Improvement of bioanalytical parameters through automation: suitability of a hand-like robotic system.

Commercial automation systems for small- and medium-sized laboratories, including research environments, are often complex to use. For liquid handling systems (LHS), development is required not only for the robot's movements but also for adapting the bioanalytical method to the automated system. This study investigates whether a more human-like automation strategy-using a robotic system (RS)-is more suitable for research laboratories than a professional automation approach utilizing a commercial automated LHS. We conducted a series of measurements for protein determination using a Bradford assay manually, with a fully automated LHS, and with our human-like RS. Although the hand-like RS approach requires more than twice the time of the LHS, it achieved the best standard deviation in this setup (RS = 0.5, manual = 0.71, LHS = 0.86). Due to the low limit of detection (LOD) and limit of quantification (LOQ), most protein samples could be quantified with the RS (samples below LOQ = 9.7%, LOD = 0.23; LOQ = 0.25) compared to manual (samples below LOQ = 28.8%, LOD = 0.24; LOQ = 0.26) and the LHS (samples below LOQ = 36.1%, LOD = 0.27; LOQ = 0.31). In another time-dependent enzymatic assay test, the RS achieved results comparable to the manual method and the LHS, although the required time could be a constraint for short incubation times. Our results demonstrate that a more hand-like automation system closely models the manual process, leading easier to accurate bioanalytical results. We conclude that such a system could be more suitable for typical research environments than a complex LHS.

New insights into the paradox between smoking and the risk of SARS-CoV-2 infection (COVID-19): Insufficient evidence for a causal association.

AIMS: Previous studies have reported a 'smoker's paradox', where people who smoke appear to be protected against Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection (COVID-19). This conflicts with well-established evidence that people who smoke are generally more vulnerable to respiratory infections. In this study, we aimed to validate the association between smoking and SARS-CoV-2 infection in a general Dutch population, and to evaluate the evidence underlying the possible causal relationship between smoking and SARS-CoV-2 infection by applying a modern adaptation of the Bradford Hill criteria. METHODS: In total, 57,833 participants from the Lifelines Cohort Study were included in the analysis. Smoking status, including never smoker, current smoker, and former smoker, was derived from the Lifelines general assessment between 2014 and 2017, while SARS-CoV-2 infection status was derived from an additional COVID-19 questionnaire from 2021 to 2022. Logistic regressions were used for the association between smoking status and infection status. The adapted Bradford Hill's criteria, including the strength of association (including an analysis of plausible confounding), plausibility, temporality and study design suitability, were applied to evaluate the existing literature. RESULTS: We found, compared with never smokers, an increased risk of SARS-CoV-2 infection for former smokers (odds ratio (OR)=1.07, 95% confidence interval (CI)=1.01-1.13), but a reduced risk for current smokers (OR=0.85, 95% CI=0.79-0.92), after adjusting for several relevant covariates. However, we discerned a possible explanation of the smoker's paradox since we observed that current smokers were more likely to be non-responders to the COVID-19 questions and, more importantly, these non-responders were more likely to have other established risk factors for SARS-CoV-2 infection. CONCLUSIONS: There is insufficient evidence to suggest that smoking protects against SARS-CoV-2 infection. According to the adapted Bradford Hill's criteria, we observed a high inconsistency between study results, a high possibility for residual confounding and no clear evidence for biological plausibility. Future studies should include linkage with the confirmed testing results from national healthcare registries to mitigate avoidable bias.

Pediatric acute kidney injury and adverse health outcomes: using a foundational framework to evaluate a causal link.

Acute kidney injury (AKI) is a major global health problem, expensive to manage, and its associations with negative pediatric health outcomes have been clearly demonstrated. One of the most fundamental questions to consider as we use previous epidemiological information to advance research and care paradigms is the strength of the causal link between pediatric AKI and health outcomes. In this review, we apply the foundational framework of the Bradford Hill criteria to evaluate the extent to which a causal link exists between AKI and the associated adverse outcomes in children. Available data in children support a causal link between AKI and short-term outcomes including mortality, length of stay, and ventilation time. Clarifying the causal nature of longer term associations requires further high-quality observational studies in children, careful consideration of what defines the most meaningful and measurable longer term outcomes after pediatric AKI, and integration of evolving biological data related to mechanisms of disease. Preventing or mitigating AKI should lead to improved outcomes. Demonstrating such reversibility will solidify confidence in the causal relationship, improve child health, and highlight an aspect which is highly relevant to clinicians, scientists, and policy makers.

Children's behavioural and emotional wellbeing during the COVID-19 pandemic: Findings from the Born in Bradford COVID-19 mixed methods longitudinal study.

Background: The COVID-19 pandemic led to a multitude of immediate social restrictions for many across the world. In the UK, the lives of children and young people were quickly impacted when COVID-19 restrictions led to school closures for most children and restrictions on social interactions. The Born in Bradford COVID-19 longitudinal research study explored the impact of the COVID-19 pandemic on the lives of children and their families living in Bradford. Methods: Surveys were administered during the first wave of the pandemic (March to June 2020) and compared to findings from before the pandemic. The current study examined the social and emotional wellbeing of children from before to during the pandemic, measured using the parent completed Strengths and Difficulties questionnaire (SDQ). Regression analyses looked at associations between a range of social determinants of health and changes in SDQ scores. Results: The results showed that those children most likely to experience difficulties during the pandemic were boys, younger children, those from White British ethnicity (compared to Pakistani heritage children) and those living in the most deprived areas. There were associations between experiencing difficulties and: food insecurity; financial worry; getting below recommended levels of physical activity; and having less than the recommended amount of sleep. Conclusions: The effect of COVID-19 restrictions are likely to have had negative consequences on children that could, in time, have long-lasting impacts on the health, wellbeing and development of children in the UK.

The COVID-19 pandemic caused immediate and long-lasting social restrictions to be implemented here in the UK and across the world. In the UK, children and young people were quickly affected by these restrictions that led to school closures and other restrictions that prevented these individuals from socialising in person with one another. This study explored the impact that the pandemic had on the wellbeing of children by comparing data from before the pandemic with data collected during the pandemic. The data that has been collected looks at the behavioural strengths and difficulties that children are displaying. Our exploration found that children that were most likely to experience difficulties during the pandemic were boys, younger children, those who were White British and those who lived in the most deprived areas. The effect of the COVID-19 restrictions are likely to have had a negative impact on children and young people which in time may impact the health and development of children living here in the UK.

Correlation of calpain sensitivity, Bradford assay instability, and electrophoretic mobility in phosphomimetic mutants of GlyT2 N-terminus.

The glycine transporter GlyT2 plays an important role in glycine-inhibitory neurotransmission of the hindbrain and spinal cord. Its special feature is the extended N-terminus, which contains a large number of potentially phosphorylated serine and threonine residues. Due to its unstructured nature, it is difficult to address the changes introduced by potential phosphorylation. Here, we used relatively simple methods such as calpain sensitivity, Bradford instability, and SDS electrophoretic mobility shift to investigate the effect of multiple phosphomimetic mutations versus neutral mutations on GlyT2N properties. The replacement of several serines or threonines with neutral alanines did not have a significant effect on the studied GlyT2N properties. Replacement of the same residues with phosphomimetic aspartate resulted in significant alterations in calpain cleavage patterns, Bradford instability, and SDS gel protein mobility. Interestingly, a correlation between the relative intensity of the measured effects was observed, indicating that they all reflect similar structural changes introduced by potential phosphorylation in vivo. Results indicate that a potential single or multiple phosphorylation significantly alters the proteomic properties of the glycine transporter GlyT2 N-terminus. Assays can be helpful in the first screening of structurally significant and possibly phosphorylated residues in the N-terminus of GlyT2.

Born in Bradford's Age of Wonder cohort: protocol for adolescent data collection.

