Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant.
We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5′UTR and first 47 amino acidsof FliC from E. coli (but no 3′UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L−1, in low cell density culture.
We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.
A persistent goal of biotechnology is to produce recombinant protein at reduced cost, while maintaining high product quality and yield. For many protein biologics, production in prokaryotes (chiefly in E. coli) is favoured. Major advantages include fast growth to high cell densities on cheap carbon sources, and simple scale up [1, 2]. However, as in many industrial biotechnology (IB) processes, downstream processing accounts for a high proportion of costs [3–5]. If protein product was secreted by the expression organism into the growth media, these costs could be reduced, as purification could be simplified, i.e. eliminating the need to lyse cells, or to remove cellular contaminants, which can compromise product purity, and may elicit an immune response in humans (e.g. lipopolysaccharide in E. coli) [6–8]. In addition, product yield and quality may be increased, as secretion would bypass cytoplasmic and periplasmic proteases. Extracellular secretion may also improve protein folding and solubility, while also reducing the formation of inclusion bodies, thus increasing product yield and quality [9–12].
Secretion of heterologous protein in E. coli has been utilised, however the majority of work concerns secretion into the periplasm via Sec or Tat-dependent systems . While this strategy can yield titres in the mg L−1 range (e.g. 30 mg L−1 human growth hormone , 60 mg L−1 GFP ), this is only achieved following purification from the periplasm. Alternatively extracellular localisation of protein can be achieved In E. coli without directed secretion to the extracellular space via strategies that make use of 'leaky strains' , where heterologous proteins are fused to a secretion tag (for example ompA, ompC, pelB [16, 17]), to enable directed secretion to the periplasm, followed by leaking into the supernatant. While titres may be favourable, the presence of additional periplasmic proteins adds complexity to purification, e.g. while 550 mg L−1 cell culture of lipase B tagged with the pelB signal sequence was found in the extracellular fraction of E. coli, high concentrations of periplasmic proteins were also detected, and only one-fifth of the product was able to be recovered . To date, true secretion (i.e. to the extracellular space via a defined secretion system, rather than through leaking or lysis) of heterologous protein by prokaryotes has been achieved at up to approximately 50 mg L−1, in organisms such as Bacillus and Pseudomonas [19–21], as opposed to the g L−1 titres achieved in eukaryotes such as Pichia pastoris . In E. coli, attempts to secrete proteins directly into the media have been limited, with evidence of low levels (i.e. 1 µg to 1 mg L−1 of heterologous protein) via type I secretion system (T1SS) using either the Haemolysin or lipase transporting ABC transporter systems [23–28]. Another option for direct secretion into the media, is the bacterial flagellar type III secretion system, which has been utilised in E. coli [29–32], Bacillus  and Salmonella enterica sv. Typhimurium [33–36]. In addition, secretion of up to 30 mg L−1 of a range of polymers , such as tropo-elastin and spider silk, have also been reported using the injectisome type III secretion system of S. enterica [37–39].
In this report we focus on the E. coli FT3SS, due to a preference in IB for both E. coli, and non-pathogenicity associated secretion systems. E. coli have 4–10 flagellum of around 20 μm in length, each comprising of up to 30,000 flagellin (FliC) monomers [40–43]. The FT3SS, despite having evolved to build a flagellum and provide motility to the cell, is effectively an efficient protein secretion machineable to assemble a multi-component structure composed of several thousand subunits on its surface. Proteins initially assemble at the inner membrane, resulting in a pore of about 2.0 nm diameter, through which the majority of the remaining flagellar proteins are exported, unfolded, to the distal end of the existing flagellar structure . Therefore in the context of biotechnology, the FT3SS may provide a one step, high capacity route for protein export.
The basic flagellar structure is comprised of the basal body (motor and secretion apparatus), hook (universal joint), and filament (propeller), which are assembled in an ordered manner, controlled by well-understood checkpoints . One key feature is the existence of a flagella master regulator (FlhD4C2-class I) that activates class II flagellar genes. These class II genes transcribe the basal body and FT3SS apparatus, along with the sigma factor FliA, which in turn promotes the transcription of the class III genes (for the filament, motor, hook associated and hook proteins, along with the chaperone and chemotaxis proteins) [46, 47]. Furthermore, transcription is also coupled to assembly, as levels of FliA are modulated by the anti-sigma factor (FlgM), which is secreted upon hook-completion [48–50]. Additionally, given the energetic cost of motility, expression of the master regulator is modulated by many environmental cues that alter gene expression of the flhDC operon or activity of FlhD4C2 [51, 52].
Here, we aimed to capitalise on the fact that upon mechanical shearing or breakage, flagellar continue to secrete unfolded FliC monomers which are transported to the distal growing tip, allowing regrowth under the FliD cap [53–55]. In addition, in the absence of the FliD cap, or other structural proteins (e.g. FlgKL), secretion of FliC monomers into the media without polymerisation occurs [56, 57]. In addition, the N-terminal secretion signals of FT3SS subunits are well characterised and have been exploited to direct non-FT3SS subunits for secretion through the FT3SS in E. coli [29–32] and (their close relatives) Salmonella [34–36]. We aimed to develop an improved, modular FT3SS E. coli strain for high yielding secretion of a range of therapeutic recombinant proteins. Our eventual aim is to surpass the secretion titres reported in other E. coli secretion systems, by achieving extracellular secreted titres in the high mg L−1 range. We hope to accomplish this using controlled secretion to the extracellular space, combined with the absence of cellular contaminants. Here we report progress towards these targets via strain development, secretion signal optimisation, rigorous use of controls, and development of a high-throughput assay to measure FT3SS secretion.
In this study we used E. coli MC1000 as the parent strain, partly for biosafety given its leucine auxotrophy, but also given the presence of an IS5 insertion sequence upstream of flhD. This reduces negative transcriptional regulation on flhDC, by disrupting promoter binding sites for the negative regulators LrhA and OmpR (for annotated sequence see Additional file 1: Fig. S1) [58–60]. Previous studies of FT3SS dependent protein secretion in E. coli employed a ΔfliD 'cap-less' strain [29–31, 36]. We also considered alternative strategies, given the potential for this strain to influence and suppress FlhD4C2 activity via post-translational protein–protein interactions of the FlhDC complex with FliT (the chaperone of FliD) [61, 62]. Specifically, we created a 'HAP-less' (ΔflgKL) strain, which would not incur feedback on FlhD4C2−, but would theoretically decouple secretion from assembly, i.e. as opposed to the ΔfliD strain where more free FliT would potentially repress FlhD4C2 activity, and thus flagellar gene expression . In addition, it has also been established that in ΔflgKL mutants, unpolymerised FliC monomers are secreted into the media, rather than assembling into a filament . To lessen competitive secretion, strains also lacked fliC. We found that the HAP-less and cap-less strains displayed identical growth curve parameters (Additional file 1: Fig. S2). We then compared their ability to export a 39 kDa shortened version of FliC that lacked the central D3 domain (residues 191–283) so that it could be easily distinguished from native FliC. This secretion substrate was termed FliC-ΔD3 and was inserted into plasmid pTrc99a-FF to create pTrc-FliC-ΔD3, and expressed by addition of IPTG. This substrate was efficiently secreted (Fig. 1) and also produced a fully motile strain when provided in trans in a ΔfliC strain (Additional file 1: Fig. S3).
As shown in Fig. 1a, b when FliC-ΔD3 was expressed with induction using 0.05 mM IPTG, we observed approximately twofold more FliC-ΔD3 (on average 7.4 mg L−1) in the secreted fraction of the HAP-less strain in comparison to the cap-less. Notably, we also observed increased amounts of FliC intracellularly in the HAP-less strain (Fig. 1a, c) but found overall that the HAP-less strain was 1.3 times more efficient at secreting protein (secreted FliC-ΔD3 as a function of total FliC-ΔD3, i.e. 7.4 mg L−1 divided by 98 mg L−1 for the HAP-less strain). Based on the absence of additional proteins in the cell supernatant (Fig. 1a—Coomassie), we infer that the improved titre of secreted FliC-ΔD3 was due to FT3SS secretion, as cell lysis was not prevalent in either of these strains. We therefore used the HAP-less, ΔfliC ΔflgKL strain in subsequent experiments as our prototype FT3SS secretion strain.
Before further experimentation, we defined optimal parameters for expression and production of proteins via the FT3SS-assessing that the optimal OD600 for harvest of cells for maximal secretion of FliC-ΔD3, is between an OD600 of 1 and 2, i.e. the mid-late exponential phase (Fig. 2a and inset). We then examined the effects of varying the inducer (IPTG) concentration on expression and secretion of FliC-ΔD3. We harvested cells at an OD600 of 1.0 and show that 0.05 mM IPTG produced the highest concentration of both intracellular and secreted FliC-ΔD3 (Fig. 2b), therefore this procedure forms the basis of experiments.
To establish whether secretion via the FT3SS could be improved from this basal level, we considered a series of mutations that might boost overall flagellar gene expression. We then compared their effect on secretion to a number of negative controls, to establish the absolute specificity of this system. As seen in Fig. 3a, b, use of an flhDC null mutant, with no FT3SS present, resulted in complete lack of FliC-ΔD3 secretion even when overproduced in the cytoplasm (by addition of 1 mM IPTG). In parallel we also tested secretion of FliC-ΔD3 in a (ΔfliC ΔflgKL) ∆flgDE strain, in which secretion of 'late' filament substrates (i.e. FliC) is abrogated due to the absence of substrate specificity switching [57, 63], and observed a complete lack of secretion in this strain. Further to these controls, we also tested for the presence of the strictly cytoplasmic, GroEL chaperonin protein. Aside from a negligible amount in the ∆flhDC strain, the cytoplasmic contaminant GroEL was absent in secreted fractions (Fig. 3c). This demonstrates that cell lysis was not prevalent, and that the two secretion strains are no more 'leaky' that the negative control strains. Together, this confirmed that the presence of FliC-ΔD3 monomers in the secreted fraction was due to targeted FT3SS secretion alone.
We then examined the effect of mutations that might upregulate flagellar gene expression. Our initial target was the protein quality control protein, ClpX, a component of the ClpXP protease complex which acts on FlhD4C2 . In previous studies the removal of clpX in Salmonella and enterohaemorrhagic E. coli resulted in an accumulation of FlhD4C2, an increase in the amount of FliC subunits secreted, and increased cell motility [65–67]. Here we show that the removal of clpX from wildtype E. coli MC1000 cells resulted in an increase in filament proteins and increased motility (Additional file 1: Fig. S4a–d). The removal of clpX from the prototype strain resulted in an increase in both intracellular and secreted FliC-ΔD3 (Fig. 3a, b). The secretion efficiency (secreted protein as a proportion of the total (intracellular and secreted) protein) did not alter, however we did measure a 1.6-fold average increase in the titre of secreted protein, and a maximum estimated secretion of 34 mg FliC-ΔD3 L−1 culture medium achieved from ΔfliC ΔflgKL ∆clpX (calculated by comparison to a FliC-ΔD3 standard). We also noted that ΔfliC ΔflgKL ΔclpX cells were on average 1.2 times longer (p < 0.001. t-test) than ΔfliC ΔflgKL cells, with a higher abundance of very long cells (> 0.8 µm) (Additional file 1: Fig. S4e–g). As we detected significantly more (p < 0.05) FlhA in ΔclpX cells (Fig. 5c), this may suggest that elongated cells harbour a larger number of FT3SS basal bodies, thus allowing increased secretion, though we have no experimental evidence for this at this stage. Of note is that we also produced a ΔfliC ΔflgKL ΔdksA strain, which in contrast to what was anticipated based on published literature [68, 69], did not result in any noticeable increases in FT3SS secretion. As a result, we moved on with the ΔfliC ΔflgKL ∆clpX strain as a Mark II FT3SS strain to test additional strain modifications.
In order to move towards our goal of production and secretion of heterologous proteins via the FT3SS, we designed and tested a synthetic prototype modular vector. This contained elements aimed at optimal secretion of proteins via the FT3SS, alongside their subsequent detection and ultimately purification (FLAG and Streptavidin, TEV protease. See Additional file 1: Fig. S5). These were produced as plasmids in the IP-Free pJEXpress backbone (DNA2.0), with the first 47 amino acids of FliC (the secretion signal ), the fliC 5′UTR (which also harbours the native fliC promoter, thus allowing both T5 based-IPTG inducible, and flagellar mediated (class III, via FliA [71, 72]) gene expression), alongside the fliC 3′UTR, both of which have been implicated in influencing secretion through the FT3SS [30, 31]. Finally, restriction enzyme sites were incorporated throughout to allow modular modification of the secretion construct; notably EcoRI and PstI sites allow for the insertion of any cargo gene for secretion.
We initially chose a heterologous protein target for secretion which was non-bacterial, commercially relevant and assayable. This enabled ease of screening for secretion via an activity assay, thus facilitating higher throughput screening for improved FT3SS strains. Our choice was the well-characterised 20.8 kDa cutinase from the unicellular fungus Fusarium solani, which had previously been expressed in E. coli intracellularly [73, 74], and for which a well-established activity assay based on its esterase activity is known . Advantageously, cutinase is also relevant in IB processes, e.g. degradation of plastics [76, 77]. An E. coli codon optimised cutinase was synthesised (GeneArt®) and inserted into the pJex-fliC47-empty plasmid resulting in pJex-fliC47-cutinase, which is predicted to yield a 30.6 kDa peptide.
Following expression of pJex-fliC47-cutinase in the ∆fliC ∆flgKL strain, both intracellular and secreted cutinase were detected, via the secretion construct incorporated FLAG tag (Fig. 4a). We next developed a simple fluorimetric secretion assay, based on cleavage of 4-methylumbelliferyl butyrate (MUB) by cutinase to yield a fluorescent 4-methylumbelliferone (4-MU) . Cell-free supernatant was mixed with MUB in the presence of an appropriate buffer, and release of 4-MU was measured. This could be qualitatively observed on a UV-transilluminator to allow quick visual screening (Fig. 4b). We also established conditions for a quantitative plate-reader based version of the assay (excitation 302 nm, emission 446 nm). To test the specificity of the assay, pJex-fliC47-cutinase was transformed into negative control strains (∆flhDC and ΔfliC ΔflgKL ∆flgDE) as in Fig. 3. After subtraction of background MUB cleavage no activity was detected in strains containing pJex-fliC47-empty or LB only controls (Fig. 4c). Absence of secretion was verified by immunoblotting of the same samples (Fig. 4d). Reassuringly, we observed increased concentrations of secreted cutinase (twofold higher AU) in the Mark II ΔfliC ΔflgKL ΔclpX strain compared to the ΔfliC ΔflgKL prototype strain. We used this assay to detect a range of concentrations of secreted cutinase (0.04–0.20 mg L−1), and note that the assay could detect cutinase concentrations is excess of this. With the assay established, it was possible to screen a larger number of strains and secretion signal variants, with the aim of generating a higher capacity, Mark III secretion strain.
