Hypermutator emergence in experimental Escherichia coli populations is stress-type dependent.

Genotypes exhibiting an increased mutation rate, called hypermutators, can propagate in microbial populations because they can have an advantage due to the higher supply of beneficial mutations needed for adaptation. Although this is a frequently observed phenomenon in natural and laboratory populations, little is known about the influence of parameters such as the degree of maladaptation, stress intensity, and the genetic architecture for adaptation on the emergence of hypermutators. To address this knowledge gap, we measured the emergence of hypermutators over ~1,000 generations in experimental Escherichia coli populations exposed to different levels of osmotic or antibiotic stress. Our stress types were chosen based on the assumption that the genetic architecture for adaptation differs between them. Indeed, we show that the size of the genetic basis for adaptation is larger for osmotic stress compared to antibiotic stress. During our experiment, we observed an increased emergence of hypermutators in populations exposed to osmotic stress but not in those exposed to antibiotic stress, indicating that hypermutator emergence rates are stress type dependent. These results support our hypothesis that hypermutator emergence is linked to the size of the genetic basis for adaptation. In addition, we identified other parameters that covaried with stress type (stress level and IS transposition rates) that might have contributed to an increased hypermutator provision and selection. Our results provide a first comparison of hypermutator emergence rates under varying stress conditions and point towards complex interactions of multiple stress-related factors on the evolution of mutation rates.

Tradeoff breaking as a model of evolutionary transitions in individuality and limits of the fitness-decoupling metaphor.

Evolutionary transitions in individuality (ETIs) involve the formation of Darwinian collectives from Darwinian particles. The transition from cells to multicellular life is a prime example. During an ETI, collectives become units of selection in their own right. However, the underlying processes are poorly understood. One observation used to identify the completion of an ETI is an increase in collective-level performance accompanied by a decrease in particle-level performance, for example measured by growth rate. This seemingly counterintuitive dynamic has been referred to as fitness decoupling and has been used to interpret both models and experimental data. Extending and unifying results from the literature, we show that fitness of particles and collectives can never decouple because calculations of fitness performed over appropriate and equivalent time intervals are necessarily the same provided the population reaches a stable collective size distribution. By way of solution, we draw attention to the value of mechanistic approaches that emphasise traits, and tradeoffs among traits, as opposed to fitness. This trait-based approach is sufficient to capture dynamics that underpin evolutionary transitions. In addition, drawing upon both experimental and theoretical studies, we show that while early stages of transitions might often involve tradeoffs among particle traits, later-and critical-stages are likely to involve the rupture of such tradeoffs. Thus, when observed in the context of ETIs, tradeoff-breaking events stand as a useful marker of these transitions.

Development and testing of the eye-pointing classification scale for children with cerebral palsy.

PURPOSE: The aim of this study was to develop and test a new classification scale to describe looking behaviours (gaze fixations and gaze shifts) in relation to eye-pointing. METHODS: The Eye-pointing Classification Scale (EpCS) was developed and tested following established procedures for the construction and evaluation of equivalent scales, and involved 2 phases: Drawing on research literature, Phase 1 involved initial drafting of the scale through a series of multi-disciplinary group discussions; evaluation of the scale through a survey procedure, and subsequent expert group evaluation. Phase 2, was an examination of scale reliability and relationships between child characteristics and level of EpCS classification. RESULTS: In Phase 1, an initial draft of the scale was developed and then evaluated by 52 participants in 10 countries, leading to its refinement. Subsequent expert evaluation of content, style and structure indicated that no further refinement was required. In Phase 2, the scale achieved excellent levels of reliability in clinical testing. A significant relationship was identified between level of child motor ability and EpCS classification, and level of child language understanding and EpCS classification.Implications for rehabilitationNon-speaking children with severe bilateral cerebral palsy who have limited upper limb movement may communicate by using controlled looking behaviours to point to objects and people, referred to as eye-pointing.However, there is little consensus as to which looking behaviours represent eye-pointing and which do not.The Eye-pointing Classification Scale (EpCS) was developed to describe looking behaviours related to eye-pointing in this population of childrenThe EpCS provides a new robust tool for clinical management and research with children with cerebral palsy.

Read between the Lines: Diversity of Nontranslational Selection Pressures on Local Codon Usage.

Protein coding genes can contain specific motifs within their nucleotide sequence that function as a signal for various biological pathways. The presence of such sequence motifs within a gene can have beneficial or detrimental effects on the phenotype and fitness of an organism, and this can lead to the enrichment or avoidance of this sequence motif. The degeneracy of the genetic code allows for the existence of alternative synonymous sequences that exclude or include these motifs, while keeping the encoded amino acid sequence intact. This implies that locally, there can be a selective pressure for preferentially using a codon over its synonymous alternative in order to avoid or enrich a specific sequence motif. This selective pressure could-in addition to mutation, drift and selection for translation efficiency and accuracy-contribute to shape the codon usage bias. In this review, we discuss patterns of avoidance of (or enrichment for) the various biological signals contained in specific nucleotide sequence motifs: transcription and translation initiation and termination signals, mRNA maturation signals, and antiviral immune system targets. Experimental data on the phenotypic or fitness effects of synonymous mutations in these sequence motifs confirm that they can be targets of local selection pressures on codon usage. We also formulate the hypothesis that transposable elements could have a similar impact on codon usage through their preferred integration sequences. Overall, selection on codon usage appears to be a combination of a global selection pressure imposed by the translation machinery, and a patchwork of local selection pressures related to biological signals contained in specific sequence motifs.

Germ lines and extended selection during the evolutionary transition to multicellularity.

The major evolutionary transitions from unicellular organisms to multicellularity resulted in a profusion of complex life forms. During the transition from single cells to multicellular life, groups of cells acquired the capacity for reproduction as discrete units; however, the selective causes and underlying mechanisms remain debated. One perspective views the evolution of multicellularity as a shift in the timescale at which natural selection primarily operates-from that of individual cells to the timescale of reproducing groups of cells. Therefore, a distinguishing feature of multicellular reproduction, as opposed to simple growth of a multicellular collective, is that the capacity for reproduction must develop over a timescale that is greater than the reproductive timescale of a single cell. Here, I suggest that the emergence of specialized reproductive cells (the germ line) was an essential first stage of the evolutionary transition to multicellularity because it imposed the necessary "delay"-allowing natural selection to operate over the longer timescale of a multicellular life cycle, ultimately resulting in the evolution of complex multicellular organisms. This perspective highlights the possibility that the ubiquity of a germ-soma distinction among complex multicellular organisms reflects the fact that such life cycles, on first emergence, had the greatest propensity to participate in Darwinian evolution.

Meta-population structure and the evolutionary transition to multicellularity.

The evolutionary transition to multicellularity has occurred on numerous occasions, but transitions to complex life forms are rare. Here, using experimental bacterial populations as proxies for nascent multicellular organisms, we manipulate ecological factors shaping the evolution of groups. Groups were propagated under regimes requiring reproduction via a life cycle replete with developmental and dispersal (propagule) phases, but in one treatment lineages never mixed, whereas in a second treatment, cells from different lineages experienced intense competition during the dispersal phase. The latter treatment favoured traits promoting cell growth at the expense of traits underlying group fitness - a finding that is supported by results from a mathematical model. Our results show that the transition to multicellularity benefits from ecological conditions that maintain discreteness not just of the group (soma) phase, but also of the dispersal (germline) phase.

Pre-hospital anaesthesia and assessment of head injured patients presenting to a UK Helicopter Emergency Medical Service with a high Glasgow Coma Scale: a cohort study.

OBJECTIVES: Patients who sustain a head injury but maintain a Glasgow Coma Scale (GCS) of 13-15 may still be suffering from a significant brain injury. We aimed to assess the appropriateness of triage and decision to perform prehospital rapid sequence induction (RSI) in patients attended by a UK Helicopter Emergency Medical Service (HEMS) following head injury. DESIGN: A retrospective cohort study of patients attended by Kent Surrey & Sussex Air Ambulance Trust (KSSAAT) HEMS. SETTING: A mixed urban and rural area of 4.5 million people in South East England. PARTICIPANTS: GCS score of 13, 14 or 15 on arrival of the HEMS team and clinical findings suggesting head injury. Patients accompanied by the HEMS team to hospital ('Escorted'), and those that were 'Assisted' but conveyed by the ambulance service were reviewed. No age restrictions to inclusion were set. PRIMARY OUTCOME MEASURE: Significant brain injury. SECONDARY OUTCOME MEASURE: Recognition of patients requiring prehospital anaesthesia for head injury. RESULTS: Of 517 patients, 321 had adequate follow-up, 69% of these were Escorted, 31% Assisted. There was evidence of intracranial injury in 13.7% of patients and clinically important brain injury in 7.8%. There was no difference in the rate of clinically important brain injury between Escorted and Assisted patients (p=0.46). Nineteen patients required an RSI by the HEMS team and this patient group was significantly more likely to have clinically important brain injury (p=0.04). CONCLUSION: In patients attended by a UK HEMS service with a head injury and a GCS of 13-15, a small but significant proportion had a clinically important brain injury and a proportion were appropriately recognised as requiring prehospital RSI. For patients deemed not to need a HEMS intervention, differentiating between those with and without clinically important brain injury appears challenging. LEVEL OF EVIDENCE: V.

