain Glu418, which in the case of the di-peptide may interact directly as in PepTSt, lessening the requirement for the Asn347 interaction.
The co-crystal structure also identified the acyl chain portion of the thioalcohol group was accommodated within an extended pocket, formed near the extracellular side of the binding site (Figure 4E). To test the importance of this pocket on S-Cys-Gly-3M3SH transport, we made a Tyr411Trp variant, which would block the cavity. Uptake was noticeably reduced, but still measurable at ~19% compared to WT (Figure 5A). The same mutation resulted in a similar large decrease in the biotransformation of S-Cys-Gly-3M3SH, while a Tyr411Ala mutation had wild-type biotransformation rates (Figure 5B). It is clear that the hydrophobic pocket in PepTSh plays an important role in facilitating the recognition of the thioalcohol group in the binding pocket and, as discussed below, is a potential site for inhibitor design.
Taken together the current data lend further support to our model of an adaptable binding site within the POT family that can accommodate chemically diverse peptides and their derivatives through the use of both side chain and water mediated interactions with conserved residues and different specificity pockets to accommodate the different chemical side chains.
The production of malodour by humans is an evolutionary ancient olfactory process that likely was involved in mate selection and evolutionary processes that relied on chemical signalling (Stoddart, 1990). Evidence for the microbial origins of chemical signalling molecules is also seen in other animals such as foxes, deer, cats and beetles (Wyatt, 2003), but the details of this process in humans is less well understood. The thioalcohol 3M3SH, a major component of body odour, is produced in the human axilla as a glutathione-conjugated precursor and is processed to a unique dipeptide structure (Figure 1). The discovery that axilla-isolated strains of S. hominis, S. lugdunensis and S. heamolyticus are able to produce 3M3SH prompted the search for the molecular basis for this process (Bawdon et al., 2015). While S-Cys-Gly-3M3SH is a highly modified and uniquely human dipeptide, we reasoned that this moiety might dictate the uptake mechanism into malodour producing bacteria and here we identify a peptide transporter, PepTSh (SH1446), from the POT family of secondary active transporters as being responsible for this process. Each of these strains have a POT family transporter that is closely related to PepTSh (Figure 4—figure supplement 2), and we would predict they will also be able to uptake S-Cys-Gly-3M3SH.
The genetic intractability of S. hominis dictated that we use a biochemical approach to demonstrate that SH1446 is likely the sole route of uptake of S-Cys-Gly-3M3SH. First, this staphylococci has a similar repertoire of peptide transporters in S. aureus, in that it has a single POT transporter and one Opp ABC transporter, and previous work in S. aureus has demonstrated that the POT transporter, DtpT, is essential for growth on dipeptides and some tripeptides, while the ABC transporter (Opp3) has a function in transporting longer oligopeptides (Hiron et al., 2007). The use of L-Ala containing peptides as inhibitors in this study demonstrates an inhibitory effect on 3M3SH production that is consistent with the properties of a POT family transporter. Several attempts were made to genetically disrupt SH1446 in S. hominis, using tools developed for S. aureus and S. epidermidis, but these were unsuccessful, most likely due to the presence of multiple species-specific restriction modification (RM) systems which render this organism, as with many other coagulase negative (CoNS) species, genetically intractable.
The specific role for POT transporters in the transport of these unusually modified dipeptides is supported from the inability of the E. coli ABC transporters for oligopeptides and dipeptides, Opp and Dpp, to recognise and transport S-Cys-Gly-3M3SH. Given that S-Cys-Gly-3M3SH is effectively a dipeptide with a highly extended R-group on the first amino acid, it is noteworthy that only the promiscuous nature of the POT binding site is able to recognise this ligand. For OppA and DppA, the substrate binding proteins that initially recognise peptides transported by the ABC systems, the binding pocket is well defined and makes general main chain-main chain interactions to ensure that the protein can bind peptides in a sequence independent manner (Tame et al., 1995; Sleigh et al., 1997; Sleigh et al., 1999). However, the pocket has evolved to accommodate regular amino acid side chains and the unusually large structure of the thioalcohol attachment would likely be sterically incompatible with recognition by either OppA or DppA. The data presented here further support the idea that POT transporters have an unusually promiscuous binding site that may play an evolutionary advantageous role in accepting modified peptides as substrates.
Recently, the human POT family transporters, PepT1 and PepT2, have gained additional attention due to their role in the absorption and retention of several important classes of drug molecule (Smith et al., 2013), including peptide modified pro-drugs. Similar to S-Cys-Gly-3M3SH, these pro-drugs are drug molecules conjugated to a peptide or amino acid (Brandsch, 2013). Pro-drugs are able to compete with natural peptides for transport via PepT1 and PepT2, and are therefore actively transported into the body (Ganapathy et al., 1998; Gupta et al., 2013; Terada and Inui, 2012). The promiscuous nature of the POT family binding site has been shown to derive, in part, from their ability to recognise peptides in different conformations and through the availability of different specificity pockets, which are able to accommodate chemically different classes of peptides (Newstead, 2017). Our results here provide the first evidence that specificity pockets can also accommodate exotic chemical extensions. The observation that water plays a role in facilitating the interaction between the S-Cys-Gly-3M3SH peptide and PepTSh may also indicate a broader role for water in facilitating the recognition of xenobiotic peptides. In particular, the observation that water may bridge interactions between the binding site and either the amino or carboxy group of the ligand would greatly increase the number of conformations a peptide could adopt in the transporter and still trigger proton coupled transport.
