Patients’ stories of their illnesses help bridge the divide between patients and providers, facilitating more humane medical care. Illness narratives have been classified into three types: restitution (expectation of recovery), chaos (suffering and loss), and quest (unexpected positive effect from illness). Undiagnosed patients have unique illness experiences and obtaining their narratives would provide insights into the medical and emotional impact of living with an undiagnosed illness. Adults and children with undiagnosed diseases apply to be evaluated by the Undiagnosed Diseases Network (UDN). Written illness narratives from 40 UDN applicants, including 20 adult probands who applied for themselves and 20 parents who applied for their children, were analyzed for: 1) narrative content and 2) narrative type.Narrative content: could be grouped into three themes: 1) Expectations of the UDN: the majority felt they had no further healthcare options and hoped the UDN would provide them with a diagnosis, with the adults expecting to return to their previously healthy life and the parents wanting information to manage their child’s healthcare. 2) Personal medical information: the narratives reported worsening of symptoms and some offered opinions regarding the cause of their illness. The proband narratives had few objective findings, while parental narratives had detailed objective information. 3) Experiences related to living with their undiagnosed illness: frustration at being undiagnosed was expressed. The adults felt they had to provide validation of their symptoms to providers, given the lack of objective findings. The parents worried that something relevant to their child’s management was being overlooked.Narrative type: All the narratives were of the chaos type, but for different reasons, with the probands describing loss and suffering and the parents expressing fear for their child’s future. The parental narratives also had elements of restitution and quest, with acceptance of “a new normal”, and an emphasis on the positive aspects of their child’s illness which was absent from the probands.These narratives illustrate the chaos that coexists with being undiagnosed. The differences between the proband and parental narratives suggest that these two groups have different needs that need to be considered during their evaluation and management. Undiagnosed diseases are common, affecting approximately 30 million Americans, and are associated with high rates of morbidity and mortality –3. They https://undiagnosed.hms.harvard.edu). Currently, seven academic medical centers have been named as UDN clinical sites and receive applications for evaluation of undiagnosed adult and pediatric patients from all over the country. The review of each application prior to acceptance includes a comprehensive examination of the medical records for the medical history, family history and prior laboratory tests and procedures. Many UDN applicants whose medical records are under review contact the clinical sites to provide incremental bits of information through multiple emails and phone calls that they believe will facilitate their acceptance into the network. At the Duke University UDN clinical site a process was initiated to systematically request written narratives from all UDN applicants, to gather the patient/parent’s perspective of the illness and prior experiences all at one time.Both children and adults are affected with undiagnosed diseases, though possibly with different experiences. Parents of pediatric patients continue to struggle and make their way through the “diagnostic odyssey” of repeated clinical consultations and testing, with the purpose of finding information about their child’s long term health and recurrence risk information for the family . In contThe restitution narrative is the least frequent among chronically ill individuals. This narrative reflects a transient nature of the illness with the patient expecting to recover from it. The narrative type usually occurs in the early stages of a chronic illness, before its longstanding nature becomes evident  has started to have seizures bi-weekly to weekly basis. Lately, I see where she is having more seizures during the night time……. (Narrative 24) ParentFrequently (19/40) the narratives noted that symptoms were increasing in severity.No one knows why I have these symptoms. …. My PCP [primary care physician] is sending me to everyone she can think of to help me. (Narrative 10) ProbandThey described how medical providers had been unable to identify a unifying diagnosis or a medical explanation for their own symptoms or their child’s symptoms.The skin burning had become quite unbearable. Wearing a shirt was painful, my pants, shoes, earrings and putting makeup on my eyelids was 6–7 pain scale on 0–10. (Narrative 10) ProbandDistinctive Features of Proband Narratives: The majority of proband narratives described symptoms of pain (13/20) and/or (9/20) fatigue, often with limited objective findings (17/20).I have had issues with my eyes, hips, bones, hands and a general feeling of malaise. (Narrative 13) ProbandThere were also descriptions of many different symptoms that were difficult to connect to one another.My last healthy day was *month/*day/*year. (Narrative 10) ProbandThese narratives often describe a very specific event, which was seen as the triggering event or a moment when symptoms were noted when previously there were none.We started [my daughter] on [medication] and on day 2 of the medicine she had the respiratory side effect of bronchial swelling. If she would have had the 3rddose I know she would not be here right now. Heart rate of 190–200 resting and 45–50 BPM [beats per minute] and retracting along with tracheal pulling and stridor. ….02is a PRN [pro re nata] around here. 95% of the time (she) is on room air and SATS [oxygen saturation] 98/110 resting. (Narrative 38) ParentDistinctive Features of Parent Narratives: The parental narratives described more objective findings such as seizures, intellectual disability, and congenital heart disease. It was striking that many of the parent narratives were written with a high level of medical literacy, suggesting that parents were becoming “medicalized” through their experiences with their child’s illness/disorder.We welcomed (our son) into the world after a normal pregnancy… He began to meet some of his first few milestones over the first few months. By month 5–6, we began to notice that [son] was falling behind in his milestones – he was not progressing with crawling, he could not roll over with consistency, could not track things visually, seemed to have difficulty with recognizing sounds, and did not have much verbal communication. (Narrative 36) ParentThe parent narratives typically followed a chronological order, beginning with pregnancy and birth history, frequently describing their first concern about their child and how that occurred.Experiences related to living with their undiagnosed illness:We appreciate your time in reading our concerns. (Narrative 26) ParentThank you for taking time to review my health history. This document contains my story from the last three and a half years. (Narrative 14) ProbandCommonalities: The process and opportunity to “tell their own story” seemed to be appreciated by both the probands and parents.As I sit to summarize [daughter] I find myself tearing up and very emotional. Nine years of chronic medical crisis’ and so many failed treatments leaves so much to recap. (Narrative 38) ParentAnd for some, the process of revisiting the story was emotional.The years of searching for answers that have only lead to more questions have been extremely challenging. (Narrative 21) ProbandAdvocating for her to have the best quality of life and to help improve her medically has been an extremely challenging and tiring process. I have to set limits on what is realistic in this journey. It is like I get on a hamster wheel and can’t get off. (Narrative 38) ParentBoth proband and parent narratives described how emotionally challenging it was to continue to look for answers.I saw twenty doctors who could not help me. I slowly went up and down with some recovery to feeling sick over the course of 14 years. (Narrative 9) ProbandBoth proband and parent narratives described previous experiences with health providers and diagnostic evaluations, including frustration with the process and lack of a diagnosis.The last neurologist I saw ran a *** that had abnormal results…. He also ordered nerve conduction studies that I was told showed slower conduction. I felt redeemed.(Narrative 5) ProbandDistinctive Features of Proband Narratives: The source of the frustration found in the proband narratives for many, was that multiple medical evaluations only led to disappointment since no definite findings had been identified to explain the symptoms and laboratory testing did not validate their symptoms. In a few (3/20) there was expressed validation at an abnormal finding that confirmed that their symptoms were real.n =3/20) probands reported either being told of the possibility of or the recommendation for investigation of a psychological component to their symptoms.My current PCP, had me trial (medication) thinking that stress could be a contributing factor. (Narrative 11) ProbandAnd some  ProbandThey also expressed that health providers don’t ‘hear” them or they felt misunderstood, or were labeled as “difficult”.My symptoms not only persisted but worsened. I was sent to ***. That neurologist also ran no tests. After a brief exam, he turned to his fellow and said, “Tell her she’ll live,” and strode out of the room. (Narrative 5) ProbandTreatment attempts were reported as not successful or to result in only marginal improvements for a short time. Probands also expressed dissatisfaction if they felt they needed additional or specific tests to diagnose their disorder and the health provider disagreed.Since my illness, I am unable to do a lot of the things I used to enjoy. Working a normal job has become virtually impossible. Enjoying social and physical activities has also been more difficult. (Narrative 13) ProbandPrior to this, I was healthy and active, ran marathons and worked long hours as a [professional]. (Narrative 7) ProbandThe overwhelming majority of the proband narratives reported loss; loss of the person they used to be and being unable to work or enjoy life since the onset of their illness or disorder. Many provided specific examples of athletic ability, professional or career success that have been negatively affected and being unable to meet family responsibilities.So many physicians dismissed things because she “looked” okay when she was little or later because she was outside of their comfort level…. I commonly get the “she is beyond me” answer or that the physician relies on me as I know her so well… This is where the frustration always comes into play. (Narrative 38) ParentDistinctive Features of Parental Narratives: The parents’ narratives differed from the proband narratives regarding the source of their frustrations. Although a few of the parent narratives also described not being “heard” by the health providers, the parents expressed worry about the possibility that something would be overlooked that might be important, and that physicians had reached the limit of what they could offer..She has eight standing appointments each week . She also sees peds [pediatrician], neuro [neurology], gastro [gastroenterology], a dietician, ophthalmology, genetics and an orthopedic specialist regularly. (Narrative 37) ParentThe majority of parents (14/20) described the complexity of care required for daily living for their childHe was wasting away in front of us and nothing we did seemed like it could stop it. (Narrative 23) ParentI can’t explain the fear and rush of emotions we felt as parents knowing nothing about seizures or Epilepsy at the time. We truly thought we were losing him. (Narrative 32) ParentFor some of the parents, there was an expressed helplessness and distress at being unable to improve their child’s outcome.My son has suffered but yet he doesn’t complain. He is the picture of a strong and kind young man. He has grace and courage where others would and do fail to. He deserves a chance at having answers or paving the way for answers for others. (Narrative 23) ParentThe majority of the parent narratives emphasized positive attributes of the child when describing how the illness/disorder has affected their child.But our sweet boy, he’s just so precious. He is such a sweetheart. He gives the best hugs and snuggles. He is absolutely adorable when he smiles at you. He is easy-going and hard-working and laid-back and persistent. He is so resilient. He rarely gets frustrated. He is a pleasure to be around and brings joy to our lives. I just wish we could figure out what is going on. (Narrative 31) ParentIn these descriptions, the love for the child comes through.As parents we want nothing more in the world to figure out what is going on with her. … She has determination that is mind blowing, she is our hero. (Narrative 25) ParentThe parent narratives suggested that parents are advocates for their children and are determined to do everything they can to help improve their child’s quality of life, even if there is no cure.She is