Abiotic Stimuli-Responsive Protein Affinity Reagent for IgG.

We describe an approach for the discovery of protein affinity reagents (PARs). Abiotic synthetic hydrogel copolymers can be "tuned" for selective protein capture by the type and ratios of functional monomers included in their polymerization and by the polymerization conditions (i.e., pH). By screening libraries of hydrogel nanoparticles (NPs) containing charged and hydrophobic groups against a protein target (IgG), a stimuli-responsive PAR is selected. The robust carbon backbone synthetic copolymer is rapidly synthesized in the chemistry laboratory from readily available monomers. The production of the PAR does not require living cells and is free from biological contamination. The capture and release of the protein by the copolymer NP is reversible. IgG is sequestered from human serum at pH 6.5 and following a wash step, the purified protein is released by elevating the pH to 7.3. The binding and release of the protein occur without denaturation. The abiotic material functions as a selective PAR for the F(ab')2 domain of IgG for pull-down and immunoprecipitation experiments and for isolation and purification of proteins from complex biological mixtures.

Abiotic Mimic of Endogenous Tissue Inhibitors of Metalloproteinases: Engineering Synthetic Polymer Nanoparticles for Use as a Broad-Spectrum Metalloproteinase Inhibitor.

We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP: a group that interacts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket, and fine-tuning of the chemical composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy provides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.

Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280FRGSAQGIEGS290, 368RRPFNIGINNQQ379 and 436FRSGFSNSSVSIIR449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368RRPFNIGINNQQ379. This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865NITIHITDTNNK876, a specific recognition site in insect receptor Bt-R1, to 368RRPFNIGINNQQ379. The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368RRPFNIGINNQQ379. This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

Engineering the Protein Corona of a Synthetic Polymer Nanoparticle for Broad-Spectrum Sequestration and Neutralization of Venomous Biomacromolecules.

Biochemical diversity of venom extracts often occurs within a small number of shared protein families. Developing a sequestrant capable of broad-spectrum neutralization across various protein isoforms within these protein families is a necessary step in creating broad-spectrum antivenom. Using directed synthetic evolution to optimize a nanoparticle (NP) formulation capable of sequestering and neutralizing venomous phospholipase A2 (PLA2), we demonstrate that broad-spectrum neutralization and sequestration of venomous biomacromolecules is possible via a single optimized NP formulation. Furthermore, this optimized NP showed selectivity for venomous PLA2 over abundant serum proteins, was not cytotoxic, and showed substantially long dissociation rates from PLA2. These findings suggest that it may show efficacy as an in vivo venom sequestrant and may serve as a generalized lipid-mediated toxin sequestrant.

Biomimetic Design of Mussel-Derived Bioactive Peptides for Dual-Functionalization of Titanium-Based Biomaterials.

Specific cell adhesion and osteogenicity are both crucial factors for the long-term success of titanium implants. In this work, two mussel-derived bioactive peptides were designed to one-step dual-biofunctionalization of titanium implants via robust catechol/TiO2 coordinative interactions. The highly biomimetic peptides capped with integrin-targeted sequence or osteogenic growth sequence could efficiently improve the biocompatibilities of titanium implants and endow the implants with abilities to induce specific cell adhesion and enhanced osteogenicity. More importantly, rationally combined use of the two biomimetic peptides indicated an enhanced synergism on osteogenicity, osseointegration and finally the mechanical stability of Ti implants in vivo. Therefore, the highly biomimetic mussel-derived peptides and the dual-functional strategy in this study would provide a facile, safe, and effective means for improving clinical outcome of titanium-based medical implants.

The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo.