Finally, we can ask the questions, why do these bacteria transport S-Cys-Gly-3M3SH and can this process be inhibited to provide effective malodour control? The presumed biochemical pathway for breakdown of the S-Cys-Gly-3M3SH is outlined in Figure 6. The firststep is the likely removal of the glycine by a dipeptidase and then the lyase reaction that cleaves the carbon-sulphur bond to release pyruvate and ammonia in addition to 3M3SH. The route of export of 3M3SH is unknown, but currently assumed to be diffusion across the bacterial inner membrane. The bacterium will gain a nutritional benefit from the catabolism of S-Cys-Gly-3M3SH via the release of both nitrogen and carbon as glycine, pyruvate and ammonia (Figure 6), which may have been the evolutionary trigger for the adaptation to catabolise this chemical. Asian individuals with single nucleotide polymorphisms (SNPs) in ABCC11, the transporters involved in the secretion of S-Cys-Gly-3M3SH (Figure 1) have been studied (Nakano et al., 2009; Baumann et al., 2014b). Recently, a microbiological investigation of individuals with wild-type alleles of ABCC11 compared to heterozygous and double mutant genotypes revealed that the proportion of Staphylococcus sp. is higher in the underarms of humans that produce the precursor (Harker et al., 2014). While these studies are limited in that they do not provide species level identification, they are at least consistent with the idea that the production of the precursor positively correlates with the presence of Staphylococcus sp. in the underarm. The discovery of the transporter and molecular understanding of the mode of S-Cys-Gly-3M3SH binding now suggests a specific route to develop inhibitors of malodour production by directly inhibiting the transporter SH1446 (Grice, 2014). Importantly, such intervention would not impact on the overall axillar microbiome, but instead specifically target the reduction in body odour production.
Escherichia coli K-12 was used as the model organism throughout this study. Escherichia coli K-12 single gene deletions were obtained from the in-house E. coli Keio collection at University of York. Bacterial strains and plasmids used in this study are listed in Supplementary file 1 and 2. All strains were cultured in LB (1% tryptone, 0.5% yeast extract, 1% NaCl) unless otherwise stated, Antibiotics were used at the following concentrations Ampicillin 100 μg/mL and Kanamycin 50 μg/mL; L-arabinose concentrations are indicated. PCR, plasmid extractions, and transformations were carried out as per standard protocols. For routine cloning, genomic DNA was extracted from BW25113 and high fidelity KOD polymerase was used (Novagen). Correct clones were confirmed by DNA sequencing. Specific details of plasmid constructions are detailed below.
Multiple gene knockouts were created in single keio deletion strains. To create the double KO strain DB1 (ΔoppC ΔdppC), the KanR cassette was removed from a target gene using pCP20 (encoding Flp recombinase) at 30°C, loss of antibiotic cassette was confirmed by PCR and sequencing. The second gene to be disrupted was PCR amplified with 100 bp homology including the FRT flanked KanR cassette from the single gene deletion strain in the keio collection. Targeted gene disruptions were created using λ Red recombination as previously described (Datsenko and Wanner, 2000).
Overexpression of the E. coli POT transporters were cloned into pBADcLIC2005 vector under the control of arabinose. Full-length genes were amplified from E. coli BW25113 genomic DNA using high fidelity KOD polymerase (Merck) using the corresponding primers listed in Supplementary file 3. In silico analysis of the available Staphylococcus hominis SK1119 genome identified STAH0001_1446 (SH1446) as a POT family member which orthologues present in all sequenced staphylococci and a MFS transporter STAH0001_0415 (SH0415) present in only S. hominis and S. haemolyticus. SH0415 and SH1446 genes were amplified from S. hominis genomic DNA using high fidelity KOD polymerase (Merck) using primers listed in Supplementary file 3. All PCR products were purified using Wizard SV Gel and PCR Clean-Up System (Promega), cloned into pBADcLIC2005 vector using standard ligation-independent cloning (LIC) techniques. All fragments were confirmed by PCR and DNA sequencing (GATC Biotech).
The wildtype STAHO0001_1446 (SH1446) cloned onto the expression plasmid pBADcLIC2005 was used to individually generate the Y411W, Y411A, Y41A, Y41F, N167A and N347A mutants. To introduce the mutation, high fidelity inverse PCR was performed using divergent primers with one per pair being mutagenic (Supplementary file 3). The resultant products were then circularised by performing a blunt end ligation and transformed into E. coli BW25113. The mutant sequences were verified by DNA sequencing (GATC Biotech).
S-Benzyl-L-cysteinylglycine and (S)-(1-(2-hydroxyethyl)−1-methylbutyl)-L-cysteinylglycine (S-Cys-Gly-3M3SH) (Peakdale Molecular) were prepared at a stock concentration of 10 mM in M9 salts (6 g Na2HPO4 g/L, 3 g KH2PO4 g/L, 0.5 g NaCl g/L) and stored at 4°C.
E. coli were grown overnight in LB, cells were harvested by centrifugation at 3, 500 rpm 10 min and resuspended in sterile M9 salts. Where appropriate, E. coli strains harbouring inducible genes on the pBADcLIC2005 plasmid were pre-induced during overnight growth with 0.0001% L-arabinose. To a sterile Eppendorf the following was added; 2 mM substrate (S-Benzyl-L-cysteinylglycine or S-Cys-Gly-3M3SH), cells to an OD650nm of 5 and M9 salts up to 200 μL. Cells only (no added substrate) and substrate only (no added cells) reactions were also included in every individual experiment. The reactions were incubated at 37°C for up to 48 hr with samples taken at appropriate time points. Liberated thiols were quantified using either MTS/PMS or DTNB labelling as detailed below.
Liberated benzyl mercaptan was labelled as follows, 500 μl from each reaction was taken at the appropriate time point and centrifuged at 13 000 r.p.m. for 2 min. To chemically label benzyl-mercaptan, 50 µl of labelling reagent, consisting of 4.09 mM 3-(4,5-dimethylthiazol-2-yl)−5- (3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium (MTS) (Promega) and 0.2 mM phenazine methosulfate (PMS) (Sigma) (Goodwin et al., 1995), dissolved in dH2O, was aliquoted into either a 96 well microplate or 0.5 ml Eppendorf tubes. For reactions using a 96 well microplate, 200 µl reaction supernatant was added to the pre- aliquoted 50 µl MTS/PMS. The plate was sealed with a semi-permeable film lid, incubated at room temperature for 50 min and the A492nm recorded on a Biotek PowerWave XS microplate spectrophotometer. The data was captured by KC junior and exported to Microsoft Excel. For reactions in Eppendorf tubes, 20 µl - 50 µl reaction supernatant was added to 50 µl MTS/PMS. The reaction was incubated at room temperature for 50 min. 20 µl was subsequently removed and diluted in 980 µl dH2O in a 1.5 ml disposable plastic cuvette. The A492nm was recorded.
To quantify free 3M3SH, a 75 µl volume from each reaction (isolated as described above) was taken at the appropriate time point and centrifuged at 13, 000 rpm for 2 min. 950 µl labelling solution, consisting of: 800 µl dH2O, 100 µl UltraPure Tris (pH 8.0) and 50 µl DTNB (Ellman, 1958) stock solution (50 mM sodium acetate, 2 mM DTNB, dissolved in dH2O) was added to a 1.5 ml disposable cuvette. 50 µl reaction supernatant was added to the cuvette, mixed by pipetting and incubated at room temperature for 5 min. The A412nm was recorded on a Jenway 6305 spectrophotometer. (Van Horn, 2003). Where appropriate, E. coli strains harbouring inducible genes on the pBADcLIC2005 plasmid were pre-induced during overnight growth with 0.0001% L-arabinose. In all experiments cells only (no added substrate) and substrate only (no added cells) were also included.
The gene encoding S. hominis SH1446 was cloned into a C-terminal octa histidine GFP fusion vector (pWaldo-GFPe) (Drew et al., 2006) and subsequently transformed into Escherichia coli C43(DE3) cells (Miroux and Walker, 1996). Mutations were introduced using site directed mutagenesis followed by DpnI digestion. An overnight starter culture was used to inoculate 4 L of Terrific Broth (TB). Cells were left to grow at 37°C and aerated by shaking at 250 rpm. Overexpression of the fusion protein was induced when the culture reached an OD600 of ~0.6, by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. Following induction, the temperature was reduced to 25°C. Cells were harvested 16 hr later, and resuspended in phosphate buffered saline (PBS) containing deoxyribonuclease I from bovine pancreas, and stored at −80°C.Unless stated, the following steps were carried out at 4°C. Cells were thawed and disrupted at 32 kpsi (Constant Systems, UK). Cellular debris was removed by centrifugation at 30,000 x g for 30 min, and membranes were isolated by ultracentrifugation at 230,000 x g for 120 min. Membranes were resuspended in PBS using a dounce homogeniser, flash frozen, and stored at −80°C. Thawed membranes were diluted with 1x PBS (10 mL per gram of membrane), 20 mM imidazole (pH 8.0), 150 mM NaCl, and 1% n-Dodecyl-β-D-maltoside (DDM) and left to solubilise for 60 min, stirring. Non-solubilised membranes were removed by ultracentrifugation at 230,000 g for 60 min. Nickel-NTA resin (ThermoFisher Scientific) was added to the sample, using a ratio of 1 mL resin per gram of membrane, and left to bind, stirring, for approximately 90 min. The resin was then packed into a glass econo-column (BioRad, USA) under gravity. The resin was washed with 20 column volumes (CV) of wash buffer (1x PBS, 20 mM imidazole (pH 8.0), 150 mM NaCl and 0.05% DDM). The resin was further washed with 10CV of wash buffer containing 30 mM imidazole. The bound GFP-SH1446 fusion protein was eluted with wash buffer containing 250 mM imidazole. The eluent was combined with an equivalent concentration of hexa histidine tagged TEV protease and dialysed overnight against 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.03% DDM. The cleaved protein was filtered using a 0.22 μm filter (Millipore, USA) and passed through a Ni-NTA 5 mL HisTrap column (GE Healthcare) to remove uncleaved GFP-SH1446 protein and TEV protease. The flow through was collected and concentrated to 300 μl using a 50 kDa MWCO concentrator (Vivapsin 20, Sartorius AG) and applied to a size exclusion column (Superdex 200 10/30, GE Healthcare) pre-equilibrated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.03% DDM. Fractions containing purified SH1446 protein were pooled and concentrated to a final concentration of 15–20 mg.ml−1.
For expression and purification of TnaA, overnight cultures were grown in LB at 37°C, 200 rpm. 1 L of LB was inoculated 1:50 with the starter culture and grown to an OD600nm ~0.6–0.8, target protein expression was induced with 0.001% arabinose. Following induction the culture was further incubated at 37°C for ~8 hr. Cells were harvested by centrifugation at 4,500 rpm for 15 min and resuspended in 35 mL resuspension buffer (50 mM Kpi, 20% Glycerol, 200 mM NaCl and 10 mL Imidazole, pH 7.8) and stored at −80°C. All subsequent steps were carried out at 4°C, for protein purification cells were thawed and lysed using sonication with 3 s pulsed and 7 s pause for 3mins. Lysates were clarified by centrifugation 25,000 rpm ~30 min. The clarified supernatant was loaded onto a 5 mL HisTrap column (GE Healthcare) and affinity purified on an AKTA Start (GE Healthcare) using a standard protocol as per manufacturer’s instructions.
Crystals of SH1446 were obtained in-meso phase. Briefly, concentrated protein was mixed with 9.9 monoacylglycerol (Monoolein, Sigma-Aldrich) at a protein:lipid ratio of 2:3, in a mechanical syringe mixer at 20°C (Ai and Caffrey, 2000). 50 nL of mesophase and 800 nL of precipitant solution was dispensed on to a 96-well glass plate using an in meso robot (Gryphon, Art Robbins Instruments). SH1446 S-Cys-Gly-3M3SH complex was crystallised in precipitant solution containing 26–27% (v/v) PEG 200, 220 mM (NH4)2HPO4, 110 mM sodium citrate (pH 5.0) and 5 mM S-Cys-Gly-3M3SH (Peakdale Molecular) and left overnight at 4°C, prior to crystallisation. Crystals appeared after 2–3 days, and grew to its maximum size after 10 days. Crystallisation wells were accessed using a tungsten-carbide glasscutter, and crystals were directly mounted onto 50–100 μm microloops (MiTeGen) and flash frozen at 100 K.
X-ray diffraction data were collected at ID23-2 at the European Synchrotron Radiation Facility on a Pilatus3 2M detector with an exposure setting of 0.05 s with a 0.2° oscillation range over 360° Diffraction data was indexed and integrated in XDS (Kabsch, 2010) and merged in Aimless (Evans, 2011).
The ligand bound complex was solved by molecular replacement in PHASER (McCoy et al., 2007) using a modified search model of the bacterial peptide transporter from Streptococcus thermophilus, PepTSt (Lyons et al., 2014), PDB: 4D2B. Iterative rounds of manual model building in COOT (Emsley et al., 2010) followed by refinement in autoBUSTER (Bricogne G. 2017) followed by refinement in Phenix (Adams et al., 2010), resulted in a final model, which was validated using the Molprobity server (Chen et al., 2010). Additional difference density was observed in the binding site and matched the stereo chemical properties and size of S-CG3M3SH. Ligand restraints were generated using the Grade Web Server (http://grade.globalphasing.org). Figures were prepared using PyMOL (Schrödinger, LLC).
Purified SH1446 was detergent exchanged into n-Decyl-β-D-maltopyranoside (DM, Anatrace) using a size exclusion column (Superdex 200 10/30, GE Healthcare) pre-equilibrated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.3% DM. Lipids (POPE:POPG in a 3:1 ratio) were mixed with the eluted protein at a ratio of 60:1. The protein:lipid mixture was rapidly diluted with 50 mM KPO4 (pH 7.0) buffer to below the CMC value of DM. Proteoliposomes were harvested by ultracentrifugation (234,000 xg) for 120 min. The pellet was resuspended in 50 mM KPO4 and dialysed overnight against 3L of 50 mM KPO4 (pH 7.0). Proteoliposomes were recovered and subjected to three rounds of freeze thawing before storage at −80°C.
