Analysis of the Airway Microbiota of Healthy Individuals and Patients with Chronic Obstructive Pulmonary Disease by T-RFLP and Clone Sequencing

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.     

Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation

Recently, interest has focused on hepatocytes’ implantation to provide end stage liver failure patients with a temporary support until spontaneous recovery or a suitable donor becomes available. To avoid cell damage and use of an immunosuppressive treatment, hepatic cells could be implanted after encapsulation in a porous biomaterial of bead or capsule shape. The aim of this study was to compare the production and the physical properties of the beads, together with some hepatic cell functions, resulting from the use of different material combinations for cell microencapsulation: alginate alone or combined with type I collagen with or without poly-L-lysine and alginate coatings. Collagen and poly-L-lysine increased the bead mechanical resistance but lowered the mass transfer kinetics of vitamin B12. Proliferation of encapsulated HepG2/C3A cells was shown to be improved in alginate-collagen beads. Finally, when the beads were subcutaneously implanted in mice, the inflammatory response was reduced in the case of alginate mixed with collagen. This in vitro and in vivo study clearly outlines, based on a systematic comparison, the necessity of compromising between material physical properties (mechanical stability and porosity) and cell behavior (viability, proliferation, functionalities) to define optima hepatic cell microencapsulation conditions before implantation.     

CT Air Trapping Is Independently Associated with Lung Function Reduction over Time

Purpose

We aimed to study the association between lung function decline and quantitative computed tomography (CT) air trapping.

Materials and Methods

Current and former heavy smokers in a lung cancer screening trial underwent volumetric low-dose CT in inspiration and expiration. Spirometry was obtained at baseline and after 3 years. The expiratory to inspiratory ratio of mean lung density (E/I-ratio_MLD) was used to quantify air trapping. CT emphysema was defined as voxels in inspiratory CT below −950 Hounsfield Unit. Linear mixed modeling was used to determine the association between CT air trapping and lung function.

Results

We included 985 subjects with a mean age of 61.3 years. Independent of CT emphysema, CT air trapping was significantly associated with a reduction in forced expiratory volume in one second (FEV₁) and the ratio of FEV₁ over the forced vital capacity (FEV₁/FVC); FEV₁ declines with 33 mL per percent increase in CT air trapping, while FEV₁/FVC declines 0.58% per percent increase (both p<0.001). CT air trapping further elicits accelerated loss of FEV₁/FVC (additional 0.24% reduction per percent increase; p = 0.014).

Conclusion

In a lung cancer screening cohort, quantitatively assessed air trapping on low-dose CT is independently associated with reduced lung function and accelerated decline of FEV₁/FVC.     

Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.     

Semi-Automatic Quantification of Subsolid Pulmonary Nodules: Comparison with Manual Measurements

Rationale

Accurate measurement of subsolid pulmonary nodules (SSN) is becoming increasingly important in the management of these nodules. SSNs were previously quantified with time-consuming manual measurements. The aim of the present study is to test the feasibility of semi-automatic SSNs measurements and to compare the results to the manual measurements.

Methods

In 33 lung cancer screening participants with 33 SSNs, the nodules were previously quantified by two observers manually. In the present study two observers quantified these nodules by using semi-automated nodule volumetry software. Nodules were quantified for effective diameter, volume and mass. The manual and semi-automatic measurements were compared using Bland-Altman plots and paired T tests. Observer agreement was calculated as an intraclass correlation coefficient. Data are presented as mean (SD).

Results

Semi-automated measurements were feasible in all 33 nodules. Nodule diameter, volume and mass were 11.2 (3.3) mm, 935 (691) ml and 379 (311) milligrams for observer 1 and 11.1 (3.7) mm, 986 (797) ml and 399 (344) milligrams for observer 2, respectively. Agreement between observers and within observer 1 for the semi-automatic measurements was good with an intraclass correlation coefficient >0.89. For observer 1 and observer 2, measured diameter was 8.8% and 10.3% larger (p<0.001), measured volume was 24.3% and 26.5% larger (p<0.001) and measured mass was 10.6% and 12.0% larger (p<0.001) with the semi-automatic program compared to the manual measurements.

Conclusion

Semi-automated measurement of the diameter, volume and mass of SSNs is feasible with good observer agreement. Semi-automated measurement makes quantification of mass and volume feasible in daily practice.     

Establishment and Characterization of Primary Glioblastoma Cell Lines from Fresh and Frozen Material: A Detailed Comparison

Background

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. High molecular heterogeneity of GBM tumors is well recognized, forming the rationale for molecular tests required before administration of several of the novel therapeutics rapidly entering the clinics. One model that has gained wide acceptance is the primary cell culture model. The laborious and time consuming process is rewarded with a relative high success rate (about 60%). We here describe and evaluate a very simple cryopreservation procedure for GBM tissue prior to model establishment that will considerably reduce the logistic complexity.

Methods

Twenty-seven GBM samples collected ad hoc were prepared for primary cell culture freshly from surgery (#1) and after cryopreservation (#2).

Results

Take rates after cryopreservation (59%) were as satisfactory as from fresh tissue (63%; p = 1.000). We did not observe any relevant molecular or phenotypic differences between cell lines established from fresh or vitally frozen tissue. Further, sensitivity both towards standard chemotherapeutic agents (Temozolomide, BCNU and Vincristine) and novel agents like the receptor tyrosine kinase inhibitor Imatinib did not differ.

Conclusions

Our simple cryopreservation procedure facilitates collection, long-time storage and propagation (modeling) of clinical GBM specimens (potentially also from distant centers) for basic research, (pre-) clinical studies of novel therapies and individual response prediction.     

Harvest site influences the growth properties of adipose derived stem cells

The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague–Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.     

Effects of management and structural connectivity on the plant communities of organic vegetable field margins in South Korea

Field margins are an important component of the agri-environment as they contribute to maintaining ecosystem functions and protecting biodiversity. Field margin structure, landscape attributes, and management practices have been examined as determinants of plant species diversity and composition for mainly cereal field margins; however, relatively little is known about their influence on vegetable field margins. We selected three types of field margins (each n = 4; non-managed connected to forests, non-managed isolated, and isolated and managed margins with mowing and organic herbicide) adjacent to organic vegetable crop fields and recorded the species richness and abundance of all vascular plants. The effects of structural connectivity, weed control management, and margin width on the community composition, species richness, and diversity were examined using multivariate statistical techniques. Plant community composition was clearly explained by structural connectivity between field margin and forest, as well as by weed control management. In contrast, species richness of functional guilds was influenced by connectivity and margin width, but not by weed control management. All communities had similar numbers of summer and fall blooming nectar- and pollen-producing plants, an important source of pollination services. In addition, each community of field margin types, despite different species composition, had similar levels of Shannon diversity and evenness. Our results suggest that habitat arrangement is important for determining community composition in field margins. Management practices may be important in determining dominance patterns of individual species. A combination of various margin types and widths may be beneficial for biodiversity conservation and ecosystem services.     

Discovery of (R)-N-(3-(7-methyl-1H-indazol-5-yl)-1-(4-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide (BMS-742413): A potent human CGRP antagonist with superior safety profile for the treatment of migraine through intranasal delivery

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.