Synthetic polymer nanoparticles (NPs) that bind venomous molecules and neutralize their function in vivo are of significant interest as "plastic antidotes." Recently, procedures to synthesize polymer NPs with affinity for target peptides have been reported. However, the performance of synthetic materials in vivo is a far greater challenge. Particle size, surface charge, and hydrophobicity affect not only the binding affinity and capacity to the target toxin but also the toxicity of NPs and the creation of a "corona" of proteins around NPs that can alter and or suppress the intended performance. Here, we report the design rationale of a plastic antidote for in vivo applications. Optimizing the choice and ratio of functional monomers incorporated in the NP maximized the binding affinity and capacity toward a target peptide. Biocompatibility tests of the NPs in vitro and in vivo revealed the importance of tuning surface charge and hydrophobicity to minimize NP toxicity and prevent aggregation induced by nonspecific interactions with plasma proteins. The toxin neutralization capacity of NPs in vivo showed a strong correlation with binding affinity and capacity in vitro. Furthermore, in vivo imaging experiments established the NPs accelerate clearance of the toxic peptide and eventually accumulate in macrophages in the liver. These results provide a platform to design plastic antidotes and reveal the potential and possible limitations of using synthetic polymer nanoparticles as plastic antidotes.

Synthetic polymer nanoparticle-polysaccharide interactions: a systematic study.

The interaction between synthetic polymer nanoparticles (NPs) and biomacromolecules (e.g., proteins, lipids, and polysaccharides) can profoundly influence the NPs fate and function. Polysaccharides (e.g., heparin/heparin sulfate) are a key component of cell surfaces and the extracelluar matrix and play critical roles in many biological processes. We report a systematic investigation of the interaction between synthetic polymer nanoparticles and polysaccharides by ITC, SPR, and an anticoagulant assay to provide guidelines to engineer nanoparticles for biomedical applications. The interaction between acrylamide nanoparticles (~30 nm) and heparin is mainly enthalpy driven with submicromolar affinity. Hydrogen bonding, ionic interactions, and dehydration of polar groups are identified to be key contributions to the affinity. It has been found that high charge density and cross-linking of the NP can contribute to high affinity. The affinity and binding capacity of heparin can be significantly diminished by an increase in salt concentration while only slightly decreased with an increase of temperature. A striking difference in binding thermodynamics has been observed when the main component of a polymer nanoparticle is changed from acrylamide (enthalpy driven) to N-isopropylacryalmide (entropy driven). This change in thermodynamics leads to different responses of these two types of polymer NPs to salt concentration and temperature. Select synthetic polymer nanoparticles have also been shown to inhibit protein-heparin interactions and thus offer the potential for therapeutic applications.

Light-triggered charge reversal of organic-silica hybrid nanoparticles.

A functional nanoparticle with light-triggered charge reversal based on a protected amine-bridged polysilsesquioxane was designed. An emulsion- and amine-free sol-gel synthesis was developed to prepare uniform nanospheres. Photolysis of suspensions of these nanoparticles results in a reversal of the ζ potential. This behavior has been used to trigger nanoparticle self-assembly, nanocomposite hydrogel formation, and nanoparticle release, showing the potential of this material in nanoscale manipulation and nanoparticle therapy.

Engineered synthetic polymer nanoparticles as IgG affinity ligands.

A process for the preparation of an abiotic protein affinity ligand is described. The affinity ligand, a synthetic polymer hydrogel nanoparticle (NP), is formulated with functional groups complementary to the surface presentation of the target protein. An iterative process is used to improve affinity by optimizing the composition and proportion of functional monomers. Since the polymer NPs are formed by a kinetically driven process, the sequence of functional monomers in the polymer chain is not controlled; only the average composition can be adjusted by the stoichiometry of the monomers in the feed. To compensate for this the hydrogel NP is lightly cross-linked resulting in chain flexibility that takes place on a submillisecond time scale allowing the polymer to "map" onto a protein surface with complementary functionality. In this study, we report a lightly cross-linked (2%) N-isopropyl acrylamide (NIPAm) synthetic polymer NP (50-65 nm) incorporating hydrophobic and carboxylate groups that binds with high affinity to the Fc fragment of IgG. The affinity and amount of NP bound to IgG is pH dependent. The hydrogel NP inhibits protein A binding to the Fc domain at pH 5.5, but not at pH 7.3. A computational analysis was used to identify potential NP-protein interaction sites. Candidates include a NP binding domain that overlaps with the protein A-Fc binding domain at pH 5.5. The computational analysis supports the inhibition experimental results and is attributed to the difference in the charged state of histidine residues. Affinity of the NP (3.5-8.5 nM) to the Fc domain at pH 5.5 is comparable to protein A at pH 7. These results establish that engineered synthetic polymer NPs can be formulated with an intrinsic affinity to a specific domain of a large biomacromolecule.

