Classification of established atopic dermatitis in children with the in vivo imaging methods

Atopic dermatitis (AD) is a cutaneous disease resulting from a defective barrier and dysregulated immune response. The severity scoring of atopic dermatitis (SCORAD) is used to classify AD. Noninvasive imaging approaches supplementary to SCORAD were investigated. Cr:forsterite laser‐based microscopy was employed to analyze endogenous third‐harmonic generation (THG) and second‐harmonic generation (SHG) signals from skin. Imaging parameters were compared between different AD severities. Three‐dimensional reconstruction of imaged skin layers was performed. Finally, statistic models from quantitative imaging parameters were developed for predicting disease severity. Our data demonstrate that THG signal intensity of lesional skin in AD were significantly increased and was positively correlated with AD severity. Characteristic gray level co‐occurrence matrix (GLCM) values were observed in more severe AD. In the 3D reconstruction video, individual dermal papilla and obvious fibrosis in the upper papillary dermis were easily identified. Our estimation models could predict the disease severity of AD patients with an accuracy of nearly 85%. The THG signal intensity and characteristic GLCM patterns are associated with AD severity and can serve as quantitative predictive parameters. Our imaging approach can be used to identify the histopathological changes of AD objectively, and to complement the SCORAD index, thus improving the accuracy of classifying AD severity.      

SOX2-Dependent Transcription in Clock Neurons Promotes the Robustness of the Central Circadian Pacemaker

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Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.     

Stable isotope analysis reveals ontogenetic feeding shifts in Pacific blue marlin (Makaira nigricans) off eastern Taiwan

To gain a better understanding of the trophic ecology of Pacific blue marlin Makaira nigricans off eastern Taiwan, nitrogen and carbon stable isotopes (δ¹⁵N and δ¹³C) and Bayesian mixing models were used to explore trophic dynamics and potential ontogenetic feeding shifts across M. nigricans of different size classes. Makaira nigricans samples from east of Taiwan (n = 213) and Palau (n = 37), as well as their prey (n = 70), were collected during 2012 and 2013. Results indicated increases in δ¹⁵N with size, with values of larger size classes (> 200 cm eye-to-fork length; L_EF) significantly higher than those < 200 cm L_EF. Values of δ¹³C were negatively correlated with size. Makaira nigricans > 200 cm L_EF had the highest estimated trophic position (4.44) and also exhibited ontogenetic changes in trophic position. Large M. nigricans fed more on dolphinfish Coryphaena hippurus and hairtail Trichiurus lepturus, while smaller M. nigricans consumed smaller forage fish (e.g., moonfish Mene maculata) and cephalopods. These changes may relate to greater swimming speeds and vertical habitat use in larger M. nigricans, allowing capture and consumption of larger prey items at higher trophic positions. The high trophic level of M. nigricans east of Taiwan confirms its important role as an apex predator in marine food webs and how ecological role changes with size.     

Long-term Therapeutic Effects of Extracorporeal Shock Wave-Assisted Melatonin Therapy on Mononeuropathic Pain in Rats

We evaluated the ability of extracorporeal shock wave (ECSW)-assisted melatonin (Mel) therapy to offer an additional benefit for alleviating the neuropathic pain (NP) in rats. Left sciatic nerve was subjected to chronic constriction injury (CCI) to induce NP. Animals (n?=?30) were randomized into group 1 (sham-operated control), group 2 (CCI only), group 3 (CCI?+?ECSW), group 4 (CCI?+?Mel) and group 5 (CCI?+?ECSW?+?Mel). By days 15, 22 and 29 after CCI, the thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but they showed no difference between the later two groups (all p?<?0.0001). The protein expressions of inflammatory (TNF-α, NF-κB, MMP-9, IL-1ß), oxidative-stress (NOXs-1, -2, -4, oxidized protein), apoptotic (cleaved-caspase3, cleaved-PARP), DNA/mitochondrial-damaged (γ-H2AX/cytosolic-cytochrome C), microglia/astrocyte activation (ox42/GFAP), and MAPKs [phosphorylated (p)-p38, p-JNK, p-ERK] biomarkers in dorsal root ganglia neurons (DRGs) and in spinal dorsal horn were exhibited an opposite pattern of TPWL among the five groups (all p?<?0.0001). Additionally, protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve exhibited an identical pattern to inflammation among the five groups (all p?<?0.0001). The numbers of cellular expressions of MAPKs (p-ERK1/2+/peripherin?+?cells, p-ERK1/2+/NF200?+?cells and p-JNK+/peripherin?+?cells, p-JNK+/NF200?+?cells) and voltage-gated sodium channels (Nav.1.8+/peripherin?+?cells, Nav.1.8+/NF200?+?cells, Nav.1.9+/peripherin?+?cells, Nav.1.9+/NF200?+?cells) in small and large DRGs displayed an identical pattern to inflammation among the five groups (all p?<?0.0001). In conclusion, the synergistic effect of combined ECSW-Mel therapy is superior to either one alone for long-term improvement of mononeuropathic pain-induced by CCI in rats.

     

Determination of nalbuphine by high-performance liquid chromatography with ultraviolet detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with ultraviolet detection was developed for the determination of nalbuphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of nalbuphine and ethylmorphine (internal standard) were greater than 86%. Calibration graphs were linear over the concentration range 0.75–150 ng/ml with a coefficient of variation, both within-day and between-day, of less than 10% at any level. The limit of quantitation was 0.75 ng/ml of plasma based on a signal-to-noise ratio of 3. Seven other clinically used analgesics were investigated to check for potential interferences and their analytical conditions. The specificity of this assay was checked with a metabolite of nalbuphine (noroxymorphine). Nalbuphine in plasma did not decompose significantly at −20°C for six weeks. Pharmacokinetic application in three surgical patients and four rabbits revealed that nalbuphine followed a linear three-compartment model with two distribution phases. The two distribution and one elimination half-lives and the plasma clearance of nalbuphine were 0.9, 5.8 and 157 min and 370 ml/min in human, and 3.5, 28 and 117 min and 21 166 ml/min in rabbits.