Bioinspired Pseudozwitterionic Hydrogels with Bioactive Enzyme Immobilization via pH-Responsive Regulation.

Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N, N-methylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors.

Toward Antibiofouling PVDF Membranes.

Membranes for biologically and biomedically related applications must be bioinert, that is, resist biofouling by proteins, human cells, bacteria, algae, etc. Hydrophobic materials such as polysulfone, polypropylene, or poly(vinylidene fluoride) (PVDF) are often chosen as matrix materials but their hydrophobicity make them prone to biofouling, which in turn limits their application in biological/biomedical fields. Here, we designed PVDF-based membranes by precipitation from the vapor phase and zwitterionized them in situ to reduce their propensity to biofouling. To achieve this goal, we used a copolymer containing phosphorylcholine groups. An in-depth physicochemical characterization revealed not only the controlled presence of the copolymer in the membrane but also that bicontinuous membranes could be formed. Membrane hydrophilicity was greatly improved, resulting in the mitigation of a variety of biofoulants: the attachment of Stenotrophomonas maltophilia, Streptococcus mutans, and platelets was reduced by 99.9, 99.9, and 98.9%, respectively. Besides, despite incubation in a plasma platelet-poor medium, rich in plasma proteins, a flux recovery ratio of 75% could be measured while it was only 40% with a hydrophilic commercial membrane of similar structure and physical properties. Similarly, the zwitterionic membrane severely mitigated biofouling by microalgae during their harvesting. All in all, the material/process combination presented in this work leads to antibiofouling porous membranes with a large span of potential biomedically and biologically related applications.

Impact of growth mode, phase, and rate on the metabolic state of the extremely thermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus is emerging as a metabolic engineering platform for production of fuels and chemicals, such that more must be known about this organism's characteristics in bioprocessing contexts. Its ability to grow at temperatures from 70 to greater than 100°C and thereby avoid contamination, offers the opportunity for long duration, continuous bioprocesses as an alternative to batch systems. Toward that end, we analyzed the transcriptome of P. furiosus to reveal its metabolic state during different growth modes that are relevant to bioprocessing. As cells progressed from exponential to stationary phase in batch cultures, genes involved in biosynthetic pathways important to replacing diminishing supplies of key nutrients and genes responsible for the onset of stress responses were up-regulated. In contrast, during continuous culture, the progression to higher dilution rates down-regulated many biosynthetic processes as nutrient supplies were increased. Most interesting was the contrast between batch exponential phase and continuous culture at comparable growth rates (∼0.4 hr-1 ), where over 200 genes were differentially transcribed, indicating among other things, N-limitation in the chemostat and the onset of oxidative stress. The results here suggest that cellular processes involved in carbon and electron flux in P. furiosus were significantly impacted by growth mode, phase and rate, factors that need to be taken into account when developing successful metabolic engineering strategies.

Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation.

The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.

Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cells.

Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

Photocaged t7 RNA polymerase for the light activation of transcription and gene function in pro- and eukaryotic cells.

A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.

Plant cell calcium-rich environment enhances thermostability of recombinantly produced alpha-amylase from the hyperthermophilic bacterium Thermotoga maritime.

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

Gel-like ionic complexes for antimicrobial, hemostatic and adhesive properties.

Ion-specific effects offer a great opportunity to construct intelligent macromolecular systems with diverse architectures, on-demand controlled release behaviors and interfacial responsiveness. Herein, we developed gel-like polyelectrolyte/counterion complexes by ionotropic gelation of poly((trimethylamino)ethyl methacrylate chloride-co-sulfobetaine methacrylate) (poly(TMAEMA-co-SBMA)) and kosmotropic polyphosphate (PP). The strong water-mediated ionic crosslinking between the cationic poly(TMAEMA) and multivalent anionic PP leads to ionic association and formation of stable dispersive colloids and gel-like complexes. Zwitterionic SBMA possesses charge balance and strong hydration as well as insusceptibility to the presence of PP. The unique features of SBMA were applied to finely adjust the physical and biological properties of gel-like complexes. Accordingly, the molar composition of poly(TMAEMA-co-SBMA) was varied to evaluate its effects on the formation of the ionic complexes, water content, gel volume, ion-exchange capability, and viscoelastic recovery upon intermittent shear stress. The state diagrams of the poly(TMAEMA-co-SBMA) solutions as a function of the PP concentration were scrutinized in order to discover the relation between the ionic association and complex formation. The stability of the polymeric ionic complex structures was determined by the cationic molecular composition in the polymers and ionic strength. In terms of applications, the poly(TMAEMA-co-SBMA)/PP gel-like complexes served as an antimicrobial agent to inactivate pathogenic bacteria via leaching and contact killing approaches. The hemostasis of the complex gels in a tail-bleeding assay using Wistar rats was verified to ensure the potential in medical applications. Moreover, the gel-like complex was applied onto various substrates as an adhesive in comparison with commercial superglue gel, revealing the robust, substrate-independent, water-based, repeatable and removable adhesive property of the ionic complex glue. Consequently, this study was carried out in an attempt to explore the structure-property relation of ionically crosslinked polymer networks for a wide spectrum of applications.

Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels.

Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T(opt)> or =80 degrees C) actively participate in the production of H(2) in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4 mol of H(2) produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H(2) through conversion to H(2)S and CH(4) by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H(2) in hyperthermophiles are needed.

Functional-genomics-based identification and characterization of open reading frames encoding alpha-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus.

Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on alpha-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 beta-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative alpha-glucan-processing enzymes.

Synthesis of non-linear protein dimers through a genetically encoded Thiol-ene reaction.

Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.

Control of protein function through optochemical translocation.

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applications-light-triggered transcription and light-triggered protease cleavage-both based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology.

Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction.

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

Temperature, not LuxS, mediates AI-2 formation in hydrothermal habitats.

Quorum sensing provides the basis for coordinating community-wide, microbial behaviors in many mesophilic bacteria. However, little attention has been directed toward the possibility that such phenomena occur in extremely thermal microbial environments. Despite the absence of luxS in hyperthermophile genomes, autoinducer-2 (AI-2), a boronated furanone and proposed 'universal' interspecies mesophilic bacterial communication signal, could be formed by Thermotoga maritima and Pyrococcus furiosus through a combination of biotic and abiotic reaction steps. AI-2 did not, however, induce any detectable quorum-sensing phenotypes in these organisms, although transcriptome-based evidence of an AI-2-induced stress response was observed in T. maritima. The significance, if any, of AI-2 in hydrothermal habitats is not yet clear. Nevertheless, these results show the importance of considering environmental factors, in this case high temperatures, as abiotic causative agents of biochemical and microbiological phenomena.

Impact of substrate glycoside linkage and elemental sulfur on bioenergetics of and hydrogen production by the hyperthermophilic archaeon Pyrococcus furiosus.

Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h(-1) at 90 degrees C. In the absence of S(0), cellobiose-grown cells generated twice as much protein and had 50%-higher specific H(2) generation rates than maltose-grown cultures. Addition of S(0) to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H(2) to H(2)S. In contrast, the presence of S(0) in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H(2) to H(2)S production, with 2.5 times more H(2) than H(2)S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in alpha- or beta-glucan uptake and processing were up-regulated in an S(0)-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S(0) in cellobiose-grown cells also responded to S(0) in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S(0) was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S(0) metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.

Responses of wild-type and resistant strains of the hyperthermophilic bacterium Thermotoga maritima to chloramphenicol challenge.

Transcriptomes and growth physiologies of the hyperthermophile Thermotoga maritima and an antibiotic-resistant spontaneous mutant were compared prior to and following exposure to chloramphenicol. While the wild-type response was similar to that of mesophilic bacteria, reduced susceptibility of the mutant was attributed to five mutations in 23S rRNA and phenotypic preconditioning to chloramphenicol.

Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-alpha-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of alpha-glucan substrates by P. furiosus.