Sexual and reproductive health knowledge among primary school students in Malaysia.

BACKGROUND: There is limited research on sexual and reproductive health (SRH) knowledge level among very young adolescents aged between 10 and 14 years. Policy makers and educators are unable to address very young adolescents' SRH needs without proper understanding of their SRH knowledge. Thus, the objective of this study is to explore very young adolescents' SRH knowledge level and the influences of demographic variables on the respondents' SRH knowledge level in Malaysian primary schools. MATERIALS AND METHODS: This cross-sectional study involved 617 primary five students who were recruited through multistage sampling method from the state of Johor, Malaysia. A self-administered questionnaire (Cronbach's α = 0.81) which comprised of 38 items related to puberty, HIV/AIDS and sexually transmitted diseases, prevention of child sexual abuse and reproductive systems and reproduction was used. Multivariate logistic regression analysis was applied to examine the association between demographic variables and student's SRH knowledge. RESULTS: Based on the results, 28.5% of the respondents had unsatisfactory level of SRH knowledge. Among the four dimensions, the respondents had more knowledge on prevention of child sexual abuse but displayed poorer knowledge on HIV/AIDS and sexually transmitted diseases topic. The findings indicated female (odd ratio [OR] = 1.464) and Indian respondents (OR = 3.208) are more likely to exhibit poor SRH knowledge which suggested demographic factors exert some extent of influence on primary school children's SRH knowledge. CONCLUSION: The findings provide useful insights for the policy makers and educators seeking to improve the comprehensiveness of culturally accepted sexuality education which are essential in promoting young adolescents' well-being.

Synthesis of a High Affinity Complementary Peptide-Polymer Nanoparticle (NP) Pair Using Phage Display.

Peptide-polymer complementary pairs can provide useful tools for isolating, organizing, and separating biomacromolecules. We describe a procedure for selecting a high affinity complementary peptide-polymer nanoparticle (NP) pair using phage display. A hydrogel copolymer nanoparticle containing a statistical distribution of negatively charged and hydrophobic groups was used to select a peptide sequence from a phage displayed library of >1010 peptides. The NP has low nanomolar affinity for the selected cyclic peptide and exhibited low affinity for a panel of diverse proteins and peptide variants. Affinity arises from the complementary physiochemical properties of both NP and peptide as well as the specific peptide sequence. Comparison of linear and cyclic variants of the peptide established that peptide structure also contributes to affinity. These findings offer a general method for identifying polymer-peptide complementary pairs. Significantly, precise polymer sequences (proteins) are not a requirement, a low information statistical copolymer can be used to select for a specific peptide sequence with affinity and selectivity comparable to that of an antibody. The data also provides evidence for the physiochemical and structural contributions to binding. The results confirm the utility of abiotic, statistical, synthetic copolymers as selective, high affinity peptide affinity reagents.

Acid and NaCl limits to growth of Listeria monocytogenes and influence of sequence of inimical acid and NaCl levels on inactivation kinetics.

