Duplications and functional divergence of ADP-glucose pyrophosphorylase genes in plants

<p>Abstract</p> <p>Background</p> <p>ADP-glucose pyrophosphorylase (AGPase), which catalyses a rate limiting step in starch synthesis, is a heterotetramer comprised of two identical large and two identical small subunits in plants. Although the large and small subunits are equally sensitive to activity-altering amino acid changes when expressed in a bacterial system, the overall rate of non-synonymous evolution is ~2.7-fold greater for the large subunit than for the small subunit. Herein, we examine the basis for their different rates of evolution, the number of duplications in both large and small subunit genes and document changes in the patterns of AGPase evolution over time.</p> <p>Results</p> <p>We found that the first duplication in the AGPase large subunit family occurred early in the history of land plants, while the earliest small subunit duplication occurred after the divergence of monocots and eudicots. The large subunit also had a larger number of gene duplications than did the small subunit. The ancient duplications in the large subunit family raise concern about the saturation of synonymous substitutions, but estimates of the absolute rate of AGPase evolution were highly correlated with estimates of ω (the non-synonymous to synonymous rate ratio). Both subunits showed evidence for positive selection and relaxation of purifying selection after duplication, but these phenomena could not explain the different evolutionary rates of the two subunits. Instead, evolutionary constraints appear to be permanently relaxed for the large subunit relative to the small subunit. Both subunits exhibit branch-specific patterns of rate variation among sites.</p> <p>Conclusion</p> <p>These analyses indicate that the higher evolutionary rate of the plant AGPase large subunit reflects permanent relaxation of constraints relative to the small subunit and they show that the large subunit genes have undergone more gene duplications than small subunit genes. Candidate sites potentially responsible for functional divergence within each of the AGPase subunits were investigated by examining branch-specific patterns of rate variation. We discuss the phenotypes of mutants that alter some candidate sites and strategies for examining candidate sites of presently unknown function.</p

Enhancement of Heat Stability and Kinetic Parameters of the Maize Endosperm ADP-Glucose Pyrophosphorylase by Mutagenesis of Amino Acids in the Small Subunit With High B Factors

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in starch synthesis and previous studies showed that the heat lability of this enzyme is a determinant to starch synthesis in the maize endosperm and, in turn, seed yield. Here, amino acids in the AGPase endosperm small subunit with high B-factors were mutagenized and individual changes enhancing heat stability and/or kinetic parameters in an Escherichia coli expression system were chosen. Individual mutations were combined and analyzed. One triple mutant, here termed Bt2-BF, was chosen for further study. Combinations of this heat stable, 3-PGA-independent small subunit variant with large subunits also heat stable yielded complex patterns of heat stability and kinetic and allosteric properties. Interestingly, two of the three changes reside in a protein motif found only in AGPases that exhibit high sensitivity to 3-PGA. While not the 3-PGA binding site, amino acid substitutions in this region significantly alter 3-PGA activation kinetics

Mixed Bacterial Growth in Prenatal Urine Cultures; An Investigation into Prevalence, Contributory Factors and the Impact of education-based Interventions

PURPOSE: Undiagnosed urinary tract infections (UTIs) in pregnancy are associated with adverse perinatal outcome. Urine microbiology cultures reported as 'mixed bacterial growth' (MBG) frequently present a diagnostic dilemma for healthcare providers. We investigated external factors contributing to elevated rates of (MBG) within a large tertiary maternity centre in London, UK, and assessed the efficacy of health service interventions to mitigate these. DESCRIPTION: This prospective, observational study of asymptomatic pregnant women attending their first prenatal clinic appointment aimed to establish (i) the prevalence of MBG in routine prenatal urine microbiology cultures, (ii) the association between urine cultures and the duration to laboratory processing and (iii) ways in which MBG may be reduced in pregnancy. Specifically we assessed the impact of patient-clinician interaction and that of an education package on optimal urine sampling technique. ASSESSMENT: Among 212 women observed over 6 weeks, the negative, positive and MBG urine culture rates were 66%, 10% and 2% respectively. Shorter duration from urine sample collection to laboratory arrival correlated with higher rates of negative cultures. Urine samples arriving in the laboratory within 3 hours of collection were most likely to be reported as culture negative (74%), and were least likely to be reported as MBG (21%) or culture positive (6%), compared to samples arriving > 6 hours (71%, 14% and 14% respectively; P < 0.001). A midwifery education package effectively reduced rates of MBG (37% pre-intervention vs 19% post-intervention, RR 0.70, 95% CI 0.55 to 0.89). Women who did not receive verbal instructions prior to providing their sample had 5-fold higher rates of MBG (P < 0.001). CONCLUSION: As many as 24% of prenatal urine screening cultures are reported as MBG. Patient-midwife interaction before urine sample collection and rapid transfer of urine samples to the laboratory within 3 hours reduces the rate of MBG in prenatal urine cultures. Reinforcing this message through education may improve accuracy of test results

Strategies to Reduce Non-Ventilator-Associated Hospital-Acquired Pneumonia: A Systematic Review

Background Point prevalence studies identify that pneumonia is the most common healthcare associated infection. However, non-ventilator associated healthcare associated pneumonia (NV-HAP) is both underreported and understudied. Most research conducted to date, focuses on ventilator associated pneumonia. We conducted a systematic review, to provide the latest evidence for strategies to reduce NV-HAP and describe the methodological approaches used. Methods We performed a systematic search to identify research exploring and evaluating NV-HAP preventive measures in hospitals and aged-care facilities. The electronic database Medline was searched, for peer-reviewed articles published between 1st January 1998 and 31st August 2018. An assessment of the study quality and risk of bias of included articles was conducted using the Newcastle–Ottawa Scale. Results The literature search yielded 1551 articles, with 15 articles meeting the inclusion criteria. The majority of strategies for NV-HAP prevention focussed on oral care (n = 9). Three studies evaluated a form of physical activity, such as passive movements, two studies used dysphagia screening and management; and another study evaluated prophylactic antibiotics. Most studies (n = 12) were conducted in a hospital setting. Six of the fifteen studies were randomised controlled trials. Conclusion There was considerable heterogeneity in the included studies, including the type of intervention, study design, methods and definitions used to diagnose the NV-HAP. To date, interventions to reduce NV-HAP appear to be based broadly on the themes of improving oral care, increased mobility or movement and dysphagia management

Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize

Helitrons are a family of mobile elements that were discovered in 2001 and are now known to exist in the entire eukaryotic kingdom. Helitrons, particularly those of maize, exhibit an intriguing property of capturing gene fragments and placing them into the mobile element. Helitron-captured genes are sometimes transcribed, giving birth to chimeric transcripts that intertwine coding regions of different captured genes. Here, we perused the B73 maize genome for high-quality, putative Helitrons that exhibit plus/minus polymorphisms and contain pieces of more than one captured gene. Selected Helitrons were monitored for expression via in silico EST analysis. Intriguingly, expression validation of selected elements by RT–PCR analysis revealed multiple transcripts not seen in the EST databases. The differing transcripts were generated by alternative selection of splice sites during pre-mRNA processing. Selection of splice sites was not random since different patterns of splicing were observed in the root and shoot tissues. In one case, an exon residing in close proximity but outside of the Helitron was found conjoined with Helitron-derived exons in the mature transcript. Hence, Helitrons have the ability to synthesize new genes not only by placing unrelated exons into common transcripts, but also by transcription readthrough and capture of nearby exons. Thus, Helitrons have a phenomenal ability to “display” new coding regions for possible selection in nature. A highly conservative, minimum estimate of the number of new transcripts expressed by Helitrons is ∼11,000 or ∼25% of the total number of genes in the maize genome

The endorsement of cognitive distortions: comparing child pornography offenders and contact sex offenders

This study examined the endorsement of cognitive distortions in child pornography offenders (CPOs), using an established assessment tool, the Abel and Becker Cognition Scale. The scale was expanded to include cognitions specific to child pornography offending, extracted from Howitt and Sheldon’s Children and Sexual Activities Inventory (C&SA). Three samples of CPOs, child sex offenders and offenders with both offence types responded to the cognition items. An exploratory Principal Component Analysis suggested six main components of the scale. CPOs were significantly less likely to endorse these statements in general, and this was more pronounced on items that project blame onto the child or other people, describe a need for power and consider children as sexually active. The statements extracted from C&SA did not differentiate between the groups. These findings are discussed under consideration of the relationship between cognitive distortions and contact sex offending, and in reference to the general criticism concerning the definition and appropriate measurement of cognitive distortion

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression

Integrating ecosystem markets to co-ordinate landscape-scale public benefits from nature

Ecosystem markets are proliferating around the world in response to increasing demand for climate change mitigation and provision of other public goods. However, this may lead to perverse outcomes, for example where public funding crowds out private investment or different schemes create trade-offs between the ecosystem services they each target. The integration of ecosystem markets could address some of these issues but to date there have been few attempts to do this, and there is limited understanding of either the opportunities or barriers to such integration. This paper reports on a comparative analysis of eleven ecosystem markets in operation or close to market in Europe, based on qualitative analysis of 25 interviews, scheme documentation and two focus groups. Our results indicate three distinct types of markets operating from the regional to national scale, with different modes of operation, funding and outcomes: regional ecosystem markets, national carbon markets and green finance. The typology provides new insights into the operation of ecosystem markets in practice, which may challenge traditionally held notions of Payment for Ecosystem Services. Regional ecosystem markets, in particular, represent a departure from traditional models, by using a risk-based funding model and aggregating both supply and demand to overcome issues of free-riding, ecosystem service trade-offs and land manager engagement. Central to all types of market were trusted intermediaries, brokers and platforms to aggregate supply and demand, build trust and lower transaction costs. The paper outlines six options for blending public and private funding for the provision of ecosystem services and proposes a framework for integrating national carbon markets and green finance with regional ecosystem markets. Such integration may significantly increase funding for regenerative agriculture and conservation across multiple habitats and services, whilst addressing issues of additionality and ecosystem service trade-offs between multiple schemes

Home-based pre-surgical psychological intervention for knee osteoarthritis (HAPPiKNEES): a feasibility randomized controlled trial

Objective: To determine the feasibility of conducting a trial of a pre-surgical psychological intervention on pain, function, and mood in people with knee osteoarthritis listed for total knee arthroplasty. Design: Multi-centre, mixed-methods feasibility randomized controlled trial of intervention plus usual care versus usual care. Setting: Participants’ homes or hospital. Participants: Patients with knee osteoarthritis listed for total knee arthroplasty and score >7 on either subscales of Hospital Anxiety and Depression Scale. Intervention: Up-to 10 sessions of psychological intervention (based on cognitive behavioural therapy). Main measures: Feasibility outcomes (recruitment and retention rates, acceptability of trial procedures and intervention, completion of outcome measures), and standardized questionnaires assessing pain, function, and mood at baseline, and four and six months post-randomisation. Results: Of 222 people screened, 81 did not meet inclusion criteria, 64 did not wish to participate, 26 were excluded for other reasons, and 51 were randomized. A total of 30 completed 4-months outcomes and 25 completed 6-month outcomes. Modal number of intervention sessions completed was three (range 2–8). At six-month follow-up, mood, pain, and physical function scores were consistent with clinically important benefits from intervention, with effect sizes ranging from small (d = 0.005) to moderate (d = 0.74), and significant differences in physical function between intervention and usual care groups (d = 1.16). Feedback interviews suggested that participants understood the rationale for the study, found the information provided adequate, the measures comprehensive, and the intervention acceptable. Conclusion: A definitive trial is feasible, with a total sample size of 444 people. Pain is a suitable primary outcome, but best assessed 6 and 12 months post-surgery