Investigation of Thermal-Induced Changes in Molecular Order on Photopolymerization and Performance Properties of a Nematic Liquid-Crystal Diacrylate.

Polymerization shrinkage and associated stresses are the main reasons for dental restorative failure. We developed a series of liquid crystal diacrylates and dimethacrylates which have markedly low polymerization shrinkage. In order to fully understand the effects of temperature-induced changes of molecular order on the photopolymerization process and performance properties of the generated polymers, the photopolymerization of a difunctional acrylate, 2-t-butyl-1,4-phenylene bis (4-(6-(acryloyloxy)hexyloxy)benzoate), which exists in the nematic liquid crystalline phase at room temperature, was investigated as a function of photopolymerization temperature over the nematic to isotropic range. Morphological studies suggested that a mesogenic phase was immediately formed in the polymer even if polymerization in thin films occurred above the nematic-to-isotropic (N→I) transition temperature of the monomer (Tn-i = 45.8 °C). Dynamic mechanical analysis of 2 × 2 mm cross-section bar samples polymerized at 60 °C showed reduced elastic moduli, increased glass transition temperature and formation of a more crosslinked network, in comparison to polymers formed at lower polymerization temperatures. Fractography analysis showed that polymers generated from the nematic liquid crystalline phase underwent a different fracture pattern in comparison to those generated from the isotropic phase. Volumetric shrinkage (2.2%) found in polymer polymerized from the nematic liquid crystalline phase at room temperature was substantially less than the 6.0% observed in polymer polymerized from an initial isotropic phase at 60 °C, indicating that an organized monomer can greatly contribute to reducing cure shrinkage.

Fast Identification and Quantification of Uropathogenic E. coli through Cluster Analysis.

Rapid diagnostic tools to detect, identify, and enumerate bacteria are key to maintaining effective antibiotic stewardship and avoiding the unnecessary prescription of broad-spectrum agents. In this study, a 15 min agglutination assay is developed that relies on the use of mannose-functionalized polymeric microspheres in combination with cluster analysis. This allows for the identification and enumeration of laboratory (BW25113), clinical isolate (NCTC 12241), and uropathogenic Escherichia coli strains (NCTC 9001, NCTC 13958, J96, and CFT073) at clinically relevant concentrations in tryptic soy broth (103-108 CFU/mL) and in urine (105-108 CFU/mL). This fast, simple, and efficient assay offers a step forward toward efficient point-of-care diagnostics for common urinary tract infections.

Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase.

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for the LHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.

An Effective Electric Dipole Model for Voltage-induced Gating Mechanism of Lysenin.

Lysenin is a pore-forming toxin, which self-inserts open channels into sphingomyelin containing membranes and is known to be voltage regulated. The mechanistic details of its voltage gating mechanism, however, remains elusive despite much recent efforts. Here, we have employed a novel combination of experimental and computational techniques to examine a model for voltage gating, that is based on the existence of an "effective electric dipole" inspired by recent reported structures of lysenin. We support this mechanism by the observations that (i) the charge-reversal and neutralization substitutions in lysenin result in changing its electrical gating properties by modifying the strength of the dipole, and (ii) an increase in the viscosity of the solvent increases the drag force and slows down the gating. In addition, our molecular dynamics (MD) simulations of membrane-embedded lysenin provide a mechanistic picture for lysenin conformational changes, which reveals, for the first time, the existence of a lipid-dependent bulge region in the pore-forming module of lysenin, which may explain the gating mechanism of lysenin at a molecular level.

Adhesive force between graphene nanoscale flakes and living biological cells.

We report on a measurement technique that quantifies the adhesive force between multi-layers of graphene flakes and the cell wall of live Escherichia coli cells using atomic force microscopy (AFM) in-fluid Peak Force- Quantitative Nanomechanical Mapping mode. To measure the adhesive force, we made use of the negative charge of E. coli cells to allow them to stick to positively charged surfaces, such as glass or silicon, that were covered by poly-L-Lysine. With this approach, cells were held in place for AFM characterization. Both pristine graphene (PG) flakes and functionalized graphene (FG) flakes were put on the E. coli cells and measurements of lateral size, flake thickness, and adhesion were made. Using this approach, the measured values of the adhesive force between multi-layers of graphene flakes (total thickness of 50 nm) and E. coli was determined to be equal or greater than 431 ± 65pN for (PG) and 694 ± 98pN for the (FG). More interestingly, the adhesive force of a graphene flake (thickness 1.3 nm) with the cell is determined to be equal or greater than 38.2 ± 16.4pN for the (PG) and 34.8 ± 15.3pN for the (FG). These interaction values can play an important role in determining and understanding the possible toxicity of graphene flakes. Copyright © 2017 John Wiley & Sons, Ltd.

