Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes.

BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

Single-pot extraction-analysis of dyed wool fibers with ionic liquids.

Analytical capabilities to identify dyes associated with structurally robust wool fibers would critically assist crime-scene and explosion-scene forensics. Nondestructive separation of dyes from wool, removal of contaminants, and dye analysis by MALDI- or ESI-MS, were achieved in a single-pot, ionic liquid-based method. Ionic liquids (ILs) that readily denature the wool α-keratin structure have been identified and are conducive to small volume, high-throughput analysis for accelerated threat-response times. Wool dyed with commercial or natural, plant-based dyes have unique signatures that allow classification and matching of samples and identification of dyestuffs. Wool released 0.005 mg of dye per mg of dyed wool into the IL, allowing for analysis of single-thread sample sizes. The IL + dye mixture promotes sufficient ionization in MALDI-MS: addition of common MALDI matrices does not improve analysis of anionic wool dyes. An inexpensive, commercially available tetrabutylphosponium chloride IL was discovered to be capable of denaturing wool and was determined to be the most effective for this readily fieldable method.

Methods to capture proteomic and metabolomic signatures from cerebrospinal fluid and serum of healthy individuals.

Discovery of reliable signatures for the empirical diagnosis of neurological diseases-both infectious and non-infectious-remains unrealized. One of the primary challenges encountered in such studies is the lack of a comprehensive database representative of a signature background that exists in healthy individuals, and against which an aberrant event can be assessed. For neurological insults and injuries, it is important to understand the normal profile in the neuronal (cerebrospinal fluid) and systemic fluids (e.g., blood). Here, we present the first comparative multi-omic human database of signatures derived from a population of 30 individuals (15 males, 15 females, 23-74 years) of serum and cerebrospinal fluid. In addition to empirical signatures, we also assigned common pathways between serum and CSF. Together, our findings provide a cohort against which aberrant signature profiles in individuals with neurological injuries/disease can be assessed-providing a pathway for comprehensive diagnostics and therapeutics discovery.

Tetraalkylphosphonium-based ionic liquids for a single-step dye extraction/MALDI MS analysis platform.

Room temperature ionic liquids, or RTILs, based on tetraalkylphosphonium (PR(4)(+)) cations were used as the basis of a platform that enables separation of dyes from textiles, extraction of dyes from aqueous solution, and identification of the dyes by MALDI-MS in a single experimental step for forensic purposes. Ionic liquids were formed with PR(4)(+) cations and ferulate (FA), α-cyano-4-hydroxycinnamate (CHCA), and 2,5-dihydroxybenzoate (DHB) anions. The use of tetraalkylphosphonium-based ionic liquids in MALDI-MS allowed detection of small molecule dyes without addition of a traditional solid MALDI matrix.

A quantitative correlation of the effect of density distributions in roller-compacted ribbons on the mechanical properties of tablets using ultrasonics and X-ray tomography.

Enabling the paradigm of quality by design requires the ability to quantitatively correlate material properties and process variables to measureable product performance attributes. In this study, we show how heterogeneities in compacted ribbon densities quantitatively correlate to tablet mechanical properties. These density variations, which have been purposely modulated by internal and external lubrications, are characterized longitudinally and transversally by nondestructive ultrasonic and X-ray micro-computed tomography measurements. Subsequently, different transversal regions of the compacted ribbon are milled under the same conditions, and granules with nominally the same particle size distribution are utilized to manufacture cylindrical tablets, whose mechanical properties are further analyzed by ultrasonic measurements. We consider three different ribbon conditions: no lubrication (case 1); lubricated powder (case 2); and lubricated tooling (hopper, side sealing plates, feed screws, and rolls) (case 3). This study quantitatively reveals that variation in local densities in ribbons (for case 1) and process conditions (i.e., internal case 2 and external lubrication case 3) during roller compaction significantly affect the mechanical properties of tablets even for granules with the same particle size distribution. For case 1, the mechanical properties of tablets depend on the spatial location where granules are produced. For cases 2 and 3, the ribbon density homogeneity was improved by the use of a lubricant. It is demonstrated that the mechanical performances of tablets are decreased due to applied lubricant and work-hardening phenomenon. Moreover, we extended our study to correlate the speed of sound to the tensile strength of the tablet. It is found that the speed of sound increases with the tensile strength for the tested tablets.

Development of Anti-Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

BACKGROUND: Yersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness. METHODS: Here, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays. RESULTS: Two of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM). CONCLUSION: These antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.

PEGylation of a maltose biosensor promotes enhanced signal response when immobilized in a silica sol-gel.

