Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.     

A novel diagnostic technique to determine uterine health of Holstein cows at 35 days postpartum

The objectives were (1) to evaluate the association of uterine lavage sample optical density (ULSOD) with uterine health, and (2) to estimate and evaluate a threshold value that will maximize the accuracy of ULSOD as a diagnostic tool for clinical endometritis. The study enrolled 1,742 cows from 3 dairy farms located near Ithaca, New York. The samples were collected at 35 ± 3 d in milk (DIM) by using low-volume uterine lavage. Cows with a purulent or mucopurulent secretion in the sample were diagnosed with clinical endometritis, whereas a subgroup of all studied cows was examined for cytological evidence of inflammation by endometrial cytology. Data for ULSOD measured at different wavelengths (200, 352, 620, 790, 860, and 960 nm) were available for 554 cows; all 1,742 cows had data for ULSOD measured at 620 nm (ULSOD(620)). Incidences of clinical endometritis, metritis, and retained placenta were 10, 15.2, and 5.6%, respectively. The ULSOD(620) was associated with clinical endometritis. Receiver operating characteristic (ROC) analysis of the accuracy of optical density in the detection of clinical endometritis was conducted for ULSOD measured at different wavelengths; ULSOD(620) was selected for further analysis because it presented the best ROC curve to detect clinical endometritis. The recommended threshold for ULSOD(620) ROC was 0.058, where the sensitivity and specificity were 76.3 and 78.3%, respectively. The ROC analysis of the accuracy of optical density in the detection of endometritis defined as a percentage of neutrophils in the uterine lavage samples higher than 18% was conducted for ULSOD(620). The recommended threshold was 0.059, where the sensitivity and specificity were 100 and 82.2%, respectively. Cows with ULSOD(620) ≤ 0.058 were 1.21 times more likely to conceive than cows with ULSOD(620) >0.058; moreover, the median calving-to-conception interval for cows that had ULSOD(620) ≤ 0.058 was 122 d compared with 148 d for cows that had ULSOD(620) >0.058. Cows that were positive for Arcanobacterium pyogenes, diagnosed with metritis, or had retained placenta had 4.0, 1.4, and 1.7 times higher odds of having ULSOD(620) >0.058, respectively. Cows with ULSOD(620) >0.058 had a higher percentage of neutrophils in the uterine lavage samples than cows with ULSOD(620) ≤ 0.058. Uterine lavage sample optical density measured at 620 nm can be used as an objective indicator of uterine health in dairy cows, principally for clinical endometritis.     

Impact of frozen storage on polyacetylene content, texture and colour in carrots disks

This study investigated the effect of freezing method (slow or blast freezing) with or without blanching during storage at −20 °C on the levels of three polyacetylenes, falcarinol (FaOH), falcarindiol (FaDOH), falcarindiol-3-acetate (FaDOAc) in carrot disks. The quality of the carrot disks was also assessed using instrumental texture and colour measurements. Blast frozen carrot disks retained higher amounts of polyacetylenes compared to their slow frozen counterparts. Whilst the levels of retention of total polyacetylenes was higher in unblanched than blanched disks prior to freezing there was a sharp decrease in the levels of polyacetylenes in unblanched frozen carrots during the storage period for 60 days at −20 °C. FaDOH was observed to be the most susceptible to degradation during frozen storage of unblanched carrot disks, followed by FaOH and FaDOAc. The changes in the level of polyacetylenes during storage were adequately described by using Weibull model. The texture and colour were also found to decrease during frozen storage compared to fresh carrots.     

Effects of the nature of the doping salt and of the thermal pre-treatment and sintering temperature on Spark Plasma Sintering of transparent alumina

A slurry of α-Al₂O₃ was doped with Mg, Zr and La nitrates or chlorides, in various amounts in the range 150-500 wt ppm and then freeze-dried to produce nanosized doped powder (∼150 nm). The powder was sintered by SPS to yield transparent polycrystalline alpha alumina. The influence of the nature of the doping element and the starting salt, the thermal treatment before sintering and the sintering temperature on the transparency of the ceramics were investigated. The transparency of the ceramics of nanosized Al₂O₃ was shown to depend mainly on the way the powder was prepared, the nature of the doping salt also had an effect. Finally, a high real inline transmittance, reaching 48.1% was achieved after optimization.     