The MUB-cutinase assay was next utilised to assess the secretion capacity of a number of alternative FT3SS secretion strains. These were engineered to upregulate flagellar expression, or reduce metabolic burden via the deletion of redundant genes. These included the clpX knockout strain outlined previously, and three additional targets: ∆flgMN (FlgM a negative regulator of class III flagellar gene regulation, and FlgN chaperone for FlgK and regulator of FlgM [50, 79, 80]), ∆fliDST (FliT—an anti-FlhD4C2 factor and FliST—chaperones of FliCD [61, 81]) and ∆motAB (MotAB responsible for energising flagellar rotation, but not required for secretion [82, 83]). Following their construction and establishing that growth of all strains was comparable to ∆fliC ∆flgKL (Additional file 1: Fig. S6), the supernatant was analysed for the presence of cutinase via the MUB fluorescence assay (Fig. 5a). Of all the combinations tested, only ∆fliC ∆flgKL ∆motAB ∆fliDST and ∆fliC ∆flgKL ∆clpX ∆motAB resulted in a significant improvement to secretion, in comparison to the original ∆fliC ∆flgKL strain: 1.64- and 1.67-fold respectively, (p < 0.05 and < 0.01). To ensure that poor secretion was not a factor of poor cutinase expression, the presence of intracellular cutinase in all strains was confirmed with immunoblotting (Fig. 5a—intracellular).
Having identified high capacity secretion strains that could secrete active fungal cutinase, we now wished to test a human recombinant protein: an E. coli codon optimised version of the gene encoding human 'CH2' (CH2) ScFv antibody fragment (23 kDa, donated by J. Pandhal, University of Sheffield [84, 85]), which is used as an adjuvant in human antibody drugs. This was inserted into our modular secretion vector. We compared secretion of CH2 and the native substrate, FliC-ΔD3, in the most promising secretion strains, and observed (similarly to cutinase) improved secretion in ΔfliC ΔflgKL ∆clpX ∆motAB strain: 2.57× (CH2) and 3.75× (FliC-ΔD3) over that observed for the prototype strain (Fig. 5b). For both substrates this amounted to a 2.8-fold improvement to secretion efficiency. In contrast, the ΔfliC ΔflgKL ∆motAB ∆fliDST strain did not perform in a similar manner to cutinase, when secreting CH2 and FliC-ΔD3, with no improvement to secretion over the prototype. We thus concentrated on ΔfliC ΔflgKL ∆clpX ∆motAB, as a Mark III strain.
To gain more information on the varying success of these two strains, we used an antibody directed against the FlhA protein (which is present as part of the FT3SS export apparatus) as a proxy indicator of the number of flagellar export apparatus in the cell population. Since its gene expression is under the control of FlhD4C2, FlhA is also an indicator of total flagellar gene expression. Whole cell lysates were examined by immunoblot and quantified by densitometry analysis (Fig. 5c). Mutant strains representing the individual knockout strategies were also included as controls. Overall, these data indicate that deletion of clpX results in an increase in FlhA levels by approximately 1.5-fold in both the ΔfliC ΔflgKL and ΔfliC ΔflgKL ΔmotAB backgrounds (p < 0.05). In contrast, deletion of motAB alone seems to reduce FlhA levels (0.75-fold; p < 0.01). Overall, it is evident that the absence of clpX in strains upregulates FlhA levels, and by extension, other class II and III genes, which may explain the improvements to secretion we observed.
In parallel to the studies aimed at strain engineering for increased FT3SS capacity, we also examined the influence of the secretion signal employed in the plasmid-based modular secretion vector. The reasoning here was that the literature reports that the inclusion of the first 47 residues of FliC , the 5′ and 3′UTRs [29, 30, 35], or a truncated FliC 26–47 construct with either the Salmonella or E. coli version of residues 26–28 [34, 35], can direct export of protein through a truncated FT3SS. All secretion construct (denoted SC) variants were inserted in frame with the cutinase gene, with the predicted size in kDa shown (Fig. 6). These studies were conducted using the prototype ∆fliC ∆flgKL strain, with a ∆flhDC strain and empty vector as negative controls. Expression of the prototype secretion construct (labelled as SC0 here) in the Mark II ∆fliC ∆flgKL ∆clpX strain served as an additional positive comparator. The data show that while all constructs produced cutinase (Fig. 7a), some constructs neither expressed nor secreted well (SC3, 5, 7), while others had low intracellular but significantly higher secreted levels of cutinase (SC1, p < 0.005), or, significantly high intracellular cutinase (p < 0.001) with none secreted (SC8) (see Fig. 7b). From these data it is possible to infer that the 3′UTR—while putatively influencing intracellular stability of cutinase-seems to actually reduce export (SC3, 5, 7). Whereas the intact 5′UTR and FliC 1–47 alone (SC1) seem to permit the highest levels of secretion, as this exports cutinase at 1.8-fold higher concentrations than the prototype (SC0). This led us to examine the possibility that the FliC 5′UTR alone, has the ability to direct secretion-as has been indicated by other workers . However, when we inserted the cutinase gene into plasmids containing only the 5 and 3′UTRs (SC10) or the 5′UTR alone (SC11), we observed that despite good expression in all strains (Additional file 1: Fig. S7), cutinase secretion was at least fourfold lower (Fig. 7c). With this evidence, the plasmid harbouring SC1 was taken forward (for the construction of a Mark IV secretion strain) as the best candidate for increased secretion through the FT3SS.
As a final step to investigate the optimal secretion signal, we compared secretion of our 'late' FliC signal with early flagellar substrate signal sequences, namely the hook (FlgE) and hook-cap (FlgD) proteins. In order to compare these signals, we constructed cutinase harbouring secretion signal sequence variants using the first 100 amino acids of FlgD or FlgE (Additional file 1: Fig. S8a) for testing in both an early-locked, hook-less [ΔfliC ΔflgKL ∆flgDE (∆clpX)], and a substrate switched [ΔfliC ΔflgKL (ΔclpX)] background. The FlgE signal did not permit secretion in either strain, and the FlgD signal was four- to fivefold less effective at export than the FliC signal (Additional file 1: Fig. S8b). This indicated that despite the streamlined nature of the hook-less strain, it is in fact less efficient for secretion than the HAP-less strain. We did observe low-levels of cutinase secretion with SC13 (FlgD signal) in the ΔfliC ΔflgKL ΔclpX strain, presumably due to higher expression of secretion apparatus, but still at a much lower level (fourfold) than with the intact FliC signal (SC1).
Finally combination of the improvements to the chassis and modular secretion vector were investigated in comparison to the prototype versions. Both strains contained more intracellular cutinase following expression of SC1 over the SC0 prototype (Fig. 8a). Additionally there was more intracellular cutinase in the Mark III strain than the prototype-irrespective of secretion construct. Detection of the FLAG-tag by immunoblotting allowed visualisation of secreted cutinase by a means other than the MUB assay. In agreement with previous results (Fig. 5a, b), the Mark III strain secreted a higher concentration of substrate in comparison to the ΔfliC ΔflgKL strain when expressing the prototype secretion construct (Fig. 8b, d), this translated to a 3.83-fold improvement in secretion efficiency (Fig. 8a, b). However, the highest concentrations of secreted cutinase were seen when either strain expressed SC1, with ΔfliC ΔflgKL ΔclpX ΔmotAB resulting in the highest. Given the low abundance of GroEL in ΔfliC ΔflgKL ΔclpX ΔmotAB cell supernatant (Fig. 8c), we show that the increase in extracellular cutinase in this modified strain is not due to cell lysis or membrane leakage. In fact the lower level of cytoplasmic leakage in ΔfliC ΔflgKL ΔclpX ΔmotAB suggests that the clpX allele may in fact destabilise the cell envelope, although this would require further investigation. We also note that the α-GroEL antibody is very sensitive and the Coomassie stained gel (not shown) did not indicate cell leakage in the ΔfliC ΔflgKL strain either. Using the same samples, a MUB protein secretion assay was carried out (Fig. 8e). The improvement attributed to the optimised strain (ΔfliC ΔflgKL ΔclpX ΔmotAB) was 7.68-fold, and for the secretion construct (SC1) 15.70. Upon combination (Mark IV strain) these improvements were additive, resulting in a 23.78-fold increase in fluorescence in comparison to the prototypes (all p < 0.001). Densitometry analysis of secreted cutinase measured by immunoblotting (Fig. 8a, b), showed that the strain was 5.64 times more efficient at secreting cutinase than the prototype. Despite immunoblotting determining the combined fold change in the Mark IV strain to be less pronounced (Fig. 8d), the overall trend is consistent with the MUB fluorescence assay (Fig. 8e). The Mark IV strain had an average titre of 0.16 mg L−1 secreted cutinase (highest 0.19 mg L−1) (Fig. 9).
Finally we investigated secretion of a number of pharmaceutically relevant, human proteins in the Mark IV FT3SS strain. These included E. coli codon optimised versions of, the aforementioned CH2 ScFv fragment, and also human growth hormone (hGH) and thioredoxin (TrxA)—both of which were kindly gifted following an ongoing collaboration with FUJIFILM Diosynth Biotechnologies, UK. Genes were PCR amplified with EcoRI and PstI restriction site ends and inserted into the SC1 harbouring plasmid. All were secreted into the media, with respective average titres of 0.25, 0.15 and 0.21 mg L−1 from an OD600 1.0 culture (Fig. 9, Additional file 1: Fig. S9).
Despite the clear benefits to protein production, to date, true secretion of recombinant protein by bacterial expression systems into the media is only possible in the mg L−1 cell culture range, even in high density culture [19–21]. For commercial use this requires improvement, especially when compared to eukaryotic expression organisms—such as Pichia which can reach 15 g L−1 . We focus on an E. coli chassis, due to its preferential use in IB, for a host of beneficial reasons. In this study, we set out to engineer an E. coli with the ability to secrete recombinant protein into the extracellular media, in one direct step. A clear candidate for this was the one step FT3SS, and while recombinant protein has been secreted through the FT3SS previously [29–31, 34, 36], we aimed to make improvements to improve secretion capacity, without leakage, though strain engineering and design of a modular secretion and purification system.
We established that a prototype ∆fliC ∆flgKL strain outperformed a ∆fliCD strain (as utilised in other studies [29–31], in both protein production and secretion (Fig. 1). One explanation might be that FliD binds FliT, and so in the FliCD strain, more FliT is free to interact with, and suppress FlhD4C2 activity, thus reducing FT3SS expression and secretion . In contrast removal of FlgKL frees up the chaperone FlgN to promote translation-coupled secretion of FlgM-further raising expression of FT3SS components (including the fliC-UTR harbouring modular secretion construct) , but whether this occurs in E. coli is not clear at present. We next investigated whether strain engineering could improve secretion, and found that removal of clpX (a component of the ClpXP protease complex), which is known to actively bind to, and degrade FlhD4C2 , improved the yield of secreted FliC by 1.6-fold, to 34 mg FliC-ΔD3 L−1 culture medium at OD600 1.0 (Fig. 3). This became our Mark II strain. We also show total absence of FliC-ΔD3 in the secreted fractions of FT3SS null strains, along with very low abundance of cytoplasmic contamination (absence of GroEL) (Fig. 3). This observation is consistent for recombinant proteins (Fig. 4d). Furthermore we show that, in the absence of the FliC secretion signal peptide, protein is not exported (Fig. 7b, c and Additional file 1: Fig. S7). Combined, this demonstrates that in our system, the truncated FT3SS shows excellent selectivity and that leakage is not prevalent.
Development of a high-throughput, enzyme based, secretion assay (Fig. 4) enabled us to screen a range of engineered strains and secretion signal constructs (Figs. 5, 7). As enzyme activity requires correct protein folding, the assay also highlights that secreted heterologous cutinase correctly folds in the extracellular media, thus allowing functionality. This is very attractive when considering the proposed biotechnological applications of this truncated FT3SS secretion platform. Through this screen we established the ∆flgKL ∆fliC ∆clpX ∆motAB as the superior (Mark III) secretion strain (Fig. 5). Several lines of evidence supported that clpX deletion has a positive effect on secretion through the FT3SS [64–67]. Further to this, we observed increased filament production and motility in ΔclpX cells in a wild-type background (Additional file 1: Fig. S4), suggesting increased flagellar gene expression. We confirmed this notion in our truncated secretor strain, as more FlhA was detected in ΔclpX mutants (Fig. 5c), suggesting an increase in the number of basal bodies (secretion apparatus). In contrast, the concentration of FlhA was not increased in motAB mutants, indicating that improved secretion in these strains was not due to higher abundance of secretion apparatus. A probable explanation is that this strain has reduced drain on the proton motive force [82, 83], which would usually provide energy to drive flagellar rotation [86, 87], but also has implications for secretion and growth.
We also established, surprisingly, that combining the fliDST mutation with the Mark III ∆flgKL ∆fliC ∆clpX ∆motAB strain increased secretion of cutinase, but not other proteins (Fig. 5b), indicating that there may be some protein specific factors in determining secretion capacity. In fact the removal of fliDST, or flgMN were generally not beneficial to secretion. This may be attributed to the fact that there are contrasting reports on the effect of FlgM(N) deletion on flagellar expression in the literature [50, 79]. This is complicated by the fact that, FliS also binds FlgM prior to fliA expression, suppressing secretion of FlgM upon hook-basal body completion , while removal of FliDST per se has been shown to result in an increase in secreted FlgM . In addition, FlgN is also implicated here, as a positive regulator of FlgM translation , further complicating this picture, and making these data both hard to predict and interpret.
We show that a construct containing the 5′UTR and FliC1–47 secretion signal (SC1) was the most efficient for secretion of recombinant proteins (Fig. 7b). In agreement with previous work [30, 33], we did not find inclusion of the 3′UTR to be necessary for secretion—as an extension to this, we also show that exclusion of the 3′UTR dramatically increases secretion, despite potentially reducing internal levels (Fig. 7a). An explanation may be linked to the evidence that the 3′UTR influences levels and stability of the fliC mRNA , which in turn has been shown to reduce FlgM secretion . If this is affected by the 3′UTR region specifically, then this could explain improved secretion in the absence of the 3′UTR, as FlgM might be more readily secreted from the FT3SS—however at this time we have no evidence to support this theory. In contrast, the 5′UTR and FliC1–47 are able to both direct secretion and ensure adequate protein expression. One reason for this might be that the signal peptide confers some level of solubility or reduces formation of inclusion bodies—though again we have not tested this directly. Notably, our evidence (Fig. 7b, Additional file 1: Fig. S7) does not match that of other reports where the FliC 5′UTR [30, 31], or 26–47 residues of the FliC signal alone [34, 35], direct efficient secretion in E. coli. Neither does it match evidence that the 5′UTR is not necessary for secretion , and we can only attribute these differences to potential variances in strain background or experimental conditions. It would seem that modification of the secretion signal had a larger effect on secretion capacity than the strain improvements (15.70- and 7.68-fold improvements respectively). Most compelling, was our finding that upon combination, these improvements have an additive effect on the secretion capacity of the FT3SS (Fig. 8). The overall 23.78-fold improvement to cutinase secretion through the FT3SS is a step-forward in terms of moving towards an E. coli strain capable of high capacity secretion of recombinant protein into the extracellular media, in one step.
In our lab, the best secreted yield of protein achieved (with cell density OD600 < 1.5) in our truncated FT3SS (maximum of 240 mg L−1, average 80–90 mg L−1, of FliC-ΔDE3 secreted into the extracellular media), surpasses that reported for alternative E. coli systems, which also enable secretion into the media. For example in the T1SS: typically in the µg L−1 range, but up to 3 mg−1 OD unit cell culture at high cell density [23–28, 91] and FT3SS (ΔfliCD): up to 12 mg L−1 (although under unspecified high density cell culture conditions, ). We also exceed reported yields achieved for secretion into the E. coli periplasm by the type II secretion system at low cell density (30–60 mg L−1 [13, 14]), without the requirement for periplasmic extraction or the risk of proteolytic enzymes compromising protein quality.
However as we are proposing the use of our strain as a secretion platform in a biotechnological context, we finally set out to investigate the performance of the Mark IV strain when secreting a broad range of industrial and pharmaceutically relevant recombinant proteins. At low cell density (OD600 1.0) we achieved average yields of 0.16, 0.25, 0.15 and 0.21 mg L−1 for cutinase, a CH2 ScFv fragment, human growth hormone and thioredoxin respectively (Fig. 9). The secreted titres we report for heterologous protein are lower than those which we measure for native protein (FliC-ΔDE3). This was anticipated, as aside from a small truncation, FliC-ΔDE3 is a natural substrate of the FT3SS, whereas the heterologous proteins are non-native to both the organism and the secretion system. We acknowledge that our titres of heterologous protein are lower compared to other bacterial systems (where up to 50 mg L−1 was achieved) [20, 21]. However our results are still compelling, given that (unlike the majority of literature examples) we have achieved these titres from (1) culture supernatant, (2) at low cell density in E. coli, and (3) showed excellent control of secretion to the extracellular space, with no evidence for cell leakage. When comparing our final Mark IV strain to other strains that meet the majority of these credentials, our current strain secretes around four times less heterologous protein than the best titre reported in the T1SS (cell density unclear) , and around 50 times less than an alternative FT3SS system (carried out at a higher cell density and with different proteins) .
To match these reported titres in the future, we aim to further boost the titres of secreted protein, with a modified cell culture strategy and more strain engineering. For example, while our supernatant was harvested at a maximum cell density of OD600 1.5; it is noted that transfer to high cell density culture (OD600 > 180) can greatly improve yields . Indeed we have begun to test these strains at higher cell density in bioreactors with no obvious growth defects (unpublished data). Finally, the modular secretion vector has not only been optimised to enable high levels of expression (with the removal of the 3′UTR), and flexibility of protein cargo, but also serves as a versatile platform technology which could be easily adapted for specific applications, i.e. by exchanging the purification or antigen tags. We are currently focussing on how the modular secretion vector performs in terms of recovery of purified protein, free of the accessory tags.
We have used a combinatorial approach, incorporating strain design and a genetic modular secretion vector, to achieve improved extracellular titres, of a range of biotechnologically relevant protein products, by secretion through a truncated E. coli FT3SS. This may enable simplified downstream processing of higher quality product, free of cellular contaminants, all of which are of great interest to the biopharmaceutical and IB communities. Furthermore, this opens up the possibility of cell cultures that secrete protein continuously, without the need to sacrifice cell culture to retrieve protein product. During development of the truncated secretion construct, a number of findings provided new information on the roles of the secretion signal peptide and UTRs of FliC monomers. We also introduce a high-throughput assay, enabling quick and accurate measurement of the secretion output of truncated FT3SS strains. We are currently using this in inverse genetic engineering approaches to identify factors that might boost secretion further. We suggest that the body of this work serves as a pilot feasibility study, and are working on further improvements (high cell density culture, purifying recombinant protein from the media), with which we envision that our FT3SS secretion platform could become a forerunner for use in the production of biopharmaceuticals and IB products.
E. coli MC1000 was used for the construction of all mutants in this work (Additional file 1: Table S1). E. coli strain MG1655 clpX::Tn5(KmR) was kindly gifted by J. Green, University of Sheffield and was used as a donor for transduction in the construction of clpX strains. Strains were grown in LB broth or on LB agar plates, supplemented with ampicillin (100 µg mL−1), chloramphenicol (25 µg mL−1) or kanamycin (50 µg mL−1) or Isopropyl β-d-1-thiogalactopyranoside (IPTG) (concentration stated in figure legends) as appropriate. Prior to assaying protein secretion by SDS-PAGE or enzymatic secretion assay, fresh media was inoculated with cells from a liquid overnight culture and induced. Bacteria were grown with shaking at 180 rpm, 37 °C.
Phage transduction was carried out by the method outlined by Lennox . Following strain construction, the eradication of phage from the strain was ensured by passaging on LB agar plates with 10 mM sodium citrate. Absence of phage was then confirmed by Evans Blue-Uranine assay, as outlined by Tiruvadi Krishnan et al. .
Plasmids utilised for chromosomal manipulations or for cloning are listed in Additional file 1: Table S2. High fidelity and diagnostic polymerase chain reaction (PCR) was carried out with Phusion High Fidelity polymerase (New England Biosciences) and DreamTaq DNA Polymerase (Thermo Scientific) respectively. Templates and custom primers (Sigma-Aldrich) are listed in Additional file 1: Table S3. Chromosomal modifications were carried out, as described by Datsenko and Wanner . To enable sequential mutagenesis, pCP20 was utilised to excise FRT flanked antibiotic resistance cassettes. Modifications were PCR verified, as was the maintenance of other lesions on the chromosome, if the strain underwent sequential deletions.
A genetic secretion construct was designed, synthesised and then incorporated into the pJexpress-404 plasmid backbone by DNA 2.0. This plasmid is referred to as pJex-fliC47-empty (Additional file 1: Fig. S4a) and forms the base cloning vehicle here. All restriction digests and ligation reactions were performed with NEB restriction enzymes and T4 DNA ligase (New England Biosciences). Following ligation, reaction mixes were transformed into NEB 5-alpha (New England Biosciences), as described in the supplier's instructions. The synthetic gene for Fusarium solani cutinase (accession number: K02640), was codon optimised and synthesised by GeneArt® Strings DNA Fragments (Life Technologies). hGH and TrxA gene fragments were obtained from FUJIFILM Diosynth Biotechnologies.
Following cell growth for secretion, 1 OD unit of culture samples were centrifuged for 15 min (16,000g) to ensure separation of cells and supernatant. Supernatant fractions were prepared by precipitation in 10% v/v trichloroacetic acid and washing in acetone [57, 95]. Both supernatant precipitate and cell pellets were suspended in 2× SDS buffer (10 mL glycerol, 1 g SDS, 0.1 g bromophenol blue, 200 mM DTT, to a volume of 50 mL in 100 mM Tris–HCl, pH 6.8) to a final volume of 50 µL and 200 µL respectively. Both cell lysate and supernatant samples underwent SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were either stained with InstantBlue™ (Expedeon) or prepared for Western immunoblotting by electro-transfer onto nitrocellulose membrane (GE Healthcare Life Science) and blocked in 3% w/v bovine serum albumin in TBS (24 g L−1 Tris base, 88 g L−1 NaCl plus 0.1% v/v Tween). The following primary antibodies were diluted 1 in 1000 in TBS/Tween: H48 monospecific H rabbit antiserum (Statens Serum Institut), monoclonal ANTI-FLAG®M2 antibody produced in mouse (Sigma-Aldrich), E. coli GroEL rabbit IgG (Sigma-Aldrich). If required, following washing and re-blocking, anti-rabbit HRP (Cell Signalling Technology) was diluted 1 in 3000. All blocking and incubation steps were carried out for 1 h. Immunoblots were visualised using Pierce® ECL Western Blotting Substrates (Thermo Scientific) and a Compact X4 X-ray Film Processor (Xograph) or C-DiGit® Chemiluminescent Western Blot Scanner (LI-COR Biosciences). Analysis by densitometry was carried out using ImageJ. For FlhA detection, the same protocol was followed except blocking was carried out in 5% w/v skimmed milk in PBS (8 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2HPO4, 0.24 g L−1 KH2PO4) with 0.05% v/v Triton X100. Polyclonal anti-FlhA antisera was diluted 1 in 1000 in PBS/Triton along with E. coli ΔflhDC soluble cell lysate (to minimise non-specific binding) and following washing, membranes were incubated with a 1 in 5000 dilution of IRDye 800CW Donkey anti-rabbit secondary antibody (LI-COR Biosciences). Membranes were analysed using an Azure c500 (Azure Biosystems) imaging system and quantified with LI-COR Image Studio Lite (LI-COR Biosciences).
To prepare purified monomers of truncated FliC, MC1000 ΔfliC harbouring pTrc-FliC-ΔDE3 were scraped from semisoft agar plates (1% w/v tryptone, 0.5% NaCl, 0.8% w/v bacteriological agar), following 24 h incubation at 37 °C. After resuspension in 50 mM Tris–HCl, pH 7.8, cells were agitated vigorously in a laboratory blender (Waring Laboratory Science) for 3 min, then centrifuged at 16,000 g for 5 min at 4 °C. The supernatant was then centrifuged at 67,500 g for 15 min at 4 °C. The pellet was resuspended in fresh buffer, and centrifugation repeated. In fresh buffer, the pellet underwent sonication for 20 s, followed by heating to 50 °C for 10 min to cause monomerisation. Finally, centrifugation was repeated and supernatant collected.
Protein standards of CH2, TrxA and hGH were purified from the intracellular fraction of E. coli BL21 (DE3) after induction with 1 mM IPTG. Protein was purified using the secretion construct incorporated Streptavadin II tag on a 1 mL StrepTrap™ HP column (GE Healthcare Life Science) according to the manufacturer's instructions. Following elution with 2.5 mM desthiobiotin, 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8, protein underwent dialysis overnight to yield purified protein in 100 mM Tris–HCl, pH 8 and quantified with a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Proteins standards were aliquoted and stored at − 20 °C.
An assay based on cleavage of 4-methylumbelliferyl butyrate (MUB) was developed to measure the activity of secreted cutinase. 10 mL cultures of cells expressing cutinase were induced with 0.05 mM IPTG and grown to OD600 1.0. 250 mM MUB (prepared fresh daily) was dissolved in dimethylformamide with 1% Triton X-100. This mixture was then diluted in 50 mM phosphate citrate (pH 5) buffer to a working concentration of 500 μM MUB. 40 μL supernatant was added to 96 well plate wells, the reaction was initiated with the addition of 160 μL MUB and incubated at 30 °C for 30 min. Following incubation, fluorescence was measured, either by imaging 96 well plates under UV light (G:BOX, Syngene), or using a Tecan Infinite 200 Pro plate reader-excitation 302 nm, emission 446 nm (Tecan Group Ltd.).
CAG generated the majority of strains and plasmids, performed and optimised secretion experiments, including SDS-PAGE and immunoblotting, developed the cutinase secretion assay, designed experiments and interpreted data, alongside NK, EC, MH and OJB. CAG and GS lead the project design and manuscript writing. CL provided the hGH and TrxA coding sequence and insight into industrial protein production processes. PCW, GS, MH and CAG contributed to the study design and inception. All authors read and approved the final manuscript.
We would like to thank J. Green and J. Pandhal for strains and the CH2 gene respectively. We thank FUJIFILM Diosynth Biotechnolgies for provision of the coding sequence of human Growth Hormone (hGH) and Thioredoxin (TrxA).
CAG was funded by a University of Sheffield Ph.D. scholarship, NK by a Ph.D. scholarship from the Government of India, and EC by a BBSRC CBMNet Business Innovation voucher to GS and CL. MH was funded by BBSRC Grant to GS and PCW (BB/J016322/1). OJB was funded by a John Lucas Walker studentship awarded by the Department of Pathology, University of Cambridge, and GMF was funded by BBSRC Project Grant BB/M007197/1.
12934_2019_1058_MOESM1_ESM.pdf Additional file 1: Table S1. Escherichia coli strains used or generated in this study. Table S2. Plasmids used or generated in this study. Table S3. Polymerase chain reaction primers used in the study. Fig. S1. Annotation of the genetic region upstream of the flhD operon in E. coli MC1000. Fig. S2. Comparison of growth of the HAP-less and cap-less strains. Growth curve data for the aforementioned strains. Fig. S3. Functional flagellar despite the absence of the FliC D3 domain. SDS-PAGE of secreted protein and motility assay. Fig. S4. The effect of the deletion of clpX from E. coli MC1000. Comparison of the strains by: abundance of secreted flagellin, motility assay and phenotype. Fig. S5. Plasmid maps of (a) pJex-fliC47-empty and (b) pJex-fliC47-cutinase, along with (c) the prototype genetic synthetic modular secretion construct of pJex-fliC47-cutinase. Fig. S6. Comparison of growth of truncated FT3SS secretion strains. Growth curves for all relevant strains. Fig. S7. The FliC secretion signal is required to enable secretion. Immunoblots of secreted and intracellular cutinase. Fig. S8. Comparison of the ''late' FliC and 'early' secretion signals. Schematic of secretion constructs and immunoblots of secreted and intracellular cutinase. Fig. S9. Secretion of a range of substrates through the optimised secretion strain. Immunoblots of secreted protein alongside a protein standard.