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle.

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates.

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.

A novel antimicrobial orthodontic band cement with in situ-generated silver nanoparticles.

OBJECTIVE: To develop an antimicrobial orthodontic band cement for the prevention of white spot lesions using a novel process that generates silver nanoparticles (AgNP) in situ. MATERIALS AND METHODS: Twenty-seven groups of AgNP-loaded Opal Band Cement (OBC) and two control groups were formulated with varying concentrations of additional benzoyl peroxide (0.5, 1.0, 1.5, or 2.0 wt%) and 2,2-(p-Tolylimino) diethanol (0.5 or 1 wt%). Rockwell15T hardness and near-infrared FTIR were used to assess degree of cure, three-point bending was used to determine modulus and ultimate transverse strength (UTS), and Ag(+) ion release was measured for up to 4 months in vitro using atomic absorption spectroscopy. Antimicrobial activity against Streptococcus mutans and Lactobacillus acidophilus was tested in vitro by counting colony-forming units for up to 28 days. Biocompatibility was evaluated following ISO specifications 7405 (2008), 10993-3 (2003), 10993-5 (2009), and 10993-10 (2010). RESULTS: Most of the experimental groups had hardness, modulus, and UTS values similar to those of the control group. Ag(+) ion release was observed for all AgNP-loaded groups for up to 4 months. Increase in Ag loading increased Ag(+) ion release and in vitro antimicrobial effect. The biocompatibility of the optimal AgNP-loaded OBC was comparable to that of negative controls. CONCLUSION: A novel antimicrobial orthodontic band cement was developed that has comparable mechanical properties to controls, controlled and sustained Ag(+) ion release, significant bacterial inhibition in vitro, and excellent biocompatibility.

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers.

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17-19 d in the 3 highest dose groups and volume of distribution of 5-6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use.

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.

A model for the hysteresis observed in gating of lysenin channels.

The pore-forming toxin lysenin self-inserts to form conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels exhibit voltage regulation and hysteresis of the macroscopic current during the application of positive periodic voltage stimuli. We explored the bi-stable behavior of lysenin channels and present a theoretical approach for the mechanism of the hysteresis to explain its static and dynamic components. This investigation develops a model to incorporate the role of charge accumulation on the bilayer lipid membrane in influencing the channel conduction state. Our model is supported by experimental results and also provides insight into the temperature dependence of lysenin channel hysteresis. Through this work we gain perspective into the mechanism of how the response of a channel protein is determined by previous stimuli.

Radiation-induced galectin-1 by endothelial cells: a promising molecular target for preferential drug delivery to the tumor vasculature.

The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature-targeting ligand, anginex, to improve the therapeutic ratio for strategies developed against solid tumors. Our findings indicate that galectin-1 is (a) one of the major receptors for anginex (b) overexpressed by tumor neovasculature and (c) further specifically upregulated in endothelial cells in response to radiation exposure as low as 0.5 Gy. An investigation of [18]-F-labeled anginex biodistribution in SCK tumors indicates that anginex is an effective targeting molecule for image and radiation-guided therapy of solid tumors. An anginex-conjugated liposome capable of being loaded with drug was shown to selectively target endothelial cells post-radiation. The presence of endothelial cells in a three-dimensional co-culture system with tumor cells developed to study tumor/endothelial cell interactions in vitro led to higher levels of galectin-1 and showed a further increase in expression upon radiation exposure when compared to tumor cell spheroids alone. Similar increase in galectin-1 was observed in tumor tissue originating from the tumor-endothelial cell spheroids in vivo and radiation exposure further induced galectin-1 in these tumors. The overall results suggest feasibility of using a clinical or subclinical radiation dose to increase expression of the galectin-1 receptor on the tumor microvasculature to promote delivery of therapeutics via the anginex peptide. This approach may reduce systemic toxicity, overcome drug resistance, and improve the therapeutic efficacy of conventional chemo/radiation strategies.