Proteoliposomes were thawed and harvested by ultracentrifugation at 150,000 g. The resulting pellet was carefully resuspended in INSIDE buffer (120 mM KCl, 2 mM MgSO4, 5 mM HEPES pH 6.8) and 1 mM pyranine (trisodium 8-hydroxypyrene-1,3,6-trisulfonate). Proteoliposomes underwent eleven freeze-thaw cycles in liquid nitrogen before the sample was extruded through a 0.4 μm polycarbonate membrane and harvested by ultracentrifugation as before. Excess pyranine dye was removed with a Microspin G-25 column (GE Healthcare) pre-equilibrated with INSIDE buffer without pyranine. For the assay, proteoliposomes were diluted into OUTSIDE buffer (120 mM NaCl, 5 mM HEPES (pH 6.8), 2 mM MgSO4).
A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) equipped with a cuvette and magnetic flea was used to carry out the transport assay. Dual fluorescence excitation was set to 460/415 nm with emission at 510 nm. Transport was initiated with 1 μM valinomycin, the resulting data was exported and analysed using Prism (GraphPad Software). Transport data was normalised to one to facilitate comparison with data collected from multiple conditions.
Proteoliposomes were harvested as before, the resulting pellet was resuspended in INSIDE buffer, subjected to three freeze-thaw cycles in liquid nitrogen, extruded through a 0.4 μm polycarbonate membrane and harvested by ultracentrifugation once more. For the assay, proteoliposomes were diluted in external buffer (110 mM NaCl, 10 mM NaPO4 and 2 mM MgSO4) supplemented with 50 μM di-alanine peptide containing trace amounts of tritiated 3H-di-alanine (specific activity 30 Ci/mmol) and the competing substrate at increasing concentrations as indicated. Competition assays were performed at 30°C, samples were taken at specified time points and transport was stopped by addition of 2 mL H2O. Proteoliposomes were immediately filtered onto a 0.22 μm cellulose filter (Merck Millipore) using a vacuum manifold and subsequently washed twice more with 2 mL H2O. The amount of peptide transported into the liposomes was calculated based on the specific activity for each peptide as detailed by the manufacturer and counting efficiency for the radioisotope in Ultima Gold (PerkinElmer) counted in a Wallac scintillation counter (3H, 45% counting efficiency). Transport assays were performed a minimum of three times to generate an overall mean and S.E.M.
Atomic coordinates for the atomic models have been deposited in the Protein Data Bank under accession numbers 6EXS.
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Olga BoudkerReviewing Editor; Weill Cornell Medicine, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "Structural basis of malodour precursor transport in the human axilla" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
The manuscript reports the identification and crystal structure determination of a transporter protein for S-Cys-Gly-3M3SH in Staphylococcus hominis. The protein is proposed to import the compound into the bacteria for the processing to 3M3SH, which is released as an underarm odor in humans. The crystallographic work is accompanied by transport and functional assays in whole cell E. coli and proteoliposomes. Flexibility of amino acid side chains at the substrate binding site is proposed to be the main factor that allows the transporter to recognize multiple types of peptide-derivatives. To identify the transporter, the authors side-step the genetic intractability of Staphylococcus spp. by transferring the relevant biochemical process to E. coli and then utilizing both the well characterized functional annotation and the knock-out libraries to dissect the pathways, moving back and forth between S. hominis and E. coli proteins.
The reviewers agreed that the work is important and should be seen by the broad readership of eLife. However, all reviewers agreed that it is not clear from the experiments presented whether the identified transporter is the only or even the main transporter of S-Cys-Gly-3M3SH in S. hominis. They felt that to make this conclusion, the authors would need to conduct a knockout experiment. Corresponding genetic tools do exist (e.g. PMID:27531908; PMID: 25104751; PMCID: PMC3709823).
Additional specific comments are:
1) The affinity for the modified peptide appears to be relatively low in high μM range. Is this affinity compatible with levels of peptide likely encountered under physiological conditions?
2) How evolutionarily broad is the ability of Staph. species to transport S-Cys-Gly-3M3SH? Are there PepTSh homologues that are likely unable to transport the peptide because of a mutated binding site? If yes, are these species still able to take up the peptide
3) Authors may want to be more transparent in the limitations of this study as the major components of this analysis are inferred from non-skin colonizing organisms. For example, the genomic searches were targeted and could not obviate additional proteins playing alternative pathways.
4) Does the 50% reduction of model substrate 5BLCG in E. coli-ΔdtpB cells suggest the existence of one more importer? Such an importer would probably help to explain the discrepancies seen with some mutant activity between proteoliposomes and whole cells. Does such an importer have a homolog in Staphylococcus hominis?
5) The average B-factor of the structure is 57.0, which is fairly low and indicates rigidity for the protein. If the residues at the substrate binding site are flexible as suggested by the authors, do they display higher B-factor values?
6) From the data presented, is it possible to estimate the turnover rate of the transporter?
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "Structural basis of malodour precursor transport in the human axilla" for further consideration at eLife. Your revised article has been favorably evaluated by Richard Aldrich (Senior Editor) and a Reviewing Editor.
There is only one small remaining issue. One of the reviewers has requested that you estimate the turnover rate of the transporter. You have provided the values in your response in nmol/min/mg. I believe that the reviewer wanted to see the numbers in mol/mol/min. It seems to me that this number comes up to approximately 1 mol/mol/min for the wild type transporter (assuming MW ca 50 kDa) unless I made an arithmetic error. This seems a bit slow. Could you please verify the turnover rate and include a comment on it in the manuscript.https://doi.org/10.7554/eLife.34995.027
- Simon Newstead
- Gavin H Thomas
- Gavin H Thomas
- Simon Newstead
- Daniel Bawdon
- A Gordon James
- Gavin H Thomas
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank the staff of beamlines I24 Diamond Light Source, UK, and ID23eh2 at the European Synchrotron Radiation Facility (ESRF) for assistance. This work was supported by a Wellcome award (102890/Z/13/Z) to SN and BBSRC LINK funding to GHT (BB/N006615/1) and was initiated by CBMNnet proof of concept funding (BB/L013703/1) to GHT and SN. DB was supported by a BBSRC CASE studentship (BB/H016201/1).
- Olga Boudker, Reviewing Editor, Weill Cornell Medicine, United States
© 2018, Minhas et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

