Characterization of CD34+ cells isolated from human fetal lung

The large capillary mass of the newborn lung demands the presence of endothelial cell precursors in lung tissue before development of the pulmonary capillary bed. The objective of this investigation was to isolate and characterize putative endothelial cell precursors from developing human lung. CD34, a cell surface marker for hematopoietic progenitor cells, endothelial precursor cells, and small vessel endothelial cells, was employed as an immunological "handle" for the selection of the desired cells. When CD34+ cells were isolated from midtrimester human fetal lung tissue, then maintained in culture, the isolated cells expressed immunoreactivity for the endothelial cell marker von Willebrand factor and the vascular endothelial growth factor receptors KDR and Flt-1. However, only 5% or fewer of the cells expressed PECAM, an important factor in cell-cell interactions and a marker for endothelial cells associated with vessels. The CD34+ cells endocytosed acetylated low-density lipoprotein and formed capillary-like structures when incubated in a cushion of Matrigel. RT-PCR analysis of mRNA for endothelial cell-related proteins Flt-1, Tie-2, and endothelial nitric oxide synthase demonstrated expression of these mRNAs by the isolated cells for at least 16 cell passages. These observations demonstrate that capillary endothelial cell precursors can be isolated from developing human lung and maintained in cell culture. These cells represent a potentially important tool for investigating the regulation of mechanisms governing development of the air-blood barrier in the human lung.     

Evolutionary genetics of self-incompatibility in the Solanaceae

The self-incompatibility (S) gene in flowering plants has long been appreciated as an example of extreme allelic polymorphism maintained by frequency-dependent selection. Recent studies of population samples of S-allele sequences obtained by RT-PCR from five species of Solanaceae now reveal a picture of conspicuous inter-specific variation in both S-allele number and age. Explanations for this variation are examined with reference to current theory. We propose that changes in species' effective population size, particularly those associated with the evolution of different life histories, best account for interspecific differences in both the number and average age of S alleles.     

Structure,Function, and Insights into the Biosynthesis of a Head-to-Head Hydrocarbon in Shewanella oneidensis Strain MR-1

A polyolefinic hydrocarbon was found in nonpolar extracts of Shewanella oneidensis MR-1 and identified as 3,6,9,12,15,19,22,25,28-hentriacontanonaene (compound I) by mass spectrometry, chemical modification, and nuclear magnetic resonance spectroscopy. Compound I was shown to be the product of a head-to-head fatty acid condensation biosynthetic pathway dependent on genes denoted as ole (for olefin biosynthesis). Four ole genes were present in S. oneidensis MR-1. Deletion of the entire oleABCD gene cluster led to the complete absence of nonpolar extractable products. Deletion of the oleC gene alone generated a strain that lacked compound I but produced a structurally analogous ketone. Complementation of the oleC gene eliminated formation of the ketone and restored the biosynthesis of compound I. A recombinant S. oneidensis strain containing oleA from Stenotrophomonas maltophilia strain R551-3 produced at least 17 related long-chain compounds in addition to compound I, 13 of which were identified as ketones. A potential role for OleA in head-to-head condensation was proposed. It was further proposed that long-chain polyunsaturated compounds aid in adapting cells to a rapid drop in temperature, based on three observations. In S. oneidensis wild-type cells, the cellular concentration of polyunsaturated compounds increased significantly with decreasing growth temperature. Second, the oleABCD deletion strain showed a significantly longer lag phase than the wild-type strain when shifted to a lower temperature. Lastly, compound I has been identified in a significant number of bacteria isolated from cold environments.Currently, there is industrial interest in nongaseous microbial hydrocarbons for specialty chemical applications and, more recently, as high-energy biofuels (20, 27, 34). Microbes produce hydrocarbons of different types, for example, aliphatic isoprenoid compounds (20) and alkanes from fatty aldehyde decarbonylation (10). Fatty aldehyde decarbonylation is not well understood but offers a clean route to diesel fuels from fatty acids.Certain microbes also make a distinctly different class of long-chain hydrocarbons, generally C₂₅ to C₃₃ in chain length, that contain a double bond near the middle of the chain (1, 3, 5, 15, 30, 31, 33, 34). These long-chain olefinic hydrocarbons are thought to derive from processes different than isoprene condensation and decarbonylation mechanisms. This class of hydrocarbons has been shown by carbon-14-labeling studies (2) to derive from fatty acids. The process, described in 1929 by Channon and Chibnall (9), has become known as head-to-head hydrocarbon biosynthesis. Albro and Ditmar (3) defined the head-to-head condensation as coupling of the head (C₁) and the α-carbon (C₂) of two fatty acids with decarboxylation, a reaction that should not be confused with an acyloin-like carboxyl carbon-to-carboxyl carbon coupling. Products of the head-to-head mechanism have been identified in Gram-positive bacteria such as Micrococcus luteus (29, 30) and Arthrobacter aurescens (13) and in Gram-negative bacteria such as Stenotrophomonas maltophilia (28). Micrococcus and Arthrobacter strains produce fatty acids that are methyl branched terminally and subterminally (8, 29, 30). The long-chain olefinic hydrocarbons from those strains similarly contain a mixture of terminal and subterminal methyl group branching (2, 13, 31).Albro and Ditmar (3, 4) acquired direct evidence for the head-to-head mechanism occurring in microbial whole organisms and cell extracts. In cell extracts, it was shown that one of the fatty acid carboxyl groups is lost as carbon dioxide, with the remaining carbon atoms being retained in the resultant hydrocarbon (4). The hydrocarbons contain a double bond at the point of condensation. More recently, Beller et al. described the genes encoding head-to-head fatty acid condensation pathway enzymes from Micrococcus luteus, which are known as ole genes for the olefin products formed (5). Three genes from Micrococcus luteus were shown to confer on Escherichia coli the ability to make long-chain olefinic hydrocarbons. Two recent patent applications by L. Friedman et al. (18 September 2008, WO2008/113041; 4 December 2008, WO2008/147781) also described a three- or four-gene cluster as being involved in head-to-head hydrocarbon biosynthesis to make olefins. The patent applications identified homologs to ole genes in different bacteria, including strains of Shewanella.Bacteria of the genus Shewanella have been heavily studied over the last decade because they are widespread and have the ability to use a startling variety of electron acceptors for respiration (11). There are more than 20 completed genome sequences for Shewanella strains. The model system for studying Shewanella is S. oneidensis MR-1. The genome sequencing of S. oneidensis MR-1 was reported in 2002 (16), and the organism has been shown to be highly amenable to genetic manipulation (11).The present study used Shewanella oneidensis strain MR-1 as a model system to investigate hydrocarbon biosynthetic genes and the possible biological function of the proteins they encode. The hydrocarbon produced by the Ole proteins in S. oneidensis MR-1 was found to be very different from hydrocarbons previously identified as deriving from a head-to-head condensation mechanism (28, 29, 32). The product was identified here as 3,6,9,12,15,19,22,25,28-hentriacontanonaene by chemical modification studies, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Previously, a similar polyolefin had been identified in many Antarctic bacteria (22). Cloning of a heterologous oleA gene into S. oneidensis MR-1 was found to produce a completely different set of products. A hydrocarbon deletion mutant showed a distinctly longer growth lag than wild-type cells when shifted to a lower temperature, suggesting that the ole genes in S. oneidensis MR-1 may aid the cells in adapting to a sudden drop in temperature.     

Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors

Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.     

Altered expression of insulin receptor isoforms in breast cancer

Purpose

Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies.

Experimental Design

mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized.

Results

The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes.

Conclusions

The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic.     

Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development

To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, we have used a new mouse line, T-Cre, to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. Our data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis.