Inequity aversion is thought to act as a mechanism to ensure cooperation and has been studied in many different species, consistently revealing inter-individual variation. Inhibitory control has been proposed to act as one factor responsible for this variation since individuals need to inhibit performing the required action and/or refuse rewards in order to exhibit inequity aversion. Here, we investigated if dogs’ sensitivity to inequity is affected by their capacity for inhibitory control, assessed in a test battery and questionnaire. Overall, dogs showing high compulsivity scores (i.e. repetitive behaviours independent of feedback) were more motivated to participate in the inequity task independent of the rewarding scheme. Dogs were more sensitive to inequity and individual contrast if they exhibited a slower decision speed in the inhibition tasks. Furthermore, less persistent and more impulsive dogs were more sensitive to reward inequity, potentially due to having a lower tolerance level for frustration. Results indicate that aspects of inhibitory control can explain the variation in dogs’ inequity response, highlighting one of the mechanisms underlying responses to inequity. Emphasising the importance to design paradigms, which allow us to disentangle capacities to recognise inequity from the inability to react to it due to poor inhibitory control abilities. Inequity aversion seems to be present across many different animal taxa (i.e. apes (e.g. ref.2), monkeys (e.g. ref.3), corvids4, rodents5, canids6) indicating that this behaviour might be a basic trait shared across different social species7. Typically, inequity aversion has been assessed by requiring pairs of individuals to alternately perform an action  in order to receive rewards, which are either equally or unequally distributed between partners. If an individual refuses to continue participating in the task after witnessing that their partner received a reward of better quality for the same amount of work (by either stopping to perform the required action or refusing to accept the reward), it is considered to show inequity aversion.Cooperation with other individuals in order to achieve a goal that could not be reached alone seems to be clearly advantageous. However, cooperative interactions are mostly beneficial if the payoff is shared more or less equally among all individuals that contributed to the action. If this is not the case, unequally treated individuals may stop their participation and look for other, potentially better, cooperative partners. Therefore one mechanism likely to stabilize cooperation is inequity aversion, the negative reaction to unequal treatment8 for a review) and only recently the individual variation in the reaction to unequal treatment has been given attention (see ref.9 for a review). Considering the nature of inequity aversion as a mechanism to stabilize cooperation, it comes as no surprise that an animal’s reaction to inequity may differ depending on the relationship with its social partner . But also individual characteristics, such as age17, sex15 (but see ref.18), and certain aspects of personality12 seem to play a role in the response to unequal treatment.Inequity aversion research has focused mainly on investigating the existence of this behaviour across different species (see ref.20). Inhibitory control is referred to as the ability to overcome the temptation of performing an immediately rewarding behaviour in favour of a delayed but more appropriate (and ultimately more rewarding) one. While social as well as some individual factors might account for the susceptibility to inequity, inhibitory control is always necessary for exhibiting an aversion to it. In order to show an aversion to inequity, an animal needs to inhibit delivering a token, pulling a tray or giving the paw, despite having been consistently rewarded for carrying out this action or refuse to take a food reward of lesser quality than the partner. Thus, the ability to inhibit cooperative interactions in unequal situations might be an important cognitive prerequisite involved in the expression of inequity aversion, and could potentially account for the wide-ranging individual variation consistently observed across studies.Another factor that has consistently been suggested to influence an individual’s reaction to inequity is inhibitory control . Of particular interest is a study by Harris and Madden23 that found a relation between cooperative interactions in a prisoner’s dilemma game and individual variation in inhibitory control. Accordingly, more impulsive people, as measured in a delay-discounting task, defect unequal offers more often than less impulsive people, who try to maintain offers for longer. In non-human animals, only one study investigated the link between inequity aversion and inhibitory control. Capuchins monkeys were found to exhibit better inhibition in a detour-reaching task than tamarins and authors related these findings to results from inequity aversion studies showing that while capuchins do show inequity aversion, tamarins do not react to unequal treatment19. However, Lakshminaryanan and Santos did not obtain measurements of inequity aversion from the same monkeys that were tested in the inhibitory control task and consequently, the authors could only make inferences about a possible relationship between these factors without having the actual data from both inequity and inhibition task to test this hypothesis directly.To our knowledge, no study so far has investigated the relationship between inhibitory control and inequity aversion directly. However, some studies looked at other cooperative behaviours likely linked to inequity aversion in humans . For example, human’s inhibitory abilities cannot be easily captured with a single task, which raises concerns in regard to the above-mentioned studies (e.g. refs23). Similarly, in a recent study with dogs26, we found that inhibitory control abilities in this species also vary across tasks, and hence it cannot be considered a unified concept measurable in a single test. Nonetheless, we could show that dogs’ inhibitory control abilities, which were assessed across five inhibition tasks, are likely explained by three components: persistency (i.e. a measure of dogs’ persistence in manipulating apparatuses), compulsivity (i.e. a measure of dogs’ consistently choosing the same option despite of not being rewarded) and decision speed (i.e. the speed with which dogs made their choices).One problem that arises in testing the involvement of inhibitory control in other behaviours is its strong context-specificity . Measures of inhibitory control were obtained from a test battery consisting of five frequently used inhibition tasks26, including a validated impulsivity questionnaire28. We hypothesized that inhibitory control is linked to inequity aversion, in particular predicting that dogs showing better inhibitory control abilities are more sensitive to inequity but also to individual contrast. However, considering that inhibitory control is a complex construct, which is best described by three components in dogs , we expected these components to be differentially involved in the expression of inequity aversion.The aim of the current study was therefore to investigate the role of inhibitory control  in the expression of inequity aversion in dogs. Dogs’ reaction to inequity was measured in a paw-giving paradigm, which has elicited inequity aversion in dogs in previous studiesb = 0.23, SE = 0.06, z = 3.74, p < 0.001), the persistency component was not significant . Accordingly, dogs showing more compulsive behaviours in the inhibition tests gave their paw more often than dogs showing fewer of these behaviours. This indicates that dogs, which consistently stuck with their choices in the inhibition tests independent of the feedback ( = compulsivity component), reacted less strongly to the differential rewarding schemes in the inequity test.Model reduction revealed that the persistency and compulsivity components had an influence on the overall number of times the paw was given, independent of test condition. While the compulsivity component had a significant positive effect on paw giving in the whole inequity paradigm (GLMM: The reduced model for the dyadic conditions (equity condition as baseline) included the coefficients decision speed, and the impulsivity score derived from the DIAS questionnaire. Both coefficients showed significant interactions with some but not all test conditions  in relation to the baseline (ET), while dogs with a faster decision time continued giving their paw for more trials. Thus dogs that took longer to make a decision in the inhibition tests also showed a stronger response to inequity, but also to contrast. Additionally, the DIAS impulsivity score, showed a significant interaction with the dogs’ paw-giving behaviour but only in the reward inequity condition  accounted for individual variation in the reward inequity condition. Less persistent dogs showed a stronger aversive reaction to reward inequity than more persistent dogs. Finally, the impulsivity score obtained from the owner-reported questionnaire explained some of the variation observed in the reward inequity condition.et al.12 found that the personality dimensions of openness, reactivity, agreeableness and dominance were negatively related to the overall number of refusals of chimpanzees in an inequity paradigm. Personality dimensions included behaviours like excitement, persistency, impulsivity but also inventiveness; indicating that chimpanzees scoring higher on these traits also completed more exchanges with the experimenter. In line with this, the ‘compulsive’ individuals might exhibit a higher working motivation in general. Nonetheless, it needs to be noted that we do not know how stable the inhibition measurements but also the inequity aversion measurements are over time. Despite keeping the duration between both tests to a minimum, test-retest reliability was not assessed in the current study. Future studies need to investigate how stable these behavioural traits are.The overall motivation of dogs to give the paw in the inequity test was affected by the compulsivity component indicating that more compulsive dogs had fewer difficulties in performing the same action (i.e. give paw) repeatedly. Interestingly, this compulsivity component related to the overall willingness of dogs to perform the task but not to the variation within specific conditions. This influence of compulsivity on dogs’ motivation to participate in a task that involves a repetitive action might act as a limiting factor on the initial selection of animals that are participating in inequity studies. Dogs that did not comply with the precondition for assessing the dogs’ motivation (i.e. giving paw 15 times in a row), and also dogs that failed to reach criterion for being included in the final results (i.e. giving paw at least 20 times in the assessment condition) may be those with a lower level of compulsive and persistent behaviours. Similarly, Brosnan 29). But also in non-human animals inter-individual variability in decision speed and accuracy have emerged, with individuals that make decisions very fast showing higher error rates than individuals that take more time to choose (see ref.31 for reviews). This might be due to the fact that fast-acting individuals do not recognize the important features of the given task, such as, in our study, the differences in reward quality.In order to understand the relationship between dogs’ inhibitory control abilities and their reaction to inequity we investigated the influence of certain inhibition components on the behaviour in each test condition in relation to the baseline conditions. One