truly a gift that has taught me the true meaning of unconditional love. (Narrative 38) ParentIn a few of the parent narratives we also found descriptions of an unexpected positive change resulting from the illness/disability.We identified three major themes that were common to the proband and parental narratives: We outline the commonalities in both followed by the differences between the proband and parent interviews for each of the three themes.n = 19/20), while the majority of the parent narratives included additional characteristics of quest (n = 17/20) or restitution (n = 10/20). Ten of the parent and one of the proband narratives had elements of all three typologies. The majority of the proband narratives did not include the additional elements of quest and/or restitution. The narrative typologies closely mirrored the lived illness experience theme in the content of the narratives and thus we summarize the narrative types in the table, without including overlapping quotes.We found that all 40 narratives were of the chaos type Table . HoweverThis study analyzed for the first time, the illness narratives pertinent to both adults and children with undiagnosed diseases. Overall, the burden of living with an undiagnosed condition is high, with suffering, frustration and uncertainty. We found both commonalities and important differences in the content of the narratives and in the typologies between the adult proband and parental narratives.Not surprisingly, both the probands and the parents applied to the UDN after having exhausted all other options. While both groups had high expectations of the UDN, the probands wished to resume their former lives, while the parents recognized that their child was unlikely to completely recover and wanted information how to better medically manage their child. This may be a reflection of the early onset and severe manifestations seen in children with difficult to diagnose disorders; for the probands who often developed symptoms abruptly, a clear awareness of the demarcation between being well and falling sick persisted, such that they yearned to become well again.The medical information in both groups reflected important reasons for why they applied to the UDN- for many the symptoms were getting worse and for all there was no answer. A striking difference between the proband and parental narratives was the lack of objective findings in most of the proband narratives, in contrast to the detailed objective findings in the parental accounts, again reflective of the multiple and serious features of pediatric undiagnosed diseases. The narratives were especially informative in providing information that was not in the medical records, such as the proband/parent’s impression of potential causes/diagnoses or a more accurate timeline of when certain events or symptoms presented. This information could be used by the UDN team to either obtain additional records that could be relevant or to address these problems specifically during the review and evaluation process.The narratives provided insight into the lived illness experiences of the proband or the experience as described by the parent. Narrators appreciated being given an opportunity to tell their story in their own words, perhaps attesting to the power of narrative medicine . ParentaAnalyses of the 40 narratives revealed that all were of the chaos type, but elements of all three of Frank’s types of illness narratives  were present in a fourth of the narratives—more so in the parental narratives, with only a few of the proband narratives reflecting restitution and only one quest. However, the restition in the proband narratives was exclusivly related to the initial expectation that health would be restored. The narratives then became dominated by chaos, when health was not restored. The chaos in the proband narratives was mainly due to the suffering and loss  and disorganization of the narrative with a multitude of difficult to connect symptoms. This disorganization is consistent with Frank’s description of chaos being “beyond words” . It is aIn contrast, the chaos associated with the parent narratives was related to fear and uncertainty about their child’s future and an inability to make plans. Most of the research on parental narratives has focused on diagnosed childhood chronic illness or cancer and thus these experiences are different from those of parents of children who apply to the UDN , 28. ForThe UDN clinical teams are put in the difficult position of having to make a decision regarding acceptance of applications from patients/parents who believe the network is their last hope for help. The decision is made almost exclusively by medical record review with very little, if any, interaction with the applicant prior to a decision. In general, if an applicant is not accepted to the UDN , there could be very little closure for the applicant and the belief of not being heard may be reinforced. While indicating that acceptance was not appropriate, an acknowledgement of the pain and suffering that had taken place would be important. This may be helpful to some of the adult probands in their transition from chaos to quest or restitution- acceptance of a new normal. For parents of children with undiagnosed illness, reassurance that they have done everything they possibly could have for their child’s medical care and encouraging their role as an advocate for their child are important. It may also be important to consider inclusion of a social worker or another mental health provider to facilitate communication with the applicants.This was a retrospective analysis of self- reported and open-ended patient/parent narratives and since it was not an interview, in which follow-up questions could be asked, there may be aspects of the illness story that could be missing. As these narratives were in a written format, the proband or parent would have had the opportunity revise or review their narrative prior to it being submitted and thus relevant information could have been lost/edited in the revision process. Additionally, there may be differences in an individual’s ability to write as opposed to verbally tell a story.The written narratives of UDN applicants, both probands and parents, are powerful and allow for an initial connection with the medical team. Important medical information and a chronology of events may be gleaned from the narratives. It is evident that having an undiagnosed disease results in emotional distress, in addition to suffering from the symptoms and signs of the illness. Probands and parents differ in their experiences with the undiagnosed disease and their expectations of the UDN. Further study of the psychological aspects of undiagnosed diseases is slated to be an important future direction for the UDN  and this


This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed the  With a label-free mass spectrometry approach, in order to quantify ubiquitinated proteins, we detected substrates of these two E3 ligases in Drosophila. This is an example of how flies can be used to reveal physiological substrates of disease-associated proteins. The results, using Drosophila as a validated model for neuronal disorders, provide new leads towards the cellular roles of these two disease causing E3 ligases.Both Parkin (PARK2) and UBE3A are E3 ubiquitin ligases for which mutations result in severe brain dysfunction, Familial Parkinson's Disease (PD), and Angelman Syndrome (AS). In order to unravel the molecular mechanisms leading to these neurological dysfunctions it is necessary to identify and understand the role of their ubiquitinated substrates. Several substrates of UBE3A and Parkin have been surveyed mostly using cell culture overexpression models in combination with proteasome inhibitors. But more recently, a more physiological setting has been achieved by using anThe human genome contains ~20.000 protein-coding genes , but theUbiquitin (Ub) is a small protein (~8.5 kDa) that is specifically attached to target proteins through a sequential enzymatic cascade . ClassicAutophagy refers to the process in which cells engulf their own contents into double-membrane structures (autophagosomes) that ultimately fuse with lysosomes, where cargo is degraded and basic biomolecules are recycled back to the cytosol  8]. Lar. Lar8]. Neurons particularly require a tight spatiotemporal regulation of their proteome. The cell body or soma is typically distant from axonal and synaptic connections; and they are constantly receiving, decoding, and transmitting information via synaptic communication. Regulation of protein interaction, sorting, and activity is not only critical for the wellbeing of the neuron itself, but it is also necessary for proper coordinated transfer of the information. Thus, right balance between protein synthesis and degradation is essential for neuronal homeostasis, both for correct neurodevelopment and, at later stages in aged neurons, to protect against stochastic proteotoxicity .The first evidence of the involvement of the UPS in the nervous system homeostasis came from the discovery that ubiquitin is present in neurofibrillary tangles of various neurodegenerative diseases , 23. HerDrosophila has been a valuable tool to shed light on our understanding of the role of ubiquitination in the nervous system. In fact, evidence of a link between UPS and synapse formation has often come first from experiments performed in flies. For example, in the early 90s, the fat facets (faf) gene was found to encode a DUB involved in fly eye development [ bendless gene was shown to regulate neuronal connectivity [ highwire were later reported to have a defective synaptic overgrowth and function in larval neuromuscular junction (NMJ) [ Drosophila studies have reported evidence of the involvement of the UPS in the nervous system development and function [elopment , 41, whiectivity , 43. Flyon (NMJ) . Similaron (NMJ) . Over thfunction –50. Drosophila as a model organism since the early years of the 20th century can be explained by its many advantages. First of all they are suitable for genetic studies as their fast reproductive cycle coupled to a great capacity to provide a large amount of eggs guarantees abundant offspring in short periods of time [ in vivo model for the study of their role in the context of many diseases, including neurodevelopmental and neurodegenerative diseases [The broad use of of time , 52. The of time , which g of time , provididiseases , 56. in vitro studies catalyses attachment of K48-linked ubiquitin chains to its substrates, consequently targeting them for proteasomal degradation [ UBE3A gene has been associated with autism [ in vitro  and therefore cannot be concluded to be neuronal targets of UBE3A [ in vivo validation of any of these candidates has been unsuccessful so far.Angelman Syndrome (AS) is a rare neurodevelopmental disorder with a prevalence of approximately 1/15.000 individuals , charactradation . Interesh autism –65. Manyof UBE3A –70, whilof UBE3A –74. MostDrosophila UBE3A (Ube3a) is ubiquitously expressed during embryogenesis and is broadly detectable in the adult nervous system, particularly in the mushroom bodies, which represent the key region for learning and memory [ UBE3A duplication-based autism cases, reporting that Ube3a mutant flies mimic characteristics of human AS [ Ube3a null mutant flies display locomotor impairment, abnormal circadian rhythms, and learning and memory defects, with a particular effect on long-term memory [ UBE3A alleles of AS patients have been reported to act as loss-of-function mutations also in its Drosophila homologue [ Drosophila melanogaster 3rd instar larvae, also inducing in both cases defects in glutamatergic signalling [d memory . Differehuman AS –78. Ube3m memory . Furtherm memory  and decrm memory . In addiomologue . Fly modgnalling .