A robust method to immobilize a maltose biosensor is described using an engineered maltose periplasmic binding protein (PBP) covalently coupled to NBDamide, an environmentally sensitive fluorophore. A mesoporous silica sol-gel derived from diglycerylsilane (DGS) was constructed to embed the maltose biosensor, and the ligand reporting fluorescence properties were measured. When sequestered in the DGS-derived silica matrix, the biosensor retained maltose-dependent fluorescence sensing capability with micromolar affinity, which is consistent with the protein free in solution. The MBP-NBD conjugate was further modified by covalent conjugation with poly(ethylene glycol)-5000 (PEG) to promote the retention of water molecules around the protein and to reduce possible steric effects between the silica matrix and protein. Bioconjugation with PEG molecules does not significantly affect the signaling response of the protein in solution. When immobilized in the DGS polymer, a consistent increase in fluorescence intensity was observed as compared to the protein not functionalized with PEG. To our knowledge, this report presents the first successful method to embed a PBP biosensor in a polymerized matrix and retain signaling response using an environmentally sensitive probe. The immobilization method presented here should be easily adaptable to all conformation-dependent biosensors.

Biosynthesis of the 3,4-dihydroxybenzoate moieties of petrobactin by Bacillus anthracis.

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional (1)H and (13)C NMR spectroscopy, as well as (13)C-(13)C DQFCOSY spectroscopy. Isotopic enrichment and (13)C-(13)C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.

Micromachining of Polyurethane Membranes for Tissue Engineering Applications.

Engineered tissue barrier models offer in vitro alternatives in toxicology and disease research. To mimic barrier-tissue microenvironment, a porous membrane that can approach the stiffness of physiological basement membranes is required. While several biocompatible membranes with micrometer range thickness (10 µm) and a stiffness less than polystyrene (3 GPa) or polyethylene (PET, 2 GPa), have been developed, there has been little effort to optimize the process to enable rapid and reproducible pore production in these membranes. Here, we investigate the use of laser irradiation with femtosecond (fs) pulses because the combination of high-precision and cold-ablation causes minimal damage to polymeric membranes. This process enables automated, high-throughput and reproducible fabrication of thin, microporous membranes that can be utilized to culture cells at air-liquid interface (ALI), a unique culture technique that simulates the tissue-barrier microenvironment. We show the optimization of laser parameters on a thin polyurethane membrane and patterned pores with an average diameter of 5 µm. Tissue was cultured at ALI for 28 days to demonstrate the membrane's utility in constructing a tissue barrier model. These results confirm the utilization of fs laser machining as a viable method for creating a porous barrier substrate in tissue engineering platforms.

Proteomic analysis of Bacillus anthracis Sterne vegetative cells.

Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism.

Surface-assisted laser desorption/ionization mass spectrometry.

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.

Structurally Defined 3D Nanographene Assemblies via Bottom-Up Chemical Synthesis for Highly Efficient Lithium Storage.

Functionalized 3D nanographenes with controlled electronic properties have been synthesized through a multistep organic synthesis method and are further used as promising anode materials for lithium-ion batteries, exhibiting a much increased capacity (up to 950 mAh g-1 ), three times higher than that of the graphite anode (372 mAh g-1 ).

Comparison of tapentadol with tramadol for analgesia after cardiac surgery.

BACKGROUND: Tapentadol is a relatively new analgesic. We decided to compare it with tramadol for their various effects after cardiac surgery. SETTING: A study in a tertiary care hospital. MATERIALS AND METHODS: Sixty adults undergoing cardiac surgery were divided into 2 groups of 30 each by computerized random allotment (Group X = tapentadol 50 mg oral and Group Y = tramadol 100 mg oral). Informed Consent and Institutional Ethics Committee approval were obtained. The patients were given either drug X or drug Y after extubation in this single blinded study, wherein the data collectors and analyzers were blinded to the study. All patients received oral paracetamol qds and either drug X or drug Y tds. The pain score was noted on a Visual Analog Scale before each drug dose, 3 h later and on coughing. Heart rate, respiratory rate, and blood pressure were recorded before the drug dose and 3 h later. Postoperative nausea or vomiting (PONV), temperature, and modified Glasgow Coma Scale readings were recorded. The above readings were obtained for 6 doses (up to 48 h after extubation). STATISTICS: t-test, Pearson Chi-square test, Fisher exact test, and Mantel-Haenszel test were used for statistics. RESULTS: Tapentadol group patients had significantly better analgesia 3 h after the drug and "on coughing" than tramadol group. The difference in their effects on blood creatinine levels, temperature, hemodynamics, oxygen saturation, and respiratory rate were not clinically significant. Tapentadol produced lesser drowsiness and lesser vomiting than tramadol. CONCLUSIONS: Tapentadol, due to its norepinephrine reuptake inhibition properties, in addition to mu agonist, is a better analgesic than tramadol and has lesser PONV.

