Alternatives for the intermediate recovery of plasmid DNA: Performance,economic viability and environmental impact

Robust cGMP manufacturing is required to produce high-quality plasmid DNA (pDNA). Three established techniques, isopropanol and ammonium sulfate (AS) precipitation (PP), tangential flow filtration (TFF) and aqueous two-phase systems (ATPS) with PEG600/AS, were tested as alternatives to recover pDNA from alkaline lysates. Yield and purity data were used to evaluate the economic and environmental impact of each option. Although pDNA yields =90% were always obtained, ATPS delivered the highest HPLC purity (59%), followed by PP (48%) and TFF (18%). However, the ability of ATPS to concentrate pDNA was very poor when compared with PP or TFF. Processes were also implemented by coupling TFF with ATPS or AS-PP. Process simulations indicate that all options require large amounts of water (100–200 tons/kg pDNA) and that the ATPS process uses large amounts of mass separating agents (65 tons/kg pDNA). Estimates indicate that operating costs of the ATPS process are 2.5-fold larger when compared with the PP and TFF processes. The most significant contributions to the costs in the PP, TFF and ATPS processes came from operators (59%), consumables (75%) and raw materials (84%), respectively. The ATPS process presented the highest environmental impact, whereas the impact of the TFF process was negligible.     

Aqueous biphasic cancer cell migration assay enables robust,high‐throughput screening of anti‐cancer compounds

Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high‐throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy‐to‐implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two‐phase system to pattern a monolayer of cells containing a cell‐excluded gap that serves as the migration niche. We adapted this technology to a standard 96‐well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti‐migratory drug screening.     

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

Abstract

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

A Phenazine based colorimetric and fluorescent chemosensor for sequential detection of Ag+ and I− in aqueous media

A new colorimetric and fluorescent probe MNTPZ based on 1H‐imidazo[4,5‐b]phenazine derivative has been designed and synthesized for successive detection of Ag⁺ and I^?. The probe MNTPZ shows selective colorimetric response by a change in color from yellow to orange and “turn‐off” fluorometric response upon binding with Ag⁺ in DMSO: Water (pH = 7, 1:1, v/v) over other cations. The binding mode of probe MNTPZ to Ag⁺ was studied by Job's plot, ¹H NMR studies, FT‐IR spectroscopy and DFT calculations. Moreover, the situ generated probe MNTPZ + Ag⁺ complex acted as an efficient fluorometric “turn‐on” probe for I^? via Ag⁺ displacement approach. The detection limit of probe MNTPZ for Ag⁺ and the resultant complex probe MNTPZ + Ag⁺ for I^? were determined to be 1.36 μmol/L and 1.03 μmol/L respectively. Notably, the developed probe was successfully used for quantitative determination of I^? in real samples with satisfactory results.     

Ultrasound-assisted extraction of flaxseed oil using immobilized enzymes

An aqueous enzymatic process assisted by ultrasound extraction (AEP-UE) was applied to the extraction of oil from flaxseed (Linum usitatissimum L.). The highest oil recovery of 68.1% was obtained when ground flaxseed was incubated with 130 U/g of cellulase, pectinase, and hemicellulase for 12 h, at 45 °C and pH 5.0. The IC₅₀ values of oil obtained by AEP-UE and organic solvent extraction (OSE), as measured by DPPH scavenging activity essay, were 2.27 mg/mL and 3.31 mg/mL. The AEP-UE-derived oil had a 1.5% higher content of unsaturated fatty acids than the OSE-derived oil. AEP-UE is therefore a promising environmentally friendly method for large-scale preparation of flaxseed oil.     

米曲霉氨基酰化酶的双水相萃取研究

本文利用聚乙二醇和磷酸盐组成的水溶液双相系统,从米曲霉(Aspergillus oryzae)中提取氨基酰化酶。通过实验对影响氨基酰化酶分配的各参数进行了研究,确定了最适体系:PEC-1540 10％(W/V),K_2HPO_418％(W/V),pH8.5;PEG-154010％(W/V),K_2HPO_412％(W/V),NaCl 0.5mol/L,pH8.5。二步萃取收率90％,纯化倍数9。为实验室和工业上采用双水相系统萃取氨基酰化酶提供了一个新方法。     

A method for microbial cell surface fingerprinting based on surface plasmon resonance

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).     

Microwave-assisted preparation of affinity medium

Microwave assistance was used for preparing polyethylene glycol (PEG)-Cibacron blue 3GA and Sepharose CL-4B-Cibacron blue 3GA affinity materials. The former was used as the affinity macroligand in a PEG-dextran aqueous two-phase system for purification of alcohol dehydrogenase and EcoRI. The Sepharose CL-4B-Cibacron blue 3GA was used for affinity chromatography of the above two enzymes. It was found that microwave assistance could reduce the time of PEG-dye preparation to 5 min (from 7h). Similarly, Sepharose CL-4B-Cibacron blue 3GA preparation time could be reduced to 21 min (from 3.5h). The performances of affinity macroligand PEG-dye and the affinity medium Sepharose-dye prepared by conventional methods and with microwave assistance were similar during purification of these enzymes.     

Inhibition effect of sugar-based amphiphiles on eutectic formation in the freezing-thawing process of aqueous NaCl solution

DSC and simultaneous XRD-DSC measurements were carried out to clarify the interaction among the ingredients in a ternary aqueous solution composed of NaCl, a sugar-based amphiphile or free sugar, and water. Two aspects of the inhibition of eutectic formation were suggested through the addition of the sugar amphiphile. One was the retention of the glass state of the eutectic phase, and the other was the trapping of NaCl hydrate into the sugar moiety of the amphiphilic aggregate. The difference between the free sugar and the amphiphilic one in terms of the trapping of NaCl hydrate was attributable to their dissimilarity in the dissolution state. The results indicated that the free sugars in water could interact with NaCl hydrate on the basis of their various hydroxyl groups. On the other hand, the sugar-based amphiphiles generated a self-assembly aggregate in the system, and interacted with NaCl hydrate by a salting-in effect with their sugar moiety in the freezing-thawing process. It was confirmed that the number of sugar units played an important role in trapping NaCl hydrate in the system. The effects of the structural isomerism in the sugars were slight with regard to the inhibition of eutectic formation.