α-synuclein [Parkinson's Disease (PD) is the second most common neurodegenerative disease after Alzheimer's. It is considered to affect 1% of people older than 60 years and up to 4% older than 80 years , 82. Parynuclein , 84. Howynuclein . Mutatioynuclein –89. Accoynuclein , 90–94. Drosophila have been critical to improve our understanding of PD pathophysiology and Parkin function. In fact, the first hint that Parkin was involved in mitochondrial homeostasis came from the analysis of Parkin null flies (generated through ablation of endogenous parkin gene through P-element mutagenesis). parkin deficient flies display decreased dopamine content and dopaminergic neurodegeneration; they also reduced longevity, motor deficits, and male sterility [ parkin null flies revealed that mitochondrial electron transport chain genes, as well as genes involved in oxidative stress and innate immune responses, were upregulated [ in vitro and in vivo studies [ Pink1 null flies display the same defective phenotypes as parkin null flies, and Parkin overexpression can rescue Pink1 loss but not vice versa [ Drosophila genetic studies showed that Pink1 and parkin interact with the mitochondrial fission and fusion machinery to regulate mitochondrial dynamics [Extensive studies performed employingterility –98. Ultrterility , 98. Traegulated . In addiegulated . Ever si studies  and, con studies . However studies –104. Sem studies , acts ince versa –108. Subdynamics –111. Drosophila S2R+ cells [ in vivo Drosophila models have reinforced mammalian cellular discoveries. Functional studies revealed that Parkin is phosphorylated by Pink1 in Drosophila cells, leading to Parkin activation. Parkin phosphorylation status modifies phenotypes typically affected in Pink1 and parkin null mutant flies [ Pink1 null associated defects, supporting the requirement of both ubiquitin and Parkin phosphorylation for Parkin activation in the Pink1/Parkin pathway [ in vivo and that defects in the disposal of dysfunctional mitochondria are involved in the progression of the PD.Mammalian cell culture studies first established that PINK1 accumulates on depolarised or damaged mitochondria to recruit and activate latent overexpressed Parkin and dispose of dysfunctional mitochondria via mitophagy , 113. ThR+ cells  and in vnt flies ; and mit pathway . Neverth Fbxo7 genetically interacts with parkin in Drosophila and is involved in PINK1/Parkin-dependent mitophagy in mammalian cells [ parkin has been shown to further genetically interact with LRRK2 and Vps35 in flies [ in vitro upon overexpression of the putative substrate and/or Parkin. Although several unbiased quantitative mass spectrometry studies have reported altered protein levels in Parkin deficient Drosophila and mice [ in vivo Parkin substrates have not been identified so far.Recent findings have identified additional PD-associated genes involved in Parkin-dependent mitophagy.an cells . In addiin flies , 119, alin flies . Parkin in flies , 121–124and mice –128, in  in vitro or in cellulo, but successive in vivo confirmation is required when translation towards human health is sought. Drosophila represents an ideal organism to study ubiquitin pathways in vivo. Ubiquitin is highly conserved across all eukaryotes [ Drosophila Ub being 100% identical to the human protein. In humans Ub is encoded by four genes: UBA52, RPS27A (UBA80), UBB, and UBC [ Drosophila three homologous genes exist: RpL40 (DUb52), RpS27A (DUb80), and Ubi-p63E [Primary discoveries are usually performedkaryotes , Drosoph and UBC , 131, whUbi-p63E . Drosophila proteome is predicted to contain ~15.000 gene products, of which ~10.000 proteins have been successfully identified employing mass spectrometry (MS) [ in vivo, however, can be very challenging, particularly in neurons. Due to the low stoichiometry at which ubiquitin-modified proteins are present within cells, it is necessary to enrich the ubiquitinated fraction prior to the MS analysis [ in vivo [Thetry (MS) , 134. Stanalysis . For thianalysis , 136–140analysis . Alternaanalysis  and used in vivo , 144. Th in vivo , 146, subioUb strategy [ in vivo [ E. coli biotin holoenzyme synthetase BirA is 14 amino acids long [ in vivo isolation of ubiquitin conjugates has so far allowed the purification and enrichment of large amounts of ubiquitin conjugates from flies [To avoid the detection of false positive ubiquitinated proteins, an enrichment process under denaturing conditions is preferred over the usage of physiological buffers. This has been classically performed using poly-histidine tagging , 148. Hostrategy , based ostrategy  during tstrategy . This bi in vivo . The minids long . This caom flies , 154, miom flies , and humom flies .bioUb system relies on the in vivo expression of the bioUb construct, which is formed by six ubiquitin-coding sequences in tandem followed by the bacterial bifunctional ligase/repressor BirA enzyme (bioUb construct releasing BirA and ubiquitin and mirroring the processing of endogenous ubiquitin gene products [bioUb) that is efficiently handled by the cascade of ubiquitin-conjugating enzymes and successfully attached to target proteins together with the endogenous ubiquitin. The advantage of having ubiquitinated proteins tagged with biotin is that they can be very efficiently and specifically purified employing avidin-conjugated beads. Biotin-avidin interaction is one of the strongest identified interactions in nature [The A enzyme . Endogenproducts . Each ubn nature , 159, ann nature . Finallyn nature , 154–156 Drosophila neurons during embryonic development [ Drosophila melanogaster and identified 234 and 369 ubiquitinated proteins, respectively [ Drosophila life cycle, reinforcing the importance of using the appropriate cell type when studying ubiquitination. For example, Ube3a was found to be active in both developing and adult neurons, while Parkin was found to be enzymatically active in adult neurons only [ Drosophila under different conditions  in adult Drosophila neuron. In both cases, to detect the E3-ligase substrates, we followed a label-free quantitative proteomics approach. Ubiquitinated proteins that were enriched using the bioUb strategy in each of the experimental conditions were independently analyzed by MS. Resulting MS raw files were subsequently combined for the bioinformatic analysis in which a search engine determined the identity of the proteins in the samples as well as their relative abundance. Consequently, those proteins, which were found to be more abundant in the presence of the wild-type version of the E3 ligase rather than in the presence of the ligase-dead version of the ligase, were considered putative E3 ligase substrates.The t manner . In fact Drosophila antibodies were available and interestingly showed that most of them were monoubiquitinated by Parkin. Furthermore, in agreement with previous mammalian cellular studies [ in vivo.We successfully isolated >1.000 ubiquitinated proteins, identifying, for example, 37 proteins whose ubiquitination is affected by Parkin activity: 35 were more and 2 were less ubiquitinated . These i studies , ubiquitIn the case of Ube3a flies, several UPS and autophagy-related proteins were identified to be more ubiquitinated upon Ube3a overexpression, including two proteasomal interacting proteins (Rpn10 and Uch-L5) earlier identified by our lab as Ube3a substrates . Our pro in vitro studies showing that UBE3A catalyses preferentially the attachment of K48-linked polyubiquitin chains [ Drosophila that Ube3a induces K48 and K11 chains on its substrates. Interestingly, not all the validated substrates of Ube3a seem to be targeted for degradation [In addition, to detect E3 ligase substrates, our investigation allowed us to gather information about specific ubiquitination sites as well as types of ubiquitination linkages. In most proteomic studies, trypsin is the enzyme of choice to digest proteins and obtain suitable peptides that are further analyzed by MS. When the conjugated ubiquitin is cleaved with trypsin, it leaves a Gly-Gly dipeptide remnant on the conjugated lysine residues that serve as a signature of ubiquitination and allows depicting the specific site of modification. In agreement withn chains , 161, weradation  as one w in vivo, which might be involved in pathways beyond mitophagy. Our proteomic analysis of Parkin substrates revealed that Parkin ubiquitinates a wide range of proteins with no obvious functional connectivity, although endocytic trafficking components, such as Vps35, Vps4, or PDCD6IP/ALiX, were overrepresented. Interestingly, parkin has been recently shown to genetically interact with Vps35 in Drosophila [Despite the fact that we identified some outer mitochondrial membrane proteins that have been reported to be ubiquitinated by Parkin during mitophagy, such as VDAC1/2/3, TOM70, and CISD1/2, mitochondrial proteins were not particularly enriched compared to previous studies , 163. Thosophila , and sevosophila , 165. Ad Drosophila results, in contrast to previous studies, have not required promotion of Pink1 activity, and therefore we might have identified some Parkin substrates that are Pink1-independent. This opens the question of how Parkin can be activated then. It could be possible that the cleaved cytosolic Pink1 fragment may have a role in the activation of Parkin for other purposes than mitophagy. It can neither be discarded that other kinases have the ability to activate Parkin. Further studies depicting the requirement of Pink1 for the activation and ubiquitination of Parkin will clarify these questions.It is important to note that our in vivo unbiased proteomics approach we have performed has provided for the first time a list of putative Ube3a substrates, whose ubiquitination is enhanced by Ube3a. Additionally, our findings corroborate previous reports performed in cells, indicating that Ube3a interacts with the proteasome and its degradative activity, which results in the accumulation of tens of ubiquitinated proteins of which many are most likely not direct targets of Ube3a. The ubiquitination of proteasomal subunits by UBE3A had been previously reported, but this Drosophila study is pioneer in reporting in vivo evidence of their ubiquitination in neuronal cells. Complementing previous observations, it appears that the ubiquitination of proteasomal subunits by Ube3a/UBE3A ([ manuscript under review) places this E3 ligase as a pivotal regulator of the proteasome and proteostasis. This finding opens a new perspective in which the ubiquitination of other proteins, and thus their levels or activity, can be affected as a downstream effect. The existing working model that UBE3A substrates are targeted for degradation does therefore need to be revised.Thea/UBE3A (, 166; RabioUb approach has been successfully applied for the MS analysis of the ubiquitin landscapes of the embryonic nervous system and Drosophila photoreceptor cells, but it has the potential to be implemented to any fly tissue at any stage during the development. The nature of the bioUb strategy allows also discerning by Western blot whether such identifications correspond to proteins that are mono- or polyubiquitinated in vivo. And most importantly, for the first time it is possible to obtain a list of candidate substrates for any Drosophila E3 ligase in vivo.The


Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension‐module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be “toggled” off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly‐line catalysis and may explain examples of apparent non‐colinearity in other modular PKS systems.Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove  Modular Type I polyketide synthases (PKSs) catalyze the biosynthesis of numerous pharmacologically relevant natural products that exhibit antibacterial, antifungal, anthelmintic, antitumor, or immunosuppressive activities.Streptomyces sp. 211726, which has been isolated from mangrove rhizosphere soil.Streptomyces malaysiensis DSM 4137,S. malaysiensis genome.Streptomyces sp. 211726 revealed a cluster (GenBank accession number: KY484834) that is highly similar, both in size and organization, to the azalomycin cluster in DSM 4137.The 36‐membered macrocyclic antifungal azalomycins (AZLs) are the main products of 1 domain in vivo . Liquid chromatography electrospray ionization high‐resolution mass spectrometry (LC‐ESI‐HRMS) analysis showed that the ΔDH1 mutant strain no longer produces known AZLs, and instead significant new peaks were found  and the presence of two additional O‐bound protons at δH 3.65 and δH 4.04  compared to the known azalomycins. In agreement with this, the DEPT analysis showed a lack of two olefinic carbon atoms at δC 128.9 and δC 131.0 Figure S6). Clearly, inactivation of the DH1 domain prevents dehydration in both the first and second extensions, thus providing direct evidence that in the AZL PKS, module 1 is used twice.In order to test the hypothesis that extension‐module 1 acts iteratively and supplies the missing full reduction module governing the second extension, a site‐specific mutation of the essential histidine residue to alanine was introduced into the active site of the DH Figure S. Liquid  Figure S. Liquid 1), which bears the same site‐directed mutation in the DH1 domain as above, was successfully expressed in soluble form in Escherichia coli BAP11 domain. In a one‐pot reaction with 4‐guanidinobutyric acid, purified Azl4 (ligase), Azl5 (acyltransferase), and AzlA(ΔDH1), the starter unit 4‐guanidinobutyryl‐CoA was produced and loaded onto the ACPL domain that comprises the LM in AzlA(ΔDH1). In order to achieve multiple turnovers of polyketide synthesis on the AzlA PKS multienzyme, our strategy1 domain. Recombinant holo‐ACP1 domain was expressed as a stand‐alone protein and purified from E. coli BAP1 and added to the incubations to compete with the integral ACP1 domain of module 1 . The polyketide‐chain‐bearing ACP1 reaction products were monitored by LC‐ESI‐HRMS. In the presence of holo‐ACP1 and malonyl‐CoA in the above one‐pot reaction, a peak with a mass corresponding to the expected product of two rounds of extension  was observed  and extension‐module 1. To further confirm the above result by in vitro analysis, the recombinant PKS AzlA, was used. Since module 1 contains a full set of reductive enzyme domains , and recruits malonyl‐CoA as extender unit, the 4‐guanidinobutyryl starter unit should be extended with two acetate units and both newly‐formed β‐keto groups should be fully reduced. However, LC‐ESI‐HRMS analysis of the one‐pot reaction showed that the molecular weight of the acyl‐ACP1 species produced was 2 Da less than expected for the product of two rounds with full reduction  should be addressed to the authors.SupplementaryClick here for additional data file.


