Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

Purification and characterization of extracellular lipases from <Emphasis Type="Italic">Pseudomonas monteilii</Emphasis> TKU009 by the use of soybeans as the substrate

A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40°C, 8–11, and 50°C; and of F1 were 6, 40°C, 6–7, and 50°C, respectively. F2 was completely inhibited by EDTA and slightly by Mg²⁺, Fe²⁺, Mn²⁺, and SDS. F1 was completely inhibited by EDTA and Fe²⁺ and strongly by Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺, and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X–100 and Tween 40, especially for F1. F2 preferably acted on substrates with a long chain (C10–C18) of fatty acids, while F1 showed a broad spectrum on those with chain length of C4–C18. The marked activity of F2 in organic solvents makes it an ideal choice for application in a water-restricted medium including organic synthesis. Li-June Ming is a visiting Professor at the National Cheng Kung University.     

2,3,4,5-tetrahydro- and 2,3,4,5,11,11a-hexahydro-1H-[1,4]diazepino[1,7-a]indoles: new templates for 5-HT(2C) agonists

The design and synthesis of the novel 2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,7-a]indole 5 is described. This azepinoindole has excellent affinity for 5-HT(2C) (K(i) 4.8 nM) and modest selectivity over 5-HT(2A) ( approximately 4-fold). Several N- and C(11)-substituted analogues of 5 were prepared, as were a number of biaryl indoline derivatives. The anxiolytic potential for the azepinoindole template 5 is demonstrated by activity in a mouse shock-aggression assay.     

A rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by 1,n-alkane-di-N-butylcarbamates

Alkane-1-N-butylcarbamate-n-ols (1-7) and 1,n-alkane-di-N-butylcarbamates (8-14) are potent pseudo-substrate inhibitors of acetylcholinesterase. For inhibitors 1-7, the pre-steady state -logK(s) values and steady state -logK(i), values are linearly correlated with the tether length (N). However, for inhibitors 8-14, correlation of the -logK(s) or -logK(i) values against N deviates from linearity. A discontinuity of the -logK(s) versus N plot, concave downwards, is indicative of a rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by inhibitors 8-14.     

A novel TLR2‐triggered signalling crosstalk synergistically intensifies TNF‐mediated IL‐6 induction

Toll‐like receptors (TLR) recognize pathogens and trigger the production of vigorous pro‐inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2‐mediated synergy, through a MyD88‐independent crosstalk, which enhances subsequent TNF‐mediated nuclear factor‐kappa B activation and interleukin‐6 induction. Membrane‐associated adaptor MAL conduces the link between TNF receptor‐associated factor 6 (TRAF6) and TNFR‐associated death domain, leading to a distinctive K63‐ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF‐mediated responses, indicating an innovative target for inflammation manipulation.     

YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO)

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.     

Relationship between Lung Function and Metabolic Syndrome

Although the link between impaired lung function and cardiovascular events and type 2 diabetes mellitus has been recognized, the association between impaired lung function and metabolic syndrome has not been comprehensively assessed in the United States (U.S.) population. The aim of our study was to explore the association between impaired lung function and metabolic syndrome in a nationally representative sample of men and women. This cross-sectional population-based study included 8602 participants aged 20–65 years in the Third National Health and Nutrition Examination Survey (NHANES III). We examined the relationship between the different features of metabolic syndrome and lung function, including forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1). After adjusting for potential confounders such as age, body mass index, inflammatory factors, medical condition, and smoking status, participants with more components of metabolic syndrome had lower predicted values of FVC and FEV1 (p for trend <0.001 for both). Impaired pulmonary function was also associated with individual components of metabolic syndrome, such as abdominal obesity, high blood pressure, high triglycerides, and low high density lipoprotein (HDL) cholesterol (p<0.05 for all parameters). These results from a nationally representative sample of US adults suggest that a greater number of features of metabolic syndrome is strongly associated with poorer FVC and FEV1. In clinical practice, more comprehensive management strategies to address subjects with metabolic syndrome and impaired lung function need to be developed and investigated.     

Systemic pentraxin-3 levels reflect vascular enhancement and progression in Takayasu arteritis

Introduction

Progression of arterial involvement is often observed in patients with Takayasu arteritis (TA) thought to be in remission. This reflects the failure of currently used biomarkers and activity criteria to detect smouldering inflammation occurring within arterial wall. Pentraxin-3 (PTX3) is a soluble pattern recognition receptor produced at sites of inflammation and could reveal systemic as well as localized inflammatory processes. We verified whether the blood concentrations of PTX3 and of C-reactive protein (CRP) in patients with Takayasu arteritis (TA) might reflect vascular wall involvement, as assessed by signal enhancement after contrast media administration, and the progression of arterial involvement.

Methods

A cross-sectional single-centre study was carried out on 42 patients with TA that comprised assessment of PTX3, of CRP and erythrocyte sedimentation velocity (ESR). In total, 20 healthy controls and 20 patients with Systemic Lupus Erythematous (SLE) served as controls. Vascular imaging was carried out by magnetic resonance angiography, doppler ultrasonography and computed tomography angiography.

Results

Patients with TA and SLE had higher plasmatic PTX3 and CRP concentrations than healthy controls (P = 0.009 and 0.017, respectively). PTX3 levels did not correlate with those of CRP. Patients with active systemic TA had significantly higher concentrations of CRP but similar levels of PTX3 than patients with quiescent disease. In contrast, patients with vascular inflammation detectable at imaging had higher PTX3 concentrations (P = 0.016) than those in which vessel inflammation was not evident, while CRP levels were similar. The concentration of PTX3 but not that of CRP was significantly higher in TA patients with worsening arterial lesions that were not receiving antagonists of tumor necrosis factor-α or interleukin-6.

Conclusions

Arterial inflammation and progression of vascular involvement influence plasma PTX3 levels in TA, while levels of CRP accurately reflect the burden of systemic inflammation. These results support the contention that PTX3 reflects different aspects of inflammation than CRP and might represent a biomarker of actual arteritis in TA.     

Plasma levels of transforming growth factor-beta 1 before and after removal of low- and high-grade astrocytomas

Transforming growth factor-beta 1 (TGF-β1) has been reported to be a possible marker for a number of tumors, including brain tumors. The aim of this study was to measure the plasma levels of TGF-β1 in patients with low- and high-grade astrocytomas before and after surgery. This prospective study included 14 patients with low-grade astrocytomas and 25 with high-grade astrocytomas who underwent tumor removal and 13 controls (patients who underwent cranioplasty for skull bone defects). Plasma levels of TGF-β1 were measured in all subjects using enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curve analysis showed that when the level of TGF-β1 before tumor removal was ?2.52 ng/ml, astrocytoma was predicted with a sensitivity of 94.9% and specificity of 100%. The mean plasma level of TGF-β1 in both the low-grade and high-grade astrocytoma groups significantly decreased after tumor removal (p < 0.05); there was no significant change in TGF-β1 plasma level of the controls following surgery. Patients with high-grade astrocytomas had a significantly higher mortality rate than patients with low-grade astrocytomas (p = 0.019) and significantly shorter survival (p = 0.008). A positive correlation between TGF-β1 level after tumor removal and tumor volume was only found in the high-grade astrocytoma group (γ = 0.597, p = 0.002). The findings show that plasma TGF-β1 level was increased in patients with low-grade and high-grade astrocytoma, and that the levels significantly decreased after tumor removal in both groups. The results provide additional evidence that TGF-β1 might be useful as a tumor marker for astrocytomas.