The diversity challenge in directed protein evolution

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships. This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and cost-effectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle). We hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.     

Universal space in the Cartesian product of Peano curves without free arcs

We prove that there is the universal space for the class of n-dimensional separable metric spaces in the Cartesian product K₁×?×Kn+1 of Peano curves without free arcs. It is also shown that the set of embeddings of any n-dimensional separable metric space X into this universal space is a residual set in C(X,K₁×?×Kn+1). Other properties of product of Peano curves without free arcs are also proved.     

The molecular structure of selected minerals of the rosasite group – An XRD,SEM and infrared spectroscopic study

Minerals in the rosasite mineral group namely rosasite, glaucosphaerite, kolwezite, mcguinnessite have been studied by powder X-ray diffraction, scanning electron microscopy and infrared spectroscopy. X-ray diffraction shows the minerals to be complex mixtures with more than one rosasite mineral observed in each sample. SEM analysis shows the minerals to be fibrous in nature and the use of EDAX enabled the chemical composition of the minerals to be determined. The spectral patterns for the minerals rosasite, glaucosphaerite, kolwezite and mcguinnessite are similar to that of malachite implying the molecular structure is similar to malachite. A comparison is made with the spectrum of malachite. The rosasite mineral group is characterised by two OH stretching vibrations at ∼3401 and 3311 cm⁻¹. Two intense bands observed at ∼1096 and 1046 cm⁻¹ are assigned to ν₁ (CO₃)²⁻ symmetric stretching vibration and the δ OH deformation mode. Multiple bands are found in the 800–900 and 650–750 cm⁻¹ regions attributed to the ν₂ and ν₄ bending modes confirming the symmetry reduction of the carbonate anion in the rosasite mineral group as C_2v or C_s. A band at ∼560 cm⁻¹ is assigned to a CuO stretching mode.     

Eine einfache und flexible Synthese von Pyrrolen aus α,β-ungesättigten Sulfonen

A Simple and Flexible Synthesis of Pyrroles from α , β -Unsaturated Sulfones: The addition of alkyl isocyanoacetates and isocyanoacetonitrile to α,β-unsaturated sulfones affords a convenient and broad access to pyrroles with unusual substitution patterns (see Scheme 2). The α,β-unsaturated sulfones required as starting materials are easily obtained from different olefines.     

Spectroscopic methods for determination of catecholamines: A mini-review

Catecholamines are biogenic compounds that perform a variety of vital functions, playing a key physiological role in humans and animals. They are important markers in diagnosis of diseases and dysfunctions as well as widespread components of pharmaceutical formulations. Therefore, development of highly sensitive, rapid, and economically efficient methods for the determination of catecholamines is of great interest. The spectroscopic analytical methods have a good potential in this respect. This mini-review summarizes in a concise manner some advances and trends in the determination of catecholamines by spectroscopic methods, including spectrophotometry, fluorescence spectroscopy, immunoassays with spectroscopic detection, Raman spectroscopy, and other methods. Information on mainly determined compounds and analyzed samples is given and discussed. Analytical strategies and performance for the quantitation of catecholamines in various samples are described.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Mathematical modeling of the first phase of morphogenesis of mechanoreceptors in <Emphasis Type="Italic">D. melanogaster</Emphasis>

The mechanoreceptors (in particular, macrochaetes) of drosophila pass three stages in its development (morphogenesis) whose genetic support is described in terms of gene networks. The key object of the gene networks of macrochaete morphogenesis is the achaete-scute (AS-C) complex of genes. Each mechanoreceptor develops from one parent cell distinguished by high concentration of the AS-C protein. The activity of this complex, which ensures the protein concentration critical for initiating morphogenesis, is determined by the central regulatory contour which includes a system of interactions among certain objects of the networks (genes and their products).     

Effect of Freon matrices on the intermediates stabilized in X-ray irradiated 1,7-dioxaspiro[5,5]undecane solutions at 77 K

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