Drosophila melanogaster Cdk5 regulates basal autophagy, a key mechanism suppressing neurodegeneration. In a targeted screen, Cdk5 genetically interacted with Acinus (Acn), a primarily nuclear protein, which promotes starvation-independent, basal autophagy. Loss of Cdk5, or its required cofactor p35, reduces S437-Acn phosphorylation, whereas Cdk5 gain-of-function increases pS437-Acn levels. The phospho-mimetic S437D mutation stabilizes Acn and promotes basal autophagy. In p35 mutants, basal autophagy and lifespan are reduced, but restored to near wild-type levels in the presence of stabilized AcnS437D. Expression of aggregation-prone polyQ-containing proteins or the Amyloid-β42 peptide, but not alpha-Synuclein, enhances Cdk5-dependent phosphorylation of S437-Acn. Our data indicate that Cdk5 is required to maintain the protective role of basal autophagy in the initial responses to a subset of neurodegenerative challenges.Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health. The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood. Here, we show that in  Cells have a problem that we recognize from our own homes: if nobody cleans up, garbage accumulates. Unwanted material in cells can include proteins that clump together and can no longer carry out their normal tasks. If left to build up, these protein aggregates can damage the cell and even kill it. Many neurodegenerative disorders, including Huntington’s disease and Alzheimer's disease, arise when such faulty proteins accumulate inside brain cells.437 that can be modified by the attachment of a phosphate group, in a process known as phosphorylation. However, the significance of this modification was not clear.Autophagy is a process that can destroy protein aggregates and other defective material to keep cells healthy. Understanding how cells regulate autophagy is thus of great interest to scientists. A protein called Acinus promotes autophagy and is found in many organisms including fruit flies and humans. All Acinus proteins share a common feature; they contain a site called SerineDrosophila melanogaster. Flies were engineered such that the normal Acinus protein was replaced with a mutant version that mimics the phosphorylation at Serine437. These mutant flies had higher levels of Acinus, showed more autophagy, and lived longer when compared to normal flies.Nandi et al. have now asked if this phosphorylation event is important for the role of Acinus in autophagy. The experiments were carried out in the fruit fly, 437. Making Cdk5 inactive using experimental tools led to lower levels of autophagy in brain cells and shortened the flies’ life span. Moreover, some aggregation-prone proteins linked to neurodegenerative diseases can enhance the activity of Cdk5 towards Acinus, thereby reducing their own accumulation through elevated autophagy.Further work identified a protein called Cdk5 as being responsible for attaching phosphate to Acinus at Serine437 stabilizes Acinus to boost autophagy. This may lead to new ways to adjust the levels of autophagy to benefit different organisms.Together these findings show that phosphorylation of Acinus by Cdk5 maintains healthy brain cells and improves life span by enhancing autophagy. The next step is to understand how phosphorylation at Serine Cdk5 shares strong homology with other members of the family of cyclin-dependent kinases (Cdks), but it is distinct in its modes of regulation and function . Unlike Autophagy, here short for macroautophagy, is a key cellular process for maintaining the health of neurons . CytoproDrosophila  as a novel regulator of basal autophagy in osophila . Mammaliosophila . In mamm complex . ASAP in complex  and part complex . Antagon complex  and genec manner .Drosophila , SCA3.Q78, and Amyloid-beta peptide 42 (Aβ42). These findings offer new insights into the complex mechanisms balancing the effects of loss and gain-of-function of Cdk5 on neurodegenerative diseases.Drosophila , we generated flies in which endogenous Acn was replaced by N-terminally Myc-tagged AcnAcnS437A . Expressc region . All genn levels . The preAcnS437D . From heWT larval eye discs with the phospho-specific pS437-Acn antibody revealed a dynamic expression pattern consistent with staining for the Myc epitope tag  reduced staining for pS437-Acn but not the Myc epitope . Previous work has shown that fat bodies from fed wild-type larvae contain predominately lysosomes smaller than 400 µm, while starved fat bodies contain lysosomes and autolysosomes larger than 1 µm e.g. . When weto AcnWT . The meaD larvae  and the D larvae .S437D flies. When challenged with starvation stress, AcnS437D flies survived significantly longer than AcnWT or AcnS437A flies . Other fly strains used were Δ45p38b, a null generated by transposon excision and removing most of the p38b coding region, UAS-p38b, UAS-p38bK53R  or Myc , guinea pig anti-Acn -P A R N R A S and double-affinity purified. For Western blots, five 96 hr larvae were crushed in 300 µl lysis buffer  at 95°C, boiled for 2 min, and spun for 10 min at 20,000x402-527 fusion proteins (WT or S437A mutant) were expressed in a 30-ml culture of BL21 bacteria and immobilized and purified on 10 µl glutathione sepharose beads using standard procedures  for 20 min at 30°C. Immobilized Acn proteins were washed twice with PBS containing 0.1% Triton-X100, eluted with 20 mM glutathione and analyzed by western blots using antibodies against Acinus  14–477) in 30 µl kinase assay buffer , and images were captured with an 11-megapixel camera . From TEMs, measurements of autolysosomal diameters and areas were obtained using Macnification software (Orbicule).For TEM, size-matched 96 hr fed larvae were dissected and processed as described earlier in Whole-mount tissues were prepared for immunofluorescence staining as previously described . BrieflyFor phosphatase treatment, dissected third instar larval carcasses were fixed in periodate-lysine-paraformaldehyde and treated with 130 U/ml Calf Intestinal Phosphatase  for 3 hr at 37°C with 1X protease inhibitor tablet (Roche) dissolved in 1X PBS, pH 7.5. Subsequently, tissues were processed and stained, and eye disc mounted as described above. For autophagy flux experiments, 72-hr-old larvae were transferred to fresh medium containing 3 mg/ml chloroquine (Sigma) as described .Drosophila media (Gibco), inverted to expose fat bodies, and incubated in 100 µM LysoTracker Red DND-99 for 1 min. Inverted carcasses were then washed in 1X PBS and fat bodies were mounted onto a droplet of Vectashield (Vector Laboratories). Samples were imaged immediately on an inverted confocal microscope . Z-projections of three optical sections of fat body tissue, each 1 µm apart were used to quantify LysoTracker and Atg8a punctae in fat bodies using Imaris software (Bitplane). The number of punctate was quantified per fat body cell. Digital images for display were imported into Photoshop (Adobe) and adjusted for gain, contrast, and gamma settings.LysoTracker staining  of size-matched 90–96 hr fat bodies from fed and starved larvae was performed as previously described . In brieWT. Integrated densities for pS437-Acinus and polyQ in eye discs were quantified using Image J software. Identical areas posterior to the morphogenetic furrow were quantified, thereby excluding dividing cells close to the furrow that were strongly stained for pS437-Acinus.Integrated intensities of Atg8a punctae in fat bodies were determined using Image J and normalized to AcnAll immunofluorescence experiments were repeated at least three times with at least three samples each.Polyglutamine aggregates were detected with a modified filter assay . BrieflyStatistical significance was determined in Prism using log-rank for survival assays, chi square analysis for eye roughness frequencies, and one-way analysis of variance for multiple comparisons, followed by Tukey’s test. To separate effects of treatment and genetic background we used two-way analysis of variance for multiple comparisons, followed by Bonferroni’s test for individual comparisons. All bar graphs resulting from these analysis show means ±SD. For quantifications of fluorescence images and Western blots, at least three independent experiments were used. P values smaller than 0.05 are considered significant, and values are indicated with one (<0.05), two (<0.01), three (<0.001), or four (<0.0001) asterisks. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.437" for consideration by eLife. Your article has been favorably evaluated by Ivan Dikic (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Stress-Induced Cdk5 Activity Enhances Cytoprotective Basal Autophagy by Phosphorylating Acinus at SerineThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.S437D promotes basal autophagy and rescues defects  of p35 mutants. You further demonstrate enhancement of the Cdk5/Acn-pS437 pathway in response to polyQ protein accumulation. Cdk5 was previously implicated in maintenance of neuronal health. This study identifies Acinus as a relevant target of Cdk5 activity as a compelling basis for this role. Overall, the findings are interesting and potentially important. The experiments are well executed and most conclusions are well supported by the data presented in this work. This manuscript will be suitable for publication in eLife after appropriate revisions.Autophagy is a critical mediator of neuronal cell health. Ongoing basal clearance of aggregated or damaged proteins is associated with protection from neurodegeneration in both flies and humans. Previous studies from the authors identified a role for the Acinus (Acn) protein in promoting basal autophagy. In this manuscript, you provide evidence that Cdk5 genetically interacts with Acn to regulate basal level of autophagy, and that Cdk5 phosphorylates Acn at S437 to modulate its stability. The phospho-mimetic AcnEssential revisions:1) In vitro kinase assays with purified proteins to show that Cdk5 kinase can directly phosphorylate Acinus.S437D larval fat bodies. The autolysosomes in AcnS437D larval fat bodies are much larger and more abundant compared to AcnWT. The mean diameter of lysosomal/autolysosome structures is even increased more than 8-fold! Inhibition of autophagy at the step of autophagosome maturation and/or degradation results in the same phenotype. Does starvation-induced autophagy also cause accumulation of enlarged autolysosomes in fat bodies? Additional characterization of autophagy flux would strengthen the interpretations of autophagy induction induced by Acn-pS437. This could be achieved with assays using the tandem fluorescent Atg8 reporter and/or Atg8-II levels in animals fed BafilomycinA.2) The current data does not sufficiently support the conclusion that autophagy activity is induced by S437-Acn. The authors showed that the number of Atg8a puncta and LysoTracker-positive structures is increased in Acn Essential revisions:1) in vitro kinase assays with purified proteins to show that Cdk5 kinase can directly phosphorylate Acinus.We performed two sets