NOS-2 signaling and cancer therapy

The role of NO and cGMP signaling in tumor biology has been extensively studied during the past three decades. However, whether the pathway is beneficial or detrimental in cancer is still open to question. We suggest several reasons for this ambiguity: first, although NO participates in normal signaling (e.g., vasodilation and neurotransmission), NO is also a cytotoxic or apoptotic molecule when produced at high concentrations by inducible nitric-oxide synthase (iNOS or NOS-2). In addition, the cGMP-dependent (NO/sGC/cGMP pathway) and cGMP-independent (NO oxidative pathway) components may vary among different tissues and cell types. Furthermore, solid tumors contain two compartments: the parenchyma (neoplastic cells) and the stroma (nonmalignant supporting tissues including connective tissue, blood vessels, and inflammatory cells) with different NO biology. Thus, the NO/sGC/cGMP signaling molecules in tumors as well as the surrounding tissue must be further characterized before targeting this signaling pathway for tumor therapy. In this review, we focus on the NOS-2 expression in tumor and surrounding cells and summarized research outcome in terms of cancer therapy. We propose that a normal function of the sGC-cGMP signaling axis may be important for the prevention and/or treatment of malignant tumors. Inhibiting NOS-2 overexpression and the tumor inflammatory microenvironment, combined with normalization of the sGC/cGMP signaling may be a favorable alternative to chemotherapy and radiotherapy for malignant tumors.     

DNA Methylation Profiles of Airway Epithelial Cells and PBMCs from Healthy, Atopic and Asthmatic Children

Background

Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression.

Objectives

To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects.

Methods

PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR.

Results

We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects.

Discussion

We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma.     

Longer Telomere Length in COPD Patients with α1-Antitrypsin Deficiency Independent of Lung Function

Oxidative stress is involved in the pathogenesis of airway obstruction in α₁-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α₁-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α₁-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α₁-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10⁻²⁹). Telomere length was not related to lung function in α₁-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α₁-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.     

Biomechanics and mechanobiology in functional tissue engineering

The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of “functional tissue engineering” has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements.     

Effects of histidine and N-acetylcysteine on diclofenac-induced anti-inflammatory response in acute inflammation in rats

The intra-plantar injection of carrageenan elicited an inflammatory response characterized by increase of the paw thickness and infiltration of neutrophils in paw tissues. Histidine, n-acetylcysteine and diclofenac decreased paw thickness, and neutrophil infiltration in the paw tissues. The anti-inflammatory effect induced by co-administration of histidine and n-acetylcysteine with diclofenac, was more than that obtained from histidine and n-acetylcysteine administered alone. The results suggested that histidine, n-acetylcysteine and diclofenac produced anti-inflammatory activities by reducing paw edema and neutrophil infiltrationin induced by carrageenan. Inhibition of cyclooxygenase products such as prostaglandins may be involved in the anti-inflammatory effects induced by histidine and n-acetylcysteine.     

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that self-organizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7, 14, 43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the difficulty in obtaining an atomic resolution structural analysis for any S layer with standard structural methods, such as X-ray crystallography. It has been assumed that the difficulty in obtaining three-dimensional crystals is the consequence of the propensity for two-dimensional assembly, which prevents the proteins from being sufficiently well behaved for crystallization. Despite that, there are a few examples of limited success for portions of S layers (38, 39). Moreover, many studies have been conducted on isolated in vitro S-layer sheets using negative-stain electron microscopy. This approach removes the interaction of the S layer with other cell wall components, which makes it more difficult to understand how a crystalline structure develops on a growing bacterium. Defects in structure that occur during the introduction of newly secreted subunits or to accommodate covering areas of strong curvature may well not be appreciated in isolated fragments, where rearrangements of the two-dimensional array are likely to occur. This may result in a more regular structure and even assist image analysis methods but does not represent what is occurring on the dynamic cell surface. Quoting Engelhardt (17): “Functional aspects have usually been investigated with isolated S-layer sheets or proteins, which disregards the interactions between S-layers and the underlying cell envelope components.”Imaging technologies such as freeze-etch and negative-stain microscopy of whole cells (48) can obtain quality images of the S layer directly on the cell. However, each of these techniques presents drawbacks, which would make impossible to extract the conclusions summarized in this paper. For example, it is very difficult to combine freeze-etching with site-specific labeling methods and is impossible with any label in the size range of Nanogold (NG). Moreover, freeze-etch images do not contain three-dimensional (3D) information. Negative staining of S layers on intact cells is difficult and generally can be imaged only on lysed (eviscerated) cells (48). In this case, resolution is hampered by overlying double S layers, membrane debris, and the stain itself. Labeling with the typical 5- to 10-nm colloidal gold probes with either approach is not amenable to averaging techniques designed to localize the labels.Prior work has shown that C. crescentus, a Gram-negative bacterium, has an S-layer subunit composed of a single highly expressed (27) protein (RsaA), secreted by a type I mechanism, such that there is no cleaved N-terminal signal leader but there is an uncleaved C-terminal secretion signal (4, 11). Six RsaA monomers (12) form the characteristic hexagonal core with p6 symmetry seen by image analysis (47, 48), and the 2D lattice is completed by hexagonal cores connected at junction points with p3 symmetry (Fig. ​(Fig.11 A). Secretion and subsequent self-assembly require calcium (35), and it is assumed that the RTX repeat domain (characteristic of type I secreted proteins) that is located adjacent to the C-terminal secretion signal is responsible for at least some of the calcium interaction; whether there is a second domain to complete subunit-subunit interactions is unknown. The S layer is attached to the outer membrane (OM) surface by interaction of an N-terminal attachment domain of approximately 200 amino acids (18) with the fraction of lipopolysaccharide (LPS) that is modified with an O-polysaccharide (3, 52).Open in a separate windowFIG. 1.(A) Schematic from a previous publication (12) showing how six RsaA monomers build a hexagonal S-layer subunit in C. crescentus. The six-point star shows the center of 6-fold symmetry, and the triangle indicates the center of 3-fold symmetry. The figure is based on results presented previously (35). (B) Cross section of a C. crescentus tomogram to show the cell wall components. The effect of the missing wedge blurring the features on the top and bottom of the cell is obvious.In this paper, we present a quantitative analysis of the C. crescentus S layer that sheds light on the overall S-layer organization as well as improves our understanding of the structure within the RsaA monomer, in advance of achieving true atomic resolution, by combining cryo-electron tomography (CET) of intact cells with statistical image-processing algorithms. CET is an ideal imaging technology with which to obtain a view of intact prokaryotic cells at molecular resolution (28, 29, 30). This technology allows the visualization of S-layer architecture directly as it exists on the dynamic, growing cell surface. The sample in the microscope is kept close to the native state without staining artifacts, while projections are obtained from different tilt angles to reconstruct a 3D density map of the sample. However, due to low contrast and a generally high sample thickness, CET images have a low signal-to-noise-ratio (SNR). In particular, C. crescentus has a diameter of approximately 600 nm, which is considered close to the thickness limit of CET imaging for these kinds of samples. Statistical image-processing techniques and target samples as thin as possible are needed to overcome the low SNR and to perform quantitative analysis of the S-layer characteristics in situ.To obtain structural information from within the RsaA monomer, we introduced unique cysteine residues at locations ranging from the N terminus to as close as possible to the C terminus (without disrupting the secretion signal), followed by labeling with maleimide-coupled Nanogold particles. At 1 to 2 nm in size, the Nanogold is not visible in individual CET images of intact cells. However, it is possible to extract thousands of small volumes containing S-layer subunits from CET images and combine them using subtomogram averaging techniques to produce higher-resolution structures. In these averages, we identified locations of high-density areas representing the Nanogold, effectively using the regularity of the S-layer structure to increase the resolution of CET imaging.In short, we found that the long-range order is substantially lower than we had expected and that there were areas of double layers, especially when RsaA was overexpressed. By comparing the site-specific Nanogold labeling to the 2-nm-resolution structure that is available (47), we have begun to correlate the primary sequence to positions within the averaged hexagonal structure, which represents a significant step toward having a rational basis for site selection for heterologous protein insertions in nanotechnology applications.     

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid ��-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ_3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD.     

The importance of rare species: a trait‐based assessment of rare species contributions to functional diversity and possible ecosystem function in tall‐grass prairies

The majority of species in ecosystems are rare, but the ecosystem consequences of losing rare species are poorly known. To understand how rare species may influence ecosystem functioning, this study quantifies the contribution of species based on their relative level of rarity to community functional diversity using a trait‐based approach. Given that rarity can be defined in several different ways, we use four different definitions of rarity: abundance (mean and maximum), geographic range, and habitat specificity. We find that rarer species contribute to functional diversity when rarity is defined by maximum abundance, geographic range, and habitat specificity. However, rarer species are functionally redundant when rarity is defined by mean abundance. Furthermore, when using abundance‐weighted analyses, we find that rare species typically contribute significantly less to functional diversity than common species due to their low abundances. These results suggest that rare species have the potential to play an important role in ecosystem functioning, either by offering novel contributions to functional diversity or via functional redundancy depending on how rare species are defined. Yet, these contributions are likely to be greatest if the abundance of rare species increases due to environmental change. We argue that given the paucity of data on rare species, understanding the contribution of rare species to community functional diversity is an important first step to understanding the potential role of rare species in ecosystem functioning.     

Conversion of antibacterial quinolone drug levofloxacin to potent cytotoxic agents

Levofloxacin, the optical S-(-) isomer of ofloxacin, is a broad-spectrum antibacterial agent widely used to control various infections caused by Gram-positive and Gram-negative bacteria. While the COOH group is necessary for antibacterial activity, its modification can offer anticancer activity to the fluoroquinolone framework. Therefore, several levofloxacin carboxamides 11a-j and 12 containing 5-substituted-1,3,4-thiadiazole residue were synthesized and screened in vitro for their anticancer activity. The in vitro MTT viability assay revealed that the most compounds had significant activity against cancer cells MCF-7, A549, and SKOV3. In particular, the 3-chloro- and 4-fluoro- benzyl derivatives ( 11b and 11h ), with IC₅₀ values of 1.69–4.76 μM were as potent as or better than doxorubicin. It should be noted that the mother quinolone levofloxacin showed no activity on the tested cancer cell lines. The SAR analysis demonstrated that the 3-chloro or 4-fluoro substituent on the S-benzyl moiety had positive effect on the activity. Further in vitro evaluations of the most promising compounds 11b and 11h by flow cytometric analysis and comet test revealed the ability of compounds in the induction of apoptosis and blockage of the cell proliferation at the G1-phase by nuclear fragmentation and DNA degradation in cancer cells. The obtained results demonstrated that the alteration of 6-COOH functional group in the levofloxacin structure and conjugation with a proper heterocyclic pharmacophore is a good strategy to obtain new anticancer agents.     

Lentiviral Expression of Rabies Virus Glycoprotein in the Rat Hippocampus Strengthens Synaptic Plasticity

Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two μl (10⁸ T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat’s brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.