domain: chemistry


text:
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The taste is good.
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clean umeboshi - no chemicals!
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domain: food


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Copyright registration legally establishes a creator's exclusive rights to reproduce, distribute, and display their original work. Copyright registration is a crucial step in protecting your intellectual property right that benefits you in the long run irrespective of your location or time. It plays a vital role in safeguarding your creative work by providing legal protection and exclusive rights.
In today's digital age, protecting intellectual property is crucial for creators and artists. One of the most effective ways to secure your creative work is through copyright registration. Let us discuss further about this process, its key terms, considerations, and other relevant aspects associated with the same.
Importance of Copyright Registration
Copyright registration provides legal protection and exclusive rights, ensuring that your work is safeguarded from unauthorized use or reproduction. The following points further root for its importance in the legal world.
- Preserving Ownership and Establishing Rights: Copyright registration establishes a clear record of ownership for your creative work. It serves as concrete evidence that you are the original creator and holds legal weight in case of disputes. By registering your copyright, you can confidently assert your rights and protect your work from unauthorized use.
- Deterrence and Unauthorized Use Prevention: Registering your copyright acts as a deterrent for potential infringers. When others see that your work is officially registered, they are more likely to respect your rights and seek proper permission before using it. Copyright registration sends a strong message that you take your work seriously and are prepared to take legal action to protect it.
- Access to Legal Remedies: Copyright registration provides you with valuable legal remedies in the event of infringement. If someone unlawfully uses your copyrighted work, registration enables you to file a lawsuit and seek damages. Without registration, you may be limited in the legal options available to you. By registering your copyright, you ensure that you have the necessary tools to enforce your rights and seek appropriate compensation.
- Public Notice and Proof of Validity: When you register your copyright, it becomes a matter of public record. This public notice serves as a warning to potential infringers that your work is protected and that unauthorized use may result in legal consequences. Additionally, copyright registration provides proof of the validity and existence of your work, which can be crucial when asserting your rights in a legal setting.
- International Protection: Copyright registration can provide international protection for your creative work. Many countries have reciprocal agreements and treaties that recognize copyrights registered in other jurisdictions. This means that by registering your copyright in your home country, you may also enjoy protection in other countries, making it easier to defend your rights on a global scale.
Key Aspects of Copyright Laws and Regulations
We will now explore the key aspects of copyright laws and regulations, including what copyright protects, the duration of copyright, fair use, and the consequences of copyright infringement.
- What Copyright Protects: Copyright protects original authorship works or tasks fixed in a tangible medium. This includes literary works, music, art, photography, films, software, and other creative expressions. Copyright gives the creator exclusive rights to reproduce, distribute, display, and perform their work, as well as the right to create derivative works based on the original.
- Duration of Copyright: Copyright protection lasts for the entire life of the creator plus a certain number of years after their death. In many countries, including the United States, copyright protection lasts for the creator's lifetime plus an additional 70 years. After this period, the work enters the public domain, meaning it can be freely used by anyone without permission.
- Fair Use: Fair use helps in limiting the use of copyrighted material without permission from the owner. It takes into account factors such as the purpose and character of the use, the nature of the copyrighted work, the amount used, and the effect on the market for the original work. Fair use allows for activities such as criticism, commentary, news reporting, teaching, and research.
- Copyright Infringement and Consequences: Copyright infringement occurs when someone violates the exclusive rights of a copyright owner without permission or a valid legal defense. Consequences of copyright infringement can include legal action, financial damages, injunctions, and the seizure or destruction of infringing copies. It is important to respect copyright laws and obtain proper permits or licenses when using copyrighted material to avoid legal repercussions.
- International Copyright Protection: Copyright laws vary from country to country, but international agreements and treaties aim to provide some level of harmonization and protection across borders. The Berne Convention for the Protection of Literary and Artistic Works is one of the key international agreements that establish minimum standards for copyright protection among its member countries. Creators should be aware of the copyright laws in their own country as well as any international treaties that may apply to their work.
Benefits of Copyright Registration for Creators
Registering your copyright offers several advantages.
- It provides legal evidence of your ownership, making it easier to establish your rights in case of infringement disputes.
- Copyright registration grants you the ability to file a lawsuit and seek damages against infringers.
- It allows you to record your copyright with customs authorities to prevent the importation of infringing copies.
- Registering your copyright can act as a deterrent, as potential infringers are more likely to respect your rights when they see that your work is officially registered.
Tips for a Successful Copyright Registration
To ensure a smooth copyright registration process, here are some valuable tips:
- Register Your Work Promptly. Register your copyright as soon as possible after creating your work to establish a clear timeline and preserve your rights.
- Keep Comprehensive Records. Maintain detailed records of your creative process, including drafts, revisions, and dates of creation. This documentation can be valuable evidence in case of disputes.
- Consider Professional Assistance. If you have complex copyright needs or face challenges during the registration process, consulting with a copyright attorney or intellectual property expert can provide guidance and expertise.
- Respect Others' Copyrights. While protecting your work, it is essential to respect the copyrights of others. Obtain proper licenses or permissions when using copyrighted materials to avoid infringing on someone else's rights.
- Stay Informed about Copyright Laws. Copyright laws evolve over time, so it is crucial to stay updated with any changes or amendments. Regularly check for updates and consult legal resources to ensure compliance with current regulations.
Key Terms for Copyright Registrations
- Intellectual Property: Copyright registration is a legal process that helps protect intellectual property, which includes original works of authorship such as books, music, art, and software.
- Originality: Copyright registration requires that the work being registered is original and not a copy of someone else's work, emphasizing the importance of creativity and uniqueness.
- Exclusive Rights: Copyright registration grants the creator exclusive rights to reproduce, distribute, display, and perform their work, allowing them to control its usage and determine how it is shared or monetized.
- Public Domain: Copyright registration ensures that the creator's work is not in the public domain, meaning it is free for anyone to use without permission or restrictions.
- Legal Protection: Copyright registration provides legal protection, enabling creators to take legal action against those who infringe upon their copyrighted work, ensuring their rights are upheld in a court of law.
Final Thoughts on Copyright Registrations
Copyright registration is a crucial step for creators to protect their original works and assert their rights in the digital age. It provides legal evidence of ownership and serves as a deterrent to potential infringers. By registering your copyright, you gain the exclusive rights to reproduce, distribute, display, and perform your work, allowing you to control its usage and determine how it is shared or monetized.
Moreover, copyright registration grants you the ability to take legal action against those who infringe upon your rights, ensuring that you have the means to enforce your ownership and seek appropriate remedies for any damages caused. It strengthens your position in negotiations and licensing agreements, providing you with leverage and protection in the marketplace.
If you want free pricing proposals from vetted lawyers that are 60% less than typical law firms, click here to get started. By comparing multiple proposals for free, you can save the time and stress of finding a quality lawyer for your business needs.

domain: law


Mercedes-Benz plans to make China a central focus in its upcoming electric vehicle (EV) campaign starting in 2025, according to CEO Ola Kaellenius. The company aims to master electric propulsion and digitalization to meet customer expectations.
Under its strategy outlined in 2021, Mercedes-Benz will produce only EVs on all new vehicle platforms from 2025 onwards. To better cater to local customers in China, the automaker is reviewing the models based on the upcoming MB.EA platform, with a particular focus on space and digital content.
China is a crucial market for Germany’s automakers, but local brands currently dominate the Chinese EV market, holding an 81% share in 2022, as per Counterpoint Research.
Notably, China has a significant ownership stake in Mercedes-Benz, with Beijing Automotive Group Co Ltd and Geely Chairman Li Shufu being its top two shareholders.
Meanwhile, Germany is aiming to reduce its economic exposure to China, prompted by efforts to derisk the economy after Russia’s invasion of Ukraine highlighted the country’s dependence on a single dominant gas supplier.
Mercedes-Benz’s decision to prioritize China in its EV campaign reflects the importance of the Chinese market in the global automotive industry. As the world’s largest auto market, China offers immense growth potential for electric mobility. However, local Chinese brands currently hold a strong grip on the EV market, presenting a challenge for international automakers like Mercedes-Benz.
Given China’s significant stake in Mercedes-Benz through its top shareholders, it is vital for the German automaker to strengthen its position in the Chinese market and tailor its EV offerings to meet the specific needs and preferences of Chinese consumers. This approach will enable Mercedes-Benz to compete effectively with local brands and gain a larger share of the Chinese EV market.
On the other hand, Germany’s efforts to derisk its economy and reduce exposure to China’s economy may have broader implications for the global economic landscape. As geopolitical tensions and supply chain vulnerabilities come to the forefront, international businesses will likely reassess their dependencies on specific countries and explore diversification strategies.
Mercedes-Benz strategic focus on China for its EV campaign reflects the country’s pivotal role in the automotive industry. The company’s efforts to address local market needs and strengthen its position in China are essential steps in its pursuit of success in the highly competitive and rapidly growing Chinese EV market. Simultaneously, Germany’s efforts to derisk its economy underscore the broader challenges and complexities of global economic interdependencies.


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In a significant boost to Prime Minister Narendra Modi’s vision for self-reliance in the defence manufacturing sector, India has cleared weapon purchases worth ₹28,732 crore, including armed drone swarms, carbines and bullet-proof jackets that will be designed and developed in the country, the Union ministry of defence said on Tuesday.
The close-quarter carbines will be for front-line soldiers deployed along India’s borders, including the Line of Actual Control (LAC) with China where the two countries have been locked in a tense standoff since May 2020, officials familiar with the matter said on conditions of anonymity.
The defence acquisition council (DAC), India’s apex weapons procurement body, accorded its acceptance of necessity (AoN) for the capital acquisition proposals at a meeting chaired by defence minister Rajnath Singh. Under India’s defence procurement rules, AoN by the council is the first step towards buying military hardware.
The procurement of autonomous surveillance and armed drone swarms was cleared to boost the army’s capability in modern warfare in the backdrop of drone technology proving to be a force multiplier in military operations and recent conflicts around the world, the defence ministry said in a statement.
Last year, the Defence Research and Development Organisation (DRDO) showcased indigenous capability to carry out offensive missions in enemy territory with scores of drones working in assorted formations to identify, encircle and strike targets, with loitering munitions being developed to meet a key military requirement of our soldiers.
Surveillance and armed drone swarms feature on a new list of ‘Make in India’ projects that the army is pursuing in partnership with the defence industry, the officials said. Drones within a swarm can carry out a wide range of missions, including strikes against tanks, infantry combat vehicles, ammunition holding areas, fuel dumps and terror launch pads.
The DAC also approved the purchase of around 400,000 close-quarter carbines “to combat the current complex paradigm” of conventional warfare, hybrid warfare and counter-terrorism at the borders, the defence ministry statement said.
“This is set to provide a major impetus to the small arms manufacturing industry in India and enhance aatmanirbharta (self-reliance),” the ministry said.
India has taken a raft of measures to boost self-reliance in the defence manufacturing sector over the past two to three years, including curbing imports and allocating funds for domestic procurement.
“India is making planned, steady and focused progress to achieve aatmanirbharta in the defence sector and several projects have been sanctioned for the local industry. Frontline soldiers urgently need carbines,” said former director general of military operations Lieutenant General Vinod Bhatia (retd).
The bullet-proof jackets will offer enhanced protection to soldiers deployed along the Line of Control (LoC) with Pakistan.
“Considering the demand of enhanced protection against the threat of enemy snipers to our troops deployed along the LoC, and in close combat operations in counter-terrorism scenario, the DAC accorded AoN for bullet-proof jackets with Indian Standard BIS VI level of protection,” the ministry said.
The defence ministry earmarked 64% of the capital acquisition budget for the domestic industry in 2021-22, but it was able to “overachieve this target” and local military purchases accounted for 65.5% of the capital budget, as previously reported.
For 2022-23, India has earmarked ₹84,598 crore – 68% of the military’s capital acquisition budget – for the purchase of locally produced weapons and systems, besides setting aside 25% of the defence research and development (R&D) budget for private industry, start-ups and academia.
The DAC also approved a navy proposal to procure an indigenously-upgraded 1250KW-capacity marine gas turbine generator for power generation on board the Kolkata-class of warships, and 14 fast patrol vessels for the Indian Coast Guard with 60% indigenous content.
The government has imposed a phased ban on the import of 310 different types of weapons and systems. These include light weight tanks, naval utility helicopters, artillery guns, missiles, destroyers, ship-borne cruise missiles, light combat aircraft, light transport aircraft, long-range land-attack cruise missiles, basic trainer aircraft, multi-barrel rocket launchers, assault rifles, sniper rifles, specified types of helicopters, next-generation corvettes and airborne early warning and control (AEW&C) systems.

domain: news


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When worlds collide December 31st 2011 The sun smiles down on me and pierces through my sunglasses as I peer up accusingly looking for the source of all the heat.My jeans are soaked, I am sweating from parts of my body I never knew could produce salty beads, but under the circumstances we are close to the equator and I am inappropriately dressed. I cannot blame the sun. I’d moan more if it was raining. I hate ... read more
Asia » Singapore Singapore was founded as a British trading colony in 1819. It joined the Malaysian Federation in 1963 but separated two years later and became independent. It subsequently became one of the world's most prosperous countries with strong international ... ... read more
In a nutshell; I am Jess. I was 27 years old, and left a wonderful teaching job in search of unemployment and volunteer opportunities across the other side of the world. Some say it's foolish, I was inclined to agree but these experiences altered me in ways I never imagined possible. I accomplished dreams, re-found lost passions, set up a charity in Sapa, taught in Cambodia, established a teaching course in Vietnam and published articles and photos for magazines and books.
... full info

domain: geography


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Dumplin’ meets Daisy Jones & the Six in this split-POV love song to country idols, romantic road trips, and queer love.
They say never meet your idols. But nothing about digging up their deepest secrets.
Seventeen-year-old aspiring journalist Darren Purchase has been a lifelong fan of country music legend Decklee Cassel, who’s as famous for her classic hits as she is for her partnership with songwriter Mickenlee Hooper. The same Mickenlee who mysteriously backed out of the limelight at the height of their careers, never to be heard from again. Now Decklee’s televised funeral marks the unveiling of her long-awaited time capsule. But when it’s revealed to be empty, a trail of scavenger-hunt clues unfolds, leading to a whopping cash prize for whoever finds the real capsule. Darren knows there’s a story there–and she’s going to be the one to break it. Even if it means a spontaneous road trip with her coworker Kendall.
Flashback to 1963, when a young runaway Decklee has her sights set on fame and glory. As she claws her way to the top over the years that follow, it’s Mickenlee’s lyrics that help rocket her to stardom. But as their relationship evolves beyond the professional, it threatens everything Decklee has worked for. What else will she sacrifice to hold on to her dreams?
Told in alternating perspectives, Every Time You Hear That Song is a queer coming-of-age story celebrating country music, complicated women, and living authentically. There’s more to Decklee’s story than Darren ever could have guessed, but the real story she has to tell is her own.