crucial component seems to be decision speed , while less persistent dogs simply give up. This persistency aspect might be of less importance in rewarded conditions because those are less frustrating; hence there might be no need for explicit coping strategies.In contrast to the decision speed component, an effect of persistency only emerged when using the asocial no-reward condition as baseline. Consequently, the less persistent dogs might get frustrated more easily in strongly unequal situations, whereas more persistent dogs try to perform the required action for longer, regardless of payoff. Similarly, Range 28 indicating that more impulsive dogs were more sensitive to reward inequity, which is in contrast to our predictions. When investigating inhibitory control in dogs26, we found that only one sub-score (‘responsiveness’) obtained from the DIAS questionnaire was negatively correlated with the ‘decision speed’ component, but not with the other two inhibition components derived from the test battery. Accordingly, dogs that were rated more responsive and aware of their environment also made faster decisions in the inhibition test battery. Nonetheless, this general lack of correlations suggests that different aspects of inhibition are assessed with the questionnaire compared to the battery. Indeed, many questions refer to daily situations, in which the dog may be excited , hence capturing the dogs’ ability to cope with arousal rather than their inhibitory control abilities. Consequently, those dogs that cannot cope well in taxing situations during daily life might be more sensitive/reactive and thus also react more strongly to inequity. Considering the nature of inequity aversion paradigms involving the distribution of unequal food rewards, tasks assessing inhibitory control in a food context might be more appropriate to assess the effect of inhibition on cognitive tasks than questionnaires assessing inhibitory control in daily situations, often outside of the food context.Interestingly, dogs’ sensitivity to reward inequity was also positively related to the impulsivity score obtained from the DIAS questionnaireshowing this sensitivity in the current setup where aversion is measured in terms of refusal, or abstaining from action. If an individual cannot refrain from obeying the given command and/or from taking the offered reward, it cannot show an aversion to the unequal treatment, even though it might very well recognize the inequity being perpetrated. It therefore becomes important to devise additional, simpler ways to measure an individual’s sensitivity to inequity. One such method was used in Brucks et al.27, in which the dogs’ social behaviour towards the better-rewarded partner dog and the experimenter who caused the inequity was observed following the various test conditions. There we found that dogs, which did not show a primate-like reaction to quality inequity, still avoided the experimenter but also the conspecific partner after the quality inequity condition. In addition, the unequally treated dogs showed reduced co-feeding behaviour with their better-rewarded partner following the inequity test27. This indicated that dogs were likely not able to show their aversion to inequity in the test context due to various reasons (e.g. lack of inhibitory control), even though they noticed the inequity. Consequently, the sensitivity to unequal treatment can be observed also with tasks that do not require high levels of inhibitory control . Since this incorporation of additional measures has so far only been done in dogs, it could be an interesting avenue for future research and might reveal that inequity aversion is present but hindered by a lack of inhibition control, also in other species.Overall our results show that inhibitory control abilities can affect an individual’s reaction to inequity. This however, does not necessarily mean that the more impulsive individuals are less sensitive to unequal treatment; rather, they may have greater difficulties in 27 (mean age ± SE: 4.9 ± 0.48 yrs.) were subsequently tested in an inhibitory control battery  we tried to keep the duration between the inequity test and the inhibition test battery to a minimum (1.8 ± 0.1 years). All procedures were discussed and approved by the ethics commission of the University of Veterinary Medicine Vienna in accordance with Good Scientific Practice guidelines and national legislation .Twenty-four dogs (10 M/14 F) that participated in the initial inequity study34), the pre-condition for participating in this paw-giving paradigm was that each dog within a dyad would give its paw for 15 times in a row, with and without being rewarded . During the test, the dogs were sitting next to each other attached by leashes to the wall. A wooden block was placed on the ground in-between the dogs as a ‘physical’ barrier to prevent dogs from changing sides. The owner was standing behind the dogs facing straight ahead without interacting with them. An experimenter was kneeling in front and facing the dogs at a distance of 50 cm. A food bowl containing an equal amount of high-value rewards (HVR) and low-value rewards (LVR) was in-between the experimenter’s legs . Only reward types, which both dogs within a dyad clearly distinguished and showed the same preference for, were used in the inequity test. The dogs needed to be in a sitting position before the experimenter asked them to give their paw, by holding her hand outstretched in front of the dog and by giving the verbal command ‘paw’  the same reward (equity condition), 2) rewards of different quality , 3) the same reward but showing the more preferred reward beforehand (food control/frustration condition) or 4) one dog received a reward, while the other dog did not (reward inequity condition) (see Table 26 for detailed descriptions of procedures).The test battery consisted of two motor inhibition tasks (box test and middle cup test), one cognitive inhibition task , a delay of gratification task and one newly- designed task with both motor and cognitive inhibition aspects (buzzer test). Dogs were tested only in one test per day and the order of tests was randomized , and contains 18 questions, which are rated on a 5-point Likert scale. One overall impulsivity score was calculated from the questionnaire (see ref.28 for details).In addition to the inhibition test battery, dog owners were asked to fill in a questionnaire about their dogs’ impulsivity in daily situations. This questionnaire has previously been validated . The inhibition data was taken from Brucks et al.26. As measures of inhibition, we took the factors obtained from a principle component analysis . We ran generalized linear mixed models (GLMM) with a Poisson distribution and log-link error function . The number of times a paw was given on command in the inequity test was used as response variable and the dog identity as random effect. As fixed effects the scores of each inhibition component , and the impulsivity score obtained from the DIAS questionnaire were entered. In order to investigate whether these inhibition components could serve as an explanation for the observed variation in paw-giving behaviour across test conditions, we analysed the data in three models. First, to assess whether dogs’ inhibition abilities alone predict how often they would give the paw independent of the test condition, we ran one model testing for the main effects of the inhibition measurements on the overall number of paws given across all test conditions . Subsequently, to investigate if inhibitory control plays a differential role in the different test conditions, we assessed the interaction between inhibition measures and the paw-giving behaviour per test condition in relation to a baseline condition. The equity condition (ET), in which both dogs receive the same reward, served as baseline condition for the dyadic test conditions (i.e. food control (FC), quality inequity (QI), reward inequity (RI)). Whereas the asocial no-reward condition (NR) served as an additional baseline for the reward inequity condition in controlling for the effect of the partner dog’s presence in the unrewarded conditions. We ran two separate models: The first model explored the interaction between paw-giving behaviour in the food control, quality inequity, and reward inequity condition and the components derived from the inhibitory control battery compared to the equity condition , inhibition components, DIAS score). The second model assessed the interaction between paw-giving in the reward inequity condition and the inhibition measures compared to the no-reward condition , inhibition components, DIAS score). We used the Akaike information criterion (AIC) to reduce the full model in order to find the best fit.The data from the inequity test was obtained from Brucks Supplementary Material


Hypertension affects more than 30% of the world’s adult population and thiazide (and thiazide-like) diuretics are amongst the most widely used, effective, and least costly treatments available, with all-cause mortality benefits equivalent to angiotensin-converting enzyme inhibitors or calcium channel antagonists. A minority of patients develop thiazide-induced hyponatremia (TIH) and this is largely unpredictable at the point of thiazide prescription. In some cases, TIH can cause debilitating symptoms and require hospital admission. Although TIH affects only a minority of patients exposed to thiazides, the high prevalence of hypertension leads to TIH being the most common cause of drug-induced hyponatremia requiring hospital admission in the UK. This review examines current clinical and scientific understanding of TIH. Consideration is given to demographic associations, limitations of current electrolyte monitoring regimens, clinical presentation, the phenotype evident on routine clinical blood and urine tests as well as more extensive analyses of blood and urine in research settings, recent genetic associations with TIH, and thoughts on management of the condition.Recent genetic and phenotyping analysis has suggested that prostaglandin E2 pathways in the collecting duct may have a role in the development of TIH in a subgroup of patients. Greater understanding of the molecular pathophysiology of TIH raises the prospect of pre-prescription TIH risk profiling and may offer novel insights into how TIH may be avoided, prevented and treated.The rising prevalence of hypertension and the widespread use of thiazides mean that further understanding of TIH will continue to be a pressing issue for patients, physicians, and scientists alike for the foreseeable future. Hypertension is the most common modifiable cause of cardiovascular disease—the leading cause of mortality worldwide , 2. At lThe association of hypertension with salt is intriguing however; sodium chloride intake correlates with the prevalence of hypertension even in disparate populations , and sodThiazide and thiazide-like diuretics share a common mechanism of action in the distal convoluted tubule of the nephron by binding to the luminal side of the thiazide-sensitive sodium chloride co-transporter, NCC. NCC is responsible for around 5–7% of renal sodium reabsorption  and mimiSince the demonstration of their antihypertensive effect in 1958 , thiazidDespite the clinical success of thiazides, they are not a panacea; they are often ineffective as monotherapy for essential hypertension  and causIn this review, we will concentrate on two principal sources of clinical data: a systematic review of all published TIH cases up to 2013 •• and a A minority of patients exposed to thiazides develop a sufficiently reduced serum sodium concentration to be defined as having TIH. However, because hypertension is such a prevalent condition, TIH is the most common cause of drug-induced hyponatremia requiring admission to hospital •. Classi2 . H•. H20•].This makes it difficult to determine what the optimal timing of a single post-thiazide initiation blood test for serum urea and electrolytes (U&E) should be. There is little consistency in the timing of this safety blood test but it is usually undertaken within a week or two of thiazide initiation. Whether undertaking more than one U&E blood test, e.g. at a few days and at 2–4 weeks, would improve detection of TIH before severe symptoms necessitate hospitalization is unclear. It is important however that all patients started on a thiazide receive at least one serum electrolyte check within a few weeks and it would seem prudent to undertake additional blood tests if symptoms of TIH occur before or after the routine blood test.It is usual to undertake blood and spot urine tests for U&E and other potential causes of hyponatremia when assessing a patient with suspected TIH. Our systematic review found that average mean trough serum U&E results in patients with TIH were sodium 117 mM (95% CI 114 to 120), potassium 3.3 mM (95% CI 3.0 to 3.5), and reduced osmolality of 242 mOsm/kg (95% CI 238 to 246 mOsm/kg) ••. PlasmSpot urinary U&E in the systematic review demonstrated less than maximal urinary dilution with urinary sodium concentration at 62 mM (95% CI 45 to 80 mM) and mean urine osmolality greater than plasma at 400 mOsm/kg (95% CI 366 to 434 mOsm/kg) ••. The 2The phenotype of TIH is therefore similar to the criteria for SIADH outlined by Bartter and Schwartz including (1) decreased plasma osmolality (< 275 mOsm/kg), (2) inappropriately concentrated urine (> 100 mOsm/kg), (3) euvolemia on clinical examination, and (4) elevated urine Na (> 20 mEq/L) with (5) normal thyroid and adrenal function . HoweverIn addition to the routine blood and spot urine tests undertaken in routine clinical care, TIH patients in the 2017 case series also underwent additional blood testing and 24 h urine collections both at the time of acute hyponatremia and after recovery to normonatremia following thiazide cessation ••. HyponTIH cases demonstrated an exaggerated free water reabsorption when taking thiazides compared to controls, which also supports a volume-expanded, diluted state. The continued production of urine which is more concentrated than plasma and which contains more than minimal sodium salt is physiologically inappropriate in the context of acute hyponatremia. Whilst 24 h urinary excretion of sodium and chloride was lower in acute TIH cases than in controls and increased from the acute state when convalescent, it is difficult to know whether such data indicates reduced salt intake in acute TIH cases given the dynamic pathophysiological state they are in. Convalescent TIH cases had slightly lower 24 h sodium and chloride excretion than controls but this was not statistically significant. There remains the possibility that TIH cases could have a slightly lower intake of dietary salt than controls but this remains unclear.Given the similarity in phenotype between SIADH and TIH, antidiuretic hormone (ADH) was measured and was surprisingly lower in acute TIH cases than in controls or convalescent cases. The phenotype of TIH cases suggested ADH excess but in fact they had less ADH than controls, raising the question whether something other than ADH may be stimulating distal nephron water absorption.Thiazide therapy does not meaningfully alter average serum sodium concentration within the treated population with essential hypertension, suggesting that there are defined subgroups of patients who are susceptible to substantial reduction in sodium on exposure to thiazides. Genetic predisposition to TIH is supported by a high degree of reproducibility on single dose thiazide rechallenge where environmental factors such as sodium intake were controlled •. A prioP < 10−5 was used. In total, 17 SNPs within 14 regions were identified as showing association with TIH at this level. The prostaglandin E2 (PGE2) transporter , was chosen for further study because TIH has a phenotype resembling SIADH, and it was the candidate with the best known function in regulating water reabsorption in the collecting duct via the AQP2 pathway. Sequencing confirmed the presence of a non-synonymous variant encoding p.A396T (rs34550074), which was in complete linkage disequilibrium with the sentinel GWAS SNP rs4854769, and the genetic association was replicated using a second cohort of TIH cases hospitalized with severe TIH [A pilot genome-wide association study (GWAS) was undertaken in a discovery cohort of 48 patients hospitalized with severe TIH and compared to patients from the 1958 British birth cohort ••. Givenvere TIH ••.Immunohistochemistry of human cadaveric kidneys confirmed that PGT was expressed in the collecting duct and co-localized with AQP2. Using an in vitro cell expression system, PGT containing the phospho-mimic p.A396E demonstrated loss of function. This would be expected to reduce reuptake of PGE2 from the urinary lumen and indeed increased urinary PGE2 was seen in TIH patients with the PGT p.A396T variant compared to TIH patients who were wild type, suggesting that the PGT variant may have a functional effect in patients. Differences in urinary PGE2 resolved following thiazide cessation.Taking these findings together, a hypothesis was proposed in which the combined effect of thiazide-induced impairment of renal free water generation, together with genetically determined PGE2-mediated increased water permeability of the collecting ducts, produces a combination of natriuresis and excessive water reabsorption sufficient to lead to a substantial decline in serum sodium concentration and presentation with severe TIH  were found to be associated with increased probability of overly rapid correction of serum sodium [Our experience of managing TIH has been that cessation of the thiazide and avoidance of excessive water intake result in recovery of serum sodium at a clinically acceptable rate of a few millimoles per liter per day in most patients. Parenteral infusion of saline under closely monitored conditions is usually reserved for TIH cases with severe or life-threatening features. In support of this approach, an observational study of thiazide -associated hyponatremia found that progressively more aggressive strategies to elevate sodium in addition to thiazide cessation (including fluid restriction, parenteral infusion of normo- or hyper-tonic saline and the use of Vm sodium . It is iThiazide-induced hyponatremia is an important medical condition which is seen regularly in internal medicine and which causes substantial morbidity to often elderly frail patients. At present, the risk of developing TIH is largely unpredictable at the point of thiazide initiation. TIH is also an interesting and important condition scientifically as a relatively little studied paradigm of human sodium and water dysregulation which may provide an opportunity to learn more about the molecular processes underlying these essential aspects of renal physiology.What is the optimal timing of a blood electrolyte test after starting thiazides, or should there be more than one electrolyte check?Is it possible to identify a subgroup of patients at increased risk of TIH by developing a risk score based on their age, gender, weight, baseline blood, and urine tests, and genetic profile? If so, can this be used either to select alternative antihypertensive treatment or to focus additional monitoring if such patients are exposed to thiazides?What is the optimal management of patients with TIH?Pressing clinical issues include:What causes TIH in those who do not carry the PGT p.A396T mutation?What role do the other genetic associations identified play in the pathophysiology of TIH?What effect do drugs which alter the production of PGE2 or which antagonize the EP4 receptor have on TIH pathophysiology?Is the cause of TIH consistent across ethnicities? Thiazides are used worldwide and yet most studies have been in Caucasians to dateWhat is the physiological response to non-thiazide diuretics in those with a history of TIH?There are also many scientific questions remaining including:Given the effectiveness of thiazides in lowering blood pressure and preventing cardiovascular disease and their very low cost, it seems likely that thiazide-induced hyponatremia will be an important long-term issue in medicine; there are a potentially large number of patients who may benefit from greater understanding of this condition.


Drosophila. Su(H) operates as molecular switch on Notch target genes: within activator complexes, including intracellular Notch, or within repressor complexes, including the antagonist Hairless. Mass spectrometry identified phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. To address the biological significance, we generated phospho-deficient [Su(H)S269A] and phospho-mimetic [Su(H)S269D] variants: the latter displayed reduced transcriptional activity despite unaltered protein interactions with co-activators and -repressors. Based on the Su(H) structure, Ser269 phosphorylation may interfere with DNA-binding, which we confirmed by electro-mobility shift assay and isothermal titration calorimetry. Overexpression of Su(H)S269D during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H)R266H. As both are able to bind Hairless and Notch proteins, Su(H)S269D and Su(H)R266H provoked dominant negative effects upon overexpression. Our data imply that Ser269 phosphorylation impacts Notch signalling activity by inhibiting DNA-binding of Su(H), potentially affecting both activation and repression. Ser269 is highly conserved in vertebrate CSL homologues, opening the possibility of a general and novel mechanism of modulating Notch signalling activity.Notch signalling activity governs cellular differentiation in higher metazoa, where Notch signals are transduced by the transcription factor CSL, called Suppressor of Hairless [Su(H)] in  One such pathway is the Notch signalling pathway, which takes place directly between neighbouring cells, steering them into alternate fates. Appropriate cellular differentiation, therefore, strictly depends on Notch signalling in many instances, explaining its impact on human health (for review6). The serious consequences of aberrant Notch signalling have triggered large-scale efforts aimed at understanding the regulatory mechanisms underlying Notch activity. One hallmark of these studies is that regulatory mechanisms can occur at multiple steps of signal transduction, ranging from the selection and variation of ligands, crosstalk with other signalling cascades, and various context and tissue specific modification of Notch signalling components9. Such investigative studies, however, are complicated by genetic redundancy in vertebrates, e.g. mammals have four Notch receptors and five ligands, highlighting the importance of using model organisms like Drosophila melanogaster to elucidate novel mechanisms of regulation.Metazoan development depends on a small number of fundamental, conserved signalling pathways that are employed repeatedly to govern cellular differentiation. Not surprisingly, their dysregulation is a major cause of congenital diseases, underlying the pathophysiology of a multitude of different cancers, including solid tumours and leukemias. Together, these fundamental pathways coordinate developmental processes by building up complex signalling networks with extensive molecular cross-talk11). CSL acts as a molecular switch on Notch target genes as it can either assemble activator or repressor complexes depending on its binding partners10. The structure of these complexes has been analysed in several organisms: CSL contains three domains, an N-terminal domain (NTD), a beta-trefoil domain (BTD), and a C-terminal domain (CTD). DNA binding is mediated by NTD and BTD, whereas BTD and CTD are involved in the formation of the activator complex by binding to NICD and Mam, respectively11. In vertebrates, several co-repressors compete with NICD for the binding of BTD10. In Drosophila, repressor complex formation relies on the general Notch antagonist Hairless. Hairless binds with high affinity to the CTD of Su(H), thereby precluding the binding of NICD13. In addition, Hairless recruits the two general co-repressors Groucho and C-terminal binding protein to silence Notch target genes14.The principles of Notch signal transduction are rather simple: the binding of ligands presented on the surface of the signalling cell triggers the cleavage of the Notch receptor in the adjacent signal receiving cell, releasing the intracellular domain NICD (Notch Intracellular Domain). NICD now becomes a transcriptional co-activator: together with co-activators of the Mastermind family (Mam) it assembles a ternary activator complex with the transcription factor CSL (CBF-1/Suppressor of Hairless/Lag-1) on Notch target genes . As several of these kinases are also an integral part of other signalling pathways they have the potential to act as the mediators of crosstalk. A prime example is the crosstalk between the epidermal growth factor receptor (EGFR) and Notch pathways in Drosophila. These two signalling pathways have a complex relationship, either acting in a cooperative or antagonistic manner, depending on the developmental context18. Pivotal to EGFR signalling is the Mitogen-activated protein kinase (MAPK), and two well established substrates of MAPK in Drosophila are the Notch signalling components Groucho (Gro) and Suppressor of Hairless [Su(H)], which are both restricted in their activity by MAPK dependent phosphorylation19. Nonetheless, there are likely to be other Notch signalling components that are modified in response to other signalling pathways.As cells are under the influence of several developmental pathways, molecular cross-talk is required for their coordinationS269D is impaired in transcriptional regulation. We find that protein complexes with NICD and Hairless form normally; however, we find that DNA binding is affected in Su(H)S269D. Moreover, overexpression analyses during fly development provide evidence for a restricted ability of Su(H)S269D to activate and repress Notch target genes, revealing dominant negative effects at the same time. In contrast, the phospho-deficient mutant Su(H)S269A behaves similarly to the wild type protein. As Ser269 is highly conserved, we propose a new mode of Notch signal regulation at the level of affecting DNA binding by the transcription factor CSL.Here, we report the identification of an additional phosphorylation site in Su(H) by a mass spectrometry approach. The identified phospho-serine 269 is located in the beta-trefoil domain (BTD) of Su(H), leaving the possibility of influencing the association of Su(H) with NICD and/or with DNA. Using phospho-site specific mutants we show that the phospho-mimetic Su(H)19 sparked our interest to search for further phosphorylation sites in Su(H) in order to identify additional cross-talk between Notch and as of yet unknown signalling pathways. To this end, we took a mass spectrometry approach and isolated Su(H) protein from Drosophila Schneider S2 cells. Myc-tagged Su(H) protein and activated RasV12 were ectopically induced in S2 cell culture followed by immunoprecipitation of Su(H) protein with anti-Myc antibodies. The upper of two Su(H) protein bands was excised and in-gel digested with trypsin sin Fig. . Resultasin Fig. . The frasin Fig. .Figure 122. Therefore, phosphorylation at this site might influence the high-affinity interaction with NICD, interfering with activator complex formation. Similarly, Nemo-like kinase (NLK) inhibits ternary complex formation by phosphorylation of NICD23. Alternatively, phosphorylation might generally affect the binding of Su(H) to target sites on the DNA. Ser269, as well as the sequence surrounding it, is absolutely conserved between Drosophila, C. elegans and mammals, including humans, and lies within a region known to make both specific and nonspecific contacts with the DNA  isoform by replacing Ser269 with alanine (S269A), and a phospho-mimetic Su(H) isoform by replacing Ser269 with aspartic acid (S269D)  of Su(H), which is known to interact with the RAM domain of the intracellular domain of Notch (NICD) and to mediate contact with DNA Fig. 20–22. ThDNA Fig. 20. To ad9D) Fig. . In addit24 Fig. . The mutDrosophila Schneider S2 cells by transient transfection with respective inducible constructs. As a read out, luciferase activity was measured from a Notch response element reporter construct (NRE-luc)25. NRE-reporter gene activity is induced by transfection of NICD, which can be further raised about threefold by co-transfection with wild type Su(H)wt wt, the phospho-deficient Su(H)S269A mutant construct resulted in a slightly enhanced activation, whereas Su(H)S269D resulted in a considerably lower reporter activation. As expected, the Su(H)R266H mutant had no impact on the NICD-mediated activation of the NRE-luc reporter  is phosphorylated in S2 cells, the increased activity of Su(H)S269A suggests that repression of Notch signalling activity by phosphorylation is in fact taking place in these cells even in the absence of activated RasV12.First, we addressed the transcriptional activity of the phospho-mutant Su(H) isoforms in S269D mutant. To this end, a co-transfection of the respective constructs with NICD and Hairless (H) was performed. Transfection of H results in a repression of NICD-mediated reporter gene activity by about two-fold26. In combination with either wild type Su(H)wt or Su(H)S269A values are similar to NICD overexpression alone. In contrast neither Su(H)S269D nor Su(H)R266H had a significant effect on H-mediated repression S269A was not significant in this assay, the tendency of differential activity is apparent. Hence, we propose that phosphorylation of Su(H) at Ser269 may mitigate transcriptional activity, affecting both activation and repression of Notch target genes alike.The Su(H) transcriptional regulator not only activates, but also represses Notch target genes, depending on its bound co-factors. We therefore wanted to determine whether repression activity was likewise affected in the Su(H)S269D in the above luciferase assay could result from a failure to assemble activator complexes together with NICD and Mam, or repressor complexes together with H. To assay protein complex formation of the Su(H) mutant isoforms, yeast two- and three-hybrid assays were performed, addressing the direct interaction of Su(H) with either H or NICD, as well as the assembly of the Notch activator complex, which includes Mam. As shown in Fig. S269A and Su(H)S269D mutants as well as the DNA binding-deficient Su(H)R266H mutant proteins, performed very similar to wild type Su(H) in the binding of either H or NICD, or in the three-hybrid assay together with NICD and Mam. These results imply that phosphorylation of Su(H) at Ser269 does not interfere with the assembly of protein complexes. We next addressed the possibility that the mutations might affect protein expression, stability or nuclear localization of Su(H) protein. To this end, expression of the Su(H) isoforms was induced by a heat pulse and detected 6 hours later on Western blots. We observed little variance in the levels of the induced proteins in vitro transcribed and translated, and subjected to an electro-mobility shift assay (EMSA) using a radiolabelled E(spl)m8-S1 oligo as target DNA27 S269A, the phospho-mimetic Su(H)S269D isoform was strongly impaired in DNA binding. As expected, DNA-binding was completely abrogated in the Su(H)R266H mutant S269D wt or Su(H)S269A – DNA binding  protein isoforms, wild type as well as the two phospho-specific mutants were A27 Fig. . For comant Fig. . Howeverin vivo consequences of Ser269 phosphorylation, the Su(H) mutant constructs  mutant variants and the wild type form was induced with omb-Gal4 in the central wing anlagen. Notch in vivo activity was monitored with the vgBE-lacZ reporter line, which is induced in a stripe along the dorso-ventral boundary of the presumptive wing blade S269A isoform wt and Su(H)S269A S269D-NICD protein binding and simultaneous failure of DNA binding, i.e. dysfunctional activator complexes are formed that cannot localize to Notch target genes. Hence, overexpression of Su(H)S269D acts in a dominant negative fashion on activated NICD molecules. Accordingly, overexpression of the Su(H)R266H variant – which completely fails to bind to DNA in vitro – showed an even stronger repression of vgBE-lacZ or Cut, and also no increase in disc size . As H directly competes with Notch for interacting with Su(H) protein, gain of H equalizes loss of Notch activity. Accordingly, overexpression of H results in repression of Notch target gene expression, which can be visualized with either vgBE-lacZ or Cut, and is accompanied by a reduction of tissue size32  results in an extreme inhibition of Notch activity as a result of excessive repressor complex formation34. As a consequence many of the affected cells are lost presumably by apoptosis37, and only a small remainder of the expression domain is observed accompanied by a total repression of Notch target gene expression  activity. A considerably weaker phenotype was seen upon combined overexpression of H with Su(H)S269D, and even more so with Su(H)R266H  activity as a consequence of lowered DNA binding.Due to its role as a molecular switch, Su(H) is required for both Notch target gene activation and repression. With the goal to analyse the ability of the different phospho-specific Su(H) variants during repression, co-expression experiments with H were performed. Overexpression of H interferes with many Notch-dependent processes during larval development, for example the formation of the peripheral nervous system like mechano-sensory organs or during wing development . For example, during wing vein development Notch signalling is required to restrict vein competence, thereby limiting the width of the vein to its normal size38. We used this process to address the activity of the various Su(H) isoforms. Using vgQE-Gal4, the respective UAS-constructs were overexpressed exclusively within the presumptive wing blade, sparing the morphogenetic antero-posterior and dorso-ventral boundaries30. Both, Su(H)wt and Su(H)S269A overexpression caused a loss of vein material, in agreement with an enhancement of vein fate restriction due to increased Notch activity. Notably, the distal parts of the longitudinal L4 and L5 veins were affected  overexpression can be largely explained by a Su(H)-H titration effect. In fact, overexpression of just the Su(H) CTD, which is capable of binding to H but is itself inactive in Notch signal transduction, is sufficient to induce Notch gain of function phenotypes41. One might have expected a similar increase in Notch activity when overexpressing Su(H)S269D or Su(H)R266H since both are able to titrate H. This was not observed. In contrast, overexpression of Su(H)S269D and Su(H)R266H caused predominantly thickened veins and deltas at the margin  isoforms are hampered in DNA-binding, any activator complex formed together with NICD will be non-functional, i.e. both isoforms also titrate active Notch, providing an explanation for the observed phenotypes. In addition to thickened veins, overexpression of Su(H)S269D also caused distinct vein gaps in the longitudinal vein 4 wt or Su(H)S269A, and a decrease when inducing Su(H)S269D or Su(H)R266H, reflecting primarily gain of