FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations

Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >105 reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations.Organismic and Evolutionary Biolog

The Ability of Flux Balance Analysis to Predict Evolution of Central Metabolism Scales with the Initial Distance to the Optimum

The most powerful genome-scale framework to model metabolism, flux balance analysis (FBA), is an evolutionary optimality model. It hypothesizes selection upon a proposed optimality criterion in order to predict the set of internal fluxes that would maximize fitness. Here we present a direct test of the optimality assumption underlying FBA by comparing the central metabolic fluxes predicted by multiple criteria to changes measurable by a 13C-labeling method for experimentally-evolved strains. We considered datasets for three Escherichia coli evolution experiments that varied in their length, consistency of environment, and initial optimality. For ten populations that were evolved for 50,000 generations in glucose minimal medium, we observed modest changes in relative fluxes that led to small, but significant decreases in optimality and increased the distance to the predicted optimal flux distribution. In contrast, seven populations evolved on the poor substrate lactate for 900 generations collectively became more optimal and had flux distributions that moved toward predictions. For three pairs of central metabolic knockouts evolved on glucose for 600–800 generations, there was a balance between cases where optimality and flux patterns moved toward or away from FBA predictions. Despite this variation in predictability of changes in central metabolism, two generalities emerged. First, improved growth largely derived from evolved increases in the rate of substrate use. Second, FBA predictions bore out well for the two experiments initiated with ancestors with relatively sub-optimal yield, whereas those begun already quite optimal tended to move somewhat away from predictions. These findings suggest that the tradeoff between rate and yield is surprisingly modest. The observed positive correlation between rate and yield when adaptation initiated further from the optimum resulted in the ability of FBA to use stoichiometric constraints to predict the evolution of metabolism despite selection for rate

Development of an Optimized Medium, Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.Organismic and Evolutionary Biolog

Percutaneous versus surgical strategy for tracheostomy: protocol for a systematic review and meta-analysis of perioperative and postoperative complications

Background: Tracheostomy is one of the most frequently performed procedures in intensive care medicine. The two main approaches to form a tracheostoma are the open surgical tracheotomy (ST) and the interventional strategy of percutaneous dilatational tracheotomy (PDT). It is particularly important to the critically ill patients that both procedures are performed with high success rates and low complication frequencies. Therefore, the aim of this systematic review is to summarize and analyze existing and relevant evidence for peri- and postoperative parameters of safety. Methods/design: A systematic literature search will be conducted in The Cochrane Library, MEDLINE, LILACS, and Embase to identify all randomized controlled trials (RCTs) comparing peri- and postoperative complications between the two strategies and to define the strategy with the lower risk of potentially life-threatening events. A priori defined data will be extracted from included studies, and methodological quality will be assessed according to the recommendations of the Cochrane Collaboration. Discussion: The findings of this systematic review with proportional meta-analysis will help to identify the strategy with the lowest frequency of potentially life-threatening events. This may influence daily practice, and the data may be implemented in treatment guidelines or serve as the basis for planning further randomized controlled trials. Considering the critical health of these patients, they will particularly benefit from evidence-based treatment. Systematic review registration: PROSPERO CRD4201502196

Improving Adherence to Essential Birth Practices Using the WHO Safe Childbirth Checklist With Peer Coaching: Experience From 60 Public Health Facilities in Uttar Pradesh, India.

