'Rewritable' and 'liquid-specific' recognizable wettability pattern.

Bio-inspired surfaces with wettability patterns display a unique ability for liquid manipulations. Sacrificing anti-wetting property for confining liquids irrespective of their surface tension (γLV), remains a widely accepted basis for developing wettability patterns. In contrast, we introduce a 'liquid-specific' wettability pattern through selectively sacrificing the slippery property against only low γLV (<30 mN m-1) liquids. This design includes a chemically reactive crystalline network of phase-transitioning polymer, which displays an effortless sliding of both low and high γLV liquids. Upon its strategic chemical modification, droplets of low γLV liquids fail to slide, rather spill arbitrarily on the tilted interface. In contrast, droplets of high γLV liquids continue to slide on the same modified interface. Interestingly, the phase-transition driven rearrangement of crystalline network allows to revert the slippery property against low γLV liquids. Here, we report a 'rewritable' and 'liquid-specific' wettability pattern for high throughput screening, separating, and remoulding non-aqueous liquids.

Covalent crosslinking chemistry for controlled modulation of nanometric roughness and surface free energy.

Smooth interfaces embedded with low surface free energy allow effortless sliding of beaded droplets of selected liquids-with homogeneous wettability. Such slippery interfaces display low or moderate contact angles, unlike other extremely liquid repellent interfaces (e.g. superhydrophobic). These slippery interfaces emerged as a promising alternative to extremely liquid repellent hierarchically rough interfaces that generally suffer from instability under severe conditions, scattering of visible light because of the hierarchically rough interface, entrapment of fine solid particulates in their micro-grooves and so on. However, a controlled and precise modulation of surface free energy and nanometric roughness is essential for designing a more compelling solid and dry antifouling interface. Here, we have unprecedentedly demonstrated the ability of covalent cross-linking chemistry for precise and simultaneous modulation of both essential surface free energy (∼49 mN m-1 to ∼22 mN m-1) and roughness (root mean square roughness from 30 nm to 3 nm) of a solid interface for achieving liquid, substrate, and process independent, robust slippery properties. The strategic selection of ß-amino-ester linkage through a 1,4-conjugated addition reaction between amine and acrylate groups of a three component reaction mixture (dominated by a 61% (w/w) crosslinker) under ambient conditions provided a facile basis for associating various important and relevant properties-including self-cleaning ability, anti-smudge properties (against both water and oil-based inks), thermal stability (>300 °C), chemical stability, physical durability, optical transparency (∼95%) and so on. The embedded slippery properties of the coating remained unaffected at both low (0 °C) and high (100 °C) temperatures. Thus, the prepared coating would be appropriate to maintain the unperturbed performance of commercially available solar cell modules and other relevant objects under outdoor conditions.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.

BACKGROUND: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. RESULTS: Polysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5'UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5'uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. CONCLUSIONS: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks.

BACKGROUND: Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A(2a) adenosine receptor (hA(2a)R), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). RESULTS: Functional hA(2a)R was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA(2a)R and GFP were still produced in the pre-induction phases. Both hA(2a)R and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake-flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. CONCLUSIONS: The production of recombinant hA(2a)R, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum.

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.

Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.

Identification of a glycosyltransferase from Mycobacterium marinum involved in addition of a caryophyllose moiety in lipooligosaccharides.

Deletion of Mycobacterium marinum MMAR2333 resulted in the loss of three of four subclasses of lipooligosaccharides (LOSs). The mutant was unable to extend an intermediate (LOS-II*) by addition of caryophyllose. These data and the predicted domain structure suggest that MMAR2333 is a glycosyltransferase involved in the generation of a lipid-linked caryophyllose donor.