Engineering Alkaline-Stable Barley Stripe Mosaic Virus-Like Particles for Efficient Surface Modification.

Viruses and virus-like particles are powerful templates for materials synthesis because of their capacity for precise protein engineering and diverse surface functionalization. We recently developed a recombinant bacterial expression system for the production of barley stripe mosaic virus-like particles (BSMV VLPs). However, the applicability of this biotemplate was limited by low stability in alkaline conditions and a lack of chemical handles for ligand attachment. Here, we identify and validate novel residues in the BSMV Caspar carboxylate clusters that mediate virion disassembly through repulsive interactions at high pH. Point mutations of these residues to create attractive interactions that increase rod length ~2 fold, with an average rod length of 91 nm under alkaline conditions. To enable diverse chemical surface functionalization, we also introduce reactive lysine residues at the C-terminus of BSMV coat protein, which is presented on the VLP surface. Chemical conjugation reactions with this lysine proceed more quickly under alkaline conditions. Thus, our alkaline-stable VLP mutants are more suitable for rapid surface functionalization of long nanorods. This work validates novel residues involved in BSMV VLP assembly and demonstrates the feasibility of chemical functionalization of BSMV VLPs for the first time, enabling novel biomedical and chemical applications.

Stagnation Point of Surface Flow during Drop Evaporation.

Capillary flow and Marangoni flow influence flow patterns of an evaporating liquid drop. While it is obvious that Marangoni stress on the drop surface affects the surface flow direction, we found that capillary flow also has an impact. The numerical results of this study showed a stagnation point near the contact line, which was further explained by the lubrication theory. The stagnation point is produced by the competing effects of Marangoni flow and capillary flow and emerges when the contact angle is small because the divergence of the capillary flow near the contact line increases as the contact angle decreases. The radial position of the stagnation point from the numerical results ( rnumerical ≈ 0.995) agreed with the experimentally observed stagnation point ( rexperimental > 0.992).

BSMV as a Biotemplate for Palladium Nanomaterial Synthesis.

The vast unexplored virus biodiversity makes the application of virus templates to nanomaterial synthesis especially promising. Here, a new biotemplate, Barley stripe mosaic virus (BSMV) was successfully used to synthesize organic-metal nanorods of similarly high quality to those produced with Tobacco mosaic virus (TMV). The mineralization behavior was characterized in terms of the reduction and adsorption of precursor and nanocrystal formation processes. The BSMV surface-mediated reduction of Pd(2+) proceeded via first-order kinetics in both Pd(2+) and BSMV. The adsorption equilibrium relationship of PdCl3H2O- on the BSMV surface was described by a multistep Langmuir isotherm suggesting alternative adsorbate-adsorbent interactions when compared to those on TMV. It was deduced that the first local isotherm is governed by electrostatically driven adsorption, which is then followed by sorption driven by covalent affinity of metal precursor molecules for amino acid residues. Furthermore, the total adsorption capacity of palladium species on BSMV is more than double of that on TMV. Finally, study of the BSMV-Pd particles by combining USAXS and SAXS enabled the characterization of all length scales in the synthesized nanomaterials. Results confirm the presence of core-shell cylindrical particles with 1-2 nm grains. The nanorods were uniform and monodisperse, with controllable diameters and therefore, of similar quality to those synthesized with TMV. Overall, BSMV has been confirmed as a viable alternate biotemplate with unique biomineralization behavior. With these results, the biotemplate toolbox has been expanded for the synthesis of new materials and comparative study of biomineralization processes.

Universal scaling laws for the disintegration of electrified drops.

Drops subjected to strong electric fields emit charged jets from their pointed tips. The disintegration of such jets into a spray consisting of charged droplets is common to electrospray ionization mass spectrometry, printing and coating processes, and raindrops in thunderclouds. Currently, there exist conflicting theories and measurements on the size and charge of these small electrospray droplets. We use theory and simulation to show that conductivity can be tuned to yield three scaling regimes for droplet radius and charge, a finding missed by previous studies. The amount of charge Q that electrospray droplets carry determines whether they are coulombically stable and charged below the Rayleigh limit of stability (Q(R)) or are unstable and hence prone to further explosions once they are formed. Previous experiments reported droplet charge values ranging from 10% to in excess of (Q(R)). Simulations unequivocally show that electrospray droplets are coulombically stable at the instant they are created and that there exists a universal scaling law for droplet charge, Q = 0.44 (Q(R)).

The inexorable resistance of inertia determines the initial regime of drop coalescence.

Drop coalescence is central to diverse processes involving dispersions of drops in industrial, engineering, and scientific realms. During coalescence, two drops first touch and then merge as the liquid neck connecting them grows from initially microscopic scales to a size comparable to the drop diameters. The curvature of the interface is infinite at the point where the drops first make contact, and the flows that ensue as the two drops coalesce are intimately coupled to this singularity in the dynamics. Conventionally, this process has been thought to have just two dynamical regimes: a viscous and an inertial regime with a cross-over region between them. We use experiments and simulations to reveal that a third regime, one that describes the initial dynamics of coalescence for all drop viscosities, has been missed. An argument based on force balance allows the construction of a new coalescence phase diagram.

Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase.

Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 µM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 µM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Barriers to entry or exit: which ones are you building?

Unknowingly, we often create barriers that make it difficult for patients to enter or doctors to refer to our medical practices. Additionally, we make it too easy for patients and referring physicians to seek care elsewhere. This article will focus on these barriers to entry (which we need to knock down) and exit (which we want to build up) for a medical practice. We will demonstrate how you can identify and eliminate your entry barriers to make it easy for patients to become a part of your practice, and how you can build defenses to make it unlikely that patients will leave you and to prevent referring physicians from calling anyone else.

Synthesis and application of virus-based hybrid nanomaterials.

A virus is a nanoscaled biomolecular substance composed of genes, protecting capsid proteins, and envelopes. The nanoscale dimensions and surface functionalities of virions have been exploited to attract and assemble inorganic and organic materials to produce functional nanomaterials with large surface areas. Genetic modifications of virus capsid proteins lead to the selective deposition and controlled growth of inorganic substances producing organized virus-based hybrid materials. Due to these properties, viruses hold promise for development as platforms for the creation of hybrid materials with multiple functionalities. This article reviews the characteristics of commonly used viruses and their fabrication into virus-based hybrid materials that have been applied in engineering applications such as nanowires and catalysts.

Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.

BACKGROUND: Confocal endomicroscopy has revolutionized endoscopy by offering subcellular images of the GI epithelium; however, the field of view is limited. Multiscale endoscopy platforms that use widefield imaging are needed to better direct the placement of high-resolution probes. DESIGN: Feasibility study. OBJECTIVE: This study evaluated the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high-resolution imaging to characterize the morphologic changes associated with a variety of GI conditions. SETTING: The University of Texas MD Anderson Cancer Center, Houston, Texas, and Mount Sinai Medical Center, New York, New York. PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASUREMENTS: Resected specimens were obtained from 15 patients undergoing EMR, esophagectomy, or colectomy. Proflavine hemisulfate, a vital fluorescent dye, was applied topically. The specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. The images were compared with histopathologic examination. RESULTS: Widefield fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy, and crowding. High-resolution imaging of widefield abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with the histopathologic features. LIMITATIONS: This imaging approach must be validated in vivo with a larger sample size. CONCLUSIONS: Multiscale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of GI conditions. Distorted glandular features seen with widefield imaging could serve as a critical bridge to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital dye may facilitate point-of-care decision making by providing real-time, in vivo diagnoses.

Modification of crystallization behavior in drug/polyethylene glycol solid dispersions.