Share the post "Figureheads on campus back change of SU model"
Figures of authority on campus have revealed their desire for the UCD Students' Union (SU) to move from its current format to a structure that resembles the model most SUs in the United Kingdom observe.
Problems with the infrastructure of UCDSU have been highlighted since the turn of the decade when the organisation was shown to be in excess of €1 million in debt due to financial mismanagement. These fiscal shortcomings underlined for many a need to change the current system in order to avoid such an issue arising in the future.
Furthermore, students felt UCDSU's efforts focus was too national and while committing to fighting fees with USI, it ignored prominent issues on campus such as the issues with students had with UCD Residences.
A shift to this UK Students' Union structure would see UCDSU fall in line with a more localised ideology that focuses primarily on campaigning for better student services on campus.
Students' Unions in the UK that apply this localised ethos to their work served find such efforts boost student involvement and see further progress made in improving student activities and services on campus, such as more affordable accommodation, more recreational activities and cheaper alcohol on campus.
Speaking about the possible shift to the UK Students' Union model, UCDSU President, Mícheál Gallagher said, "I would definitely like to see UCDSU move in a direction that sees it constantly adopting international, cutting edge examples of best practice.
New UCD President Andrew Deeks echoed Gallagher's sentiments, saying he hopes they can be more concerned with the experience of students on campus.
"Coming from the Australian system, the Students' Unions went from a situation where they were a breeding ground for future politicians to a situation where they were much more concerned with student facilities, the student experience on campus, with representing the student body to senior management.
During the first week of semester two, UCDSU released their first audited accounts to students, something Gallagher noted as a move towards this complete fiscal transparency that is the backbone of UK Students' Unions operations.
In order to maximize UCD students' involvement in such an overhaul of structures in the SU, Gallagher hopes that a survey being conducted over the next few weeks will allow students to have their say in what shape they UCDSU.