Notch activity in the former two and loss in the latter  variants on fly development. This is hampered by the fact that a manipulation of Notch signalling activity during larval development most often results in larval or pupal lethality, limiting the processes and tissues to be analysed. Lateral inhibition is one of the best-studied processes mediated by the Notch pathway, a process in which a few cells are selected from a group of equipotent cells to assume the primary cell fate, whereas the others are forced into a secondary fate R266H resembled those resulting from the sole H overexpression with regard to size and residual intervein material, although the distal portions of the longitudinal veins were clearly thicker  isoforms exclusively on the repression of Notch signalling activity, we simultaneously overexpressed H within the wing anlagen. As expected, sole overexpression of H with ins Fig. , a phenoate Fig. . In contin vivo. A phospho-mimetic mutation of this site [Su(H)S269D] results in strongly reduced DNA binding, which is accompanied by a mitigation of Notch signalling readout in several different developmental contexts tested. As Su(H) acts as a switch in the Notch pathway, being the nuclear effector for both activator and repressor complexes, it follows that Su(H)S269D could either affect the transcriptional activation or the repression of Notch target genes, depending on the cellular context. If Su(H) phosphorylation occurred simultaneously with Notch receptor activation, transcriptional activation would be lessened by the reduced DNA binding affinity of Su(H). At other times, however, Su(H) phosphorylation would result in a release of target gene inhibition, since binding of the Su(H)-H repressor complex would be mitigated. Consistent with this premise, our overexpression studies demonstrate that both, transcriptional activation and repression are affected in vivo. Bearing the limitations of overexpression studies in mind, the respective contribution of Ser 269 phosphorylation during normal development and signalling situations, however, remains to be determined.In this study we identified phosphorylation of Su(H) at Serine 269 by nano-LC-ESI-MS/MS and provide evidence for the function of this modification 20. The NTD makes major-groove sequence specific contacts to the second half of the binding site (5′-GGGA-3′) in a manner similar to other Rel-DNA complexes. The BTD also makes specific DNA contacts via a beta-hairpin loop that inserts into the minor-groove sequence, thereby selecting for the 5′ pyrimidine followed by a G (5′-C/t G)20. CSL-DNA crystal structures have been determined from human, mouse, C. elegans and Drosophila proteins with DNA corresponding to the consensus HES-1 binding site, and remarkably, the structures are very similar despite the huge evolutionary distance of the studied species (for review11). Interestingly, the beta-hairpin loop of the BTD involved in DNA contacts appears to be flexible as it can adopt alternate conformations42: in the preferred conformation the backbone carbonyl of Ser269 and the side chain of Gln270 make sequence specific contacts [numbering is from Drosophila Su(H)]. In alternative conformations, however, Ser269 can move into the place of Gln270 to make side chain-hydrogen bonds28. Alanine replacement of Ser269 should likewise allow for the backbone carbonyl contacts but not for side chain-hydrogen bonds, which would likely have little impact on Su(H)-DNA complex stability, but may affect specificity. Whereas aspartate at this position is expected to interfere with DNA binding altogether due to its negative charge. Perhaps the flexibility of the beta hairpin-loop is an important aspect of phospho-Ser269 regulation, since it is conceivable that its conformation influences the accessibility of Ser269 by protein kinases. This flexibility may also underlie the molecular basis for why Su(H)S269D retains some DNA binding in our EMSA data, whereas Su(H)R266H does not. Moreover, there are indications that the conformation of the BTD beta-hairpin loop may correlate with target sequences bound by CSL28. In this case, Ser269 phosphorylation of Su(H) might be target gene specific and would result in a specific rather than a general downregulation of Notch signalling activity within a cell.Structure analyses revealed that CSL transcription factors recognize their cognate DNA binding site [C/tGTGGGAA] through extensive sequence-specific and non-specific DNA contacts mediated by the BTD and NTD19. Also the MAPK-target site is absolutely conserved between fly and vertebrates. In mouse, phosphorylation of RBPJ at the corresponding Thr339 by the stress kinase p38 MAPK results in a destabilization of RBPJ43. In Drosophila we have, however, no indications to date that phosphorylation regulates Su(H) protein stability, despite the fact that Su(H) protein stability is an important means of regulating its availability44. The relative protein levels of either Su(H)wt, Su(H)S269D or Su(H)S269A were very similar after overexpression. If phosphorylation at this site resulted in a destabilisation or even degradation of the protein, we might have expected to see differences in stability. There is ample evidence for the influence of phosphorylation on the stability and the half-life of a protein. A prime example is the Notch receptor itself, as NICD is phosphorylated and degraded upon signalling45.Our new results uncover extensive crosstalk between the Notch signalling pathway and other signalling cascades by means of phosphorylation. They reveal a second example of CSL phosphorylation. Earlier, we have shown that Su(H) is targeted within its C-terminus at Thr426 by MAPK, resulting in an attenuation of Notch signalling readout46. Although we have identified the phosphorylation of Ser269 in cultured S2 cells in the presence of activated RasV12, the sequence surrounding Ser269 does not have a typical MAPK signature. Perhaps phosphorylation of Ser269 is mediated by a kinase, which itself is activated upon high levels of Ras signalling. As active Ras controls key aspects of growth, cell death, metabolism and mediates inter-cellular stress responses47 it might also pilot Notch signalling activity via Su(H) phosphorylation by an as yet unknown kinase. Alternatively, Ser269 phosphorylation may be independent of Ras activity. To our knowledge, no murine or human homologue phosphorylated at the corresponding Ser has been identified in high throughput screens (http://www.phosphosite.org/). It is conceivable that Ser269 in Su(H) is phosphorylated constitutively, and dephosphorylated specifically by an unknown phosphatase in a context specific manner. As this phosphorylation target site is absolutely conserved in all mammalian CSL orthologues it is tempting to speculate that – as in Drosophila - this site is used for crosstalk in mammals as well, opening the possibility for a new strategy to achieve tissue homeostasis and to consequently coordinate and react on cellular stress signals.According to computational predictions based on the phosphorylation site database algorithm (PHOSIDA) several kinases may be considered to recognize Ser269 as target siteS269A, Su(H)S269D and Su(H)R266H. Sequence verified constructs were cloned as Acc65I/XbaI fragments into the pUAST-attB vector. Transgenic flies were established with the PhiC31 integrase-based system29 using the ΦX-58A or ΦX-96E strain harbouring the landing site at 58A and 96E, respectively. Correct integration was tested by PCR. Other fly stocks used: ombmd65-Gal4/FM748, vgQE-Gal419, vgBE-lacZ30, Sco/CyO hs-Gal4 UAS-GFP (BL5702), UAS-GFP (BL4776), UAS-lacZ (BL8529), UAS-H at 68E12. For co-overexpression experiments UAS-Su(H) wild type12 or mutant constructs at 96E were recombined with UAS-H at 68E. Flies were raised on standard cornmeal at 18 °C and crosses kept at 18 °C or 25 °C as indicated.Su(H) cDNA in Bluescript vector was used for QuickChange site-directed mutagenesis  to generate Su(H)19. The coomassie stained Su(H) protein was in-gel-digested with trypsin and mass spectrometry analysis was performed on a Thermo LTQ-Orbitrap instrument  equipped with a nanoelectrospray ion source as described before49. Survey spectra (m/z = 300–1800) were detected in the Orbitrap at a resolution of 60,000 at m/z = 400. Database searches were performed with MASCOT search algorithm to identify the Su(H)-phosphopeptide, verified by manual inspection of the MS/MS spectra49.Myc-tagged Su(H) protein was derived from stably transfected Schneider S2 cells by immuno-precipitation as described before35. The following constructs were used: pEG-ICN I (codons 1827–2259)50, pEG-Mam (codons 118–194), pESC-NICD (codons 1762–2176)13, pEG-H 51. The Su(H)S269A, Su(H)S269D and Su(H)R266H mutant variants were cloned as full length constructs into pJG vector52 and were sequence verified.Yeast two- and three-hybrid experiments were performed as described before12. Cells were transiently transfected with 1 μg NRE-luciferase reporter25 and 0.2 μg of control Renilla plasmid for normalization. cDNA of Su(H)wt, Su(H)R266H, Su(H)S269A and Su(H)S269D was cloned as 1.8 kb Eco RI/Kpn I fragment into pRmHa3 (pMT) vector53. In addition, the constructs were tagged C-terminally with myc. Protein expression was induced with CuSO4 6 h after transfection and luciferase activity was measured 18 h later using dual-luciferase reporter assay  with Lumat LB 9507 . Su(H) myc-tagged protein was precipitated from about 108 Schneider S2 cells stably transfected with pMT-rasV12, pMT-Gal4 and UAS-Su(H)-myc19. The expression was induced with 0.7 mM CuSO4 and extracts were prepared 18-22 hours after induction.Schneider S2 cell culture reporter assays were performed at least in triplicate according to ref.19 with a double stranded DNA-oligomer harbouring the E(spl)m8-S1 Su(H) binding site 27. Su(H) protein isoforms were produced by coupled in vitro transcription / translation with TNT-Coupled Reticulocyte Lysate System  and 5 µl used for the reaction. The binding buffer contained 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.0275 µg/µl salmon sperm DNA.Electro mobility shift assays were performed as described before13. ITC experiments were carried out using a MicroCal VP-ITC microcalorimeter exactly as outlined earlier using double stranded DNA with the Hes-1 consensus 28. All Su(H)-DNA experiments were performed at 10 °C in 50 mM sodium phosphate pH 6.5 and 150 mM NaCl. Su(H) was degassed and buffer-matched using dialysis and size exclusion chromatography. A typical Su(H)-DNA binding experiment contained 10 μM Su(H) in the cell and 100 μM DNA in the syringe. The data were analyzed using ORIGIN software and fit to a one-site binding model.For isothermal titration calometry (ITC), recombinant Su(H) (98–523) was overexpressed and purified from bacteria, as previously describedombmd65-Gal448. Wing discs were isolated from late third instar larvae and stained as described before54. Expression of vestigial (vg) was determined using the vgBE-lacZ reporter line30 and anti-β-galactosidase antibodies . For Western blots, hs-Gal4 was crossed to the respective UAS-Su(H) lines, and expression induced in embryos with one hour heat shock at 37 °C as described earlier51. Other antisera used were: anti-Cut , anti-H-A55 or anti-Su(H) . S2 cells transiently transfected with myc-tagged pMT-Su(H) constructs were stained as outlined before56 with anti-myc , anti-Su(H)39 and anti-Pzg57 as nuclear marker. Secondary antibodies with minimal cross-reactivity coupled to DTAF, Cy3 and Cy5 generated in donkey were purchased from Jackson Immuno-Research Laboratories . Discs were mounted in VectaShield  and documented with a Bio-Rad MRC1024 confocal system using LaserSharp 2000 imaging software coupled to a Zeiss Axiophot microscope . Pictures were assembled with Corel-PhotoPaint and CorelDRAW Version 9.0 software.Overexpression in wing anlagen was induced with ImageJ programme.Adult wings were dehydrated in ethanol and mounted in Euparal , pictures of wings were taken with an ES120 camera  using Pixera viewfinder software version 2.0. Wing size was measured with Statistical significance of probes was evaluated by ANOVA using a two-tailed Tukey-Kramer approach for multiple comparisons  p > 0.05).All relevant data are within the manuscript and its supplementary information files.Supplementary Information


As a case study, we detected five nsSNPs that potentially influenced heterotrimeric G proteins phosphorylation status in rice, indicating that genetic polymorphisms showed impact on the signal transduction by influencing the phosphorylation status of heterotrimeric G proteins. The results in this work could be a useful resource for future experimental identification and provide interesting information for better rice breeding.Single nucleotide polymorphisms (SNPs) are widely used in functional genomics and genetics research work. The high-quality sequence of rice genome has provided a genome-wide SNP and proteome resource. However, the impact of SNPs on protein phosphorylation status in rice is not fully understood. In this paper, we firstly updated rice SNP resource based on the new rice genome Ver. 7.0, then systematically analyzed the potential impact of Non-synonymous SNPs (nsSNPs) on the protein phosphorylation status. There were 3,897,312 SNPs in Ver. 7.0 rice genome, among which 9.9% was nsSNPs. Whilst, a total 2,508,261 phosphorylated sites were predicted in rice proteome. Interestingly, we observed that 150,197 (39.1%) nsSNPs could influence protein phosphorylation status, among which 52.2% might induce changes of protein kinase (PK) types for adjacent phosphorylation sites. We constructed a database, SNP_rice, to deposit the updated rice SNP resource and phosSNPs information. It was freely available to academic researchers at  The draft sequences of two main cultivated rice genomes, ndica 93–1 and Jape genome ,5. This Oryza sativa L.), SNP mutations causing protein-coding changes or gene expression alterations both have the potential to account for rice agronomic traits [SNPs are DNA sequence variations occurring when a single nucleotide in the genome differs between members of a biological species . SNPs mic traits ,9. In adc traits .Non-synonymous SNPs that cause coding amino acid change would have the potential to influence protein phosphorylation status ,12. ErxlIn this paper, we performed a genome-wide analysis of SNPs that potentially impacted the protein phosphorylation status in rice. Firstly, we updated the rice SNPs resource based on the new rice genome data (Ver. 7.0) and predicted protein phosphorylation sites in rice by using NetPhosK 1.0 and Rice_phospho 1.0. After that, these two data were integrated to analyze the relationship between SNPs and protein phosphorylation sites. Finally, using heterotrimeric G protein as a case study, we interpreted the impact of nsSNP on phosphorylation sites and the function of heterotrimeric G protein in rice.http://www.ncgr.ac.cn/RiceHap2. Then, they were mapped to rice genome Ver. 7.0 by BLASTn. Finally, a total 3,907,374 SNPs were detected in rice genome Ver. 7.0, which was lower than that in rice genome Ver. 4.0 , Gβ subunit (LOC_Os03g46650) and Gγ2 subunit (LOC_Os02g04520), respectively . The relIn indica (9311) and japonica (Nipponbare) [indica (9311) and japonica (Nipponbare). We found only 9.9% of SNPs were nsSNPs. Ren et al. indicated that a very small proportion of human SNPs were nsSNPs (<1%) [indica (9311) and japonica (Nipponbare), which could be accessed at http://bioinformatics.fafu.edu.cn/SNP-rice. A genome-wide SNP resource was comprised of 4.11 million loci polymorphism between the two major cultivated rice subspecies, ponbare) ,18. Thisponbare) . With thPs (<1%) . FinallyProtein phosphorylation plays essential roles in the majority of biological pathways. Genome-wide prediction of phosphorylopathies in rice might provide a highly valuable resource for further experimental identifications. In this research work, we predicted 2,508,261 phosphorylation sites in rice proteome by using the combination of NetPhosK1.0 and Rice_phospho 1.0. After conducting a systematic analysis, we observed that 150,197 (39.1%) nsSNPs could affect protein phosphorylation. In particular, 52.2% of phosSNPs were Type III phosSNPs, which induced changes of PK types for adjacent phosphorylation sites rather than creating or removing phosphorylation sites. This was consistent with the results in the research work of Ren et al. . They foHeterotrimeric G protein signaling cascades is one of the primary sensing mechanisms between the cell and environment in metazoans. In our previous research work, we detected heterotrimeric G-protein subunits in rice were phosphorylated to transduct abscisic acid (ABA) and drought stress signal ,20. Our http://www.ncgr.ac.cn/RiceHap2/index.html), while DNA sequences of rice genome Ver. 4.0 (IRGSP Build 4.0) and 7.0 (MSU Release 7.0) were downloaded from RAP-DB (http://rapdb.dna.affrc.go.jp/download/build4.html) and MSU-RAP (ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/), respectively. SNPs data were first subjected to rice genome Ver. 4.0 to get the DNA sequences including SNPs and then mapped to rice genome Ver. 7.0 to create SNPs in the new version rice genome. Meanwhile, the rice genome Ver. 7.0 annotation files, including allseq, cds (coding sequences) and all gff3, were downloaded.SNPs data were downloaded from RiceHap2 database . The cSNPs were further grouped into non-synonymous SNPs (nsSNPs) or synonymous SNP (sSNPs), which was defined according to whether a cSNP could cause an amino acid substitution or not. Then, sSNPs that result in Premature Termination Codons (PTCs) were removed according to Ren et al. .http://www.cbs.dtu.dk/services/NetPhosK/) was used to scan the benchmark protein sequences and variant protein sequences for the phosphorylation sites and specific kinases with the high threshold, respectively. The predicting phosphorylation sites were confirmed by using Rice_phospho1.0 (http://bioinformatics.fafu.edu.cn), which was a rice-specific phosphorylation sites predictor.First, we took the protein sequence in rice genome Ver. 7.0 as benchmark sequence data. Then, we made changes to a protein sequence, one of its nsSNPs at a time, to prepare a variant sequence. NetPhosK 1.0 , which was defined as a phospho-Ser, phospho-Thr, or phospho-Tyr flanked by 7 residues upstream and 7 residues downstream, were extracted from the protein sequences and constructed as a dataset. By comparing results of the two kinds of phosphorylation site datasets, the phosSNP that might influence protein phosphorylation status could be detected based on the definition.According to the definitions given by Ren et al. and Ryu et al. ,14, we dWe downloaded the heterotrimeric G protein sequences from NCBI and searched SNPs in the G protein sequences against the rice genome Ver. 7.0 SNP dataset. We also predicted phosphorylation sites in G protein sequences by using NetPhosK 1.0 and Rice_phospho1.0 tools, then identified different phosSNPs in the heterotrimeric G proteins according to the above protocol.http://bioinformatics.fafu.edu.cn. The database will be continuously updated when new phosphorylation sites and other SNP data become available.We used the JAVA to develop the database, SNP-rice 1.0, which was accessible at http://bioinformatics.fafu.edu.cn. As a case study, we detected five nsSNPs that potentially influenced heterotrimeric G protein phosphorylation status in rice, indicating that genetic polymorphisms had an impact on the signal transduction between the cell and the environment by influencing the phosphorylation status of heterotrimeric G proteins.In this paper, we attained an updated rice SNP resource, among which 9.9% of SNPs were nsSNPs. Whilst, we observed that 150,197 (39.1%) nsSNPs could potentially influence protein phosphorylation status, among which 52.2% might induce changes of protein kinase (PK) types for adjacent phosphorylation sites. Finally, we constructed a database, SNP_rice, to deposit the updated rice SNP resource and phosSNPs information, which could be freely available for academic researchers at


Microsurgical subinguinal varicocelectomy (MSV) is generally considered the gold standard nowadays in view of the lower risk of complications and recurrence. To achieve complete ligation of veins while preserving testicular artery (TA) during the procedure remains challenging despite the application of high power optical magnification and micro-Doppler ultrasonography. The use of intraoperative indocyanine green angiography (ICGA) with infrared fluorescence operative micro-scope in MSV potentially lowers the incidence of TA injury and shortens the learning curve of novice surgeons. We present our initial experience in the application of the technique in nine patients and explore the potential of the new adjunct. Subinguinal microsurgical varicocelectomy (MSV) became the gold standard technique for varicocelectomy nowadays in view of the lower rate of recurrence and complications compared with open or laparoscopic techniques , 2. On oBetween September 2016 and January 2017, nine patients had unilateral MSV and ICGA performed on left grade 2 to 3 varicoceles in our unit. Four of the nine patients suffered from infertility with abnormal semen parameters. Two patients who presented with incidental finding of grade 3 left varicocele and oligozoospermia preferred surgical intervention after counseling. One patient was operated on due to bothersome discomfort associated with left grade 3 varicocele. Two varicocelectomies in adolescents were performed in view of testicular size discrepancy.The procedures were performed under general anaesthesia. The infrared fluorescence operative microscope  was brought into the field after incision of skin and spermatic fasciae. The vas deferens and its vessels were protected. ICGA was performed when the possible TA was identified. A pack of 25mg of indocyanine green (ICG)  was dissolved in 10mL of water. Each angiography required 5mL (12.5mg) of ICG solution which was prepared and administered by the anaesthetist in a bolus via a peripheral line. The Infrared 800 mode of the microscope was activated and the fluorescence angiography was recorded and analyzed. ICGA was repeated if necessary and at the end of the procedure to confirm a successful TA preservation.Testicular artery was clearly identified by ICGA in all patients . Two tesAll patients were discharged the same day after the operation. No adverse reaction was observed after injection of ICG. No clinical recurrence and complication was recorded upon follow-up at 4 weeks after the operation.50 of 50-80mg/kg in animals , the microscope can be shared among different specialties in the setting of a multi-disciplinary hospital. The running cost of ICGA is much lower than micro-Doppler for each procedure. A pack of 25mg Diagnogreen costs around USD $43 in our locality and usually one to two packs were consumed for each procedure while a disposable micro-Doppler probe costs USD $386.In conclusion, the use of intraoperative ICGA is safe and consistently provides objective assessment of testicular artery. The technique facilitates early identification and preservation of TA, and may decrease the incidence of TA injury during MSV. ICGA is potentially superior to and provides additional information compared to the current technique of TA identification with direct visualization of pulsation under high power magnification and micro-Doppler.


The core pluripotency transcription factor NANOG is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Although NANOG is phosphorylated at multiple residues, the role of NANOG phosphorylation in ESC self-renewal is incompletely understood, and no information exists regarding its functions during reprogramming. Here we report our findings that NANOG phosphorylation is beneficial, although nonessential, for ESC self-renewal, and that loss of phosphorylation enhances NANOG activity in reprogramming. Mutation of serine 65 in NANOG to alanine (S65A) alone has the most significant impact on increasing NANOG reprogramming capacity. Mechanistically, we find that pluripotency regulators  are transcriptionally primed and preferentially associated with NANOG S65A at the protein level due to presumed structural alterations in the N-terminal domain of NANOG. These results demonstrate that a single phosphorylation site serves as a critical interface for controlling context-dependent NANOG functions in pluripotency and reprogramming.  •NANOG phospho-residues are evolutionarily conserved in mammals•Phosphorylation promotes NANOG function in sustaining ESC self-renewal•Loss of phosphorylation improves NANOG function in reprogramming•Pluripotency regulators are preferentially associated with NANOG S65A in pre-iPSCs Wang and colleagues outline the role of phosphorylation in regulating NANOG function in ESCs and during reprogramming. Phosphorylation promotes NANOG function in maintaining ESC self-renewal, whereas loss of phosphorylation increases NANOG activity in reprogramming. Mutation of NANOG S65 results in transcriptional priming of pluripotency genes and their preferential association with NANOG S65A at the protein level, resulting in increased reprogramming efficiency. The phospho-proteome in pluripotent stem cells has been extensively and systematically studied , and hasOne study suggested that phosphorylation is important for maintaining NANOG stability in ESCs . This stWe performed IP-MS of endogenous NANOG in J1 mESCs A, and idNanog−/− setting. We utilized doxycycline (Dox)-inducible Nanog conditional knockout (NgcKO) ESCs  and phospho-mimic (3E) mutants A could mKO) ESCs  to generKO) ESCs B and tesWe seeded NgcKO ESCs at clonal density, withdrew LIF, and added Dox for 5 days, then performed alkaline phosphatase (AP) staining to assess pluripotency status based on colony morphology C and 2D.Nanog−/− somatic cells (pre-iPSCs) to naive induced pluripotent stem cells (iPSCs)  A. As exp (iPSCs) B. We als (iPSCs) B and 3C.Nanog−/− pre-iPSCs, reproducibly generating ∼3.5-fold more AP + iPSC colonies than NANOG WT  reprogramming  Figure F. AdditiCollectively, these results indicate that phosphorylation is dispensable for NANOG activity in reprogramming, and that blocking phosphorylation at S65 can enhance NANOG reprogramming activity.To determine whether the increased reprogramming activity of S65A over WT NANOG was due to enhanced protein stability, we treated NANOG WT and NANOG S65A pre-iPSCs with the translation inhibitor cycloheximide over a 6-hour time course. We did not observe any differences in the degradation rates or the protein half-lives of NANOG WT and NANOG S65A A and 4B,Next, we performed immunofluorescence for NANOG WT and NANOG S65A in pre-iPSCs and found that both were predominantly nuclear, with no obvious differences in overall distribution C, which Taken together, we conclude that the enhanced reprogramming activity of NANOG S65A is not due to increased protein stability or altered subcellular distribution.Esrrb and Oct4 in pre-iPSCs, to promote reprogramming efficiency   D. Westeror Tet1) E, suggesNanog−/− pre-iPSCs (2 ratio ≈ 0). Most importantly, however, we detected a preferential enrichment of all the pluripotency regulators tested in NANOG can physically interact and synergize with pluripotency regulators such as TET1 to promote pre-iPSC reprogramming . To overre-iPSCs F. As expTogether, we conclude that loss of phosphorylation may endow NANOG S65A with increased affinity with other pluripotency-associated regulators, leading to enhanced reprogramming see D.Here we report our findings on the role of NANOG phosphorylation in the maintenance and establishment of pluripotency. While phosphorylation was not essential for NANOG to maintain pluripotency of ESCs, it seemed to be beneficial for NANOG to sustain ESC self-renewal. Conversely, however, we found that loss of phosphorylation promoted NANOG function in reprogramming.Nanog−/− pre-iPSCs -derived pre-iPSCs were generated and used for reprogramming as described by 4 pre-iPSCs were seeded after selection onto gelatin-coated 12-well plates on top of irradiated MEFs and grown in serum + LIF for 2 days before medium switch to 2i + LIF medium . On day 10 in 2i + LIF, plates were stained for AP activity and iPSC colonies were counted under bright-field microscopy.4 reprogrammable MEFs containing a Dox-inducible OKSM cassette were infected with retroviral NANOG WT, NANOG S65A, or EV control. The next day 34,000 infected MEFs/well were seeded on top of a feeder layer of irradiated MEF feeders on a 6-well plate coated with gelatin, in “Dox + 3c”-containing ESC medium. On day 6 the medium was switched to ESC medium without Dox or 3c, and plates were stained for AP activity on day 10.MEF reprogramming was performed as described by Nanog−/− pre-iPSCs expressing 3xFLAGNANOG WT or 3xFLAGNANOG S65A were each expanded to 8 15-cm dishes after culturing for 2 weeks in SILAC ESC medium supplemented with either light or heavy lysine and arginine. Pre-iPSC nuclear extracts were pre-cleared with Protein G agarose beads rotating overnight at 4°C. The next day, 3xFLAGNANOG WT or 3xFLAGNANOG S65A were immunoprecipitated, washed, and eluted from α-FLAG beads with a 3xFLAG peptide solution. Eluted protein was then concentrated, quantified, mixed in a 1:1 ratio for each sample, and subjected to SDS-PAGE. Finally, whole lanes were excised from the gel and subjected to quantitative liquid chromatography-tandem mass spectrometry analysis.SILAC IP-MS was performed as described by A.S. designed and performed experiments, analyzed data, and wrote the manuscript; D.L. performed experiments and analyzed data; F.F. and X.H. provided bioinformatics support and technical assistance; M.F., D.G., J.D., F.Y., Y.X., and H.Z. provided technical assistance, reagents, and helpful discussions; J.W. conceived the project, designed the experiments, analyzed data, and prepared and approved the manuscript.


Shipping contributes primary and secondary emission products to the atmospheric aerosol burden that have implications for climate, clouds, and air quality from regional to global scales. In this study we exam the potential impact of ship emissions with regards to ice nucleating particles. Particles that nucleate ice are known to directly affect precipitation and cloud microphysical properties. We have collected and analyzed particles for their ice nucleating capacity from a shipping channel outside a large Scandinavia port. We observe that ship plumes amplify the background levels of ice nucleating particles and discuss the larger scale implications. The measured ice nucleating particles suggest that the observed amplification is most likely important in regions with low levels of background particles. The Arctic, which as the sea ice pack declines is opening to transit and natural resource exploration and exploitation at an ever increasing rate, is highlighted as such a region. Mineral dust, biological particles, and other primary particles have all been identified as ice nucleators, yet the abundance of such INP does not match the observed macroscopic features of clouds and precipitation5. Although, some mechanisms of ice multiplication are known8, the “closure” between abundance and composition of cloud-active aerosol, cloud ice, and precipitation remains a significant scientific hurdle.Cloud radiative properties, precipitation, and cloud electrification all respond to and are largely controlled by the formation and evolution of cloud ice. Ice Nucleating Particles (INP) are those atmospheric particles, which lower the free energy barrier that exists to spontaneous nucleation9, and they have therefore been used as a proxy for calculating global INP climatologies10. Laboratory studies of soot particles acting as INP have been more ambiguous, with many investigators failing to find any measurable nucleating capabilities above homogeneous freezing temperatures14. That said it is known that soot has a morphology that evolves considerably with aging, as can surface absorbents and therefore hygroscopicity. There is some evidence that increased hygroscopicity will lead to more favorable ice nucleation conditions15, and field measurements of aged and mixed soot have shown it to be an active ice nucleator17. Further complicating the picture is that there is not necessarily a strong link between liquid condensation and ice nucleation18, and some soot-surface coatings may act to suppress ice formation19.It is thought that globally mineral dust particles are the most common INP21. For most of the year the Arctic is well isolated from the middle and lower latitudes and is thus characterized by very low particle concentrations22. From May through September nearly 70% of the central Arctic is covered by low-level stratiform clouds23 -the radiative properties of which depend upon their microphysics24. Thus small changes in terms of regional sources and/or large scale transport of particulate may have significant impacts25. Furthermore, in the Arctic the sea-ice albedo feedback is highly sensitive to the radiative forcing of clouds21, and mixed-phase systems, in which solid ice and liquid water sustain coexistence, predominate – suggesting changes in INP may have amplified environmental effects. Therefore, it is important to understand the physical and chemical characteristics of INP that contribute to Arctic climate, and to its potential perturbation.In polar regions, which are some of the most remote and pristine areas of the world, what dominates ice nucleation may also be unique. As a deep ocean covered by a thin veneer of ice, the Arctic in particular is a unique and important area that demonstrates sensitive responses to cloud and radiative feedbacks26. At the turn of the millennium polar shipping was dominated by community re-supply, fishing, and tourism27, but future projections foresee increases in both destination traffic28, and also an inevitable push towards large cargo transit shipping as navigable northern maritime routes open due to changes in climate and therefore sea ice extent30. The northern Europe-Asia routes can be significantly shorter, and therefore save considerable fuel  relative to the traditional Suez Canal passages. Shorter transit distances and lower fuel consumption are in general positive for the global shipping footprint, but not all locations are created equal and the Arctic is a particularly unspoiled ecosystem31. An even stronger impact will be made by oil and gas shipping, which typically has 50% higher fuel consumption than transit shipping27. The arrival of more shipping in the Arctic basin is inevitable if even the most optimistic climate projections are correct, but the climate impact and feedbacks are not so certain. Although, many of the primary gas and particle emissions are already studied and it is known that many of them like black carbon have both long and short-lived climate forcing effects, specific effects of INP on clouds and climate have not been fully considered. For example, using the INP observations we present, Possner et al.32 recently simulated stratiform mixed-phase cloud response to INP emissions32. They show that moisture content, precipitation, and cloud-top radiative feedback all respond in non-linear, a-systematic ways, that can be sensitive to the cloud history and background state.Arctic shipping, including maritime natural resource exploration and exploitation, is an area of both economic growth and an obvious source of anthropogenic particles and pollutants Fig. 26. At thHerein we present the results of ship plume measurements made in 2013 and 2014 in a Scandinavian shipping channel. The results demonstrate the ability of ship emissions to amplify the number of atmospheric INP. The results are compared to background conditions to illustrate that even low absolute levels of introduced INP may contribute significantly in relatively pristine areas. The implications for Arctic clouds and climate are discussed.34. Outside of the ECAs the IMO regulatory limit on FSC is currently 3.5% and will drop to 0.5% in 2020. Thus, the Risholmen location allows for measurements of plumes from ships operating under near open water conditions and that largely comply with the future regulatory structures of global shipping.The Port of Gothenburg is a river and deep water sea port and is the largest Scandinavian cargo port with approximately 11,000 vessel calls annually. The port’s container, cargo, and ferry terminals all lie well inside the Gothenburg archipelago, where a relatively narrow shipping lane threads between many islands and peninsulas. The measurements described here were made from the Risholmen peninsula , soot characterization, gas and particle mass spectrometry, cloud condensation nuclei (CCN), and in 2013 and 2014 INP characterization  and other methods in order to chemically and physically characterize captured particles.From 2013–2015 annual measurement campaigns were undertaken at Risholmen that focused on the physical and chemical characteristics of ship plume gas and particle emissionsThe prevailing meteorology during INP sampling varied between ship passages but was generally representative of late-fall Nordic maritime climatic conditions. Diurnal variability was limited by the strong marine influence, resulting in conditions dominated by the passage of frontal cyclones. During INP sampling wind speeds typically ranged from 3–13 m/s with temperatures from 3–12 °C. Relative humidities at the sea shore stayed high (60–85%) with periods of fog and rain. Plume travel times between 30 seconds and 3.5 minutes can be estimated from the wind direction/speed and the distance from the shipping channel. Given the dominant wind vectors most plumes sampled for INP had travel times at the lower end of this interval (30–60 s.), where ships were sampled when approaching to within 300 meters of the station.36. The sample collection proceeded directly via a particle inlet for the time period that the individual plumes transited the sampling site. The presence of plume particles was anticipated using ship tracking software, with the plume arrival detected by measuring particle counts using a CPC  and an optical particle counter  were used to characterize particles within the capturing chamber. Measurements showed stable particle size distributions for ≈30 minutes at 10 L/min sampling flows, confirming that INP sampling losses from sedimentation and/or diffusion to the chamber walls were negligible during PEAC7 sampling increments. Fifty to sixty litres of air were sampled for each wafer in 2014.Subsequent to the 2013 measurements the deposition wafers were analyzed and yielded measurable but low absolute INP counts. In an effort to increase statistical reproducibility the results of 2013 prompted an updated 2014 sampling protocol to increase the total sampling volume and thus the absolute number of collected INP irrespective of the plume transit time. Thus in 2014 a 450 L plume capturing chamber  measured by the FRIDGE apparatus for all ships in 2013 and 2014 are shown in Fig. 36. Thus the standard error of the mean given by the solid bars is indicative of the large ship-to-ship variation in emitted INP. In both years the variability between individual ships far outweighed any identifiable trends based on ship type, and as can be seen from the overlapping solid error bars in Fig. The mean absolute number concentrations (INP LAlthough it might be expected that secondary (aging) processes could influence the ice nucleation ability of ship emissions no significant correlations between INP concentration and ambient conditions were observed. This suggests that the short plume travel time and variable but narrowly constricted ambient conditions did not impact the results. The effect of plume aging remains an open scientific question that should be further investigated.−1) above the measured background state is plotted for both 2013 and 2014. The intensification is taken to be the difference in concentration The observed effect of ship emissions on INP is shown in Fig. EF may be used. An INP emission factor EFINP relates the number of emitted INP to the total mass of fuel comsumed. For combustion plumes CO2 concentration has been shown to be a reliable tracer of fuel consumption and thus can be used as a basis for calculating gas and particle emissions as a function of mass of fuel burned38. Such calculations also depend on the conversion efficiency of the hydrocarbon fuel to CO2, where here we assume the mass of emitted CO2 per kg fuel burned is 40. Thus given EFINP is determined by the ratio between the increase of INP concentration ΔINP to the corresponding increase of CO2 referenced to standard temperature and pressure ΔCO2,In order to extrapolate from these land-based, stationary measurements, emission factors 2 concentration, temperature, and local pressure were measured continuously with one second time resolution. Thus ΔCO2 could be straightforwardly calculated by integrating the plume signal minus the background concentration.During both measurement campaigns COi condition and averaged as in Figs 5 to ≥107 are plotted in Fig. 38.Emission factors were calculated for individual ships at each T and S36, were investigated on four different wafers. From the background measurements 13 of 15 particles had substantial carbon (16–79 wt%) and oxygen content (6–81 wt%), which may be indicative of oxygen associated with carbonaceous material. However, more than 50% (8 of 15) had an O/C ratio above 0.5 and/or a substantial inorganic content , and thus inorganic phases like mineral dust and even sea salt appear to be present in many background INP. Conversely, of the analyzable single particles, those from the shipping wafers were only identified as soot.One advantage of utilizing the FRIDGE technique is that subsequent to INP analysis the deposition wafers can be analyzed using SEM to study chemical composition, size and morphology of single particles. A SEM analysis of a limited set of 2013 wafers was done using energy-dispersive x-ray spectroscopy (EDX), with the aim of qualitatively evaluating the chemical composition of particles acting as INP. Fifty-one INP positions, identified from the FRIDGE image analysis18. Some such results may be attributed to experimental resolution, as many studies demonstrate that fresh, laboratory generated soot is weakly ice nucleating. Schill et al.14. observed activated fractions from off-road diesel emissions of between 10−6 and 10−8. Likewise, maximum activated fractions of ≤10−5 have been observed for automobile diesel emissions41, which are similar to those observed from a propane diffusion flame (≤10−4)13. Dymarska et al.12 posit an upper limit for ice production from a range of experimentally measured soot types that corresponds to an activated fraction of ≈0.5 × 10−6. While these low magnitudes have largely been dismissed as unlikely to be important on a global scale, our measurements demonstrate that within the context of a low background of INPs – such point sources may notably shift the ambient condition.We see two interesting results from this study. First we do observe ice nucleation from ship plume particles that are most likely to be fresh soot particles. Given that the cursory single particle analysis on 2013 wafers pinpointed only unremarkable carbonaceous soot particles, no exhaustive particle characterization was completed. However, it is not unforeseen that some subset of soot particles do have nucleating capacity, and given the FRIDGE’s ability to detect low concentrations due to the flexibility of the sampling protocol, we see the result manifested here. This is not entirely at odds with previous measurements of the ice nucleating potential of combustion particles, although previously observations of combustion soot nucleation have largely been limited to the homogeneous regime, or where soot particles only act as freezing nuclei after activating liquid dropletsSecond, we identify another potential indirect cloud and climate effect that should be considered for ship emissions in pristine environments. Attempts to quantify INP in polar regions have been complicated by instrument resolution, but the available records indicate low concentrations of ambient INP on the order of one per liter Table . Clearlyet al.27) of 2.9 to 5.2 megatonnes per annum27. Using the calculated emission factors from this study (≈2 × 107 INP(kg fuel)−1, Fig. 16 to 1017 new Arctic INP yr−1. Although, dispersed over an entire region the signal may remain small it is important to note that such emissions will be point (or line) contributions, with much greater average intensities over channels of shipping traffic. Others have already shown that aerosol emissions from ships can, for example, lead to lightning enhancement in narrow bands above busy traffic channels42. In particular mixed-phase Arctic stratiform clouds have significant radiative feedbacks and can be widespread and persistent43. Whether the delicate balance would be maintained with the boundary layer injection of nucleating particles is a complicated calculus related to surface coupling and cloud feedbacks .Furthermore, as the climate changes, shipping in the Arctic is already increasing dramatically. In addition to the up to 50% fuel savings that could potentially be achieved on Europe to Asia trading routes, reductions in sea ice open the Arctic to more oil, mineral, and gas exploration. Predictions suggest that as much as 2% and 5% of global sea trade could be shifted to the Arctic in 2030 and 2050 respectively. This represents an increase in annual fuel consumption in the Arctic  INP recycling that occurs when cloud particles evaporate and/or sublimate near the cloud base, and the nuclei are thus free to be reutilized, is sufficient to maintain ice production and therefore regulate liquid water content over multiple days45. The Possner et al.32 study has tried to directly assess some of the competing microphysical effects using idealized large eddy simulations, which resolve the large scale turbulent structures within clouds, to perform a case study based upon observations of single-layer mixed phase stratus with inserted ship emissions, including INP32. They find that microphysical feedbacks and cloud response are sensitive to the ice phase in non-obvious ways. The manifestation of cloud radiative and precipitation response to ship-perturbations is sensitive to background conditions and INP recycling. Furthermore, previously identified warm-phase stratus responses to emission’s perturbations may be compensated for, or diminished by, additional feedbacks involving the ice phase. For example, in warm-phase stratocumulus the cloud radiative effect of ship emissions has previously been determined to result from changes in cloud condensation nuclei increasing cloud optical thickness due to decreases in cloud droplet size and corresponding increases in liquid water path46. That effect can be somewhat mitigated when the ice phase helps to stabilize the cloud layer.Satellite observations of Arctic mixed-phase clouds show that ice water content and surface precipitation are influenced and potentially increased by shipping47. It is therefore important to consider what new effects will result due to anthropogenic encroachment, and as once distant influences become local drivers.In summary, the FRIDGE electrostatic deposition and wafer analysis system for INPs has been used to collect and study the particles from ship emissions outside of the Port of Gothenburg. The ship emissions are observed to more than double the ice nucleating capability of the background aerosol. Given the boreal location of Gothenburg, Sweden and low ambient INP concentrations, the observations can be taken as an indication of potential impact for ships in the Arctic and/or other pristine aerosol regions. Although the Arctic remains isolated and remote it is currently in a period of unprecedented climate and ecosystem change, and much of the change is Arctic response to global forcing