BACKGROUND: Adherence to evidence-based essential birth practices is critical for improving health outcomes for mothers and newborns. The WHO Safe Childbirth Checklist (SCC) incorporates these practices, which occur during 4 critical pause points: on admission, before pushing (or cesarean delivery), soon after birth, and before discharge. A peer-coaching strategy to support consistent use of the SCC may be an effective approach to increase birth attendants' adherence to these practices. METHODS: We assessed data from 60 public health facilities in Uttar Pradesh, India, that received an 8-month staggered coaching intervention from December 2014 to September 2016 as part of the BetterBirth Trial, which is studying effectiveness of an SCC-centered intervention on maternal and neonatal harm. Nurse coaches recorded birth attendants' adherence to 39 essential birth practices. Practice adherence was calculated for each intervention month. After 2 months of coaching, a subsample of 15 facilities was selected for independent observation when the coach was not present. We compared adherence to the 18 practices recorded by both coaches and independent observers. RESULTS: Coaches observed birth attendants' behavior during 5,971 deliveries. By the final month of the intervention, 35 of 39 essential birth practices had achieved >90% adherence in the presence of a coach, compared with only 7 of 39 practices during the first month. Key behaviors with the greatest improvement included explanation of danger signs, temperature measurement, assessment of fetal heart sounds, initiation of skin-to-skin contact, and breastfeeding. Without a coach present, birth attendants' average adherence to practices and checklist use was 24 percentage points lower than when a coach was present (range: -1% to 62%). CONCLUSION: Implementation of the WHO Safe Childbirth Checklist with coaching improved uptake of and adherence to essential birth practices. Coordination and communication among facility staff, as well as behaviors with an immediate, tangible benefit, showed the greatest improvement. Difficult-to-perform behaviors and those with delayed or theoretical benefits were less likely to be sustained without a coach present. Coaching may be an important component in implementing the Safe Childbirth Checklist at scale.Note: At the time of publication of this article, the results of evaluation of the impact of the BetterBirth intervention were pending publication in another journal. After the impact findings have been published, we will update this article on the effect of the intervention on birth practices with a reference to the impact findings

Advancing the research agenda on food systems governance and transformation

The food systems upon which humanity depends face multiple interdependent environmental, social and economic threats in the 21st Century. Yet, the governance of these systems, which determines to a large extent the ability to adapt and transform in response to these challenges, is underresearched. This perspective piece synthesises the findings of two recent reviews of food systems governance and transformations and proposes a comprehensive research agenda for the coming years. These reviews highlight the influence of governance on food systems, methodological obstacles to explaining the effectiveness of governance in realising food sustainability, and conditions that have historically supported food system transformations. We argue that the following steps are key to improving our knowledge of the role of governance in food systems: (1) developing more comparable research designs for building generalisable explanations of the governance elements that are most effective in realising food systems goals; (2) using the lens of polycentricity to help disentangle complex governance networks; (3) giving greater attention to the conditions and pre-conditions associated with historical food system transformations; (4) identifying adaptations that strengthen or weaken path dependency; and, (5) focusing research on how transformations can be supported by institutions that facilitate collective action and stakeholder agency

The value of health care – a matter of discussion in Germany

BACKGROUND: Interest in assessing the value of health-care services in Germany has considerably increased since the foundation of the Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, IQWiG (Institute for Quality and Efficiency in Health Care). The practical application of value assessment illustrates how problematic the process can be. In all decisions made for the provision of health care, data concerning the measurable dimensions (quantity and quality of efficacy and effectiveness, validity of the results and costs) flow into a complex and not yet standardized decision-making process concerning public financing. Some of these decisions are based on data of uncertain validity, unknown reproducibility and unclear appropriateness. DISCUSSION: In this paper we describe the theoretical aspects of value from psychological and economic viewpoints and discuss national and international approaches. Methodic details and difficulties in assessing the value of health-care services are analysed. A definition of the intangible value of health-care services will be proposed which contains only three factors: the absolute risk reduction (usually a measure of efficacy), the validity of the scientific papers examined and the type of the expected effectiveness (prevention of death and disability, restitution of well-being). The intangible value describes the additional benefit when comparing two possible actions, like treatment or observation only. CONCLUSION: The description of intangible value from the viewpoint of different stakeholders is a useful measure for subsequent steps (not discussed here) – the evaluation of costs and of patient benefit. A standardised, transparent, fair and democratic evaluation is essential for the definition of a basic benefit package