of experiments to address this concern.402-527 (WT or S437A) and exposed them to phosphorylation with commercially available Cdk5/p35 complexes . We observed Cdk5/p35-dependent S437-phosphorylation, that was not present in S437A controls  in S2 cells, immobilized and purified the proteins on Strep-Tactin beads before incubation with commercial Cdk5/p35. These experiments also showed Cdk5/p35-dependent S437-Acn phosphorylation new .In combination, we believe these experiments convincingly address the reviewers’ concerns to show that Cdk5 kinase can directly phosphorylate Acinus at S437.S437D larval fat bodies. The autolysosomes in AcnS437D larval fat bodies are much larger and more abundant compared to AcnWT. The mean diameter of lysosomal/autolysosome structures is even increased more than 8-fold! Inhibition of autophagy at the step of autophagosome maturation and/or degradation results in the same phenotype. Does starvation-induced autophagy also cause accumulation of enlarged autolysosomes in fat bodies?2) The current data does not sufficiently support the conclusion that autophagy activity is induced by S437-Acn. The authors showed that the number of Atg8a puncta and LysoTracker-positive structures is increased in AcnStarvation-induced autophagy in fat bodies has been extensively studied by multiple labs. Examples include:   . Neither of these papers quantified the changes in size from basal to starvations-induced autolysosomes. Importantly, however, all of these papers showed examples of electron micrographs from starved tissues containing large autolysosomes exceeding 1 µm or even 2 µm . When staining these chloroquine-treated fat bodies for endogenous ATG8, we observed significant increases in ATG8a punctae and ATG8a signal intensity for wild-type starved or for fed S437D phospho-mimetic mutants consistent with increased autophagic flux new .To keep this figure manageable, we have split the previous In addition, we like to point out that the beneficial effects endowed by the S437D phospho-mimetic mutation on starvation-resistance new , longevi


Chiral spin textures in ultrathin films, such as skyrmions or chiral domain walls, are believed to offer large performance advantages in the development of novel spintronics technologies. While in-plane magnetized films have been studied extensively as media for current- and field-driven domain wall dynamics with applications in memory or logic devices, the stabilization of chiral spin textures in in-plane magnetized films has remained rare. Here we report a phase of spin structures in an in-plane magnetized ultrathin film system where out-of-plane spin orientations within domain walls are stable. Moreover, while domain walls in in-plane films are generally expected to be non-chiral, we show that right-handed spin rotations are strongly favoured in this system, due to the presence of the interfacial Dzyaloshinskii–Moriya interaction. These results constitute a platform to explore unconventional spin dynamics and topological phenomena that may enable high-performance in-plane spin-orbitronics devices. Chiral domain walls in magnetic films can be electrically controlled, which makes them attractive for applications, but domain walls in ultrathin films are normally non-chiral. Here, the authors observe chiral domain walls in ultrathin Fe/Ni bilayers that are stabilized by the magnetic anisotropy. Spin textures in magnetic materials are determined by the balance of contributions to the magnetic free energy. In-plane magnetized films are a textbook case31561Si and Sj are two atomic spins on neighbouring atomic sites i and j, and Dij is the DMI vector. For in-plane Néel walls the cross-product Si × Sj points out-of-plane, and Dij is a vector that lies within the planeRecently, homochiral spin textures have been observed in the presence of the interfacial Dzyaloshinskii–Moriya interaction (DMI)10111213141516In atomically thin films, the interplay between strong interfacial DMI and exchange interaction can lead to homogeneous14In the following, we show how the thickness-dependent Néel-to-Bloch wall transition can be reversed by deliberately adjusting magnetic anisotropies, that is, stabilizing out-of-plane domain walls in ultrathin films. Moreover, we show that out-of-plane domain walls stabilized in this way are indeed right-handed chiral walls. Modelling the domain walls in Monte-Carlo simulations, we show that their stability can be understood by taking into account a uniaxial anisotropy contribution, a perpendicular anisotropy contribution as well as the contribution of the interfacial DMI.Spin-polarized low-energy electron microscopy (SPLEEM)191A much more unusual result emerges when the thickness of the Fe layer is slightly less, 3.3 ML as sketched in 1Ku, stabilizing the magnetization direction of the two domains, is along the W[001] direction; so the spins within this conventional Néel wall point into a hard-axis direction along W[1–10]. However, in most thin film systems the energy penalty associated with rotating spins into a hard axis is smaller than the dipolar energy penalty associated with rotating spins into an out-of-plane alignment. One contribution that can counter this dipolar energy penalty is perpendicular magnetic anisotropy, which is indeed present in Fe layers grown in this orientation21Now we discuss how the stability of this unusual domain wall spin texture can be understood. In the Fe/Ni/W(110) system, additional contributions to the magnetic free energy might play a role to stabilize the domain wall structure. These include perpendicular magnetic anisotropy of the Fe layer2121dFe=2.4 ML. With additional Fe, the magnetization gradually cants back towards in-plane orientation, until the double-spin reorientation transition is complete at dFe=3.1 ML. In this case of 15 ML Ni/W(110), the range of Fe film thickness dFe resulting in stable out-of-plane domain walls is estimated to be between 3.1 ML and 4 ML Fe (The strength of this perpendicular anisotropy can be demonstrated by measuring the magnetization direction in Fe/Ni/W(110) as a function of the Fe overlayer thickness . In the  4 ML Fe . SimilarIn this picture of the Fe/Ni/W(110) system, the dipolar penalty for out-of-plane spin alignment is largely balanced by the perpendicular anisotropy of the Fe layer and, as a result, out-of-plane domain wall spin texture may be energetically favoured over conventional Néel wall texture.Ku and the total effective anisotropy Keff. Results, summarized in Ku is negligible then, as a function of increasing Keff, there is a spin reorientation transition where the in-plane easy magnetization axis  films similar to the sample of Si × Sj has finite components within the plane, leading to a finite magnitude of EDM.Previously, effects of the interfacial DMI on in-plane magnetized systems have not been considered to be importantα between the W[001] direction and the magnetization direction at points within domain wall, and the angle φ between the W[001] direction and the domain wall normal vector n. Cycloidal spin spirals  and helical spin spirals  are then associated with the values φ=0 and φ=90°, respectively, as sketched in The spin structure in the domain wall surrounding a circular bubble domain is a convenient shorthand to describe the chiral magnetic properties of this system. α and φ were measured in a large ensemble of domain walls. The results are summarized in the histogram plotted in α–φ space. Brighter/darker colour indicates higher/lower count for α/φ combinations. A horizontal bright streak at α=−90° spans the entire range of 0°<φ<90°, indicating the prevalence of right-handed domain wall spin textures with significant cycloidal character. Left-handed textures (α=90°) are more rare and are essentially limited to a narrow band near φ=90°, where the spin spiral has significant helical character. For φ=90°, where the spin spiral is pure helical type, α=+90° and −90° have comparable probability: this indicates that these domain walls are non-chiral. This difference of the chirality along different substrate directions is related to the orientation of the DMI vector Dij. Interfacial DMI vectors are generally oriented perpendicular to the atomic position vectors rij=ri−rj  substrate: considering that the shape-induced uniaxial anisotropy provides a hard axis in the direction across the nanowireJ) for the effective anisotropy Keff/J=−0.01, nanowire shape-induced uniaxial anisotropy Ku/J=0.476, and strength of the DMI Dij/J=0.1, we find that right-handed chiral out-of-plane domain walls have the lowest energy in this nanowire. In the presence of the giant spin Hall effect of tungsten171822242623252627The Slonczewski-like torque that drives fast chiral domain wall motion in perpendicularly magnetized spin-orbitronics systems232526In summary, using micromagnetic imaging experiments we found an unusual type of chiral out-of-plane domain wall spin textures in an in-plane magnetized ultrathin film system. Supported by Monte-Carlo simulation, the interplay between magnetic anisotropies is understood to be the driving force stabilizing the out-of-plane wall textures. The magnetic chirality of these domain walls is stabilized along in-plane orientations and is understood to result from the interfacial DMI. Current-31323334353637394041424345464750−8 Torr O2, and final annealing at the same temperature under ultrahigh vacuum. Fe and Ni layers were deposited at room temperature by physical vapour deposition from electron beam evaporators, and the Fe film thickness dFe and Ni film thickness dNi was controlled by monitoring the image intensity oscillations associated with atomic layer-by-layer growth. The sample was annealed to 630 °C for several minutes after growth of the first 1 ML Ni to develop the well-ordered c-(1 × 7) structure as a template for subsequent growth of Ni film and Fe film in fcc(111) epitaxyThe W(110) substrate was cleaned by flash heating to 1,950 °C in 3 × 10A is proportional to the strength of the component of the magnetization M along the selected axis. The Cartesian components Mx, My and Mz of the magnetization were resolved by recording triplets of images with the electron beam spin polarization aligned along the x, y and z directions, respectivelyα–φ histogram in φ angle was separated into bins of 5°. In each bin, the average value of the magnetic chirality was estimated using the method shown in Magnetic images were measured using the SPLEEM instrument at the National Center for Electron Microscopy at Lawrence Berkeley National Laboratory172022The Monte-Carlo simulations were carried out based on a two-dimensional model described in refs The total energy term can be written asSi and Sj are spin moments located on atomic sites i and j in a two-dimensional plane, ri and rj are the position vectors of the spin blocks in sites i and j. J, DDip, Kz, Ku and Dij correspond to exchange interaction, dipole interaction, perpendicular anisotropy, uniaxial in-plane anisotropy and DMI, respectively. The effective anisotropy Keff is defined as J, DDip, Kz, Ku and Dij are used for simulating domain wall spin structures. For the simulation results summarized in J=1, DDip/J=0.1, Dij/J=0.1 were assumed, and J=1, DDip/J=0.1, Keff/J=−0.01, Ku/J=0.05 and Dij/J=0.1 were used. As described in ref. 54DDip is not considered, and Ku/J=0.476 is deduced based on the demagnetization factor of the nanowire. Initial temperature in the simulation is chosen to be lower than the Curie temperature to avoid destroying the initial setting. After annealing these spin block arrays, domain walls relax to a finite thickness and the lowest energy state is determined by comparing the energies of the three states. φ-dependent chirality in where nd  ref. . For theDij is perpendicular to the distance vector Rij=Ri−Rj between sites i and j and, as a result of the bcc(110) interface, the DMI is anisotropicDDip can be estimated as μ0=4π × 10−7 H m−1, μB=9.274 × 10−24 A m2, μNi=0.6μB, dNi=15 ML, μFe=2.6μB, dFe=3.3 ML, a||=2.49 Å. We find Dij=DDip, which provides an experimental estimate of the strength of the DMI in this system of ∼0.53 meV per atom.At W(110) interfaces, the DMI vector entation  when DijThe data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Chen, G. et al. Out-of-plane chiral domain wall spin-structures in ultrathin in-plane magnets. Nat. Commun.8, 15302 doi: 10.1038/ncomms15302 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures


Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins. PTPN4 is a non-receptor tyrosine phosphatase whose multiple functions have been linked to T-cell signalling, learning, spatial memory and cerebellar synaptic plasticity4. Its overexpression reduces cell proliferation in COS-7 (CV-1 (simian) in Origin and carrying the SV40 genetic material) cells and suppresses CrkI-mediated cell growth and mobility in HEK293T (Human embryonic kidney) cells6. Recently, PTPN4 has been identified as a specific inhibitor of the TIR-domain-containing adapter-inducing interferon-β-dependent toll-like receptor 4 pathway7.Protein-tyrosine phosphatases (PTPs) represent the largest family of human phosphatases. These enzymes are critical in regulating signal transduction and their impairment has been previously associated with human diseases8. PTPN4 is proteolysed in the cell by calpain in response to physiological stimuli, leading to enzyme activation9. In vitro, PTPN4 is cleaved and activated by both trypsin and calpain9. The active form of PTPN4 consists of the PDZ and PTP domains  is a large modular protein containing a N-terminal FERM  domain, a PDZ (PSD-95/Dlg/ZO-1) domain and a C-terminal catalytic tyrosine phosphatase domain  p38γ  as a cellular partner of PTPN4. The PBM of p38γ has the highest affinity of all PBM from endogenous partners of PTPN4 and has a similar affinity to the optimized pro-death 13-amino acids peptide Cyto8-RETEV. Both peptides are efficient inducers of cell death after their intracellular delivery12. Furthermore, we showed that the PDZ domain of PTPN4 inhibits the phosphatase activity, while a PBM bound to the PDZ domain abrogates the auto-inhibition of the catalytic activity of PTPN413.We previously showed that PTPN4 prevents the induction of cell death in glioblastoma cell lines in a PDZ-PDZ ligand  dependent manner13. Of particular interest is the observation that, in order to communicate, the PDZ and PTP domains must be covalently linked as shown by the absence of inhibition when PTPN4-PDZ is added in trans to the linker-PTP construct13. These observations suggest that the linker has a crucial functional role in the communication between the two domains. Yet, the inter-domain linker of PTPN4 is flexible and unstructured, and does not display any direct detectable interactions by NMR with either the PDZ or the PTP domains13.While it is clear that the PDZ domain regulates the activity of PTPN4, the mechanism of crosstalk between the PDZ and PTP domains remains poorly understood and appears to be rather complex. In particular, previously published NMR (nuclear magnetic resonance) data indicated that the transition from the auto-inhibited state to the active state of PTPN4 is mediated by a modification of the global dynamic behaviour of the PDZ domain when going from the unbound to the bound stateIn this study, we investigate the role of the disordered linker connecting the PDZ and PTP domains in the active PDZ-PTP bidomain construct of PTPN4 Fig. . To that14, MLRC15, PHD16 – and the consensus secondary structure prediction. Common features to all results are that a helix is predicted downstream of the PDZ domain (residues 605–614) and that the linker after residue 615 does not adopt any regular secondary structure. We have previously reported the resonance assignments of the linker (residue 605 to 637) for the PDZ-linker and the bidomain constructs  below the significant threshold  33) Fig. . Thus, iTo complement our results, we characterized PTPN4 PDZ-PTP by limited proteolysis with trypsin, chymotrypsin, and the non-specific subtilisin and papain proteases. Indeed, we identified three high specificity chymotrypsin cleavage sites and two trypsin cleavage sites in the linker from sequence analysis Fig. . The PDZC/S  and of the PDZ-linker constructs both in the absence of PBM (unliganded) and with a PBM bound (liganded) to the PDZ domain. We reported that the transverse relaxation R2 and the longitudinal relaxation R1 values showed that the PDZ domain in the PDZ-PTP construct reorients more freely in solution when liganded with dynamic characteristics of the isolated PDZ domain13. We also collected dynamical parameters of the linker. R2 values are shown in Fig. 2 of 6.5 s−1) is more flexible in the liganded bidomain PDZ-PTPC/S than in the unliganded bidomain form (R2 of 13.0 s−1), with R2 values that tend toward the values observed in the PBM-liganded PDZ-linker isolated domain (R2 of 5.1 s−1). However, the linker flexibility in the liganded PDZ-PTP construct does not reach that of the isolated domain. Although we did not identify a stable surface of interaction between PDZ and PTP domains by NMR, from this dynamic behavior, we conclude that the transient intramolecular interactions that restrain the PDZ and the linker mobility in unliganded PDZ-PTP are destabilized in the PBM-liganded PDZ-PTP.We previously collected dynamical parameters in solution of the bidomain PDZ-PTP19 that allows us to broaden the selection to homologous sequences and avoid paralogous sequences. We used a more divergent alignment with all proteins that have a PDZ-linker-PTP organization Pediculus humanus) which presents the shortest linker (only 21 residues vs 35 residues in humans), this stretch becomes very short (two residues)  in the region predicted helical by secondary structure predictions programs  to that of the F620G, Q621G, Y622G and I623G and ΔFQYI variants. First, we ensured that we obtained similar kcat and KM values for the linker-PTP that includes the inter-domain linker and the phosphatase domain, and the PTP construct that contains only the PTP domain .WT and linker-PTP  and the turnover number (kcat) values obtained by fitting the experimental data are listed in Table cat and KM values decrease when comparing linker-PTP with PDZ-PTPWT as previously reported13. KM values of Y622G and I623G mutants are similar to the one of the PDZ-PTPWT while those of F620G and Q621G are significantly higher by 1.7 fold, with intermediate values between those of PDZ-PTPWT and linker-PTP  of ΔFQYI is thus strongly increased.Kinetics experiments of PTPN4 bidomain constructs were first performed in the absence of PBM corresponding to the inhibited state of the phosphatase to determine catalytic parameters of the PTPN4 mutants. We showed that PDZ-PTP mutants dephosphorylate the pNPP substrate following Michaelis-Menten kinetics as previously reported for the PDZ-PTPPTP Fig. . In all 12 peptide were performed to determine catalytic parameters of the bidomain mutants in their “activated” state  indicating that PBM abrogates inhibition for all mutants, but those values are lower than that of PBM-liganded PDZ-PTPWT except for the PBM-liganded ΔFQYI mutant that presents a kcat 2 fold higher. Thus, all mutants are affected in the PBM-mediated release of inhibition . Interestingly, KM of F620G and Q621G are unchanged upon the addition of Cyto8-RETEV whereas KM of Y622G and I623G increase  as observed with the KM of the wild-type  that are not strongly changed in the presence or absence of the effector peptide.Both k13 Table . All kcaion Fig. . Howeverype Fig. . As prevHence, all PDZ-PTP mutants are affected in the PBM-mediated regulation of PTPN4 activity either in the modulation of the affinity for the PTP substrate or in the catalytic activity of the enzyme. This modulation of the PDZ-mediated inhibition and PBM-mediated release of inhibition of the PTPN4 bidomain suggests that the linker has a function in the inter-domain PDZ-PTP communication driving the regulation of the activity of the phosphatase PTPN4.13. Remarkably, the linker region connecting the PDZ and PTP domains is required for such a crosstalk, as the regulation is abolished when the two domains are not covalently linked. However, the 33-residue linker is flexible and unstructured, and no detectable interaction could be observed by NMR with either the PDZ or the PTP domains. These findings prompted us to perform additional investigations, in order to characterize more directly the role of the linker in the regulation of PTPN4.Previous structural investigation of PTPN4 revealed that this enzyme is regulated by a mechanism of auto-inhibition which is, at least in part, mediated by an intramolecular communication between the PDZ and PTP domains13, it is not fully solvent accessible, displaying resistance to proteolysis in both PDZ-PTP and linker-PTP constructs and that NMR dynamics parameters (R1 and R2) do not reach the values of the truncated PDZ-linker construct. This suggest a potential interaction between the linker and the PTP domain. We previously proposed that the transient intramolecular interactions which constrain the PDZ and the linker mobility in unliganded PTPN4 are destabilized in the presence of the PBM. Based on multiple sequence alignment, we identified a conserved hydrophobic region in the linker that may be involved in the regulation of PTPN4, being universally conserved in PTPN4 from different species.It is interesting to observe that, whilst the linker is largely disorderedcat and/or KM. Both the inhibition of the phosphatase activity by the PDZ domain and the abrogation of the inhibition by the PBM binding are affected, indicating that the PDZ-PBM regulation of PTPN4 is a linker-mediated mechanism.We showed that mutations in this hydrophobic patch –FQYI–, highly conserved among the PTPN4 orthologous sequences, affects the phosphatase kinetics with effects on kM and kcat of the enzyme with opposite effects. The PBM binding to the PDZ domain abolishes this auto-inhibition and restores the catalytic properties of PTPN4, most probably affecting the active site via a distal interaction occurring through a protein interface. So, the PDZ domain of PTPN4 is not only directly involved in discriminating its PBM-containing targets but also allosterically regulates the enzymatic activity of the phosphatase through the PDZ-PTP linker. Thus, both the linker sequence and PDZ domain are required for the regulation of the enzymatic activity of PTPN4  according to the manufacturer’s instructions, and the insertion was confirmed by DNA sequencing. Template DNA used for the PTP construct was a synthetic construct comprising the PTP domain cloned in a pgex6p1 expression plasmid. Expression and purification of linker-PTP and PTP construct were performed as described previously13. Bidomain PDZ-PTP variants were expressed and purified as previously described without TEV cleavage. Briefly, clarified supernatants were loaded on Ni2+ column , washed and eluted with an imidazole gradient from 40 mM to 1 M. The eluted fractions containing the protein were pooled and loaded on a gel filtration column . Proteins were concentrated using centrifugal filter devices . Protein concentration was estimated from its absorbance at 280 nm. The peptide Cyto8-RETEV (SAESHKSGRETEV) was synthesized in solid phase using a Fmoc strategy (JPT).The variants of PTPN4 named F620G, Q621G, Y52G, I623G and ΔFQYI were obtained by using the Quik-Change Mutagenesis kit (Stratagene) according to the manufacturer’s instructions, and the substitutions were confirmed by DNA sequencing. Template DNA used for PTPN4 mutants was the PDZ-PTPWT and subtilisin:PDZ-PTPWT, and 1:100 for trypsin:PDZ-PTPWT and chymotrypsin:PDZ-PTPWT at 37 °C for 0, 10, 60 min and overnight. Reaction mixtures at each time interval were stopped by mixing with SDS loading buffer and heating at 95 °C for 5 min. Protein fragments were resolved by SDS-PAGE through a 12% polyacrylamide gel and visualized by Coomassie staining. The bands corresponding of the major proteolysis products were then identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).Limited proteolysis was carried out in 50 mM Tris pH 7.5, 150 mM NaCl and 0.5 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) using a ratio (w:w) of 1:1000 for papain:PDZ-PTP19 to retrieve one single homolog per species assessed as probable ortholog through a reciprocal blast search procedure against the non-redundant database. Retrieved full-length sequences were re-aligned using MAFFT23 and displayed using Jalview24.Sequence of PTPN4 (accession number NP_002821.1) was used as query on the InterEvolAlign serverKM and kcat constants were deduced from fitting the Michaelis–Menten equation with the prism software. The equation takes into account excess substrate inhibition.Kinetics experiments were carried out on a single-mixing SX-18 stopped-flow instrument (Applied Photophysics). Absorbances values were measured continuously at 410 nm at 25 °C. Experiments were performed by mixing a constant concentration of each of the PTPN4 variants, 0.6 μM in the presence of increasing concentrations of Cyto8-RETEV peptide, ranging between 0 μM and 100 μM versus increasing concentrations of pNPP ranging from 19 μM to 10 mM. The buffer was 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 150 mM NaCl, 0.5 mM TCEP. Initial linear reaction rates were calculated during a 20 s reaction. The


Empirical research has shown performance improvement of many different technological domains occurs exponentially but with widely varying improvement rates. What causes some technologies to improve faster than others do? Previous quantitative modeling research has identified artifact interactions, where a design change in one component influences others, as an important determinant of improvement rates. The models predict that improvement rate for a domain is proportional to the inverse of the domain’s interaction parameter. However, no empirical research has previously studied and tested the dependence of improvement rates on artifact interactions. A challenge to testing the dependence is that any method for measuring interactions has to be applicable to a wide variety of technologies. Here we propose a novel patent-based method that is both technology domain-agnostic and less costly than alternative methods. We use textual content from patent sets in 27 domains to find the influence of interactions on improvement rates. Qualitative analysis identified six specific keywords that signal artifact interactions. Patent sets from each domain were then examined to determine the total count of these 6 keywords in each domain, giving an estimate of artifact interactions in each domain. It is found that improvement rates are positively correlated with the inverse of the total count of keywords with Pearson correlation coefficient of +0.56 with a p-value of 0.002. The results agree with model predictions, and provide, for the first time, empirical evidence that artifact interactions have a retarding effect on improvement rates of technological domains. J) vary widely ranging from 3 to 65 percent , McNerney et al.’s treatment [A quantitative model of the effect of such interactions on technological improvement was developed first for simple artifacts and changes in cost . For a sreatment  for unitJ is the performance of a domain J and KJ its performance improvement rate . AJ, and dJ are respectively a set of scaling and interaction parameters specific to a domain. K is domain independent, and represents the rate of growth in the number of individual operating ideas (IOI) generated through a combinatorial analogical transfer process encompassing all of science and technology. These IOI are then assimilated into the components of domain artifacts, where the influence of interaction and scaling parameters manifest. The equation predicts that KJ (improvement rate) is proportional to the inverse of the interaction parameter dJ, stating that domains with a higher average number of interactions (dJ) will be retarded leading to slower improvement rate. However, is there evidence that demonstrates artifact interactions retard improvement rates of technological domains? The goal of the study described in this paper is to investigate artifact interactions empirically and to test whether it has a retarding effect on improvement rates of technological domains. However, what are artifact interactions in a physical sense, and in what forms, do they manifest in artifacts?Considering a broader framework, Basnet and Magee  and BasnIn design of artifacts, Simon  introducUsing an in-depth qualitative analysis of two technologies, VLSI  systems and complex electro-mechanical-optical (CEMO) systems, Whitney , 16, howTwo potentially applicable approaches exist for studying artifact interactions. One method that is well recognized is the design structure matrix (DSM) , which wAnother similar technique to represent interactions is networks with nodes and edges. Complex networks allow modeling and characterizing nonlinear behaviors underlying complex systems; for some novel and efficient complex network analysis theories, see –21. For Another approach is to use documents: One set of documents potentially relevant for studying interactions are design manuals and engineering books related to specific domains, which could be analyzed using text mining techniques. Hommes and Whitney  have useAnother set of promising documents are patents. Patents are particularly attractive; as a data source, they are generalizable, objective, and publically available. They provide meta-data, and qualitative data (drawings and text). The textual data describes state-of-art prior to the inventions, and associated problems that were solved. Second, the data is available for many generations of inventions, and easily accessible from USPTO or other websites such as Google.com. Additionally, the Classification Overlap Method (COM) , 24, 25,The patent analysis using a text mining approach was conducted in two phases. In a pilot study, using patents from 5 domains—battery, wind power, solar PV, capacitors and computer tomography scanning (CT scan), feasibility for extracting data about artifact interactions from patent text was explored using Latent Semantic Analysis, Latent Dirichlet Analysis, and keyword-based techniques. Only the keyword-based technique was found to be useful in extracting data on interactions, and hence will be discussed further. In the extended study, the keyword-based technique developed in the pilot study was implemented in 27 domains.Both the pilot and extended study consist of four broad steps: (1) preparation of textual data from patents (2) identification of interactions and associated keywords (3) keyword-based text mining (4) analysis and interpretation of variations in keywords across domains. In step 1, domain-specific patents were identified, electronically retrieved from the web, and cleaned to prepare text for analysis. In step 2, patents were read in detail to identify potential generic keywords associated with interactions. In step 3 the raw data on interactions was extracted using keyword-based text mining, which was then analyzed in step 4. Subsequent sections describe these four steps. Since step 2 is the most critical among these, we describe this step first.Based on the qualitative work of Simon , Suh 1212, WeberBetween functional requirements: These interactions are consequences of the dependencies between multiple functions and design parameters . For exaBetween component and component, or between component and system: A good example of this type of interaction is the necessity to match impedance between sub-systems in order to maximize power transfer , 16.Parasitic/side effects: These represent undesirable effects exhibited by the components and sub-systems, while they fulfill their main functions , 16. Somtitle, abstract, background, and summary sections, and not include the detailed description and claims sections to maximize signal to noise ratio.Using the above framework, as part of step 2 in the pilot study, two researchers, including the current lead author and an Intern working with the author for several months, carefully read a set of 60 patents from the 5 domains noted earlier  to identify text describing technical issues that reflect interactions of the types discussed above. Three patents from each decade starting from the 1970’s until the present were selected to make a total of 12 patents in each of the 5 domains. It was observed that background or prior art sections, as expected, described problems with the state-of-art artifacts. It was also found that many patents, while summarizing the current invention, also discussed problems that were not previously discussed in the background or prior art section. In both of these sections, descriptions of problems consistent with the prior research , 15, 16 italicized) indicating interactions with associated keywords in bold are presented below in The essential part of step 2 was to extract text samples that