Some days I feel like a juggler, keeping many diverse things up in the air, balancing (sort of) perfectly but always right on the edge of control. Today was such a day. Everyone was busy in the shop. Out in the yard we were doing new fencing for the horses. We were also stocking up on supplies needed for the Sculpture Magic Workshop that starts tomorrow. In between I worked at my design table and other various jobs.
Some of the tasks we were doing were not glamorous nor flashy, but still very necessary. One such item(s) was the first batch of fence posts, now ready for the application of concrete and then sculpting.
The hard working crew has also finished almost a hundred rock forms, both welded and wired. These will be soon welded to the 'dirt bank' structures that forms the mountain before we apply the concrete in the coming weeks.
When I went to town for supplies I stopped in up at Cultus Lake to see progress on-site. The large crew up there has been very busy to say the least. The bumper boat pool bottom was poured today. Next week they will backfill the pool and begin work on the island which will support the raised deck for the giant swing ride. That will be our cue to begin our theme work on site.
Forms for the first floor (of three) are almost in place. By the end of the week those walls should be poured and then they begin work on the cast in place suspended slab. It won't be long until our work begins to build a giant mountain around the building. It's going to get to be more fun all the time as we really get into the job!


It's time for the ABC Hanukkah hunt, a hunt that will take you through the eight-day Festival of Lights. "It's Hanukkah from A to Z. / An alphabet of things to see." Can you see those latkes being fried, the donuts, the menorah, and little sister playing with the dreidel? A is for King Antiochus, a king who was not a friend to the Jews. Traveling through this ABC book there is a full rhyme on each page, many asking the young reader to find something on the page or recall a bit of history they may have learned at home or in Hebrew school. "This calendar shows Kislev's here. / Which holiday starts this time of year?" Of course most children know that Kislev is when the Festival of Lights begins.
There are many special things that happen during the Festival of Lights, otherwise known as Hanukkah. Do you know what sufganiyout or latkes are? They are very special yummy things that are served during Hanukkah. A sufganiyot is the doughnut that you just cannot resist. "Sufganiyout! This One's for me. / How many doughnuts do you see?" Hanukkah is a time to be generous and you have to think about who you will help with the coins you put in your tzedakah box. There are all kinds of fun things in this ABC book to check out. Do you know what a shamash candle is? If not you will see one in this book!
This is a marvelous rhyming ABC book that helps young children explore the events of Hanukkah. Each letter is found within the full rhyme, with the letter capitalized. For example, when we look at the letter V, it is within the poem. "All Voices sing a Hanukkah song. / What family member 'howls' along?" Of course that family member is the dog. The artwork is bright, bold, colorful, and expresses the childrens' joy as they anticipate Hanukkah events. The poems have that pep that's needed to keep it's young audience captivated. This is a perfect book to use at home or in Hebrew school to help young children learn about Hanukkah. In the back of the book is a brief descriptioin of the Hanukkah celebration.
Quill says: This is an wonderful book to introduce many of the events and things to be found during Hanukkah to the youngest participants!


