Precursor-directed biosynthesis of epothilone in Escherichia coli

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.     

Integration of self-consistent polycrystal plasticity with dislocation density based hardening laws within an implicit finite element framework: Application to low-symmetry metals

We present an implementation of the viscoplastic self-consistent (VPSC) polycrystalline model in an implicit finite element (FE) framework, which accounts for a dislocation-based hardening law for multiple slip and twinning modes at the micro-scale grain level. The model is applied to simulate the macro-scale mechanical response of a highly anisotropic low-symmetry (orthorhombic) crystal structure. In this approach, a finite element integration point represents a polycrystalline material point and the meso-scale mechanical response is obtained by the mean-field VPSC homogenization scheme. We demonstrate the accuracy of the FE-VPSC model by analyzing the mechanical response and microstructure evolution of α-uranium samples under simple compression/tension and four-point bending tests. Predictions of the FE-VPSC simulations compare favorably with experimental measurements of geometrical changes and microstructure evolution. Specifically, the model captures accurately the tension–compression asymmetry of the material associated with twinning, as well as the rigidity of the material response along the hard-to-deform crystallographic orientations.     

Magnetic Relaxometry with an Atomic Magnetometer and SQUID Sensors on Targeted Cancer Cells

Magnetic relaxometry methods have been shown to be very sensitive in detecting cancer cells and other targeted diseases. Superconducting Quantum Interference Device (SQUID) sensors are one of the primary sensor systems used in this methodology because of their high sensitivity with demonstrated capabilities of detecting fewer than 100,000 magnetically-labeled cancer cells. The emerging technology of atomic magnetometers (AM) represents a new detection method for magnetic relaxometry with high sensitivity and without the requirement for cryogens. We report here on a study of magnetic relaxometry using both AM and SQUID sensors to detect cancer cells that are coated with superparamagnetic nanoparticles through antibody targeting. The AM studies conform closely to SQUID sensor results in the measurement of the magnetic decay characteristics following a magnetization pulse. The AM and SQUID sensor data are well described theoretically for superparamagnetic particles bound to cells and the results can be used to determine the number of cells in a cell culture or tumor. The observed fields and magnetic moments of cancer cells are linear with the number of cells over a very large range. The AM sensor demonstrates very high sensitivity for detecting magnetically labeled cells does not require cryogenic cooling and is relatively inexpensive.     

Titanium‐Mediated Synthesis of Spirocyclic NH‐Azetidines from Oxime Ethers

The Ti^IV‐mediated synthesis of spirocyclic NH‐azetidines from oxime ethers using either an alkyl Grignard reagent or terminal olefin ligand exchange coupling partner is described. Through a proposed Kulinkovich‐type mechanism, a titanacyclopropane intermediate forms and serves as a 1,2‐aliphatic dianion equivalent, inserting into the 1,2‐dielectrophilc oxime ether to ultimately give rise to the desired N‐heterocyclic four‐membered ring. This transformation proceeds in moderate yield to furnish previously unreported and structurally diverse NH‐azetidines in a single step.     

Effect of Feed Strategy on Methane Production and Performance of an AnSBBR Treating Effluent from Biodiesel Production

The aim of this work was to investigate the effect of different feeding times (2, 4 and 6 h) and applied volumetric organic loads (4.5, 6.0 and 7.5 gCOD L⁻¹ day⁻¹) on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) treating effluent from biodiesel production. Polyurethane foam cubes were used as inert support in the reactor, and mixing was accomplished by recirculating the liquid phase. The effect of feeding time on reactor performance showed to be more pronounced at higher values of applied volumetric organic loads (AVOLs). Highest organic material removal efficiencies achieved at AVOL of 4.5 gCOD L⁻¹ day⁻¹ were 87 % at 4-h feeding against 84 % at 2-h and 6-h feeding. At AVOL of 6.0 gCOD L⁻¹ day⁻¹, highest organic material removal efficiencies achieved with 4-h and 6-h feeding were 84 %, against 71 % at 2-h feeding. At AVOL of 7.5 gCOD L⁻¹ day⁻¹, organic material removal efficiency achieved with 4-h feeding was 77 %. Hence, longer feeding times favored minimization of total volatile acids concentration during the cycle as well as in the effluent, guaranteeing process stability and safety.     

