On the contrary, both backyard and intensive flocks were affected by aMPV subtype B. Potential epidemiological links associated to the importation of parental birds from foreign countries could be established. These results highlight non-negligible circulation of these viruses, warranting further epidemiological studies and the evaluation of control measure implementation.Poultry production plays a relevant role in the Ethiopian economy and represents a source of poverty alleviation for several social classes. Infectious diseases can therefore significantly impact the economy and welfare. Despite infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) being present, the knowledge of their epidemiology and impact is extremely limited. In the present work, a cross-sectional study based on 500 tracheal swabs collected from 50 intensive and backyard unvaccinated flocks of the Jimma Zone was performed to investigate the circulation of these viruses and molecularly characterize them. IBV and aMPV presence was tested by molecular assays, and genotyping was carried out on positive samples. Accordingly, 6% (95% CI 2.06% to 16.22%) and 8% (95% CI 3.15% to 18.84%) of flocks tested IBV and aMPV positive, respectively. Particularly, IBV 793B (GI-13) strains were detected in backyard flocks only, and identical or closely related sequences ( Infectious bronchitis (IB) is a worldwide-distributed, acute, highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is characterized by lesions in the respiratory, reproductive, and urogenital organs ,2,3,4. ICoronaviridae, order Nidovirales, genus Gammacoronavirus, species Avian coronavirus [IBV belongs to the family onavirus ,8. Its gonavirus . Additioonavirus ,11. AmonThe spike protein is cleaved into subunits S1  and S2  . The S1 The IBV distribution and diversity differ remarkably among genotypes ; for exaIn Africa, IBV is one of the most important viral diseases that threaten chicken production . So far,Avian metapneumovirus (aMPV) has been much less investigated. However, recent studies have indicated the increasing role of aMPV in respiratory diseases in broiler farms, mainly as a primarily pathogen [Avian Metapneumovirus belongs to the family Pneumoviridae and genus Metapneumovirus, causing diseases like turkey rhinotracheitis in turkeys [pathogen . Avian M turkeys , swollen turkeys , and a d turkeys .Avian metapneumovirus contains a non-segmented, single-stranded, negative-sense RNA genome of approximately 13 kb in length, organized as 3′ leader N P M F M2 SH G L trailer 5′ [ailer 5′ . After iailer 5′ , analysiailer 5′ . aMPV-A ailer 5′ ,36, and ailer 5′ .IBV and aMPV’s epidemiology, strain diversity, molecular characterization, and regional distribution have not been comprehensively reported in Africa, particularly in Ethiopia .However, there is no doubt among veterinarians about the economic importance of IBV and aMPV in the Ethiopian poultry industry. Therefore, the purpose of this study was to molecularly characterize aMPV and IBV in Jimma Zone and determine their genetic relationships with previously known strains using sequence data analysis.Oromia Regional State is the largest state in Ethiopia, hosting nearly half of the country’s population and livestock, including poultry. The majority of the commercial poultry farms and breeding centers in Ethiopia are located in this region. Jimma is the capital city of Jimma Zone, which is located in Oromia Regional State. The total population of Jimma Zone is 2,642,114, of which 2,204,225 (88.66%) are members of rural communities engaged in agricultural activities for their livelihood. Jimma Zone is, therefore, a remarkable source of livestock which contribute to the country’s growth and domestic production: about 466,154 sheep, 194,677 goats, 1,718,284 cattle, 40,555 donkeys, 30,541 mules, 74,774 horses, and 1,774,116.36 chickens are kept in this area.https://epitools.ausvet.com.au/). Commercial poultry farms in Jimma city and backyard production systems from Jimma Zone were stratified according to production type, breed, and agro-ecological region.A stratified cross-sectional study design was used in line with the primary objectives of this study. The sample size for pooled samples was calculated with the aim to estimate the infection prevalence with a confidence level of 95% and precision of 5%, assuming a conservative prevalence of 15%. Therefore, a minimum of 45 pooled samples was determined . The samFrom the selected poultry farms, 10 tracheal swabs were randomly collected, air-dried to prevent bacterial and mold growth, put in Eppendorf tubes, and transported to the Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) Molecular Biotechnology Laboratory for storage at −80 °C. Tracheal swabs with dry ice were shipped to the Veterinary Infectious Disease Laboratory, Department of Animal Medicine, Production and Health, Padova University, Italy for PCR, sequencing, and genotyping. Whenever possible, the following data were collected: sampling date, farm geolocalization, animal category (broilers or layers), breed, age, presence of clinical signs, production (intensive and extensive) and housing system , population size, and vaccination used.Overall, tracheal swabs were taken from a total of 500 chickens located in 50 farms  during January and February 2020.Risk factors potentially associated to IBV and aMPV infection  were assessed via logistic regression, conducted using SPSS version 20 software.Individual samples were added to 500 µL of PBS and thoroughly vortexed. Then, 100 µL was collected from each of the 10 resuspended swabs collected in the same farm and pooled. The RNA was extracted from 200 µL of the obtained solution using the High Pure Viral RNA Kit  following the manufacturer’s instructions. The pools were tested for IBV presence using the SuperScript™ III Platinum™ One-Step RT-PCR Kit , amplifying a 464 bp hyper-variable region of the S1 gene using the method described by Cavanagh et al.  with somhttp://www.geospiza.com), and consensus sequences were assembled using ChromasPro . The Ethiopian IBV sequences obtained in the present study were aligned with the reference strains obtained from Valastro et al. [Chromatogram quality was evaluated using FinchTV . Handling of the study animals throughout the study period was according to the World Organization for Animal Health (OIE) animal welfare guidelines.P = 0.436), chicken breed (P = 0.053), farm type (P = 0.086), or flock size (P = 0.257) was identified on IBV and aMPV infection frequency using linear regression. IBV-positive results were found in three backyard production systems only  = 19), enteric symptoms (n = 25), watery eyes or mucus in the nares (n = 15), lameness and crooked neck (n = 5), and blindness (n = 1). Clinical signs were not detected in the IBV-positive flocks; however, the intensive farms positive for aMPV showed respiratory signs. No documented data indicated either