Theophylline Cocrystals Prepared by Spray Drying: Physicochemical Properties and Aerosolization Performance

The purpose of this work was to characterize theophylline (THF) cocrystals prepared by spray drying in terms of the physicochemical properties and inhalation performance when aerosolized from a dry powder inhaler. Cocrystals of theophylline with urea (THF-URE), saccharin (THF-SAC) and nicotinamide (THF-NIC) were prepared by spray drying. Milled THF and THF-SAC cocrystals were also used for comparison. The physical purity, particle size, particle morphology and surface energy of the materials were determined. The in vitro aerosol performance of the spray-dried cocrystals, drug-alone and a drug-carrier aerosol, was assessed. The spray-dried particles had different size distributions, morphologies and surface energies. The milled samples had higher surface energy than those prepared by spray drying. Good agreement was observed between multi-stage liquid impinger and next-generation impactor in terms of assessing spray-dried THF particles. The fine particle fractions of both formulations were similar for THF, but drug-alone formulations outperformed drug-carrier formulations for the THF cocrystals. The aerosolization performance of different THF cocrystals was within the following rank order as obtained from both drug-alone and drug-carrier formulations: THF-NIC > THF-URE > THF-SAC. It was proposed that micromeritic properties dominate over particle surface energy in terms of determining the aerosol performance of THF cocrystals. Spray drying could be a potential technique for preparing cocrystals with modified physical properties.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9883-3) contains supplementary material, which is available to authorized users.Key words: aerodynamic diameter, cocrystal, spray drying, surface energy, theophylline     

Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4–5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III₂ and Apo C-III₁ (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r²>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III₀ showed no correlation (r² = 0.01–0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.The maternal physiology during pregnancy is characterized by inflammation and hyperlipidemia. Plasma protein composition fluctuates dynamically throughout gestation to reflect these physiological changes. Apolipoproteins, a diverse subset of triglyceride transport proteins, contribute to the hyperlipidemia of pregnancy by modulating lipid homeostasis in maternal plasma (1–3). Exaggerated hyperlipidemia and peripheral apolipoprotein burden are associated with inflammatory insult and signal obstetric complications (4–5). Numerous post-translationally modified apolipoprotein isoforms are reported in plasma, but it is unclear how these modifications affect apolipoprotein function and plasma distribution. For example, changes in the glycosylation status of apolipoprotein variants predate the onset of clinical symptoms in patients with preeclampsia, a hypertensive disorder of pregnancy with clinical features in common with cardiovascular disease (6–8). The identification and functional characterization of plasma apolipoprotein isoforms and their post-translationally modified subtypes may reveal important diagnostic and/or therapeutic targets for hypertensive disorders of pregnancy (6).Mass spectrometry and targeted proteomics analyses afford unprecedented sensitivity and specificity for detecting apolipoproteins and their numerous isoforms and subtypes (9–12). Mass spectrometry approaches overcome limitations inherent in biochemical approaches (e.g. ELISA [enzyme-linked immunosorbant assays] and Western blot analysis), especially the lack of specificity of antibodies for post-translationally modified variants of plasma proteins. The objective of this study was to longitudinally evaluate maternal plasma apolipoprotein profile over gestational time by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)¹ analysis of intact proteins and a complementary targeted LTQ-Orbitrap XL MS approach. We evaluate changes in 13 post-translationally modified subtypes of the plasma apolipoproteins A-II, C-I, C-II, and C-III over gestational time.     

Lipocalin-2-mediated upregulation of various antioxidants and growth factors protects bone marrow-derived mesenchymal stem cells against unfavorable microenvironments