Viability of Using Glycerin as a Co-substrate in Anaerobic Digestion of Sugarcane Stillage (Vinasse): Effect of Diversified Operational Strategies

Vinasse, from sugar and ethanol production, stands out as one of the most problematic agroindustry wastes due to its high chemical oxygen demand, large production volume, and recalcitrant compounds. Therefore, the viability of using glycerin as a co-substrate in vinasse anaerobic digestion was tested, to increase process efficiency and biogas productivity. The effect of feeding strategy, influent concentration, cycle length, and temperature were assessed to optimize methane production. Glycerin (1.53% v/v) proved to be a good co-substrate since it increased the overall methane production in co-digestion assays. CH₄ productivity enhanced exponentially as influent concentration increased, but when temperature was increased to 35 °C, biogas production was impaired. The highest methane productivity and yield were achieved using fed-batch mode, at 30 °C and at an organic loading rate of 10.1 kg COD m⁻³ day⁻¹: 139.32 mol CH₄ m⁻³ day⁻¹, 13.86 mol CH₄ kg COD_applied, and 15.30 mol CH₄ kg COD_removed. Methane was predominantly produced through the hydrogenotrophic route. In order to treat all the vinasse produced by a mid-size sugar and ethanol plant, nine reactors with 7263.4 m³ each would be needed. The energy generated by burning the biogas in boilers would reach approximately 92,000 MW h per season and could save up to US$ 240,000.00 per month in diesel oil demand.

     

Estimation of the Kolmogorov constant by large-eddy simulation in the stable PBL

In this paper is evaluated the inertial subrange Kolmogorov constant C₀ in a stable boundary-layer. The importance of the constant C₀ is well known as predictions of turbulent dispersion by means of Lagrangian stochastic models depend upon its value. Different values of the C₀ constant has been proposed along the years, most of them determined at low Reynolds number and/or under different techniques. Here we estimate the constant C₀ by tracking an ensemble of Lagrangian particles injected in a stable planetary boundary-layer simulated with a large-eddy simulation model and analyzing the ensemble-averaged Lagrangian velocity structure function in the inertial subrange. Our estimative of C_0,w is 3.7, which is consistent with values found in literature. The evaluation of C_0,u and C_0,v cannot be easily accomplished since it is difficult to identify an inertial subrange for the wind field horizontal components.     

Electronic structure of amorphous materials under laser irradiation

The electronic structure of amorphous materials under laser irradiation is investigated in the one-electron approximation. It is shown that in this approximation the modification of the density-of-states is much less important than in the crystal case. Many-electron effects are briefly discussed.     

Use of a SQUID array to detect T-cells with magnetic nanoparticles in determining transplant rejection

Acute rejection in organ transplant is signaled by the proliferation of T-cells that target and kill the donor cells requiring painful biopsies to detect rejection onset. An alternative non-invasive technique is proposed using a multi-channel superconducting quantum interference device (SQUID) magnetometer to detect T-cell lymphocytes in the transplanted organ labeled with magnetic nanoparticles conjugated to antibodies specifically attached to lymphocytic ligand receptors. After a magnetic field pulse, the T-cells produce a decaying magnetic signal with a characteristic time of the order of a second. The extreme sensitivity of this technique, 10(5) cells, can provide early warning of impending transplant rejection and monitor immune-suppressive chemotherapy.     

Mini-Tensile Experiments of Clock-Rolled Zirconium Plate

We present our efforts to measure the tensile strength of clock-rolled pure zirconium in the through-thickness (TT) direction of the plate. Although the plate is too thin to produce standard ASTM tensile samples in the TT orientation, such measurements are relevant to benchmarking our constitutive models of hardening and texture evolution. We have designed a fixture and sample to perform tensile tests on our 9 mm thick plate. The sample is a double-ligament mini-tensile sample: 8 × 8 × 1 mm overall; each ligament has a gage section of 1 × 1 × 3 mm. In contrast, our standard “macro” tensile sample is a flat dogbone with a gage section of 3 × 1.5 × 25 mm. We validate our design by comparing the results of mechanical tests performed on samples of both geometries. Although the hardening response is nearly identical, the flow stress of the miniature samples is offset by +25 MPa at the onset of plastic yield. We present our efforts to resolve the origin of this offset.