backyard or commercial farm vaccination against IBV or aMPV among the farms included in this study.The geographical distribution of the farms is reported in Sequence analysis of the hypervariable region of the S1 gene  showed tComparison of the obtained sequences with the reference ones demonstrated a relatively high percentage of identity with the oldest known isolate of this genotype, EU914938-Morocco-1983 GI 13 (nucleotide identity of 95.4%), as well as with Z83975 United Kingdom 1991 GI-13 (nucleotide identity of 98–99%) and JQ739375|China-2011-GI-13 (nucleotide identity of 98–99%). However, when a broader 793B dataset was evaluated, a closer phylogenetic relationship was observed with strains collected in India and China .Especially in the small-scale village context, chicken farming can significantly contribute to poverty alleviation by means of income generation and household food security ,45, and Different from the serological survey by Tesfaye et al., only genotype 793B  was fouThe 793B genotype frequency in unvaccinated backyard farms was 6%, lower than in most previous studies—16.04% in Oman , 40.62% Although a low IBV infectious pressure cannot be excluded in Ethiopia, the high seroprevalence levels observed in previous studies seem to contradict this hypothesis. Many factors could have contributed to the low detection ratio, including the low animal/farm density and the old animal age. In fact, most animals were some months old, while IBV infection often occurs in early life phases. Therefore, previously acquired natural immunity could have decreased the infection frequency in the tested population. Younger animals could represent a future research target to investigate whether IBV infection is common in early life phases and to evaluate its impact and the potential usefulness of extensive vaccination protocol implementation.The dominance of 793B is not surprising, considering its widespread use as a vaccine ,56,57,58The 793B strains detected within the backyard flocks shared a high nucleotide sequence identity , and the phylogenetic analysis based on the S1 hypervariable gene sequence demonstrated a certain clustering, indicating that closely related strains may be circulating between different, distantly located, Ethiopian backyard flocks and even regions .In the absence of strict biosecurity measures, several spreading sources could be involved, including wild birds, wherein IBV-like viruses have been identified from time to time. This is particularly true in the case of backyard chickens that have access to the outdoors and live in close contact with wild birds ,60. TherComparison of the S1 hypervariable gene sequence of Ethiopian 793B strains demonstrated a percentage of identity higher than 96% with most reference strains collected from Europe, including the ancient French isolate AJ618985_France-1985-GI 13, but especially with Asian strains. Parental chicken stocks were imported into Ethiopia beginning in the early 1950s .Although genetically distinct, the relatively close relatedness demonstrated on the basis of sequence analysis with commercial homologous vaccines might indicate a higher chance of good protection . TherefoThe results reported in this study evidenced the presence of aMPV in commercial and backyard farms. The viral load of aMPV was extremely low (cp of ~40) and no sequencing was possible. However, the use of specific probes allowed us to identify the aMPV-B subtype. aMPV-B was detected in both production systems (intensive and backyard) in animals raised for dual purposes (egg and meat production). Previously, subtype aMPV-B was reported in central Ethiopia in institutional farms . In thisThe present study demonstrates the relevant circulation of two main poultry pathogens in Ethiopia and provides a molecular characterization of IBV strains. Since the old age of tested animals could have decreased infection prevalence and disease occurrence, the pathogens’ economic impact might be considered even more intense. Commercial vaccines against the reported genotypes and subtypes are widely available. This information, combined with previous study results, will be of help in the implementation of vaccination strategies directed toward the actually circulating genotypes, instead of generic ones. In the future, more focused studies should be performed to investigate the impact of these strategies and their potential benefits for the Ethiopian poultry sector’s productivity and overall economy and welfare.


S-acyltransferase family comprises 23 enzymes that mediate the S-acylation of a multitude of cellular proteins, including channels, receptors, transporters, signaling molecules, scaffolds, and chaperones. This reversible post-transitional modification (PTM) involves the attachment of a fatty acyl chain, usually derived from palmitoyl-CoA, to specific cysteine residues on target proteins, which affects their stability, localization, and function. These outcomes are essential to control many processes, including synaptic transmission and plasticity, cell growth and differentiation, and infectivity of viruses and other pathogens. Given the physiological importance of S-acylation, it is unsurprising that perturbations in this process, including mutations in ZDHHC genes, have been linked to different neurological pathologies and cancers, and there is growing interest in zDHHC enzymes as novel drug targets. Although zDHHC enzymes control a diverse array of cellular processes and are associated with major disorders, our understanding of these enzymes is surprisingly incomplete, particularly with regard to the regulatory mechanisms controlling these enzymes. However, there is growing evidence highlighting the role of different PTMs in this process. In this review, we discuss how PTMs, including phosphorylation, S-acylation, and ubiquitination, affect the stability, localization, and function of zDHHC enzymes and speculate on possible effects of PTMs that have emerged from larger screening studies. Developing a better understanding of the regulatory effects of PTMs on zDHHC enzymes will provide new insight into the intracellular dynamics of S-acylation and may also highlight novel approaches to modulate S-acylation for clinical gain.The human zDHHC  Proteins are essential functional and structural components of living organisms, and an estimated 20,000 protein coding genes have been identified in higher organisms, such as humans  and miceS-Acylation represents one of over 200 different types of PTMs , oleate (C18:1), and other longer chain fatty acids can also be utilized to a lesser extent  that contains a catalytic DHHC (apartate-histidine-histidine-cysteine) tetrapeptide , which are important to maintain the structural integrity of this domain .Traditional labeling and detection methodologies have identified PTMs that regulate many cellular proteins, albeit with a bias toward phosphorylation. Although these traditional approaches have been hugely valuable, they are time-consuming and often focus on detailed analysis of single sites of modification within a single protein of interest. In recent years, the vast increase in the use of -omics technologies to investigate cellular processes and pathways together with prediction algorithms have produced a wealth of information on the different chemical modifications that have been detected or are predicted to occur on cellular proteins. Whereas this has provided a detailed catalogue of PTMs on any single protein, it is important to note that the majority of these lack validation, and the stoichiometry of modification is unclear. The relative abundance of different PTMs in human and mouse zDHHC enzyme isoforms is highlighted in Fig. 1lization –56. SpecA). Ubiquitination is also detected on many zDHHC enzymes, whereas lysine acetylation and S-acylation are prominent in the human and mouse enzyme families, respectively. The latter is likely a consequence of the divergent proteomics screening efforts that have been, to date, conducted in these species. In both human and mouse studies, arginine and lysine methylation of zDHHC enzymes have been reported less commonly . Of the 23 zDHHC enzymes, current data suggest that zDHHC5 may be the most extensively modified, exhibiting eight different PTM types in humans and five different PTM types in mice. In the human isoform, phosphorylation and ubiquitination are the predominant modifications. However, a caveat when comparing PTMs of different zDHHC enzymes is that detection will be influenced by the relative abundance of these enzymes in the source tissue.Globally, phosphorylation represents the most studied PTM, which has also been found to be highly abundant in the human proteome . TherefoFig. 18 by ataxia telangiectasia and Rad3-related kinase enhanced its interaction with the substrate protein melanocortin 1 receptor (MC1R)   , 58. S-Apathways . Further in mice . zDHHC13 in mice . However 43–277) , and thu59 in zDHHC13 is also modified by phosphorylation (67) is part of the substrate interaction site of this enzyme. Moreover, a tyrosine-to-alanine substitution resulted in a 5-fold drop in the affinity of zDHHC17 for the SNAP25 substrate protein 59 in zDH18 by fibroblast growth factor (FGF) receptor and Tyr295/Tyr297 by Src kinase . However, following neuronal stimulation, the zDHHC5-Fyn interaction is reduced, allowing dephosphorylation of Tyr533 and subsequent recruitment of the μ subunit of the clathrin adaptor AP2, followed by clathrin-mediated endocytosis of zDHHC5 and co-localization with δ-catenin (A). Interestingly, the C-terminal tail of zDHHC5 appears to exhibit extensive serine and threonine phosphorylation  and Fyn kinase . Under tse sites . How doeic motif , 64 . Analysis of the localization of mouse zDHHC2 in neuroendocrine PC12 cells and hippocampal neurons identified two potential endocytic signals in the C-terminal tail, which when mutated led to enhanced accumulation at the plasma membrane: (i) a nonconventional dileucine motif  at amino acid positions 330–335, which differs from the conventional acidic (D/E)XXXLL motif that interacts with clathrin adaptors and (ii) an Asn-Pro (NP) motif at amino acid positions 357 and 358  at Ser330 led to increased abundance of zDHHC2 at the plasma membrane, whereas a phosphomimetic mutant (aspartic acid substitution) had no effect . The key serine of the SXXXLL motif (Ser330) has been reported as a phosphorylation site in a single phosphoproteome study of mouse pancreatic cells, whereas modification of Ser341 and Ser357 in human zDHHC2, which are present at the same position as Thr340 and Ser356 in mouse zDHHC2, has been identified in multiple mass spectrometric studies S-acylatio effect . Intereso effect . Specifi studies –71 , with reported phosphorylation at Ser446 in zDHHC14 and Ser330 of zDHHC20  motif is a conserved feature of zDHHC enzymes (TXE motif (Thr241) and the aspartate of the catalytic DHHC tetrapeptide (Asp153) (C). Importantly, alanine substitution of the two threonine amino acids to form an AAXE mutant was associated with a marked reduction in enzymatic activity (XE) motif, where a similar stabilizing hydrogen-bonding interaction between the serine and the DHHC aspartate can be predicted . Interestingly, there is proteomics evidence to support the phosphorylation of Ser552 in the TSXE motif of human zDHHC13 . This hypothesis was strengthened by modeling a phosphothreonine PTM into the zDHHC20 crystal structure TTXE motif. This model shows that hydrogen bonding between Asp153 and the phosphorylated form of Thr241 appears to be precluded . Thus, phosphorylation of the TSXE motif in zDHHC13 might be a novel mechanism of enzyme regulation, although further experiments are required to validate this phosphorylation site and the possible effects of this modification on zDHHC13 activity.Analysis of other reported phosphorylation sites in zDHHC enzymes highlights further potential for structural/conformational regulation. The C-terminal Thr-Thr-Xaa-Glu XE) motif(Asp153)  Fig. 3CCXE motifactivity . Some zDXE motif in zDHHC13 and its effects on intramolecular interactions with the DHHC domain of this enzyme. However, these discussions have covered only a fraction of the total residues and zDHHC enzymes that have so far been reported as modified by this PTM type. The effect of the majority of these phosphorylation modifications on zDHHC enzymes is still unknown, and this represents a major knowledge gap, highlighting the need for further research in the area.It is clear that phosphorylation of zDHHC enzymes can be associated with a broad range of regulatory effects. This section has focused on reported effects of phosphorylation on substrate interactions of zDHHC13 and zDHHC3 and dynamic trafficking of zDHHC2 and zDHHC5 and highlighted where these effects might extend to related enzymes. In addition, we provided a more speculative example of potential phosphoregulation of the TSS-acylation outside of their catalytic DHHC cysteine. The role of this S-acylation as a regulatory modification has been most extensively studied for zDHHC6 using a combination of kinetic measurements of protein and S-acylation t½ of WT and cysteine mutant zDHHC6 combined with mathematical modeling. zDHHC6 is S-acylated by zDHHC16 on three cysteine residues that lie within the soluble cytosolic C-terminal tail of the enzyme, namely Cys328, Cys329, and Cys343  that is pronounced further when the other two cysteines are also modified (protein t½ of 0.3 h) (S-acylated at Cys328 is rapidly degraded by the ubiquitin-dependent endoplasmic reticulum–associated degradation (ERAD) pathway or converted to a different species by acyl-protein thioesterase 2 (APT2)-mediated deacylation  . zDHHC6 cylation . In conton in t½ . Intrigue enzyme . The exad Lys371 –84  structure, each interacting with a Zn2+ ion in a tetrahedral coordination geometry , where acyl-CoA engagement takes place  could allow S-acylation of cysteines that are normally unavailable for such modification S-acylatif zDHHC3 . Intriguactivity . The autfication . Neverthorebrain  S-acylation of C-terminal cysteines can impact the stability, localization, and/or activity of zDHHC enzymes. Furthermore, as several zDHHC enzymes contain conserved S-acylation sites, the effects of this PTM may extend to other members of the zDHHC family. Research showing that cysteines involved in zinc coordination can also be targets for S-acylation suggests potentially intriguing mechanisms whereby this modification could directly affect enzyme structural integrity and activity. However, further work is required to determine whether S-acylation of these zinc-coordinating cysteines is physiologically relevant.In summary, there is convincing evidence from research on zDHHC5 and zDHHC6 that S-acyltransferases is limited. As discussed above, there is evidence that the extent of S-acylation of zDHHC6 could affect ubiquitination of specific lysines and subsequent enzyme degradation via ERAD  protein complex pair Erf2-Erf4 showed a similar stabilizing effect, with the t½ of Erf2 decreasing by approximately 6-fold in an Erf4 mutant strain  were mutated to arginines S-acyltra281 of zDHHC9 lies within the palmitoyl conserved C terminus (PaCCT) motif that has been shown to be important for S-acyltransferase catalytic activity (265) has also been reported to undergo ubiquitination  could expose lysine residues for ubiquitinatin.Lysine ubiquitination has also been reported in the vicinity of other critical domains of human LL motif , 95, 96; motif 8284, 95–99tif 8295–. Again, S-acylation is central to stability of the zDHHC6 enzyme. The detection of ubiquitination on other zDHHC enzymes suggests a wider role for this modification in controlling zDHHC degradation. In addition, as ubiquitination often occurs in regions of zDHHC enzymes linked to activity and trafficking, this PTM could also impact other aspects of zDHHC biology either by disrupting the integrity of important sequences or motifs or by directly affecting enzyme localization. Further research is required to explore these potential regulatory effects.In summary, analysis of Erf2-Erf4 has clearly shown an important role for interplay between accessory protein binding and ubiquitination-mediated zDHHC enzyme degradation, and by analogy this may also operate for zDHHC9/GCP16. In addition, interplay between ubiquitination and RRID:SCR_001837) and published  protein lysine methyltransferase in humans . By mutaf zDHHC8 , it will121 and zDHHC5 Lys97 have been shown to be both acetylation and ubiquitination sites in separate proteomics studies . However, given that several zDHHC enzymes are modified (A), exploring the effects of methylation and acetylation is expected to reveal new insight into the regulatory effects of these PTMs on zDHHC enzymes.In summary, although zDHHC8 lysine methylation has been confirmed experimentally and the modifying enzyme has been identified, the functional relevance of acetylation and methylation of any zDHHC enzyme is not yet known B. Howevemodified A, explorB). These PTMs affect many aspects of zDHHC enzyme biology, including trafficking, localization, substrate binding, activity, and stability/degradation. However, this is likely to represent only the tip of the iceberg. The available PTM proteome data sets combined with improved understanding of the structure-function relationship of zDHHC enzymes provides a wealth of information that can help generate a much better understanding of regulatory mechanisms controlling this important class of enzymes.Although our understanding of how zDHHC enzymes are regulated has lagged behind our appreciation of the substrate targets and cellular functions of zDHHC enzymes, there is emerging evidence showing important regulatory effects of PTMs B. These All data are contained within the article.


To measure the hit rate (A hit occurs when the predicted site is within 15 mm of the target) of different algorithms, 47 target sites are tested. Moreover, to evaluate the efficiency of different models in narrowing down the target range, 54 targets are verified. As a result, the proposed algorithm achieves the most hits (37/47) and fewest misses (9/47), and it narrows down the target range most, from 27.62 ± 3.47 mm to 10.72 ± 9.58 mm among 54 target sites. It is expected to be applied in the real-time prediction of the origin of ventricular activation to guide the clinician toward the target site.Pace mapping is commonly used to locate the origin of ventricular arrhythmias, especially premature ventricular contraction (PVC). However, this technique relies on clinicians’ ability to rapidly interpret ECG data. To avoid time-consuming interpretation of ECG morphology, some automated algorithms or computational models have been explored to guide the ablation. Inspired by these studies, we propose a novel model based on spatial and morphological domains. The purpose of this study is to assess this model and compare it with three existing models. The data are available from the Experimental Data and Geometric Analysis Repository database in which three  Premature ventricular contraction (PVC) is one of the most common ventricular arrhythmias encountered in clinical practice, occurring in 1–4% of the general population . FrequenIt is of therapeutical importance to localize the origin of abnormal ventricular activation before catheter ablation. The localization can be done by several approaches. Activation mapping is the most direct technique which can be applied in patients with frequent PVCs . Yet, itIn order to automatically analyze the information of pacing sites and progressively guide the clinician to the origin of PVC, several methods have been developed. One method is to train a universal model from a cohort of patients based on machine learning methods . HoweverIn addition to the image-based simulation method, some simpler but less computational models based on information of multiple pacing sites have also been investigated. Lately, the QRS integrals (QRS-Ints) of 12-lead ECG have been used to predict the 3D coordinate of the PVC origin directly . BesidesInspired by previous studies, in this paper, a novel model only based on the information of pacing sites is proposed and compared with three existing models  to Two target site selection schemes were adopted for different purposes. First, in order to evaluate the hit rate (a hit occurs when the predicted site is within 15 mm of the target), the target site is defined as the site with at least 5 adjacent sites which are greater than 15 mm and less than 35 mm away from it, and a total of 47 pacing sites meet the conditions. Secondly, in order to evaluate the efficiency of different models in narrowing down the target range, the target site is defined having at least 5 adjacent sites within the range of 35 mm of it, and a total of 54 pacing sites meet the requirement. Once a target site is selected, its adjacent sites that meet the corresponding definition serve as potential modeling sites. In this study, the modeling sites are those whose physical locations and corresponding 12-lead ECG signals are known, while the target sites are those whose 12-lead ECG signals are known, but their physical locations need to be estimated by the algorithm.Step 1: Initialize modeling sites. The 3 or 5 farthest unused potential modeling sites  from the target site are selected as initial modeling sites, and they will be removed from the list of unused potential modeling sites. For example, supposing point 0 is selected as the target site, and its adjacent points 1, 2, 3, 4, 5, and 6 match the definition of potential modeling sites. Therefore, the initial list of unused potential modeling sites is . We firsStep 2: Train the models mentioned above to predict the target site.Step 3: Termination judgment. If there are no unused potential modeling sites left, terminate. If the predicted site hits the target, terminate.Step 4: Evaluate predicted site. If the predicted site is outside 35 mm of the target site, it is not credible, skip to step 6.Step 5: Pick out a nearest site from the unused potential modeling sites if it is within 15 mm of the predicted site, then remove it from the list of unused potential modeling sites and skip to step 2. Otherwise, there is no unused potential modeling site that can replace the predicted site, turn to the next step.Step 6: Pick out a site that is farthest from the geometric center of current modeling sites to obtain as much spatial information as possible, and remove it from the list of unused potential modeling sites and skip to step 2.A total of 47 target sites are used to evaluate the hit rate of four models. As The reduced distance is equal to the minimum distance between the modeling sites and the target site minus the estimated error, and a positive reduced distance indicates a reduction in the unknown range of the target by modeling and prediction. A total of 54 target sites are used to evaluate the efficiency of different models in narrowing down the target range. P = 0.046), and the numbers of modeling sites of QIM and DEM, QIM and DDM have a significant difference , indicating that DCM has the worst estimated error and QIM used the most modeling sites.This work proposed a novel model for the localization of PVC target sites based on the mapping between the spatial domain and the morphological domain. In our study, the pacing sites are not as adjacent as those generated by clinical pace mapping, so we selected modeling sites from a larger range to predict the target site step by step. For inexperienced doctors, the results obtained by our method may provide a reference location, so that they can simply determine the most likely ablation site as soon as possible and shorten the mapping procedure. We compared our model with three existing models and found that the proposed model was slightly superior to other models by achieving the most hits, the smallest estimated errors, and the biggest reduced distances. Especially when the number of modeling sites is small, the advantages of our model are more obvious. By observation of Then, as can be observed from In addition, there is a certain relationship between DDM and DEM. In essence, DEM is to establish a scalar model through the relationship of the module length between dp and dw, while DDM directly uses the relationship between dp and dw to build a vector model. When the number of modeling sites is less than 5, the prediction effect of DEM is worse than that of DDM, which is likely due to the lack of direction information. However, when the number of modeling sites increases, the lack of direction information is gradually compensated by more and more complete distance network between points, and DEM achieves a similar result to DDM.J. Finally, in terms of computational complexity, QIM, and DDM are simpler since the fitted models can be used for prediction directly, while DEM and DCM are more complex due to the additional search for optimal solution that can minimize the cost function However, there still exist some limitations in this study. First, the way of picking out modeling sites is relatively random. Theoretically, the next modeling site should be the predicted result if it is reliable; otherwise, the next modeling site is selected by the doctor. Limited by the actual distribution of pacing sites, we take the second place and replace the predicted site with the nearest one among the unused potential modeling sites, which may cause the randomness in modeling site selection due to the different prediction results of four models, for example, when we design a fixed modeling site selection scheme, in which we choose a furthest site from the remaining potential sites in each round. Consequently, QIM, DEM, DCM, and DDM achieve 32, 35, 28, and 33 hits, respectively, indicating that the way of modeling site selection has a certain impact on the research results and the proposed DDM is more suitable for selecting modeling sites by referring to the predicted positions. Because collecting multiple-pace data in clinical practice will increase the risk of patients during operation, this kind of data is difficult to obtain. Therefore, we mainly used the data in the open database collected from three volunteers provided by Charles University in accordance with the strict experimental process. From the perspective of the number of individual patients, our sample is still relatively small, but the total number of test sites used in this paper is relatively large. In the future research, we can also consider the application of four methods to animal experimental data or retrospective clinical data analysis.To conclude, it is a desirable goal to develop an automated algorithm for the localization of PVC origins. This work provided a novel solution based on the mapping between spatial domain and morphological domain. It performs better with fewer modeling points and is expected to be used to predict the origin of ventricular activation in real-time and guide clinicians to focus on ablation targets.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of Charles University Hospital, Prague, Czechia. The patients/participants provided their written informed consent to participate in this study.KH and JS: conceptualization, formal analysis, and methodology. CY: resources, supervision, and project administration. KH, YW, and GZ: software and visualization. KH: writing—original draft preparation. CY and GZ: writing—review and editing. KH and YW: revising and correcting. JS, KH, and CY: clinical interpretation and discussion of findings and their relevance. All authors contributed to the article 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.


