The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1

Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.     

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.     

The TERESA project: from space research to ground tele-echography

Ultrasound examinations represent one of the major diagnostic modalities of future healthcare. They are currently used to support medical space research but require a high skilled operator for both probe positioning on the patient's skin and image interpretation. TERESA is a tele-echography project that proposes a solution to bring astronauts and remotely located patients on ground quality ultrasound examinations despite the lack of a specialist at the location of the wanted medical act.     

Dynamic control and conventional strength ratios of the quadriceps and hamstrings in subjects with anterior cruciate ligament deficiency

The hamstrings:quadriceps muscle strength ratio has been used as an indicator of normal balance between the knee flexors and extensors. A more functional approach to this strength ratio would be to compare opposite muscle actions of antagonistic muscle groups. The dynamic strength control ratio (DSCR) should give a more appropriate measure relating to knee function. There is a lack of normative data relating to DSCR for anterior cruciate ligament (ACL) deficient subjects. Effects of ACL deficiency on isokinetic peak torque for eccentric and concentric muscle actions of the quadriceps and hamstrings, in conjunction with isometric peak torque, were examined in 10 patients awaiting reconstructive surgery (male = 8, female = 2 ; age = 32.8 +/- 8.3 years; height = 1.77 +/- 0.08 m; mass = 72.1 +/- 12.5 kg). These variables were assessed using an isokinetic dynamometer. The results were considered in terms of the conventional ratio and DSCR. Anterior tibial drawer was measured using a knee ligament arthrometer to confirm clinical diagnosis of ACL rupture. The isokinetic peak torque data analysed were for angular velocities of 1.05 rad s(-1) (60 degrees s(-1)). Significant strength deficits were apparent between normal and injured sides for: concentric isokinetic quadriceps action (p < 0.05); isometric quadriceps action at 70 degrees of knee flexion (p < 0.05); isometric quadriceps action at 40 degrees of knee flexion (p<0.01); eccentric isokinetic hamstrings action (p < 0.05). With bilateral comparison, the conventional strength ratios showed no significant difference, as did the DSCR. The bilateral comparison of isometric strength ratios revealed significant losses in quadriceps strength for the injured side (p < 0.05) but no significant losses in hamstring strength (p > 0.05). Thus, differences can be seen in conventional ratios and DSCR for ACL-deficient subjects. This is an area of clinical interest with the increasing frequency of ACL reconstruction using hamstrings tendons.     

Registration of 3D Points Using Geometric Algebra and Tensor Voting

We address the problem of finding the correspondences of two point sets in 3D undergoing a rigid transformation. Using these correspondences the motion between the two sets can be computed to perform registration. Our approach is based on the analysis of the rigid motion equations as expressed in the Geometric Algebra framework. Through this analysis it was apparent that this problem could be cast into a problem of finding a certain 3D plane in a different space that satisfies certain geometric constraints. In order to find this plane in a robust way, the Tensor Voting methodology was used. Unlike other common algorithms for point registration (like the Iterated Closest Points algorithm), ours does not require an initialization, works equally well with small and large transformations, it cannot be trapped in “local minima” and works even in the presence of large amounts of outliers. We also show that this algorithm is easily extended to account for multiple motions and certain non-rigid or elastic transformations.     

Evaluation of D（d,n）^3 He reaction neutron source models for BNCT irradiation system design

A mathematical method was developed to calculatc the yield.energy spectrum and angular distribution of neutrons from D(d,n)3 He(D-D)reaction in a thick deuterium-titanium target for incident deuterons in energies lower than 1.0MeV.The data of energy spectrum and angular distribution wefe applied to set up the neutron source model for the beam-shaping-assembly(BSA)design of Boron-Neutron-Capture-Therapy(BNCT)using MCNP-4C code.Three cases of D-D neutron source corresponding to incident deuteron energy of 1000.400 and 150 kaV were investigated.The neutron beam characteristics were compared with the model of a 2.45 MeV mono-energetic and isotropic neutron source using an example BSA designed for BNCT irradiation.The results show significant differences in the neutron beam characteristics,particularly the fast neutron component and fast neutron dose in air,between the non-isotropic neutron source model and the 2.5 MeV mono-energetic and isotropic neutron source model.     

A Tunable-MMI-Coupler-Based Wavelength Adjustable Laser

This paper introduces a novel integrated widely tunable laser, the tunable multimode interference (T-MMI) laser, with as tunable component an MMI coupler with a wavelength adjustable transmission spectrum. Experiments demonstrate up to 150 nm of tuning range for the T-MMI component and operation of a widely tunable T-MMI laser is demonstrated over a wavelength range of 38 nm.     

Attenuated total internal reflection infrared microscopy of multilayer plastic packaging foils

Multilayer plastic foils are important packaging materials that are used to extend the shelf life of food products and drinks. Fourier transform infrared (FT-IR) spectroscopic imaging using attenuated total internal reflection (ATR) can be used for the identification and localization of different layers in multilayer foils. A new type of ATR crystal was used in combination with a linear array detector through which large sample areas (400 x 400 microm(2)) could be imaged with a pixel size of 1.6 microm. The method was tested on laminated plastic packing materials containing 5 to 12 layers. The results of the identification of the different materials using ATR-FT-IR were compared with differential scanning calorimetry (DSC) and the layer thickness of the individual layers measured by ATR-FT-IR was compared with polarized light microscopy (LM) and scanning electron microscopy (SEM). It has been demonstrated that individual layers with a thickness of about 3 microm could be identified in multilayer foils with a total thickness ranging from 100 to 150 microm. The results show a spatial resolution of about 4 microm (measured at wavenumbers ranging from 1000 to 1730 cm(-1)), which is about a factor of two better than can be obtained using transmission FT-IR imaging. An additional advantage of ATR is the ease of sample preparation. A good correspondence was found between visible and FT-IR images. The results of ATR-FT-IR imaging were in agreement with those obtained by LM, SEM, and DSC. ATR-FT-IR is superior to the combination of these techniques because it delivers both spatial and chemical information.     

The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

Abdominal aortic aneurysm (AAA) is a complex disorder that has a significant impact on the aging population. While both genetic and environmental risk factors have been implicated in AAA formation, the precise genetic markers involved and the factors influencing their expression remain an area of ongoing investigation. DNA methylation has been previously used to study gene silencing in other inflammatory disorders and since AAA has an extensive inflammatory component, we sought to examine the genome-wide DNA methylation profiles in mononuclear blood cells of AAA cases and matched non-AAA controls. To this end, we collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers (n = 11), AAA non-smokers (n = 9), control smokers (n = 10) and control non-smokers (n = 11). Methylation data were obtained using the Illumina 450k Human Methylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9), and adenylate cyclase 10 pseudogene 1 (ADCY10P1). Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9. These findings are novel and suggest DNA methylation may play a role in AAA pathobiology.