Background: Adolescence and transition into adulthood are periods shaping life-long mental health, cardiometabolic risk, and inequalities. However, they are poorly studied and understood. By extending and expanding the Born in Bradford (BiB) cohort study through this period using innovative, co-produced approaches to collect and analyse data, we aim to understand better the interplay of factors that influence health and wellbeing, and inform/evaluate interventions to improve them and reduce inequalities. Protocol: BiB Age of Wonder (AoW) is a large, whole city cohort that will capture the contemporary lived experience amongst multi-ethnic adolescents progressing into young adulthood. We will collect repeated data from existing BiB participants and their peers (N~30,000 adolescents). The protocol for the first phase of the quantitative methods, involving survey measurements and health assessments in mainstream secondary schools is described here. We describe the co-production behind these methods, and lessons learned from the first year of data collection.

Born in Bradford have been following the health and wellbeing of over 13,000 Bradford children since they were born. This group of children are now in their teenage years ­ a time that is crucial for their future mental and physical health. Age of Wonder aims to capture this journey through adolescence and early adulthood with up to 30,000 young people in Bradford over 7 years. In the first phase of this project, data collection is taking part in secondary schools in Bradford. This protocol describes how this data collection is currently being carried out in the schools. Young people aged 12-15 are being asked to complete questionnaires, covering topics such as mental and physical health. These topics have been designed with groups of young people, schools and other partners, to make sure we are capturing data on the things most important to young people. Those in Year 9 (13-14 years old), are also asked to take part in physical health measurements such as height, weight, blood pressure and a blood sample, as well as computer-based assessments of cognition (memory), movement and language. There have been a number of lessons learned from the first full year of data collection, such as how to make the research as easy as possible for schools to take part in when they have so many competing demands on their time.

A quantitative weight-of-evidence method for confidence assessment of adverse outcome pathway networks: A case study on chemical-induced liver steatosis.

The field of chemical toxicity testing is undergoing a transition to overcome the limitations of in vivo experiments. This evolution involves implementing innovative non-animal approaches to improve predictability and provide a more precise understanding of toxicity mechanisms. Adverse outcome pathway (AOP) networks are pivotal in organizing existing mechanistic knowledge related to toxicological processes. However, these AOP networks are dynamic and require regular updates to incorporate the latest data. Regulatory challenges also persist due to concerns about the reliability of the information they offer. This study introduces a generic Weight-of-Evidence (WoE) scoring method, aligned with the tailored Bradford-Hill criteria, to quantitatively assess the confidence levels in key event relationships (KERs) within AOP networks. We use the previously published AOP network on chemical-induced liver steatosis, a prevalent form of human liver injury, as a case study. Initially, the existing AOP network is optimized with the latest scientific information extracted from PubMed using the free SysRev platform for artificial intelligence (AI)-based abstract inclusion and standardized data collection. The resulting optimized AOP network, constructed using Cytoscape, visually represents confidence levels through node size (key event, KE) and edge thickness (KERs). Additionally, a Shiny application is developed to facilitate user interaction with the dataset, promoting future updates. Our analysis of 173 research papers yielded 100 unique KEs and 221 KERs among which 72 KEs and 170 KERs, respectively, have not been previously documented in the prior AOP network or AOP-wiki. Notably, modifications in de novo lipogenesis, fatty acid uptake and mitochondrial beta-oxidation, leading to lipid accumulation and liver steatosis, garnered the highest KER confidence scores. In conclusion, our study delivers a generic methodology for developing and assessing AOP networks. The quantitative WoE scoring method facilitates in determining the level of support for KERs within the optimized AOP network, offering valuable insights into its utility in both scientific research and regulatory contexts. KERs supported by robust evidence represent promising candidates for inclusion in an in vitro test battery for reliably predicting chemical-induced liver steatosis within regulatory frameworks.

Exploring attitudes and variation by sociodemographic factors in consent provided for financial data linkage in an experimental birth cohort study.

BACKGROUND: Improving our understanding of household incomes and what constitutes financial insecurity can help us to better understand how financial insecurity is experienced and how this can change over time within and between individuals and populations. However, financial circumstances are often perceived as sensitive and stigmatising, particularly within some ethnic minority groups. This research aims to explore attitudes and variation by sociodemographic factors in consent provided for financial data linkage in an experimental birth cohort study, in order to obtain validated income and benefits data and to better understand the impact of community interventions on the financial security of its participants and their families. METHODS: This research utilises an observational study design to explore consent rates, attitudes and variation in sociodemographic factors between participants of an experimental birth cohort in a deprived and ethnically diverse setting who consent and do not consent to financial data linkage. RESULTS: Overall, participants were equally likely to consent and decline consent for financial data linkage. Measures of socioeconomic insecurity were associated with being more likely to provide consent for financial data linkage. Participants who were not employed (OR 1.49 95% CI 0.93, 2.40) and were more financially insecure (OR 1.85 95% CI 1.14, 3.93) were more likely to provide consent for financial data linkage. Where the participant's first language was a language other than English, participants were also less likely to provide consent for data linkage (OR 0.65 95% CI 0.39, 0.98). The choice of consent for financial data linkage was not associated with: ethnicity; relationship factors; employment status of the participant's partner; person present at time of recruitment; and measures of health, such as general health, mental health, wellbeing and health-related quality of life. CONCLUSIONS: This research sets out an approach to obtaining validated income and benefits data, as a proxy measure for financial security, within an experimental birth cohort study in a deprived and ethnically diverse setting. It achieves good consent rates and demonstrates greater input from those who report greater potential need for financial support. Further research should be conducted to further understand the interplay of language spoken in this context.

Exercise for knee osteoarthritis pain: Association or causation?

Exercise is universally recommended as a primary strategy for the management of knee osteoarthritis (OA) pain. The recommendations are based on results from more than 100 randomized controlled trials (RCTs) that compare exercise to no-attention control groups. However, due to the inherent difficulties with adequate placebo control, participant blinding and the use of patient-reported outcomes, the existing RCT evidence is imperfect. To better understand the evidence used to support a causal relationship between exercise and knee OA pain relief, we examined the existing evidence through the Bradford Hill considerations for causation. The Bradford Hill considerations, first proposed in 1965 by Sir Austin Bradford Hill, provide a framework for assessment of possible causal relationships. There are 9 considerations by which the evidence is reviewed: Strength of association, Consistency, Specificity, Temporality, Biological Gradient (Dose-Response), Plausibility, Coherence, Experiment, and Analogy. Viewing the evidence from these 9 viewpoints did neither bring forward indisputable evidence for nor against the causal relationship between exercise and improved knee OA pain. Rather, we conclude that the current evidence is not sufficient to support claims about (lack of) causality. With our review, we hope to advance the continued global conversation about how to improve the evidence-based management of patients with knee OA.

New species in the genera Eumacrocyrtus Schultze, 1923 and Enoplocyrtus Yoshitake, 2017 from Luzon Island, Philippines (Coleoptera, Curculionidae, Entiminae, Pachyrhynchini).

We describe and illustrate two new species from two previously monotypic genera Eumacrocyrtus Schultze, 1923 and Enoplocyrtus Yoshitake, 2017 from Luzon Island, Philippines: Eumacrocyrtusrobertfoxisp. nov., and Enoplocyrtusangelalcalaisp. nov.Eumacrocyrtusrobertfoxisp. nov. serves as a new record for Luzon Island for Eumacrocyrtus which was only previously represented by E.canlaonensis Schultze, 1923 from Negros Island whereas Enoplocyrtusangelalcalaisp. nov. serves as an additional record of Enoplocyrtus in Mountain Province in Luzon Island. The discovery of these two new species from the Zoological Collections of the Philippine National Museum, collected in 1947 and 1985, respectively, highlights the value of natural history collections for the present and future generations of researchers.

Is it feasible to nest a Trial within a Cohort Study (TwiCS) to evaluate an early years parenting programme? A Born in Bradford's Better Start study protocol.

BACKGROUND: Evaluating the effectiveness of early years parenting interventions provides evidence to improve the development and wellbeing of children. This protocol paper describes a study to explore the feasibility of evaluating the Incredible Years Toddler early life intervention programme, which is offered to parents of 1-3-year-olds via the Better Start Bradford programme. The study aims to use a Trial within a Cohort Study (TwiCS) design that randomly selects individuals participating in a cohort to be offered an intervention. The TwiCS information and consent process is person-centred and aims to replicate real-world practice whereby only those who are offered the intervention are given information about the intervention. The cohort is the Born in Bradford's Better Start (BiBBS) cohort, an interventional birth cohort recruiting expectant parents in three areas of Bradford, UK. The study will assess the feasibility of TwiCS procedures, staged consent, and intervention take-up. METHODS: We will conduct a feasibility TwiCS to test study procedures. We aim to establish the following: (1) whether TwiCS methodology can be implemented to create control and intervention arms, whilst documenting any incidences of contamination within the cohort; (2) whether satisfactory rates of intervention uptake are achieved among participants allocated to the intervention; and (3) whether satisfactory rates of retention of participants in the intervention can be achieved. A Red Amber Green (RAG) rating system has been applied to support the feasibility assessment of each objective: to be rated red (not achieved), amber (partly achieved), and green (achieved). Eligible participants in the BiBBS cohort will be individually randomised 1:1 to the intervention or control arms, with stratification by child age (1 or 2 years old at the time of randomisation) and ethnicity (White British, South Asian, or other). BiBBS researchers will seek consent from participants randomised to the intervention to pass their contact details onto Incredible Years' delivery agents. DISCUSSION: This feasibility study will inform the utility of the TwiCs approach within an experimental birth cohort to evaluate interventions for infants, toddlers, and their families. TRIAL REGISTRATION: The study was prospectively registered on ISRCTN (ISRCTN16150114).

Breast implant illness: Is it causally related to breast implants?

Once believed to be completely inert implants, Silicon Breast Implants (SBIs) have been shown to be able to induce a chronic inflammatory response in the body which can lead to a variety of possible manifestations ranging from the most common capsular contraction to rarer conditions such as malignancies and autoimmune diseases. Among the latter, new syndromes have been consistently recognized: Breast Implant Illness (BII) and autoimmunity/autoinflammatory syndrome induced by adjuvants (ASIA syndrome/Shoenfeld's Syndrome). The pathophysiological mechanisms underlying such syndromes are not yet clear and the overlap they show with other common conditions have sparked an important debate in the scientific community regarding their existence and their cause-effect relationship with SBIs. In this article Professor Cohen Tervaert and Professor Bassetto, leading experts in the field, are going to present arguments in favor and against such causal relationship according to the latest scientific evidence. Professor Cohen Tervaert is going to demonstrate how the evidence available is enough to prove a causal relationship as defined by the Bradford Hill's criteria. Professor Bassetto is going to highlight how the many biases that afflict the available evidence prevent us from drawing such conclusions. Professor Shoenfeld is going to moderate the discussion with its insightful conclusions.

Relationships Between Exposure to Gestational Diabetes Treatment and Neonatal Anthropometry: Evidence from the Born in Bradford (BiB) Cohort.

OBJECTIVES: To examine the relationships between gestational diabetes mellitus (GDM) treatment and neonatal anthropometry. METHODS: Covariate-adjusted multivariable linear regression analyses were used in 9907 offspring of the Born in Bradford cohort. GDM treatment type (lifestyle changes advice only, lifestyle changes and insulin or lifestyle changes and metformin) was the exposure, offspring born to mothers without GDM the control, and birth weight, head, mid-arm and abdominal circumference, and subscapular and triceps skinfold thickness the outcomes. RESULTS: Lower birth weight in offspring exposed to insulin (- 117.2 g (95% CI - 173.8, - 60.7)) and metformin (- 200.3 g (- 328.5, - 72.1)) compared to offspring not exposed to GDM was partly attributed to lower gestational age at birth and greater proportion of Pakistani mothers in the treatment groups. Higher subscapular skinfolds in offspring exposed to treatment compared to those not exposed to GDM was partly attributed to higher maternal glucose concentrations at diagnosis. In fully adjusted analyses, offspring exposed to GDM treatment had lower weight, smaller abdominal circumference and skinfolds at birth than those not exposed to GDM. Metformin exposure was associated with smaller offspring mid-arm circumference (- 0.3 cm (- 0.6, - 0.07)) than insulin exposure in fully adjusted models with no other differences found. CONCLUSIONS FOR PRACTICE: Offspring exposed to GDM treatment were lighter and smaller at birth than those not exposed to GDM. Metformin-exposed offspring had largely comparable birth anthropometric characteristics to those exposed to insulin.

Causation in different scientific disciplines: A comparison of standards in occupational medicine and particle physics.

Occupational medical research involves the collection and analysis of data to draw conclusions about the causes and prevention of workplace injuries and diseases. However, there has been criticism that some studies lack rigour in determining causation. This article examines the similarities and differences between occupational medical research and particle physics in terms of their approach to hypothesis testing, statistical methods, and confounder control. The article also explores the use of criteria such as the Bradford Hill criteria to determine causation in occupational medical research. While particle physics is often viewed as a highly rigorous science, occupational medical research also employs rigorous scientific methods to ensure findings are accurate and reliable. However, there is room for improvement in determining causation in occupational medical research, particularly in the use of criteria such as the Bradford Hill criteria to guide the development of more robust studies. It is essential for occupational medical research to adhere to rigorous scientific methods to deliver findings that can help reduce workplace injuries and diseases. The use of criteria such as the Bradford Hill criteria can ensure that the conclusions drawn. Med Pr Work Health Saf. 2023;74(4):333-9.

Examining individual social status using the MacArthur Scale of Subjective Social Status: Findings from the Born in Bradford study.

Subjective social status is how a person perceives their social class relative to other people and has frequently been associated with people's health and well-being. A frequently used measure of subjective social status is the MacArthur Scale of Subjective Social Status that depicts social status as a 10 rung ladder, asking individuals to rank themselves on this ladder relative to other people, either in their local neighbourhood or wider society. The Born in Bradford's Better Start birth cohort study aims to understand the lives, relationships, wellbeing, and social and economic circumstances of pregnant women and their children in three inner city areas of Bradford, UK. Pregnant mothers were asked to report their subjective social status, using the MacArthur subjective social status scales, comparing themselves to other people in their local neighbourhood and in England as a whole. This paper explores the characteristics of the women who gave responses, examines associations between the MacArthur subjective social status measures and other subjective and objective measures and looks specifically at the characteristics of women who reported either very low or very high subjective social status. On average, women reported that they had a higher social status compared to others within their local neighbourhood (mean ladder rung = 6) and, although participants were from areas of Bradford with very high levels of deprivation, 23% placed themselves on the top three rungs, 8-10. Respondents reported that they had an average social status when comparing themselves to people in all of England (mean ladder rung = 5) and 13% placed themselves on the top rungs 8-10. These findings raise important questions about the interpretation of the MacArthur scale of subjective social status.

Protein Quantitation and Analysis of Purity.

The accurate quantitation of proteins and an analysis of their purity is essential in numerous areas of scientific research and is a critical factor in many clinical applications. The large number and variety of techniques employed for this purpose is therefore not surprising. The selection of a suitable assay is dependent on such factors as the level of sensitivity required, the presence of interfering agents, and the composition of the protein itself. In this chapter, protocols for the most commonly used protein determination methodologies are outlined, including an overview of the highly sensitive real-time quantitative immuno-polymerase chain reaction assay. In addition, an approach to validate the UV protein absorption assay is outlined, which can be applied to any procedure for method validation.

Associations between maternal gestational diabetes metformin or insulin treatment and offspring growth trajectories from birth to 60 months of age: Findings from the Born in Bradford (BiB) study.

AIM: To investigate the associations between gestational diabetes mellitus (GDM) metformin or insulin treatment and offspring growth trajectories from 0 to 60 months. METHODS: Participants were from the Born in Bradford birth cohort study. Using covariate-adjusted multilevel linear spline models (4 splines: 0-1.6, 1.6-6, 6-17 and 17-60 months), we compared weight, height and body mass index (BMI) z-score trajectories of: (1) 76 offspring exposed to metformin (OGDM-Metformin) and 420 offspring exposed to insulin (OGDM-Insulin); (2) OGDM-Metformin and 9171 offspring not exposed to GDM (No-GDM); (3) OGDM-Insulin and No-GDM. RESULTS: (1) OGDM-Metformin had comparable growth trajectories to OGDM-Insulin from 0 to 60 months. (2) OGDM-Metformin had a lower mean birthweight z-score than No-GDM. OGDM-Metformin had faster changes in height z-score (0.13 [95% CI 0.026, 0.24]) from 17 to 60 months and by 60 months, had comparable mean BMI z-score to No-GDM. (3) OGDM-insulin had lower mean birthweight and height z-scores than No-GDM. OGDM-Insulin had faster changes in weight (0.32 [0.021, 0.62]) and height (0.50 [0.087, 0.91]) from 1.6 to 6 months and by 60 months, had comparable mean BMI z-score to No-GDM. CONCLUSIONS: GDM metformin treatment was not associated with differences in offspring growth trajectories compared to insulin treatment. Both metformin and insulin-exposed offspring had comparable BMI z-score to No-GDM by 60 months.

Pyrus calleryana extracts reduce germination of native grassland species, suggesting the potential for allelopathic effects during ecological invasion.

Invasive plant species' success may be a result of allelopathy, or the release of secondary metabolites that are harmful for surrounding plant species. Allelopathy can be mediated through the abiotic environment by chemical sorption or transformation, so the substrate on which interactions occur can lead to differential outcomes in allelopathic potential. One aggressive invader, Pyrus calleryana, has become dominant in many ecosystems throughout Eastern US, and has reduced the abundance of native species where it invades. Thus, our goal was to identify if P. calleryana had allelopathic potential by testing the impact of leaf and flower leachate on gemination of six common grassland species (three grasses and three forbs) in either sterilized sand or field collected soils. Germination of five out of six tested species was reduced by P. calleryana leaf litter, with weaker impacts from flower leachate. This suggests that allelopathy is one mechanism driving the success of P. calleryana and that allelopathic effects may change with plant phenology. For instance, P. calleryana has late leaf senescence in the fall and copious blooming in the spring that may elongate the timeframe that allelopathic inhibition can occur. Further, germination was higher in sand than in soil, suggesting that the context of the abiotic environment can mediate this relationship. In our study, two grass species that could be overabundant in restored grasslands had higher germination rates in soil than sand and one was not altered by P. calleryana suggesting that this relationship could further promote the overabundance of grass species. Taken together, P. calleryana likely inhibits the germination of native species where it invades, but there is context dependency of this relationship with both soil chemistry and seasonality.

A Simple Biochemical Method for the Detection of Proteins as Biomarkers of Life on Martian Soil Simulants and the Impact of UV Radiation.

The search for life on other planets relies on the detection of biosignatures of life. Many macromolecules have been suggested as potential targets, among which are proteins that are considered vital components of life due to their essential roles in forming cellular structures, facilitating cellular communication and signaling, and catalyzing metabolic reactions. In this context, accurate quantification of protein signatures in soil would be advantageous, and while several proposed methods exist, which are limited by their sensitivity and specificity, their applicability needs further testing and validation. To this aim, we optimized a Bradford-based assay with high sensitivity and reproducibility and a simple protocol to quantify protein extracted from a Martian soil simulant. Methods for protein spiking, extraction, and recovery were optimized, using protein standards and bacterial proteins as representative models. The proposed method achieved high sensitivity and reproducibility. Taking into account that life remains could exist on the surface of Mars, which is subjected to UV radiation, a simulation of UV exposure was performed on a spiked soil simulant. UV radiation degraded the protein spike, thus highlighting the importance of searching for the remaining signal from degraded proteins. Finally, the applicability of the method was explored in relation to the storage of the reagent which was stable even up to 12 months, thus making its application possible for future planetary exploration missions.