Nanocarrier for Poorly Water-Soluble Anticancer Drugs—Barriers of Translation and Solutions

Many existing chemotherapeutic drugs, repurposed drugs and newly developed small-molecule anticancer compounds have high lipophilicity and low water-solubility. Currently, these poorly water-soluble anticancer drugs (PWSAD) are generally solubilized using high concentrations of surfactants and co-solvents, which frequently lead to adverse side effects. In recent years, researchers have been actively exploring the use of nanotechnology as an alternative to the solvent-based drug solubilization approach. Several classes of nanocarrier systems (lipid-based, polymer-based and albumin-based) are widely studied for encapsulation and delivery of the existing and new PWSAD. These nanocarriers were also shown to offer several additional advantages such as enhanced tumour accumulation, reduced systemic toxicity and improved therapeutic effectiveness. In this article, the recent nanotechnological advances in PWSAD delivery will be reviewed. The barriers commonly encountered in the development of PWSAD nanoformulations (e.g. formulation issues and nanotoxicity issues) and the strategies to overcome these barriers will also be discussed. It is our goal to provide the pharmaceutical scientists and clinicians with more in-depth information about the nanodelivery approach, thus, more efficacious and safe PWSAD nanoformulations can be developed with improved translational success.     

phenoVein—A Tool for Leaf Vein Segmentation and Analysis

Precise measurements of leaf vein traits are an important aspect of plant phenotyping for ecological and genetic research. Here, we present a powerful and user-friendly image analysis tool named phenoVein. It is dedicated to automated segmenting and analyzing of leaf veins in images acquired with different imaging modalities (microscope, macrophotography, etc.), including options for comfortable manual correction. Advanced image filtering emphasizes veins from the background and compensates for local brightness inhomogeneities. The most important traits being calculated are total vein length, vein density, piecewise vein lengths and widths, areole area, and skeleton graph statistics, like the number of branching or ending points. For the determination of vein widths, a model-based vein edge estimation approach has been implemented. Validation was performed for the measurement of vein length, vein width, and vein density of Arabidopsis (Arabidopsis thaliana), proving the reliability of phenoVein. We demonstrate the power of phenoVein on a set of previously described vein structure mutants of Arabidopsis (hemivenata, ondulata3, and asymmetric leaves2-101) compared with wild-type accessions Columbia-0 and Landsberg erecta-0. phenoVein is freely available as open-source software.Leaf veins are an important aspect of leaf structure and responsible for both the mechanical support of leaves and the long-distance transport of water, nutrients, and photoassimilates (Onoda et al., 2011; Malinowski, 2013). The molecular mechanisms by which vascular tissues acquire their identities are yet largely unknown (Roschzttardtz et al., 2014), and there is high interest in analyzing and evaluating traits of veins or leaf venation networks and their genetic regulation. The impact of vein density on photosynthesis is a major investigated topic (Sack and Scoffoni, 2013). During the last decade, a positive correlation between leaf venation and photosynthesis has been observed (Sack and Holbrook, 2006; Brodribb et al., 2007). An optimization of photosynthetic rates was shown to occur by spatial coordination between leaf vein and stomatal densities (Zhang et al., 2012; Carins Murphy et al., 2014; Fiorin et al., 2015). Additionally, there is interest in the impact of vein density on interveinal distances (Dengler et al., 1994; McKown and Dengler, 2009) and the effect of climate, habitat, or growth form on vein density (Sack and Scoffoni, 2013; Scoffoni et al., 2015) or vein width with respect to leaf hydraulic conductance (Feild and Brodribb, 2013; Xiong et al., 2015). Other researchers are particularly interested in the evolution from C3 to C4 plants, which requires higher vein density (Gowik and Westhoff, 2011) and led to selecting for variation of vein density within species (e.g. in a mutant collection by Feldman et al., 2014).Leaf venation studies analyzing traits of veins and venation networks are generally performed on microscopic images of leaves that are properly cleared after harvest. For very small leaves, e.g. the cotyledons or the first leaves (leaves 2–5) of Arabidopsis (Arabidopsis thaliana), basic traits, such as total vein length or vein density (vein length per leaf area), can be achieved manually. However, for larger leaves, manual vein segmentation may become tedious, and at least partially automated analysis is needed for studies on large series of leaf collections. Furthermore, the quantification of vein widths and in particular mean values of vein width of certain vein pieces of interest can hardly be achieved manually. Dedicated image processing tools are, therefore, needed to support researchers for fast and reliable data analysis.A number of software tools have been published that are either specifically made or adapted to analyze leaf veins. These programs have some common properties, like image processing functionalities for vein/areole segmentation and trait extraction. However, they differ in handling strategies or vein parameter analysis methods. A general overview on plant image analysis tools is collected in an online database at http://www.plant-image-analysis.org (Lobet et al., 2013). Programs allowing automated or semiautomated analysis of leaf venation parameters are, for example, a method to extract leaf venation patterns (Rolland-Lagan et al., 2009), the leaf extraction and analysis framework graphical user interface LeafGUI (Price et al., 2011), the leaf image analysis interface LIMANI (Dhondt et al., 2012), the user-interactive vessel generation analysis tool VESGEN (Vickerman et al., 2009; Parsons-Wingerter et al., 2014), and the software network extraction from images NEFI (Dirnberger et al., 2015). Nevertheless, for the analysis of large-scale leaf vein phenotyping experiments, there are certain needs that are only partly covered by each of the approaches and programs mentioned above. Specifically, the following properties are needed: (1) automated vein segmentation with optional manual correction; (2) invariance of the segmentation procedure to inhomogeneous illumination or brightness variations in the leaf image; (3) automated determination of total vein length and projected leaf area; (4) a well-defined and automated determination of vein widths, which is, as far as possible, independent of user chosen thresholds; (5) ability to process large high-resolution images of whole leafs; and (6) full transparency of the source code as well as offline availability of the tool. To provide these functionalities, we developed the user-friendly analysis tool phenoVein. It features automated leaf vein segmentation based on advanced image filtering techniques and includes determination of various vein traits, particularly a model-based vein width estimation. phenoVein allows easy and fast visual control and manual correction on the automatically achieved skeleton of the veins enabled by a real-time overlay of the segmented leaf vein structures on the original image. The length measurement algorithm of phenoVein was validated against complete manual segmentation. We evaluated the impact of image resolution on the results, which has recently been discussed (Price et al., 2014; Sack et al., 2014), and tested whether the orientation (angle) of a leaf on an image may affect the results as suspected from image analysis theory on binary skeleton length measurements (Russ, 2011). To show the powerful phenotyping capabilities of phenoVein, we analyzed the venation traits of leaves of Arabidopsis at different developmental stages (cotyledons, pooled leaves 1 + 2, and leaf 6) harvested from previously described venation mutants and corresponding wild-type lines: asymmetric leaves2-101 (as2-101), ondulata3 (ond3), and hemivenata2 (hve-2) versus Columbia-0 (Col-0) and Landsberg erecta-0 (Ler-0; Semiarti et al., 2001; Alonso-Peral et al., 2006; Robles et al., 2010; Pérez-Pérez et al., 2011). We offer the source code of phenoVein to the public as open-source software that can be further adapted or improved (for details, see “Materials and Methods”).     

Soret Spectroscopic and Molecular Graphic Analysis of Human Semi-β-Hemoglobin Formation

The interaction of heme-free α (α^o) and heme-containing β (β^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^?1 s^?1. This was 100-fold more rapid than the reported rate of combination of α^h and β^h proteins. This encounter-like rate of semi-β-hemoglobin (α^oβ^h) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled α^o protein superimposed over native α^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the α₁β₁ interface, was apparently responsible for the enhanced association properties of the α^o protein.     

Early-phase clinical trials of anti-HIV Drugs——understanding and discussion

Innovative anti-HIV drugs developed by local sponsors in China have come into the stage of early-phase clinical trials. How to systemically design the clinical trials of innovative anti-HIV drugs still remains a challenge for them. This article references the literature and the experience of reviewers, to introduce general considerations concerning early-phase clinical trials of innovative anti-HIV drugs.     

3D-Printed Drugs for Children—Are We Ready Yet?

The first medicine manufactured by three-dimensional (3D) printing was recently approved by the Food and Drug Administration (FDA). The advantages of printing as a manufacturing route enabling more flexibility regarding the dose, and enlarging individual treatment options, have been demonstrated. There is a particular need for flexible drug delivery solutions when it comes to children. Printing as a new pharmaceutical manufacturing technology brings manufacturing closer to the patient and can easily be adjusted to the required dosing scheme, offering more flexibility for treatments. Printing of medicine may therefore become the manufacturing route of choice to provide tailored and potentially on-demand treatments for patients with individual needs. This paper intends to summarize and discuss the state of the art, the crucial aspects which should be taken into account, and the still-open questions, in order to make 3D printing a suitable manufacturing route for pediatric drugs.     

