Imaging cells and sub-cellular structures with ultrahigh resolution full-field X-ray microscopy

Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refractive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The concluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research.     

Frequency Dependent Alterations in Regional Homogeneity of Baseline Brain Activity in Schizophrenia

Low frequency oscillations are essential in cognitive function impairment in schizophrenia. While functional connectivity can reveal the synchronization between distant brain regions, the regional abnormalities in task-independent baseline brain activity are less clear, especially in specific frequency bands. Here, we used a regional homogeneity (ReHo) method combined with resting-state functional magnetic resonance imaging to investigate low frequency spontaneous neural activity in the three different frequency bands (slow-5∶0.01–0.027 Hz; slow-4∶0.027–0.08 Hz; and typical band: 0.01–0.08 Hz) in 69 patients with schizophrenia and 62 healthy controls. Compared with controls, schizophrenia patients exhibited decreased ReHo in the precentral gyrus, middle occipital gyrus, and posterior insula, whereas increased ReHo in the medial prefrontal cortex and anterior insula. Significant differences in ReHo between the two bands were found in fusiform gyrus and superior frontal gyrus (slow-4> slow-5), and in basal ganglia, parahippocampus, and dorsal middle prefrontal gyrus (slow-5> slow-4). Importantly, we identified significant interaction between frequency bands and groups in the inferior occipital gyrus and caudate body. This study demonstrates that ReHo changes in schizophrenia are widespread and frequency dependent.     

Chemical approaches for the construction of multi-enzyme reaction systems

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Contributions of cation-π interactions to the collagen triple helix stability

Cation–π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X–Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation–π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host–guest peptide system: (Pro-Hyp-Gly)₃-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)₃, where X is an aromatic residue and Y is a cationic residue, to study the cation–π interaction in the collagen triple helix. Circular dichroism (CD) measurements and T_m data analysis show that the cation–π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation–π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation–π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π–electron density and the cation–π interaction in collagen. Our data demonstrate that the cation–π interaction could play an important role in stabilizing the collagen triple helix.     

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus

Introduction

Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.

Methods

The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10⁻D16^low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.

Results

SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10⁻CD16^low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.

Conclusions

Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.     

Image Alignment for Tomography Reconstruction from Synchrotron X-Ray Microscopic Images

A synchrotron X-ray microscope is a powerful imaging apparatus for taking high-resolution and high-contrast X-ray images of nanoscale objects. A sufficient number of X-ray projection images from different angles is required for constructing 3D volume images of an object. Because a synchrotron light source is immobile, a rotational object holder is required for tomography. At a resolution of 10 nm per pixel, the vibration of the holder caused by rotating the object cannot be disregarded if tomographic images are to be reconstructed accurately. This paper presents a computer method to compensate for the vibration of the rotational holder by aligning neighboring X-ray images. This alignment process involves two steps. The first step is to match the “projected feature points” in the sequence of images. The matched projected feature points in the - plane should form a set of sine-shaped loci. The second step is to fit the loci to a set of sine waves to compute the parameters required for alignment. The experimental results show that the proposed method outperforms two previously proposed methods, Xradia and SPIDER. The developed software system can be downloaded from the URL, http://www.cs.nctu.edu.tw/~chengchc/SCTA or http://goo.gl/s4AMx.     

Vascular endothelial growth factor: an angiogenic factor reflecting airway inflammation in healthy smokers and in patients with bronchitis type of chronic obstructive pulmonary disease?

BackgroundPatients with bronchitis type of chronic obstructive pulmonary disease (COPD) have raised vascular endothelial growth factor (VEGF) levels in induced sputum. This has been associated with the pathogenesis of COPD through apoptotic and oxidative stress mechanisms. Since, chronic airway inflammation is an important pathological feature of COPD mainly initiated by cigarette smoking, aim of this study was to assess smoking as a potential cause of raised airway VEGF levels in bronchitis type COPD and to test the association between VEGF levels in induced sputum and airway inflammation in these patients.Methods14 current smokers with bronchitis type COPD, 17 asymptomatic current smokers with normal spirometry and 16 non-smokers were included in the study. VEGF, IL-8, and TNF-α levels in induced sputum were measured and the correlations between these markers, as well as between VEGF levels and pulmonary function were assessed.ResultsThe median concentrations of VEGF, IL-8, and TNF-α were significantly higher in induced sputum of COPD patients (1,070 pg/ml, 5.6 ng/ml and 50 pg/ml, respectively) compared to nonsmokers (260 pg/ml, 0.73 ng/ml, and 15.4 pg/ml, respectively, p < 0.05) and asymptomatic smokers (421 pg/ml, 1.27 ng/ml, p < 0.05, and 18.6 pg/ml, p > 0.05, respectively). Significant correlations were found between VEGF levels and pack years (r = 0.56, p = 0.046), IL-8 (r = 0.64, p = 0.026) and TNF-α (r = 0.62, p = 0.031) levels both in asymptomatic and COPD smokers (r = 0.66, p = 0.027, r = 0.67, p = 0.023, and r = 0.82, p = 0.002, respectively). No correlation was found between VEGF levels in sputum and pulmonary function parameters.ConclusionVEGF levels are raised in the airways of both asymptomatic and COPD smokers. The close correlation observed between VEGF levels in the airways and markers of airway inflammation in healthy smokers and in smokers with bronchitis type of COPD is suggestive of VEGF as a marker reflecting the inflammatory process that occurs in smoking subjects without alveolar destruction.     

Plasmacytoid dendritic cells synergize with myeloid dendritic cells in the induction of antigen-specific antitumor immune responses

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8(+) CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8(+) T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.     

SELDI-TOF mass spectrometry of High-Density Lipoprotein

Background  

High-Density Lipoprotein (HDL), one of the main plasma lipoproteins, serves as a docking station for proteins involved in inflammation, coagulation, and lipid metabolism.