The crystallization kinetics of various active pharmaceutical ingredient/polyethylene glycol (API/PEG) solid dispersions has been investigated using wide-angle X-ray diffraction (XRD) and Raman spectroscopy. APIs with different physicochemical properties and crystallization tendency were employed to form solid dispersions with PEG. The crystallization rate of benzocaine (BZC) in BZC/PEG (20/80 wt %) solid dispersions was decreased substantially in comparison to that of the pure API, while the PEG matrix did not affect the crystallization behavior of haloperidol (HLP). The induction time for crystallization of ibuprofen (IBP) and fenofibrate (FNB) in a PEG matrix was decreased relative to the induction times for pure IBP and FNB. For the latter systems, it appears that crystalline PEG acted as a favorable heterogeneous nucleation site. The crystallization behavior of PEG in the API/PEG systems was also affected to different extents, depending on the API studied. These results suggest that PEG can delay, promote or have no influence on the crystallization kinetics of different APIs, and that any effects on crystallization behavior should be investigated in order to be able to produce a solid dispersion with consistent properties.

Time-resolved SAXS/WAXS study of the phase behavior and microstructural evolution of drug/PEG solid dispersions.

Simultaneous small-angle X-ray scattering/wide-angle X-ray scattering (SAXS/WAXS) was employed to elucidate the physical state and location of various small molecule drugs blended with polyethylene glycol (PEG), as well as the time dependent microstructural evolution of the systems. Samples were prepared by comelting physical mixtures of the drug and PEG, followed by solidification at 25 °C. The model drugs selected encompassed a wide variety of physicochemical properties in terms of crystallization tendency and potential for interaction with PEG. It was observed that compounds which crystallized rapidly and had weak interactions with PEG tended to be excluded from the interlamellar region of the PEG matrix. In contrast, drugs which had favorable interactions with PEG were incorporated into the interlamellar regions of the polymer up until the point at which the drug crystallized whereby phase separation occurred. These factors are likely to impact the effectiveness of drug/PEG systems as drug delivery systems.

Biotemplated aqueous-phase palladium crystallization in the absence of external reducing agents.

A new synthetic strategy enabling highly controlled aqueous-phase palladium crystallization on the tobacco mosaic virus (TMV) is demonstrated without the addition of external reducing agents. This low cost, solution processing method yields continuous and uniform coatings of polycrystalline palladium on TMV, creating highly uniform palladium nanowires of tens of nanometers in thickness and hundreds of nanometers in length. Our approach utilizes a palladium chloride precursor to produce metallic Pd coatings on TMV without the need for an external reducing agent. X-ray photoelectron spectroscopy and in situ transmission electron microscopy were used to confirm the reduction of the surface palladium oxide layer on the palladium metal wires during room temperature hydrogenation. This leads to metallic palladium nanowires with surfaces free of residual organics, making these structures suitable for applications in nanoscale electronics.

Structural Insights into Self-Assembled Aerosol-OT Aggregates in Aqueous Media Using Atomistic Molecular Dynamics.

In water, the surfactant dioctyl sulfosuccinate (Aerosol-OT or AOT) exhibits diverse aggregate structures, ranging from micelles to lamella. An atomic-level understanding, however, of the formation and structure of these aggregates is lacking. Herein, using atomistic molecular dynamics (MD) with microsecond-long simulations, self-assembly of AOT in water is studied for concentrations of 1, 7.2, and 20 wt % at 293 K and for 7.2 wt % at 353 K. Assembly proceeds through stepwise association and dissociation of single AOT molecules, and the fusion and fission of AOT clusters. At 293 K, AOT self-assembles into either (i) spherical micelles (1 wt %), (ii) biphasic systems consisting of rod-like and prolate spheroidal micelles (7.2 wt %), or (iii) bilayers (20 wt %). We hypothesize that the observed rod-like structure is a precursor to lamellar microdomains found experimentally in biphasic dispersions. Increasing temperature to 353 K at 7.2 wt % results in a system consisting of prolate micelles but no rod-like micelles. Simulated phase behavior agrees with previously published experimental observations. Individual aggregates formed during self-assembly are identified using graph theory. Structural metrics of these aggregates like the radius of gyration, shape anisotropy, and prolateness are presented. Trends in structural metrics quantitatively reflect how shapes and sizes of AOT aggregates vary with surfactant concentration and temperature. These simulations provide deeper insight into open questions in the scientific community and demonstrate a method to generate physics-based micelle structures that can be used to rationalize experimental observations.

Engineering Tobacco Mosaic Virus and Its Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials.

Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them is the plant tobacco mosaic virus (TMV) due to its high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV and related viruses, and their virus-like particle (VLP) derivatives, and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. How these features have been exploited in engineering applications such as, sensing, catalysis, and energy storage are briefly outlined. While control of VLP surface features is well-established, fewer tools exist to control VLP self-assembly, which limits efforts to control template uniformity and synthesis of certain templated nanomaterials. However, emerging advances in synthetic biology, machine learning, and other fields promise to accelerate efforts to control template uniformity and nanomaterial synthesis enabling more widescale industrial use of VLP-based biotemplates.

Evaluation of the microstructure of semicrystalline solid dispersions.

As a result of an increase in the number of emerging therapies with dissolution limited bioavailability, formulation strategies such as solid dispersions that enhance the rate of solubilization are of interest. In this study, the microstructure of solid dispersions prepared with polyethylene glycol (PEG) and four model compounds with different physicochemical properties was evaluated using a variety of experimental techniques. Solid dispersions were prepared by fusion and evaluated using small-angle X-ray scattering (SAXS), powder X-ray diffraction (PXRD), atomic force microscopy (AFM), optical microscopy and differential scanning calorimetry (DSC). SAXS results indicated that aceclofenac and chlorpropamide solid dispersions favored the interlamellar incorporation of the drug in the PEG matrix. Optical microscopy did not show any evidence of interspherulitic accumulation for any of the model compounds. Haloperidol was highly crystalline in the dispersions, whereas evidence of amorphous material was found for the other model compounds. Results indicated that both the crystallization tendency of the drug and its solubility in amorphous regions of PEG played important roles in determining the location (i.e., interlamellar, interfibrillar or interspherulitic regions) and size of the drug domains within the dispersion.

A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that has a tremendous impact on human health and livestock. High seroprevalence among humans and other animals is facilitated by the conversion of rapidly proliferating tachyzoites into latent bradyzoites that are housed in tissue cysts, which allow transmission through predation. Epigenetic mechanisms contribute to the regulation of gene expression events that are crucial in both tachyzoites as well as their development into bradyzoites. Acetylation of histones is one of the critical histone modifications that is linked to active gene transcription. Unlike most early-branching eukaryotes, Toxoplasma possesses two GCN5 homologues, one of which, GCN5b, is essential for parasite viability. Surprisingly, GCN5b does not associate with most of the well-conserved proteins found in the GCN5 complexes of other eukaryotes. Of particular note is that GCN5b interacts with multiple putative transcription factors that have plant-like DNA-binding domains denoted as AP2. To understand the function of GCN5b and its role(s) in epigenetic gene regulation of stage switching, we performed co-immunoprecipitation of GCN5b under normal and bradyzoite induction conditions. We report the greatest resolution of the GCN5b complex to date under these various culture conditions. Moreover, reciprocal co-IPs were performed with distinct GCN5b-interacting AP2 factors (AP2IX-7 and AP2XII-4) to delineate the interactomes of each putative transcription factor. Our findings suggest that GCN5b is associated with at least two distinct complexes that are characterized by two different pairs of AP2 factors, and implicate up to four AP2 proteins to be involved with GCN5b-mediated gene regulation.

Laparoscopic-assisted ileocolic resection for Crohn's disease.

BACKGROUND: This study reviews our experience with laparoscopic-assisted ileocolic resection in patients with Crohn's disease. The adequacy and safety of this procedure as measured by intraoperative and postoperative complications were evaluated. Special attention was paid to the group in which laparoscopy was not feasible and conversion to laparotomy was necessary. METHODS: Between 1992 and 2005, 168 laparoscopic-assisted ileocolic resections were performed on 167 patients with Crohn's ileal or ileocolic disease. Follow-up data were complete in 158 patients. RESULTS: In 38 patients (24%), conversion to laparotomy was necessary. Previous resection was not a predictor of conversion to laparotomy. Average ileal and colonic length of resected specimens was 20.9 cm and 6.5 cm, respectively, in the laparoscopic group, versus 24.9 cm and 10.6 cm in the converted group. Twenty of 120 specimens (16.6%) in the laparoscopic group were found to have margins microscopically positive for active Crohn's disease. None of the 38 specimens in the converted group had positive ileal margins. CONCLUSIONS: Laparoscopic-assisted ileocolic resection can be safely performed in patients with Crohn's disease ileitis. The finding of positive surgical margins following laparoscopic resections compared with none among conventional resections has to be thoroughly evaluated.

Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase†1.

Kinetoplastid-based infections are neglected diseases that represent a significant human health issue. Chemotherapeutic options are limited due to toxicity, parasite susceptibility, and poor patient compliance. In response, we studied a molecular-target-directed approach involving intervention of hexokinase activity-a pivotal enzyme in parasite metabolism. A benzamidobenzoic acid hit with modest biochemical inhibition of Trypanosoma brucei hexokinase 1 (TbHK1, IC50 =9.1 µm), low mammalian cytotoxicity (IMR90 cells, EC50 >25 µm), and no appreciable activity on whole bloodstream-form (BSF) parasites was optimized to afford a probe with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50 =0.28 µm; BSF, ED50 =1.9 µm). Compounds in this series also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency than toward TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple disease-causing trypanosomatid protozoa.

Percutaneous liver biopsy complicated by hemobilia-associated acute cholecystitis.

Liver biopsy is generally considered a safe and highly useful procedure. It is frequently performed in an outpatient setting for diagnosis and follow-up in numerous liver disorders. Since its introduction at the end of the 19th century, broad experience, new imaging techniques and special needles have significantly reduced the rate of complications associated with liver biopsy. Known complications of percutaneous biopsy of the liver include hemoperitoneum, subcapsular hematoma, hypotension, pneumothorax and sepsis. Other intra-abdominal complications are less common. Hemobilia due to arterio-biliary duct fistula has been described, which has only rarely been clinically expressed as cholecystitis or pancreatitis. We report a case of a fifteen year-old boy who developed severe acute cholecystitis twelve days after a percutaneous liver biopsy performed in an outpatient setting. The etiology was clearly demonstrated to be hemobilia-associated, and the clinical course required the performance of a laparoscopic cholecystectomy. The post operative course was uneventful and the patient was discharged home. Percutaneous liver biopsy is a safe and commonly performed procedure. However, severe complications can occasionally occur. Both medical and surgical options should be evaluated while dealing with these rare incidents.

Deposition of platinum clusters on surface-modified Tobacco mosaic virus.

Nanoscaled Pt conductors were prepared from genetically engineered Tobacco mosaic virus (TMV) templates through Pt cluster deposition on the outer surface of the TMV. Pt clusters were synthesized and deposited on the engineered TMV with surface-exposed cysteine via the in situ mineralization of hexachloroplatinate anions. This deposition was driven by the specific binding between thiols and the solid metal clusters. In addition, Pt-thiolate adducts are suggested to form on the engineered TMV in aqueous solutions that work as nucleation sites for the formation of the Pt clusters. The specific binding between Pt clusters and the engineered TMV template was investigated using UV/vis spectrophotometry and quartz crystal microbalance (QCM) analysis. The electric conductance of Pt-deposited TMV was greater than that of the uncoated TMV virion particles. This result suggests the application of metal cluster-deposited engineered TMV in future electrical devices such as rapid response sensors.