Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Simultaneous Measurement of Smoothened Entry Into and Exit From the Primary Cilium

Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.     

Polynitroxyl-albumin (PNA) enhances myocardial infarction therapeutic effect of tempol in rat hearts subjected to regional ischemia-reperfusion

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a low molecular weight stable nitroxyl radical (nitroxide), has been demonstrated in many in vitro and in vivo models to have protective effects against oxidative stress. The beneficial effect of TPL, however, is limited because of its short life-time in tissues. We have previously shown that polynitroxylated macromolecules such as polynitroxyl-human serum albumin (PNA) enable maintaining a sustained concentration of TPL for longer periods in tissues. PNA itself has previously been shown to inhibit ischemia-reperfusion (I/R) injury in the gut and to potentiate the activity of TPL. The aim of the present study was (i) to select an optimum formulation of PNA + TPL for therapeutic applications using in vivo EPR spectroscopy and (ii) to evaluate the efficacy of the PNA + TPL formulation in preventing I/R injury to heart, in an in vivo rat model. Rats were subjected to 45 min occlusion of the left anterior descending (LAD) coronary artery followed by 120 min reperfusion. PNA (100 mg/ml) + TPL (10 mg/ml), human serum albumin (HSA, 100 mg/ml) + TPL (10 mg/ml), or saline were injected 5 min before ischemia (3 ml/kg BW, i.v.) and 5 min before reperfusion (3 ml/kg BW, i.v.), followed by a 4 ml/kg BW infusion over 2 h reperfusion. Myocardial risk and infarct regions were then estimated. The results showed that the infarct volume, expressed as a percentage of the risk region, in the group treated with PNA + TPL was 39.7 +/- 3.1%, which was significantly smaller than for the saline (51.3 +/- 3.5%) or HSA + TPL (48.4 +/- 1.4%) groups. The results demonstrate that the PNA + TPL combination is very effective in reducing myocardial ischemia-reperfusion injury.     

Relationships between Inflammation,Adiponectin, and Oxidative Stress in Metabolic Syndrome

Metabolic syndrome (MS) represents a cluster of physiological and anthropometric abnormalities. The purpose of this study was to investigate the relationships between the levels of inflammation, adiponectin, and oxidative stress in subjects with MS. The inclusion criteria for MS, according to the Taiwan Bureau of Health Promotion, Department of Health, were applied to the case group (n = 72). The control group (n = 105) comprised healthy individuals with normal blood biochemical values. The levels of inflammatory markers [high sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6), adiponectin, an oxidative stress marker (malondialdehyde), and antioxidant enzymes activities [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)] were measured. Subjects with MS had significantly higher concentrations of inflammatory markers and lower adiponectin level, and lower antioxidant enzymes activities than the control subjects. The levels of inflammatory markers and adiponectin were significantly correlated with the components of MS. The level of hs-CRP was significantly correlated with the oxidative stress marker. The IL-6 level was significantly correlated with the SOD and GPx activities, and the adiponectin level was significantly correlated with the GPx activity. A higher level of hs-CRP (≥1.00 mg/L), or IL-6 (≥1.50 pg/mL) or a lower level of adiponectin (<7.90 µg/mL) were associated with a significantly greater risk of MS. In conclusion, subjects suffering from MS may have a higher inflammation status and a higher level of oxidative stress. A higher inflammation status was significantly correlated with decreases in the levels of antioxidant enzymes and adiponectin and an increase in the risk of MS.     

Characterization of unstable hemoglobin A1c complexes by dynamic capillary isoelectric focusing

Glucose spontaneously reacts with hemoglobin amino groups to produce unstable Schiff base complexes that can dissociate or rearrange to form stable Amadori products. We used dynamic capillary isoelectric focusing and boronate affinity chromatography to assess the formation and dissociation of unstable hemoglobin complexes in vitro. Formation was studied by incubating erythrocytes at 37°C for up to 24h in phosphate-buffered saline (PBS) supplemented with 0 to 55.6 mmol/L glucose. Dissociation was studied by incubating glucose-loaded erythrocytes in PBS without glucose. Dynamic capillary isoelectric focusing separated hemoglobin A1c into two subfractions identified as A1c1 and A1c2. The A1c1 subfraction contained both stable and unstable hemoglobin complexes. The A1c2 subfraction contained only unstable hemoglobin complexes. Both subfractions quantitatively increased in the presence of glucose and decreased in its absence. Rates of increase and decrease were faster and time to equilibrium was shorter for A1c2 (~4 h) compared with A1c1 (~20 h). Unstable hemoglobin complexes did not bind to boronate affinity columns but instead eluted intact in A1c1 and A1c2 subfractions from nonglycated affinity fractions. Cyanoborohydride reduction confirmed the presence of Schiff base complexes. Evidence of multiple unstable hemoglobin complexes with different rates of glycation suggests that new models are needed to describe nonenzymatic hemoglobin glycation.     

Proteomic analysis and identification of aqueous humor proteins with a pathophysiological role in diabetic retinopathy

Diabetic retinopathy (DR) can cause irreversible blindness and is the severest microvascular complication in the eyes of patients with diabetic mellitus (DM). The identification of susceptibility factors contributing to development of DR is helpful for identifying predisposed patients and improving treatment efficacy. Although proteomics analysis is useful for identifying protein markers related to diseases, it has never been used to explore DR-associated susceptibility factors in the aqueous humor (AH). To better understand the pathophysiology of DR and to identify DR-associated risk factors, a gel-based proteomics analysis was performed to compare AH protein profiles of DM patients with and without development of DR. MALDI-TOF MS was then performed to identify protein spots that were differentially expressed between the two groups and western blot analysis was used to validate the expressional change of protein demonstrated by proteomics. Our proteomics and bioinformatics analysis identified 11 proteins differentially expressed between DR and control groups. These proteins are linked to biological networks associated with nutrition transport, microstructure reorganization, angiogenesis, anti-oxidation, and neuroprotection. The data may provide potential AH biomarkers and susceptibility factors for predicting DR development, and provide an insight into the underlying pathophysiological mechanisms of DR. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Assessing recruitment of lung diffusing capacity in exercising guinea pigs with a rebreathing technique.

Noninvasive techniques for assessing cardiopulmonary function in small animals are limited. We previously developed a rebreathing technique for measuring lung volume, pulmonary blood flow, diffusing capacity for carbon monoxide (Dl(CO)) and its components, membrane diffusing capacity (Dm(CO)) and pulmonary capillary blood volume (Vc), and septal volume, in conscious nonsedated guinea pigs at rest. Now we have extended this technique to study guinea pigs during voluntary treadmill exercise with a sealed respiratory mask attached to a body vest and a test gas mixture containing 0.5% SF(6) or Ne, 0.3% CO, and 0.8% C(2)H(2) in 40% or 98% O(2). From rest to exercise, O(2) uptake increased from 12.7 to 25.5 ml x min(-1) x kg(-1) while pulmonary blood flow increased from 123 to 239 ml/kg. The measured Dl(CO), Dm(CO), and Vc increased linearly with respect to pulmonary blood flow as expected from alveolar microvascular recruitment; body mass-specific relationships were consistent with those in healthy human subjects and dogs studied with a similar technique. The results show that 1) cardiopulmonary interactions from rest to exercise can be measured noninvasively in guinea pigs, 2) guinea pigs exhibit patterns of exercise response and alveolar microvascular recruitment similar to those of larger species, and 3) the rebreathing technique is widely applicable to human ( approximately 70 kg), dog (20-30 kg), and guinea pig (1-1.5 kg). In theory, this technique can be extended to even smaller animals provided that species-specific technical hurdles can be overcome.     

Retinoic acid induces nonuniform alveolar septal growth after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) enhances compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 4 mo, the remaining lung was fixed by tracheal instillation of fixatives at a constant airway pressure for detailed morphometric analysis. After RA treatment compared with placebo, lung volume was slightly but not significantly lower. Volume density of septum to lung was 37% higher because of a 50 and 25% higher volume density of capillary and septal tissue, respectively. Mean septal thickness was 27% higher. Absolute volumes of endothelial cells and capillary blood were 31-37% higher, whereas epithelial and interstitial volumes were not different between groups. Absolute alveolar-capillary surface areas did not differ between groups, and alveolar septal surface-to-volume ratio was 20% lower in RA-treated animals. RA treatment exaggerated interlobar differences in morphometric indexes and caused alveolar capillary morphology to revert to a more immature state. Thus RA treatment during early post-R-PNX adaptation preferentially enhanced alveolar capillary and endothelial cell volumes consistent with formation of new capillaries, but the associated septal distortion precluded a corresponding increase in gas-exchange surface or morphometric estimates of lung diffusing capacity.