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

Comparison of voluntary food intake and palatability of commercial weight loss diets in healthy dogs and cats

Background Obesity in dogs and cats is usually managed by dietary energy restriction using a purpose-formulated weight loss diet, but signs of hunger and begging commonly occur causing poor owner compliance. Altering diet characteristics so as to reduce voluntary food intake (VFI) can improve the likelihood of success, although this should not be at the expense of palatability. The aim of the current study was to compare the VFI and palatibility of novel commercially available canine and feline weight loss diets. Methods The relative performance of two canine (C1 and C2) and two feline (F1 and F2) diets was assessed in groups of healthy adult dogs and cats, respectively. Diets varied in energy, protein, fibre, and fat content. To assess canine VFI, 12 (study 1) and 10 (study 2) dogs were offered food in 4 meals, for 15 min on each occasion, with hourly intervals between the meals. For feline VFI, 12 cats were offered food ad libitum for a period of 18 h per day over 5 consecutive days. The palatability studies used separate panels of 37 dogs and 30 cats, with the two diets being served, side-by-side, in identical bowls. Results In dogs, VFI was significantly less for diet C1 than diet C2 when assessed on energy intake (study 1, 42% less, P = 0.032; study 2, 28% less, P = 0.019), but there was no difference in gram weight intake (study 1: P = 0.964; study 2: P = 0.255). In cats, VFI was 17% less for diet F1 than diet F2 when assessed by energy intake (P < 0.001), but there was again no difference in gram weight (P = 0.207). There was no difference in palatability between the two canine diets (P = 0.490), whilst the panel of cats diet preferred F1 to F2 (P < 0.001). Conclusion Foods with different characteristics can decrease VFI without affecting palatability in both dogs and cats. The effects seen could be due to decreased energy content, decreased fat content, increased fibre content, different fibre source, and increased protein content. Further studies are now needed to determine whether similar findings occur in obese dogs and cats on controlled weight loss programmes

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

This is the final version. Available from National Academy of Sciences via the DOI in this recordUltrahigh-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 dehydro-genase 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.Biotechnology and Biological Sciences Research CouncilEuropean Research CouncilEngineering and Physical Sciences Research CouncilEuropean Commissio

Screening of yeast strains with pectinolytic activity

The present study was undertaken to find yeast strains with high pectinolytic activity. Yeast strains isolated from citrus fruits, were screened for pectinase production. The species with the best pectinase activity was selected to evaluate their ability to produce pectic enzymes by submerged fermentation processes. The effect of different carbon sources on polygalacturonase (PGase) production was studied. Different vegetable tissues were used for tests of maceration with the crude enzyme extract. Among 154 yeast strains isolated, yeast Nº 111 was positive for pectinase activity and was identified as Geotrichum sp. Maximum PGase production obtained was 90.7 EU/ml when this yeast grown in shake flasks under the presence of glucose and citrus pectin as carbon sources. All tissues studied were macerated by the enzyme extract, microscopic examination showed a suspension of loose single cells. Lysis of cells was not observed.El objetivo del presente trabajo fue el aislamiento de cepas de levaduras con buena actividad pectinolítica. Las levaduras aisladas a partir de frutas cítricas fueron seleccionadas en base a sus actividades pectinolíticas. Se evaluó la capacidad de producción de enzimas pécticas por fermentación en medio líquido de la levadura con mayor actividad pectinolítica. Se estudió la influencia de diferentes fuentes de carbono sobre la producción de poligalacturonasa (PGasa) y la capacidad macerante de los extractos enzimáticos sobre tejidos vegetales. De 154 levaduras aisladas, la cepa Nº 111 mostró actividad pectinolítica y fue identificada como Geotrichum sp. La máxima producción de PGasa obtenida fue de 90,7 EU/ml, cuando esta levadura creció en medio líquido con glucosa y pectina de citrus como fuentes de carbono. Los tejidos vegetales estudiados fueron macerados por el extracto enzimático. El examen microscópico mostró células simples liberadas, no observándose lisis por destrucción de la pared celular.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Screening of yeast strains with pectinolytic activity

The present study was undertaken to find yeast strains with high pectinolytic activity. Yeast strains isolated from citrus fruits, were screened for pectinase production. The species with the best pectinase activity was selected to evaluate their ability to produce pectic enzymes by submerged fermentation processes. The effect of different carbon sources on polygalacturonase (PGase) production was studied. Different vegetable tissues were used for tests of maceration with the crude enzyme extract. Among 154 yeast strains isolated, yeast Nº 111 was positive for pectinase activity and was identified as Geotrichum sp. Maximum PGase production obtained was 90.7 EU/ml when this yeast grown in shake flasks under the presence of glucose and citrus pectin as carbon sources. All tissues studied were macerated by the enzyme extract, microscopic examination showed a suspension of loose single cells. Lysis of cells was not observed.El objetivo del presente trabajo fue el aislamiento de cepas de levaduras con buena actividad pectinolítica. Las levaduras aisladas a partir de frutas cítricas fueron seleccionadas en base a sus actividades pectinolíticas. Se evaluó la capacidad de producción de enzimas pécticas por fermentación en medio líquido de la levadura con mayor actividad pectinolítica. Se estudió la influencia de diferentes fuentes de carbono sobre la producción de poligalacturonasa (PGasa) y la capacidad macerante de los extractos enzimáticos sobre tejidos vegetales. De 154 levaduras aisladas, la cepa Nº 111 mostró actividad pectinolítica y fue identificada como Geotrichum sp. La máxima producción de PGasa obtenida fue de 90,7 EU/ml, cuando esta levadura creció en medio líquido con glucosa y pectina de citrus como fuentes de carbono. Los tejidos vegetales estudiados fueron macerados por el extracto enzimático. El examen microscópico mostró células simples liberadas, no observándose lisis por destrucción de la pared celular.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

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

The weakest link: competence and prestige as constraints to referral by isolated nurses in rural Niger

BACKGROUND: For a health district to function referral from health centres to district hospitals is critical. In many developing countries referral systems perform well below expectations. Niger is not an exception in this matter. Beyond obvious problems of cost and access this study shows to what extent the behaviour of the health worker in its interaction with the patient can be a barrier of its own. METHODS: Information was triangulated from three sources in two rural districts in Niger: first, 46 semi-structured interviews with health centre nurses; second, 42 focus group discussions with an average of 12 participants – patients, relatives of patients and others; third, 231 semi-structured interviews with referred patients. RESULTS: Passive patients without 'voice' reinforce authoritarian attitudes of health centre staff. The latter appear reluctant to refer because they see little added value in referral and fear loss of power and prestige. As a result staff communicates poorly and show little eagerness to convince reluctant patients and families to accept referral proposals. CONCLUSIONS: Diminishing referral costs and distance barriers is not enough to correct failing referral systems. There is also a need for investment in district hospitals to make referrals visibly worthwhile and for professional upgrading of the human resources at the first contact level, so as to allow for more effective referral patterns

How well do adolescents recall use of mobile telephones? Results of a validation study

<p>Abstract</p> <p>Background</p> <p>In the last decade mobile telephone use has become more widespread among children. Concerns expressed about possible health risks have led to epidemiological studies investigating adverse health outcomes associated with mobile telephone use. Most epidemiological studies have relied on self reported questionnaire responses to determine individual exposure. We sought to validate the accuracy of self reported adolescent mobile telephone use.</p> <p>Methods</p> <p>Participants were recruited from year 7 secondary school students in Melbourne, Australia. Adolescent recall of mobile telephone use was assessed using a self administered questionnaire which asked about number and average duration of calls per week. Validation of self reports was undertaken using Software Modified Phones (SMPs) which logged exposure details such as number and duration of calls.</p> <p>Results</p> <p>A total of 59 adolescents participated (39% boys, 61% girls). Overall a modest but significant rank correlation was found between self and validated number of voice calls (ρ = 0.3, P = 0.04) with a sensitivity of 57% and specificity of 66%. Agreement between SMP measured and self reported duration of calls was poorer (ρ = 0.1, P = 0.37). Participants whose parents belonged to the 4^thsocioeconomic stratum recalled mobile phone use better than others (ρ = 0.6, P = 0.01).</p> <p>Conclusion</p> <p>Adolescent recall of mobile telephone use was only modestly accurate. Caution is warranted in interpreting results of epidemiological studies investigating health effects of mobile phone use in this age group.</p