Acrocephalus sechellensis) to investigate the impact of maternal and paternal age on offspring life span and lifetime reproductive success. We found negative effects of maternal age on female offspring life span and lifetime reproductive success, which were driven by within‐mother effects. There was no difference in annual reproductive output of females born to older versus younger mothers, suggesting that the differences in offspring lifetime reproductive success were driven by effects on offspring life span. In contrast, there was no association between paternal age and female offspring life span or either maternal or paternal age and male offspring life span. Lifetime reproductive success, but not annual reproductive success, of male offspring increased with maternal age, but this was driven by between‐mother effects. No paternal age effects were found on female offspring lifetime reproductive success but there was a positive between‐father effect on male offspring lifetime reproductive success. We did not find strong evidence for environment‐dependent parental age effects. Our study provides evidence for parental age effects on the lifetime fitness of offspring and shows that such effects can be sex dependent. These results add to the growing literature indicating the importance of intergenerational effects on long‐term offspring performance and highlight that these effects can be an important driver of variation in longevity and fitness in the wild.Parental age can have considerable effects on offspring phenotypes and health. However, intergenerational effects may also have longer term effects on offspring fitness. Few studies have investigated parental age effects on offspring fitness in natural populations while also testing for sex‐ and environment‐specific effects. Further, longitudinal parental age effects may be masked by population‐level processes such as the selective disappearance of poor‐quality individuals. Here, we used multigenerational data collected on individually marked Seychelles warblers ( In virtually all animals, an individual's health and condition deteriorate with age (senescence), which impacts their survival and the number of offspring produced in later life. Importantly, the quality of offspring produced, as measured through their physiological condition, survival, and reproductive success, may also be impacted by their parents’ ageing. This may have considerable health and evolutionary implications. Most of the evidence for intergenerational effects of parental age comes from studies on laboratory animals that are kept under artificial conditions. However, little is known about such effects in natural populations, where a myriad of different stresses and strains act upon individuals. Furthermore, it remains unclear whether parental age effects on offspring fitness are sex specific or affected by environmental conditions. Our study reveals that, in a wild‐living bird, parental age effects on offspring life span and lifetime fitness depend on both the sex of the parent and the sex of the offspring. Cross‐sectionally, we found negative effects of maternal age, but no effects of paternal age, on the life span and lifetime reproductive success of female offspring. In contrast, although there were no parental age effects on male offspring life span, there was a positive between‐mother and between‐father effect on male offspring lifetime reproductive success. Our study highlights that not including such intergenerational age effects, not separating within‐ and between‐parental effects, and not testing for sex‐specific effects may underestimate or obscure important components of ageing and senescence in wild populations. These intergenerational effects could have important implications for population dynamics, particularly in age‐structured populations, for example, in terms of conservation management and evolutionary dynamics.Early‐life environmental conditions can have considerable consequences for later‐life individual fitness. Parental effects, whereby an offspring's phenotype is influenced by their mother's or father's phenotype, above and beyond the genes they inherit, are widespread in natural populations , and bird  populations  population on Cousin Island. This facultatively cooperative‐breeding bird lives in and defends territories in which a dominant male and female reside. Around half of these territories may also contain subordinate individuals, of which approximately one third help with parental care in any given year  on Cousin Island  has been monitored intensively, although a small number of birds on the island were ringed before the project began Komdeur . The domFieldwork is carried out during the breeding seasons, when as many birds as possible are caught using mist nests or captured in the nest. The age of unringed birds is estimated based on lay, hatch, or fledge dates and/or using eye color .To investigate the influence that early‐life conditions have on parental age effects, we investigated the effect of natal territory quality, group size, and helper presence in the offspring's natal territory. We used an index of insect abundance as a measure of territory quality as warblers are almost entirely insectivorous (Komdeur and Daan N = 43 of 1321), helper presence in the natal territory was included as a binary variable (present/absent).We used natal group size and helper presence as proxies for the early‐life social environment. Natal group size was the number of independent individuals  in the natal territory (range: 2−6). Helpers are subordinates observed to help incubate or provision offspring in a specific breeding season (0−3 per territory). Due to few instances of >1 helper in the dataset . We investigated parental age effects with genetic parents identified using MasterBayes version 2.5.2 .Life span was calculated for all individuals who survived to at least 1 year of age. Because the resighting probability is high, an individual's death year was assigned as the last sighting year. Life span analyses were performed using Poisson generalized linear mixed models (GLMM) in glmmTMB version 1.0.2.1 .LRS, calculated as the sum of offspring produced who survived to at least 1 year of age, was calculated for all individuals who survived to at least 1 year of age, were not translocated, and whose last seen year was before 2018 (last year of the pedigree). Analyses were performed using a zero‐inflated Poisson GLMM in glmmTMB. It included the same fixed and random effects as the life span models. As for the life span models, we subsequently tested for maternal × paternal age interactions and environment‐dependent parental age effects by including interactions between parental age and natal group size, helper presence, and natal territory quality. To test whether any effects of parental age may be mediated by offspring life span, we included offspring life span in our models. Alternatively, parental age effects on offspring LRS may be mediated by differences in annual reproductive output of offspring. To test this, we also ran models of annual reproductive success (see Methods in the Supporting Information for full details). This method centers all measurements of the parent around their mean age, thus eliminating between‐subject variation. Between‐parent effects could be caused by anything driving differences in a parent's mean age. For example, positive between‐parent effects could be caused by parents surviving to or starting to breed at older ages having offspring with higher fitness. After accounting for between‐parent effects, the within‐parent effects subsequently reflect true age‐specific variation in offspring life span and LRS within individual parents.Any parental age effects observed at the cross‐sectional level may not reflect age‐specific variation within parents because both within‐ and between‐parental effects can drive patterns at the population level. In wild populations, there is often variation between individuals in the onset of breeding as well as life span. Such variation can mask parental ageing effects, or could generate spurious results if a parent's life span rather than ageing per se is related to offspring life span and LRS . The correlation between the age of the genetic father and dominant male was moderate , which was unsurprising given that 59% of offspring are sired by the dominant male within the territory  age2 effects and interactions were removed from the models sequentially, in order of least significance, to ensure their inclusion did not affect interpretation of the first‐order effects. All dropped terms were retested against the base model using likelihood ratio tests.Correlations between age of the mother and the age of the genetic and/or social father were weak, which allowed them to be included in the same model . There was no significant association between maternal age and life span for male offspring . In contrast, there was a weaker but positive association between maternal age and the LRS of male offspring  = 0.057, P = 0.812; life span β = 1.743 ± 0.117 SE, χ2(1) = 204.930, P < 0.001), which suggests that differences in LRS between female offspring born to different age mothers were driven by differences in longevity rather than by differences in annual reproductive output. In support of this, we found no association between parental age and the annual reproductive success of either female or male offspring  and common terns (Sterna hirundo) female, but not male, offspring born to older mothers had lower LRS , daughters born to older mothers had greater annual reproductive success, which translated to higher LRS , a complex pattern was observed , natal environmental conditions impacted the life span and breeding success of males but not females  and lifetime reproductive success  in the Seychelles warbler for females  and males  who survived to one year and had complete lifetime data Figure S2. Scatterplot of raw data showing the correlation between (genetic) maternal and paternal ages of Seychelles warbler offspring at hatching .Figure S3. Within‐individual maternal age effects (‘Delta age mother’) on the lifespan of female offspring in the Seychelles warbler, using within‐subject centering (van de Pol and Wright 2009)Figure S4. Within and between genetic parental age effects on the LRS of female (A) and male (B‐C) offspring in the Seychelles warbler using within‐subject centering (van de Pol and Wright 2009)Figure S5. Scatterplot of raw data showing the correlation between lifespan and lifetime reproductive success  in the Seychelles warbler for all individuals who survived to one year and had complete lifetime data (n=925)Figure S6. Interaction plot describing the marginally significant interaction between the age of the genetic father and territory quality on female offspring lifetime reproductive success (LRS) in the Seychelles warbler (Table S11)Figure S7. Interaction plot describing the marginally significant interaction between the age of the genetic father and territory quality on female offspring lifetime reproductive success (LRS) in the Seychelles warbler (Table S11).Figure S8. Interaction plot describing the marginally significant interaction between the age of the dominant male and territory quality on female offspring lifetime reproductive success (LRS) in the Seychelles warbler (Table S11).Figure S9. Interaction plot describing the marginally significant interaction between the age of the dominant male and territory quality on female offspring lifetime reproductive success (LRS) in the Seychelles warbler (Table S11).Table S1. Generalised linear mixed model (GLMM) results investigating associations between the status of the female and provisioning rate (feed counts) in the Seychelles warblerTable S2. Generalised linear mixed model (GLMM) results investigating the associations between parental age and offspring lifespan and LRS where age of the father and age of the dominant male were included in separate modelsTable S3. Cox proportional hazards mixed effects model results for parental age effects on offspring lifespan, for males and females separately, in the Seychelles warblerTable S4. Generalised linear mixed model results investigating between versus within maternal and paternal age effects on offspring lifespan, in each offspring sex separately, in the Seychelles warbler using the within‐subject centering method (van de Pol and Wright 2009).Table S5. Generalised linear mixed model results investigating between versus within maternal and paternal age effects on offspring lifespan, in each sex separately, in the Seychelles warbler, using the within‐subject centering method (van de Pol and Wright 2009).Table S6. Significance of maternal and paternal age interactions, quadratic parental age effects and environment by parental age interactions on female offspring and male offspring lifespan, in the Seychelles warbler using a GLMMTable S7. Significance of maternal and paternal age interactions, quadratic parental age effects and environment by parental age interactions on female offspring and male offspring lifespan, in the Seychelles warbler, in the Cox proportional hazards mixed effects modelTable S8. Generalised linear mixed model results investigating associations between parental age and offspring reproductive lifespan in the Seychelles warbler for each sex separatelyTable S9. Generalised linear mixed model results investigating between versus within maternal and paternal age effects on offspring LRS in each offspring sex separately in the Seychelles warbler using the within‐subject centering method (van de Pol and Wright 2009)Table S10. Generalised linear mixed model results investigating between‐ versus within‐ maternal and paternal age effects on offspring lifetime reproductive success, in each sex separately, in the Seychelles warbler using the within‐subject centering method (van de Pol and Wright 2009)Table S11. Significance of maternal and paternal age interactions, quadratic parental age effects and environment by parental age interactions on female offspring and male offspring lifetime reproductive success in the Seychelles warblerTable S12. Generalised linear mixed model results investigating parental age effects on offspring annual reproductive success (measured as the number of offspring in the pedigree who survived to 1 year), in each sex separately, in the Seychelles warblerTable S13. Generalised linear mixed model results investigating between versus within maternal and paternal age effects on female offspring lifespan in the Seychelles warbler, using the within‐subject centering method (van de Pol and Wright 2009)Table S14. Generalised mixed model results investigating between versus within maternal and paternal age effects on female offspring lifetime reproductive success in the Seychelles warbler using the within‐subject centering method (van de Pol and Wright 2009)Click here for additional data file.


Plasmids are extrachromosomal genetic elements capable of autonomous replication within a host cell. They play a key role in bacterial ecology and evolution, facilitating the mobilization of accessory genes by horizontal gene transfer. Crucially, plasmids also serve as valuable tools in modern molecular biology. Here, we highlight recent articles aimed at implementing standardized plasmid assembly techniques and plasmid repositories to promote open science as well as to improve experimental reproducibility across laboratories. Research focused on assisting these fundamental aims is a further step towards improving standardization in molecular and synthetic biology. The availability of plasmid repositories, together with improved DNA assembly techniques for the generation of recombinant plasmids, is accelerating the development and reproducibility of molecular biology, synthetic biology and biotechnology approaches. Indeed, the number of naturally occurring bacterial plasmids continues to grow rapidly with the advancement of new sequencing technologies. Currently, there are more than 34 000 bacterial plasmids listed in the NCBI Reference Sequence (RefSeq) database; which represents an almost twofold increase within the 2020–2021 period  compositional standard, a set of rules for the physical assembly of three basic components of plasmid vectors , which are interconnected by invariant sequences. These rules were bolstered through the creation of the SEVA platform (http://seva‐plasmids.com/), a web‐based resource and clone repository designed to assist users regarding optimal plasmid vector selection, and the SEVA database (SEVA‐DB), which allows users to customize plasmid design for their specific needs, deciding on the optimal configuration of the above basic components. This valuable resource has been used to answer a plethora of fundamental and applied questions in prokaryotic biology – primarily in Gram‐negative bacteria but also in Gram‐positive bacterial hosts  to create genetic constructs optimized for different biotechnological and synthetic biology applications  and the functionality of the final vectors was demonstrated in P. putida KT2440 and P. aeruginosa PAO1 , ‘pSTDesR’ (a RhaRS/PrhaB expression system) and ‘pBGDes’  to independently co‐express three different proteins in KT2440 and PAO1. Although expression was achieved in both hosts, distinct expression responses were noted for these two pseudomonads  and ‘Addgene’ (http://www.addgene.org/). These two repositories rely on donations from the scientific community and store more than 460 000 and 106 000 plasmids, respectively. The work by Brkljacic et al. was motivated by the observation that several deposited plasmids had unexpected restriction patterns. All deposited plasmids in ABRC and Addgene passed exhaustive quality controls that included: (i) the analysis by restriction endonuclease digestion of various individual colonies (ABRC); or (ii) plasmid sequencing and the annotation of transposable elements like insertion sequences (IS) (Addgene). However, the re‐analysis of various plasmid stocks by restriction digestion, PCR and sequencing revealed the insertion of undesired IS elements in plasmids with diverse backbones. This IS acquisition was found to occur at both prior to stock deposition and during host culturing at repositories (Brkljacic et al., Escherichia coli hosts for plasmid propagation prevents the insertion of IS elements (Brkljacic et al., 1A and IS10R being by far the most frequent (Brkljacic et al., Maintaining large collections of standardized plasmids can result in a few surprising challenges. In a recent report published in c et al.  investigá la carte plasmid design and accessibility, while also facilitating extensive variation within a plasmid assembly. It will be exciting to see how this approach will be used to probe Pseudomonas core architecture, and to expand the capability of different hosts in synthetic biology applications. In contrast, the use of IS‐free hosts and additional controls will hopefully slow unwanted variation in plasmid sequences during plasmid construction and propagation. Future research in this field will facilitate the generation of new molecular tools for the use in a broad spectrum of model bacteria, including non‐standard hosts.These two studies highlight the enormous potential and some challenges that lie ahead for plasmid research and standardization. ‘SEVAtile’ upholds the clear advantages of The authors declare that there is no conflict of interest.


Saturated fatty acids (FAs) are the main component of high‐fat diets (HFDs), and high consumption has been associated with the development of insulin resistance, endoplasmic reticulum stress and mitochondrial dysfunction in neuronal cells. In particular, the reduction in neuronal insulin signaling seems to underlie the development of cognitive impairments and has been considered a risk factor for Alzheimer's disease (AD).This review summarized and critically analyzed the research that has impacted the field of saturated FA metabolism in neurons.We reviewed the mechanisms for free FA transport from the systemic circulation to the brain and how they impact neuronal metabolism. Finally, we focused on the molecular and the physiopathological consequences of brain exposure to the most abundant FA in the HFD, palmitic acid (PA).Understanding the mechanisms that lead to metabolic alterations in neurons induced by saturated FAs could help to develop several strategies for the prevention and treatment of cognitive impairment associated with insulin resistance, metabolic syndrome, or type II diabetes. Saturated fatty acids induce insulin resistance in neurons. Neuronal metabolism of palmitic acid alters mitochondrial function. Epidemiologic studies from 2016 indicate that 13% of adults and 18% of children and adolescents under 19 years are obese worldwide,Due to the complex nature of FA effects on the brain, in this review, we describe the current knowledge of the mechanisms involved in their transport from the circulation to the brain as well as the main metabolic routes activated in neurons, including evidence for neuronal β‐oxidation. Finally, we highlight the major mechanisms by which exposure to high levels of saturated FA can lead to neuronal dysfunction.2Fatty acids are made up of a hydrocarbon chain with a terminal carboxyl group. They are classified as saturated when composed of single aliphatic chain bonds, monounsaturated with one double bond, and polyunsaturated with two or more double bonds. According to the chain length, FAs are divided into short‐chain FAs (2–4 carbon atoms), medium‐chain FAs (6–12 carbon atoms), and long‐chain FAs (14–18 carbon atoms).FAs travel in circulation mostly bound to albumin (95%), esterified into lipoproteins, and as unbound FAs .314CO2 as an indicator of the β‐oxidation process.It is known that 20% of the total energy requirement in the adult brain is obtained from the oxidation of FA.Similar to other cells, neurons have another option to metabolize FAs. While short‐, medium‐ and long‐chain FAs are β‐oxidized in mitochondria, very‐long‐chain FAs (26:0) are metabolized in peroxisomes.411C]‐palmitate and [18F]‐fluoro‐6‐thia‐heptadecanoic acid in obese patients with metabolic syndrome, increased uptake and accumulation of FAs in different brain regions was found in comparison with healthy subjects.2+ uptake capacity that was accompanied by a decrease in hypothalamic neuronal excitability and consequent impaired function of energy control in the hypothalamus during obesity.There is a strong correlation between chronic intake of HFD and the development of neuroinflammation and brain insulin resistance. The last effect was reported to be dependent on the increased phosphorylation of residue S307 of insulin receptor substrate‐2 in the hypothalamus5PA is a 16‐carbon long‐chain saturated FA that is the most abundant saturated FA in the human body (65% of saturated FAs) and represents the main component of HFD. PA can be provided by the diet from vegetables (10%–40%) and animal fat (20–30%)5.1Ceramides are signalling molecules involved in neuronal development, neuronal death and cellular senescence. De novo ceramide synthesis is controlled by the availability of palmitoyl‐CoA, which activates the rate‐limiting enzyme serine‐palmitoyl transferase.5.2+ contents and an increase in ATP levels after exposure to high but not toxic doses of PA, suggesting that neurons can utilize PA as an energy substrate when exposed to high concentrations of saturated FAs.+ was correlated with the blunted effects of PA on insulin‐induced metabolic activation as well as with the inhibition of the insulin/PI3K/Akt pathway. The participation of ROS production in this effect was demonstrated by the inhibition of mitochondrial ROS with the mitochondria‐targeted antioxidant mitoTEMPO, which prevented PA‐dependent insulin resistance.2+ channels. The resulting increase in intraneuronal Ca2+ concentrations activates Ca2+‐dependent cPKC, which phosphorylates Akt in inhibitory residues, leading to its inactivation.The role of insulin in neuronal function as a metabolic, growth, synaptic and survival modulator has been extensively validated.5.3In addition to the above effects, PA exerts damaging consequences through enhances mitochondrial oxidative stress. PA treatment increased the production of superoxide dismutase after 8 h of exposure and decreased the production of ATP at 24 h, suggesting mitochondrial damage that correlates with the development of insulin resistance in neuroblastoma Neuro‐2a cells.How might lipid‐overloaded mitochondria induce mitochondrial stress and insulin resistance? Although this question has not been fully resolved, it has been proposed that chronic elevation of fatty acids leads to persistent pressure on the electron transport chain, resulting in disruption of redox balance and ROS‐signalling.5.4It has been demonstrated that HFDs trigger excessive endoplasmic reticulum (ER) stress and exert opposite influences on the expression of plasticity‐related proteins such as BDNF, synaptophysin and NMDA receptors in the rat prefrontal cortex.6In addition to the consequences of the metabolic oxidation of PA on neurons, other effects can also be carried out through the activation of free fatty acid metabotropic receptors (FFARs). FFARs are G‐protein coupled receptors (GPCRs) located in the cell membrane that are mainly involved in metabolic regulation. FFARs are a family of four members: FFAR2  and FFAR3 (GPR41) are activated by short‐chain FFAs, whereas FFAR1 (GPR40) and FFAR4 (GPR120) are activated by medium‐ and long‐chain FFAs.Activation of GPR40 regulates insulin secretion in pancreatic β‐cells, but its role in the CNS is not yet clear.In general, polyunsaturated FAs prevent and/or reverse the metabolic alterations induced by saturated FAs and manifest beneficial effects in neurodegenerative diseases.7Comorbidities associated with a high intake of saturated fat, such as obesity and type II diabetes, are risk factors for the development of cognitive impairments and even AD.AppNL−F/NL−F, long‐term HFD intake was associated with insulin resistance, poor cognitive performance, increased deposition of Aβ and the presence of neuroinflammation and oxidative stress markers.In fact, new evidence supports the relationship between obesity and dementia from a review of 19 longitudinal studies including people aged 35–65 years.These data demonstrate the effects of brain exposure to high levels of saturated FAs and point to the connection between lipid dyshomeostasis and the risk for AD.8The consumption of saturated FAs is strongly associated with morphological and functional changes in neurons. Accumulating evidence describes consistent dysregulation of neuronal metabolism induced by PA that leads to insulin resistance, decreased glycolysis, altered mitochondrial function and ER stress. These effects seem to contribute to cognitive decline. Recent interesting evidence supports the notion that in certain conditions, neurons can metabolize saturated long‐chain FAs through a metabolic energy pathway that sustains part of their deleterious effect. Understanding the mechanisms that lead to metabolic alterations in neurons could help to develop several strategies for the prevention, early detection and treatment of cognitive impairment associated with high consumption of saturated FAs or associated with insulin resistance, metabolic syndrome or diabetes. Although a variety of potential mechanisms have been explored, the underlying molecular cascade responsible for dietary fat‐induced neuronal dysfunction and behavioural changes remain elusive, and more research is needed to understand the signalling pathways that are activated in a specific metabolic context. An interesting avenue of studies to clarify the mechanisms downstream of the activation of GPR40 in neurons by polyunsaturated and saturated fatty acids is now open, as well as interrogation of the conditions that determine the metabolic routes that FAs follow into the brain to induce or avoid lipotoxicity.Karina Sánchez‐Alegría: Conceptualization ; data curation ; formal analysis ; funding acquisition (supporting); investigation ; methodology ; project administration (supporting); resources (supporting); software ; supervision (supporting); validation (supporting); visualization (supporting); writing – original draft ; writing – review and editing (supporting). Clorinda Arias: Conceptualization ; data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration (lead); resources ; software ; supervision (lead); validation (lead); visualization (lead); writing – original draft ; writing – review and editing (lead).The authors declare no conflict of interest.The authors declare that neither animals nor human subjects were involved in the creation of this manuscript.


Proper spindle orientation is essential for cell fate determination and tissue morphogenesis. Recently, accumulating studies have elucidated several factors that regulate spindle orientation, including geometric, internal and external cues. Abnormality in these factors generally leads to defects in the physiological functions of various organs and the development of severe diseases. Herein, we first review models that are commonly used for studying spindle orientation. We then review a conservative heterotrimeric complex critically involved in spindle orientation regulation in different models. Finally, we summarize some cues that affect spindle orientation and explore whether we can establish a model that precisely elucidates the effects of spindle orientation without interfusing other spindle functions. We aim to summarize current models used in spindle orientation studies and discuss whether we can build a model that disturbs spindle orientation alone. This can substantially improve our understanding of how spindle orientation is regulated and provide insights to investigate this complex event. The division process is associated with the emergence of diverse cellular functions and assembly of three‐dimensional tissue structures, which rely partially on the orientation of spindle fibres.Microtubule remodelling occurs during the formation of the specialized bipolar structure of the spindle fibres. Chromosomes are attached to the spindle microtubules at the kinetochore, which appears as a bridge between the poles. The astral microtubules interact with cortical proteins linking the spindle poles to the cell cortex. Despite research progress in regulating spindle orientation in the past decades, the lack of a suitable universal model has been a key limitation in the study of spindle orientation, suggesting a need to develop an appropriate model. This review presents several causes of spindle misorientation and discusses the possible solutions.2Drosophila and Caenorhabditis elegansA correlation between the orientation of the division axis and cell fate has been discovered in However, in vitro models have more advantages in observing intracellular changes using microscopic analysis. The frequently used in vitro models include cells cultured on fibronectin‐coated plates on micropatterns, or using three‐dimensional (3D) culture methods Figure . Among t3A conserved heterotrimeric complex, Gɑi‐LGN‐NuMA, is reportedly involved in regulating spindle orientation both in vivo and in vitro.Drosophila and GOA1/GPA16‐GPR1/2‐LIN5 in C. elegans , a structure composed of proteins and polysaccharides secreted by cells, is located on the cell surface or between cells and affects spindle orientation directly or indirectly.4.3Drosophila wing is not dependent on its shape.Drosophila follicular epithelium.In the past, cells were considered to divide along their longest axis. This is called the ‘Hertwig rule’. It has been indicated that the spindle in a mitotic cell can perceive cell shape changes to realign itself along the longest axis.5The findings on the molecular mechanisms that control the orientation of the mitotic spindle reveal that the conservative heterotrimeric complex Gɑi‐LGN‐NuMA regulates spindle orientation in different species. However, the molecular interplay to regulate the recruitment and maintenance of LGN to the cellular cortex is still unknown. The knockdown of LGN or NuMA results only in weak spindle orientation phenotypes,A specialized bipolar spindle orientation machinery plays an irreplaceable role in regulating spindle angles.Drosophila.Aurora‐A kinase can regulate αPKC/Numb cortical polarity and spindle orientation to inhibit neuroblast self‐renewal in Adenosine‐5′‐monophosphate‐activated protein kinase has been found to regulate mitotic spindle orientation through the phosphorylation of the myosin regulatory light chain.Previous studies have compared cell division in vitro and tissue development in vivo under controlled spindle orientation.Tao Zhong: Software ; writing – original draft (lead); writing – review and editing (lead). Xiaoxiao Gongye: Writing – original draft . Minglei Wang: Software ; writing – original draft . Jinming Yu: Conceptualization (lead); funding acquisition (supporting); project administration (lead); writing – review and editing (lead).The authors confirm that there are no conflicts of interest.


In the published article, there was an error in the article title. Instead of “An Robust Rank Aggregation and Least Absolute Shrinkage and Selection Operator Analysis of Novel Gene Signatures in Dilated Cardiomyopathy,” it should be “Robust Rank Aggregation and Least Absolute Shrinkage and Selection Operator Analysis of Novel Gene Signatures in Dilated Cardiomyopathy.”guichun@yahoo.com,” it should be “gui_chun@163.com.”In the published article, due to the previous corresponding author's email address becoming unavailable, it has been updated. Instead of “The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


A significant decrease in the granularity, indicative of neutrophil activation, was observed both in the exposure measurements and the exposure minus control measurements. Earlier EMF publications show immune function increase in isolated cells and more effective immune responses in animals with infections. This result, therefore, supports the thesis that the exposure can activate the innate immune system in humans, speed up the innate immune response, and may have potential beneficial effects in infectious disease. © 2022 Bioelectromagnetics Society.This research aims to demonstrate in a randomized, placebo‐controlled crossover design study that a nominal 5 μT low‐frequency electromagnetic field (LF‐EMF) signal for 30 min activates neutrophils in vivo in humans. Granularity of neutrophils was measured in blood samples of healthy human volunteers ( Neutrophilic granulocytes are the most numerous white blood cells in humans (~2 billion in the bloodstream and 8–50 billion in tissue). They are first responders to a new infection. Neutrophils crucially provide early innate immune responses against a range of extracellular and intracellular bacteria but also control certain viral and fungal infections. Neutrophils display phenotypic and functional heterogeneity and regulate antimicrobial function and the innate immune response. An important recently discovered function is that neutrophils are able to activate and facilitate the response to an infection by other more specialized immune cells, and thereby connect the innate to the adaptive immune system , The question ‘how and why cells detect low strength LF‐EMF signals and respond to them’ is beyond the scope of this article, although there is abundant evidence that they do [Rosado et al., The short‐term exposure, of which possible effects can be rapidly detected, allowed for a randomized, placebo‐controlled crossover design which could be completed in a short time. The primary outcome variable was the activation state of the neutrophils that could be determined in a routine setting in the clinical chemistry laboratory of a hospital. Several confounding factors, however, have to be considered, as this could help avoid loss of samples as happened in our study due to these unforeseen issues. First, different Sapphire analyzers, even when duly calibrated to give precise blood cell counts, have a different baseline for Nimn. Second, due to the high sensitivity of neutrophils to environmental conditions, a delay in analyzing after blood sampling induced substantial shifts in Nimn. Therefore, all samples must be analyzed as soon as possible, and delays should be avoided. Third, using tubes, there might be a risk on blood sample coagulation which requires drawing an extra blood sample. Also, inside  buildings the ambient static field can be significantly changed in direction and strength due to construction iron. Moreover, when working in an area (or building) with an earth (or ambient) magnetic field strength different from The Netherlands (47 µT), the frequencies may have to be changed (linearly) to achieve a similar resonance potential. Lastly, it is crucial to maintain sufficient distance between individuals receiving the exposure and the control exposures, as even very small signals (down to 3 nT) can still have an effect on neutrophils [Cuppen et al., The results of this study indicate that the LF‐EMF treatment activates neutrophils in vivo in humans, aged between 50 and 70. Therefore, LF‐EMF exposure with the CBT signal may have the potential to be applied in providing a treatment modality in infectious diseases, especially when applied early in the infection by speeding up the immune response that could keep the infection smaller and reduce its clinical effects on the patient.JC originally identified immune stimulation with EMF as a research subject inspired by work by Dr. Georges Vanroy (†2003) in chronic infectious disease using combined electric and electromagnetic stimuli. JC designed the signal, the device hardware and software, and designed and supervised the study. HS originally identified neutrophils as a target for research in EMF immune stimulation and the granularity measurement as a method of interest. CG did the statistics, and BR coordinated and managed the data collection with support from AW. JC and HS wrote the article. TV and JC declare to own stock in Umani Medical BV, which owns the IP concerning the method and devices used. CG and AW are coworkers of Umani Medical BV.


Nail disorders in general are difficult to treat and often frustrating, and this is also the case with nail psoriasis, especially when it is limited to the nails, and not affecting joints. The quality of life of patients with nail psoriasis is negatively affected, owing to the chronic course of the disease and frequent relapses. The purpose of this study was to compare treatment response and maintenance of response during follow‐up of 12 patients with nail matrix psoriasis limited to a few nails, who were treated with intralesional injections of either methotrexate (MTX) 25 mg/mL or triamcinolone acetonide 10 mg/mL. Patients were treated every 6 weeks for 24 weeks and followed up for 6 months. Photographic documentation and assessment by Nail Psoriasis Severity Index were performed during each treatment session and at each follow‐up visit. At the end of the four treatment sessions, all patients had improvement of their disease, which continued during follow‐up, especially for the MTX‐treated group. The purpose of this study was to compare the response to treatment and follow‐up of 12 patients with nail matrix psoriasis, limited to few nails, treated with intralesional injections of methotrexate 25 mg/mL or triamcinolone acetonide 10 mg/mL. Nail disorders in general are difficult to treat and often frustrating, and this is also the case with nail psoriasis, especially when it is limited to the nails. Based on recent expert consensus,We report our own experience with intralesional injections of MTX compared with TA in adult patients with nail‐matrix psoriasis, but with a more severe clinical presentation than patients previously reported in the literature.The study was approved by the University of Bologna  and all patients provided signed, written informed consent before participating in the study.Inclusion criteria were nail psoriasis present for > 1 year; nail involvement exclusively; no use of topical or systemic treatment for psoriasis in the 3 months preceding the study; and failure of the disease to respond to conventional treatments and/or severe relapse after their discontinuation. Exclusion criteria were pregnancy and lactation; debilitating haematological, hepatic, infectious, neoplastic or neurological conditions; and immunodepression of any kind.In total, 12 adult patients  with biopsy‐proven nail‐matrix psoriasis limited to 3 nails, were enrolled in the study starting from January 2019, with the study ending in September 2020. All patients had comparable medical and personal history verified to avoid potential biases. Patients were divided into two groups of six patients each: Group 1 was treated with MTX 25 mg/mL and Group 2 with TA 10 mg/mL. Each group had a baseline Nail Psoriasis Severity Index (NAPSI) of 5.3.All necessary investigations were performed before and at each visit during MTX injections and patients were asked to take folic acid 5 mg once weekly (not on the day of injection) to reduce MTX‐associated AEs and toxicities. No investigations were considered necessary for patients undergoing TA injections.In total, 20 nails in the 12 patients were treated. Depending on the group, either MTX or TA was injected into each affected nail, following the De Berker techniqueAt the end of the four sessions, all patients showed improvement of their nail psoriasis Figs  and 2 anAll patients were satisfied with the procedure despite procedural pain in all of them that was, however, well tolerated. No major AEs were reported. Subungual haematoma occurred in one patient treated with MTX and in one patient treated with TA. Hypopigmentation of the proximal nail fold was instead reported in two of six patients treated with TA.MTX, a folic acid antagonist with anti‐inflammatory and immunosuppressive properties, has been successfully used for the systemic treatment of multiple inflammatory diseases including psoriasis and psoriatic arthritis. MTX exerts its cytotoxic effects, especially on rapidly multiplying tissues with high growth fraction, inducing a reduced release of inflammatory cytokines such as tumour necrosis factor‐α, interferon‐gamma, interleukin‐12 and interleukin‐6.According to the literature systemic MTX is well known to improve nail psoriasis where mainly the matrix is affected.In the presence of nail matrix psoriasis limited to few nails, high potency steroid as clobetasol propionate 0.05% cream is a valid option,Nail psoriasis is a difficult to treat disorder, especially in its isolated form.The quality of life of affected patients is markedly reduced due to the chronic course of the disease and frequent relapses.Intralesional TA injections are considered a safe and effective method for the treatment of nail psoriasis.Intralesional MTX injections have also been reported as a safe and effective treatment.In this study, nail‐matrix psoriasis responded very well to both treatments, but there were fewer AEs and more stability at follow‐up for the MTX‐treated patients.and larger studies are warranted to draw definite and statistically relevant conclusions.Larger studies are however necessary to establish the optimal dosage, number and frequency of injections to clear up the condition, and maximum duration of treatment. A long‐term follow‐up is also needed to detect possible relapses after drug withdrawal. A placebo arm should be also useful to draw definitive conclusions and to understand if improvement is due to the injected drug or to a reverse Koebner phenomenon (disappearance of a dermatosis after the site of injury).


SAD) were of particular interest because of their known role in the conversion of fully saturated into unsaturated fatty acids. Two SAD genes were more highly expressed in seeds from Canadian populations, consistent with the observation of increased levels of unsaturated fatty acids in seeds from that region. We also constructed a gene co‐expression network to investigate regional variation in network modules. The results of this analysis revealed regional differentiation for eight of 12 modules but no clear relationship with oil biosynthesis. Overall, the differential expression of SAD genes offers a partial explanation for the observed differences in seed oil composition between Texas and Canada, while the expression patterns of other metabolic genes suggest complex regulation of fatty acid production and usage across latitudes.Seed oil composition, an important agronomic trait in cultivated sunflower, varies latitudinally across the native range of its wild progenitor. This pattern is thought to be driven by selection for a higher proportion of saturated fatty acids in southern populations compared with northern populations, likely due to the different temperatures experienced during seed germination. To investigate whether these differences in fatty acid composition between northern and southern populations correspond to transcriptional variation in the expression of genes involved in fatty acid metabolism, we sequenced RNA from developing seeds of sunflowers from Texas, USA, and Saskatchewan, Canada  grown in a common garden. We found 4,741 genes to be differentially expressed between Texas and Canada, including several genes involved in lipid metabolism. Several differentially expressed lipid metabolism genes also colocalized with known oil quantitative trait loci (QTL). The genes producing stearoyl‐ACP‐desaturases ( Across their native range, wild sunflower populations vary latitudinally in the proportion of saturated FAs in their seed oil; that is, southern populations tend to produce seeds with a greater proportion of saturated FAs than northern populations , which is one of the world's most important oilseed crops. Cultivated sunflower was initially domesticated over 4,000 years ago in what is now the east‐central USA as a source of edible seeds and for cultural purposes  Are differentially expressed genes (DEGs) related to oil metabolism found within known oil QTL? and (4) Are DEGs related to oil metabolism found within co‐expression network modules associated with geographic regions? To answer these questions, we sequenced the transcriptomes of developing seeds from the same crosses of Texan and Canadian sunflower populations that produced the seed for the oil phenotyping performed in McAssey et al. . The ground tissue was then transferred into a tube and placed in liquid nitrogen to keep it frozen. After removing individual samples from the liquid nitrogen for processing, 1 ml of Trizol was added to each tube. The contents were then mixed and allowed to incubate at room temperature for 5 min. Chloroform (300 μl) was then added to each sample, and the tubes were manually shaken and then centrifuged at 12,000 × G for 10 min. The aqueous phase of each sample was then removed via pipetting and transferred to a new tube. After mixing with a 0.53X volume of 100% ethanol, RNA was purified by processing the solution following the Qiagen RNeasy Plant Mini Kit  protocol with on‐column DNase digestion.http://dna.uga.edu; Athens, GA). The resulting sequence data are available via the Short Read Archive (SRA) under BioProject PRJNA706177.RNA quality was assessed using an Agilent Bioanalyzer . Nine samples from Texas and 11 from Canada had RIN values ≥8.5 out of 10 and therefore passed our minimum quality thresholds for use in library construction (Table https://github.com/EDitt/Sunflower_RNAseq). Adapter sequences were first trimmed using Trimmomatic v0.36 with default settings . GOseq v1.34.1  terms for each of the sunflower genes were obtained from the blast2go . Using a Wilcoxon rank sum test to compare differences in the TPM of the two SAD genes and most highly expressed FAD gene between TEX and CAN, we found that there were significant differences in expression for all three genes between regions saturation in seed oils  genes are more highly expressed in samples from Texas versus Canada. KCS genes are involved in the elongation step during the synthesis of very‐long‐chain FAs , while within any individual oil QTL, DEGs were not significantly enriched.We also asked if DEGs were enriched within known sunflower oil QTL from previous mapping studies. We determined the genomic location of DEGs relative to 51 oil QTL from previously published mapping studies in cultivated sunflower and eight oil QTL from a wild × cultivated sunflower cross , and the other differentially expressed FAD gene is the most highly expressed FAD gene in our data set discussed earlier (HanXRQChr14g0452931). One differentially expressed KCS gene is also found within a palmitic acid content QTL on chromosome 8. As noted earlier, KCS genes contribute to the production and elongation of very‐long‐chain FAs and are involved in abiotic stress tolerance  found on chromosome 6 (HanXRQChr06g0172791); this gene corresponds to the rate‐limiting enzyme producing lysophosphatidic acid (an important intermediate in the formation of acyl‐lipids) in plants .The five genes with the highest connectivity within each module were identified as “hub” genes (supporting information Data Set While genes related to oil metabolism were found within co‐expression modules, we did not find large groups of oil metabolism genes that were co‐expressed and also varied between regions. This suggests that the variation in seed oil composition between regions is not the result of coordinated gene regulation within a co‐expression module. Instead, variation in seed oil composition is likely the result of several independent processes that do not necessarily act in a coordinated manner.4SAD genes in Canadian sunflower populations compared with Texan sunflower populations could play a role in altering seed oil composition and contribute to observed patterns of latitudinal variation in oil composition. Furthermore, several other genes related to lipid metabolism are differentially expressed across the range and can be found within oil QTL. While these genes could influence the FA composition of seeds, they could also be involved in other important biological processes related to lipid metabolism. Overall, our results provide insights into the possible role of transcriptomic variation of many genes involved in oil metabolism, and specifically SAD and FAD genes, in shaping variation in seed oil composition across the range of wild sunflower while also providing avenues for future studies of this ecologically and agronomically important trait.Taken together, our results suggest that the observed differences in seed oil composition between regions likely result from the complex regulation of numerous genes involved in FA metabolism. Evidence suggests that the higher expression of The authors did not report any conflict of interest.MHB analyzed the data and wrote the paper. EVM designed the experiment jointly with JMB, grew the plants, extracted RNA, prepared the sequencing libraries, and contributed to writing the paper. ELD assisted with analyses and contributed to writing the paper. JMB designed the experiment jointly with EVM and contributed to interpretation of analyses and writing of the paper.Data S1. Supporting InformationClick here for additional data file.Data S2. Supporting InformationClick here for additional data file.Data S3. Supporting InformationClick here for additional data file.Data S4. Supporting InformationClick here for additional data file.Data S5. Supporting InformationClick here for additional data file.Data S6. Supporting InformationClick here for additional data file.Figure S1. Number of reads mapping to the sunflower genome for each sample in our data.Click here for additional data file.


However, far fewer studies have attempted to translate the findings from cellular and molecular studies into a human model. In this review, we focus on the role that serotonin (5‐HT) plays in muscle activation in humans. 5‐HT is a potent regulator of neuronal firing rates, which can influence the force that can be generated by muscles during voluntary contractions. We firstly outline structural and functional characteristics of the serotonergic system, and then describe how motoneurone discharge can be facilitated and suppressed depending on the 5‐HT receptor subtype that is activated. We then provide a narrative on how 5‐HT effects can influence voluntary activation during muscle contractions in humans, and detail how 5‐HT may be a mediator of exercise‐induced fatigue that arises from the central nervous system.Ionotropic inputs to motoneurones have the capacity to depolarise and hyperpolarise the motoneurone, whereas neuromodulatory inputs control the state of excitability of the motoneurone. Intracellular recordings of motoneurones from  Abstract figure legend Inputs to neuromodulatory receptors on motoneurones, such as those involved in the serotonergic system, modify the motoneurones’ responsiveness to ionotropic input. The release of serotonin (5‐HT) into the spinal cord is linked to the level of motor activity being performed, where 5‐HT can increase the discharge rate of motoneurones via excitatory 5‐HT receptors on the soma and dendrites. This in turn can lead to increased voluntary muscle activation (VA) and maximal force generation. However, intense release of 5‐HT onto motoneurones may lead to a spillover of 5‐HT into extracellular compartments to activate inhibitory 5‐HT receptors on the axon initial segment. This can cause a reduction in motoneurone discharge rate, thus decreasing VA and maximal force generation. To gain insight into the serotonergic contributions to muscle activation in humans, pharmacological interventions have been employed to enhance the concentration of 5‐HT in the central nervous system or activate selective 5‐HT receptors. The final common pathway in the motor system is the motor unit, which consists of the α‐motoneurone in the spinal cord and all muscle fibres that it innervates. Given that the motor unit links the central nervous system to skeletal muscle, efficient and purposeful movement can only be achieved by tightly controlled motoneurone activity. The firing pattern of a motoneurone is produced by converting synaptic inputs into action potentials, where the regulation of input to output is determined by the intrinsic electrical properties of motoneurones Kernell, . Hence, in vitro and in situ animal preparations have provided considerable insight into how electrical properties of motoneurones are influenced by neuromodulators. However, fewer studies have attempted to translate findings from intracellular experiments into humans, as there are considerable challenges in reproducing reductionist experiments in an intact human neuromuscular system.Synaptic inputs to motoneurones will typically activate two classes of receptors: ionotropic and neuromodulatory. In a general sense, inputs to ionotropic receptors are command signals from descending motor pathways, peripheral afferents, or spinal interneurons, which have the capacity to depolarise and hyperpolarise the motoneurone. Typically, ionotropic receptors are ligand‐gated ion channels which directly gate ion flow into cells to generate either an excitatory or inhibitory response in postsynaptic neurons  is a monoamine neurotransmitter that modulates the intrinsic properties of neurons in the central nervous system (CNS). In the periphery, it is synthesised in enteric mucosal cells, influences gut motility among other functions, and is stored in platelets  and L‐type voltage‐gated calcium channels (Cav1.2 and Cav1.3) on somato‐dendritic surfaces of motoneurones  play a critical role in setting the gain of motoneurones. PICs are mediated by voltage‐gated sodium channels (NaF). Discharge rates are recorded from intramuscular electromyography electrodes , in whom ∆F increased with ingestion of an SSRI or decreased with a 5‐HT2 receptor antagonist  and inhibitory  synaptic activity that can be modulated by 5‐HT Fig. . Adding In vivo cat experiments have revealed that reciprocal inhibition may be especially critical for regulating PIC amplitude during functional motor activity. Even minor rotations of the ankle joint that cause barely detectable changes in reciprocal inhibition of motoneurones will reduce PICs by ∼50% . Participant safety is an additional challenge in human experiments that use pharmacology to manipulate neurotransmitter activity. Human studies must operate within a window of safe drug administration, and typically use therapeutic doses of 5‐HT‐modulating medications. Thus, very little is known about how dosage effects influence 5‐HT activity in humans. Pharmacological interventions are also non‐specific, and will not have 100% effectiveness when competitively agonising, antagonising or inhibiting 5‐HT reuptake in humans. Individual variation in pharmacokinetics and drug responses also poses a challenge when studying humans, but variability in 5‐HT responses is also a prevalent feature of None.Both authors contributed to the conception and the design of this work, as well as the drafting and final approval of the manuscript. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.No funding was received for this work.Peer Review HistoryClick here for additional data file.


Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms  and embed them into building blocks  to construct native‐like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging. This Minireview covers some of the recent advances in the development of small chemical tags for optical bioimaging, from their chemical synthesis and characterization to their application in different biological systems, such as cells, tissues and whole organisms. It focuses on the design of fluorescent and Raman labels and features examples of metabolites, amino acids, nucleic acids as well as organelle and cell‐targeting probes. Among different modalities, optical imaging features multiplexing capabilities, low cost, and the possibility to directly acquire molecular information from cells, tissues and organisms with high spatial and temporal resolution.In the recent years, multiple chemical approaches have been devised to miniaturize fluorescent and Raman‐active tags for minimally invasive labeling of biomolecules while retaining sensitivity and compatibility with fluorescence and Raman microscopy Figure .[2] InThis Minireview article summarizes recent advances in chemical methodologies to reduce the size of optical labels to a few atoms  and accelerate the construction of native‐like supramolecular structures. In addition to the design and synthesis of minimal chemical reporters, we will also review examples of how they can be integrated into metabolites and biomolecules to prepare smart molecular entities  that were previously inaccessible Figure .2Fluorescent molecules have been extensively researched over the last decades and revolutionized the way we investigate biological processes in vitro and in vivo. While the chemical structures of fluorophores can vary widely,The oldest example of small sized fluorophores are coumarins, which were first synthesized by Perkin in 1868.Nitrobenzoxadiazoles (NBD) are another family of small sized fluorophores commonly used for biological imaging. Building from the original NBD chloride, N‐substituted NBD amines have been described both as environmentally sensitive dyes and as turn‐on or turn‐off probes.Another emerging class of small sized fluorophores are single benzene ring carbocycles. Fully carbocyclic fluorescent molecules such as aminonaphthalenes and azulene had long been known,3Nucleic acids are key biomolecules to define cell function. Due to their complex interactions, fluorescent nucleic acids (FNAs) must have minimal interference in the molecular recognition properties of the native counterparts. Nucleobases have been derivatized to produce collections of fluorescent analogues Figure ,[25] iThe derivatization of fluorescent nucleobases can be grouped into three types of molecular architectures: isomorphic, expanded and extended. Approaches that modify the heterocyclic cores of nucleobases are referred to as isomorphic. One of the most prominent isomorphic nucleobases is thienoguanosine, where the imidazole ring of guanine is replaced by a thiophene.The most widespread application of FNAs is their use for optical imaging, including the direct visualization of intracellular processes in live cells ,5, 38 Our group developed Trp‐BODIPY as the first BODIPY‐based fluorogenic amino acid to be used as a Trp surrogate in antimicrobial peptides for imaging fungal pathogens in ex vivo human lungs.Trp‐based amino acids including smaller chemical modifications to boost their optical properties have been reported. Although they are typically not as bright as BODIPY‐containing amino acids, they still display enhanced optical properties when compared to the natural Trp. Some of these amino acids include azatryptophans (emission ≈400–420 nm)Alternative scaffolds have been successfully employed to prepare fluorescent amino acids with suitable properties for in vitro biomolecular and bioimaging studies Figure . These iThe miniaturized size of fluorescent amino acids has also been exploited to utilise the machinery of living cells for site‐specific labeling of proteins under physiological conditions. Examples of these structures include the seminal work by the groups of Schultz, Chin and others on genetically encoded amino acids ,5Whereas several Raman microscopy reports have demonstrated label‐free detection of proteins,−1 using spontaneous Raman spectroscopy. Subsequently, stimulated Raman scattering (SRS) microscopy, which offers faster image acquisition rates, has been identified as a versatile imaging modality for biological applications using alkyne probes. For instance, alkyne tag Raman imaging was employed to visualize small molecule drugs,4‐fold higher signals compared to single alkyne species were described for rapid visualization of cellular organelles using SRS microscopy.−1—and two‐photon fluorescence via aggregation‐induced emission.The first example of alkyne tag Raman imaging was demonstrated with the thymidine analogue EdU, a probe that incorporates into nascent DNA to monitor cell proliferation.C. elegans.Owing to their small size, alkynes and nitriles are interesting minimal groups to track biometabolites. Early examples included the detection of nucleic acids using EdU and EU.−1) as demonstrated with the visualization of a wide variety of biomolecules, including proteins,7 as a precursorIn parallel to triple bond labels, isotopic substitution strategies are a powerful labeling method due to their extremely small size. Deuterium labeling is attractive because C−D bonds generate Raman shifts within the cell silent region  to 968 cm−1 (13C‐Phe) enabled ratiometric measurements.13C‐labeled alkynes , which in principle allow for the identification of tens of molecular species at the same time.Multiplexed optical analysis is challenging because of the limited readouts that can be simultaneously detected in complex biological samples. Raman vibrational tags provide transitions with narrow peak widths  was prepared, with each probe exhibiting a discrete spectral frequency within 2017–2280 cm−1 for simultaneous imaging of organelle structures and combinatorial barcoding using polymer beads. The strategy was further optimized by incorporating polyyne Raman tags inside polystyrene nanoparticles (termed “Rdots”) for improved sensitivity in the picomolar range.The group of Min reported conjugated polyyne scaffolds to detect 20 different targets in live cells Figure .[91] A5‐fold). This process was referred to as electronic pre‐resonance (epr)‐SRS microscopy.An interesting alternative design for multiplexed miniaturized probes was recently reported via coupling of Raman‐active alkyne and nitrile moieties to NIR chromophores.p‐trifluoromethoxyphenylhydrazone.−1 to 2207 cm−1) was gradually observed at increasing pH values.One of the first applications of small molecule ratiometric Raman sensing demonstrated intracellular pH measurement using the pH‐sensitive C≡N stretching frequency of carbonylcyanide 2S) and ionic species has been possible using Raman‐based techniques. Notably, a ratiometric sensor for H2S was reported based on a bisarylbutadiyne probe containing an aromatic azide group that was reduced to aniline in the presence of intracellular H2S.2+ in live cells was demonstrated using a chelation‐based Raman probe,The ratiometric detection of small cellular messengers including hydrogen sulfide  and an intensity increase via epr‐SRS. Photoswitchable SRS imaging was achieved at 2194 cm−1 (DTE‐Ph‐Mito) in live cells through the incorporation of a pyridinium group for mitochondrial targeting, whilst multicolor SRS imaging using other Raman probes  was demonstrated.The narrow peaks of Raman tags represent an advantage over fluorescent structures for multicolor photoswitchable imaging. For instance, photoswitchable SRS imaging of cis‐1,2‐dicyano‐1,2‐bisethene was reported, where its two isomers  were interchangeable by irradiation with UV or visible light, respectively.12C/13C and 14N/15N. The authors simultaneously detected 4 targeted enzymes in live A549 cells, highlighting the potential of this technique to visualize protein heterogeneity in complex samples. In an alternative strategy, chemical activation of epoxy ketones to yield a terminal alkyne via Eschenmoser–Tanabe fragmentation using p‐toluenesulfonyl hydrazine was reported.−1. Altogether, these studies demonstrate that activatable Raman probes offer a unique approach for multiplexed imaging with potential applications to investigate the complex interplay of biological systems.Ozeki, Kamiya and co‐workers reported some of the first activatable Raman probes for multiplexed enzyme detection Figure .[108] 8In parallel to label‐free imaging, the miniaturization of chemical labels has become an integral challenge in optical probe development Table . The finThe authors declare no conflict of interest.Sam Benson received an MSc in Medicinal and Biological Chemistry from the University of Nottingham in 2016. Sam carried out his PhD under the supervision of Prof Marc Vendrell at the University of Edinburgh developing novel theranostics for cancer, including the synthesis of small fluorophores and photosensitizers. He has subsequently remained in the same group as a postdoctoral research associate, where his work focuses on the development of new systems for photodynamic therapy of glioblastoma.Fabio de Moliner obtained his PhD in Organic Chemistry from the University of Genova in 2010 under the guidance of Prof Andrea Basso. After graduation, he joined the research group of Prof Christopher Hulme at the University of Arizona (Tucson) as a postdoctoral research assistant to focus on the design and application of chemical methods based on multicomponent and domino processes. He joined the Vendrell group at the University of Edinburgh in 2015. His main research interests are the development of optical probes for in vivo imaging with a focus on the preparation of fluorescent amino acids and the labeling of small metabolites.William Tipping obtained his PhD in Chemistry from the University of Edinburgh in 2017 under the guidance of Prof Alison Hulme and Prof Valerie Brunton. After a brief postdoctoral position in the same group, he joined the University of Strathclyde in 2019 to work with Prof Duncan Graham and Prof Karen Faulds. His research interests include the development of imaging probes for Raman microscopy, and the development of stimulated Raman scattering microscopy for biomedical imaging applications.Marc Vendrell graduated in Chemistry at the University of Barcelona in 2007. He then joined the Singapore Bioimaging Consortium to work in synthetic fluorophores for optical imaging. In 2012 he started his independent career at the University of Edinburgh to develop optical probes for imaging immune function in humans. His research has led to the commercialization of several fluorescent probes and to the use of different molecular reagents for first‐in‐human imaging studies, which has been recognized internationally with several awards and distinctions. Since 2020 he is appointed as Chair of Translational Chemistry and Biomedical Imaging at the College of Medicine in Edinburgh.


Biological traits analysis (BTA) links community structure to both ecological functions and response to environmental drivers through species’ attributes. In consequence, it has become a popular approach in marine benthic studies. However, BTA will reach a dead end if the scientific community does not acknowledge its current shortcomings and limitations: (a) uncertainties related to data origins and a lack of standardized reporting of trait information; (b) knowledge gaps on the role of multiple interacting traits on driving the organisms’ responses to environmental variability; (c) knowledge gaps regarding the mechanistic links between traits and functions; (d) a weak focus on the spatial and temporal variability that is inherent to the trait expression of species; and, last but not least, (e) the large reliance on expert knowledge due to an enormous knowledge gap on the basic ecology of many benthic species. BTA will only reach its full potential if the scientific community is able to standardize and unify the reporting and storage of traits data and reconsider the importance of baseline observational and experimental studies to fill knowledge gaps on the mechanistic links between biological traits, functions, and environmental variability. This challenge could be assisted by embracing new technological advances in marine monitoring, such as underwater camera technology and artificial intelligence, and making use of advanced statistical approaches that consider the interactive nature and spatio‐temporal variability of biological systems. The scientific community has to abandon some dead ends and explore new paths that will improve our understanding of individual species, traits, and the functioning of benthic ecosystems. The Biological traits analysis (BTA) is attractive as it is easily applied to past and present datasets, transcending the taxonomic composition of the study area and thus supporting inter‐regional comparisons; however, BTA is not exempt from challenges, and we propose solutions to bring us out of some dead ends and walk on new paths. Providing this information enables sensitivity assessments of the trait data, i.e., analysis of the effect of expert opinion/lack of data, to be more readily conducted. While Hewitt et al.  and ThruThere is also a need for scientific consensus on the use of common terms, as there is a great diversity in the language used for traits, hindering their general application and even leading to misinterpretation , behavior , and morphology  , but also over shorter time scales   could be analyzed utilizing statistical analyses that focus on non‐linearities and in detecting change points . Plant ecology has much to share in terms of approaches for better elucidating intraspecific variability, with methods for comparing trait expression across the entirety of environmental gradients and for combining field observations with transplant experiments (“common garden experiments” in plant ecology terms) to tease apart genetic variation and trait plasticity .The BTA should incorporate a sensitivity analysis (including uncertainty in the input data), and a confidence assessment of the data sources each time BTA is used.Points 1 and 2 should be the basic requirement for any BTA.Researchers should explore new technologies, intelligent re‐use of survey outputs, non‐traditional data sources accompanied by a quality check (including citizen science initiatives), and the acquisition (and management) of big data to generate more baseline natural history data.Benthic ecology needs to embrace traits’ variability  beginning with the interpretation and discussion of results, but eventually incorporating variability in a multi‐dimensional species–traits matrices.The current open‐access data initiatives can contribute to a more transparent and standardized naming, selection, and assignment of traits if they keep evolving with the required scientific needs. And to achieve this, scientists need to make a consolidated effort to reach a consensus for a unified data standard that would allow the exchange and validation of traits data. Such an initiative would not only facilitate data exchange between research groups but would also make it easier for scientists to update and contribute to these databases. Despite recent advances  ; Writing—original draft (lead); Writing—review & editing (lead). Julie Bremner: Conceptualization (supporting); Writing—original draft ; Writing—review & editing . Judi Hewitt: Writing—original draft ; Writing—review & editing . Anna Törnroos: Conceptualization (supporting); Writing—original draft (supporting); Writing—review & editing . Maria Cristina Mangano: Writing—original draft (supporting); Writing—review & editing (supporting). Simon Thrush: Writing—original draft (supporting); Writing—review & editing . Hilmar Hinz: Conceptualization (lead); Writing—original draft ; Writing—review & editing .The authors declare no competing interests.Fig S1‐Table S1‐S2Click here for additional data file.


To establish age‐related, normal limits of monocular and binocular spatial vision under photopic and mesopic conditions.Plus (AP) test. The experiments were carried out on participants (age range from 10 to 86 years), who met pre‐established, normal sight criteria. Mean and ± 2.5σ limits were calculated within each 5‐year subgroup. A biologically meaningful model was then fitted to predict mean values and upper and lower threshold limits for VA and CT as a function of age. The best‐fit model parameters describe normal aging of spatial vision for each of the 16 experimental conditions investigated.Photopic and mesopic visual acuity (VA) and contrast thresholds (CTs) were measured with both positive and negative contrast optotypes under binocular and monocular viewing conditions using the Acuity‐p < 0.004).Out of the 382 participants recruited for this study, 285 participants passed the selection criteria for normal aging. Log transforms were applied to ensure approximate normal distributions. Outliers were also removed for each of the 16 stimulus conditions investigated based on the ±2.5σ limit criterion. VA, CTs and the overall variability were found to be age‐invariant up to ~50 years in the photopic condition. A lower, age‐invariant limit of ~30 years was more appropriate for the mesopic range with a gradual, but accelerating increase in both mean thresholds and intersubject variability above this age. Binocular thresholds were smaller and much less variable when compared to the thresholds measured in either eye. Results with negative contrast optotypes were significantly better than the corresponding results measured with positive contrast (This project has established the expected age limits of spatial vision for monocular and binocular viewing under photopic and high mesopic lighting with both positive and negative contrast optotypes using a single test, which can be implemented either in the clinic or in an occupational setting. Boundaries for healthy aging of spatial vision have been established under both photopic (daylight) and low mesopic (twilight) lighting for 16 different stimulus conditions.Equations that describe normal aging limits and full measures of variability in spatial vision have been produced using monocular and binocular thresholds measured with increments and decrements in luminance.The normal aging limits for spatial vision obtained in this study with standardized stimulus conditions have immediate use in the clinic and visually demanding occupational environments.Photopic visual acuity (VA) measurements are common in clinical practice and determine the patient's ability to resolve fine detail in high contrast.The rate of loss of visual sensitivity with decreasing retinal illuminance is also indicative of age‐related changes and/or the presence of early‐stage retinal disease.2 were used to investigate whether mesopic VA is a predictor for car crash involvement.2, although considered low in this study, is higher than the working luminance levels encountered in a number of occupations. In addition, measurements under mesopic conditions are considered to be more sensitive in medical selection.Normal aging affects both VA and CS.Binocular vision yields significant improvements in many aspects of vision, particularly in the mesopic range. A knowledge of binocular VA and CT limits of visual performance as a function of age is of great interest in occupational environments.This study employed optimized parameters for the assessment of spatial vision based on preliminary investigations and results from earlier studies reported in the literature.A total of 382 participants with age range between 10 and 86 years were recruited at three different sites in the Netherlands: (1) a private eye clinic (Damme Optometrie in Kesteren); (2) a university eye clinic  and (3) a normal working environment . The inclusion of a primary care setting, educational institution and workplace environment was a conscious choice to maximize random sampling in diverse populations. The study was approved by the Research and Ethics Committee at the City, University of London and the Medical Ethical Committee at the University Medical Centre, Utrecht, the Netherlands. All participants provided written consent. In cases where participants were younger than 16 years old, the consent form was, in accordance with Dutch law, signed by the participants' parents/legal guardians (10–11 years) or by the participant (child) and parents/legal guardians (12–15 years).precision‐vision.com) using the updated prescription. The original ETDRS illuminator cabinet was used to generate a chart luminance of 160 cd/m2. The 3 m distance was selected for convenience and to match the viewing distance employed in the Acuity‐Plus test. The anterior segment was assessed using a Topcon SL‐7F slit lamp  or CSO SL9900 5X‐D , and the transparency of the lens was noted for each participant and classified according to the Optometric Grading Scales.topcon.com]) or Canon CX‐1 .A detailed medical history was taken: all participants or parent/guardian answered questions about their general health, use of medications, ocular health and general and ocular family history. A full objective and subjective refraction were conducted at a test distance of 3 m. VA was then measured monocularly and binocularly in logMAR units with the 2000 series revised Early Treatment Diabetic Retinopathy Study (ETDRS) chart 2 . This test of spatial vision is one of a series of Advanced Vision and Optometric Tests (AVOT) developed at the City, University of London for use in occupational health and in the clinic. The Acuity‐Plus test employs a stable, high‐resolution, 10‐bit dynamic range visual display  which the participant views from a distance of 3 m. The room was darkened at all three locations and the low mesopic ambient lighting was attributed to the light produced by the operator's monitor and the stimulus background field on the visual display. This ensured that the ambient lighting remained very similar across the three testing sites. The testing display is fitted with a hood to minimize ambient lighting, and initial adjustments were carried out by the manufacturer to minimize the black light level and to achieve a maximum luminance of ~146 cd/m2 in native colour mode. Furthermore, the stimulus display was checked for luminance calibration periodically and recalibration of each primary colour was performed when required using the LMT‐1009 luminance meter  and the LUMCAL calibration program . A high‐performance laptop drove the display via a VESA DisplayPort interface, which supports 10‐bit output graphics needed to match the dynamic range of the visual display. All participants performed the Acuity‐Plus test with full updated correction for the testing distance of 3 m, which was provided in a trial frame, to ensure that testing conditions for all participants were equivalent with respect to spectacle properties. The VA and CTs were measured using a four‐alternative, spatially‐aided, forced‐choice procedure based on four, randomly interleaved, two‐up/one‐down, staircases with variable step sizes. The chance probability of a correct response is 1/16 and the thresholds measured correspond to 71% probability of a correct response.Plus test. Short breaks separated successive tests to minimize fatigue and the participants were also encouraged to take additional breaks whenever needed during the session. The mesopic protocol was always preceded by ~10 min of adaptation to the low luminance screen employed in the mesopic condition. For this protocol, the participants wore spectrally calibrated, ‘neutral density’ sunglasses . The program employed the known spectral transmittance of the sunglasses to ensure that when viewed through the glasses, the stimulus display had the correct luminance and chromaticity. A ‘learning mode’ option preceded any measurements of VA or CTs. This brief test required 100% correct responses and ensured that every applicant was familiar with and understood the task. Every participant carried out the initial learning test under binocular viewing conditions. The order of testing  was randomized for both photopic and mesopic conditions. The study employed the Acuity‐Plus standard photopic and mesopic protocols. The standard photopic protocol measured VA and CTs at the fovea with both positive and negative contrast stimuli for a screen luminance of 32 cd/m2 and CIE— chromaticity coordinates of 0.305, 0.323. The standard mesopic protocol measures the same four parameters using light of the same chromaticity but with a screen luminance of 2 cd/m2. The choice of adapting background luminance was based on typical luminances encountered in mesopic work environments when good spatial vision is still needed in order to carry out the visual tasks. Similar light levels are also found in many lit spaces at night and in traffic situations when safety remains an important requirement.High contrast VA and CTs with optotypes of both positive and negative contrast were measured in each participant using the Acuity‐10 (MAR), where MAR is the size of the gap (in minutes of arc) needed at the threshold to locate its position. Alternatively, when using the most common Snellen notation, VASnellen ~ 6/(6 × MAR). This means that a Snellen VA of 6/6 corresponds to a MAR of 1′. Doubling this to 2′ makes the Snellen acuity 6/12. The VA results in this paper are presented in log units (LogMAR) and shown in all tables and graphs. A linear scale is also provided for convenience. CTs, measured in percentage contrast, are plotted on a log scale and the corresponding values are also given in log units.The stimulus was presented for 160 ms to avoid letter scanning and hence multiple fixations. When this presentation time is employed, the majority of normal trichromats achieved a spatial resolution better than 1 min of arc (1′) acuity, i.e., a Landolt ring size of 5′ outer diameter converts to an equivalent Snellen VA of 6/6 and a LogMAR VA of 0.0. The conversions are simple; LogMAR = log t‐test analyses were performed to determine significant differences between participants with and without each of the selected conditions. If there was no difference between the selected chronic condition subgroup and the remaining subjects with no such conditions, the participants were included in the study. This was the case for participants with hypertension who rarely exhibit significant loss of spatial vision. However, those diagnosed with diabetes were excluded since this systemic condition is known to affect several aspects of vision.Absence of a medical history of ocular or systemic conditions known to affect vision. Participants were arranged into groups by type of chronic condition. Absence of current signs of ocular disease, conventional or refractive laser surgery, corneal dystrophies or lens extraction. Participants with nuclear, cortical and posterior subcapsular lens opacities graded 3 or higher  were excluded.A new filter was developed and applied to detect those with potential subclinical, but yet unidentified, visual problems. The filter relies on the comparison of thresholds measured with the same stimulus parameters in each of the two eyes. Since changes caused by either the optics of the eye or diseases of the retina rarely affect both eyes in exactly the same way, participants with abnormal differences in VA and/or CTs between the two eyes were not included in the analysis for normal age limits. The index employed to describe the interocular difference (IOD) between the log values of the measured thresholds in the two eyes was, IOD = ABS(Log RE − Log LE), with RE and LE referring to the right and left eyes, respectively. Figure In addition, the results for the remaining study participants were reanalysed per decade and all outliers with log thresholds outside the ±2.5σ range with respect to the corresponding mean threshold values were removed from the analysis. This filter was applied separately to each measurement condition. At this last stage of screening for normal healthy vision, the study participants eliminated from the analysis varied from 0% to just under 2.5%, depending on the stimulus condition.The principal aim of this study was to establish mean values and upper normal limits of spatial vision as a function of age. In order to achieve this aim, a number of filters were employed to ensure that the participants included in the study had ‘normal visual performance’ for the corresponding age. Each of the included participants fulfilled the following requirements:ibm.com) and JMP . Distributions and frequencies of results for categorical variables were examined in each decade. Skewness, kurtosis and Shapiro–Wilk were conducted as normality tests. The filters described made it possible to identify those participants who failed to perform the spatial vision tasks within the expected, normal statistical limits for the corresponding age. Paired samples t‐tests were used to compute differences between the positive and negative contrast measurements per decade, and a one‐way analysis of variance (ANOVA) was conducted to compare the photopic and mesopic measurements between the different test locations.Statistical analyses were carried out using SPSS . Hypertension was therefore not used as an exclusion criterion. The subgroups with other systemic and ocular diseases were too small to analyse reliably and were therefore excluded from the normal group. The effects of applying the various filters to the study population are illustrated in Figure Three hundred and eighty‐two participants were enrolled in this study. Independent Plus test for both the photopic and the mesopic conditions. Paired t‐tests were carried out to assess significant differences between the two eyes. All statistical comparisons carried out employed Bonferroni correction to account for multiple comparisons. No significant differences were found between the right and left eye VA and CT results (p > 0.01). The order in which the two eyes were tested had no significant difference in the results for both VA and CTs (p > 0.01). Within the same eye, the measured differences between positive and negative contrast optotypes were found to be statistically significant for all VA and CT data sets (p < 0.008). Overall, lower thresholds for both VA and CT were obtained with negative contrast. Photopic VA thresholds measured with negative contrast optotypes were also compared against the equivalent ETDRS VA data. Paired t‐tests revealed significant differences between the two tests with slightly larger VA thresholds when measured with the Acuity‐Plus test (p < 0.02). A one‐way ANOVA was conducted to compare the photopic and mesopic measurements between the testing sites. There were no statistically significant differences per decade between groups for all measurements as determined by one‐way ANOVA (p > 0.004).Table Figures Mean photopic thresholds and overall variability remain largely invariant below 50 years of age. Above 50 years, both mean thresholds and the observed intersubject variability increase with age Figures  and 5a–dt‐tests reveal significant differences (p < 0.004) between mean thresholds per decade and the mean threshold for the subsequent decade only when above 50 years of age.In the mesopic conditions, both VA and CTs start with much larger values  and the thresholds start to increase more rapidly above 30 years of age, particularly for optotypes of positive contrast. The fitted curves capture well the observed effects of normal aging for both photopic and mesopic conditions. Independent ‐Plus test fulfils many of the requirements of this study. The test was initially designed to assess the effects of corneal refractive surgery on visual performance under photopic and mesopic lighting,The principal aim of this study was to establish how the healthy normal aging of the eye and visual pathways affect spatial vision under photopic and high mesopic lighting conditions. To achieve this aim, we needed a sensitive and efficient test of spatial vision to measure VA and CTs at photopic and high mesopic light levels. The AcuityThe largest thresholds were measured with optotypes of positive contrast and correspond to the monocular viewing condition. The best performance for both VA and CTs is achieved with negative contrast optotypes in binocular viewing. VA and CTs in the photopic range appear to be stable up to 50 years of age. These results are in general consistent with findings from other studies.Both mean thresholds and intersubject variability are reduced significantly in binocular viewing, and this was particularly evident for both VA and CTs in the mesopic range. The majority of participants exhibit large binocular summation, which is in agreement with earlier findings.2 for use in the Acuity‐Plus protocol is consistent with typical residential street lighting and other mesopic working environments where adequate spatial vision is required.2 are considered to be more representative of the mesopic range,2 and also less useful clinically because of the much increased within and intersubject variabilities. The largest variability in both VA and CTs corresponds to monocular measurements with positive contrast optotypes and hides the significant increases in mean thresholds during the first few decades of life.The choice of 2 cd/mThe results show that even in the high mesopic range, both VA and CTs are more susceptible to aging than the corresponding findings in the photopic range. A number of different factors may contribute to the more rapid worsening of spatial vision with increasing age in the mesopic range. Increased pupil miosis and greater absorption and scattering of light by the lens cause a significant reduction in retinal illuminance, and this results in a greater loss in retinal sensitivity to contrast in older subjects in the upper mesopic range.‐Plus test may well yield similar limits to those reported here. If so, then the use of the spatial limits obtained in this study that describe the effects of aging under standardized conditions could be extended to other tests. The normal age limits reported here are described fully, and equations are provided for each of the 16 stimulus viewing conditions. This makes it possible to compare our limits to those obtained with other instruments, in addition to the FrACT test. However, the validation studies may not be without challenges since the Acuity‐Plus test employs a fully calibrated 10bit display and spectrally calibrated ND glasses for use in the mesopic protocol. Although this approach is of value in order to achieve standardized conditions, we acknowledge that in terms of general use, the more expensive calibrated equipment and the much higher dynamic range may limit the availability of the test. Although not included in this study, similar measurements carried out in patients with diabetes and other ocular conditions reveal much larger differences in both VA and CTs when comparing results measured with equivalent optotypes of opposite luminance contrast. The combination of the four measurements into one single test has other significant advantages. In previous studies, VA and sensitivity to contrast were assessed using different test charts and in different experimental sessions.Plus test is the stimulus presentation time of ~160 ms. This short time eliminates multiple fixations and minimizes within‐subject variability. In general, the use of a short presentation time results in slightly higher VA thresholds when compared to the same measurements under continuous viewing on ETDRS test charts.Plus test measures VA and CTs, the measured parameters are also sensitive to changes in the temporal response characteristics of the retina. A limitation of this study may be the selection of the study population in three different settings. This choice has, however, ensured the exposure of study participants to a variety of occupations and work‐related visual tasks. This can also be considered important when establishing normal age‐related limits for occupational use. Furthermore, all participants were Caucasian and hence the use of the limits derived from this study with other ethnicities rests on the assumption that any differences in spatial vision are small. Uncorrected refractive errors and astigmatism in particular have been shown to affect VA and CS.The results measured with negative contrast optotypes were significantly better than the corresponding thresholds measured with positive contrast, in agreement with findings from earlier studies.Plus test is simple to carry out and the availability of upper normal age limits for each of the four measured parameters makes this assessment more valuable as part of the standard optometric examination. The measure of CTs introduced and investigated in this study relies on the measurement of only one luminance contrast for a fixed stimulus size. Since visually demanding tasks rarely employ alphanumeric characters smaller than three times the average acuity limit , the Landolt ring employed in this contrast threshold test was selected to have an outer diameter of 15′ with a 3′ gap size. The ability to resolve and locate a 3′ gap size in low contrast is functionally important in many visual tasks. The combined assessment of VA and CTs using photopic and high mesopic light levels with optotypes of positive and negative contrast provides a better description of the participant's spatial vision. Combining the four parameters and the availability of normal age limits can help in the early detection of retinal disease and may justify the use of the test in clinical practice. In particular, the mesopic measurements and the upper normal age limits established in this study may be of interest in the early detection of retinal disease.Currently, visual function testing is often limited to photopic high contrast VA, simply because measures of CS are too demanding and require the investigation of several parameters using sinusoidal gratings, making the test often too long, complex and impractical in clinical practice.In conclusion, this study establishes upper, normal age limits for monocular and binocular viewing under photopic and high mesopic lighting with both positive and negative contrast optotypes using a single, efficient test that can be used in many occupational settings and in the clinic.Arjan Keuken: Conceptualization ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration ; resources ; validation ; writing – original draft (lead); writing – review and editing . Ahalya Subramanian: Conceptualization ; methodology ; supervision ; validation ; writing – review and editing . Sigrid Mueller‐Schotte: Formal analysis ; methodology ; supervision ; validation ; writing – review and editing . John L. Barbur: Conceptualization ; formal analysis ; methodology ; resources ; supervision ; validation ; writing – review and editing .John Barbur is the inventor of the Advanced Vision Optometric Tests (AVOT), some employed in this study; an employee of City, University of London; and a director of City Occupational Ltd. . Other authors: None.Table S1Click here for additional data file.