Journey towards universal viral load monitoring in Maputo, Mozambique: many gaps, but encouraging signs.

Introduction: Viral load (VL) monitoring for people on antiretroviral therapy (ART) is extremely challenging in resource-limited settings. We assessed the VL testing scale-up in six Médecins Sans Frontières supported health centres in Maputo, Mozambique, during 2014-15. Methods: In a retrospective cohort study, routine programme data were used to describe VL testing uptake and results, and multi-variate logistical regression to estimate predictors of VL testing uptake and suppression. Results: Uptake of a first VL test was 40% (17 236/43 579). Uptake of a follow-up VL test for patients with a high first VL result was 35% (1095/3100). Factors associated with a higher uptake included: age below 15 years, longer time on ART and attending tailored service delivery platforms. Virological suppression was higher in pregnant/breastfeeding women and in community ART Group members. Patients with a high first VL result (18%; 3100/17 236) were mostly younger, had been on ART longer or had tuberculosis. Out of 1095 attending for a follow-up VL test, 678 (62%) had virological failure. Of those, less than one-third had started second line ART. Conclusion: This was the first study describing the uptake and results of VL testing scale-up in Mozambique. Identified gaps show patient and programmatic challenges. Where service delivery was customized to patient needs, VL monitoring was more successful.

Life cycles, fitness decoupling and the evolution of multicellularity.

Cooperation is central to the emergence of multicellular life; however, the means by which the earliest collectives (groups of cells) maintained integrity in the face of destructive cheating types is unclear. One idea posits cheats as a primitive germ line in a life cycle that facilitates collective reproduction. Here we describe an experiment in which simple cooperating lineages of bacteria were propagated under a selective regime that rewarded collective-level persistence. Collectives reproduced via life cycles that either embraced, or purged, cheating types. When embraced, the life cycle alternated between phenotypic states. Selection fostered inception of a developmental switch that underpinned the emergence of collectives whose fitness, during the course of evolution, became decoupled from the fitness of constituent cells. Such development and decoupling did not occur when groups reproduced via a cheat-purging regime. Our findings capture key events in the evolution of Darwinian individuality during the transition from single cells to multicellularity.

Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae).

The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a horticultural pest of Australia and New Zealand that has more recently invaded Hawaii, Europe, and California. A 2,216-bp region of the mitochondrial genome containing the cytochrome oxidase I and II genes was sequenced from 752 individuals. Haplotype network analyses revealed a major split between a predominantly Western Australian clade and all other samples, suggestive of either a deep genetic divergence or a cryptic species. Nucleotide and haplotype diversity were highest in the country of origin, Australia, and in New Zealand populations, with evidence of haplotype sharing between New Zealand and Tasmania. Nucleotide and haplotype diversity were higher in California than within the British Isles or Hawaii. From the total of 96 haplotypes, seven were found in California, of which four were private. Within California, there have been at least two introductions; based on genetic diversity we were unable to assign a likely source for a single moth found and eradicated in Los Angeles in 2007; however, our data suggest it is unlikely that Hawaii and the British Isles are sources of the major E. postvittana population found throughout the rest of the state since 2006.

Selective sweeps at the organophosphorus insecticide resistance locus, Rop-1, have affected variation across and beyond the α-esterase gene cluster in the Australian sheep blowfly, Lucilia cuprina.

