In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2, *3, *4, and *10

Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower k_cat values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased K_m values and diminished k_cat values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.     

Correlation between micro-computed tomography and histomorphometry for assessment of new bone formation in a calvarial experimental model

Conventional histologic or histomorphometric evaluation provides clear evidence of the bone healing process. However, the sample preparation process is tedious and destructive, and the three-dimensional (3D) anisotropic information of the bone trabeculae is compromised. Micro-computed tomography (microCT) has been introduced as an alternative to these traditional evaluation methods. microCT is noninvasive and provides a faster approach to evaluate and quantify cancellous bone. Most previous studies that used microCT have focused on studying trabecular structures of cancellous bone. In this study, we used microCT to analyze the micro-architecture of the regenerated membranous bone using a rabbit cranial defect model. Two 1 cm diameter circular bony defects were created in 12 New Zealand white rabbits. Specimens were harvested at 6 weeks and 12 weeks after surgery and were scanned using a MicroCT machine (Skyscan 1072, Aartselaar, Belgium). The specimens were then sectioned and stained with Goldner's trichrome. Bone volume density (BV/TV), bone surface density (BS/BV), and trabecular thickness (TbTh) were determined from histomorphometric and two-dimensional (2D) and 3D microCT analysis. Pearson's correlation coefficient (gamma), paired t-tests, and intraclass correlation coefficients from measurements between the 2D and 3D microCT and histomorphometry were calculated. There were very strong positive correlations of BV/TV between histomorphometric and 2D or 3D microCT measurements. Correlation between histomorphometric and 2D microCT measurements for BS/BV was moderate, whereas correlation between histomorphometric and 3D microCT measurements was weak. Weak correlations in TbTh among the three methods were found. In conclusion, the present study suggests that, in evaluating micro-architectures in regenerated bones, the correlation between measuring methods vary according to the features measured.     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Portable low-power thermal cycler with dual thin-film Pt heaters for a polymeric PCR chip

Polymerase chain reaction (PCR) has been widely used for major definite diagnostic tool, but very limited its place used only indoor such as hospital or diagnosis lab. For the rapid on-site detection of pathogen in an outdoor environment, a low-power cordless polymerase chain reaction (PCR) thermal cycler is crucial module. At this point of view, we proposed a low-power PCR thermal cycler that could be operated in an outdoor anywhere. The disposable PCR chip was made of a polymeric (PI/PET) film to reduce the thermal mass. A dual arrangement of the Pt heaters, which were positioned on the top and bottom of the PCR chip, improved the temperature uniformity. The temperature sensor, which was made of the same material as the heater, utilized the temperature dependence of the Pt resistor to ensure simple fabrication of the temperature sensor. Cooling the PCR chip using dual blower fans enabled thermal cycling to operate with a lower power than that of a Peltier element with a high power consumption. The PCR components were electrically connected to a control module that could be operated with a Li-ion battery (12 V), and the PCR conditions (temperature, time, cycle, etc.) were inputted on a touch screen. For 30 PCR cycles, the accumulated power consumption of heating and cooling was 7.3 Wh, which is easily available from a compact battery. Escherichia coli genomic DNA (510 bp) was amplified using the proposed PCR thermal cycler and the disposable PCR chip. A similar DNA amplification capability was confirmed using the proposed portable and low-power thermal cycler compared with a conventional thermal cycler.     

Analysis of pelvic compensation for dynamic sagittal imbalance using motion analysis

To analyze pelvic compensation during walking in patients with severe sagittal plane deformity by using motion analysis. A total of 44 patients with sagittal plane deformity who were scheduled to undergo surgery were included. Motion analysis was performed 3 consecutive times during walking to estimate the anterior pelvic tilt (Ant-PT) angle, trunk kyphosis (TK) angle, and distance of the center of gravity (CoG) from the center of mass (CoM) of the pelvic segment, and hip and knee joint angles during gait. The patients were classified into Ant-PT+/Ant-PT−, TK+/TK−, and CoG+/CoG− groups according to the changes in Ant-PT angle, TK angle, and distance of the CoG from the CoM of the pelvic segment. Increases and decreases in the values of the variables from the first trial to the third trial were indicated with “+” and “−” signs, respectively. The mean Ant-PT angle, TK angle, and distance of the CoG from the CoM of the pelvic segment increased progressively, and the differences in the values of these variables from the first to the third trials were statistically significant (P = 0.046, P = 0.004, and P = 0.007 for the Ant-PT angle, TK angle, and distance of the CoG from the CoM of pelvic segment, respectively). Among the 44 patients, 27 and 34 were classified into the Ant-PT+ and CoG+ groups, respectively. Older age and higher body mass index (BMI) were significantly associated with the Ant-PT+ group. The CoG+ group demonstrated a significantly higher height and weight than the CoG− group. Higher BMI, height, and weight are risk factors for progressive worsening of dynamic sagittal imbalance. These slides can be retrieved under Electronic Supplementary Material.     

Evaluation of MRI resolution affecting trabecular bone parameters: Determination of acceptable resolution

The objective of this study is to evaluate the effect of MR image resolution on trabecular bone parameters and to determine the acceptable resolution that can be accurately analyzed to assess structural parameters. Ten distal femoral condyle specimens of 1 × 1 × 1 cm³ were scanned with a 4.7‐T Bruker BioSpec MRI scanner using a three‐dimensional fast large‐angle spin‐echo sequence with various iso‐cubic voxels sizes (65, 130, 160, 196, 230, and 260 μm). Otsu thresholding was applied to identify voxels containing bone. Conventional bone parameters, structural bone parameters, and skeleton‐based local trabecular thickness (slTB.Th) were evaluated. The Bland–Altman method and correlation indicated that the conventional and structural bone parameters were preserved with an iso‐cubic voxel size up to 230 μm (r > 0.932 and r > 0.843, respectively). In addition, slTB.Th derived from the highest resolution images (65 μm iso‐cubic voxel size), correlated well (r > 0.833) with the values computed from lower resolution images, up to 230 μm, which is twice typical human trabecular thickness range (100–150 μm). The outcome of this study suggests that the various bone parameters were well preserved up to 230 μm images. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Pharmacokinetics and bioavailability of carbovir, a carbocyclic nucleoside active against human immunodeficiency virus, in rats.