Toxoplasmosis and Epilepsy — Systematic Review and Meta Analysis

Background

Toxoplasmosis is an important, widespread, parasitic infection caused by Toxoplasma gondii. The chronic infection in immunocompetent patients, usually considered as asymptomatic, is now suspected to be a risk factor for various neurological disorders, including epilepsy. We aimed to conduct a systematic review and meta-analysis of the available literature to estimate the risk of epilepsy due to toxoplasmosis.

Methods

A systematic literature search was conducted of several databases and journals to identify studies published in English or French, without date restriction, which looked at toxoplasmosis (as exposure) and epilepsy (as disease) and met certain other inclusion criteria. The search was based on keywords and suitable combinations in English and French. Fixed and random effects models were used to determine odds ratios, and statistical significance was set at 5.0%.

Principal findings

Six studies were identified, with an estimated total of 2888 subjects, of whom 1280 had epilepsy (477 positive for toxoplasmosis) and 1608 did not (503 positive for toxoplasmosis). The common odds ratio (calculated) by random effects model was 2.25 (95% CI 1.27–3.9), p = 0.005.

Conclusions

Despite the limited number of studies, and a lack of high-quality data, toxoplasmosis should continue to be regarded as an epilepsy risk factor. More and better studies are needed to determine the real impact of this parasite on the occurrence of epilepsy.     

Physicochemical characterization of γ-crystallins from bovine lens—Hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the γ-crystallin of the bovine lens. Sedimentation study indicates that γ-crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10^?7 cm²/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated γ-crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of γ-crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these γ-crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, γ-crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for γ-crystallins showed a minimum at about 217 nm, characteristic of a β-sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for γ-crystallin II.     

Cross-linked hydroxypropyl-β-cyclodextrin and γ-cyclodextrin nanogels for drug delivery: Physicochemical and loading/release properties

Due to their size and high surface-to-volume ratio, nanogels can give some unique drug delivery opportunities. A novel technique to prepare cyclodextrin (CD) nanogels, in which the cross-linking takes place simultaneously with an emulsification/solvent evaporation process, has been implemented. The aqueous phase consisted of γ-cyclodextrin (γCD) or hydroxypropyl-β-cyclodextrin (HPβCD) at a fix concentration of 20% (w/w) with or without hydroxypropyl methylcellulose (HPMC) or agar at various concentrations. The incorporation of the cross-linking agent, ethyleneglycol diglycidyl ether (EGDE), was essential for the nanogel formation. By contrast, nanogels could be formed in the absence of surfactant such as Span 80, which can be attributed to the emulsion stabilizing effect of CDs by forming inclusion complexes with the organic solvent at the interface. Gas chromatography-mass spectrometry (GC-MS) analysis of the nanogels confirmed that dichloromethane levels were below the safety limit and, therefore, that these conditions of the organic solvent evaporation (60 °C for 180 min) led to nanogels that satisfy residual solvent requirements. Infrared analysis (IR), transmission electron microscopy (TEM) and dynamic light scattering (DLS) provided information about the cross-linking degree, the size and the size distribution of the nanogels. The ability of the nanogels to host a molecule that can form inclusion complexes and to sustain its release was tested using 3-methylbenzoic acid (3-MBA) as a probe with a high affinity for both β-cyclodextrin (βCD) and γCD. Permeability tests confirmed that 3-MBA was indeed taken up by the nanogels and then slowly released.     

Selecting Essential Information for Biosurveillance—A Multi-Criteria Decision Analysis

The National Strategy for Biosurveillancedefines biosurveillance as “the process of gathering, integrating, interpreting, and communicating essential information related to all-hazards threats or disease activity affecting human, animal, or plant health to achieve early detection and warning, contribute to overall situational awareness of the health aspects of an incident, and to enable better decision-making at all levels.” However, the strategy does not specify how “essential information” is to be identified and integrated into the current biosurveillance enterprise, or what the metrics qualify information as being “essential”. Thequestion of data stream identification and selection requires a structured methodology that can systematically evaluate the tradeoffs between the many criteria that need to be taken in account. Multi-Attribute Utility Theory, a type of multi-criteria decision analysis, can provide a well-defined, structured approach that can offer solutions to this problem. While the use of Multi-Attribute Utility Theoryas a practical method to apply formal scientific decision theoretical approaches to complex, multi-criteria problems has been demonstrated in a variety of fields, this method has never been applied to decision support in biosurveillance.We have developed a formalized decision support analytic framework that can facilitate identification of “essential information” for use in biosurveillance systems or processes and we offer this framework to the global BSV community as a tool for optimizing the BSV enterprise. To demonstrate utility, we applied the framework to the problem of evaluating data streams for use in an integrated global infectious disease surveillance system.     

Analysis of plastid and nuclear DNA data in plant phylogenetics——evaluation and improvement

Correct combination of plastid(cp)and nuclear(nr)DNA data for plant phylogenetic reconstructions is not a new issue,but with an increasing number of nrDNA loci being used,it is of ever greater practical concern.For accurately reconstructing the phylogeny and evolutionary history of plant groups,correct treatment of phylogenetic incongruence is a vital step in the proper analysis of cpDNA and nrDNA data.We first evaluated the current status of analyzing cpDNA and nrDNA data by searching all articles published in the journal Systematic Botany between 2005 and 2011.Many studies combining cpDNA and nrDNA data did not rigorously assess the combinability of the data sets,or did not address in detail possible reasons for incongruence between the two data sets.By reviewing various methods,we outline a procedure to more accurately analyze and/or combine cpDNA and nrDNA data,which includes four steps:identifying significant incongruence,determining conflicting taxa,providing possible interpretations for incongruence,and reconstructing the phylogeny after treating incongruence.Particular attention is given to explanation of the cause of incongruence.We hope that our procedure will help raise awareness of the importance of rigorous analysis and help identify the cause of incongruence before combining cpDNA and nrDNA data.     

‘Glocal’ Robustness Analysis and Model Discrimination for Circadian Oscillators

To characterize the behavior and robustness of cellular circuits with many unknown parameters is a major challenge for systems biology. Its difficulty rises exponentially with the number of circuit components. We here propose a novel analysis method to meet this challenge. Our method identifies the region of a high-dimensional parameter space where a circuit displays an experimentally observed behavior. It does so via a Monte Carlo approach guided by principal component analysis, in order to allow efficient sampling of this space. This ‘global’ analysis is then supplemented by a ‘local’ analysis, in which circuit robustness is determined for each of the thousands of parameter sets sampled in the global analysis. We apply this method to two prominent, recent models of the cyanobacterial circadian oscillator, an autocatalytic model, and a model centered on consecutive phosphorylation at two sites of the KaiC protein, a key circadian regulator. For these models, we find that the two-sites architecture is much more robust than the autocatalytic one, both globally and locally, based on five different quantifiers of robustness, including robustness to parameter perturbations and to molecular noise. Our ‘glocal’ combination of global and local analyses can also identify key causes of high or low robustness. In doing so, our approach helps to unravel the architectural origin of robust circuit behavior. Complementarily, identifying fragile aspects of system behavior can aid in designing perturbation experiments that may discriminate between competing mechanisms and different parameter sets.     

Physicochemical and structural studies onAcinetobacter calcoaceticus RAG-1 and MR-481—Two standard strains in hydrophobicity tests

Acinetobacter calcoaceticus RAG-1 and MR-481, two standard strains used in microbial adhesion to hydrocarbons (MATH), were characterized by contact angles, pH-dependent zeta potentials, elemental surface composition by X-ray photoelectron spectroscopy (XPS), and molecular composition by infrared spectroscopy (IR). Negatively stained (methylamine tungstate) and ruthenium red-stained cells were studied by transmission electron microscopy to reveal the absence or presence of surface appendages. Despite the fact thatA. calcoaceticus RAG-1 is known to be extremely hydrophobic in MATH, whereas MR-481 is a completely non-hydrophobic mutant, neither XPS nor IR indicated a significant difference in chemical composition of the cell surfaces. Contact angles with polar liquids, water and formamide, were considerably higher on RAG-1 than on MR-481, in accordance with their relative hydrophobicities as measured by MATH. However, no significant differences in contact angles were observed between the two strains with apolar liquids like diiodomethane,-bromonaphthalene, and hexadecane. Fibrous extensions on RAG-1, observed after ruthenium red staining, were absent on the non-hydrophobic mutant MR-481. Tentatively, these extensions could be held responsible for the hydrophobicity ofA. calcoaceticus RAG-1.