Variability in growth limits of Listeria monocytogenes in response to low pH (adjusted with HCl) or high salinity (NaCl) was evaluated for 127 strains in brain heart infusion broth at 25 degrees C. Over 95% of strains habituated at pH 5.0 grew subsequently at pH 4.2, while 25% were able to grow at pH 4.1. More than 85% of strains preadapted to growth at 8.5% NaCl (wt/vol) subsequently grew in the presence of 11.3% NaCl, while 25% were able to grow at 13% NaCl, and 4.7% grew in the presence of 13.9% NaCl. The results extend the generally accepted growth limits for L. monocytogenes in response to these hurdles. Two strains, one of which was relatively tolerant of both hurdles, and another that was less tolerant of both hurdles, were subjected to different sequences of lethal acid (pH 3.5) and NaCl (14%, wt/vol) stresses to determine whether survival was affected by growth limits, or by sequence of application of treatment. There was no significant difference in the inactivation kinetics of the two strains, but inactivation rates were affected by different treatments. For both strains, the inactivation rates, from fastest to slowest, resulted from: (i) lethal pH and then by lethal water activity, or lethal water activity and then by lethal pH; (ii) lethal pH and water activity applied simultaneously; (iii) lethal pH; and (iv) lethal water activity. The results demonstrated that the sequence of lethal stress application strongly influences L. monocytogenes inactivation, and that L. monocytogenes growth limits are not good predictors of survival in inimical environments.

Characterization and composition of heavy metals and persistent organic pollutants in water and estuarine sediments from Gao-ping River, Taiwan.

The objective of this study was to determine the concentrations and possible sources of heavy metals and persistent organic pollutants (POPs) in water and estuarine sediments from Gao-ping River in order to evaluate the environmental quality of aquatic system in southern Taiwan. High concentrations of heavy metals including Cr, Zn, Ni, Cu and As, ranging from 10.7 to 180 mg/kg-dry weight (dw), were detected in sediments from Gao-ping River. When normalized to the principal component analysis (PCA), swinery and electroplating wastewaters were found to be the most important pollution sources for heavy metals. Of various organochlorine pesticide (OCP) residues detected, aldrin and total-hexachlorocyclohexane (HCH) were frequently found in sediments. The total concentrations of OCPs were in the range 0.47-47.4 ng/g-dw. Also, the total-HCH, total-cyclodiene, and total-dichlorodiphenyltrichloroethane (DDT) were in the range 0.37-36.3, 0.21-19.0, and 0.44-1.88 ng/g-dw, respectively. The polychlorinated biphenyl (PCB) concentrations in sediments from Gao-ping River ranged between 0.37 and 5.89 ng/g-dw. The PCB concentrations are positively correlated to the organic contents of the sediment particles. alpha-HCH was found to be the dominant compound of HCH in the sediments, showing that long-range transport may be the possible source for the contamination of HCH in sediments from Gao-ping River. In summary, trace amounts of POPs in estuarine sediments from Gao-ping River were detected, showing that there still exist a wide variety of POP residues in the river sediments in Taiwan. These POP residues may be mainly from long-range transport and weathered agricultural soils, while heavy metal contamination is primarily from the swinery and industrial wastewaters.

Engineered nanoparticles bind elapid snake venom toxins and inhibit venom-induced dermonecrosis.

Envenomings by snakebites constitute a serious and challenging global health issue. The mainstay in the therapy of snakebite envenomings is the parenteral administration of animal-derived antivenoms. Significantly, antivenoms are only partially effective in the control of local tissue damage. A novel approach to mitigate the progression of local tissue damage that could complement the antivenom therapy of envenomings is proposed. We describe an abiotic hydrogel nanoparticle engineered to bind to and modulate the activity of a diverse array of PLA2 and 3FTX isoforms found in Elapidae snake venoms. These two families of protein toxins share features that are associated with their common (membrane) targets, allowing for nanoparticle sequestration by a mechanism that differs from immunological (epitope) selection. The nanoparticles are non-toxic in mice and inhibit dose-dependently the dermonecrotic activity of Naja nigricollis venom.

Efficient capture, rapid killing and ultrasensitive detection of bacteria by a nano-decorated multi-functional electrode sensor.

In this work, we demonstrated a nano-decorated porous impedance electrode sensor for efficient capture, rapid killing and ultrasensitive detection of bacteria. The multi-functional sensor was prepared by a facile sonochemical method via in situ deposition of antibacterial prickly Zn-CuO nanoparticles and graphene oxide (GO) nanosheets on a Ni porous electrode. Due to the surface burr-like nanostructures, the nano-decorated impedance sensor exhibited very good bacterial-capture efficiency (70 - 80% in 20min) even at a low concentration of 50 CFU mL-1, rapid antibacterial rate (100% killing in 30min) and high detection sensitivity (as low as 10 CFU mL-1). More importantly, the nano-decorated sensor has proven to be highly effective in quantitative detection of bacteria in a biological sample, for example, a rat blood sample spiked with E. coli. Despite the complexity of blood, the sensor still exhibited excellent detection precision within 30min at bacteria concentrations ranging from 10 - 105 CFU mL-1. The simplicity, rapidity, sensitivity, practicability and multifunctionality of this impedance sensor would greatly facilitate applications in portable medical devices for on-the-spot diagnosis and even the possibility for simultaneous therapy of diseases caused by bacterial infections.

Functionalized Vesicles by Microfluidic Device.

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165).

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

Polymer nanoparticle-protein interface. Evaluation of the contribution of positively charged functional groups to protein affinity.

Cationic-functionalized polymer nanoparticles (NPs) show strikingly distinct affinities to proteins depending on the nature of the cationic functional group. N-Isopropylacrylamide (NIPAm) polymer NPs incorporating three types of positively charged functional groups (guanidinium, primary amino, and quaternary ammonium groups) were prepared by precipitation polymerization. The affinities to fibrinogen, a protein with an isoelectric point (pI) of 5.5, were compared using UV-vis spectrometry and a quartz crystal microbalance (QCM). Guanidinium-containing NPs showed the highest affinity to fibrinogen. The observation is attributed to strong, specific interactions with carboxylate groups on the protein surface. The affinity of the positively charged NPs to proteins with a range of pIs revealed that protein-NP affinity is due to a combination of ionic, hydrogen bonding, and hydrophobic interactions. Protein affinity can be modulated by varying the composition of these functional monomers in the acrylamide NPs. Engineered NPs containing the guanidinium group with hydrophobic and hydrogen bonding functional groups were used in an affinity precipitation for the selective separation of fibrinogen from a plasma protein mixture. Circular dichroism (CD) revealed that the protein was not denatured in the process of binding or release.

In vitro synergistic antimicrobial effect of imipenem and colistin against an isolate of multidrug-resistant Enterobacter cloacae.

BACKGROUND/PURPOSE: Enterobacter cloacae is an important nosocomial pathogen responsible for various infections. Little is known about the synergistic effects of imipenem and colistin against multidrug-resistant E. cloacae. Therefore, we investigated the in vitro effects of imipenem and colistin against a clinical isolate of multidrug-resistant E. cloacae. METHODS: A strain of E. cloacae, designed Ent 831, was isolated from the sputum of a woman who developed severe pneumonia in a medical intensive care unit. Minimal inhibitory concentrations (MICs) of imipenem and colistin were determined by the agar dilution method. The synergistic effects were investigated using the time-kill method. RESULTS: MICs of imipenem and colistin for E. cloacae strain Ent 831 were 0.5 microg/mL and 1.0 microg/mL, respectively. Using a standard inoculum (5 x 10(5)) CFU/mL), synergism was shown with a concentration of two times the MICs of imipenem and colistin. Furthermore, four times the MIC of imipenem completely inhibited bacterial growth for more than 48 hours, but four times the MICs of colistin resulted in re-growth after 4 hours. There was no synergism between imipenem and colistin at two times the MICs against a high concentration inoculum (6.24 x 10(6)) CFU/mL). Nevertheless, imipenem, with or without colistin, at a concentration of four times MICs could inhibit the growth of bacteria for more than 48 hours. CONCLUSION: High-dose imipenem, alone or in combination with colistin, is effective against multidrug-resistant E. cloacae. Colistin alone, even at a high dose, is not effective. However, in vitro susceptibility to antimicrobial compounds does not always correlate with clinical success. Thus further testing of these antibiotic combinations in animal models is needed in order to predict their suitability for clinical use.