Fluid and Resistive Tethered Lipid Membranes on Nanoporous Substrates.

Cell membranes perform important biological roles including compartmentalization, signaling, and transport of nutrients. Supported lipid membranes mimic the behavior of cell membranes and are an important model tool for studying membrane properties in a controlled laboratory environment. Lipid membranes may be supported on solid substrates; however, protein and lipid interactions with the substrate typically result in their denaturation. In this report, we demonstrate the formation of intact lipid membranes tethered on nanoporous metal thin films obtained via a dealloying process. Uniform lipid membranes were formed when the surface defect density of the nanoporous metal film was significantly reduced through a two-step dealloying process reported here. We show that the tethered lipid membranes on nanoporous metal substrates maintain both fluidity and electrical resistivity, which are key attributes to naturally occurring lipid membranes. The lipid assemblies supported on nanoporous metals provide a new platform for investigating lipid membrane properties, and potentially membrane proteins, for numerous applications including next generation biosensor platforms, targeted drug-delivery, and energy harvesting devices.

Proteomic analysis of detergent resistant membrane domains during early interaction of macrophages with rough and smooth Brucella melitensis.

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide the basis for novel ways of treating or diagnosing Brucellosis.

Stable and fluid multilayer phospholipid-silica thin films: mimicking active multi-lamellar biological assemblies.

Phospholipid-based nanomaterials are of interest in several applications including drug delivery, sensing, energy harvesting, and as model systems in basic research. However, a general challenge in creating functional hybrid biomaterials from phospholipid assemblies is their fragility, instability in air, insolubility in water, and the difficulty of integrating them into useful composites that retain or enhance the properties of interest, therefore limiting there use in integrated devices. We document the synthesis and characterization of highly ordered and stable phospholipid-silica thin films that resemble multilamellar architectures present in nature such as the myelin sheath. We have used a near room temperature chemical vapor deposition method to synthesize these robust functional materials. Highly ordered lipid films are exposed to vapors of silica precursor resulting in the formation of nanostructured hybrid assemblies. This process is simple, scalable, and offers advantages such as exclusion of ethanol and no (or minimal) need for exposure to mineral acids, which are generally required in conventional sol-gel synthesis strategies. The structure of the phospholipid-silica assemblies can be tuned to either lamellar or hexagonal organization depending on the synthesis conditions. The phospholipid-silica films exhibit long-term structural stability in air as well as when placed in aqueous solutions and maintain their fluidity under aqueous or humid conditions. This platform provides a model for robust implementation of phospholipid multilayers and a means toward future applications of functional phospholipid supramolecular assemblies in device integration.

Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.

Characterization of reaction intermediate aggregates in aniline oxidative polymerization at low proton concentration.

Aggregates of reaction intermediates form during the early stages of aniline oxidative polymerization whenever the initial mole ratio of proton concentration to aniline monomer concentration is low ([H(+)](0)/[An](0)

Synthesis and characterization of ethylene glycol substituted poly(phenylene vinylene) derivatives.

We report the synthesis of a series of water-soluble, fluorescent, conjugated polymers via the Gilch reaction with an overall yield greater than 40%. The yield for the Gilch reaction decreases with the increase in the length of the side chain (ethylene glycol repeat units), presumably due to the steric effects inhibiting the linking of monomeric units. The hydrophilic side chain enhances the solubility of the polymer in water and concomitantly leads to a side-chain-dependent conformation and solvent-dependent quantum efficiency. An increase in the ethylene glycol repeat units on the polymer side chain structure results in changes in chain packing; hence, the crystallinity evolves from semicrystalline to liquid crystalline to completely amorphous. An increase in the length of the side chain leads to changes in the polymer-solvent interaction as manifested in the photophysical properties of these polymers. These novel polymers exhibit two glass transition temperatures, which can be readily rationalized by differences in microstructure when casted from hydrophobic and hydrophilic solvents. Cyclic voltammograms of polymer 1d-3d suggest two-electron transfer, as compared to P1 which has one complete redox pair. The potential of having a nanoscaled domain structure and stabilizing two electrons on a polymer chain signifies the potential of these polymers in fabricating electronic and photovoltaic devices.