Defining DNA-based operational taxonomic units for microbial-eukaryote ecology

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 75 (2009): 5797-5808, doi:10.1128/AEM.00298-09.DNA sequence information has been increasingly used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, the autecology of ecologically relevant species, and the identification and enumeration of species of interest to human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is partly a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes, and partly the problematic nature and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximate species-level distinctions. The approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small subunit ribosomal RNA (18S) gene sequences and partial gene sequences obtained from GenBank for morphologically described protistan species. The program was tested using environmental 18S data sets from two oceanic ecosystems. A total of 388 operational taxonomic units were observed among 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific.Support for this manuscript was provided by National Science Foundation grants MCB-0732066, MCB-0703159 and OCE-0550829 and a grant from the Gordon and Betty Moore Foundation

Regional remodeling strain and its association with myocardial apoptosis after myocardial infarction in an ovine model

ObjectiveProgressive left ventricular remodeling after myocardial infarction has been viewed as an important contributor to progressive heart failure. The objective of this study was to investigate the relationship between myocardial apoptosis and strain during progressive cardiac remodeling.MethodsBefore creation of an anterolateral left ventricular infarction by ligation of diagonal arteries, 16 sonomicrometry transducers were placed in the left ventricular free wall of 8 sheep to assess regional deformation in the infarct, adjacent, and normally perfused remote myocardial regions over 8 weeks' duration. Hemodynamic, echocardiographic and sonomicrometric data were collected before infarction and then 30 minutes and 2, 6, and 8 weeks after infarction. At the end of the study, regional myocardial tissues were collected for apoptotic signaling proteins.ResultsAt terminal study, an increase in left ventricular end-diastolic pressure of 8.1 ± 0.1 mm Hg, a decrease in ejection fraction from 54.19% ± 5.68% to 30.55% ± 2.72%, and an end-diastolic volume increase of 46.08 ± 5.02 mL as compared with the preinfarct values were observed. The fractional contraction at terminal study correlated with the relative abundance of apoptotic protein expressions: cytochrome c (r2 = 0.02, P < .05), mitochondrial Bax (r2 = 0.27, P < .05), caspase-3 (r2 = 0.31, P < .05), and poly (adenosine diphosphate–ribose) polymerase (r2 = 0.30, P < .05). These myocardial apoptotic activities also correlated with remodeling strain: cytochrome c (r2 = 0.02, P < .05), mitochondrial Bax (r2 = 0.28, P < .05), caspase-3 (r2 = 0.43, P < .05), and poly (adenosine diphosphate–ribose) polymerase (r2 = 0.37, P < .05).ConclusionIncrease in regional remodeling strain led to an increase in myocardial apoptosis and regional contractile dysfunction in heart failure

Characterization of a murine model of monocrotaline pyrrole-induced acute lung injury

<p>Abstract</p> <p>Background</p> <p>New animal models of chronic pulmonary hypertension in mice are needed. The injection of monocrotaline is an established model of pulmonary hypertension in rats. The aim of this study was to establish a murine model of pulmonary hypertension by injection of the active metabolite, monocrotaline pyrrole.</p> <p>Methods</p> <p>Survival studies, computed tomographic scanning, histology, bronchoalveolar lavage were performed, and arterial blood gases and hemodynamics were measured in animals which received an intravenous injection of different doses of monocrotaline pyrrole.</p> <p>Results</p> <p>Monocrotaline pyrrole induced pulmonary hypertension in Sprague Dawley rats. When injected into mice, monocrotaline pyrrole induced dose-dependant mortality in C57Bl6/N and BALB/c mice (dose range 6–15 mg/kg bodyweight). At a dose of 10 mg/kg bodyweight, mice developed a typical early-phase acute lung injury, characterized by lung edema, neutrophil influx, hypoxemia and reduced lung compliance. In the late phase, monocrotaline pyrrole injection resulted in limited lung fibrosis and no obvious pulmonary hypertension.</p> <p>Conclusion</p> <p>Monocrotaline and monocrotaline pyrrole pneumotoxicity substantially differs between the animal species.</p

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model

<p>Abstract</p> <p>Background</p> <p>Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions <it>in vitro </it>in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays.</p> <p>Methods</p> <p>One family of peptides with high activity is derived from the α-fibrils of type IV collagen. Based on the results from the <it>in vitro </it>screening, we have evaluated the ability of a 20 amino acid peptide derived from the α5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed <it>in vivo </it>angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line.</p> <p>Results</p> <p>Pentastatin-1 decreased the invasion of vessels into angioreactors <it>in vivo </it>in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density <it>in vivo </it>in a small cell lung cancer xenograft model.</p> <p>Conclusions</p> <p>The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.</p

Optimization of MicroCT Imaging and Blood Vessel Diameter Quantitation of Preclinical Specimen Vasculature with Radiopaque Polymer Injection Medium

Vascular networks within a living organism are complex, multi-dimensional, and challenging to image capture. Radio-angiographic studies in live animals require a high level of infrastructure and technical investment in order to administer costly perfusion mediums whose signals metabolize and degrade relatively rapidly, diminishing within a few hours or days. Additionally, live animal specimens must not be subject to long duration scans, which can cause high levels of radiation exposure to the specimen, limiting the quality of images that can be captured. Lastly, despite technological advances in live-animal specimen imaging, it is quite difficult to minimize or prevent movement of a live animal, which can cause motion artifacts in the final data output. It is demonstrated here that through the use of postmortem perfusion protocols of radiopaque silicone polymer mediums and ex-vivo organ harvest, it is possible to acquire a high level of vascular signal in preclinical specimens through the use of micro-computed tomographic (microCT) imaging. Additionally, utilizing high-order rendering algorithms, it is possible to further derive vessel morphometrics for qualitative and quantitative analysis

Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate

© 2016 The Authors. Published under the terms of the CC BY 4.0 licenseLung cancer (LC) is the leading cause of cancer-related deaths worldwide. Early LC diagnosis is crucial to reduce the high case fatality rate of this disease. In this case–control study, we developed an accurate LC diagnosis test using retrospectively collected formalin-fixed paraffin-embedded (FFPE) human lung tissues and prospectively collected exhaled breath condensates (EBCs). Following international guidelines for diagnostic methods with clinical application, reproducible standard operating procedures (SOP) were established for every step comprising our LC diagnosis method. We analyzed the expression of distinct mRNAs expressed from GATA6 and NKX2-1, key regulators of lung development. The Em/Ad expression ratios of GATA6 and NKX2-1 detected in EBCs were combined using linear kernel support vector machines (SVM) into the LC score, which can be used for LC detection. LC score-based diagnosis achieved a high performance in an independent validation cohort. We propose our method as a non-invasive, accurate, and low-price option to complement the success of computed tomography imaging (CT) and chest X-ray (CXR) for LC diagnosis

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%–50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH

Transcriptional profiling unveils molecular subgroups of adaptive and maladaptive right ventricular remodeling in pulmonary hypertension

Right ventricular (RV) function is critical to prognosis in all forms of pulmonary hypertension. Here we perform molecular phenotyping of RV remodeling by transcriptome analysis of RV tissue obtained from 40 individuals, and two animal models of RV dysfunction of both sexes. Our unsupervised clustering analysis identified ‘early’ and ‘late’ subgroups within compensated and decompensated states, characterized by the expression of distinct signaling pathways, while fatty acid metabolism and estrogen response appeared to underlie sex-specific differences in RV adaptation. The circulating levels of several extracellular matrix proteins deregulated in decompensated RV subgroups were assessed in two independent cohorts of individuals with pulmonary arterial hypertension, revealing that NID1, C1QTNF1 and CRTAC1 predicted the development of a maladaptive RV state, as defined by magnetic resonance imaging parameters, and were associated with worse clinical outcomes. Our study provides a resource for subphenotyping RV states, identifying state-specific biomarkers, and potential therapeutic targets for RV dysfunction