Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity

The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r = -0.52, p = 0.005 and r = -0.61, p = 0.023, respectively) compared to patients with coexisting metabolic syndrome (r = -0.25, p = 0.47 and r = -0.15, p = 0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.     

Dendritic morphology of hippocampal and amygdalar neurons in adolescent mice is resilient to genetic differences in stress reactivity

Many studies have shown that chronic stress or corticosterone over-exposure in rodents leads to extensive dendritic remodeling, particularly of principal neurons in the CA3 hippocampal area and the basolateral amygdala. We here investigated to what extent genetic predisposition of mice to high versus low stress reactivity, achieved through selective breeding of CD-1 mice, is also associated with structural plasticity in Golgi-stained neurons. Earlier, it was shown that the highly stress reactive (HR) compared to the intermediate (IR) and low (LR) stress reactive mice line presents a phenotype, with respect to neuroendocrine parameters, sleep architecture, emotional behavior and cognition, that recapitulates some of the features observed in patients suffering from major depression. In late adolescent males of the HR, IR, and LR mouse lines, we observed no significant differences in total dendritic length, number of branch points and branch tips, summated tip order, number of primary dendrites or dendritic complexity of either CA3 pyramidal neurons (apical as well as basal dendrites) or principal neurons in the basolateral amygdala. Apical dendrites of CA1 pyramidal neurons were also unaffected by the differences in stress reactivity of the animals; marginally higher length and complexity of the basal dendrites were found in LR compared to IR but not HR mice. In the same CA1 pyramidal neurons, spine density of distal apical tertiary dendrites was significantly higher in LR compared to IR or HR animals. We tentatively conclude that the dendritic complexity of principal hippocampal and amygdala neurons is remarkably stable in the light of a genetic predisposition to high versus low stress reactivity, while spine density seems more plastic. The latter possibly contributes to the behavioral phenotype of LR versus HR animals.     

Beneficial effects of Murraya koenigii leaves on antioxidant defense system and ultra structural changes of pancreatic beta-cells in experimental diabetes in rats

Oxidative stress and oxidative damage to tissues are common end points of chronic diseases such as atherosclerosis, diabetes, and rheumatoid arthritis. Oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can further increase the deleterious effects of free radicals. The aim of the present study was to evaluate the possible protective effects of Murraya koenigii leaves extract against beta-cell damage and antioxidant defense systems of plasma and pancreas in streptozotocin induced diabetes in rats. The levels of glucose and glycosylated hemoglobin in blood and insulin, Vitamin C, Vitamin E, ceruloplasmin, reduced glutathione and TBARS were estimated in plasma of control and experimental groups of rats. To assess the changes in the cellular antioxidant defense system such as the level of reduced glutathione and activities of superoxide dismutase, catalase and glutathione peroxidase were assayed in pancreatic tissue homogenate. The levels of glucose, glycosylated hemoglobin, insulin, TBARS, enzymatic and non-enzymatic antioxidants were altered in diabetic rats. These alterations were reverted back to near control levels after the treatment of M. koenigii leaves extract. Transmission electron microscopic studies also revealed the protective nature of M. koenigii leaves on pancreatic beta-cells. These findings suggest that M. koenigii treatment exerts a therapeutic protective nature in diabetes by decreasing oxidative stress and pancreatic beta-cell damage. The antioxidant effect of the M. koenigii extract was compared with glibenclamide, a well-known hypoglycemic drug.     

Enhanced xylanase and endoglucanase production from Beauveria bassiana SAN01, an entomopathogenic fungal endophyte

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.     

The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes

Rationale

Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous disease. In COPD, the presence of emphysema is associated with increased mortality and risk of lung cancer. High resolution computed tomography (HRCT) scans are useful in quantifying emphysema but are associated with radiation exposure and high incidence of false positive findings (i.e., nodules). Using a comprehensive biomarker panel, we sought to determine if there was a peripheral blood biomarker signature of emphysema.

Methods

114 plasma biomarkers were measured using a custom assay in 588 individuals enrolled in the COPDGene study. Quantitative emphysema measurements included percent low lung attenuation (%LAA) ≤ −950 HU, ≤ − 910 HU and mean lung attenuation at the 15^th percentile on lung attenuation curve (LP15A). Multiple regression analysis was performed to determine plasma biomarkers associated with emphysema independent of covariates age, gender, smoking status, body mass index and FEV₁. The findings were subsequently validated using baseline blood samples from a separate cohort of 388 subjects enrolled in the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study.

Results

Regression analysis identified multiple biomarkers associated with CT-assessed emphysema in COPDGene, including advanced glycosylation end-products receptor (AGER or RAGE, p < 0.001), intercellular adhesion molecule 1 (ICAM, p < 0.001), and chemokine ligand 20 (CCL20, p < 0.001). Validation in the TESRA cohort revealed significant associations with RAGE, ICAM1, and CCL20 with radiologic emphysema (p < 0.001 after meta-analysis). Other biomarkers that were associated with emphysema include CDH1, CDH 13 and SERPINA7, but were not available for validation in the TESRA study. Receiver operating characteristics analysis demonstrated a benefit of adding a biomarker panel to clinical covariates for detecting emphysema, especially in those without severe airflow limitation (AUC 0.85).

Conclusions

Our findings, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema, and when combined with clinical covariates, may be useful clinically in predicting the presence of emphysema compared to just using covariates alone, especially in those with less severe COPD. Ultimately biomarkers may shed light on disease pathogenesis, providing targets for new treatments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0127-9) contains supplementary material, which is available to authorized users.     

Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone

The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA–ArsD interface that affect their interactions.     

Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl(meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme-inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.     

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34⁺ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4⁺ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4⁺ and CD8⁺ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4⁺ and CD8⁺ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4⁺ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.An ideal animal model of human immunodeficiency virus (HIV) infection remains elusive. Nonhuman primates that are susceptible to HIV infection typically do not develop immunodeficiency (63), and although the simian immunodeficiency virus (SIV) infection of rhesus macaques has provided many critically important insights into retroviral pathogenesis (30), biological and financial considerations have created some limitations to the wide dissemination of this model. The great need for an improved animal model of HIV itself recently has been underscored by the disappointing results of human trials of MRKAd5, an adenovirus-based HIV type 1 (HIV-1) vaccine. This vaccine was not effective and actually may have increased some subjects'' risk of acquiring HIV (53). In the wake of these disappointing results, there has been increased interest in humanized mouse models of HIV infection (54). The ability of humanized mouse models to test candidate vaccines or other immunomodulatory strategies will depend critically on the ability of these mice to generate robust anti-HIV human immune responses.Mice have provided important model systems for the study of many human diseases, but they are unable to support productive HIV infection, even when made to express human coreceptors for the virus (7, 37, 52). A more successful strategy to humanize mice has been to engraft human immune cells and/or tissues into immunodeficient severe combined immunodeficiency (SCID) or nonobese diabetic (NOD)/SCID mice that are unable to reject xenogeneic grafts (39, 42, 57). Early versions of humanized mice supported productive HIV infection and allowed investigators to begin to address important questions in HIV biology in vivo (23, 40, 43-45). More recently, human cord blood or fetal liver CD34⁺ cells have been used to reconstitute Rag2^−/− interleukin-2 receptor γ chain-deficient (γ_c^−/−) and NOD/SCID/γ_c^−/− mice, resulting in higher levels of sustained human immune cell engraftment (27, 29, 61). These mice have allowed for stable, disseminated HIV infection (2, 4, 24, 65, 67), including mucosal transmission via vaginal and rectal routes (3). These mice recently have been used to demonstrate an important role for Treg cells in acute HIV infection (29) and to demonstrate that the T-cell-specific delivery of antiviral small interfering RNA is able to suppress HIV replication in vivo (31). These mice also have demonstrated some evidence of adaptive human immune responses, including the generation of HIV-specific antibody responses in some infected mice (2, 65), and some evidence of humoral and cell-mediated responses to non-HIV antigens or pathogens (24, 61). Most impressively, Rag2^−/− γ_c^−/− mice reconstituted with human fetal liver-derived CD34⁺ cells have generated humoral responses to dengue virus infection that demonstrated both class switching and neutralizing capacity (32). In spite of these advances, however, these models have not yet been reported to generate de novo HIV-specific cell-mediated immune responses, which are considered to be a crucial arm of host defense against HIV infection in humans.In contrast to humanized mouse models in which only human hematopoietic cells are transferred into immunodeficient mice, the surgical implantation of human fetal thymic and liver tissue has been performed in addition to the transfer of human hematopoietic stem cells (HSC) to generate mice in which human T cells are educated by autologous human thymic tissue rather than by the xenogeneic mouse thymus. Melkus and colleagues refer to mice they have reconstituted in this way as NOD/SCID-hu BLT (for bone marrow, liver, and thymus), or simply BLT, mice (41). We previously referred to mice that we have humanized in a similar way as NOD/SCID mice cotransplanted with human fetal thymic and liver tissues (Thy/Liv) and CD34⁺ fetal liver cells (FLC) (33, 60) but now adopt the designation BLT mice as well. BLT mice demonstrate the robust repopulation of mouse lymphoid tissues with functional human T lymphocytes (33, 41, 60) and can support the rectal and vaginal transmission of HIV (13, 59). Further, BLT mice demonstrate antigen-specific human immune responses against non-HIV antigens and/or pathogens (41, 60). The ability of these mice to generate human immune responses against HIV, however, has not yet been reported. In this study, we investigated whether the provision of autologous human thymic tissue in BLT mice generated by the cotransplantion of human fetal Thy/Liv tissues and CD34⁺ FLC would allow for the maturation of human T cells in humanized mice capable of providing improved cellular responses to HIV as well as providing adequate help for improved humoral responses. To describe the cells contributing to human immune responses in BLT mice, we also characterized the phenotypes of multiple subsets of T cells, B cells, dendritic cells (DCs), and monocytes/macrophages present in uninfected humanized mice. The generation of robust HIV-directed human cellular and humoral immune responses in these mice would further demonstrate the ability of humanized mice to provide a much needed platform for the evaluation of HIV vaccines and other novel immunomodulatory strategies.     

A coding measure scheme employing electron-ion interaction pseudopotential (EIIP)

In this paper, a revision for the existing method of locating exons by genomic signal processing technique employing four binary indicator sequences is presented. The existing method relies on the pronounced period three peaks observed in the Fourier power spectrum of the exon regions which are absent in non-coding regions. The authors have abandoned the four sequences all together and adopted a single 'EIIP indicator sequence' which is formed by substituting the electron-ion interaction pseudopotentials (EIIP) of the nucleotides A, G, C and T in the DNA sequence, reducing the computational overhead by 75%. The power spectrum of this sequence reveals period three peaks for exon regions. Also a number of exons have been identified which exhibit period three peaks when mapped to 'EIIP indicator sequence' and which do not show the same when the binary indicator sequences are employed. We could get better discrimination between exon areas and non-coding areas of a number of genomes when the sequences are mapped to EIIP indicator sequences and the power spectra of the same are taken in a sliding Kaiser window, compared to the existing method using a rectangular window which utilizes binary indicator sequences.