Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified

Method for replica-plating Neurospora

Method for replica-plating Neurospor

Isotope discrimination by the stearoyl desaturase of Neurospora crassa

In wild type Neurospora crassa, we previously observed an unexpectedly low proportion of label in unsaturated fatty acids that should have been derived from deuterated palmitate (16:0). Interpretation of whether the position of deuteration could account for these results was inconclusive, because wild type had a low proportion of total fatty acids derived from the deuterated supplement. The N. crassa cel-1 strain is ideally suited for studies of fatty acid enrichment, because cel-1 must obtain its fatty acids exogenously. We supplemented cel-1 with 16:0\u27s deuterated at different positions. With each of the labelled 16:0\u27s, cel-1 desaturated the stearate (18:0) derived from the 16:0 more efficiently than wild type. However, when the 16:0 was deuterated at the site of future desaturation, we indeed observed effects on fatty acid composition and label distribution consistent with inhibition of the 18:0 desaturase

A systematic survey of the fatty acid composition of Neurospora strains

Numerous studies have examined aspects of lipid metabolism in Neurospora crassa, N. tetrasperma and N. sitophila, but little systematic comparison has been done on lipids of the different species. Most obviously missing, however, is comparison of the fatty acid composition of lipids among Neurospora species. Fatty acid composition (especially the production of particular polyunsaturated fatty acids) is often a key factor in genus or species identification, particularly in bacteria, but also in other fungi such as Aspergillus, Penicillium and Mortierella (Kock and Botha 1998 In Frisvad et al. (eds), Chemical Fungal Taxonomy, Marcel Dekker, NY, p. 219-246). Until this study, reports of the fatty acid composition of Neurospora lipids have been limited to a few laboratory strains of N. crassa

An all-ZnO microbolometer for infrared imaging

Cataloged from PDF version of article.Microbolometers are extensively used for uncooled infrared imaging applications. These imaging units generally employ vanadium oxide or amorphous silicon as the active layer and silicon nitride as the absorber layer. However, using different materials for active and absorber layers increases the fabrication and integration complexity of the pixel structure. In order to reduce fabrication steps and therefore increase the yield and reduce the cost of the imaging arrays, a single layer can be employed both as the absorber and the active material. In this paper, we propose an all-ZnO microbolometer, where atomic layer deposition grown zinc oxide is employed both as the absorber and the active material. Optical constants of ZnO are measured and fed into finite-difference-time-domain simulations where absorption performances of microbolometers with different gap size and ZnO film thicknesses are extracted. Using the results of these optical simulations, thermal simulations are conducted using finite-element-method in order to extract the noise equivalent temperature difference (NETD) and thermal time constant values of several bolometer structures with different gap sizes, arm and film thicknesses. It is shown that the maximum performance of 171 mK can be achieved with a body thickness of 1.1 μm and arm thickness of 50 nm, while the fastest response with a time constant of 0.32 ms can be achieved with a ZnO thickness of 150 nm both in arms and body. © 2014 Elsevier B.V. All rights reserved

Switching from an Induced-Fit to a Lock-and-Key Mechanism in an Aminoacyl-tRNA Synthetase with Modified Specificity

Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced-fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 Å resolution) to reveal the structural basis for the altered specificity. The mutations result in both the loss of important contacts with Met and the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced-fit mechanism to a lock-and-key one, thereby enhancing its catalytic efficiency

Accidental swallowing of partial denture: a case report

We describe a 42-year-old age woman who accidentally swallowed her lower denture, which was composed of eleven teeth. The daily descent of the denture was followed by plain abdominal radiography and physical examination. The image was localized at the left upper quadrant on admission day, but it stopped on its way at the right lower quadrant on day two and three. Since the patient's complaints increased we planned surgical removal of the denture. In this report, we had discussed the diagnosis, follow up and treatment options of swallowed partial denture with current literature review

A joint replenishment policy with individual control and constant size orders

We consider inventory systems with multiple items under stochastic demand and jointly incurred order setup costs. The problem is to determine the replenishment policy that minimises the total expected ordering, inventory holding, and backordering costs-the so-called stochastic joint replenishment problem. In particular, we study the settings in which order setup costs reflect the transportation costs and have a step-wise cost structure, each step corresponding to an additional transportation vehicle. For this setting, we propose a new policy that we call the (s, Q) policy, under which a replenishment order of constant size Q is triggered whenever the inventory position of one of the items drops to its reorder point s. The replenishment order is allocated to multiple items so that the inventory positions are equalised as much as possible. The policy is designed for settings in which backorder and setup costs are high, as it allows the items to independently trigger replenishment orders and fully exploits the economies of scale by consistently ordering the same quantity. A numerical study is conducted to show that the proposed (s, Q) policy outperforms the well-known (Q, S) policy when backorder costs are high and lead times are small. © 2010 Taylor & Francis

In-situ Reinforcement Processing for Laser Powder Bed Fused Ti64 Parts

The objective of this study was to investigate how the microstructure and mechanical properties of Ti-6Al-4V samples, fabricated using laser powder bed fusion (L-PBF), change when a predefined local double melting strategy is employed within each layer of the manufacturing process. The analysis primarily focused on evaluating microstructural aspects, defects, and grain size, along with the mechanical properties, specifically the Vickers hardness at various positions within the samples. The findings indicated that the integration of the predefined locally double melting scan in each layer had a significant influence on the microstructure, resulting in variations in grain size across different locations, as well as hardness values with variations of up to 10% across different areas. Moreover, these discoveries underscore the potential of employing the predefined locally double melting strategy in each layer to create fabricated components with distinctive behaviors, like composites, which could find applications in the aerospace industry.Mechanical Engineerin

Cell-selective metabolic labeling of proteins

Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected