Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS)

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD(+)-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.This research was funded by the Engineering and Physical Sciences Research Council (studentship to RH and an Impact Acceleration Account Partnership Development Award), the Biological and Biotechnological Research Council (BBSRC) and Johnson Matthey. SE and MF were supported by postdoctoral Marie-Curie fellowships

Production of Pectinolytic Enzymes by the Yeast Wickerhanomyces anomalus Isolated from Citrus Fruits Peels

Wickerhamomyces anomalus is pectinolytic yeast isolated from citrus fruits peels in the province of Misiones, Argentine. In the present work, enzymes produced by this yeast strain were characterized, and polygalacturonase physicochemical properties were determined in order to evaluate the application of the supernatant in the maceration of potato tissues. W. anomalus was able to produce PGin liquid mediumcontaining glucose and citrus pectin,whosemode of actionwas mainly of endo type.The supernatant did not exhibit esterase or lyase activity. No others enzymes, capable of hydrolyzing cell wall polymers, such as cellulases and xylanases,were detected. PGshowedmaximal activity atpH4.5 and at temperature range between 40∘Cand50∘C. It was stable in the pH range from3.0 to 6.0 and up to 50∘Cat optimumpH.The enzymatic extractmacerated potato tissues efficiently.Volume of single cells increased with the agitation speed.The results observed make the enzymatic extract produced by W. anomalus appropriate for future application in food industry, mainly for the production of fruit nectars or mashed of vegetables such as potato or cassava, of regional interest in the province of Misiones, Argentine.Fil: Martos, María Alicia. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; ArgentinaFil: Zubreski, Emilce R.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; ArgentinaFil: Garro, Oscar Alfredo. Universidad Nacional del Chaco Austral. Departamento de Ciencias Basicas y Aplicadas. Laboratorio de Microbiologia de Alimentos; ArgentinaFil: Hours, Roque Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); Argentin

Single-photon emission from the natural quantum dots in the InAs/GaAs wetting layer

Time-resolved microphotoluminescence study is presented for quantum dots which are formed in the InAs/GaAs wetting layer. These dots are due to fluctuations of In composition in the wetting layer. They show spectrally sharp luminescence lines with a low spatial density. We identify lines related to neutral exciton and biexciton as well as trions. Exciton emission antibunching (second order correlation value of g^2(0)=0.16) and biexciton-exciton emission cascade prove non-classical emission from the dots and confirm their potential as single photon sources

Identification of a yeast with maceration capacity and partial characterization of the extracellular carbohydrolases

Estudios previos demostraron que una levadura autóctona, aislada de frutas cítricas, produce extractos enzimáticos con capacidad macerante de tejidos vegetales (papa, mandioca). El objetivo del presente estudio fue identificar dicha levadura por métodos moleculares e identificar las actividades enzimáticas posiblemente involucradas en el proceso de maceración. La identificación de la levadura se realizó mediante amplificación de la región entre los genes 18S rRNA y 28S rRNA utilizando los primers ITS1 e ITS4, seguido de digestión con enzimas de restricción (Hinf I, Cfo I y Hae III). El fragmento amplificado, 5.8S-ITS rRNA, fue secuenciado para confirmar su identidad. La levadura fue identificada como Pichia anomala. En sobrenadantes de cultivo se detectaron dos isoenzimas con actividad de poligalacturonasa (PG) de tipo endo; no se detectó actividad de pectinliasa, pectatoliasa, pectinesterasa, celulasa ni xilanasa.Previous studies have demonstrated that enzymatic extracts from a wild-type yeast isolated from citrus fruit peels display macerating activity of plant tissues (i.e. potato, cassava, etc.). The aim of the present study was to identify the wild-type yeast by molecular methods and characterize the enzymatic activities possibly involved in the maceration process. Molecular identification of the wild-type yeast was performed by amplification of the 18S and 28S rRNA regions using ITS1 and ITS4 primers followed by digestion with restriction enzymes (Hinf I, Cfo I and Hae III). The species assignation was also confirmed by sequence analyses of the 5.8S-ITS rRNA region. The wild-type yeast was identified as Pichia anomala. Two isoenzymes with PG activity were found in culture supernatants, whereas cellulase, xylanase, pectinlyase, pectatelyase and pectinesterase activities were not detected.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA–pectin cryogels and enzyme release was 20.8 ± 2.1%, 63.8 ± 0.2%, 41.5 ± 3.5% and 26.0 ± 3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37 ºC after 3 h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5 ± 1.5, 65.8 ± 3.8, 57.3 ± 0.2 and 34.0 ± 4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA–pectin cryogels at pH close to the human skin (pH = 5.5) showed 15.0% free antibiotic following first order kinetic at 37 ºC after 5 h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Risk of brain tumours in relation to estimated RF dose from mobile phones: results from five Interphone countries

OBJECTIVES: The objective of this study was to examine the associations of brain tumours with radio frequency (RF) fields from mobile phones. METHODS: Patients with brain tumour from the Australian, Canadian, French, Israeli and New Zealand components of the Interphone Study, whose tumours were localised by neuroradiologists, were analysed. Controls were matched on age, sex and region and allocated the 'tumour location' of their matched case. Analyses included 553 glioma and 676 meningioma cases and 1762 and 1911 controls, respectively. RF dose was estimated as total cumulative specific energy (TCSE; J/kg) absorbed at the tumour's estimated centre taking into account multiple RF exposure determinants. RESULTS: ORs with ever having been a regular mobile phone user were 0.93 (95% CI 0.73 to 1.18) for glioma and 0.80 (95% CI 0.66 to 0.96) for meningioma. ORs for glioma were below 1 in the first four quintiles of TCSE but above 1 in the highest quintile, 1.35 (95% CI 0.96 to 1.90). The OR increased with increasing TCSE 7+ years before diagnosis (p-trend 0.01; OR 1.91, 95% CI 1.05 to 3.47 in the highest quintile). A complementary analysis in which 44 glioma and 135 meningioma cases in the most exposed area of the brain were compared with gliomas and meningiomas located elsewhere in the brain showed increased ORs for tumours in the most exposed part of the brain in those with 10+ years of mobile phone use (OR 2.80, 95% CI 1.13 to 6.94 for glioma). Patterns for meningioma were similar, but ORs were lower, many below 1.0. CONCLUSIONS: There were suggestions of an increased risk of glioma in long-term mobile phone users with high RF exposure and of similar, but apparently much smaller, increases in meningioma risk. The uncertainty of these results requires that they be replicated before a causal interpretation can be made

Occurrence of Different Species of <i>Fusarium</i> from Wheat in Relation to Disease Levels Predicted by a Weather-Based Model in Argentina Pampas Region

Fusarium head blight (FHB) is an important disease throughout many of the world wheat-growing areas that have humid to semi-humid climate. The infection happens mainly during the anthesis of the wheat, when there have been favorable conditions of moisture and temperature. The direct relation of the infection to environmental factors makes possible the formulation of mathematical models that predict the disease. The causal agent of the FHB of the spike of wheat is attributed principally to Fusarium graminearum. High economic losses due yield decrease have been recorded in Argentina. In the present work, 67 isolates of Fusarium spp. were obtained from samples of wheat grains from Pampas region from 15 locations distributed in Buenos Aires, Entre Rios, Santa Fe and Corboba provinces during 2006 and 2007 wheat-growing seasons. The identification of species from monosporic isolates was carried out by morphological characterization and use of species-specific PCR-based assays. Both identification criteria were necessary and complementary for the species determination, since in some cases the molecular identification was not specific. Scanty presence of F. graminearum was observed in 2006 wheat-growing season coinciding with the lack of favorable meteorological conditions for producing FHB infection events. High presence of F. graminearum isolates was observed in 2007 wheat-growing season, in accordance with moderate incidence of the disease according to spatial distribution of FHB incidence values. The aim of this report was to identify the causal agent of the FHB disease by different taxonomic criteria and to relate its occurrence with disease incidence values predicted by a weather-based model in Argentina.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones IndustrialesFacultad de Ciencias Naturales y MuseoInstituto de Botánica "Dr. Carlos Spegazzini

Generation and control of polarization-entangled photons from GaAs island quantum dots by an electric field

Semiconductor quantum dots are potential sources for generating polarization-entangled photons efficiently. The main prerequisite for such generation based on biexciton–exciton cascaded emission is to control the exciton fine-structure splitting. Among various techniques investigated for this purpose, an electric field is a promising means to facilitate the integration into optoelectronic devices. Here we demonstrate the generation of polarization-entangled photons from single GaAs quantum dots by an electric field. In contrast to previous studies, which were limited to In(Ga)As quantum dots, GaAs island quantum dots formed by a thickness fluctuation were used because they exhibit a larger oscillator strength and emit light with a shorter wavelength. A forward voltage was applied to a Schottky diode to control the fine-structure splitting. We observed a decrease and suppression in the fine-structure splitting of the studied single quantum dot with the field, which enabled us to generate polarization-entangled photons with a high fidelity of 0.72±0.05