Background. After the 2011 terror attack on Utøya Island, a collective visit was organized for bereaved families. There is limited knowledge whether bereaved families can benefit from such visits after terror.Objective. This study aims to explore how bereaved families experienced visiting the site of death after the 2011 terror attack.Method. As part of in-depth interviews, 22 parents and 16 siblings were asked whether they had visited Utøya and, if so, how they experienced the visit. Participants’ responses were analysed using thematic analysis.Results. The results showed that for the majority of the bereaved, visiting Utøya had been important in processing their loss. Three key themes emerged as to what they considered important with the visit: ‘seeing the actual place of death’, ‘seeking factual information’, and ‘learning to know the island’. These factors were associated both with beneficial reactions  and with distressing reactions , but the benefits had outweighed the distress. Having the opportunity for multiple visits seemed to optimize the benefits.Conclusion. Bereaved families should be offered the opportunity to visit the site of death after terror. At the site, the police said where the deceased were found and answered any question that the family had related to the death. Flowers were available for everyone, and the buildings had sandboxes where the families could place candles. Later, it was possible to visit the island at anniversaries and on open days. Individual visits have also been allowed.3.Explore how the bereaved families experienced visiting the site of death after the 2011 terror attack on Utøya Island. Is it beneficial and/or distressing?Identify factors that made the visit beneficial or distressing.The aims of this paper are as follows:4.4.1.This study is part of the project, ‘Bereaved parents, siblings and friends after Utøya, 22 July 2011ʹ, a longitudinal mixed-method study initiated following the terror attack in Norway, 22 July 2011  and siblings (N = 36) who consented to be interviewed. Since the aim was to explore the diversity of experiences, we selected the sample according to different background variables . The sample selected for interviews consisted of 22 parents  and 16 siblings . The age of the parents was between 40 and 61 years (Mean = 50.1), whereas the age of the siblings was between 15 and 44 years (Mean = 24.3). Geographically, both the parents and the siblings represented all parts of Norway. The parents were fairly well educated; 64% had a higher education (beyond 12 years). The age of the deceased ranged between 15–21 years (M = 17.5).The names of parents and siblings of the deceased were accessed through the National Population Register. The interview sample was drawn from a total sample of parents , we asked the following questions: Have you been to Utøya after the terror attack? If so, can you describe your experience of the visit(s). Depending on the answers, we explored conditions associated with experiencing the visit(s) as beneficial and/or distressing.All parents and siblings were interviewed by the first and second author in their homes or at places chosen by the interviewees between October and December 2013. To synchronize the interview method, the second author conducted a trial interview in the presence of the first author. This interview was discussed prior to conducting other in-depth interviews. The length of the interviews varied between two and five hours, including required or desired breaks. The interviews were audiotaped and fully transcribed.None of the interviewees had been to Utøya before the terror attack, and 20 of 22 parents (91%) and all 16 siblings (100%) had visited Utøya after the terror attack. The majority of parents (80%) and siblings (75%) had visited the island more than once. Most had participated in collective visits and anniversaries the first and second year after the attack, but many had also visited the island on their own .4.4.Parents’ and siblings’ descriptions of how they experienced visiting Utøya were analysed according to thematic analysis .6.The first key theme, ‘seeing the actual place of death’, was related to a need to see what the site looked like. Many struggled with difficult fantasies of the site before the visit, or had strong feelings of disbelief related to the loss.6.1.The first visit was very important. I had a huge need to visit Utøya to try to grasp or comprehend that he in fact had died, that his heart stopped there.Seeing the place of death was, for many, important in order to grasp or realize that the death had occurred. A sibling said:It was important for me to see where she had been killed, it was important for me to be where she was the last few seconds of her life. So it helped a little on understanding what happened. It made it a bit more concrete, a little more real when I saw the bullet holes, saw the place she was found. But it was also terribly painful.During the first visits to the island, the bereaved could see concrete traces of the killings, e.g. bullet holes, blood stains on the floor inside the buildings. Witnessing these traces was painful, but also important in trying to grasp what had happened and to counteract the strong sense of disbelief. One sibling said:And I was there before the trial, it early spring, and I had to see the island, when it was not summer, without media attention, and to spend more time there. And I felt that it was part of comprehending what had happened, of working through the loss. I felt it was important. Still, I was always reminded when I was there of how he died. So it was painful to be there, but it was also good to be there.Many mentioned that they needed to visit Utøya several times to really comprehend what had happened, but the traumatic circumstances of the death made the process difficult. A sibling said:Others struggled with difficult fantasies of what the site looked like, and experienced that the visit had changed these disturbing images. A sibling said: ‘I had many pictures in my head of what happened that day, and they were not always correct (…) so when I visited Utøya these pictures changed’.6.2.Another beneficial effect of seeing the place of death was feeling closer to the deceased. Several bereaved experienced a special connection to the place of death at the site. One sibling said: ‘Both the island and the place where he died becomes very special to me, and I immediately felt attached to it’. Some actively sought closeness at the site, for example by touching the ground where the deceased was found, or by being on the place of death on the exact time when their loved one was killed. For some the site became sacred, and they said that they wanted to have the opportunity to visit the place of death also in the years to come. Subsequently, they felt much anger and resentment towards the owner of the island when they, shortly after the killings, recommended that several of the buildings should be destroyed, a decision that was later reversed.6.3.On some of the visits, we brought along other family members so that they also could see the island and understand more how we feel when we are there. This has been important for us, but also for them. Now we have something in common, and they can better relate to what we are saying when we talk about Utøya.Several parents said that visiting the island together with family members had been important when talking about the incident. The joint family visit had imparted a common family narrative or understanding of Utøya and what had occurred there. One mother said that it was difficult to explain to other family members what it felt like to visit Utøya. She noted the following:For some siblings the joint family visit had some challenges. This was mainly due to the responsibility and concern they felt for their parents, since they had not been able to focus on themselves during the visit. One sibling put it like this: ‘It was quite difficult because I had to relate to my parents’ grief when I had enough with my own’.6.4.It is very distressing to be on the actual site, that’s where she lived her last minutes, and that’s where she experienced the most horrible things you can ever imagine. It hurt to realize and feel that.Being at the site not only gave the bereaved a feeling of closeness to their loved one, they also came closer to the gruesome acts of violence that happened there. Many experienced intrusive thoughts or images of the killings at the site. A sibling said: ‘It was difficult, particularly to see the site where he was murdered. I could