Public Participation in Risk Management Decisions: The Right to Define, The Right to Know and the Right to Act

It is important to solicit public opinions before making decisions about Risk, but this is seen as only a first step. The author observes that it is also important that the public be involved in defining the problem, identifying needed information, interpreting information, and choosing among the options for action

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals. Introduction Enzymes are Nature's catalysts, tremendously accelerating the rates of a wide range of biochemical reactions, often with exquisite specificity. Harnessing enzymes for other purposes usually requires engineering them to improve their activity or stability. One approach to engineering enzymes is to make specific modifications, but this demands a detailed and frequently unattainable understanding of the relationship between sequence and function. Directed evolution bypasses this problem in much the same way as natural evolution, by combining mutation with selection or screening to identify improved variants. Because it is never possible to test more than an infinitesimal fraction of the vast number of possible protein sequences, it is essential to have a strategy for creating directed evolution sequence libraries that are rich in proteins with the desired enzymatic function. Such libraries can be designed by drawing on our knowledge of how proteins respond to mutation Directed evolution strategies Directed evolution works when the researcher can find at least one enzyme with improved properties in the sequence library. The most naïve strategy of creating a library of random protein sequences is not useful for most enzyme engineering goals. Although sequences with simple functions such as ATP binding Most directed evolution strategies involve making relatively small changes to existing enzymes. This takes advantage of the fact that enzymes often have a range of weak promiscuous activities that are quickly improved with just a few mutations Random mutagenesis The most straightforward strategy for library construction is to randomly mutate the full gene of an enzyme with a function close to the desired function. This approach requires no structural or mechanistic information, and can uncover unexpected beneficial mutations. Using sequential rounds of error-prone PCR to make an average of a few mutations per gene, followed by screening or selection for improved variants, is effective for a wide range of engineering goals. The creation of enantioselective catalysts from an enzyme whose structure is unknown is one such application. A single round of error-prone PCR produced several dozen cyclohexane monooxygenases with R or S selectivity Beneficial mutations found by random mutagenesis can be combined by DNA shuffling. A study with b-glucuronidase showed that beneficial mutations drive each other to extinction during recursive random mutagenesis, but that this problem can be eliminated by DNA shuffling Random mutagenesis can also uncover additional beneficial mutations in rationally designed enzymes. The Withers laboratory Targeted mutagenesis Some engineering goals, such as dramatically altering an enzyme's specificity or regioselectivity, may require mul- Random mutagenesis, targeted mutagenesis and recombination are three strategies for producing sequence libraries for directed evolution. (a) Random mutagenesis introduces amino acid substitutions throughout the protein and can uncover beneficial mutations distant from the active site. The red residues in the structure at top show four mutations uncovered by random mutagenesis that enhanced the activity of mammalian cytochrome P450 2B1 on several substrates Using a high-resolution crystal structure to target mutagenesis to three active site residues, Hill et al. [23] created a triple mutant of phosphotriesterase with a rate enhancement of three orders of magnitude for the degradation of organic triesters such as those used in chemical warfare agents. Crucially, two of the corresponding single mutants did not increase activity and so would not have been identified if they had been explored one at a time. The problem of inverting the enantioselectivity of a lipase offers an interesting comparison between full-gene random mutagenesis and targeted mutagenesis. Reetz and co-workers [24] used several rounds of full-gene random mutagenesis and DNA shuffling to invert the enantioselectivity of a lipase of unknown structure from S to R. Another lipase was engineered for the same goal by simultaneous mutation of four active site residues A variety of other enzymes have recently been engineered by targeted mutagenesis. Mutating three active site residues of penicillin acylase created six variants with improved activity, five of which were triple mutants [27]. Juillerat et al. [28] targeted four active site residues to engineer an O6-alkylguanine-DNA alkyltransferase for the efficient in vivo labeling of fusion proteins. They developed a selection system that allowed them to examine over 20,000 mutants and found that the best variants were triple mutants, suggesting the importance of simultaneously exploring multiple mutations. Novel DNA and RNA polymerases have also been engineered by targeted mutagenesis. Chelliserrykattil and Ellington [29] mutated four amino acids in RNA polymerase to engineer the enzyme to transcribe 2 0 -O-methyl RNA. Using a screen that selected variants that generated more RNA, they identified several mutants that incorporated nucleotides modified at the 2 0 position. Fa et al. [30] used targeted mutagenesis to engineer a DNA polymerase to specifically incorporate 2 0 -O-methyl ribonucleoside triphosphates by mutating six amino acids and selecting improved variants using phage display. Targeted mutagenesis of two active site residues was used to engineer a thioredoxin protein to replace the disulfide bond formation system in Escherichia coli Schultz and co-workers have created tRNA synthetases that charge orthogonal tRNAs with non-natural amino acids by targeting mutagenesis to five or six amino acids involved in substrate recognition. They then performed a positive selection for recognition of the non-natural amino acid and a negative selection against recognition of other amino acids The best mutants discovered by targeted mutagenesis almost always contain multiple mutations. These mutations are often beneficial as single mutants, but evidence is accumulating that at least some of them are beneficial only in combination Recombination Recombining structurally similar proteins can access larger degrees of sequence change than random mutagenesis The family shuffling protocol relies on regions of sequence identity to create crossovers that recombine the sequences of related proteins. This protocol is therefore limited to proteins with more than 70-75% identity, because libraries created from more diverged sequences tend to yield mostly parent sequences. A variety of methods have been developed to avoid this problem in the recombination of divergent sequences by using mismatched PCR primer pairs Although the studies described above demonstrate that recombining highly diverged but homologous sequences can produce libraries of diverse folded sequences, so far there has been little work to test whether it is also a useful method for discovering new functions. A tantalizing hint is that four out of fourteen chimeras of two cytochrome P450 proteins with 64% sequence identity show new product profiles Non-homologous recombination that combines fragments of unrelated proteins is another way to introduce large sequence changes. A new methodology was used to recombine the non-homologous chorismate mutase and fumarase proteins A striking application of non-homologous recombination is Ostermeier and co-workers' creation of a protein that combines the activity of a b-lactamase with the maltoseinduced conformational change of maltose-binding protein. In one experiment, they randomly inserted the lactamase sequence into the maltose-binding protein and screened for mutants with enhanced lactamase activity in the presence of maltose Conclusions Directed evolution is now an established method to engineer enzymes for a wide range of uses. Full-gene random mutagenesis continues to be a straightforward and powerful tool, and studies using this approach repeatedly illustrate that beneficial mutations can occur at unexpected sites. Targeted mutagenesis and recombination can extend directed evolution to the engineering of enzyme properties that require more than a few uncoupled changes in a protein's sequence (which are easily obtained by sequential rounds of random mutagenesis and screening). The increasing incorporation of structural and chemical knowledge will undoubtedly enhance the utility of these methods. The growing use of rational design in conjunction with directed evolution offers the exciting promise of generating libraries containing a high frequency of sequences with the desired functional properties. Update Recent work has emphasized the tendency of directed evolution to improve weak promiscuous functions by broadening specificity, as discussed i

Genetically Determined Plasma Lipid Levels and Risk of Diabetic Retinopathy: A Mendelian Randomization Study.

Results from observational studies examining dyslipidemia as a risk factor for diabetic retinopathy (DR) have been inconsistent. We evaluated the causal relationship between plasma lipids and DR using a Mendelian randomization approach. We pooled genome-wide association studies summary statistics from 18 studies for two DR phenotypes: any DR (N = 2,969 case and 4,096 control subjects) and severe DR (N = 1,277 case and 3,980 control subjects). Previously identified lipid-associated single nucleotide polymorphisms served as instrumental variables. Meta-analysis to combine the Mendelian randomization estimates from different cohorts was conducted. There was no statistically significant change in odds ratios of having any DR or severe DR for any of the lipid fractions in the primary analysis that used single nucleotide polymorphisms that did not have a pleiotropic effect on another lipid fraction. Similarly, there was no significant association in the Caucasian and Chinese subgroup analyses. This study did not show evidence of a causal role of the four lipid fractions on DR. However, the study had limited power to detect odds ratios less than 1.23 per SD in genetically induced increase in plasma lipid levels, thus we cannot exclude that causal relationships with more modest effect sizes exist

Re: Impact of holding the baby following stillbirth on maternal mental health and well-being: findings from a national survey. BMJ Open 2016;6(8):e010996

Response to 'Impact of holding the baby following stillbirth on maternal mental health and well-being: findings from a national survey' BMJ Open 2016, 6(8), e010996 (9pp). doi: 10.1136/bmjopen-2015-010996. Response available at: https://bmjopen.bmj.com/content/6/8/e010996.responses (Accessed: 19 January 2023)Dear Editor: We were interested in the recent article by Redshaw et al. which reported higher rates of mental health and relationship difficulties among women who held their stillborn baby.1 We agree this is an important topic, but after reviewing the article in depth, we would like to raise several concerns. (1) We note that this was a retrospective survey with a 30.2% response rate in which just 3% of women did not see and 16% did not hold their baby; these limitations were acknowledged but we believe they also restrict the ability to draw broad conclusions. (2) There was little exploration into the reasons why women did not hold their babies and if they had any regrets about their decisions. While four out of five women reported they did not hold because they could not or did not want to, the study did not account for the fact that women who declined may be fundamentally different at baseline, so that mental health outcomes may be due to underlying differences in mothers rather than their choices or experiences at birth. (3) While the authors emphasize that holding was associated with a trend toward worse mental health outcomes, their actual multivariable analyses show that at 9 months, the only statistically significant difference was higher odds of anxiety. Pre-existing anxiety could contribute to a woman's hesitance to hold the baby after delivery and separately serves as a predictor of postpartum mental health. (4) Even though there are many validated, widely-tested measures to assess postpartum depression,2-5 anxiety,6 and PTSD,7, 8 in both live birth and bereaved mothers, this study used non-validated self-report measures which leads to the need for very cautious interpretation of the results. (5) The factors which have been demonstrated to be strong predictors of postpartum depression and PTSD include prior mental health conditions, interpersonal violence, and lack of social support.9-12 This study did not measure or control for any of these factors. (6) Another issue not addressed in this article is the well-acknowledged preference by parents to be given the option to see or hold their baby and strong evidence that the majority of women are satisfied with their decision.10, 13 Events surrounding the birth of a stillborn baby can have lasting impact on how a mother experiences, remembers, and copes with this event.14 The decision to see or hold a stillborn baby warrants additional investigation, but research must adjust for the known confounders which have been shown to predict development of mental health problems. Moreover, there should be recognition that the experience of a mother at the time of delivery is complex, and multiple pre-existing and intrapartum factors may affect subsequent outcomes and grief. In summary, we believe it is not possible to reach a conclusion from this study about whether the decision to see or hold a stillborn baby is detrimental or helpful to bereaved parents and urge research to gain a more nuanced understanding of the factors which contribute to parental experiences at the time of delivery and which may influence long-term mental health outcomes. We strongly urge health care providers to continue to offer women the option to hold their stillborn baby, and to make this offer in a respectful, supportive, and normative manner

Stillbirths: recall to action in high-income countries.

Variation in stillbirth rates across high-income countries and large equity gaps within high-income countries persist. If all high-income countries achieved stillbirth rates equal to the best performing countries, 19,439 late gestation (28 weeks or more) stillbirths could have been avoided in 2015. The proportion of unexplained stillbirths is high and can be addressed through improvements in data collection, investigation, and classification, and with a better understanding of causal pathways. Substandard care contributes to 20-30% of all stillbirths and the contribution is even higher for late gestation intrapartum stillbirths. National perinatal mortality audit programmes need to be implemented in all high-income countries. The need to reduce stigma and fatalism related to stillbirth and to improve bereavement care are also clear, persisting priorities for action. In high-income countries, a woman living under adverse socioeconomic circumstances has twice the risk of having a stillborn child when compared to her more advantaged counterparts. Programmes at community and country level need to improve health in disadvantaged families to address these inequities.Mater Research Institute – The University of Queensland provided infrastructure and funding for the research team to enable this work to be undertaken. The Canadian Research Chair in Psychosocial Family Health provided funding for revision of the translation of the French web-based survey of care providers.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/S0140-6736(15)01020-

Care in subsequent pregnancies following stillbirth: An international survey of parents

Objective: To assess the frequency of additional care, and parents' perceptions of quality, respectful care in pregnancies subsequent to stillbirth. Design: Multi-language web-based survey. Setting: International. Population: 2,716 parents, from 40 high- and middle-income countries. Methods: Data were obtained from a broader survey of parentsâ experiences of stillbirth. Data were analyzed using descriptive statistics and stratified by geographical region. Subgroup analyses explored variation in additional care by gestational age at index stillbirth. Main outcome measures: Frequency of additional care, and perceptions of quality, respectful care. Results: The majority (66%) of parents conceived their subsequent pregnancy within one year of stillbirth. Additional antenatal care visits and ultrasound scans were provided for 67% and 70% of all parents, respectively, although there was wide variation across geographical regions. Care addressing psychosocial needs was less frequently provided, such as visits to a bereavement counsellor (10%) and access to named care provider's phone number (27%). Compared to parents whose stillbirth occurred at 29 weeks' gestation or less, parents whose stillbirth occurred at 30 weeksâ gestation or greater were more likely to receive various forms of additional care, particularly the option for early delivery after 37 weeks. Around half (47-63%) of all parents felt that elements of quality, respectful care were consistently applied, such as spending enough time with parents and involving parents in decision-making. Conclusions: Care in pregnancies subsequent to stillbirth appears inconsistent. Greater attention is required to providing thoughtful, empathic, and collaborative care in all pregnancies following stillbirth. Training for health professionals is needed

The global burden of adolescent and young adult cancer in 2019:a systematic analysis for the Global Burden of Disease Study 2019

Background In estimating the global burden of cancer, adolescents and young adults with cancer are often overlooked, despite being a distinct subgroup with unique epidemiology, clinical care needs, and societal impact. Comprehensive estimates of the global cancer burden in adolescents and young adults (aged 15-39 years) are lacking. To address this gap, we analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, with a focus on the outcome of disability-adjusted life-years (DALYs), to inform global cancer control measures in adolescents and young adults. Methods Using the GBD 2019 methodology, international mortality data were collected from vital registration systems, verbal autopsies, and population-based cancer registry inputs modelled with mortality-to-incidence ratios (MIRs). Incidence was computed with mortality estimates and corresponding MIRs. Prevalence estimates were calculated using modelled survival and multiplied by disability weights to obtain years lived with disability (YLDs). Years of life lost (YLLs) were calculated as age-specific cancer deaths multiplied by the standard life expectancy at the age of death. The main outcome was DALYs (the sum of YLLs and YLDs). Estimates were presented globally and by Socio-demographic Index (SDI) quintiles (countries ranked and divided into five equal SDI groups), and all estimates were presented with corresponding 95% uncertainty intervals (UIs). For this analysis, we used the age range of 15-39 years to define adolescents and young adults. Findings There were 1.19 million (95% UI 1.11-1.28) incident cancer cases and 396 000 (370 000-425 000) deaths due to cancer among people aged 15-39 years worldwide in 2019. The highest age-standardised incidence rates occurred in high SDI (59.6 [54.5-65.7] per 100 000 person-years) and high-middle SDI countries (53.2 [48.8-57.9] per 100 000 person-years), while the highest age-standardised mortality rates were in low-middle SDI (14.2 [12.9-15.6] per 100 000 person-years) and middle SDI (13.6 [12.6-14.8] per 100 000 person-years) countries. In 2019, adolescent and young adult cancers contributed 23.5 million (21.9-25.2) DALYs to the global burden of disease, of which 2.7% (1.9-3.6) came from YLDs and 97.3% (96.4-98.1) from YLLs. Cancer was the fourth leading cause of death and tenth leading cause of DALYs in adolescents and young adults globally. Interpretation Adolescent and young adult cancers contributed substantially to the overall adolescent and young adult disease burden globally in 2019. These results provide new insights into the distribution and magnitude of the adolescent and young adult cancer burden around the world. With notable differences observed across SDI settings, these estimates can inform global and country-level cancer control efforts. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems