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

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

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

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

Systematic review of epidemiological studies on health effects associated with management of solid waste

Background: Management of solid waste (mainly landfills and incineration) releases a number of toxic substances, most in small quantities and at extremely low levels. Because of the wide range of pollutants, the different pathways of exposure, long-term low-level exposure, and the potential for synergism among the pollutants, concerns remain about potential health effects but there are many uncertainties involved in the assessment. Our aim was to systematically review the available epidemiological literature on the health effects in the vicinity of landfills and incinerators and among workers at waste processing plants to derive usable excess risk estimates for health impact assessment.Methods: We examined the published, peer-reviewed literature addressing health effects of waste management between 1983 and 2008. For each paper, we examined the study design and assessed potential biases in the effect estimates. We evaluated the overall evidence and graded the associated uncertainties.Results: In most cases the overall evidence was inadequate to establish a relationship between a specific waste process and health effects; the evidence from occupational studies was not sufficient to make an overall assessment. For community studies, at least for some processes, there was limited evidence of a causal relationship and a few studies were selected for a quantitative evaluation. In particular, for populations living within two kilometres of landfills there was limited evidence of congenital anomalies and low birth weight with excess risk of 2 percent and 6 percent, respectively. The excess risk tended to be higher when sites dealing with toxic wastes were considered. For populations living within three kilometres of old incinerators, there was limited evidence of an increased risk of cancer, with an estimated excess risk of 3.5 percent. The confidence in the evaluation and in the estimated excess risk tended to be higher for specific cancer forms such as non-Hodgkin's lymphoma and soft tissue sarcoma than for other cancers.Conclusions: The studies we have reviewed suffer from many limitations due to poor exposure assessment, ecological level of analysis, and lack of information on relevant confounders. With a moderate level confidence, however, we have derived some effect estimates that could be used for health impact assessment of old landfill and incineration plants. The uncertainties surrounding these numbers should be considered carefully when health effects are estimated. It is clear that future research into the health risks of waste management needs to overcome current limitations