domain: artistic


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Elvis Presley Posters
Elvis Presley Posters is inordinately transcendent and makes a excellent wall decoration, the item dimension is 24 x 18 inch and the price is $259.99. If you're trying to chase top recommended Elvis Presley Posters with reasonable price, the poster is the magnificent deals for you.
Type: Framed Memorabilia
Dimension: 24 x 18 inch
Elvis Presley Posters is very brawny to frame but it's an stimulating poster. The item is radically cheap and arrived in the last few days in durable packaging that protected the one very well. This produce itself is gorgeous and radically high resolution. This item looks riveting, and overall a immensely terrific experience since it was so simple and easy. A transcendent zombie poster! Theis luxury for displaying, not too big, nor too small. The unit stocks competitive pricing to consumers. This produce comes in sizeand is amazing. Elvis Presley Posters measuring , these are also printed on cardboard.


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Comedy Podcasts >
American Playboy: An Ode To Hugh Hefner
Hugh Hefner, founder of Playboy magazine, civil rights activist, and American Icon, has passed away at the age of 91. Tonight, I say a few words about the man, the myth, the legend, and share my thoughts on how this man, who so many just thought of as a peddler of smut, was actually an innovator, a civil rights activist, and a visionary. There is no more fitting example of the American Dream than Hugh Hefner. After this podcast, I think you will agree.

domain: sociology


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Francis Herman Evison is the 5th oldest and 3rd son of William Evison and Ellen Wickham. He is my great uncle — brother to my great grandfather, William St. Thomas Evison.
Francis was commonly known as “Frank” and he was named probably for his grandfather’s brother, Francis Evison, and his grandmother’s illegitimate son, Herman Frost.
On 11 Jan 1871, Frank was born in Westminster Township, Middlesex County (Ontario). He lived with his family until he married Charlotte Hill on 10 May 1893 in Lambeth, Middlesex County (Ontario).
Charlotte is the daughter of George and Mary Hill and she was born in Yorkville, York County (Ontario) on 24 Apr 1859 — making Charlotte almost 12 years older than Frank!
Update on 13 Dec 2013
Before she married Frank, Charlotte had a daughter named Marion who was born in 1885. Marion lived with her grandfather, George Hill, in Delaware (Ontario) until she married Harry Edwin Reynolds Davis in Alberta (Canada) in 1912.
Between 1894 and 1900, Frank and Charlotte had 4 children, all born in Delaware, Middlesex County (Ontario):
- Hannah Frances born on 25 Feb 1894
- Norman Edward born on 30 Oct 1895
- Richard Herman born on 20 Mar 1898
- Charlotte Ellen “Nellie” Evison (also known as Irene Olive) born on 6 Aug 1900
In 1901, the family was living together in Delaware and by 1911, the family had moved to Lobo Township (probably to Komoka).
Frank was 45 years old when he enlisted in the Canadian army in Strathroy (Ontario) on 23 Feb 1916. He was assigned to the 135th O.S. Battalion – C.E.F. and was promoted to Corporal — but was wounded during the fighting and returned home.
The photograph below was sent to me by a distant relative of Frank and Charlotte’s. Frank’s Attestation Papers described him as being 5 ft 7 in tall (with a dark complexion, grey eyes and brown hair). Looking at the photo and how tall Charlotte is compared to Frank, I would guess that Charlotte was about 5 ft tall.
And below are scanned copies of “paintings” (or maybe colourized photographs?) of Frank and Charlotte that were given to me by their granddaughter, Shirley. Shirley confirmed that Charlotte was a tiny woman and that she rarely saw her grandmother standing up — she most frequently sat in a chair.
Charlotte died in 1939 in her 80th year and Frank in 1944. He was 73 years old. They are buried together in Oakland Cemetery in Delaware, Middlesex County (Ontario).
Update on 5 Nov 2013
The 1921 Canadian Census has now been uploaded and indexed by Ancestry.ca. I found Frank and Charlotte living in Zone Township, Lambton County (Ontario) in 1921.

domain: history


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Please tell me it's a joke.
I saw this in the classified section in the most recent issue of ArtNews under "Job Opportunities:"
"60-year-old male major artist in Queens seeks female assistant, collaborator, organizer for learning experience of a lifetime. All mediums. 2 studios. No money involved. 917-214-4899."
Sign me up!
It's gotta be a joke...


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Jul 22, 2022
Are you looking for an effective way to transform your hotel's digital presence by achieving the maximum result? Now you can easily take full control of your hotel operation with advanced Hotel Management Software.
Mar 05, 2022
A AsiaTech hotel booking engine is a way for hotels to fight the OTA dominance, necessary though it may be. AsiaTech hotel booking engine can be simply added to your existing website enabling guests to book rooms directly from your hotel's website.
Apr 17, 2021
Are you running a travel business? Do you want to be online to expand your customer base? It is necessary to join hands with the AsiaTech Inc travel technology company in Noida. Call For Channel Manager 7840-800-800
Aug 10, 2016
Do you own a Hotel or a Travel Agency? Then you must build a website at affordable price which is easy to use and will also make you feel quite amazing about the experience. The visitors coming to your website must feel enigmatic while browsing.
Jun 25, 2016
Asia Tech Inc is one of the emerging hotel software companies which provide many online services such as Channel Manager, Hotel Booking Engine, Channel Management System, Travel Agent Website Development, Web Development Services, SEO and many more.

domain: travel


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European Holocaust Research Infrastructure - The Austrian Consortium
EHRI-AT connects archives, museums, commemorative organisations and research institutions in the field of Holocaust research, documentation and education in Austria.
EHRI-AT works towards making source material on National Socialist crimes in Austria findable and accessible through a digital research infrastructure.
EHRI-AT fosters cross-border cooperation and promotes research on the various groups of victims of the Holocaust.Learn More
More about EHRI's mission and vision
Below are some of the innovative services created by EHRI for use by all.
The full-day workshop “EHRI and Microarchives in Austria” took place at the Vienna Wiesenthal Institute for Holocaust Studies on March 4, 2024. The Institute presented EHRI’s plans to improve support for microarchives and invited Austrian microarchive owners and experts to discuss the opportunities and challenges of collaboration. Read More


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- monthly budget planner mod apkAPK v3.3.9
A CERTAIN MAN made a wooden image of Mercury and offered it for sale. When no one appeared willing to buy it, in order to attract purchasers, he cried out that he had the statue to sell of a benefactor who bestowed wealth and helped to heap up riches. One of the bystanders said to him, "My good fellow, why do you sell him, being such a one as you describe, when you may yourself enjoy the good things he has to give?' "Why," he replied, "I am in need of immediate help, and he is wont to give his good gifts very slowly."Secret of the Pendulum Mod Secret of the Pendulum v184.108.40.206 mod Features:Modify coins and diamonds is unlimited.Following a series of clues, join our female heroines Lily and Madison. Together you must help solve the murders that transpire in this chilling time-tale mystery. Journey through this thrilling story-driven game navigating the clues that you discover.GREAT FEATURES:• Uncover hidden objects from beautifully hand crafted scenes.• Investigate crimes scenes to bring you closer to the truth.• Engaging and secretive characters that have their own agendas.• Enjoy playing with all your friends, share gifts, items, help your friends get items they need to move on, and compete with them in the game Leaderboards if you want.• Each Episode/Memory brings you closer to unravelling the mystery that lays before you.• Challenging puzzles that leads you to discover new clues. • Catch the killers and bring them to justice.• Adventurous new Episodes/Memories delivered with each major update.PLEASE NOTE – Secret of the Pendulum is completely free to play, however some game items can also be purchased for real money. If you don't want to use this feature, please disable in-app purchases in your device's settings.**CUSTOMER REVIEWS**5/5 Review by CrowofCarnage“Great animation, great plot line, puzzles aren't too hard or too easy, and it's completely free! There should be more apps like this in the App Store, you guys should make more games like this and free too!!! Super awesome, everyone should get this app it's super fun!!!”5/5 Review by Rachel4554“Fun and challenging”5/5 Review by Deastrid“Enough challenges to keep you busy!”**Highly recommended for fans of adventure games like Secret of Monkey Island and hidden object games like Pearl's Peril.At GamesCafe we worked hard to make Secret of the Pendulum™ the best game possible. We really hope that you enjoy this game and tell your friends and family! :)Twitter: https://twitter.com/secretpendulumFacebook: https://www.facebook.com/SecretofthePendulum

Clash Royale's Poison spell has been a formidable force ever since it was first added to the game, and even a nerf several months back hasn't slowed it down too much.

But if you've yet to find a way to make it work for you – or stop other people from destroying you with it – our resident Clash Royale expert Josh Jones has a few ideas on what you need to do.

Reading Comprehension Kids App Mod Reading Comprehension Kids App Mod (paid) 1.7c Features:paid for freeReading comprehension fun is an educational application for younger ones. It's aim is to make your children learn how to read the passages and answer the given question of that paragraph. Read through a range of different stories and test your comprehension with a variety of questions about the selected passages. This app has a fun learning way that the kids will love playing while learning new things and getting new skills. Children are encouraged in this comprehension app through interactions with a simple and beautiful graphical interface with great sounds. It is designed for young children targeting grades 1, 2 and 3. To make students learn and know English better and build a strong communication skills is the aim taken along making this app. It targets young children to strengthen their basic knowledge skills which they take along themselves for the rest of their lives. This app is aimed at helping individuals who need extra practice with reading comprehension and learning how to recall specific details of stories. Engaging, colorful, and smooth child-friendly game play and controls which makes playing this game more exciting and learning a fun thing for kids.You don't need to look for activities and passages scrolling down the internet as you can all of it in one.Reading Comprehension Fun Game Features:•Read and test your comprehension skills.•Reading comprehension for early reader.•Engaging high-interest passages.•Read and answer questions about each passage.•It's never too early to improve your reading skills.•Paragraphs for young children of grade 1, 2 and 3.•Check wrong and right answers.A child's comprehension must be strong in order to make them confident enough to be able to communicate to others. It is important to teach them basics from an early age and also very time consuming to maintain their interest along. Young students tend to get distracted much quicker and this app is surely to provide ease to teachers and parents in keeping them stick to the learning routine. The overall content and interface is highly child friendly and appropriate for kids to improve their comprehension skills. We have come up with this app for kids with the purpose of making education fun, interactive and easier for them. Parents can leave their kids to play with and they will learn new things on their own. Kids will enjoy and have fun along this app and is the best way to make them learn without struggling with getting their attention.Many more learning apps and games for kids on: https://www.thelearningapps.com/'O father, what a hell of witchcraft lies
Jet Airplane War - Fighter Air Combat Mod Jet Airplane War - Fighter Air Combat v 1.1 Mod Features:Enter the game to give a lot of moneyHere you are a pilot, you have to fly fighter jet planes in the sky, and you have to defeat your enemies in air combat by shooting bullets and missiles at them.In air combat, every time, you will be given different types of missions like destroying enemy warships, buildings, fighter planes, etc.Along with you, there will be few other fighter jet planes, who will be fighting from your side, so as a team, you can defeat the enemies.You will get notified during the air combat if any enemy has locked a missile on your plane so you can save yourself from the hit.In every level of the game, you will get some stars out of three, and you will earn some XP and coins. To unlock a new map, you will require a certain number of stars.You can unlock new modern fighter jet planes using coins and gems and use them in air combat.Fighter jet plane controls:- From the bottom left side of your screen, you can control the direction.- From the bottom right side of your screen, you can control speed.- Using the cross, you can aim at your targets.- You will also see the altitude and speed of your plane.- Using the machine gun, you can continuously fire bullets.- You can lock the enemy target and fire a missile.- You can see the enemy location from the radar.Download the game, fly your fighter jet planes in the sky, do an airstrike on enemy targets, and defeat them in war.

domain: game


The VDB-L-QTW (WIRED) boompole, measuring 80cm (31.5 inches) collapsed and 384cm (151 inches) extended.
- Rigid, light and strong. VDB Boom Poles benefit from the recent advances in carbon fiber winding and 30 years of crafting one of the most internationally recognized boom poles.
- Greater length from a reduced size. The six section design offers an increased length, from a reduced collapsed size.
- Reduced hand noise. A polished surface on the outer tube reduces handling noise dramatically.
- Silent. Between each section of the pole is a precision machined internal incompressible joint, offering the
minimum space to achieve a soft slide. It also contributes to the rigidity of the system and prevents the tubes from rattling when rotating or swinging quickly.
- Instant optional cabling. straight or coiled internal microphone cable can be set or removed instantly in all the models without technical intervention.
- A quick release top screw.


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On Saturday, August 26th, 2023, Remember Everyone Deployed (RED) will be hosting a Candlelight Vigil in honor of the 13 Military Members that were killed in Kabul on August 26th, 2021, marking the 2nd Anniversary of their ultimate sacrifice. All are welcome to attend. The ceremony will begin at 1900 at the Vietnam Memorial Gardens in Jacksonville, NC. This year, the memorial will be hosted by retired U.S. Marine Corps Sgt. Maj. Raquel Painter. The appointed keynote speakers will be Corporal Krysten Kagels and Corporal David Austin, Marines who served and knew Sgt Nicole Gee. Speeches will also be given by North Carolina Senator Michael A. Lazzara, Swansboro Mayor John Davis, and Onslow City Commissioner Cindy Edwards.
We look forward to seeing you all there to honor these fallen heroes.