Bi-stability, hysteresis, and memory of voltage-gated lysenin channels.

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages. We report on the consequences of slow voltage-induced gating of lysenin channels inserted into a planar Bilayer Lipid Membrane (BLM), and demonstrate that these pore-forming proteins constitute memory elements that manifest gating bi-stability in response to variable external voltages. The hysteresis in macroscopic currents dynamically changes when the time scale of the voltage variation is smaller or comparable to the characteristic conformational equilibration time, and unexpectedly persists for extremely slow-changing external voltage stimuli. The assay performed on a single lysenin channel reveals that hysteresis is a fundamental feature of the individual channel unit and an intrinsic component of the gating mechanism. The investigation conducted at different temperatures reveals a thermally stable reopening process, suggesting that major changes in the energy landscape and kinetics diagram accompany the conformational transitions of the channels. Our work offers new insights on the dynamics of pore-forming proteins and provides an understanding of how channel proteins may form an immediate record of the molecular history which then determines their future response to various stimuli. Such new functionalities may uncover a link between molecular events and macroscopic processing and transmission of information in cells, and may lead to applications such as high density biologically-compatible memories and learning networks.

Potential analytical applications of lysenin channels for detection of multivalent ions.

Transmembrane protein transporters possessing binding sites for ions, toxins, pharmaceutical drugs, and other molecules constitute excellent candidates for developing sensitive and selective biosensing devices. Their attractiveness for analytical purposes is enhanced by the intrinsic amplification capabilities shown when the binding event leads to major changes in the transportation of ions or molecules other than the analyte itself. The large-scale implementation of such transmembrane proteins in biosensing devices is limited by the difficulties encountered in inserting functional transporters into artificial bilayer lipid membranes and by the limitations in understanding and exploiting the changes induced by the interaction with the analyte for sensing purposes. Here, we show that lysenin, a pore-forming toxin extracted from earthworm Eisenia foetida, which inserts stable and large conductance channels into artificial bilayer lipid membranes, functions as a multivalent ion-sensing device. The analytical response consists of concentration and ionic-species-dependent macroscopic conductance inhibition most probably linked to a ligand-induced gating mechanism. Multivalent ion removal by chelation or precipitation restores, in most cases, the initial conductance and demonstrates reversibility. Changes in lipid bilayer membrane compositions leading to the absence of voltage-induced gating do not affect the analytical response to multivalent ions. Microscopic current analysis performed on individual lysenin channels in the presence of Cu(2+) revealed complex open-closed transitions characterized by unstable intermediate sub-conducting states. Lysenin channels provide an analytical tool with a built-in sensing mechanism for inorganic and organic multivalent ions, and the excellent stability in an artificial environment recommend lysenin as a potential candidate for single-molecule detection and analysis.

Multivalent ions control the transport through lysenin channels.

We report the effect of different ions on the conducting properties of lysenin channels inserted into planar lipid bilayer membranes. Our observations indicated that multivalent ions inhibited the lysenin channels conductance in a concentration dependent manner. The analysis performed on single channels revealed that multivalent ions induced reversible sub-conducting or closed states depending on the ionic charge and size. Good agreement is reported between experimental results and a theoretical model that is proposed to describe the interaction between divalent ions and lysenin channels as a simple isothermal absorption process.

A dynamic cpSRP43-Albino3 interaction mediates translocase regulation of chloroplast signal recognition particle (cpSRP)-targeting components.

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.

Controlled gating of lysenin pores.

Lysenin forms unitary large conductance pores in artificial bilayer membranes containing sphingomyelin. A population of lysenin pores inserted into such a bilayer membrane exhibited a dynamic negative conductance region, as predicted by a simple two-state model for voltage-gated channels. The recorded I-V curves demonstrated that lysenin pores inserted into the bilayer are uniformly oriented. Additionally, the transition between the two-states was affected by changes in the monovalent ion concentration and pH, pointing towards an electrostatic interaction governing the gating mechanism.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.