Methylotrophic yeasts as factories for the production of foreign proteins

In this contribution we discuss the potential of methylotrophic yeasts as hosts for the high level production of valuable foreign proteins. Recent relevant achievements on the intracellular production or secretion of proteins are summarized. Special attention is paid to a specific advantage of the use of methylotrophic yeasts, namely the possibility of accumulating the foreign gene products inside peroxisomes. This approach may be of major advantage when the protein product is toxic for the host cell and, also, to protect these proteins these proteins from undesired side-effects such as proteolysis or aggregation.     

Proinflammatory Cytokines Perturb Mouse and Human Pancreatic Islet Circadian Rhythmicity and Induce Uncoordinated β-Cell Clock Gene Expression via Nitric Oxide,Lysine Deacetylases,and Immunoproteasomal Activity

Pancreatic β-cell-specific clock knockout mice develop β-cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine interleukin-1β (IL-1β) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1β and interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/β agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast, low (<5,0 μM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1β mediated β-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3, and immunoproteasome activity.     

Temporal change analysis for characterization of mass lesions in mammography

In this paper, we present a fully automated computer-aided diagnosis (CAD) program to detect temporal changes in mammographic masses between two consecutive screening rounds. The goal of this work was to improve the characterization of mass lesions by adding information about the tumor behavior over time. Towards this goal we previously developed a regional registration technique that finds for each mass lesion on the current view a location on the prior view where the mass was most likely to develop. For the task of interval change analysis, we designed two kinds of temporal features: difference features and similarity features. Difference features indicate the (relative) change in feature values determined on prior and current views. These features may be especially useful for lesions that are visible on both views. Similarity features measure whether two regions are comparable in appearance and may be useful for lesions that are visible on the prior view as well as for newly developing lesions. We evaluated the classification performance with and without the use of temporal features on a dataset consisting of 465 temporal mammogram pairs, 238 benign, and 227 malignant. We used cross validation to partition the dataset into a training set and a test set. The training set was used to train a support vector machine classifier and the test set to evaluate the classifier. The average A(z) value (area under the receiver operating characteristic curve) for classifying each lesion was 0.74 without temporal features and 0.77 with the use of temporal features. The improvement obtained by adding temporal features was statistically significant (P = 0.005). In particular, similarity features contributed to this improvement. Furthermore, we found that the improvement was comparable for masses that were visible and for masses that were not visible on the prior view. These results show that the use of temporal features is an effective approach to improve the characterization of masses.     

Biocathodic nitrous oxide removal in bioelectrochemical systems

Anthropogenic nitrous oxide (N(2)O) emissions represent up to 40% of the global N(2)O emission and are constantly increasing. Mitigation of these emissions is warranted since N(2)O is a strong greenhouse gas and important ozone-depleting compound. Until now, only physicochemical technologies have been applied to mitigate point sources of N(2)O, and no biological treatment technology has been developed so far. In this study, a bioelectrochemical system (BES) with an autotrophic denitrifying biocathode was considered for the removal of N(2)O. The high N(2)O removal rates obtained ranged between 0.76 and 1.83 kg N m(-3) net cathodic compartment (NCC) d(-1) and were proportional to the current production, resulting in cathodic coulombic efficiencies near 100%. Furthermore, our experiments suggested the active involvement of microorganisms as the catalyst for the reduction of N(2)O to N(2), and the optimal cathode potential ranged from -200 to 0 mV vs standard hydrogen electrode (SHE) in order to obtain high conversion rates. Successful operation of the system for more than 115 days with N(2)O as the sole cathodic electron acceptor strongly indicated that N(2)O respiration yielded enough energy to maintain the biological process. To our knowledge, this study provides for the first time proof of concept of biocathodic N(2)O removal at long-term without the need for high temperatures and expensive catalysts.     

Femtosecond laser-inscribed fiber Bragg gratings for strain monitoring in power cables of offshore wind turbines

A fiber Bragg grating sensor system used for monitoring the effects of strain on the power cable of an offshore wind turbine is presented. The Bragg grating structure was inscribed into coated nonphotosensitive standard telecommunication fibers using an IR femtosecond laser and the point-by-point writing technique. Because of the presence of the protective coating of the fiber, the mechanical stability of the resultant sensor device is better than that of a sensor consisting of a bare fiber. A system containing this sensing element was to our knowledge for the first time successfully installed and tested in an offshore wind turbine prototype (REpower 6M, REpower Systems, AG, Germany) in February 2010, near Ellh?ft (Germany). The fabrication process of the fiber Bragg gratings, measurement results of the online monitoring, and a comparison between the sensor signal and commonly used sensing techniques are presented.     

Dual-pulse frequency compounded superharmonic imaging

Tissue second-harmonic imaging is currently the default mode in commercial diagnostic ultrasound systems. A new modality, superharmonic imaging (SHI), combines the third through fifth harmonics originating from nonlinear wave propagation through tissue. SHI could further improve the resolution and quality of echographic images. The superharmonics have gaps between the harmonics because the transducer has a limited bandwidth of about 70% to 80%. This causes ghost reflection artifacts in the superharmonic echo image. In this work, a new dual-pulse frequency compounding (DPFC) method to eliminate these artifacts is introduced. In the DPFC SHI method, each trace is constructed by summing two firings with slightly different center frequencies. The feasibility of the method was established using a single-element transducer. Its acoustic field was modeled in KZK simulations and compared with the corresponding measurements obtained with a hydrophone apparatus. Subsequently, the method was implemented on and optimized for a setup consisting of an interleaved phased-array transducer (44 elements at 1 MHz and 44 elements at 3.7 MHz, optimized for echocardiography) and a programmable ultrasound system. DPFC SHI effectively suppresses the ghost reflection artifacts associated with imaging using multiple harmonics. Moreover, compared with the single-pulse third harmonic, DPFC SHI improved the axial resolution by 3.1 and 1.6 times at the -6-dB and -20-dB levels, respectively. Hence, DPFC offers the possibility of generating harmonic images of a higher quality at a cost of a moderate frame rate reduction.     

Morphological, structural, optical and dielectric properties of 91SiO2:4Li2O:4Nb2O5:1Dy2O3 (% mole) glass prepared by sol–gel

We report on the structural, morphological and optical characterization of glass and glass–ceramic samples, with nominal composition of 91SiO₂:4Li₂O:4Nb₂O₅:1Dy₂O₃ (% mole). The samples were heated treated in air in the temperature range between 500 °C and 800 °C. The glass–ceramics samples start to crystallize at temperatures above 600 °C and besides the SiO₂ and LiNbO₃ crystalline phases, sub micron DyNbO₄ precipitates were identified by X-ray diffraction and scanning electron microscopy for temperatures above 750 °C. Using Raman spectroscopy was possible to identify the vibration modes assigned to the crystalline phases detected by the XRD analysis. Also, the performed Photoluminescence Spectroscopy analysis suggest that the optical activation of Dy³⁺ ions in the glass and glass–ceramics hosts was found to be dependent of the heat treatment temperature. Intraconfigurational transitions between the ⁴F_9/2 and ⁶H_{J(=15/2, 13/2)} multiplets of Dy³⁺ ions were only observed for samples without the presence of the DyNbO₄ crystalline phase. Moreover, for samples heat treated between 500 °C and 700 °C the intensity ratio of the ⁴F_9/2⁶H_13/2 and ⁴F_9/2⁶H_15/2 transitions of the lanthanide ion changes, suggesting different ion environments which are dependent on the thermal annealing temperature. The dielectric measurements revealed that ε′ value depends mainly in the number of network modifier ions and the relaxation phenomenon observed at frequencies above 10 kHz, in all samples, can be assigned to the increase of the volume ratio between the LiNbO₃ particles and the glass network.     

Speciation-dependent microbial reduction of uranium within iron-coated sands

Transport of uranium within surface and subsurface environments is predicated largely on its redox state. Uranyl reduction may transpire through either biotic (enzymatic) or abiotic pathways; in either case, reduction of U(VI) to U(IV) results in the formation of sparingly soluble UO2 precipitates. Biological reduction of U(VI), while demonstrated as prolific under both laboratory and field conditions, is influenced by competing electron acceptors (such as nitrate, manganese oxides, or iron oxides) and uranyl speciation. Formation of Ca-UO2-CO3 ternary complexes, often the predominate uranyl species in carbonate-bearing soils and sediments, decreases the rate of dissimilatory U(VI) reduction. The combined influence of uranyl speciation within a mineralogical matrix comparable to natural environments and under hydrodynamic conditions, however, remains unresolved. We therefore examined uranyl reduction by Shewanella putrefaciens within packed mineral columns of ferrihydrite-coated quartz sand under conditions conducive or nonconducive to Ca-UO2-CO3 species formation. The results are dramatic. In the absence of Ca, where uranyl carbonato complexes dominate, U(VI) reduction transpires and consumes all of the U(VI) within the influent solution (0.166 mM) over the first 2.5 cm of the flow field for the entirety of the 54 d experiment. Over 2 g of U is deposited during this reaction period, and despite ferrihydrite being a competitive electron acceptor, uranium reduction appears unabated for the duration of our experiments. By contrast, in columns with 4 mM Ca in the influent solution (0.166 mM uranyl), reduction (enzymatic or surface-bound Fe(III) mediated) appears absent and breakthrough occurs within 18 d (at a flow rate of 3 pore volumes per day). Uranyl speciation, and in particular the formation of ternary Ca-UO2-CO3 complexes, has a profound impact on U(VI) reduction and thus transport within anaerobic systems.