Molecular Cloning, Expression and Site-Directed Mutagenesis of a Complementary DNA Encoding Rabbit Muscle Fructose 6-Phosphate-1-Kinase.

This dissertation contains three parts based on my research project which investigates three aspects of rabbit muscle phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11; PFK). The first part reports the isolation and characterization of two full-length rabbit muscle PFK cDNAs. The DNA sequences of these two cDNAs (cDNA-A and cDNA-B) show an identical coding sequence but heterogeneous 5\sp\primeuntranslated regions. cDNA-A is formed by removal of a 1.7 Kb upstream intron while cDNA-B retains the 3\sp\primeregion of this intron. The second part describes the expression of the cDNA in several bacterial hosts. Recombinant rabbit muscle PFK has been expressed at a significant level in a PFK deficient E. coli strain DF1020 using the plasmid pPL2 as the expression vector. This expression system provides a visible selection for clones which express rabbit muscle PFK. The recombinant PFK resembles the enzyme purified from rabbit muscle with respect to its affinity for substrates, its allosteric behavior and its physical properties. The third part shows the structural and functional role of Gln-200 in rabbit muscle PFK (RMPFK). The comparison of the amino acid sequence of RMPFK to that of the PFK from Bacillus stearothermophilus suggests that Gln-200 of RMPFK is located in a region corresponding to the 6-F loop. This loop plays a critical role in the R-T transition of BsPFK. Mutation of RMPFK at position 200 affects the apparent affinity of the enzyme for Fru 6-P under the conditions for which RMPFK behaves allosterically. Three mutations were made at position 200: Gln-200 $\to$ Arg (Q200R), Gln-200 $\to$ Glu (Q200E), and Gln-200 $\to$ Ala (Q200A). The \rm S\sb{0.5 (Fru 6-P)} of the Q200R mutant is 4-fold lower than that of the wild type RMPFK. The \rm S\sb{0.5 (Fru 6-P)} of the Q200E mutant is increased about 14-fold. The Q200A mutant cannot achieve half-saturation, even at a concentration of Fru 6-P over 200 mM. Sigmoidal kinetics with respect to Fru 6-P were observed with Hill coefficients of 2.4, 1.9, and 1.0 for the wild type RMPFK and the Q200R and Q200E mutants, respectively. The data demonstrate that Gln-200 is a residue important for the cooperative binding of Fru 6-P to RMPFK

Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen

Exposure to protein allergen epicutaneously, inducing a Th2-dominant immune response, sensitizes the host to the development of atopic disease. Antigen-driven bystander effect demonstrates that polarized T cells could instruct naïve T cells to differentiate into T cells with similar phenotype. In this study, we aimed to determine the contribution of antigen-driven bystander effect on epicutaneous sensitization with a newly introduced protein allergen. BALB/c mice were immunized intraperitoneally with BSA emulsified in alum, known to induce a Th2 response, three weeks before given BSA and OVA epicutaneously. Lymph node cells from these mice restimulated with OVA secreted higher levels IL-4, IL-5 and IL-13 as compared with cells from mice without BSA immunization. In addition, BALB/c mice immunized subcutaneously with BSA emulsified in complete Freund's adjuvant, known to induce a Th1-predominant response, also induced higher Th1 as well as Th2 cytokine response when restimulated with OVA as compared with mice without immunization. We demonstrated that subcutaneous immunization with BSA in CFA induced Th2 as well as Th1 response. The threshold of epicutaneous sensitization to OVA was also reduced, possibly due to increased expressions of IL-4 and IL-10 in the draining lymph nodes during the early phase of sensitization. In conclusion, antigen-driven bystander effect, whether it is of Th1- or Th2-predominant nature, can accelerate epicutaneous sensitization by a newly introduced protein allergen. These results provide a possible explanation for mono- to poly-sensitization spread commonly observed in atopic children

Modified empirical fitting of the discharge behavior of LiFePO4_4 batteries under various conditions

A mathematical model is developed by fitting the discharge curve of a new LiFePO$_4$ battery and then used to investigate the relationship between the discharge time and the closed-circuit voltage. This model consists of exponential and polynomial terms where the exponential term dominates the discharge time of a battery and the polynomial term dominates the change in the closed-circuit voltage. Time shift and time scale processes modify the exponential and polynomial terms, respectively, so that the model is suitable for batteries under various conditions. References W. Su, H. Eichi, W. Zeng and M.-Y. Chow, A survey on the electrification of transportation in a smart grid environment, IEEE Intl. Conf. Ind. I. 8:1–10, 2012. doi:10.1109/TII.2011.2172454 J. Wang, Z. Sun and X. Wei, Performance and characteristic research in LiFePO$_4$ battery for electric vehicle applications, IEEE Vehicle Power 1657–1661, 2009. doi:10.1109/VPPC.2009.5289664 A. Shafiei, A. Momeni and S. S. Williamson, Battery modeling approaches and management techniques for plug-in hybrid electric vehicles, IEEE Vehicle Power 1–5, 2011. doi:10.1109/VPPC.2011.6043191 P. Bai, D. A. Cogswell and M. Z. Bazant, Suppression of phase separation in LiFePO$_4$ nanoparticles during battery discharge, Nano Lett. 11:4890–4896, 2011. doi:10.1021/nl202764f H. L. Chan and D. Sutanto, A new battery model for use with battery energy storage systems and electric vehicle power systems, IEEE Power Eng. Soc. 1:470–475, 2000. doi:10.1109/PESW.2000.850009 T. Kim and W. Qiao, A hybrid battery model capable of capturing dynamic circuit characteristics and nonlinear capacity effects, IEEE T. Energy Conver. 26:1172–1180, 2011. doi:10.1109/TEC.2011.2167014 D. N. Rakhmatov and S. B. K. Vrudhula, An analytical high-level battery model for use in energy management of portable electronic systems, IEEE ICCAD 488–493, 2001. doi:10.1109/ICCAD.2001.968687 V. Srinivasan and J. Newman, Discharge model for the lithium iron-phosphate electrode, J. Electrochem. Soc. 151:A1517–A1529, 2004. doi:10.1149/1.1785012 V. Rao, G. Singhal, A. Kumar and N. Navet, Battery model for embedded systems, VLSI Des. 105–110, 2005. doi:10.1109/ICVD.2005.61 S. Dargavillez and T. W. Farrell, Predicting active material utilization in LiFePO$_4$ electrodes using a multiscale mathematical model, J. Electrochem. Soc. 157:A830–A840, 2010. doi:10.1149/1.3425620 R. Rao, S. Vrudhula and D. N. Rakhmatov, Battery modeling for energy-aware system design, Computer 36:77–87, 2003. doi:10.1109/MC.2003.1250886 M. Chen and G. A. Rincon-Mora, Accurate electrical battery model capable of predicting runtime and i-v performance, IEEE T. Energy Conver. 21:504–511, 2006. doi:10.1109/TEC.2006.874229 L. Gao, S. Liu and R. A. Dougal, Dynamic lithium-ion battery model for system simulation, IEEE T. Compon. Pack. T. 25:495–505, 2002. doi:10.1109/TCAPT.2002.803653 V. Agarwal, K. Uthaichana, R. A. DeCarlo and L. H. Tsoukalas, Development and validation of a battery model useful for discharging and charging power control and lifetime estimation, IEEE T. Energy Conver. 25:821–835, 2010. doi:10.1109/TEC.2010.2043106 B. Schweighofer, K. M. Raab and G. Brasseur, Modeling of high power automotive batteries by the use of an automated test system, IEEE T. Instrum. Meas. 52:1087–1091, 2003. doi:10.1109/TIM.2003.81482

Association between metabolic body composition status and vitamin D deficiency: A cross-sectional study

This study aimed to investigate the risk of vitamin D deficiency in a relatively healthy Asian population, with (i) metabolically healthy normal weight (MHNW) (homeostasis model assessment-insulin resistance [HOMA-IR] < 2. 5 without metabolic syndrome [MS], body mass index [BMI] < 25), (ii) metabolically healthy obesity (MHO) (HOMA-IR < 2.5, without MS, BMI ≥ 25), (iii) metabolically unhealthy normal weight (MUNW) (HOMA-IR ≥ 2.5, or with MS, BMI < 25), and (iv) metabolically unhealthy obesity (MUO) (HOMA-IR ≥ 2.5, or with MS, BMI ≥ 25) stratified by age and sex. This cross-sectional study involved 6,655 participants aged ≥ 18 years who underwent health checkups between 2013 and 2016 at the Chang Gung Memorial Hospital. Cardiometabolic and inflammatory markers including anthropometric variables, glycemic indices, lipid profiles, high-sensitivity C-reactive protein (hs-CRP), and serum 25-hydroxy vitamin D levels, were retrospectively investigated. Compared to the MHNW group, the MHO group showed a higher odds ratio (OR) [1.35, 95% confidence interval (CI) 1.05–1.73] for vitamin D deficiency in men aged < 50 years. By contrast, in men aged > 50 years, the risk of vitamin D deficiency was higher in the MUO group (OR 1.44, 95% CI 1.05–1.97). Among women aged < and ≥ 50 years, the MUO group demonstrated the highest risk for vitamin D deficiency, OR 2.33 vs. 1.54, respectively. Our study revealed that in women of all ages and men aged > 50 years, MUO is associated with vitamin D deficiency and elevated levels of metabolic biomarkers. Among men aged < 50 years, MHO had the highest OR for vitamin D deficiency

A delta-doped quantum well system with additional modulation doping

A delta-doped quantum well with additional modulation doping may have potential applications. Utilizing such a hybrid system, it is possible to experimentally realize an extremely high two-dimensional electron gas (2DEG) density without suffering inter-electronic-subband scattering. In this article, the authors report on transport measurements on a delta-doped quantum well system with extra modulation doping. We have observed a 0-10 direct insulator-quantum Hall (I-QH) transition where the numbers 0 and 10 correspond to the insulator and Landau level filling factor ν = 10 QH state, respectively. In situ titled-magnetic field measurements reveal that the observed direct I-QH transition depends on the magnetic component perpendicular to the quantum well, and the electron system within this structure is 2D in nature. Furthermore, transport measurements on the 2DEG of this study show that carrier density, resistance and mobility are approximately temperature (T)-independent over a wide range of T. Such results could be an advantage for applications in T-insensitive devices

T Lymphocytes Amplify the Anabolic Activity of Parathyroid Hormone through Wnt10b Signaling

SummaryIntermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism

Verapamil protects dopaminergic neuron damage through a novel anti-inflammatory mechanism by inhibition of microglial activation

Verapamil has been shown to be neuroprotective in several acute neurotoxicity models due to blockade of calcium entry into neurons. However, the potential use of verapamil to treat chronic neurodegenerative diseases has not been reported. Using rat primary mesencephalic neuron/glia cultures, we report that verapamil significantly inhibited LPS-induced dopaminergic neurotoxicity in both pre- and post-treatment experiments. Reconstituted culture studies revealed that the presence of microglia was essential in verapamil-elicited neuroprotection. Mechanistic studies showed that decreased production of inflammatory mediators from LPS-stimulated microglia underlay neuroprotective property of verapamil. Further studies demonstrated that microglial NADPH oxidase (PHOX), the key superoxide-producing enzyme, but not calcium channel in neurons, is the site of action for the neuroprotective effect of verapamil. This conclusion was supported by the following two observations: 1) Verapamil failed to show protective effect on LPS-induced dopaminergic neurotoxicity in PHOX-deficient (deficient in the catalytic subunit of gp91phox) neuron/glia cultures; 2) Ligand binding studies showed that the binding of [3H]Verapamil onto gp91phox transfected COS-7 cell membranes was higher than the non-transfected control. The calcium channel-independent neuroprotective property of verapamil was further supported by the finding that R(+)-verapamil, a less active form in blocking calcium channel, showed the same potency in neuroprotection, inhibition of pro-inflammatory factors production and binding capacity to gp91phox membranes as R(-)-verapamil, the active isomer of calcium channel blocker. In conclusion, our results demonstrate a new indication of verapamil-mediated neuroprotection through a calcium channel-independent pathway and provide a valuable avenue for the development of therapy for inflammation-related neurodegenerative diseases

Automatic segmentation of meningioma from non-contrasted brain MRI integrating fuzzy clustering and region growing

<p>Abstract</p> <p>Background</p> <p>In recent years, magnetic resonance imaging (MRI) has become important in brain tumor diagnosis. Using this modality, physicians can locate specific pathologies by analyzing differences in tissue character presented in different types of MR images.</p> <p>This paper uses an algorithm integrating fuzzy-c-mean (FCM) and region growing techniques for automated tumor image segmentation from patients with menigioma. Only non-contrasted T1 and T2 -weighted MR images are included in the analysis. The study's aims are to correctly locate tumors in the images, and to detect those situated in the midline position of the brain.</p> <p>Methods</p> <p>The study used non-contrasted T1- and T2-weighted MR images from 29 patients with menigioma. After FCM clustering, 32 groups of images from each patient group were put through the region-growing procedure for pixels aggregation. Later, using knowledge-based information, the system selected tumor-containing images from these groups and merged them into one tumor image. An alternative semi-supervised method was added at this stage for comparison with the automatic method. Finally, the tumor image was optimized by a morphology operator. Results from automatic segmentation were compared to the "ground truth" (GT) on a pixel level. Overall data were then evaluated using a quantified system.</p> <p>Results</p> <p>The quantified parameters, including the "percent match" (PM) and "correlation ratio" (CR), suggested a high match between GT and the present study's system, as well as a fair level of correspondence. The results were compatible with those from other related studies. The system successfully detected all of the tumors situated at the midline of brain.</p> <p>Six cases failed in the automatic group. One also failed in the semi-supervised alternative. The remaining five cases presented noticeable edema inside the brain. In the 23 successful cases, the PM and CR values in the two groups were highly related.</p> <p>Conclusions</p> <p>Results indicated that, even when using only two sets of non-contrasted MR images, the system is a reliable and efficient method of brain-tumor detection. With further development the system demonstrates high potential for practical clinical use.</p