Hypertonic saline reduces inflammation and enhances the resolution of oleic acid induced acute lung injury

Background

The association between bronchial obstruction severity and mortality in Chronic Obstructive Pulmonary Disease (COPD) is well established, but it is unknown whether disease-specific health status measures and multidimensional assessment (MDA) have comparable prognostic value.

Methods

We analyzed data coming from the Salute Respiratoria nell'Anziano (Respiratory Health in the Elderly – SaRA) study, enrolling elderly people attending outpatient clinics for respiratory and non-respiratory problems. From this population we selected 449 patients with bronchial obstruction (77.3% men, mean age 73.1). We classified patients' health status using tertiles of the Saint George Respiratory Questionnaire (SGRQ) and a MDA including functional (the 6' walking test, WT), cognitive (Mini-Mental State Examination, MMSE) and affective status (Geriatric Depression Scale, GDS). The agreement of the classification methods was calculated using the kappa statistic, and survival associated with group membership was evaluated using survival analysis.

Results

Pulmonary function, expressed by the FEV1, worsened with increasing SGRQ or MDA scores. Cognitive function was not associated with the SGRQ, while physical performance and mood status were impaired only in the highest tertile of SGRQ. A poor agreement was found between the two classification systems tested (k = 0.194). Compared to people in the first tertile of SGRQ score, those in the second tertile had a sex-adjusted HR of 1.22 (0.75 – 1.98) and those in the third tertile of 2.90 (1.92 – 4.40). The corresponding figures of the MDA were 1.49 (95% CI 1.02 – 2.18) and 2.01 (95% CI: 1.31 – 3.08). After adjustment for severity of obstruction, only a SGRQ in the upper tertile was associated with mortality (HR: 1.86; 95% CI: 1.14 – 3.02).

Conclusion

In elderly outpatients with mild-moderate COPD, a disease-specific health status index seems to be a better predictor of death compared to a MDA.     

C-2-aryl O-substituted HI-236 derivatives as non-nucleoside HIV-1 reverse-transcriptase inhibitors

Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2 cell cultures. The compounds were synthesized in order to fine-tune the activity of HI-236 as well as to gain insight into spatial characteristics in the pocket pertaining to the positional choice of tether in the design of [NRTI]-tether-[HI-236] bifunctional inhibitors. Two of the thiourea derivatives bearing a butynyl (6c) or hydroxyethyl tether (6n) were endowed with improved anti-HIV activity compared to HI-236. NNRTI activity was confirmed by a cell-free RT assay on six of the derivatives in which 6c returned an IC(50) of 3.8 nM compared to 28 nM for HI-236, establishing it as an improved lead for HI-236. The structure-activity profile is discussed in terms of potential interactions in the NNRTI pocket as suggested by a docking model using AutoDock, which have a bearing on the bifunctional drug design.     

Resveratrol induces catalytic bioscavenger paraoxonase 1 expression and protects against chemical warfare nerve agent toxicity in human cell lines

Current advances in enzyme bioscavenger prophylactic therapy against chemical warfare nerve agent (CWNA) exposure are moving towards the identification of catalytic bioscavengers that can degrade large doses of organophosphate (OP) nerve agents without self destruction. This is a preferred method compared to therapy with the purified stoichiometric bioscavenger, butyrylcholinesterase, which binds OPs 1:1 and would thus require larger doses for treatment. Paraoxonase-1 (PON-1) is one such catalytic bioscavenger that has been shown to hydrolyze OP insecticides and contribute to detoxification in animals and humans. Here we investigated the effects of a common red wine ingredient, Resveratrol (RSV), to induce the expression of PON-1 in the human hepatic cell line HC04 and evaluated the protection against CWNA simulants. Dose-response curves showed that a concentration of 20 microM RSV was optimal in inducing PON-1 expression in HC04 cells. RSV at 20 microM increased the extracellular PON-1 activity approximately 150% without significantly affecting the cells. Higher doses of RSV were cytotoxic to the cells. Resveratrol also induced PON-1 in the human lung cell line A549. RSV pre-treatment significantly (P = 0.05) protected the hepatic cells against exposure to 2x LD(50) of soman and sarin simulants. However, lung cells were protected against soman simulant exposure but not against sarin simulant exposure following RSV treatment. In conclusion, these studies indicate that dietary inducers, such as RSV, can up-regulate PON-1, a catalytic bioscavenger, which can then hydrolyze and protect against CWNA-induced toxicity, providing a prospective new method to protect against CWNA exposure.     

An improved procedure for routine determination of reserve-K in pastoral soils

Non-exchangeable K (reserve K) can be an important source of K for plant uptake in micaceous soils but testing for reserve K is laborious and expensive and involves use of potentially hazardous chemicals. Our objectives were to (1) identify a cheaper and more reliable reserve K test that is amenable for routine use by commercial laboratories, and (2) quantify the response of grass-clover pasture to applied K in soils differing in reserve K. We measured the release of reserve K using different combinations of the K-precipitant, sodium tetraphenyl boron (0.033 to 0.2?M), and extraction time (1 to 16 h). Copper chloride was used to decompose the K-tetraphenyl boron precipitate rather than the more toxic mercuric chloride or acetone. Based on data obtained for soils with a wide range of reserve K values, a test involving extraction of soil (1-g) in 0.1 M Na-tetraphenyl boron for 4 h was selected as the best option for routine use because it generally gave similar values to the current test (thus the same diagnostic criteria may be applicable) and it provided a better fit with operational requirements of commercial laboratories. Pasture K responses, in the presence and absence of applied N, were measured over a single growing season (pasture harvested at intervals of 1–2 months, depending on growth rate) at seven sites in Canterbury, New Zealand with reserve K (measured using the current test) ranging from 0.5 to 2.3 cmol_c/kg (all sites were low in exchangeable K, <0.33 cmol_c/kg). The size of the K response depended on the clover content of the pasture and only one site with a high clover content responded consistently to K throughout the season. Clover content increased at all sites following K addition, confirming that a major benefit of pasture K fertilization is in improving clover production. Our results suggest that there may not be a unique critical threshold for reserve K that can be applied to all pastures; rather, the value below which a production response to K is obtained may depend on the amount of clover in the pasture.     

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.     

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.