A major theoretical consequence of selection at a locus is the genetic hitchhiking of linked sites (selective sweep). The extent of hitchhiking around a gene is related to the strength of selection and the rate of recombination, with its impact diminishing with distance from the selected site. At the Rop-1 locus of the sheep blowfly, Lucilia cuprina, polymorphisms at two different sites within the LcαE7 gene encode forms of the protein that confer organophosphorus insecticide resistance. To assess the impact of selection at these two sites on variation around LcαE7, we sequenced regions within six other genes along chromosome IV across isogenic (IV) strains of L. cuprina. High levels of linkage disequilibrium, characterized by low haplotype number (K) and diversity (H), and significant R(2) values were observed for two genes, LcαE1 and LcαE10, both members of the same α-esterase gene cluster as LcαE7. A significant R(2) value was also observed for a gene predicted to be the next closest to LcαE7, AL03, but not for any of the other genes, LcRpL13a, Lcdsx, or LcAce. Skews in the site frequency spectra toward high-frequency variants were significant for LcαE1 (Fay and Wu's H = -2.91), LcαE10 (H = -1.85), and Lcdsx (H = -2.00). Since the selective sweeps, two forms of likely returning variation were observed, including variation in microsatellites in an intron of LcαE10 and a recombination event between LcαE7 and LcαE10. These data suggest that two incomplete soft sweeps have occurred at LcαE7 that have significantly affected variation across, and beyond, the α-esterase gene cluster of L. cuprina. The speed and impact of these selective sweeps on surrounding genomic variation and the ability of L. cuprina to respond to future environmental challenges are discussed.

A study into the children's palliative care educational needs of health professionals in Uganda.

STUDY AIM: To research the children's palliative care (CPC) educational needs of health professionals in Uganda. METHODOLOGY DESIGN AND SETTING: Mixed quantitative and qualitative survey set in three hospice sites in Uganda. INTERVENTIONS: Self-rating survey, log book of problem cases, focus group of students of a CPC course. MAIN OUTCOME MEASURES: Self-rated "usefulness of further training" scores for CPC subject areas; thematic analysis of log books; thematic analysis of focus group findings. RESPONDENTS: All health professionals (n = 50) were invited and 48 (96%) consented to participate. ETHICAL CONSIDERATIONS: The study was approved by the Hospice Africa Uganda (HAU) Research and Ethics Committee. RESULTS: Communication with children rated highest in all three arms of the study. SELF-RATING SURVEY: Average score = 8.3 of 10; range = 6.4 of 10 to 9.5 of 10. Communication with children, pain management, and psychological issues rated highest, and technical subject areas predominated. LOG BOOK ANALYSIS: Strongest themes were communicating with children and families, team-working, and managing personal stress. FOCUS GROUP ANALYSIS: Strongest themes were communicating with children, assessment and management planning, and managing personal stress. DISCUSSION AND CONCLUSION: There is educational need for all CPC subject areas across the board, but communication with children is the most pressing. There are disparities between recognized learning needs (technical skills predominating) and unrecognized learning needs (interpersonal and intrapersonals skills predominating). While the broad subject areas for CPC may be similar in resource-rich and resource-poor settings, educational resources developed for the specific context of African and other resource poor settings are required.

The beginnings of children's palliative care in Africa: evaluation of a children's palliative care service in Africa.

AIM: To evaluate a children's palliative care service designed specifically for a resource-poor sub-Saharan African setting. METHODOLOGY: The study used mixed quantitative and qualitative methodology: quantitative retrospective, comparative survey and cross-sectional, noninterventional interview survey. RESULTS: Evaluation showed increases in referrals, proportion of children on program, morphine and chemotherapy prescriptions, and improved compliance for a cost of $100 per child. The most valued service strengths were free drugs, food, play, learning, and staff attitude. Weaknesses included insufficiency of strengths listed above, as well as poor hospital staff attitude, lack of school fees and poor treatment compliance rates. Suggestions included more of the strengths as well as more accessible service locations. DISCUSSION: The study suggests affordable, nurse-led, volunteer-supported children's palliative care services are both achievable and effective in sub-Saharan African. The study suggests that palliative care units should provide a specialized service focused on children. Such a service would clearly identify children in need of children's palliative care and should provide medication for symptom control; food and basic needs support; play and learning facilities; child protection; and systems for patient education, communication and follow up. Staff lack confidence and/or competence and this is a significant barrier to children's palliative care that should be addressed in Africa.

Biotinylation of glycan chains in beta2 glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9.

Binding of beta2GPI (beta2 glycoprotein I), a human plasma protein, to AnPLs (anionic phospholipids) plays a key role in the formation of antiphospholipid antibodies involved in autoimmune diseases like antiphospholipid syndrome or systemic lupus erythematosus. We recently showed that binding of beta2GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of beta2GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass beta2GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized beta2GPI and in beta2GPI-bh (beta2GPI-biotin-hydrazide), but were absent in native beta2GPI; (ii) binding of beta2GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native beta2GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer beta2GPI. However, in solution at 1 microM beta2GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of beta2GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native beta2GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to beta2GPI-bh but did not bind to monomeric native and oxidized beta2GPI. It is likely that the dimeric quaternary structure of beta2GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody.