Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of D -Amino acid oxidase from Rhodotorula gracilis ( Rg DAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between Rg DAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a Rg DAAOx variant library containing ∼10 6 clones while lowering the D -Ala substrate concentration over three sorting rounds in order to isolate variants with low K m . After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower K m than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the Rg DAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes

Microcephaly and macrocephaly. A study on anthropometric and clinical data from 308 subjects

Head circumference is the auxological parameter that most correlates with developmental anomalies in childhood. Head circumference (HC) two standard deviations (SD) below or above the mean defines microcephaly and macrocephaly, respectively. The aim of this retrospective study was to explore anthropometric parameters and clinical characteristics among subjects with abnormalities in HC who had been referred for developmental assessment. One hundred and sixty four subjects with microcephaly and 144 subjects with macrocephaly were enrolled from birth to 18 months of age. Head circumference at birth and the association with variables related to maternal health status, gestational age, growth pattern, brain imaging and clinical characteristics were analyzed. In some cases, an etiological diagnosis was made. In the two considered conditions, we found different anthropometric and clinical associations, some of which were statistically significant, with implications for ongoing neurodevelopmental surveillance

High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering

Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma. Typical enhancements sought in enzyme engineering and in vitro evolution campaigns include improved folding stability, catalytic activity, and/or substrate specificity. Despite significant progress in recent years in the areas of high-throughput screening and DNA sequencing, our ability to explore the vast space of functional enzyme sequences remains severely limited. Here, we review the currently available suite of modern methods for enzyme engineering, with a focus on novel readout systems based on enzyme cascades, and new approaches to reaction compartmentalization including single-cell hydrogel encapsulation techniques to achieve a genotype–phenotype link. We further summarize systematic scanning mutagenesis approaches and their merger with deep mutational scanning and massively parallel next-generation DNA sequencing technologies to generate mutability landscapes. Finally, we discuss the implementation of machine learning models for computational prediction of enzyme phenotypic fitness from sequence. This broad overview of current state-of-the-art approaches for enzyme engineering and evolution will aid newcomers and experienced researchers alike in identifying the important challenges that should be addressed to move the field forward

Studi Optimasi Analisis Logam Co dan Ni secara Voltammetri Striping Adsorptif (Adsv) untuk Penentuan Logam dalam Konsentrasi Runut

The research of the determination of Co(II) and Ni(II) by adsorptive stripping voltammetry have been done. The method used to determine the concentration of cobalt and nickel in trace (ultra trace) with calcon for Co (II) and DMG for Ni (II) as a complexing agent. The parameters studied were variation of calcon or DMG concentration, pH, accumulation potential and accumulation time. In this study, the optimum conditions were calcon concentration of 0.7 mM for Co (II), DMG concentration of 0.8 mM for Ni (II), pH 6 for Co (II) while pH 9 for Ni (II), accumulation potential -0.3 V and accumulation time of 70 s for the Co (II) and 220 s for the Ni (II). At optimum conditions the relative standard deviation were 0.75% and 1.54% for Co (II) and Ni (II) respectively for ten replicates (n= 10) measurement of 10 µg /L standard solution Co(II) and Ni (II). The method was applied to the direct determination of Co(II) and Ni(II) in the Stones River water rot, tap water and sea water Bungus Padang, with recovery of 106.5% and 102%. for Co(II) and Ni(II) respectively

Glutamate-evoked redox state alterations are involved in tissue transglutaminase upregulation in primary astrocyte cultures

AbstractThe aim of this study was to evaluate the involvement of oxidative stress in glutamate-evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose-dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor-mediated, as demonstrated by inhibition with GYKI 52466. The pre-incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate-increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate

Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4`-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects

Enzyme-mediated hydrogel encapsulation of single cells for high-throughput screening and directed evolution of oxidoreductases

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol cross-coupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H; 2; O; 2; -producing enzymes many folds

Genetically encoded stimuli-responsive cytoprotective hydrogel capsules for single cells provide novel genotype-phenotype linkage

Modification of cell surfaces with synthetic polymers is a promising approach for regulating cellular behavior. Here, we describe a genetically controlled strategy for selectively encapsulating single yeast cells in synthetic microniches comprising cross-linked phenol-modified alginate and chitosan hydrogel capsules. Our system links inducible gene expression with enzyme-mediated hydrogel polymerization and provides a novel genotype–phenotype linkage whereby only cells carrying a requisite gene encoding a flavin adenine dinucleotide-dependent oxidoreductase undergo autonomous enzyme-mediated surface polymerization, resulting in the formation of hydrogel capsules. The composition of the hydrogel capsules is highly tunable and the capsule sizes are pH-responsive, allowing for control of capsule porosity and shell diameters over a range of 15–80 μm. The hydrogel capsules prevent extracellular proteins from reaching the cell surface, thereby conferring cellular immunity to lytic enzyme cocktails and rendering the hydrogel capsules cytoprotective against osmotic shock. We demonstrate the utility of this genetically controlled artificial hydrogel-encapsulated cell phenotype by isolating and enriching uniform eukaryotic cell lineages from genetically heterogeneous cell mixtures at 95–100% efficiency. The encapsulated cells remained viable and were capable of dividing and breaking free from their hydrogel capsules, allowing further propagation of selected cells. Our bottom-up approach to cellular compartmentalization links inducible intracellular genetic components with an artificial extracellular matrix that resists enzymatic lysis and mediates communication with the surrounding environment through a size-tunable and permeable hydrogel capsule

Comparing the use of ERA5 reanalysis dataset and ground-based agrometeorological data under different climates and topography in Italy

Study region: The study region is represented by seven irrigation districts distributed under different climate and topography conditions in Italy. Study focus: This study explores the reliability and consistency of the global ERA5 single levels and ERA5-Land reanalysis datasets in predicting the main agrometeorological estimates commonly used for crop water requirements calculation. In particular, the reanalysis data was compared, variable-by-variable (e.g., solar radiation, R-s; air temperature, T-air; relative humidity, RH; wind speed, u(10); reference evapotranspiration, ET0), with in situ agrometeorological obser-vations obtained from 66 automatic weather stations (2008-2020). In addition, the presence of a climate-dependency on their accuracy was assessed at the different irrigation districts. New hydrological insights for the region: A general good agreement was obtained between observed and reanalysis agrometeorological variables at both daily and seasonal scales. The best perfor-mance was obtained for T-air, followed by RH, R-s, and u(10) for both reanalysis datasets, especially under temperate climate conditions. These performances were translated into slightly higher accuracy of ET0 estimates by ERA5-Land product, confirming the potential of using reanalysis datasets as an alternative data source for retrieving the ET0 and overcoming the unavailability of observed agrometeorological data

Use of small scale electrical resistivity tomography to identify soil-root interactions during deficit irrigation

Plant roots activity affect the exchanges of mass and energy between the soil and atmosphere. However, it is challenging to monitor the activity of the root-zone because roots are not visible from the soil surface, and root systems undergo spatial and temporal variations in response to internal and external conditions. Therefore, measurements of the activity of root systems are interesting to ecohydrologists in general, and are especially important for specific applications, such as irrigation water management. This study demonstrates the use of small scale three-dimensional (3-D) electrical resistivity tomography (ERT) to monitor the root-zone of orange trees irrigated by two different regimes: (i) full rate, in which 100% of the crop evapotranspiration (ETc) is provided; and (ii) partial root-zone drying (PRD), in which 50% of ETc is supplied to alternate sides of the tree. We performed time-lapse 3-D ERT measurements on these trees from 5 June to 24 September 2015, and compared the long-term and short-term changes before, during, and after irrigation events. Given the small changes in soil temperature and pore water electrical conductivity, we interpreted changes of soil electrical resistivity from 3-D ERT data as proxies for changes in soil water content. The ERT results are consistent with measurements of transpiration flux and soil temperature. The changes in electrical resistivity obtained from ERT measurements in this case study indicate that root water uptake (RWU) processes occur at the 0.1 m scale, and highlight the impact of different irrigation schemes. (C) 2017 Elsevier B.V. All rights reserved