Carbovir is a novel carbocyclic nucleoside which has been shown to have potent in vitro activity against human immunodeficiency virus, the causative agent of acquired immunodeficiency syndrome. Sprague-Dawley male rats were used to investigate the pharmacokinetics and bioavailability of carbovir. Six rats received carbovir (20 mg/kg of body weight) through the jugular vein, and blood samples were collected through the femoral vein 2, 5, 10, 15, 20, 30, 45, 60, 75, 90, 120, 150, 180, and 240 min after the dose. Four of these rats also received a 60-mg/kg oral dose of carbovir, and a similar blood sampling schedule was followed. Whole-blood samples were prepared by solid-phase extraction, and the carbovir concentration in the samples was analyzed by reversed-phase high-pressure liquid chromatography. The profile of carbovir concentration in blood versus time after the intravenous dose was biexponential, with a very rapid distribution phase. Terminal elimination half-life was 21.4 +/- 4.37 min, and total body clearance was 55.2 +/- 13.8 ml/min per kg, which was within the range of the hepatic blood flow. The volume of distribution at steady state was 1,123 +/- 250 ml/kg. The blood/plasma ratio and the plasma protein binding of carbovir in rat blood were determined in vitro by ultrafiltration. The plasma protein binding of carbovir was only 20% and was not concentration dependent. However, the blood/plasma ratio decreased significantly as concentration increased, indicating saturable binding sites in erythrocytes. After the oral dose, the terminal half-life was 81.0 +/- 67.6 min, indicating that oral carbovir followed "flip-flop" kinetics, with absorption being much slower than elimination of the drug from the body. Oral bioavailability was 0.101 +/- 0.035. Double peaks were present in the concentration-time profile for each rat receiving the oral dose, indicating either a delay in stomach emptying of the drug or slow dissolution of precipitated carbovir in the stomach and upper small intestine.     

Effectiveness of raloxifene on bone mineral density and serum lipid levels in post-menopausal women with low BMD after discontinuation of hormone replacement therapy

OBJECTIVE: To evaluate the effect of raloxifene on bone mineral density (BMD) and serum lipid levels in post-menopausal women who had discontinued hormone replacement therapy (HRT). METHODS: Thirty-four post-menopausal women with low BMD who had taken 60 mg of raloxifene daily for 12 months after discontinuing HRT were evaluated retrospectively. Information about their demographics, fracture history, BMD, lipid profiles and adverse events were collected from medical records and intranet database. The outcome measures were changes in the spine (L2-L4) and femur BMD, serum lipid concentrations, fracture rate and tolerability. RESULTS: The post-menopausal women had a significant increase in their spine (L2-L4) and femur BMD from their baseline BMD [spine, 2.9 +/- 4.6% (P < 0.001); femur, 3.0 +/- 6.6% (P = 0.01)]. Serum low-density lipoprotein (LDL) cholesterol was significantly reduced by 22.6% below baseline after 12 months (P = 0.007). No fractures were observed during therapy. Raloxifene was well tolerated. The most common adverse event was hot flash, which was generally mild. CONCLUSIONS: Raloxifene increases BMD at important skeletal sites, and lowers LDL cholesterol with tolerable adverse events.     

Prevalence and comorbidities of bronchiolitis in adults: A population-based study in South Korea

Bronchiolitis generally refers to inflammation and/or fibrosis of the non-cartilaginous small airways located approximately from the 8th airway generation down to the terminal and respiratory bronchioles. In contrast to young children, the frequency of small airway infection in adult bronchiolitis appears less frequent and a number of other pathophysiological conditions have been implicated in adult bronchiolitis. However, little information is available on the exact medical burden of bronchiolitis such as its prevalence and comorbidities in the adult population. The aim of this study is to elucidate the prevalence and comorbidities of bronchiolitis. We used the Korea National Health Insurance Service-National Sample Cohort, which provides data for 1,000,000 individuals out of the entire population by 2% stratified random sampling according to age, sex, residential area, and level of household income. We defined the cause of bronchiolitis other than acute infection as a patient with diagnostic code J448 or J684 and over 20 years of age who visited a clinic or hospital in South Korea. Then, 1:1 propensity score matching was performed to define a non-bronchiolitis (control) group to compare the comorbidities and mortality in the 2 groups. The overall prevalence of bronchiolitis was 688 cases/1,000,000 population during the study period (95% confidence interval, 625–751). The most common comorbid clinical condition in adults with bronchiolitis was rhinitis (52.3%), followed by bronchial asthma (52.23%), hypertension (43.69%), gastroesophageal reflux disease (30.56%), sinusitis (28.72%), diabetes (22.77%), and osteoporosis (17.85%). Other common bronchiolitis-associated comorbidities were cerebrovascular disease (16.86%), angina (14.37%), peripheral vascular disease (13.42%), congestive heart failure (11.9%), and malignancy in any organ (10.6%). Healthcare costs for bronchiolitis increased steeply during the same period. Malignancy in any organ was the leading cause of mortality in the patient group, followed by bronchiolitis itself. Further larger prospective multiethnic cohort studies should be carried out in the near future.