Rohit joined the David Adamson Group in 2015 and is a Director based in India as part of the Quantity Surveying Division for the India operations.
Since joining, Rohit has been instrumental in expanding the David Adamson Group India office to its current operational size, capability and resource, and making it one of the pre-eminent Construction Consultancy firms in India.
Rohit has in excess of 15 years professional experience, engaged on large scale prestigious projects in the building and civil engineering sectors.


Founded by Edgar Aver in 1985 as a small iron workshop, the company today is managed by the second generation of Aver family. Since 2010, Aver Metals has been working to transform light in art and today is recognized in Brazil for doing luxury light fixtures with original design.
Accord was born in the countryside of southern Brazil, from the dream of three brothers to work together with the legacy of their family: carpentry. Our founders were carpenters, and with them, we learned our passion for wood, from which comes our history and our essence. Using wood as the main raw material, all of our products are the result of a careful production process that combines technology with the irreplaceable manual finishing, involving the meticulous handwork of at least 30 of our employees in the production of one single piece. We always believe in the elegance of timeless design and the richness of craftsmanship to produce the best wooden luminaires in Brazil. Now we want to bring light and sophistication all over the world, one lamp at a time.
Designers: Bruno Diego Felippe and Micheli Beal Ribeiro.
Using her background as an architect, Alessandra is dedicated to furniture and objects design since 1998. Modernism is a strong influence on her creations, carried with functionality and timelessness. Her personal mission is to share the experience of a passionate drawing, in 100% authorial products. Her office brand, called Alessandra Delgado Design, is based in São Paulo, Brazil, where the creations can also be appreciated at the exclusive store named by the brand.
Active 25 years in the furniture business with projects in architectural, landscaping design and decoration, Araucari creates furniture designs and solutions, both indoor and outdoor, for shopping centers and malls, hotels, commercial and residential buildings, parks, and public spaces. Project development, choice of prime materials, and product manufacture follows eco-efficient standards in sustainability, and aims to balance pleasing aesthetics with functionality. What makes us different is the confidence we build with our clientele, our commitment to quality in all our projects, and the constant search for innovation.
Designers: Maria Eugenia Collaço, Rafael Collaço, Ricardo Afiune and Roberto Collaço.
We are a design furniture company that produces unique handcrafted solid wood pieces. The quality and atemporality of our products are recognized both by the prizes awarded and by the best contemporary furniture stores in Brazil.
Chairs, armchairs, sofas, benches, stools/ottomans, sofas, chaises, tables, console tables, coat hangers, bowls, etc.
Asadesign is a design studio located in the city of Curitiba, Brazil. Since 2000, we have been assisting leading furniture companies to create successful cases, joining our solid design expertise to the rich tradition of Brazilian wood and upholstery craftsmanship. Our approach is rooted on a profound understanding of each manufacturer´s main abilities and vocations, and we always pursue the perfect balance between originality and workability.
Designers: Alexandre Rocha and Daniela Ferro.
Founded in 2000, Boulle is a Brazilian company dedicated to the creation of finely crafted luxury and timeless, furniture and objects.
Based in São Paulo, ABUP – Brazilian Association of Home Utility and Gift Companies (sugestão) – is a non-profit association encompassing a group of companies in the homeware industry. In charge of promoting two of the largest Latin American fairs in this industry – Home & Gift, and Têxtil & Home – ABUP launches products by main players in our industry, with the best of independent Brazilian design, and presents a complete panorama of trends for the decoration, tableware, home utilities, and textile markets. Through the exchange of ideas and connections, ABUP fosters and encourages entrepreneurship to its associates. Promoting partnerships and holding events, the most important national business shows of the sector, the association propels its exhibitors internationally and contributes to the growth of Brazilian economy.
It is with great pleasure that I present some of my creations with semi-precious stones such as rock crystal, amethyst and more rare and exclusive amber and citrine Quartz. All pieces are considered collectable "Organic Modern" design. Concept: "Create design pieces that enhance the natural beauty of Brazilian stones. Letting the energy of stones and light show through them, giving a unique sensory experience." My inspiration comes from natural quartz formations that I see in my field trips. I work with raw natural quartz points, some with natural inclusions and imperfections. All pieces are hand assembled, with a technique that I developed using balance and patience and liquid resin. The construction can be made on stainless steel, brass, copper or aluminum bases. Some are made in foundry with casted metals. All pieces are unique, and the production can vary in size, shape and weight.
Embraer arose from the dream to transform Brazil in a country with thelatest in technology. From these dreams and challenges we have turned Embraerinto one of the largest commercial aircraft companies in the world. Bystretching the limits of what is possible, and embracing opportunities from dayone of our history, we have done the unimaginable in the sectors of commercialand executive aviation, defense and security. What motivates us is the genuinepassion for what we do. Maybe this spirit is why we resonate in the hearts ofso many Brazilians, collaborators and partners. We use design, imaginativeengineering, and technology as the principle tools to create new perspectivesand findsolutions that bring excellence and performance to each of our clientsand partners.
Original design, formal simplicity and elegance at all costs. This sentence is the hallmark of the work of two architects from São Paulo, who graduated from Universidade Mackenzie. Flavio Borsato and Mauricio Lamosa went to school together and founded estudiobola sixteen years ago around this concept, creating products with personality and simplicity studied to exhaustion. A work free from fads and trends requires rigorous research on proportions and niching in order to provide products with timeless results, which increase the lifespan of the pieces, making them pleasing to the eyes for years. This concept is applied to all the production of estudiobola, from creating a chair to designing lighting and upholstery projects.
Designer: Flavio Borsato and Mauricio Lamosa.
Geo Luz & Cerâmica is a manufacturer of high-end 100% handmade ceramic luminaires by Brazilian designer Maurício D'Ávila. The products are available for indoors and outdoors, and they combine ecology and design. Valued for their unique look, texture, and niches. Geo Luz & Cerâmica has become one of the most recognized companies in the lighting segment in Brazil. The company was first developed for architects who demanded unique pieces for their projects. Since then, the company has tremendously grown its portfolio to offer abroad a product line with unique design and lighting effects. With exclusive techniques and accuracy, Geo's products can adapt to any style and project.
With a philosophy focused on the development of architectural design as a global product that meets the wishes and needs of our customers, the office of Ivan Rezende Arquitetura has been working since 1982 in several areas where the intervention of the architecture professional is needed for the purposes of understanding and developing the space in a better way, the architectural object and its relationship with the surroundings. Creating projects in Brazil and abroad, the curriculum of Ivan Rezende Arquitetura includes awards from the Institute of Architects of Brazil (Instituto dos Arquitetos do Brasil – IAB), Brazilian Association of Architecture Offices (Associação Brasileira dos Escritórios de Arquitetura - Asbea), and the Brazilian House Museum (Museu da Casa Brasileira – MCB), among others.
European-trained, Rio de Janeiro based designer Brunno Jahara is an energetic champion of a fresh new Brazilian design scene. With bright and beautiful products and furniture that speaks about Brazil in an international language, he is helping this vast and multicultural country build its own design identities, as a place of abundant resources, innovation and craftsmanship.
Konsepta is the synthesis of my diversified ways of creating and producing design. It's how I combine my skills as an artist and a graphic designer to apply them to the ceramic and objects creation. The studio mission is to employ a singular and unique aesthetic language that can be plural at the same time regarding the possibilities of use and decor. The process incorporates sustainability in the use of materials because I believe that "beauty" in any form can and should be aware and socially responsible. In this sense, some objects are produced from the use of discarded woods and marbles. This allows us to bring a new look and meaning to these noble fragments generating unique creations and supporting the artisan Brazilian suppliers at the same time. In just one and a half year, Konsepta work and objects are already found in the most famous design and decoration shops in the country.
La Lampe was established in 1985 transcending the typical store design of the time, which offered lighting concepts restricted to chandeliers and lamps. La Lampe strove to integrate lighting and architecture. The company also imagined a concept that offered complete solutions, from design to final installation – a significant innovation. For the past years the company made a strong commitment to foster local designers, promoting genuinely Brazilian creations. This work is carefully done by studio Nada Se Leva, our designers and also curators for the brand, helping us select prominent national designers for La Lampe. The combination of avantgarde vision and impeccable product quality has been largely responsible for lifting La Lampe to the rank of leader in the lighting market in Brazil, granting the brand the award for Top of Mind lighting company in the country more than 10 times. We are also present in important commercial and hospitality projects as Belmond Copacabana Palace, Fasano São Paulo, Grand Hyatt Rio, Trump Rio de Janeiro Hotel, Four Seasons and Palacio Tangará, a new development of the Oetker Collection.
Designer: Nada Se Leva studio.
Against the general tendency of large-scale production and homogenization, we develop unique and exclusive designs. Our creative process claims on rational and functional solutions that explore the essence of shapes in the conception of the object and/or space. We seek to develop a timeless aesthetic, which translates into the disposal of excess and the exploration of key spatial issues such as proportion, light and shadow, materials and textures. The studio activities include various fields of architecture – residential, commercial, institutional and interior projects – as well as furniture design. In all these spheres, the detail is considered essential, being directly associated to the project design.
For more than 15 years, Luxion has been a synonym for innovation, withhigh quality products and design. Luxiondevelops lighting products that satisfy the user, both for visual comfort andenergy performance, providing customized and value-added solutions for theproject. Luxion develops, produces and sells products for commercial,residential and corporate lighting. With appropriate technology, high quality and design, the company aimsto satisfy customers and employees, contributing to the well-being of theindividual and respecting the environment.
Designers: Roque Frizzo and ViniciusSiega.
A company with tradition that is increasingly consolidating its brand in the domestic market and setting new standards for design and experience in school furniture and is now expanding its business into the corporate environment. A successful career track due to the aggressiveness and entrepreneurship of its founder, who has always believed in his business and never stopped investing in that ideal, and is now reinventing its history with an innovative, modern and participatory management, adding value to its pool of over 600 collaborators. Current investments in projects and expansion are building a portfolio for a new culture, concepts, new products and new management, turning our strategies into true success stories that each day differentiate us from the competition, and strongly expand our brand in the market. Success accrues over time, along with maturity and experience.
Designers: Luciana Sobral and José Machado.
Our dream is to make your home the best place in the world! We work to surprise customers, be innovative and always putting people first. Our effort is to act fairly within a supportive work ethic. We strive to constantly improve while working lean, clever with a clear workflow.
MOBLE applies architecture in the development of furniture made for a world that is not disposable. We explore different scales, shapes and materials in search of authentic, intelligent and functional solutions to comfort and inspire everyday life. With rigor and technical ingenuity, each piece is carefully crafted to offer quality, optimal performance and well-being.
Designers: Bruno Chiarioni Thomé and Thiago Natal Duarte.
Munclair Lighting Brazil has been present in the world of decorative lighting since 1966. Our product lines have models signed by renowned Brazilian designers and are handcrafted with premium quality. Asa Lamp, design by Erlon Tessari with exclusivity for Munclair.
Noemi Saga Atelier is a Brazilian product brand established in 2013 by Nomina Design. Beyond aesthetics and functionality, Noemi Saga Atelier pursues emotional connection between people and products. Research on arts, History, trends and cultural expressions are important inspiration sources. They also combine new materials and technology with traditional techniques to make unique art-furniture. The company is based in São Paulo and Noemi Saga is the founder of Nomina Design and CCO of Noemi Saga Atelier. Fernando Ikeda is architect and designer of Atelier Noemi Saga.
Designers: Noemi Saga and Fernando Ikeda.
Plataforma4 was created with the aim of aligning ideas, adding experiences and proposing new ways of working with design. It is a platform for collaboration and exchange, where each of the designers applies their specific skills and contributes to the creative process.
Designers: Amélia Tarozzo, Camila Fix, Flávia Pagotti Silva and Rejane Carvalho Leite.
Reboh Design develops intense, functional, unique long-lasting projects and products that carry cultural references and affective memories with meaning and history. The concept of "design to last" is about the responsibility of bringing a new product to the world. Each product created, an act of love and responsibility. Cases and wards are a consequence. Laureate in Design by Bezalel, the designer won various awards, such as IF Design Award + 60years of IF best projects, Objeto Brasil Internacional Award, Brasil Excelence Award, Museu do Objeto Brasileiro, and contributed in expositions such as the NY Design Week, London Design Festival, São Paulo Design Week, Brazilian Design Biennial, Europalia Belgium, Milan Design Week and has her work published in many design publications worldwide.
Renata Rubim is a surface designer and color consultant. Having attended RISD (Rhode Island School of Design) in Providence, RI, for three terms on a Fulbright scholarship, she's the head of her own design office which bears her name (Renata RubimDesign & Cores). She has been responsible for industrial projects in different segments. She has participated of several shows and has also been awarded with the Bornancini Award in 2008, the Idea Brasil Award in 2009 and three different international iF Product Design Award (Munich) - with coatings in 2012 and 2014.
The award-winning design studio Fahrer has as their main feature the bent wood. Sergio and Jack Fahrer use sustainable materials and the development of new techniques for their creations.
Designers: Sergio Fahrer and Jack Fahrer.
Sette 7 is a design studio that uses the richness of native Brazilian raw materials to create unique and high-quality pieces, based on the aspects of contemporary design, presenting a universal and minimalist style. The creation of the brand had, for a long time, been one of Vivian Coser's dream, which she was able to accomplish in a partnership with her sister Erika Coser. The creative process of Sette7 focus on functionality and timelessness, resulting in a clean design shape.
"Brazilian by birth, Parisian by adoption, intermittently Italian by intermittently. Artist and Architect." is how Daniela Busarello introduces herself. Exploring her work, we discover the influence of her cosmopolitan environment and a cocktail mix of elements that combine the passion, light, nature and vitality of Brazil with the thoughtfulness, precision and refinement that she has integrated since her arrival in Europe in 2007. In the different spheres in which she expresses her creativity, the themes of introspection and freedom constantly reoccur. Abundance, vitality and generosity permeate her multifaceted career : trained as an architect, a landscape designer, teacher and artist. For the rigorous lines and limpid volumes of her houses, she draws her inspiration from infinitely varied urban environments and modernist architecture. One house, in steel and glass, won the 2007 National prize for Architecture in Brazil. She is also a scenographer, which realizations in Paris includes: GASTROMOTIVA at Pavillon Ledoyen , PROJETO RAIZES at Brazilian Embassy , LVMH at Jardin d'Acclimation , Cassina  among others. Whatever the medium she uses, always hand-made, her creations are at the same time inspired, nourished but also anchored in nature, silence, the passage of time, the genius loci and environmental manifestos. Murano Glass : Mose and Igarapé; Textile : Cosmographie rugs and tapestries in collaboration with Manufacture Cogolin; Parfum Bottles for Ex Nihilo,; Landscape-Objects : hybrid furniture, etc... With her works she develops "her own language". Abundance, vitality and generosity permeate heris multi-faceted career. This year, The VIA has named her this year as one of the 100 Designers of objects and space that makes The French Design Touch shines internationally. More thaen a prize, a responsiability to keep creating refined works and art.
Since 2004, we realized that, in order to grow, investing in a concept, identity and quality were necessary. From then on, we have chosen to use designing as a strategic tool and integrating element of our production chain. This decision has led SOLLOS to become known as an innovative company. We seek to integrate the best of our culture to international references, through the rational use of wood as a raw material, a method and production control which that are in agreement accordance with all the ecological requirements, and the investment in the use cutting- edge technological equipment along with artisanal techniques. Our wide and consistent production presents a balanced variety of typologies, which seek the simplicity and perfection of shapes. This concern results in timeless pieces that have been recognized for their elegance and novelty.
St. James is a Brazilian manufacturer of high end, metal home décor objects, established in 1976.
Designers: Nino Bauti, Vivian Coser and Luis Pons.
A native of São Paulo who grew up in Belo Horizonte, Minas Gerais, where he learned to see architecture, folklore and art in a playful and intimate way. In childhood he attended the Guignard Faculty of Fine Arts Guignard with his mother, where he learned to enjoy the natural drawing of painting and sculpture. Architect and town planner State University of Londrina UEL. 2002. He has been working in the area since 2003 with architectural, interior and furniture design projects. It seeks beauty, sustainability, the balance of materials, and affective memory in his works. Its main source of inspiration is Brazilian architecture.
We are an integrated design center genuinely Brazilian, in which all areas interact simultaneously, resulting in customized and strategic projects.For over 10 years, we have accumulated expertise in several segments, acting in the design, communication, furniture and accessories retail areas in residential and corporate interior design. Focusing on the furniture and interior design segments, we make strategic design our competitive edge, making it possible to ''think different''.All these aspects together are only possible thanks to a creative multidisciplinary team , eager for challenges in creating new products, the development of graphic materials and also in interior design.


'Using personal experiences to understand other people's mental health recovery' - presented by S. Ajayi, J. Billsborough, T. Bowyer, P. Brown, A. Faulkner, A. Hicks, J.A. Larsen, P. Mailey, R. Sayers, R. Smith, Rethink. The Rethink team are in the process of writing up this research and so welcome comments on this presentation until the end of July 09. Please email Sarah Ajayi.
Lydia Lewis' paper, 'Politics of Recognition: What can a Human Rights Perspective Contribute to Understanding Users' Experiences of Involvement in Mental Health Services?', will be published in a themed section of the journal Social Policy and Society, 8 (2), April 2009: Lewis, L. (Ed.), 'Mental Health and Human Rights: Social Policy and Sociological Perspectives'. Contact Lydia Lewis for further details.
Our fifth AGM took place at the Medical Sociology Group Annual Conference at the University of Sussex on Friday 5 September 2008. The event included a presentation from Dr Susie Scott, University of Sussex on her award-nominated book, Shyness and Society: The Illusion of Competence (2007, Basingstoke: Palgrave).
Second AGM at the BSA Medical Sociology Group Conference, University of York on the 16th September 2005. Click here for the full report.


This week listen to Christmas jazz and sip local wine, enjoy supper with Santa and finish your weekend with a DELICIOUS brunch.
This week is all about festive food. Our province is in full swing for the holiday season and there are many culinary experiences waiting for you!
Deck your halls and trim your trees, the holiday season has arrived. Nova Scotia is filled with incredible culinary experiences for you to enjoy this week.


IT Mailing Contacts began as an online channel for supplying quality business mailing databases. With millions of companies monitored every day for the best and the most relevant customer data, we are among the most internationally acclaimed database providers in the world. Our verified COGZ Customers Email list of COGZ Technology Users from US makes b2b communication among high-ranking executives from various types of industries responsive and result-driven.
The COGZ Technology User list is one of the cost effective data services that can help companies expand their reach to more clients every time. Our database helps to empower your marketing strategies by directing campaigns towards intended addresses, making them highly deliverable and lead generating. Additionally improve office efficiency, increase revenue dramatically, and reduce fixed costs, offering an instant Return-on-Investment by switching to our services. The COGZ Technology Users Email List that we supply to our patrons is well-enabled mailing database and is the secret of most (IT driven) businesses and we specialize at the very core. Our opt-in data will help you deliver customized offers, deals, and value-added services to the right prospect at the right time.
We give you more than just access to the people employed in the technology industry and data needed for successful targeted B2B marketing plan. With the extensive COGZ Users email database from IT Mailing Contacts marketers can promote and sell their products and services, network extensively, generate prospective leads, perform market research and do more.
Get ahead of your competition. Connect directly with COGZ Technology users. Global data with valid business emails. Lists updated every quarter. Plus our proprietary email and validation technology tests contact level accuracy at the point of delivery. You get the most current data.


Last night's Premier League fixtures span up a few shocks, with Manchester City taking a hammering, Spurs being beaten by the Hammers, and Arsenal losing at home to Swansea City.
The Citizens travelled to Liverpool on Wednesday night to try and pour more misery on the Reds, having beaten them to the Capitol One Cup trophy on Sunday, but that plan do not come through.
Liverpool won 3-0 thanks to goals from Adam Lallana, Roberto Firmino and former City midfielder James Milner, closing the gap between the Anfield side and fourth place to only six points.
Tottenham also travelled (albeit only across the city) in order to succumb to defeat, with West Ham snatching a 1-0 win, although the Hammers looked comfortable winners despite the result.
Arsenal picked up the most shocking result however, hosting a Swansea City side who are threatened with relegation, and who had only picked up two points in their last four matches.
The Gunners even took the lead in the first half, only to be pegged back before the interval, before Ashley Williams sealed the win for the dominant away side in the 74th minute.
Leicester will be more than pleased to have gained a further point on all of their league challengers, but could not have been happy with their 2-2 draw with Norwich City on Tuesday either.


A federal appeals court Thursday refused to block a controversial Arizona law that shuts down businesses for knowingly hiring illegal immigrants.
The action by the three-judge panel of the U.S. 9th Circuit Court of Appeals in San Francisco clears the way for the statute to be enforced beginning Saturday.
In a brief order, the judges said that business and immigrant rights groups had not shown an adequate need for delaying enforcement of the law.
After the measure went into effect Jan. 1, county prosecutors said they would not file any cases until March 1 to allow the courts time to decide whether to issue the injunction. A trial court judge earlier this month found the law to be constitutional.
The 9th Circuit panel will consider that issue at hearings this spring but is not expected to rule for many months.
Passed by the Arizona Legislature, the law places on probation any company found to have knowingly hired an illegal immigrant.
If the company is found in violation of the law again, its business license is automatically revoked, ending its ability to operate in the state.
At least four other states are working on bills modeled after the Arizona law. Business groups have opposed the measure, warning that it would force them to discriminate against Latinos and drive them from Arizona. Some companies have said they will not expand in the state as long as the law remains in effect.
Farrell Quinlan, a spokesman for many of the business groups challenging the Arizona law, said in a statement that getting an injunction had been a long shot.
"We remain confident that when the appeals court considers the merits of our case, it will find the Arizona law unconstitutional on multiple grounds," he said.


The number of mobile health (or mhealth) apps has reached 165,000.
That is according to figures released from IMS Institute for Healthcare Informatics. One quarter of mhealth apps target treatment management and chronic disease. Over 65% are focused on fitness and wellness. The study revealed that patient retention rates on clinical trials was much higher when the patient was prescribed with an app.
But the question remains: how can users know which are the best apps with so many out there, and which have reliable protection for their sensitive personal data?