Despite many advantages of mesenchymal stem cells (MSCs) that make them suitable for cell therapy purposes, their therapeutic application has been limited due to their susceptibility to several stresses (e.g., nutrient-poor environment, oxidative stress, and hypoxic and masses of cytotoxic factors) to which they are exposed during their preparation and following transplantation. Hence, reinforcing MSCs against these stresses is a challenge for both basic and clinician scientists. Recently, much attention has been directed toward equipping MSCs with cytoprotective factors to strengthen them against unfavorable microenvironments. Here, we engineered MSCs with lipocalin 2 (Lcn2), a cytoprotective factor that is naturally induced following exposure of cells to stresses imposed by the microenvironment. Lcn2 overexpression not only did not interfere with the multidifferentiation capacity of the MSCs but also granted many protective properties to them. Lcn2 potentiated MSCs to withstand oxidative, hypoxia, and serum deprivation (SD) conditions via antagonizing their induced cytotoxicity and apoptosis. Adhesion rate of MSCs to coated culture plates was also enhanced by Lcn2 overexpression. In addition, Lcn2 induced antioxidants and upregulated some growth factors in MSCs. Our findings suggested a new strategy for prevention of graft cell death in MSC-based cell therapy.     

SNUAGE: an efficient platform-as-a-service security framework for the cloud

In this paper we present SNUAGE, a platform-as-a-service security framework for building secure and scalable multi-layered services based on the cloud computing model. SNUAGE ensures the authenticity, integrity, and confidentiality of data communication over the network links by creating a set of security associations between the data-bound components on the presentation layer and their respective data sources on the data persistence layer. SNUAGE encapsulates the security procedures, policies, and mechanisms in these security associations at the service development stage to form a collection of isolated and protected security domains. The secure communication among the entities in one security domain is governed and controlled by a standalone security processor and policy attached to this domain. This results into: (1) a safer data delivery mechanism that prevents security vulnerabilities in one domain from spreading to the other domains and controls the inter-domain information flow to protect the privacy of network data, (2) a reusable security framework that can be employed in existing platform-as-a-service environments and across diverse cloud computing service models, and (3) an increase in productivity and delivery of reliable and secure cloud computing services supported by a transparent programming model that relieves application developers from the intricate details of security programming. Last but not least, SNUAGE contributes to a major enhancement in the energy consumption and performance of supported cloud services by providing a suitable execution container in its protected security domains for a wide suite of energy- and performance-efficient cryptographic constructs such as those adopted by policy-driven and content-based security protocols. An energy analysis of the system shows, via real energy measurements, major savings in energy consumption on the consumer devices as well as on the cloud servers. Moreover, a sample implementation of the presented security framework is developed using Java and deployed and tested in a real cloud computing infrastructure using the Google App Engine service platform. Performance benchmarks show that the proposed framework provides a significant throughput enhancement compared to traditional network security protocols such as the Secure Sockets Layer and the Transport Layer Security protocols.     

Monitoring of genotoxic damage in smokeless tobacco ((Maraş powder) consumers using micronucleated exfoliated oral cells

Mara? powder is a kind of smokeless tobacco used in the south-eastern region of Turkey. The present study was carried out to assess possible DNA damage in exfoliated oral cells of Mara? powder users by analysing the frequencies of micronuclei (MN), which is a simple and reliable biomarker for genotoxic damage and to screen for the detection of site-specific differences in the frequencies of MN. The mean (±SD) MN frequency in the inner lip mucosa site was 1.27(±0.55) % for Mara? powder users and 0.88(±0.47) % for non-smoking control subjects (p < 0.05) and 0.82(±0.40) % for the buccal site of Mara? powder users (p < 0.01). There was no significant site-specific difference between the inner lip site and the buccal mucosa site 0.73(±0.43) % in the MN frequency of non-smoking control subjects (p > 0.05). There was no significant effect of daily consumption of Mara? powder, and duration of usage on MN frequencies. The present study suggests that the oral use of smokeless tobacco represents a genotoxic hazard and also that use of MN from a single site may be misleading as a marker of genotoxic exposure. Habitual use of Mara? powder should be taken into account and could be considered unsafe.     

Cytogenetic analysis of buccal cells from shoeworkers and pathology and anatomy laboratory workers exposed to n-hexane,toluene, methyl ethyl ketone and formaldehyde

People employed in the shoe manufacture and repair industry are at an increased risk for cancer, the strongest evidence being for nasal cancer and leukaemia. A possible causal role for formaldehyde is likely for cancer of the buccal cavity and nasopharynx. Exfoliated buccal cells are good source of tissue for monitoring human exposure to inhaled and ingested occupational and environmental genotoxicants. To assess the cytogenetic damage related to occupational exposure to airborne chemicals during shoe-making and the processes in pathology and anatomy laboratories, the micronuclei (MN) count per 3000 cells was measured in buccal smears from shoe-workers (group I, n = 22) exposed to mainly n-hexane, toluene and methyl ethyl ketone (MEK) and from anatomy and pathology staff (group II, n = 28) exposed to formaldehyde (FA). Eighteen male university staff were used as controls. The mean time-weighted average (TWA) concentrations of n-hexane, toluene and MEK in 10 small shoe workshops were 58.07 p.p.m., 26.62 p.p.m. and 11.39 p.p.m., respectively. The measured air concentrations of FA in the breathing zone of the anatomy and pathology laboratory workers were between 2 and 4 p.p.m. Levels of 2,5-hexadione (2,5-HD) and hippuric acid (HA), metabolic markers of n-hexane and toluene exposure, respectively, were significantly higher in the urine of workers in group I than in control subjects (p < 0.001 and p < 0.01, respectively). The mean (±SD) MN frequencies in buccal mucosa cells from workers in group I, group II and controls were 0.62±0.45%, 0.71±0.56% and 0.33±0.30%, respectively (p < 0.05 and p < 0.05 compared with controls for group I and group II, respectively). The effects of smoking, age and duration of exposure on the frequency of micronucleated buccal cells from workers in all three groups studied were also evaluated. Overall, the results suggest that occupational exposure to organic solvents, mainly n-hexane, toluene, MEK and FA, may cause cytogenetic damage in buccal cells and that use of exfoliated buccal cells seems to be appropriate to measure exposure to organic solvents.     

Suitability indices and habitat suitability index model of Caspian kutum (Rutilus frisii kutum) in the southern Caspian Sea

The relationship between species and habitat is important in ecosystem-based fisheries management. Habitat suitability index (HSI) modeling is a valuable tool in ecology and can be used to describe the relationship between fish abundance and ecological variables in order to estimate the suitability of specific habitats. In the present study, an HSI model was applied to determine suitable habitats for the Caspian kutum (Rutilus frisii kutum), an important commercial species in the southern Caspian Sea. An arithmetic mean model (AMM) was found to be the most appropriate model for describing the relationship between two of the environmental variables investigated (depth and benthos biomass). However, a geometric mean model explained the evident relationship when all four environmental variables were used (depth, benthos biomass, photosynthetically active radiation and sea surface temperature). The areas with an HSI > 0.5 had over 85 % of the total catch indicating the reliability of the prediction of the Caspian kutum habitat using the AMM. The present study showed that depth and substrate structure are the most important environmental variables for the Caspian kutum to select its habitats, and between remotely sensed data, chlorophyll a, photosynthetically active radiation and sea surface temperature are the most critical parameters for near real-time prediction of the Caspian kutum habitat.     

A Novel Assay for Measurement of Membrane‐Protein Surface Expression using a β‐lactamase Reporter

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one‐step and potentially high‐throughput assay, using the β‐lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell‐impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well‐established paradigms of internalization and molecular chaperoning, applied to two G‐protein‐coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression .      

Myosin VI has a One Track Mind Versus Myosin Va When Moving on Actin Bundles or at an Intersection

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin‐actin) or mixed polarity (α‐actinin‐actin) bundles. The stepping dynamics of individual Qdot‐labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor .     

Modeling and analysis of soybean (Glycine max. L) Cu/Zn,Mn and Fe superoxide dismutases

Superoxide dismutase (SOD, EC 1.15.1.1) is an important metal-containing antioxidant enzyme that provides the first line of defense against toxic superoxide radicals by catalyzing their dismutation to oxygen and hydrogen peroxide. SOD is classified into four metalloprotein isoforms, namely, Cu/Zn SOD, Mn SOD, Ni SOD and Fe SOD. The structural models of soybean SOD isoforms have not yet been solved. In this study, we describe structural models for soybean Cu/Zn SOD, Mn SOD and Fe SOD and provide insights into the molecular function of this metal-binding enzyme in improving tolerance to oxidative stress in plants.