A detailed understanding of herd types is needed for animal disease control and surveillance activities, to inform epidemiological study design and interpretation, and to guide effective policy decision-making. In this paper, we present a new approach to classify herd types in livestock systems by combining expert knowledge and a machine-learning algorithm called self-organising-maps (SOMs). This approach is applied to the cattle sector in Ireland, where a detailed understanding of herd types can assist with on-going discussions on control and surveillance for endemic cattle diseases. To our knowledge, this is the first time that the SOM algorithm has been used to differentiate livestock systems. In compliance with European Union (EU) requirements, relevant data in the Irish livestock register includes the birth, movements and disposal of each individual bovine, and also the sex and breed of each bovine and its dam. In total, 17 herd types were identified in Ireland using 9 variables. We provide a data-driven classification tree using decisions derived from the Irish livestock registration data. Because of the visual capabilities of the SOM algorithm, the interpretation of results is relatively straightforward and we believe our approach, with adaptation, can be used to classify herd type in any other livestock system. A herd type can be considered as a population of individual farms that have broadly similar enterprise and management patterns6. A detailed understanding of herd types is needed for animal disease control and surveillance activities, to inform epidemiological study design and interpretation and to guide effective policy- and decision-making.Herd production type can influence the behaviour of infectious diseases in livestock populations. Depending on the nature of the pathogen under study, the risk factors for introduction and spread, the route(s) of exposure, and the speed of spread can each vary considerably between different herd types  are by far the most frequently applied techniques (e.g.10). The main objective of the PCA approach is to reduce a usually large number of input variables into a set of synthetic variables , which are then used to form clusters. The use of this approach, and an understanding of its results, may require a level of technical and mathematical understanding which is not matched by all the participants, many of whom may have been chosen primarily for their knowledge in livestock husbandry. Logically, it is helpful to use a method which combines the advantages of both approaches, including expert participation and the formal identification of important data dimensions, as well as outputs that are understandable by all participants.A number of different methods have been used to classify herd types, including expert-based and statistical approaches13). The algorithm was first proposed by14 and has since been applied as an exploratory and visualization tool for complex datasets in a variety of fields, ranging from industry and finance to medicine and the natural sciences15. As the method does not transform the data into a new data space, unlike PCA, the interpretation of results is relatively straightforward. This provides the opportunity for expert knowledge to be incorporated during the exploration of multi-dimensional data.Self-organising maps (SOMs), also known as Kohonen maps, are a machine-learning algorithm that projects high-dimensional input data into topology-preserving maps. These maps represent the distribution of the input data records according to their similarity. Thereby, clusters and patterns in complex datasets can be visualized  and infectious bovine rhinotracheitis (IBR). In this paper, we present our approach and discuss its benefits and transferability to other regions and livestock systems. Finally, we provide a decision tree for cattle herd classification in Ireland that can be used by stakeholders and decision makers to support planning in the context of animal-health decision-making.16. This information is recorded at an individual animal level, and each animal has its own unique identity (ID) or tag number, so that its sex, breed and birth date, as well as the IDs of its herd and dam, can be ascertained. Data is collected from farmers, markets , abattoirs and licensed meat and live export locations.Data for this analysis was obtained from the Animal Identification and Movement (AIM) database maintained by the Department of Agriculture, Food and the Marine (DAFM) in Ireland. In accordance with EU requirements, the AIM database comprises records on all births, movements and disposals , tracking each individual bovine in Ireland from birth or import to death or export17, we accessed the database for three dates in 2017  and extracted relevant demographic data for all cattle registered on these dates. Movement data was extracted for each day in 2017 (1st January–31st December 2017).Taking into account the patterns of calving in Ireland outlined by17, we classified animals as dairy if their dam and sire were of following breeds (i.e. not necessarily the same): Ayrshire, Brown Swiss, Holstein/Friesian, Jersey, Normande and Norwegian Red. Animals were classified as beef if their dam and sire were of the following breeds: Angus, Aubrac, Blonde d’Aquitaine, Belgian Blue, Belted Galloway, Bazadais, Charolais, Dexter, Galloway, Hereford, Highland, Irish Maol/Droimeann, Kerry, Longhorn, Limousin, Marchigiana, Montbeliarde, MRI/MRY, Piedmontese, Partenaise, Rotbunte, Romagnola, Salers, Shorthorn, Simmental. Cattle were classified as cross-bred if bred by a beef sire and a dairy dam or vice versa, based on the dairy and beef breeds listed here.We classified all bovines in the dataset as being of beef or dairy breed type according to the detailed breed information provided in the AIM system. Based on the work of the Irish Cattle Breeding Federation (ICBF) andWe then aggregated the individual animal data to herd level, initially classifying the resulting ~ 100,000 Irish cattle herds as being of beef, dairy, mixed or unknown. Following recommendations of ICBF, this was done by first considering herds to be breeding enterprises if the proportion of animals in a herd that had ever calved by September 1st 2017  was ≥ 25%. These herds were then classified as being of dairy type if ≥ 70% of their animals were dairy according to the above explanation. In cases where this value was < 30%, herds were classified as beef enterprises. All other breeding herds were classified as mixed. Herds classified as non-breeding using the ICBF criterion were not further categorised (i.e. herd type unknown).Rule extraction from self-organising-maps”. The variables reflect relevant processes for livestock management, i.e. demographic parameters and transport statistics.Table 14. It is a method for dimensionality reduction. The produced map (output layer) consists of a predefined grid of nodes that is iteratively trained according to the patterns of the input data15. Due to the structure of the algorithm, input vectors that are similar to each other will be represented by the same or a nearby node. This allows for a visual identification of clusters/classes in the output (a map). As a result, SOMs have the ability to visualize unclassified high-dimensional input data in a structured way.An SOM is an unsupervised learning algorithm intended to project high-dimensional input data onto a low-dimensional map while preserving the topological properties of the input dataK vectors, each with N elements, formed by the  data records Zk, k = 1,…, K and the variables Var 1 to Var N  each with N elements, initialised with random values. During the iterative training process, the weight vectors are altered to best represent the vectors of the input layer. For each iteration, all data vectors are processed in random order. For each vector , the most similar weight vector wkx,y of W is determined. Similarity is measured by the Euclidean distance between the input vector Zk and each weight vector wx,y, and the weight vector with minimum distance is chosen:The functionality of the SOM is based on two layers, the input layer and the output layer Fig. , and an Zk, the values of corresponding weight vector W is performed for all input vectors  in random sequence. For the subsequent iteration cycle (i.e. each input vector processed once), The most similar vector is commonly called the Best Matching Unit  as described inExtracting classification rules from a trained SOM can be divided into three main sub-tasks which are: (1) visual inspection of the structure of the SOM, 2) selecting attributes which would discriminate classes suggested by the SOM, and (3) deriving rules for assigning data records to these classes . As an example, the unclassified non-breeding herds become clearly visible in the SOM output, jointly characterised by a minimal proportion of calved animals (i.e. blue area in right panel is coincident with the grey region in the left panel). Herds with a high proportion of animals of dairy breed , i.e. herds with different proportions of dairy breed animals. This visual discrimination then leads to a search for useful sub-classifications.To provide an example on how to read the component planes of the SOM and to extract classification rules, we have trained a supervised SOM (BDK) with our pre-classified data (i.e. by the ICBF rules) using two input variables, namely als Fig. a. The lepOutMovesToSL) to our input data and trained a new SOM based on this extended dataset and the initial classification  is well suited as a means of separating fattening and store herds from each other.To continue this illustrative example, the herds that are thus far not classified were subjected to further classification in subsequent steps. In this process, expert knowledge was introduced from a wide range of stakeholders . From these experts we were told that in Ireland, non-breeding herds may be characterised by their practice to deliver animals for slaughter. Based on expert knowledge herds either sell their cattle to another herd prior to slaughter  or send their cattle to the slaughter . To represent the difference, we added a third variable  sell most of their calves, but female calves are moved to external contract rearing herds  beef herds were identified from the data see Fig. c. The fiThe suckling to beef (BSB) herds follow the full beef production cycle, from birth through to the age of slaughter. Typically, calves stay with their dams until weaning at 6–8 months. Then, the weaned calves are retained for rearing and fattening in the same enterprise. Usually, these animals are sold to slaughter prior to two and a half years of age. Some females are kept as replacement heifers. This herd type can be distinguished by its high proportion of out moves to slaughter see Fig. g.The beef suckling to youngstock (BSY) system is very similar to the BSB system, however these herds do not fatten their cattle intended for slaughter. Instead, these animals are mainly sold to fattening herds as yearlings. This herd type becomes visible in the component planes by their equal proportions of male and female youngstock between 1 and 2 years see Fig. e.Non-rearing suckling to youngstock (BSY-nR) herds are a variation of the BSY herd type, with the difference being that most female calves are sold after weaning. Its main production objective is rearing of bulls and steers for beef production. These herds mainly purchase pregnant heifers for replacement from the market or other suckling herds. BSY-nR herds are characterized by a very high proportion of males between 1 and 2 years of age see Fig. e.Beef suckler to weanling (BSW) herds are the most common beef system in Ireland. These herds sell their male and some female calves after weaning between 6 and 8 months of age to store or fattening herds. A proportion of females are kept for replacement. These herds have almost no male youngstock aged between 1 and 2 years see Fig. e.The store/rearing group only comprises herds that are non-calving. Overall, five sub-types were identified within this group, which differ in terms of the animals these herd preferentially rear (i.e. breed and sex) see Fig. d. Store This study identified three different types of beef store herds, which differ in terms of their sex composition, rather than by management practices: store beef males (Sbm), store beef females (Sbf) and store beef mixed (Sbmx) systems see Fig. d. These In rearing dairy female (Rdf) herds, young female dairy calves are introduced, reared and inseminated, before being returned to their birth herd (DnR-C) as pregnant heifers. They become visible in the SOM output due to their increased proportion of out moves back to the birth herd see Fig. h.During analysis, we could identify herds which on average consist of half pure-bred dairy animals and half animals cross-bred between dairy and beef see Fig. b,c. ThesThere are some non-breeding herds that have a high proportion of in and out moves and where the majority of animals remain in these herd for less than 30 days  buy calves, weanlings, youngstock and cows from a wide range of herd types and fatten them until slaughter. Fattening herds do not normally produce their own calves, and the majority of their outward moves go to slaughter see Fig. a,g. Afteremaining herds is presented as a histogram. Thresholds for class assignment (dashed lines) are given below the histogram. Overall, the classification comprises nine variables and 13 decisions. To classify a herd, a maximum of six of the 13 possible decisions are necessary.Through examination of the component planes of the SOM, we developed a decision tree for the classification of the identified herd types see Fig. . At eachLivestock systems, including the Irish cattle sector, are often structured by production or breeding type. This information is important, noting that system classification can facilitate informed decision-making and protocol development. Due to the complexity and specialization of modern livestock management systems, however, such classifications rarely provide the details necessary for epidemiological risk analysis and modelling.In this paper, we have combined expert knowledge and machine-learning methods to derive a sophisticated yet straightforward method to allow the Irish livestock system to be classified by characteristics derived from the AIM data, while reflecting herd management practices. To our knowledge, this is the first time that the SOM algorithm has been used to analyse and classify livestock systems. Using this approach, we have identified 17 herd types using 9 variables, applied in 13 decisions.Herd type classification seeks to group herds into classes that each have broadly similar enterprise and management patterns. Trading networks, animal movements and age distributions derived from these classifications are more representative, providing valuable input for the development of targeted interventions or epidemiological models. As an example, herds assigned to the class non-rearing dairy herds (DnR-C) do not rear their own replacement heifers and sell most calves soon after birth. At the same time, these herds have to introduce in-calf heifers from a contract rearer for replacement of older dams. Hence, there will be herds that introduce calves, rearing them until they return to their birth herd as pregnant heifers. These were identified in this study as herds that rear dairy females (Rdf). Such refined data-driven structures capture regular and recurring dependencies between herds, which is very useful for risk assessment and contingency planning. A detailed understanding of such structural patterns will contribute to an improved understanding of disease epidemiology, including the eradication and surveillance of infectious diseases.20. Especially for the bovine sector, with the new EU regulations the herd management type became an important criterion for shaping the sampling strategy of national surveillance and control programs. With regard to BoHV-1 eradication as an example, the AHL prescribes certain rules on how herds can achieve a status of disease freedom. These rules often interact with specifics of the management regime in a herd. For example, the use of bulk tank milk testing alone can only be used in herds that have a minimum proportion of lactating cows. Due to normal replacement and breeding of offspring, a typical self-breeding dairy herd would never fall into this category. Non-rearing dairy herds (DnR_C and DnR_nC), on the other hand, could be checked by bulk tank milk testing alone. Such details become even more important when models are used to estimate program costs.Another recent example would be the planning of surveillance efforts in relation to the programme to eradicate bovine viral diarrhoea (BVD) from the Irish cattle population. The measures under discussion include the regular serological screening of youngstock in a herd as a test for undetected virus circulation. The approach is useful for dairy herds which raise their own replacements, but incompatible for herds in the dairy sector that rely on contract rearing.A recent example would be the new Animal Health Law (AHL) with its implementation principles that present new challenges for the management of livestock diseases21), however, explicit decision-support , it is certainly possible that other variables would be of interest and a modified classification could emerge when using the same SOM approach.According to26). The advantage of the SOM algorithm relates to the graphical representation, which can be directly interpreted together with livestock experts. Thus, the participatory procedure is facilitated by iterative cycles that include the addition of expert knowledge rules and graphical representation of the resulting structure.The proposed approach to combine expert knowledge with machine-learning is straightforward and can be adopted to any livestock system with comprehensive existing livestock registers. Several software packages for SOMs are publicly available and can be integrated in commonly used programming languages, such as R or MATLAB (e.g.


UBE3A gene encodes the ubiquitin E3-ligase protein, UBE3A, which is implicated in severe neurodevelopmental disorders. Lack of UBE3A expression results in Angelman syndrome, while UBE3A overexpression, due to genomic 15q duplication, results in autism. The cellular roles of UBE3A are not fully understood, yet a growing body of evidence indicates that these disorders involve mitochondrial dysfunction and increased oxidative stress. We utilized bioinformatics approaches to delineate the effects of murine Ube3a deletion on the expression of mitochondrial-related genes and pathways. For this, we generated an mRNA sequencing dataset from mouse embryonic fibroblasts (MEFs) in which both alleles of Ube3a gene were deleted and their wild-type controls. Since oxidative stress and mitochondrial dysregulation might not be exhibited in the resting baseline state, we also activated mitochondrial functioning in the cells of these two genotypes using TNFα application. Transcriptomes of the four groups of MEFs, Ube3a+/+ and Ube3a−/−, with or without the application of TNFα, were analyzed using various bioinformatics tools and machine learning approaches. Our results indicate that Ube3a deletion affects the gene expression profiles of mitochondrial-associated pathways. We further confirmed these results by analyzing other publicly available human transcriptome datasets of Angelman syndrome and 15q duplication syndrome.The  UBE3A gene that encodes for the ubiquitin E3-ligase protein UBE3A is located in the q11–q13 region of chromosome 15 in humans, and at 28.65 Cm of chromosome 7 in mice. UBE3A possesses five well-characterized functional domains: a HECT domain, E6 binding domain, p53 binding domain, three nuclear receptor interaction domains, and an activation domain [UBE3A gene. While the cellular roles of UBE3A are not fully understood, it is known to play a role in neurodevelopmental disorders in a dose-dependent manner. Lack of UBE3A causes a severe neurodevelopmental disorder known as Angelman syndrome (AS) [The n domain . So far,n domain . Recent n domain ,11,12. In domain ,15, suchn domain ,17,18,19ome (AS) , while oome (AS) ,23,24,25ome (AS) ,27,28,29UBE3A showed that in dup15q autism, where UBE3A is overexpressed, there is a mitochondrial dysfunction [One of the first studies that examined the cellular functions of function . Severalfunction . This stfunction . Later ofunction , was ablfunction . Coincidfunction . The finfunction . A laterfunction . Taken tIt is possible that in the resting baseline conditions, impaired mitochondrial functioning might suffice and would not reflect altered signaling pathways. However, further challenging the mitochondria will expose additional compromised mitochondria-related pathways. One way to induce mitochondrial-related signaling pathways is to stimulate cells with the cytokine tumor necrosis factor-α (TNFα). TNFα stimulation has been shown to elicit a wide range of cellular responses in almost all cell types, including cell death, survival, differentiation, and proliferation ,38,39, aUbe3a deletion on mitochondrial-related signaling pathways might shed light on the development of autism spectrum disorders beyond AS and dup15q. In addition, mitochondrial dysfunction ,49,50,51Ube3a deletion on the expression of mitochondrial-related genes, by also utilizing TNFα stimulation as a means to further push mitochondrial-related pathways. For this, we utilized mouse embryonic fibroblasts (MEFs) that either contained both copies of the Ube3a gene (Ube3a+/+) or a complete deletion of the two copies of Ube3a gene (Ube3a−/−). Within these cell cultures, we examined the mRNA expression profiles of genes that encode for proteins directly localized to the mitochondria [Ube3a deletion effects the expression of genes localized to mitochondria, we utilized additional previously published RNA-sequencing and proteomics datasets. The first transcriptome dataset, used for this validation, is an RNA-sequencing dataset we recently generated from brains  of AS model mice and their wild-type (WT) control littermates [Given all of the above, our aim in this study was to investigate the effects of chondria  and of gtermates . The sectermates . Moreovetermates . Furthertermates . Ube3a deletion is related to mitochondrial dysfunction and dysregulation of mitochondrial-dependent oxidative stress, but the cause for that is not known. It is a possible that these mitochondrial-related impairments are due to perturbation of gene expression regulation. Nevertheless, no studies examined the effects of Ube3a deletion on the expression of mitochondrial-related genes. Using transcriptome sequencing and applying various bioinformatics tools and approaches gives the opportunity to examine such questions in a broad perspective of pathways and cellular processes, instead of looking into particular genes. To examine whether indeed there is an indication for the involvement of mitochondrial-related transcriptome, we generated poly-A RNA sequencing of Ube3a+/+ and Ube3a−/− mouse embryonic fibroblasts (MEFs) derived from the same litters. Next, we performed a broad unsupervised analysis to identify differentially expressed genes in these Ube3a−/− cells compared to Ube3a+/+ cells by utilizing DeSeq2 algorithm [Ube3a−/− cells compared to Ube3a+/+ cells  with TNFα. TNFα stimulation is known to affect mitochondrial functioning and the induction of mitochondrial-related stress. TNFα was shown to induce mitochondria translocation [To induce a mitochondrial-related perturbation, we treated the two types of cells , or to tabolism  and FoxOtabolism )  considering the two factors of genotype and TNFα treatment. This analysis identified 275 genes significantly affected by both factors: genotype and TNFα treatment (Benjamini–Hochberg adjusted for multiple comparisons p < 0.01)  . Pathway < 0.01) . All the < 0.01) . To foll < 0.01) . Examini < 0.01) . All theUbe3a deletion affects the expression of genes localized to mitochondria, we utilized two additional previously published RNA-sequencing datasets. The first dataset was a transcriptomics dataset recently generated by us from brains  of AS model mice and their control littermates [p-value < 0.05) in AS and WT mouse hippocampi   . Perform < 0.05) B.UBE3A gene. Although the genetic etiology of AS is clear, the exact cellular mechanisms underlying the AS pathophysiology remain to be discovered. Over the last two decades, a substantial body of literature has demonstrated the involvement of mitochondrial dysfunction in neurodevelopmental disorders, such as autism [Ube3a with an emphasis on the mitochondrial-related pathways.AS is a neurodevelopmental disorder caused by the lack of expression of the UBE3A protein, usually due to a deficiency of the maternal copy of the s autism , Rett sys autism ,69, and s autism . In addis autism ,33,35. TUbe3a deletion. In the past, MEFs have been shown to be a powerful discovery tool for the identification of molecular pathways relevant to neurodegenerative disorders [Ube3a may entail multiple possible transcripts, only three transcripts were shown to be active and entail differential localization and possibly differential roles [−/−Ube3a MEFs, all transcripts were deleted and no Ube3a protein was expressed [In the current study, we utilized mouse embryonic fibroblasts (MEFs) as a screening system and an exploratory tool to identify pathways that are dysregulated by isorders . Moreoveisorders . Althougal roles ,11,12. Ixpressed . Ube3a+/+ and Ube3a−/− MEFs. We found 186 genes that were differentially regulated in the Ube3a knockdown MEFs (2O2) to water (H2O) [Ube3a−/− cells coinciding with previous studies that demonstrated elevated oxidative stress levels in CA1 hippocampal neurons of AS model mice [We first compared the transcriptomes of own MEFs A compareown MEFs . These per (H2O) . Our findel mice ,33,35. Idel mice .Ube3a−/− MEFs, we analyzed the transcriptome differences in light of genes that encode for mitochondrial-localized proteins which are represented in the MitoCarta2 database [+/+Ube3a and -/-Ube3a MEFs. Performing principal component analysis based on this gene expression matrix we found that these mitochondrial-localized genes clearly differentiate between the Ube3a+/+ and Ube3a−/− MEFs  leak out and interact with oxygen producing ROS like superoxide or hydrogen peroxide, which if not eliminated properly may result in mitochondrial damage and cell death . Alteratdatabase . The Mit−/− MEFs C–D.Ube3a+/+ and Ube3a−/− MEFs. We found that TNFα induced substantial changes in both Ube3a+/+ and Ube3a−/− MEFs, and some of these differentially expressed genes were common to both Ube3a+/+ and Ube3a−/− MEFs  induces diverse signaling pathways that result in altering the spatial distribution of mitochondria, mitochondrial membrane potential (MMP), cellular respiration, and ROS production ,40,41. M−/− MEFs . Functio−/− MEFs , which c+/+ and Ube3a−/− MEFs, were also affected by genotype factor alone as shown in the previous analysis  in dup15q patients compared to healthy controls, the ‘mitochondrion’ gene ontology cluster was highly significant  and WT (Ube3a+/+) 13.5-day-old embryos were generated by breeding Ube3a−/+ mice [6 cells were cultured on 100 mm dishes at 37 °C with 5% CO2. In this study, we used MEFs within three to five passages to avoid replicative senescence. Housing, handling, and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were reviewed and approved by the University of Haifa animal ethics committee (ethics # 576/18). Mice used were all on a C57BL/6 background. The MEFs from null . TNFα was initially dissolved in PBS 0.1% BSA according to manufacturer’s protocol. Following, TNFα was dissolved in the medium to a final concentration of 25 ng/mL. We replaced the medium of half of the dishes of each genotype TNFα-containing medium and the other half of the dishes with a similar medium without TNFα. We harvested the MEFs for RNA isolation after 16 h of incubation in the mediums.Ube3a−/− and Ube3a+/+ MEFs that were used for mRNA sequencing were cultured for four passages and either treated or not treated with TNFα as abovementioned. After trypsinization, cells were collected and total RNA was isolated using the RNeasy Lipid Tissue Mini Kit, Cat No: 74804  according to the manufacturer’s instructions. The mRNA was isolated using polyA selection according to the manufacturer’s instructions  v2). The isolated mRNA concentration and quality were determined by Qubit® quantitation assay using Qubit® 2.0 fluorometer  and Agilent 4200 TapeStation System . Samples were prepared for Illumina sequencing using NEB’s Ultra RNA Library Prep Kit for Illumina (NEB#7530)  according to the manufacturer’s protocol. Libraries were sequenced in the Technion Genome Center  with a 2 × 150 bp PE run on Illumina HiSeq 2500  using a V3 flow cell.The https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/ using HiSat2 algorithm [RNA sequencing data was processed by the Tauber Bioinformatics Research Center parallelized pipeline. The raw reads were cleaned from adaptors according to manufacturer’s instructions of the kit Cat.# C05010047 CATS-mRNAseq-kit-manual (with polyA selection) v2. The cleaned reads were aligned to mouse genome assembly GRCM38:mm10 lgorithm  with ‘--lgorithm  in FPKM lgorithm .Ube3a−/− (sample 10) was discarded from further analysis due to the low number of acceptable quality reads in the sample compared to all other generated samples. Genotyping of samples was reconfirmed by analyzing the alignment of reads on Ube3a exon2  and interaction effect [p-value < 0.01.In order to identify genes differentially affected by TNFα treatment in n effect . SignifiDAVID  softwareUbe3a+/+ and Ube3a−/− MEFs). We added an iterative procedure to the standard random Forest R package [Random Forest (RF) is a classification algorithm that has been successfully applied in several recent RNA sequencing studies ,87. RF i package  to ident package  to furthUbe3a+/+ and Ube3a−/− MEFs dataset, we utilized our previously published RNA sequencing data generated from hippocampi of AS model mice and their WT control littermates (GEO project accession: PRJNA48422). The second dataset was an RNA sequencing dataset generated from patient-derived iPSC-derived neurons and compared to iPSC-derived neurons derived from healthy donors [https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/ in case of mouse derived data or to human genome assembly GRCh38 https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/ when the data was generated from human cell cultures using Bowtie2 algorithm [In order to validate our results from the y donors   in postmortem brains of dup15q patients compared to healthy controls. This gene expression table of dup15q patients was obtained from the supplementary information of a publicly available mRNA-seq dataset [Further validation of our results was by investigating the opposite condition of dup15q, where UBE3A is overexpressed. For that, we utilized DAVID functional annotation software  for func dataset .Ube3a−/− and Ube3a+/+ MEFs are available in GEO (PRJNA634721).Fastq files of RNA-seq data from treated and untreated


The molecular function of a protein relies on its structure. Understanding how variants alter structure and function in multidomain proteins is key to elucidate the generation of a pathological phenotype. However, one may fall into the logical bias of assessing protein damage only based on the variants that are visible (survivorship bias), which can lead to partial conclusions. This is the case of PNKP, an important nuclear and mitochondrial DNA repair enzyme with both kinase and phosphatase function. Most variants in PNKP are confined to the kinase domain, leading to a pathological spectrum of three apparently distinct clinical entities. Since proteins and domains may have a different tolerability to variation, we evaluated whether variants in PNKP are under survivorship bias. Here, we provide the evidence that supports a higher tolerance in the kinase domain even when all variants reported are deleterious. Instead, the phosphatase domain is less tolerant due to its lower variant rates, a higher degree of sequence conservation, lower dN/dS ratios, and the presence of more disease-propensity hotspots. Together, our results support previous experimental evidence that demonstrated that the phosphatase domain is functionally more necessary and relevant for DNA repair, especially in the context of the development of the central nervous system. Finally, we propose the term "Wald’s domain" for future studies analyzing the possible survivorship bias in multidomain proteins. About 60% of the prokaryotic proteome and 80% of eukaryote proteins are multidomain proteins . It is pMost variants we observe in patients are those that manage to be viable despite affecting protein functioning. The variation that results in unviable proteins and eventually unviable organisms, is almost never considered because we hardly see those variants in patients. For instance, it has been shown that proteins and domains vary in their tolerance to non-synonymous point variants according to their mutation rates, degree of sequence conservation and interaction networks . This scAnother interesting case was the one that occurred during World War I, after the introduction of the Brodie helmet and others , which wAnalyzing the pathogenicity of variants according to the different domains of a protein can provide significant information about the sites where it is more likely or not to find disease-causing variation . Thus, bin silico analysis, published experimental data and open sequencing data to integrate both structural and functional features.Disease-associated variants in PNKP cause a wide pathological clinical spectrum. The most severe phenotype is Microcephaly with early-onset Seizures (MCSZ) . In the First, we wanted to analyze some general features of PNKP. From all the available protein sequences in the Uniprot database, seventy sequences of twenty taxonomic groups were selected. We performed a Multiple Sequence Alignment using Fast Fourier Transform (MAFFT) against the human PNKP sequence see .Mus musculus: 80.88%). The species within the group of birds, reptiles, and amphibians had a range between 50–70% . Different species of fish showed even a lower range (50–60%). The rest of the species had percentage identities below 50% (e.g. Drosophila melanogaster: 40.16%).As expected, the group of primates was the closest to the human, with identities between 90–100%. The species belonging to the other eight orders of mammals presented a range between 80–90%  pathway with respect to ten vertebrate species. Protein sequences of PNKP, PARP1, XRCC1, POLB, and LIG3 were obtained from Uniprot and analyzed using the BlastP tool . We idenMus musculus (mouse) was almost 20%. To further evaluate this difference, an ANOVA test was performed and we confirmed that there is a smaller gradient, and thus less differences between species, for POLB protein sequence, and a larger gradient, with a notable difference between species for PNKP and XRCC1 , while no differences were found with respect to PARP1 and LIGIII (p > 0.05). No significant differences were found between PARP1, LIGIII and XRCC1 (p > 0.05). PNKP shows bigger differences in sequence identity than POLB (p = 0.0000035), PARP1  and LIG III (p = 0.013). No difference was found between PNKP and XRCC1 .2 = 22.865, df = 8, p = 0.0035; The evident differences that we found between proteins from the same DNA repair pathway, made us wonder if the degree of sequence conservation of PNKP was different between domains. To analyze this, the previous multiple sequence alignment (MSA) was used as input for the ConSurf server to get and compare the conservation scores of the amino acids within the phosphatase (from amino acid 146 to 337) and kinase domain (from 341 to 521) . There wOnce we determined that the phosphatase domain is more conserved and generated the best 3D structure of PNKP, we used the SuSPect server  to predith percentile, close to a DPS of 75  [3ZVL_A, 1UJX_A, 1YJ5_C, 2BRF_A, 3KT9_A, 1LY1_A), Phyre2 selected 7 templates  while I-TASSER used ten templates to model the structure . From all the models generated with I-TASSER, we chose the model with the lowest C-score (-2.22).Once we characterized the disease-propensity hotspots of PNKP, we wanted to combine this information with some structural analyses. For this purpose, since there is no crystal structure for the human PNKP in the RCSB Protein Data Bank, different servers were used to perform homology modeling. Fortunately, the crystal structure for the murine Phosphatase-Kinase domains is available (PDB ID: 3ZVL) , which rThe Ramachandran plots of each model were generated with the Swiss-Model Structure Assessment tool. According to these plots, the model generated by Modeller presented the highest number of amino acids in a favored position and the lowest number of outliers . For thiin silico. For this purpose, we used the 3D structured predicted with Modeller as input for the COACH [Once the chose the best structure, we wanted to analyze if predicted disease-propensity hotspots match the functional ligand-binding sites of PNKP. As these sites have been already determined at the experimental level, we wanted to determine if we were able to get comparable results he COACH  and COFAhe COACH  servers.he COACH , 28.+2 binding sites  which also matched a hotspot within the phosphatase domain (C-Score: 0.20). This is also in agreement with experimental results [For the phosphate group binding site (phosphatase active site), COFACTOR predicted with greater C-Score (0.42) the residues L172, D173, T217, N218, K260 . These r results , 28. Altin vivo and in vitro phosphorylation data. Phospho.ELM confirmed the phosphorylation of S114, T118, T122, S126. PhosphoSitePlus also confirm these sites but also reported S12, T109, T111, T122, N144, G188, S364 and S379, although some with few references. Other PTMs were reported, as acetylation in K226, K233 and K242 and ubiquitination in K142, E151, K183, K226, and K484. However, these sites do not coincide with disease-propensity hotspots nor mutation sites.To add one more layer of complexity, we also analyzed the potential post-translational modifications (PTM) sites of PNKP. NetPhos 3.1 predicted S47, S114, T118, S126, S143, S221, S280, S307, T323, S347, T407 as possible phosphorylation sites (prediction score > 0.90). To confirm this prediction, we reviewed the Phospho.ELM and PhosphoSitePlus v6.5.9.1 database, which contain As we found that the disease-propensity hotspots matched some predicted ligand binding sites, we next evaluated whether Single Amino acid Variants reported so far in patients also coincided with these hotspots. We also evaluated the structural damage of each variant using the Missense3D server. Dynamut were also used to analyze the impact of mutations on protein dynamics and stability resulting from vibrational entropy changes. We included the following missense variants : Arg462PAlthough Arg462Pro is not located within a hotspot site, according to the Missense3D prediction, the variant generates an alteration that affects nearby amino acids  leading to the expansion of cavity volume by 117.72 Å^3. These nearby amino acids are part of the ATP/ADP binding sites in the kinase domain. In addition, the change in folding free energy (ΔΔG) is predicted to be stabilizing. Lys378Thr is located within a hotspot associated with ATP/ADP binding site. Missense3D predicted a contraction of cavity volume by 74.736 Å^3 and the disruption of all sidechain/main-chain H-bond (s) formed by this buried Lys residue (RSA 3.4%). The variant is predicted to be destabilizing, increasing protein flexibility. Gly375Trp is also within a hotspot (ATP/ADP-binding site). However, it does not generate any structural alteration according to Missense3D and the change in ΔΔG is predicted to be stabilizing. The rest of the variants in the kinase domain do not present any clear tendency . Pro343LOnly three disease-associated variants have been reported in the phosphatase domain so far , 29. Leu2 = 11.449; p < 0.001). In addition, we found 12 nonsense variants in the kinase and only 3 in the phosphatase domain. From the all the frameshifts reported (31), we observed 23 in the kinase and only 8 in the phosphatase. Of all missense variants, only 12 were reported in homozygous condition, but they represented a very small portion since each variant has a high allelic count.We also wanted to analyze variants segregating in the population, so we looked for PNKP variants reported in the gnomAD v2.1.1 dataset, which includes more than 141.000 individuals. We found a total of 424 missense variants, but only 193 in the kinase domain, 132 in the phosphatase and 71 in the FHA domain. Based on these results, observed mutations in the kinase domain are higher than expected compared to the phosphatase domain . This is calculated based on the single nucleotide missense and synonymous variants (SNVs) from gnomAD in a protein-coding region. This score is later corrected for the sequence composition of the protein coding region based on the total possible missense and synonymous SNVs. When we compared the density distribution of each domain, we found a higher density of low dN/dS values in the phosphatase domain .Current bioinformatics tools provide useful After the sequence identity analysis, we performed the sequence conservation analysis and we found that as expected, the phosphatase domain was more conserved give the higher number of amino acids with higher conservation scores and less with very low conservation scores. Therefore, we believe that the evolutionary rate of the kinase domain in PNKP is higher, which is supported by the observed higher mutational tolerability. Curiously, it has been reported that kinase proteins derived from a single protein structural fold compared to protein phosphatases, that are associated with many different protein folds and catalytic mechanisms . More reAlthough the crystal structure of human PNKP has not been determined, we obtained very good results when using different servers to predict it by homology modeling. In fact, the structural alignment showed that our model was comparable (very low RMSD and high TM-Score) to the murine crystal phosphatase-kinase structure. The accuracy of our model was very useful to predicted protein ligand binding sites, which was confirmed by comparing our predictions with previous experimental results. Likewise, we were able to confirm that three-dimensional protein models are effective to infer the pathogenic effect of a variant.The three pathogenic variants reported in the phosphatase domain are associated with the most severe phenotype (MCSZ), however, none are predicted to generate a significant structural alteration. As expected, Gly292Arg and Glu326Lys presented low DPS but high conservation scores. Gly292Arg has only been reported as compound heterozygous together with Ala55Ser. The latter is a variant located in the FHA domain that also has low DPS (= 39) but high conservation score (= 8) and without predicted structural damage. The Leu176Phe variant is located within a disease-propensity hotspot and was reported to exhibit reduced levels of both DNA phosphatase and DNA kinase activity at 30°C . One mayIn the kinase domain, the Lys378Thr mutation affects the ligand-binding site and is predicted very pathogenic, only reported in MCSZ. Gly375Trp is located also in the ligand binding site (ATP/ADP) and although it does not generate any structural alteration, it is predicted pathogenic as well. However, Gly375Trp has only been associated with AOA. In this case, as both can be in homozygous condition, the genotype, and the prediction of its effect on protein activity is not related to the observed phenotype. To support this, Arg462Pro also affects nearby residues of the ATP/ADP binding site in the kinase domain but can cause MCSZ when homozygous or AOA when accompanied by Leu399Pro. Thus, variants in the active site of the kinase domain can not only be tolerated but can be found in a homozygous condition. Likewise, Leu399Pro, Cys409Trp and Pro343Leu  usually occur in a compound heterozygous condition, accompanied by a second allele with more serious insults in the kinase domain, usually deletions or frame shift variants .From all the missense variants reported in gnomAD, a higher number were located in the kinase domain. None of the homozygous variants reported in the phosphatase domain are within any hotspot. In addition, the general prediction for these variants is benign, possibly because most of them are located at the surface of the protein. This is very important since surface residues are less evolutionarily conserved and do not present a restricted conformational space, leading to potentially more viable physicochemical changes .Most of the disease-causing variants in PNKP are confined to the kinase domain. However, not all combinations are equally pathogenic as many patients harboring only kinase mutations can present either a severe (MCSZ) and/or milder phenotype (AOA4/CMT). In contrast, only three pathogenic variants have been reported in the phosphatase domain and the three of them are associated to the most severe phenotype (MCSZ). A few novel variants in the FHA domain are associated with both AOA4 and MCSZ , 35. AltAt the functional level, PNKP kinase and phosphatase activities do not act in concert on substrates containing both 5'-hydroxyl and 3'-phosphate termini, but PNKP phosphatase activity is much faster than the kinase activity. In addition, the 5'-phosphorylation of strand breaks containing a 3'-phosphate and 5'-hydroxyl depends on the preprocessing by the phosphatase activity of PNKP . LikewisIt has been shown that disease-resistant domains and proteins are more able to tolerate variation, as they show significantly higher variant rates and less degree of sequence conservation than disease-susceptible proteins and domains . In lineEnzymes from both the SSBR/DSBR pathway have variants associated with pathological phenotypes like those associated with PNKP, but only variants in PNKP appear to combine both phenotypes . The dual function of PNKP in DNA Damage Response allows the enzyme to participate in both SSBR and DSBR, but for reasons that remain unknown, the features that define which is the first affected pathway at the intracellular level in the nervous system is what determines the appearance of the clinical manifestations. Like germline deletion of other key components of base excision repair (BER), such as XRCC1, PNKP is essential for early embryogenesis . Even whAs none of the disease-causing mutations ablate the DNA 3’-phosphatase activity of PNKP, it is possible that MCSZ cells could retain residual levels of PNKP . This reAnother experiment using PNKP-deficient HCT116 and HeLa cells generated with CRISPR/Cas9 showed that cells were biochemically competent in removing both protruding and recessed 3'-phosphates from synthetic DSB substrates . HoweverEven when all the variants in the kinase domain are deleterious, we found a greater variant tolerability for this domain due to higher dN/dS ratios. Accordingly, the kinase domain presented higher variant rates in both clinical reports and gnomAD dataset. It also showed lower degree of sequence conservation and fewer sites considered as disease-propensity hotspots compared to the phosphatase domain. This means that the kinase domain can tolerate serious damage in places such as the active site and still generate a viable phenotype. However, very few patients are reported with damage in the phosphatase domain, as most mutations in this domain are very unlikely to be viable. Since the majority of pathogenic variants observed in patients are in the kinase domain, we propose that