imagine more clearly what had happened to him when he was killed’. Others said that they could almost feel the deceased’s suffering and fear while being at the site. A sibling said:What became clearer to me at the visit was all the other things that happened on the island – the other adolescents who were killed, all the screams, all those who hid, all those who were afraid – I now have more images of these in my mind.These distressing reactions were, for some, also related to becoming more aware of the magnitude of the incident, realizing the number of all who were killed at the island, and what they went through. A sibling said:I told my son that he should hide, but he said that there were no places to hide (…) When I visited Utøya, I saw that he was right; there were no places to hide there. And I realized that I had given him the worst advice I could give.For others, the visit activated grief reactions such sadness and yearning. This was particularly difficult for those who actively had tried to avoid their grief as a way to cope with the loss. A sibling said: ‘It is hard for me to visit the site because I cannot control my feelings anymore when I am there. Then I am confronted by my own grief, and that’s what I am struggling with’. Some also blamed themselves when they saw the site. A parent said that he had talked to his son on the phone only minutes before he was killed. He said:After I had seen the place where the death of my daughter occurred, my nightmares became more concrete. I had seen the landscape and the exact place, and then it got worse because they became more physical. Now I saw and recognized the place of the killings in my nightmares.Although most of these reactions were temporarily activated while being at the site, a few experienced a worsening of reactions after they had visited Utøya. A parent said that her nightmares had changed for the worse after seeing the place of the death. She said:7.The second key theme, ‘seeking factual information’, was also, for many, a reason to visit Utøya. Most of the bereaved had many unanswered questions related to what had happened and had a need for information.7.1.The first visit was the most difficult one. Then we were told his (the deceased’s) story, where he ran, and where he was found killed – all the information about what had happened. But at the same time, it also felt good. Now we had seen the place, instead of ruminating about what may have happened there.At the first collective visit the police informed each family on the site what they knew about each child/sibling. This was painful, but also helpful. A mother said:It was very important to meet with Kristin, who had been together with our daughter just minutes before she was killed. Then I managed to understand why they had stayed so long at the site. First, I thought that hiding there was like being trapped, but after I talked to Kristin I realized that it was impossible for me to understand what it was like being in that situation.For some, visiting Utøya together with a person who had been on the island on the day of the killings had given access to detailed information of where their loved one had been, and the difficult choices that they had to take. In this sense the ‘survivor’ could fill in some of the blanks and unanswered questions that many bereaved struggled with. A mother said:8.The third key theme, ‘learning to know the island’, was related to getting a more concrete impression of the whole island and to try to comprehend the choices their loved one had made, which many had ruminated about.8.1.When we looked at photographs of the island, we got a quite different view of the topography and distances than what it actually was. When we were there, we realized, for example how small the island was.In their effort to understand and put more ‘pieces in the puzzle’, many bereaved stated that they had to walk all over the island. This had given a different view of what it looked like. One father said:Prior to the visit I had all these ideas of how it looked like at Utøya, the different rooms, where they could hide, distances and so on. So when I walked around the island I got a different picture. I understood more why my son did what he did, and how difficult it was to hide.A mother said: ‘The first time I visited Utøya, I did not really understand anything’. Having the opportunity to walk around the island had changed her perceptions of it. She said further:8.2.I stood on the path, and looked down towards where my son had hidden. Then I realized that he had found a good hiding place. I had ruminated a lot about this, and I couldn’t have seen this on a picture (...) After the second visit to Utøya, things fell more into place, and I felt calmer from within. I am sure it made it easier for me to go on.Furthermore, in trying to get answers to their questions, many followed the ‘route’ where their loved one had escaped to see with their own eyes where they had tried to hide from the perpetrator. This ‘reconstruction’ had given a better understanding of the choices that they had to make. A mother said:8.3.Visiting the island had facilitated ‘integration of the deceased’s perspective’ of the island. Several parents expressed that they more easily could realize why their child wanted to go to the camp, and why they thought it was so much fun to be there. One father said: ‘The second time we were there, Utøya was more like how we imagined the adolescents experienced it, with summer and nice weather, and you could realize how fantastic a time they must have had there’. Another father said: ‘I think that the positive experience we had at the island the second time we were there pushed the bad things away. Then I got another impression of what they actually enjoyed when they were there’.9.In this paper, we explored how bereaved families experienced visiting the site of death after a terror incident. The main finding from the in-depth interviews is that visiting the site of death is beneficial but is also associated with painful and distressing reactions. Still, the benefits had outweighed the distress. The need to know what the site look like and to see in order to comprehend or make sense of what has happened was for many the major motive for visiting the site (Gilles & Neimeyer, The findings can inform both grief and trauma theory. Generally the bereaved are drawn towards places that are linked with the deceased Parkes,  and, accCognitive trauma theory posits that a central component of processing a traumatic event is facilitating integration of the trauma into autobiographical memory (Ehlers & Clark, In addition to cognitive processing of the loss, our findings also show that visiting the site of death can strengthen relational bonds in the family. Making sense of the loss always happens within the context of the family Nadeau, . As we hThe finding that seeking out the last place where a person lived can give an increased feeling of closeness to the deceased is consistent with the theory of continuing bonds (Klass, Silverman, & Nickman, 10.This study has some limitations. We do not know the representativeness of those who consented to be interviewed. Although persons bereaved by single-incident violent losses underline their need for visiting the death scene (Williams, Rheingold, McNallan, & Knowlton, 11.Our findings suggest that having the opportunity for multiple visits can optimize the benefits. Survivors who were directly exposed to the disaster can contribute with important information about the deceased’s last moments, but this can also be an extra burden on the survivors. Subsequently, more research is needed before this can be used systematically in the follow-up of bereaved families. Furthermore, careful planning, mental preparation prior to the visit, and support through the visit is necessary to try to minimize or reduce some of the distress that can be associated with such visits (Kristensen et al., Visiting the site of death may be a useful intervention for those who struggle with prolonged grief and PTSD (Murray et al., There are still research questions related to visits to the site of death that need to be addressed such as the number of visits to recommend, why some refuse to visit the site of death, and if there is anyone who should refrain from visiting.12.Bereaved families should be offered the opportunity to visit the site of death after terror, but tailored preparations are necessary to reduce some of the distress that is associated with such visits. Having the opportunity for multiple visits can optimize the benefits.