All our professionals at Portville Dental maintain the highest levels of accreditation and pursue ongoing education to stay abreast of the latest trends in dentistry.
Cheryl has been a hygienist for 23+ years. She graduated from ECC hygiene school in Buffalo, New York and teaches the dental assisting program at BOCES in Olean. Her experience in dentistry is crucial to her patients and their care. Her knowledge of dentistry is noted when she communicates with her patients. She has also been certified in dental anesthesia and Florida Probe technology. These tools allow her to maintain the dental health of her patients with experience and technology. Cheryl also has extended knowledge in TMD disorders and can fabricate a dental orthotic to help eliminate joint and muscle symptoms. Cheryl loves to sky dive and has over 300 jumps to her name. When not diving out of a plane, she loves to spend time with hergrandchildren and work with youth at her church.
Robin has been in dentistry for 36+ years and brings this knowledge to the office. She graduated from ECC in 1995 with an associates degree in applied science/dental hygiene and Houghton College in 2009 for her BS. Robin has advanced education in dental anesthesia and Florida Probe technology which allows her to treat her patients with the up most care and experience. She has vast knowledge in the area of periodontal therapy which helps patients maintain their teeth and overall oral health for a lifetime. Prior to becoming a dental hygienist she was a dental assistant for 10 years. This allows her to discuss treatment options in all aspects of dentistry. When you see Robin you know that your care will be professional and caring. Robin loves to Contradance, cook and keep healthy with Yoga.
Eileen has been a dental hygienist for 38+ years. She graduated from MCC in hygiene and works part time with our office and is a wonderful addition to our dental family. She demonstrates professionalism and care to all her patients. She is trained in Florida Probe and has a vast knowledge of dentistry. When a patient is seen by Eileen they can feel confident that their dental health will be maintained and dental goals achieved. When not in the office she loves to travel, cook and spend time with her family.
Krista graduated from BOCES dental assisting program in 2012 and has become proficient in CEREC technology using CAD/CAM designing of crowns and bridges. Krista uses her artistic ability to make each crown look life like. She also assists the Doctor in all aspects of dentistry including implants and extractions.
Her passion for dentistry shows through in her communication with her patients and the care that they receive. Krista likes to camp and spend time with her family and friends traveling to various sporting events.
Donna has been in dentistry for over 50 years and graduated from Career Academy in Washington, DC for dental assisting. Donna assists the Doctor in treating patients and helps maintain the sterilization area and CEREC equipment.
She is also proficient in taking models, X-rays, CEREC technology and all other aspects of dentistry due to her vast experience. With her dental knowledge, as a patient you know that you are getting the best care around. In her spare time she loves to cook and play with her grandchildren.
Molly has been in dentistry for 3+ years and has helped our patients achieve their dental goals through her caring personality. Prior to dentistry, Molly received her BA from the University of Buffalo and her Masters of Education in Psychology with a certificate in School Psychology from the University of Edinboro. Molly worked in Pennsylvania and also in New York at Cattauragus Little Valley, Friendship and Portville Central School. Molly helps to coordinate treatment and schedule patients in ways that are convenient to the patient and their budget. As you are checking out you can see instantly her passion and care for our patients. She always goes the extra mile to help each patient and makes you feel like part of the family. In her spare time Molly likes to spend time with her family and travel.
Judy has been in dentistry for 12+ years and helps our office flow smoothly. Her duties are vast and expand over all dentistry helping patients achieve their dental goals. This is done through processing insurance claims for our patients and helping them understand what treatment is recommended and scheduling appropriately. Judy is one of the first people that our patients see or talk to when coming into our office. You can see right away from her personality that she truly cares about each patient and makes them feel welcome. When not at the office Judy likes to Kayak, cook and travel.
Linda has been part of our office for 15+ years. Linda graduated from JCC and runs a successful accounting business in Olean. Her expertise ranges from payroll to patient management. Linda keeps our office running efficiently and has been a crucial part of our team. In her spare time Linda loves to spend time with her grandchildren, travel and tend to her horses.


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text:
Passenger Vehicle »
BMW Group sales
november 2019 sales
BMW Group sales grow 1.4% to 225,662 units in November
The German automobile group attributes the high demand for electrified vehicles behind the growth momentum. The electrified vehicles from the group have registered 17,480 units of sales, up 18.4 per cent from November 2018.ETAuto | December 06, 2019, 15:22 IST
Substantial growth in the sales for BMW 3 Series sedan and Touring have helped BMW to post an overall growth in global retail number.
New Delhi: BMW Group has announced that its sales grew in November 2019 to 225,662 units, up 1.4 per cent compared to the same month last year.
The German automobile group attributes the high demand for electrified vehicles behind the growth momentum. The electrified vehicles from the group have registered 17,480 units of sales, up 18.4 per cent from November 2018.
The BMW brand alone has registered 194,690 units last month, up 2.9 per cent compared to the same month in 2018. The automaker claims that this is due to the significant growth in the US and China markets, where it sold 34,280 units and 68,366 units respectively. BMW has registered 7.6 per cent and 12.1 per cent growth in these two markets respectively.
As the automaker further claims, substantial growth in the sales for BMW 3 Series sedan and Touring have helped it to post an overall growth in global retail number.
Commenting on this sales performance, Pieter Nota, member of the Board of Management of BMW AG responsible for Customer, Brands and Sales, said, "After renewed growth in November, we continue to approach a new record for the full year as planned. Our electrified vehicles also performed particularly well in November, with sales increasing by 18.4% to reach a new all-time sales high. Last month, one in five BMW 5 Series Sedans was a plug-in hybrid."
While the BMW brand itself has posted a marginal growth, its subsidiary British auto brand Mini has recorded 30,509 of sales in November 2019, down 6.8 per cent compared to the same month last year.
BMW India introduces Smart Repairs initiative
Tags : Passenger Vehicle, Cars, BMW Group sales, BMW 3 series, november 2019 sales, Mini, bmw
Most Read in Passenger Vehicle
Most carmakers post decline in sales in Dec, Maruti Suzuki and M&M return to positive
XUV400 could be the first SUV from Mahindra-Ford JV: All you need to know

redpajama_set_name: RedPajamaCommonCrawl


DULLES, Va.Jun. 3, 2015-- Orbital ATK, Inc. (NYSE: OA), a global leader in aerospace and defense technologies, announced today that it has been awarded a production order from the U.S. Army to fulfill additional U.S. Air Force requirements for the DSU-33D/B proximity sensor. This order, valued at approximately $27 million, is a follow-on award to the full-rate production contract signed in 2013. The sensor provides a fire pulse to the bomb fuze, enabling precise detonation to maximize the effect of the weapon. This contract marks the fourth full-rate production contract awarded to Orbital ATK and this is the second Delivery Order issued against that contract.
We have provided DSU-33 sensors to the U.S. Armed Forces since 1999. Additional production of this version is testament to their quality and dependability, said Cary Ralston, vice president and general manager of the Missile Products division of Orbital ATK s Defense Systems Group. Accuracy and dependability on the battlefield are vital and our employees give their all to meet the expectations of the warfighter.
The DSU-33D/B is an all-weather, active, radio-frequency, ranging radar that senses the height of the weapon above the target area. It is used in conjunction with FMU-139 and FMU-152 bomb fuzes to detonate the weapon at a fixed height above surface targets, maximizing the warhead's blast and fragmentation effects. Orbital ATK is the sole provider of the DSU-33 sensor family, delivering more than 154,000 sensors to the U.S. Air Force, U.S. Navy, and allied nations since starting production in 1999.
The DSU-33D/B will be produced at the Allegany Ballistics Laboratory (ABL) facility in Rocket Center, West Virginia. ABL is a U.S. Navy-owned, Orbital ATK-operated facility specializing in advanced manufacturing technologies for a variety of programs supporting current and future U.S. industrial base needs in advanced fuzing and integration, conventional munitions assemblies, solid rocket motor propulsion and advanced material structures.


A very elegant set of French dining chairs from the 1930s . The seats are covered in a red velvet and could use an update. I personally love the contrasting colors and the original fabric.
The strip of wood on the back to support the body and the blown up front legs bring an almost Orientalist feel to these chairs.


text:
Former Teachers' Union President Arrested in Virginia for Embezzlement
Yale Law School Bars Press From Free Speech Panel. Yes, Really.
Aaron Sibarium
Virginia School Board Weighs Speech Code that Would Prohibit Criticism of Equity Plan
Teachers' union concerned the code will stifle speech
Students at a Loudoun County, Va., elementary school / Getty Images
Chrissy Clark
Virginia's Loudoun County Public Schools district board will vote on whether to adopt a speech code that would prohibit teachers from criticizing the school board's racial-equity plan.
According to a draft of the proposed speech code obtained by the Washington Free Beacon, employees of the school district would not be allowed to criticize the school district's "commitment to action-oriented equity practices" in all forms of public and personal communication. The speech code is hidden inside a "professional conduct" policy proposal, which prohibits behavior that undermines "the views, positions, goals, policies, or public statements of the Loudoun County School Board or its superintendent."
"Employees are expected to support the school division's commitment to action-oriented equity practices through the performance of their job duties, as the Division engages in the disruption and dismantling of white supremacy, systemic racism, and language and actions motivated by race, religion, country of origin, gender identity, sexual orientation, and/or ability," the policy draft reads.
The code briefly acknowledges employees have a First Amendment right to engage in protected speech, but says that right "may be outweighed" by the school district's interest in "promoting internal ... and external community harmony and peace" as well as "class equity, racial equity, and the goal to root out systemic racism."
Nicole Neily, president of the free speech legal group Speech First, told the Free Beacon the directives of the school board do not supersede the First Amendment.
"It's deeply concerning that a public school system—which is a state actor and is bound to uphold the First Amendment—seems determined to police its employees' speech on matters of public concern," Neily said. "The school's plan is ... designed to chill speech with which the district disagrees."
The new policy proposal also tells employees they have a "duty to report" their colleagues' alleged speech code violations to the school district administration. Employees would be prohibited from "retaliating" against accusers, even if the accusations are false. The policy claims the district will accommodate false complaints, so long as they are found to be made in "good faith."
Loudoun County has come under fire for spending $422,500 on diversity training inspired by critical race theory, which claims racism is inherent in nearly every aspect of America. The school district also received backlash from teachers and parents for teaming up with the Southern Poverty Law Center to create a "social justice" curriculum for kindergartners.
President Donald Trump has promised to defund public schools that promote critical race theory. He also signed an executive order banning federal contractors from doing certain kinds of diversity training.
Manhattan Institute scholar Max Eden told the Free Beacon that the school district's speech code should not be permitted given the Trump administration's recent executive order. "If President Trump gets a second term, I think we could expect strong federal action against such totalitarian initiatives," Eden said. "If Biden is elected, however, I expect that he will use the power of the Department of Education's Office for Civil Rights to coerce school districts to adopt [critical race theory] initiatives."
The district's top teachers' union, Loudoun Education Association, said they are concerned with the policy's speech code. Sandy Sullivan, the president of the union, told the Free Beacon the union will meet with the school district before the Oct. 12 vote on whether to adopt the policy. Sullivan said teachers are upset with the proposed policy, which immediately drew concern from the union.
"Anything that is going to inhibit the speech of people freely expressing themselves in an appropriate manner ... concerns us," Sullivan said. "We want employees to make sure that they can be heard."
Published under: Critical Race Theory , Free Speech , Loudoun County , Teachers Unions