contained the description of the interactions. These text samples were examined for keywords that tended to appear as reliable signals of interactions but that were not domain specific. Two examples of such text  count of occurrence, (2) cross-domain usage of the keywords across the domains, and (3) relevancy of keywords in reflecting interaction. First, high occurrence of a keyword was necessary to get a statistically strong signal capable of showing variation across the domains. For example, the words ‘parasitic, problem, prevent, undesirable, requirement, failure, disadvantage, and overcome. The root of each keyword searched is shown as bold and italicized text. The root is chosen so that the searched keyword is able to identify the maximum number of instances of the keyword, but without reducing the relevancy percentage. These 8 keywords had high count and are relatively general.Data from the pilot study of 430 patents showed that majority of the 30 keywords words had low count, and hence were removed from the list. Additionally, using the cross-domain usage criteria, the remaining keywords were reduced to 8 keywords: parasitic (97%), problem (58%), prevent (83%), undesirable (94%), requirement (75%), failure (72%), disadvantage (81%), and overcome (98%). The relevancy percentage for each individual domain is presented in problem’ to describe design opportunities to improve main functions of a technology, or to take advantage of new applications. This convention is reflected in the patent text as exemplified by the example in problem’ was removed from the list.Relevancy percentages for the keywords were determined from reading of the 60 patents (described above) for identifying interactions and associated keywords. Mean relevancy percentages for these 8 keywords across the 5 domains are listed here inside the parentheses: The remaining 7 keywords were further vetted in the extended study with patents from 27 domains. The raw data from keywords showed that the keyword ‘parasitic’ did not have wide cross-domain usage. In fact, 12 domains did not use it even once, and only 5 domains—Camera Sensitivity, Capacitor, Electric Power Transmission, Fuel Cell, IC chips—used it often. the, a, it, and in. Although they are critical in natural language, they do not add any value to the data. Removing them makes it possible to focus on the important words, and to reduce computational cost as well as noise in the data. The completion of this step prepared the text for mining.The outcome of this step was the raw text from the 100 most-cited patents for each of the domains being studied. (Please see Out of the 2700 patents used for extended study (sub-step 2 above), 2400 patents were downloaded using a web-scraping tool from the Google patent database, which provides patents in searchable html files. Out of the remaining patents, we successfully downloaded 276 manually, thus resulting in 97 to 100 patents in each domain. Almost all of these manually downloaded patents either lacked proper titles describing the sections, or background and summary were merged with detailed descriptions. For these cases, the background information and summary had to be identified by reading the patents and then manually extracted.The prepared patent text for the 27 domains was mined using Python scripts for determining the count of the keywords as well as the total number of words in the patent set in each domain. The total number of words in the patent set was used for normalizing the count of keywords in each domain, and the normalized keyword count is expressed per 100 thousand words in the patent set.For analysis, we assumed a linear relationship between normalized count of keywords and artifact interactions described by the models , 9, 10. The total count of words  in patent text varies widely between the individual patents, with the ratio of total word count between the patent with the highest word count to the patent with the lowest count was more than 100. However, when the total word count of domain-specific patent sets is compared, the distribution of total word count is much narrower, shown in The count of the 6 keywords is summed, normalized with respect to total word count in the respective patent set, and finally expressed in terms of keywords per 100,000 words. dJ. The normalized keyword count is assumed to provide a relative estimate of artifact interactions encountered in generating inventions in the various domains: we assume a linear relationship between the normalized keyword count (KWJ) and the degree of interactions characteristic of a domain (dJ). With this assumption, the prediction based upon According to the model , 9, 10, Relationship 3 is expressed as a hypothesis as follows:The performance improvement rate of technological domains are positively correlated with the inverse of the normalized count of interaction keywords in a set of patents belonging to each of the domains.Hypothesis: J) positively correlate with higher values of the inverse of normalized count of the 6-keywords (KWJ).The hypothesis was tested empirically using the 6-keyword results. The improvement rate in a domain is plotted as a function of the inverse of normalized count of the 6-keywords in the same domain in The correlation coefficients using two widely used methods, Pearson (parametric method) and Spearman Rank Order (non-parametric method), have been calculated. Assuming a normality of the population of K values and of 1/normalized count of KW, the Pearson correlation coefficient calculated using EXCEL2013 is +0.56 with a p-value of 0.002. The Spearman’s Rank Order coefficient is 0.632 with p-value slightly less than 0.001. Note that the Spearman’s correlational method does not assume any distribution. Both p-values are much smaller than 0.05, a threshold value employed by many researchers; the value of 0.002 and 0.001 indicate a very strong likelihood that the correlation is not due to random effects. This result supports the theoretical prediction that domains associated with higher degree of interactions improve at a slower pace. How reliable is this correlation? We further examine the reliability of this finding using a robustness study using Pearson’s correlation coefficients.The robustness study was conducted by creating 20 groups of 14 domains , where each group was generated by randomly selecting a combination of 14 domains from the 27 domains. For each group of 14 domains, the Pearson’s correlation coefficient between K and 1/KW was calculated.The results of those 14 groups are presented in The goal of this study was to investigate artifact interactions empirically, and to test the theoretical prediction in The first methodological finding of the empirical study is that patents can be a useful resource for studying artifact interactions, and to our knowledge, this is the first time patents have been used to study interactions. This finding potentially opens up the possibility of using patents as a new generally available data source for studying artifact interactions and other similar attributes in artifacts, including those designed many decades in the past.prevent, undesirable, requirement, failure, disadvantage and overcome—signal different types of interactions: component-to-component, component-to-system, side effects, and coupling of functional requirements. The keywords have been selected based on three criteria: (1) that they have at least a moderate level of occurrence ; (2) that they are widely used across domains hence requiring them to be non-domain specific; (3) that they signal interaction most of the time in the text in which they are used. In order to satisfy criterion 2, keywords are chosen such that they describe interactions at a higher abstraction level. For an example, usage of the keyword ‘prevent’ implies prevention of some problem, but without specifying what the problem is. The further specification (such as a word ‘corrosion’) would potentially make it domain specific and not useful in the kind of cross-domain test performed here. Importantly, criterion 3 requires that the keyword is relevant, that is, the text marked by the selected keywords needs to signal interactions most of the time (true positives). The difficulty arises when the keyword used may have two (or more) meanings, such that the text in which it is used signals interaction in one case, but not in the other. The keyword ‘problem’ discussed earlier  on improvement rates across the 27 domains is as indicated by the The analysis showed that improvement rates and the inverse of normalized keyword counts are positively correlated with the Pearson coefficient (r) equal to +0.56 with p-value equal to 0.002, and Spearman rank order coefficient of 0.632 with p-value 0.001. The correlation is medium, and does not explain all the variation in the rates. This can be understood in the context of J, another very likely source is due to limited resolution of the keywords as an estimate of interactions. An additional possible source of noise might be due to the limited number of patents (100 most-cited patents) being used for the study (due to limited resolution of Classification Overlap Method (COM) in its current state . This int state . An openOverall, the correlation analysis from this empirical study strongly supports the theoretical prediction that the domain interaction parameter is a factor that can lead to variation in improvement rates, where a higher interaction parameter leads to lower improvement rates. Further, it also supports the relational form the model predicts.S1 TableThe search terms are presented in column 2 between quotes. Asterisk (*) represents a wild card character used in Python code for searching multiple characters in text strings.(DOCX)Click here for additional data file.S2 Tableprevent, undesirable, requirement, fail, disadvantage, overcome) followed by cumulative count of 6 keyword for each domain; columns 11 lists total words in each domain, then followed by normalized count of 6 keywords, and performance improvement rate (KJ).Columns 1 and 2 identify the domains; column 3 lists number of patents used in domains; columns 4–9 list count of 6 keywords ((DOCX)Click here for additional data file.S3 TableThe relevancy is the ratio of frequency of the keyword actually signaling interactions to frequency of keyword. The character ‘/’ in the table indicates that the specific keyword was not found in the text studied.(DOCX)Click here for additional data file.S4 TableS4 Table lists technological domains and their associated patents which were studied. For each technological domain, column 3 provides the range of patent identification numbers associated with the respective domain. The (XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 FigThe count of words includes count keywords and all other words in the text  from approximately 100 most-cited patents in each domain. This, however, does not include the count of stop words  removed from the text. (DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.