Enzymatic degradation of polyacrylamide in aqueous solution with peroxidase and H2O2

Polyacrylamides are often used in water‐based hydraulic fracturing for natural gas and oil production. However, residual polymer remaining in the fractured rock can limit production. A novel approach for degrading partially hydrolyzed polyacrylamide (HPAM) was investigated using hydrogen peroxide and horseradish peroxidase (HRP). This sustainable HRP/H₂O₂ system degraded the polymer in solution, reducing its viscosity in both pure water and brine solutions. Molecular weight measurements confirmed that the viscosity reduction was due to a significant degradation of the polymer backbone and not primarily by other mechanisms such as amide hydrolysis or rearrangement, and so forth. The reduction in viscosity and molecular weight was first order with respect to H₂O₂ concentration. The kinetics of viscosity reduction and molecular weight are closely correlated which would allow the quicker and simpler viscosity method to help engineer future processes. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44560.     

Dichlorophenylacrylonitriles as AhR Ligands That Display Selective Breast Cancer Cytotoxicity in vitro

Knoevenagel condensation of 3,4‐dichloro‐ and 2,6‐dichlorophenylacetonitriles gave a library of dichlorophenylacrylonitriles. Our leads (Z)‐2‐(3,4‐dichlorophenyl)‐3‐(1H‐pyrrol‐2‐yl)acrylonitrile ( 5 ) and (Z)‐2‐(3,4‐dichlorophenyl)‐3‐(4‐nitrophenyl)acrylonitrile ( 6 ) displayed 0.56±0.03 and 0.127±0.04 μm growth inhibition (GI₅₀) and 260‐fold selectivity for the MCF‐7 breast cancer cell line. A 2,6‐dichlorophenyl moiety saw a 10‐fold decrease in potency; additional nitrogen moieties (‐NO₂) enhanced activity (Z)‐2‐(2,6‐dichloro‐3‐nitrophenyl)‐3‐(2‐nitrophenyl)acrylonitrile ( 26 ) and (Z)‐2‐(2,6‐dichloro‐3‐nitrophenyl)‐3‐(3‐nitrophenyl)acrylonitrile ( 27 ), with the corresponding ‐NH₂ analogues (Z)‐2‐(3‐amino‐2,6‐dichlorophenyl)‐3‐(2‐aminophenyl)acrylonitrile ( 29 ) and (Z)‐2‐(3‐amino‐2,6‐dichlorophenyl)‐3‐(3‐aminophenyl)acrylonitrile ( 30 ) being more potent. Despite this, both 29 (2.8±0.03 μm ) and 30 (2.8±0.03 μm ) were found to be 10‐fold less cytotoxic than 6 . A bromine moiety effected a 3‐fold enhancement in solubility with (Z)‐3‐(5‐bromo‐1H‐pyrrol‐2‐yl)‐2‐(3,4‐dichlorophenyl)acrylonitrile 18 relative to 5 at 211 μg mL^?1. Modeling‐guided synthesis saw the introduction of 4‐aminophenyl substituents (Z)‐3‐(4‐aminophenyl)‐2‐(3,4‐dichlorophenyl)acrylonitrile ( 35 ) and (Z)‐N‐(4‐(2‐cyano‐2‐(3,4‐dichlorophenyl)vinyl)phenyl)acetamide ( 38 ), with respective GI₅₀ values of 0.030±0.014 and 0.034±0.01 μm . Other analogues such as 35 and 36 were found to have sub‐micromolar potency against our panel of cancer cell lines (HT29, colon; U87 and SJ‐G2, glioblastoma; A2780, ovarian; H460, lung; A431, skin; Du145, prostate; BE2‐C, neuroblastoma; MIA, pancreas; and SMA, murine glioblastoma), except compound 38 against the U87 cell line. A more extensive evaluation of 38 ((Z)‐N‐(4‐(2‐cyano‐2‐(3,4‐dichlorophenyl)vinyl)phenyl)acetamide) in a panel of drug‐resistant breast carcinoma cell lines showed 10–206 nm potency against MDAMB468, T47D, ZR‐75‐1, SKBR3, and BT474. Molecular Operating Environment docking scores showed a good correlation between predicted binding efficiencies and observed MCF‐7 cytotoxicity. This supports the use of this model in the development of breast‐cancer‐specific drugs.     

Wall slip heating

When molten plastic is extruded, the upper limiting throughput is often dictated by fine irregular distortions of the extrudate surface. Called sharkskin melt fracture, plastics engineers spike plastics formulations with processing aids to suppress these distortions. Sharkskin melt fracture is not to be confused with gross melt fracture, a larger scale distortion arising at throughputs higher than the critical throughput for sharkskin melt fracture. Sharkskin melt fracture has been attributed to a breakdown of the no slip boundary condition in the extrusion die, that is, adhesive failure at the die walls, where the fluid moves with respect to the wall. In this article, we account for the frictional heating at the wall, which we call slip heating. We focus on slit flow, which is used in film casting, sheet extrusion, curtain coating, and when curvature can be neglected, slit flow is easily extended to pipe extrusion and film blowing. In slit flow, the magnitude of the heat flux from the slipping interface is the product of the shear stress and the slip speed. We present the solutions for the temperature rise in pressure‐driven slit flow and simple shearing flow, each subject to constant heat generation at the adhesive slip interface, with and without viscous dissipation in the bulk fluid. We solve the energy equation in Cartesian coordinates for the temperature rise, for steady temperature profiles. For this simplest relevant nonisothermal model, we neglect convective heat transfer in the melt and use a constant viscosity. We arrive at a necessary dimensionless condition for the accurate use of our results: Pé?1. We find that slip heating can raise the melt temperature significantly, as can viscous dissipation in the bulk. We conclude with two worked examples showing the relevance of slip heating in determining wall temperature rise, and we show how to correct wall slip data for this temperature rise. POLYM. ENG. SCI., 55:2042–2049, 2015. © 2014 Society of Plastics Engineers     

Investigation of Trypanothione Reductase as a Drug Target in Trypanosoma brucei

There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62 000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel‐compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.     

The capacity and distance constrained plant location problem

This article introduces a new problem called the Capacity and Distance Constrained Plant Location Problem. It is an extension of the discrete capacitated plant location problem, where the customers assigned to each plant have to be packed in groups that will be served by one vehicle each. The constraints include two types of capacity. On the one hand plants are capacitated, and the demands of the customers are indivisible. On the other hand, the total distance traveled by each vehicle to serve its assigned customers in round trips plant–customer–plant is also limited. The paper addresses different modeling aspects of the problem. It describes a tabu search algorithm for its solution. Extensive computational tests indicate that the proposed heuristic consistently yields optimal or near-optimal solutions.     

Hierarchical virtual screening for the discovery of new molecular scaffolds in antibacterial hit identification

One of the initial steps of modern drug discovery is the identification of small organic molecules able to inhibit a target macromolecule of therapeutic interest. A small proportion of these hits are further developed into lead compounds, which in turn may ultimately lead to a marketed drug. A commonly used screening protocol used for this task is high-throughput screening (HTS). However, the performance of HTS against antibacterial targets has generally been unsatisfactory, with high costs and low rates of hit identification. Here, we present a novel computational methodology that is able to identify a high proportion of structurally diverse inhibitors by searching unusually large molecular databases in a time-, cost- and resource-efficient manner. This virtual screening methodology was tested prospectively on two versions of an antibacterial target (type II dehydroquinase from Mycobacterium tuberculosis and Streptomyces coelicolor), for which HTS has not provided satisfactory results and consequently practically all known inhibitors are derivatives of the same core scaffold. Overall, our protocols identified 100 new inhibitors, with calculated K_i ranging from 4 to 250 μM (confirmed hit rates are 60% and 62% against each version of the target). Most importantly, over 50 new active molecular scaffolds were discovered that underscore the benefits that a wide application of prospectively validated in silico screening tools is likely to bring to antibacterial hit identification.