variants in this domain represent “the areas where an airplane could suffer damage and still return" but the phosphatase domain, that is rarely mutated, represents the areas that “if attacked, would cause the plane to be lost” . This caFinally, we propose the term "Wald’s domain" for future studies analyzing the effect of variants in multidomain proteins and the possible survivorship bias. Based on our results, Wald’s domains would be characterized by lower O/E variants ratio, higher degree of sequence conservation, higher DPS and lower dN/dS ratios. Hence, our work may constitute a new approach to evaluate the effect of variants on protein structure and function.From Wald’s perspective, the regions mutated within the kinase domain are under survivorship bias since these sites represent points where the protein can be damaged and still generate a viable phenotype. It is also possible that factors other than the affected domain can influence the specific pathological phenotype observed . Taken together, our results support previous experimental evidence that demonstrates that the phosphatase domain is functionally more necessary and relevant for the repair of DNA insults, especially in the context of the development of the central nervous system. Our data illustrate the value and accuracy of available bioinformatics tools to determine the relationship between protein structure and function when analyzing the effect of multiple variants. We suggest considering survivorship bias when analyzing the role of variants in multidomain proteins.http://www.uniprot.org) [https://www.ncbi.nlm.nih.gov/protein). We chose the most representative species of each taxonomic group: 42 mammals, 8 bony fish, 5 reptiles, 3 amphibians, 3 birds, 2 arthropods, 2 apicomplexan, 1 amoebozoan, 1 cnidaria, 1 Mollusca, 1 bacterium and 1 virus (mimivirus). Uniprot: Homo sapiens (Q96T60), Mus musculus (Q9JLV6), Rattus norvegicus (A0A0G2JUH4), Pan troglodytes (K7BAU7), Bos taurus (F1N3Q9), Anolis carolinensis (G1KMT2), Pongo abelii (H2NZP9), Ictidomys tridecemlineatus (I3N446), Otolemur garnettii (H0WRT3), Chlorocebus sabaeus (A0A0D9S429), Loxodonta africana (G3TPU6), Macaca fascicularis (A0A2K5X601), Saimiri boliviensis boliviensis (A0A2K6TGX4), Rhinopithecus bieti (A0A2K6JZR0), Colobus angolensis palliatus (A0A2K5JFF3), Propithecus coquereli (A0A2K6FTG9), Aotus nancymaae (A0A2K5F2F6), Cebus capucinus imitator (A0A2K5R4X3), Mesocricetus auratus (A0A1U7R368), Dipodomys ordii (A0A1S3G4D4), Mandrillus leucophaeus (A0A2K5YE36), Cavia porcellus (H0V4R0), Cricetulus griseus (G3I715), Myotis lucifugus (G1PW59), Ailuropoda melanoleuca (G1M4Y9), Capra hircus (A0A452FU49), Felis catus (A0A337SAT2), Sus scrofa (F1RHU1), (Ursus maritimus (A0A452TNF4), Ursus americanus (A0A452QHV0), Mustela putorius furo (M3XT89), Callithrix jacchus (F6RVI4), Drosophila melanogaster (Q9VHS0), Danio rerio (Q08BP0), Oryzias latipes (H2MLN4), Xenopus tropicalis (B1WBK3), Equus caballus (F6QG90), Xenopus laevis (A9UMQ0), Canis lupus familiaris (E2R0U3), Fopius arisanus (A0A0C9R1P0), Latimeria chalumnae (H3B245), Electrophorus electricus (A0A4W4HJQ6), Acanthamoeba castellanii str. (L8GVC5), Nothobranchius furzeri (A0A1A7ZB78), Alligator sinensis (A0A1U7R7V0), Austrofundulus limnaeus (A0A2I4BRQ3), Balaenoptera acutorostrata scammoni (A0A384ACF1), Ictalurus punctatus (A0A2D0SWM3), Gorilla gorilla gorilla (G3R3W6), Acanthamoeba polyphaga mimivirus (Q5UQD2), Nothobranchius kuhntae (A0A1A8JP17), Physeter macrocephalus (A0A455AU26), Alligator mississippiensis (A0A151NJZ6), Erinaceus europaeus (A0A1S3AK18), Hydra vulgaris (T2M7D9), Castor canadensis (A0A250XY04), Plasmodium malariae (A0A1A8W4X2), Neovison vison (U6D1K9), Callorhinus ursinus (A0A3Q7NQ21), Plasmodium gallinaceum (A0A1J1GNL7), Mycobacterium sp. (A0A1A0X7E1), Odobenus rosmarus divergens (A0A2U3WM26), Tarsius syrichta (A0A1U7TJR7), Vulpes vulpes (A0A3Q7U9N3), Delphinapterus leucas (A0A2Y9P3J8). NCBI: Gopherus evgoodei (XP_030400898.1), Microcaecilia unicolor (XP_030053814.1), Podarcis muralis (XP_028557931.1), Falco cherrug (XP_027653441.1), Lonchura striata domestica (XP_021405459.1), Gallus gallus (XP_025001234.1), Aplysia californica (XP_005090552.1).Human PNKP protein sequence and other seventy sequences from different species were collected, sixty-three obtained from Uniprot (rot.org)  and seveMultiple sequence alignment was performed with the EMBL-EBI’s server for Multiple Alignment using Fast Fourier Transform (MAFFT)  as it isPan troglodytes, Equus caballus, Sus scrofa, Canis lupus, Felis catus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster). The refseq_protein database (Max target sequences = 1000) was used and the sequences with the lowest E value, highest coverage and the highest similarity were chosen for each species. The difference in the sequence percentage identity was calculated and compared using the ANOVA test.The human protein sequences of PNKP, PARP1, XRCC1, PARP1, and LIG3 were obtained from Uniprot  Table 2Table 2. The Multiple Sequence Alignment previously generated was used as input in the ConSurf 2016 server  to estimBased on both sequence and structure (Modeller) we analyzed the mutational sensitivity of the amino acids from the phosphatase and kinase domain using the SuSPect server . This tohttps://www.schrodinger.com/pymol). Structure alignment was performed with the TM-align server [As there is no crystal structure of the human PNKP protein in the RCSB Protein Data Bank (only the FHA domain based on the mouse’s structure) different servers were used to predict its 3D structure. We used Modeller , SWISS-Mn server  using thin vivo and in vitro phosphorylation data.We used the COACH  and COFAWe evaluated the structural damage of missense variants reported in PNKP using the 3D structure predicted with Modeller as input for the Missense3D server . Dynamuthttps://gnomad.broadinstitute.org/). We only included variants from the canonical transcript (ENST00000322344.3). We especially analyzed “Missense” variants to compare the incidence between domains and analyze those cases reported as homozygous. Low confidence variants were not considered. To further obtain information about the mutational tolerability of PNKP, we used the MetaDome server [All reported (non-intronic) variants for PNKP were collected from both exome sequencing and whole genomes in the gnomAD v2.1.1 dataset   Ramachandran plots of the three models generated on each platform. B) Comparison of the model with a non-redundant set of PDB structures. C) Local quality estimate according to the local similarity to the target. The comparison of each model with a non-redundant set of PDB structures showed that this model has the lowest value for the Qualitative Model Energy ANalysis (QMEAN) (-4.34). This indicates whether the QMEAN score of the model is comparable to what one would expect from experimental structures of similar size. The QMEAN for the model generated in Phyre2 was -7.06 and for I-TASSER was -10.21 . The “Local Quality” plot shows for each residue of the model (reported on the x-axis), the expected similarity to the native structure (y-axis).(TIF)Click here for additional data file.S2 FigTwelve mutations were found in homozygous condition in specific regions of the world.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Graphical Abstract(TIF)Click here for additional data file.


Plasmodium falciparum harbors group 1 and group 2 chaperonin systems to mediate the folding of cellular proteins in different cellular locations. Two distinct group 1 chaperonins operate in the organelles of mitochondria and apicoplasts, while group 2 chaperonins function in the cytosol. No structural information has been reported for any chaperonin from plasmodium. In this study, we describe the crystal structure of a double heptameric ring Plasmodium falciparum mitochondrial chaperonin 60 (Cpn60) bound with ATP, which differs significantly from any known crystal structure of chaperonin 60. The structure likely represents a unique intermediate state during conformational conversion from the closed state to the opened state. Three of the seven apical domains are highly dynamic while the equatorial domains form a stable ring. The structure implies large movements of the apical domain in the solution play a role in nucleotide-dependent regulation of substrate binding and folding. A unique 26–27 residue insertion in the equatorial domain of Plasmodium falciparum mitochondrial chaperonin greatly increases both inter-ring and intra-ring subunit–subunit interactions. The present structure provides new insights into the mechanism of Cpn60 in chaperonin assembly and function. Group 1 chaperonins are found in the cytosol of bacteria, such as GroEL from Escherichia coli or Cpn60 from Thermus Thermophilus, while in eukaryotic organisms, they are located in the chloroplast or mitochondria3. Group 2 chaperonins include the archaeal thermosome, the eukaryote tailless complex polypeptide 1 (CCT), and the eukaryotic tailless complex polypeptide 1 ring complex (TRiC)4. In both chaperone groups, each monomer is comprised of three domains: an equatorial domain, an intermediate domain, and an apical domain. Seven or eight monomers assemble together to form a heptameric or octameric ring, and two rings interact back-to-back through the equatorial domains to form tetradecameric or hexadecameric double-ring structures4.Group 1 and group 2 chaperonins are two distinct classes of chaperones that exist in diverse organisms to assist with protein folding in an ATP-dependent manner2. The equatorial domains contain the ATP binding site, the intermediate domain serves as a hinge connecting the equatorial to the apical domain, and the apical domain is involved in substrate binding. Group 1 chaperonins require the binding of a co-chaperone Cpn10 to the apical domain to encapsulate the substrate protein (SP), while the apical domain of group 2 chaperonins utilizes a built-in lid. The binding of Cpn10 to group 1 chaperonins is critical for the folding mechanism that forms bullet-shaped (Cpn6014Cpn107), American football-shaped (Cpn6014Cpn1014), or American half-football-shaped (Cpn607Cpn107) complexes at distinct stages of the folding mechanism  . The partner co-chaperonin, GroES, is a dome-shaped heptameric ring of identical monomers with a monomeric molecular weight of approximately 10 kDa . GroEL requires ATP binding in order to shift from a closed conformational state to an open conformational state that allows for SP and GroES binding6. In this open conformation, the intermediate and apical domains of GroEL rotate and move upward to form a complex with GroES. Two cycles have been suggested for the function of GroEL. The prevailing asymmetric cycle which  and nucleotide symmetry between the rings (ATP or ADP binds both rings)17. Furthermore, the human chaperonin forms football-shaped complexes with a different inter-ring arrangement from that of GroEL17. Additionally, the crystal structure of the football complex with both rings bound to ADP suggests that human Cpn60 follows a symmetrical cycle, where both rings can undergo hydrolysis at the same time. Cryo-electron microscopy recently revealed that in addition to the full football-shaped Cpn6014Cpn1014 (PDB: 6MRC), the half-football-shaped complex consisting of Cpn607Cpn107 (PDB: 6MRD) can independently exist in solution, with both forms active in assisting the folding of imported mitochondrial proteins18; this suggests ring separation may be a possible step during the chaperonin cycle19. Similarly, the bullet-shaped structure from T. thermophilus exhibited a significant deviation from the sevenfold symmetry for the apical domain around the cis-cavity20. Studies on the chaperonin from Chlamydomonas chloroplast demonstrated a significantly different mechanism for chaperonin to capture, encapsulate, fold, and release substrate proteins. The binding and hydrolysis of ATP induce not only both positive (intra-ring) and negative (inter-ring) cooperative actions, but also promotes the partial disassembly of chaperonin into monomer, a phenomenon unique to chloroplast chaperonin22. Research on Cpn60 from Paracocus denitrificans indicated that the inter-ring interaction in this Cpn60 is weakened compared to E. coli GroEL, thus both single ring and double ring forms were observed23. In summary, due to structural and functional variations between Cpn60 orthologs, studies on Cpn60 from diverse organisms are warranted to better define the overall mechanism of Cpn60.However, there is evidence that chaperonins from organisms other than 24. Cpn60 is a ubiquitous protein that exists in nearly all domains of life including bacteriophage25 and plays an essential role in maintaining protein homeostasis26. In yeast, deletion of mitochondrial Cpn60 is lethal27. In mice, the inactivation of its homolog causes embryonic mortality28. Heat-shock proteins such as Cpn60 are often upregulated in response to stressful conditions. PfCpn60 must cope with two radically different host environments, mosquito and human, making it an interesting ortholog to study. In order to understand its mechanism in P. falciparum, we report the first crystal structure of P. falciparum Cpn60 (PfCpn60) bound with ATP, which reveals the large conformational dynamic of the apical domain relative to the intermediate and equatorial domains.Malaria remains a major public health problem worldwide resulting in nearly half a million deaths and 200 million clinic illnesses annuallyP. falciparum. Two group 1 Cpn60 genes exist, with one located on chromosome 12 that expresses a Cpn60 believed to localize to mitochondria30, and a second located on chromosome 10 with an encoded Cpn60 that is expected to function specifically inside the apicoplast31. Compared to known structures of Cpn60, the mitochondria-specific P. falciparum Cpn60 contains insertions including a 26–27 residue insert in the equatorial domain and a 10 residue insert in the apical domain  with a sequence identity of 39%20. The structure of 4V4O is a bullet-shaped complex, Cpn6014Cpn107, with one heptameric ring in closed conformation with no nucleotide bound, and the other heptameric ring in opened conformation with ADP and Cpn10 binding20. Since PfCpn60 was co-purified with PfCpn10, the opened conformation of TtCpn60 was used as the search model first. However, no molecular replacement solution was found.Matthew’s coefficient calculation estimated that there are seven subunits in an asymmetric unit in the crystal structure of PfCpn60, with a coefficient of 3.9 and an estimated solvent content of 68%Interestingly, no molecular replacement solution was found using the closed conformation of TtCpn60 as well, implying the conformation of PfCpn60 in the current structure may differ from both conformations of TtCpn60 in 4V4O. We hypothesized that the flexibility within the intermediate hinge region may have caused the positions of the equatorial and apical domains to differ from the search model. Therefore, in order to identify the molecular replacement solution, the individual domains were used as the search model. The molecular replacement solution was identified using the equatorial domain alone from both conformations of the TtCpn60 structure. Furthermore, the equatorial domain from the closed conformation ring performed better as a search model than the opened domain, indicating that the current structure is closer to the closed conformation. Careful inspection of molecular packing for the molecular replacement solution indicated that enough empty space was available for the packing of the intermediate plus apical domains, but not enough for the additional Cpn10 binding to the apical domain, suggesting that Cpn10 may not be present in the current structure. Eventually, the molecular replacement solution was successfully found by searching one copy of the heptamer of the equatorial domain alone and seven copies of the intermediate plus apical domain. The final solution gave a total likelihood gain of 7360.Inspection of the molecular replacement solution indicated that Cpn60 is well-packed in the crystal structure, confirming the absence of Cpn10. A reason for Cpn10 not co-crystallizing with Cpn60 may be due to weak interaction between Cpn10 and Cpn60 resulting in the loss of Cpn10 during purification, due to specific conditions during crystallization, or due to the ATP binding state. We performed a negative stain electron microscopy analysis which revealed that the major species existing in solution is a heptamer single ring structure of Cpn60 without the presence of Cpn10 Fig. b, even tC-axis. A second heptameric ring in an adjacent asymmetric unit that is related by twofold crystallographic symmetry results in a tetradecameric complex with the rings stacked back-to-back, as observed in the other Cpn60 structures. The seven subunits in the asymmetric unit are in slightly different conformations and differ from the closed and opened conformations typically observed for other Cpn60 molecules  with the final refined structure revealed that the current PfCpn60 structure is closest to that of ATP-bound EcGroEL structure (PDB ID: 2C7E)34, with a root mean square deviation (RMSD) of 2.3 Å.One heptameric ring was found per asymmetric unit of the PfCpn60 structure. The sevenfold NCS axis was almost parallel with the crystallographic les Fig. . The apiles Fig. b. This lE. coli and T. thermophilus Cpn60 sequence  of group 1 chaperonin currently exists, possibly due to the high flexibility of the apical domain in this conformation.Although structures of diverse Cpn60 orthologs with double heptameric rings have been determined  confirmed that the conformation of the PfCpn60 structure is a unique ATP-induced conformation distinct from both the closed and open conformation Fig. b. The whIn comparison with the sequences of other Cpn60 orthologs with known structures . This C-terminal segment further interacts with the N-terminal segment  in an anti-parallel β-strand arrangement to form a four-stranded β-sheet (β1↓β19↑β2↑β3↓) that glues the two adjacent subunits together  that forms an extended α-helix loop and contacts the left-handed intra-ring subunit , two salt-bridges, R452-E461 and van der Waals contacts among S463, V 464, and N467, form the right site contact. The van der Waals contacts among K105, A108, and A109 in the cis-ring, and A109, G110, and M111 in the trans-ring, contribute to the left site interactions. However, these residues are not conserved in PfCpn60 and the specific inter-ring interactions are not directly comparable 42; (2) ATP bound lidless double-ring Cpn6014 partially opened form (2C7E and 4AAQ)35; (3) ADP or ATP bound single lid bullet-shaped form, Cpn6014Cpn107 (1PCQ), with ADP or ATP binding only to the cis ring43; (4) ATP or ADP + Be3F bound double lidded football-shaped opened form, Cpn6014Cpn1014 (3WVL and 4PKO), with ADP + Be3F or ATP binding to both rings45. The PfCpn60 D474A mutant crystal structure revealed a symmetric double-ring complex with all subunits occupied by ATP that is similar to pre-open ATP-bound GroEL Cryo-EM structures 4AAU and 4AB235. Negative stain images reveal varying forms of PfCpn60 for the wildtype  does not display any negative inter-ring cooperativity at any point of the reaction cycle. PfCpn60 has been observed in the single ring conformation  from 69 to 631 only. After cleavage of the first 23 residues of the mitochondrial signal peptide, predicted based on the SignalP 3.0 Server48, the mature PfCpn60 should have 46 extra residues at the N-terminus before G69, and 87 extra residues at the C-terminus after E631. It is interesting to notice that there are 51 negatively charged residues of Asp + Glu at its C-terminus. The extra residues protruding from the equatorial domain towards the inner cavity in the wild-type PfCpn60 certainly block free passage between the cavities in both rings. The negative charges inside the equatorial ring in wild-type PfCpn60 may also prevent double-ring formation. Further study of PfCpn60 in the native state may be essential to fully understand the structure and mechanism of PfCpn60.As in all other Cpn60 structures, both 45. The triplet residues in PfCpn10 and L31-F32-L33 are comparable to the corresponding triplets I31-M32-L33 in human mitochondrial Cpn10 and I25-V26-L27 in GroES30. The corresponding interacting residues such as L303, E304, and L328 in Cpn60 are also conserved . Both structures are American football-shaped Cpn6014Cpn1010 structures bound with ADP. In the X-ray crystallographic structure (4PJ1), the apical domains in two of the subunits (subunit G in one ring and subunit N in the opposite ring) are very different from the conformations of the apical domains in other intra-ring subunits, exhibiting as large as 100° of counterclockwise rigid body movement of the apical domain compared to those in other subunits17. However, in the structure (6MRC) determined by cryo-EM, no such asymmetry phenomenon was observed, and all subunits are in a similar conformation. The breakage of perfect seven-fold symmetry for intra-ring subunits was reported previously but to a milder degree. In the structures of apo GroEL and ATP-bound GroEL (PDB: 1OEL and 1KP8), the conformational variations of the apical domains relative to the equatorial domains among the intra-ring subunits were apparent, with spreads of 5° and 7° for apo and ATP-bound states, respectively41. Because no distinct gap between these two states was observed, it was realized the variations were likely induced by different packings due to different lattices (C2221 for 1OEL and P21 for 1KP8). Similarly, the apical domain in the cis-ring of the bullet-shaped TtCpn6014Cpn107 complex exhibited large deviation from the seven-fold symmetry20. The conformational variations of the apical domain in group 1 chaperonins are likely to be intrinsic to encapsulate different SPs rather than a single SP.The tetradecameric assembly of PfCpn60 is mainly held together by the subunit-subunit interaction of the equatorial domains. The apical domain is highly dynamic; its position is affected easily by nucleotide binding, protein substrate binding, and potentially the crystal packing environment. Asymmetry with subunits in a differing apical domain conformation has been observed in several Cpn60 crystal structuresPlasmodium falciparum (isolate FCR-3/Gambia) mitochondria Cpn60 was retrieved from Uniprot entry number P34940 and then aligned to the crystal structure sequence of E. coli GroEL (PDB: 1AON). The flanking N and C terminal residues of P. falciparum Cpn60 were truncated to the alignment of E. coli GroEL. A D474A mutation was made in accordance to the ATP hydrolysis-deficient GroEL mutant (PDB: 3WVL)45 to inhibit ATP hydrolysis of Cpn60 and stabilize the complex for purification. Normal ATP binding is retained but ATP hydrolysis occurs at a 0.1% rate14. The mutant follows the same cycle as the wild-type but at a much slower rate, allowing for the observation of more transient intermediates. The overall structure of GroEL appears largely unaffected by the ATP hydrolysis-deficient mutation when compared to the wild-type football complex (PDB: 4PKO) with a rmsd value between the superimposed structures of 1.79 Å. The sequence was then synthesized by GenScript and cloned into a pET-28a vector with kanamycin antibiotic resistance genes. The sequence of mitochondria Cpn10 from P. falciparum was retrieved from Uniprot entry Q50JA6, synthesized by GenScript, and cloned into a pET-28a vector with the same antibiotic genes and a six-histidine tag. Expression of both constructs was accomplished by transformation of the plasmid into BL21 Rosetta (DE3) E. coli competent cells followed by initial culture growth of the transformed cells into a 50 mL flask with LB buffer and antibiotics. 20 mL of the initial culture was transferred into 1L LB flasks, incubated for 3 h at 37 °C, and induced with the addition of 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). After overnight induction at 18 °C, the cell culture was spun down at 4000g for 15 min. The pellet was resuspended with 25 mL of buffer solution A containing 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM KCl, and 10 mM MgCl2. A tablet of Pierce Protease inhibitor cocktail, 0.1 mM of PMSF, 0.1% (v/v) β-mercaptoethanol, and 5 mg of lysozyme was also added to the lysis buffer. Following sonication, the lysed cells were pelleted by centrifugation at 55,000g for 20 min. The supernatant of Cpn10 was purified using nickel-his affinity chromatography with two 10 mL washes containing buffer solution A and 10 mM imidazole. The protein was eluted from the nickel resin with 15 mL of buffer solution A containing 500 mM imidazole. The purified protein was concentrated to 1 mL using a 3 kDa cutoff Centricon and further purified with size-exclusion chromatography into buffer solution A. An average of 25 mg of Cpn10 protein was yielded from 1 L of culture. 1 mM of ATP was added to the supernatant of Cpn60 and incubated for an hour at 4 °C. 25 mg of purified Cpn10 was then mixed with the supernatant of Cpn60 and incubated for 1 h at 4 °C for complex formation. To purify the complex, a nickel-his pulldown was performed with the same procedure as the nickel-his affinity purification for Cpn10. The eluted complex was concentrated to 1 mL in a 10 kDa cutoff Centricon and further purified using size exclusion chromatography in buffer A. Fractions containing the complex were pooled and then concentrated to 20 mg/mL in a 100 kDa cutoff Centricon to remove unbound Cpn10 and used for crystallization.The full sequence of http://www.mrc-lmb.cam.ac.uk/kzhang/) and 2D classes were generated using RELION49.The sample quality and structural features were assessed by negative stain transmission electron microscopy in a Thermo Scientific Tecnai T20 microscope equipped with a charge-coupled device (CCD) camera. The protein samples (100 ng/mL) were applied to fresh plasma-cleaned carbon-coated grids (Quantifoil), followed by negative staining with 2% (w/v) uranyl acetate. Particles were auto-picked using Gautomatch  isopropanol. Optimization of conditions was then performed in 24-well hanging drop trays with a 1:1 ratio of 1 µl of protein to well buffer. After optimization of crystallization conditions 0.2 mm hexagonal prism shaped crystals grew in phosphate citrate buffer pH 4.4, 13% (v/v) isopropanol, and 0.21 mM of lithium sulfate. Crystals were cryoprotected with 30% (v/v) glycerol and then immediately submerged in liquid nitrogen for shipment to Argonne National Laboratory for data collection. X-ray diffraction data were collected at 100 K on beamline SERCAT 22-ID at the Argonne National Laboratory, Argonne, IL, USA through remote access mode. The data was recorded with an Eiger 16 M detector, processed, and scaled with the XDS package51 in Phenix package34 using the Cpn60 structure from T. thermophilus  as the search model20. The structure was manually built using graphic package COOT52 and refined using Phenix package53. During refinement, sevenfold NCS torsional-angle restraints with three NCS groups consisting of the equatorial domain (residues 69–203 and 486–631), intermediate domain (residues 204–255 and 452–485), and apical domain (residues 256–451) were applied. Secondary structure restraints with Ramachandran restraints and planar peptide restraints were also applied. In the final cycle of refinement, the TLS refinement with three TLS groups  per chain, for a total of 21 TLS groups, was performed. The final Rwork and Rfree were 22.7% and 27.7%, respectively, with reasonably good geometry (Table The structure of Cpn60mt-D474A was determined by molecular replacement with the program PhaserSupplementary Information.


Wheat is a major component of the Northern European diet and contributes significantly to dietary pesticide exposure. Here we report results of a 2-year retail survey, which compared pesticide residues in organic and conventional, whole-grain and white, common and Spelt wheat flour brands available in the UK and Germany. Pesticide residues were detected significantly more frequently in conventional (87%) than organic (25%) flour samples. Chlormequat, a plant growth regulator, was the most frequently detected compound. Total concentrations of pesticide residues were (a) ~4 times higher in conventional than organic, (b) ~100% higher in common than Spelt wheat flour and (c) ~110% higher in conventional whole-grain than white flour samples, but (d) not significantly different in organic whole-grain and white flour. Results suggest that the use of organic wheat products allows increased whole-grain cereal consumption in line with nutritional recommendations, without an increase in dietary pesticide intake. A recenTriticum spelta) was widely used in Northern Europe in the past, but is now considered a minor cereal. Spelt is currently increasing its market share and this is thought to due to (a) its ability to grow under low input conditions (which make it particularly suitable for organic farming systems) and (b) consumer perceptions that spelt wheat has a higher nutritional value than common wheat  and post-harvest processing (white vs whole-grain flour) on pesticides loads.Hypotheses:The use of organic production methods results in lower pesticide residues in wheat flour than conventional production protocols. The effect of using organic production methods on pesticide residue levels is augmented/confounded by (a) crop genetic and (b) postharvest storage/processing/milling factors.The main objectives of this study were therefore to (a) compare pesticide loads in white and whole-grain common and Spelt wheat flour brands/products available in the UK and Germany (b) quantify the effect of contrasting primary production methods  on pesticides loads in wheat flour and (c) identify potential interactions between primary production protocols, wheat species (22.1T. aestivum vs T. spelta), (3) farming system  and (4) flour type (white vs wholegrain).The retail survey of wheat flour for pesticide residue analysis was conducted over two successive years (2016 and 2017) in the UK and in Germany. The experimental design included 4 factors/variables: (1) country (UK and Germany), (2) wheat species  being used for each combination of wheat species, farming system and flour type in each year. This was primarily done to avoid pseudo-replication, since the use of more than one sample per brand could have resulted in both flour samples originating from the same batch of grain used by millers; different brands were assumed to have been made by different mills or at least from different grain batches  and Germany  and websites in UK  in the same period in each year  in 2016 and Concept Life Sciences Ltd.  in 2017 using validated extraction methods and GC-ECD, GC/MS and LCMSMS based analytical protocols. Further information on the analytical protocols is provided in the 2.3‘nlme’ package in R .Analysis of Variance (ANOVA) derived from linear mixed-effects (lme) models  were useage in R . Data onp < 0.05) interactions between factors, general linear hypothesis tests (Tukey contrasts) were performed using the ‘glht’ function of the ‘multcomp’ package (‘wilcox.test’ command in R) were used as a supplementary test to assess whether two sample means are equal or not. Means and standard errors of means for the main effect and the interaction effect tables were generated by using the ‘tapply’ function in R. When analysing the mean concentrations of pesticides, the concentration in negative samples  were set at half the concentration of the limit of detection of the analytical method as recommended in previous studies  concentration of cypermethrin residue in some positive samples were below the limit of quantification set by the laboratory and not available and (b) the detection limit for glyphosate was different in the analytical methods used in 2016 and 2017.3In 2016, eight active compounds used in crop protection products (CPPs) were detected in the 113 flour samples including deltamethrin, chlormequat, piperonyl butoxide, pirimiphos methyl, alpha-cypermethrin, pendimethanil, tebuconazole, and mepiquat. In 2017, seven active compounds used in CPPs were detected in 147 flour samples assessed, including deltamethrin, chlormequat, piperonyl butoxide, pirimiphos methyl, 2-phenylphenol, glyphosate, and chlorpyrifos methyl see .3.1The only CPP residues detected in a substantial proportion of flour samples were chlormequat (detected in 35% of flour samples) and piperonyl butoxide (detected in 20% of flour samples). Other CPP residues detected in a smaller proportion flour samples included glyphosate (detected in 14% of samples), 2-phenylphenol (detected in 8% of samples) and cypermethrin (detected in 13% of samples), and pesticides found in only very few samples included pirimiphos-methyl (detected in 5% of samples), deltamethrin (detected in 4% of samples), mepiquat (detected in 2% of samples), pendimethanlin , tebuconazole (detected in 2% of samples), and chlorpyrifos methyl (detected in 0.1% of samples).Significant main effects were detected only for farming system . The perFor chlormequat (CCC) a weak, but significant interaction between country and farming system was detected. The difference in the proportion of positive samples between organic and conventional flour was greater in the UK (5% vs 91%) than in Germany (0 vs 47%) .Table 2IFor piperonyl butoxide a weak, but significant interaction between wheat species and farming system was detected . For Spe3.2Sufficient numbers of samples with quantifiable concentrations of individual CPPs to obtain normally distributed data for ANOVA were only available for chlormequat. As a result, factorial ANOVAs to compare the proportion of positive samples and CPP concentrations between wheat species, countries, farming systems and flour types could only be carried out for chlormequat and the estimated mean total concentration of CPP residues  . ResultsFor both chlormequat and total CPP concentrations, significant main effects were detected for all four factors included in the two different 3-factor ANOVAs carried out . SignifiFor both chlormequat and total CPP residues, ANOVA detected significant two-way interaction between (a) flour type and farming system, (b) country and farming system . SignifiFor total CPP residues, ANOVA also detected significant two-way interaction between (a) wheat species and farming system . Signifi44.1Retail surveys such as the one reported here can provide an accurate estimate of (a) the mean nutritional composition of different types of flour available to consumers and (b) the effect of using certified organic production methods on pesticide residue levels in flour. However, information on location of farms, crop varieties and specific agronomic practices used by organic and conventional farmers and post-harvest drying, storage, cleaning, and milling protocols used for organic and conventional grains were not recorded and it was therefore not possible to assess to what extent these parameters affected the flour composition parameters measured. In the discussion we have therefore related the results of the survey reported here to previous experimental and farm survey based studies that focused on identifying climatic, crop genetic, agronomic, grain processing and quality assurance related parameters affecting pesticide residue levels in crops.4.2The CPP residue present at detectable levels in wheat flour were (a) the plant growth regulators chlormequat and mepiquat (both quaternary ammonium compounds), (b) glyphosate (an organophosphorus herbicide and crop desiccant), (c) pendimethalin (a dinitroaniline herbicide), (d) chlorpyrifos-methyl and pirimiphos-methyl (both organophosphorus insecticides), (e) deltamethrin and alpha-cypermethrin (both synthetic pyrethroid insecticides), (f) piperonyl butoxide , (g) tebuconazole (a triazole fungicide) and (h) 2-phenylphenol  . These cAlthough residues below the maximum residue level (MRL) set for specific active ingredients in crop protection products (CPPs) are considered safe by regulators, concern have recently been raised about negative public health impacts of chronic dietary exposure to below MRL-levels of CPPs . These cNearly all CPPs detected in wheat flour in this study  are suspected or confirmed EDCs .in vitro and animal studies demonstrating additive effects of exposure to mixtures of EDCs , and total and multiple CPP residues. Considering that (a) cereal products account for between a quarter (e.g. in Germany and the UK) and half (e.g. in China and India) of daily calorie intake  and (b) Recent epidemiological studies from the US suggest that this may particularly apply to reproductive health, since reduced dietary intake of CPPs via fruit and vegetables was reported to be linked to higher sperm quality in men  and highCCC is used in cereals production to reduce stem length/longitudinal shoot growth and thereby increasing lodging resistance and improving harvest index and yield . The MRLThe application of chlormequat, piperonyl butoxide and cypermethrin (which were detected as residues in organic flour samples) to cereal crops is prohibited under organic farming standards . HoweverThe finding that a higher proportion of organic (25%) than conventional (5%) flour samples tested positive for residues of alpha-cypermethrin residues should be further investigated. This together with the finding of piperonyl-butoxide (an activity enhancer often uses in pyrethroid formulations) in 12% of organic flour samples may indicate the use of synthetic pyrethroid products in the field or in grain storage facilities. Different to pyrethrum-CPPs , synthetic pyrethoid-based CPPs are not approved for use in organic farming .4.4However, the finding that Spelt wheat flour had only half the CCC and total CPP residue concentration compared with common wheat flour, could have been due to either (a) fewer CPPs having been applied to Spelt crops and/or (b) the husk surrounding the Spelt grain having provided partial protection against the grain becoming contaminated. Spelt wheat is often described as a more stress resistant, robust species than modern common wheat cultivars . Also, pA significant difference in CPP residues between Spelt and common wheat was only detected when conventional flour samples were compared, which suggests that switching from common to Spelt wheat consumption would result in a 50% reduction the total dietary CPP residue intake only for conventional but not organic food consumers.4.5There is increasing evidence for significant health benefits of switching from white to whole-grain cereal consumption . HoweverIn contrast, CCC and total CPP residues concentrations in organic flour were not only substantially lower, but also similar in whole-grain and white flour. This clearly demonstrates that organic whole-grain cereal consumption allows the nutritional benefits of whole-grain consumption phenol/antioxidant intakes) to be achieved without a simultaneous increase in dietary exposure to pesticide residue.4.6The slightly higher CPP residues in conventional UK compared with German wheat may suggest that the CPP spraying regimes used in Germany are less intensive than those used in the UK. However, in retail surveys such as the one reported here it is not possible to obtain information on the agronomic protocols and especially crop protection practices used to produce the wheat grain from which the flour was made. Also, a proportion of the grain used to produce flour is imported in both Germany and the UK, but for most brands it was not possible to identify whether or not they used imported grain, from the information provided on the label. As a result, it was not possible to identify the reasons for the differences in CPP-residue levels between the two countries.5Results from this study support the hypothesis that organic cereal production methods result in significantly lower pesticide residues in wheat flour and suggest that switching to organic wheat flour product may substantially reduce dietary exposure to total CPP residues. However, since residue levels in all retail flour products were below the MRLs set for specific active ingredients/substances used in CPPs they are currently considered safe by EU and national regulators.Results also provide evidence for the hypothesis that crop genetic and postharvest processing parameters affect and/or augments the impact of primary production protocols  on pesticide residue levels. Most importantly, whole-grain flour was shown to have substantially higher pesticide residues than white flour, but only when conventional flour samples were compared.Due to the uncertainty about the potential effects of exposure to mixtures of CPP residues and the lack of testing for epigenetic effects of exposure to endocrine disrupting CPP there is an urgent need to investigate the potential physiological and health impact of reducing pesticide exposure via switching to organic food consumption. There is a particular need for controlled dietary intervention studies with animal models and humans, to provide a mechanistic understanding for associations between organic food consumption and positive health outcomes associated with organic food consumption reported in recent human cohort studies .Juan Wang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Gultakin Hasanalieva: Investigation, Methodology. Liza Wood: Investigation. Christos Anagnostopoulos: Investigation, Methodology, Writing - review & editing. Georgios Ampadogiannis: Methodology, Writing - review & editing. Eleftheria Bempelou: Investigation. Maroula Kiousi: Investigation. Emilia Markellou: Conceptualization, Writing - review & editing. Per Ole Iversen: Writing - review & editing. Chris Seal: Methodology, Project administration, Resources, Supervision, Validation, Writing - review & editing. Marcin Baranski: Data curation, Formal analysis, Validation. Vanessa Vigar: Writing - review & editing. Carlo Leifert: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - original draft, Writing - review & editing. Leonidas Rempelos: Data curation, Formal analysis, Investigation, Supervision, Visualization, Writing - original draft, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The calponin homology (CH) domain is one of the most common modules in various actin-binding proteins and is characterized by an α-helical fold. The CH domain plays important regulatory roles in both cytoskeletal dynamics and signaling. The CH domain is required for stability and organization of the actin cytoskeleton, calcium mobilization and activation of downstream pathways. The CH domain has recently garnered increased attention due to its importance in the onset of different diseases, such as cancers and asthma. However, many roles of the CH domain in various protein functions and corresponding diseases are still unclear. Here, we review current knowledge about the structural features, interactome and related diseases of the CH domain. Actin is an essential cytoskeletal protein that plays a critical role in multiple cellular processes . Actin mRecent studies have reported that CCPs, including molecule interacting with CasL (MICAL), leucine-rich repeats and calponin homology containing 4 (Lrch4), smoothelin-like 1 (SMTNL1) and transgelin-2, exhibit an unappreciated functional variety and play important roles in the onset of various diseases . However10-helical turn is also present in the loop between helices IV and V. Three helices of the CH domain of calponins  form a triple-helix bundle, and helix I binds at a right angle across the surface provided by helices III and VI  than wild-type utrophin (KD = 19 μM) (in vitro co-sedimentation assays (not in live cells) showed that the same truncated utrophin binds to F-actin 30 times weaker than the full-length protein  in the linker region between the CH1-CH2 domain of dystrophin acts like a swivel to allow these conformational transitions . After t= 19 μM) . Moreove= 19 μM) . However protein .The functional diversity of different CH domains is a result of different binding partners including actin, tubulin and signaling proteins as described below .The exact mechanisms regulating binding between actin and the CH domain are still unclear. This process is associated with complex factors, including the CH domain number (single or a tandem pair), conformational differences, and flanking sequences . The CH Residue mutations can also affect binding between the CH domain and F-actin. For example, the CH2 domain mutation (L253P) of β-III-spectrin caused opening of the CH1-CH2 domains and promoted the N-terminal region of CH1 to become α-helical, thus enhancing approximately 1000-fold the actin-binding affinity . In contWhether a single CH domain binds actin is still controversial. By constructing calponin without C-terminal tandem repeats, the resulting protein with the CH domain failed to bind to actin , suggestThe CH domain can also bind other cytoskeletal proteins such as tubulin. The end-binding protein 1 (EB1) is the first example of a single CH domain that can associate with tubulin . A truncD obtained by isothermal titration calorimetry was 2.7 × 10–6 M  and calmodulin . The CH × 10–6 M . SMTNL1 × 10–6 M . However× 10–6 M . Removal× 10–6 M . However× 10–6 M .For other CCPs, the N-terminal 53–271 residues of affixin that cover the CH1 domain but not the CH2 domain are the binding sites of Cdc42/Rac1-specific guanine nucleotide exchanging factor (αPIX), as shown using co-immunoprecipitation assays . TransgeCCPs, including MICAL1/2, Lrch4 and SMTNL1, have been shown to play crucial roles in various diseases as discussed below. A summary of the involvement of CH domains in various diseases is shown in MICAL1/2 contains a CH2 domain and oxidizes methionine residues of actin to disassemble F-actin into G-actin . The MICStudies have shown that MICAL proteins are closely related to neural diseases and cancers. MICAL expression is substantially elevated in oligodendrocytes and in meningeal fibroblasts during spinal cord injury, suggesting an involvement of MICAL in neuronal regeneration . TargetiLrch4 is a plasma membrane protein abundantly expressed in the spleen and thymus, containing a single-pass transmembrane domain with nine leucine-rich repeats and a CH3 domain in its ectodomain . Recent Mycobacterium avium subsp hominissuis (Lrch4 is a novel Toll-like receptor (TLR) accessory protein as Lrch4 knockdown attenuated TNFα secretion induced by various TLR ligands . Therefoinissuis . Microarinissuis .Smoothelin-like 1, which contains a CH2 domain in the C-terminal region, is a novel member of the smoothelin protein family . DeletioSmoothelin-like 1 can modulate muscle contractility, and its biological activity may involve interaction with the contractile actin machinery . One of in vitro experiments using recombinant proteins only, it should be noted that due to the complexities of the cellular environment, other factors could be coming into play and distorting the results. With newly discovered proteins that interact with CH domains, some of the diverse functions have now been elucidated. However, many other binding proteins and functions certainly remain to be discovered, thus warranting further research into CH domain biology. Given that numerous CCPs, such as MICAL and transgelin-2, have been identified as promising therapeutic targets in diseases, it will be important to investigate in the future whether compounds can be designed to specifically target CH domains and thus improve the outcome of certain diseases.The CH domain displays high structural conservation, but shows diverse biological functions. The indispensable flanking regions and/or intrinsically unfolded protein modules may contribute to orchestrating CH domain functions. However, when comparing cell-based experiments with L-MY designed the work, wrote the manuscript, and prepared the figures. MS and C-DJ drafted and revised the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.