Hebbian plasticity, a mechanism believed to be the substrate of learning and memory, detects and further enhances correlated neural activity. Because this constitutes an unstable positive feedback loop, it requires additional homeostatic control. Computational work suggests that in recurrent networks, the homeostatic mechanisms observed in experiments are too slow to compensate instabilities arising from Hebbian plasticity and need to be complemented by rapid compensatory processes. We suggest presynaptic inhibition as a candidate that rapidly provides stability by compensating recurrent excitation induced by Hebbian changes. Presynaptic inhibition is mediated by presynaptic GABA receptors that effectively and reversibly attenuate transmitter release. Activation of these receptors can be triggered by excess network activity, hence providing a stabilising negative feedback loop that weakens recurrent interactions on sub-second timescales. We study the stabilising effect of presynaptic inhibition in recurrent networks, in which presynaptic inhibition is implemented as a multiplicative reduction of recurrent synaptic weights in response to increasing inhibitory activity. We show that networks with presynaptic inhibition display a gradual increase of firing rates with growing excitatory weights, in contrast to traditional excitatory-inhibitory networks. This alleviates the positive feedback loop between Hebbian plasticity and network activity and thereby allows homeostasis to act on timescales similar to those observed in experiments. Our results generalise to spiking networks with a biophysically more detailed implementation of the presynaptic inhibition mechanism. In conclusion, presynaptic inhibition provides a powerful compensatory mechanism that rapidly reduces effective recurrent interactions and thereby stabilises Hebbian learning. Synapses between neurons change during learning and memory formation, a process termed synaptic plasticity. Established models of plasticity rely on strengthening synapses of co-active neurons. In recurrent networks, mutually connected neurons tend to be co-active. The emerging positive feedback loop is believed to be counteracted by homeostatic mechanisms that aim to keep neural activity at a given set point. However, theoretical work indicates that experimentally observed forms of homeostasis are too slow to maintain stable network activity. In this article, we suggest that presynaptic inhibition can alleviate this problem. Presynaptic inhibition is an inhibitory mechanism that weakens synapses rather than suppressing neural activity. Using mathematical analyses and computer simulations, we show that presynaptic inhibition can compensate the strengthening of recurrent connections and thus stabilises neural networks subject to synaptic plasticity, even if homeostasis acts on biologically plausible timescales. Synaptic plasticity is widely believed to be the neuronal substrate for learning and memory. Hebbian plasticity , in partB receptors causes a reduction of neurotransmitter release + = 0. In this case the steady-state firing rate reduces tow.For external input strengths To derive the critical homeostatic timescales in the presence and absence of presynaptic inhibition, we revisit the five-dimensional dynamical system given by Eqs rE, rI and p are much faster than those of w and τE, τI, τp ≪ τw, τc), we can use a separation of timescales approach to reduce the full system to two dimensions. With respect to the slow plasticity and homeostasis dynamics, the fast variables are at their steady-state. For the excitatory firing rate we can therefore writew and wI from w on both sides givesg′ is the derivative of g with respect to rE. Solving for As the dynamics of w from the partial derivative above we reorder w, insert the result and after simplifying the expression finally obtainTo eliminate rE. Thus the firing rate rE becomes the slow dynamic variable together with its running estimate Using the chain rule rE and The system described by Eqs The characteristic polynomial then isΨ is positive for moderate external input (I < κ(1 + wI)), because g is a monotonously decreasing function and all other variables are positive. The stability condition follows immediately if the square root is imaginary. In case the square root is real, we can write the larger eigenvalue asγ is the second term of the product in the square root and κ4τc > τw then If the real part of both eigenvalues is negative, the fixed point is stable (see for example ). In theκ4τc < τw it follows thatIf, on the other hand, ΨUsing the stability condition derived above, we can identify the critical homeostatic timescale asrE = κ) to give g(wIκ)wκ = κ + wIκ + I and insert this into the To obtain a more intuitive expression, we reorder κ is guaranteed as long as the homeostatic time constant τc is smaller than a critical value g(wIκ), excitatory recurrence w, the change g′(wIκ) of release factor with inhibitory acitvity (i.e. GABA spillover) and the dependence wI of this inibitory activity on excitatory firing rate. Note that the critical homeostatic timescale needs to be seen in relation to the effective time constant of plasticity In summary, we have shown that stability of the fixed point at the target rate g(r)≡1 (implying g′(r)≡0) into Without presynaptic inhibition the two-dimensional system simplifies toe et al. . We can The recurrent spiking network we studied consists of 5000 leaky integrate-and-fire neurons (4000 excitatory and 1000 inhibitory neurons), which are randomly connected with current-based synapses . In addiVi of neuron i evolves according toVR, membrane time constant τm and synaptic input currents from excitatory (IE), inhibitory (II) and external populations (Iext). If a voltage trace crosses the threshold VT, the neuron emits a spike and the voltage is reset to VR for an absolute refractory period of τref  onto excitatory synaptic weights. However, in the spiking network we consider a more local version of the mechanism with neuron-specific release factors AGABA—for every inhibitory spike according toWe estimate local GABA levels  by accumulating a fixed amount of GABA—given by The local GABA levels are neuron-specific and affected by all inhibitory neurons projecting to the respective neuron. Thus, an inhibitory spike leads to both an inhibitory current and an increase in local GABA level at downstream excitatory neurons. The inhibition of release through GABA spillover is then again modelled by a linear decrease in release factor with increasing GABA levels, that isτp = 300 ms to roughly match the timescale of presynaptic inhibition observed experimentally [β determines the strength of presynaptic inhibition and p ≡ 1 recovers the traditionally used current-based network.We set the time constant of this process to mentally . As befoCGABA to be related to local GABA concentration, it is unitless and can not be mapped to any specific biophysical quantity. Nevertheless, we normalise AGABA by the number of ingoing inhibitory synapses, such that the local GABA level CGABA roughly relates to the inhibitory firing rate. This allows us to use a presynaptic inhibition strength paremeter β on the same order of magnitude as in the rate and mean population model . Although we consider A+ is the amount of long-term potentiation (LTP), τ+, τslow and τ− timescales of the triplet STDP model  and κ tτc and as in the rate network a crucial parameter for network stability [The overall timescale of this homoeostatic component of the metaplastic triplet STDP rule is therefore given by tability .wij < wmax during ongoing plasticity, whereas structural changes are not allowed . A summary of parameters related to plasticity is shown in τw = 2975 s  or D. background input. Bottom: Firing rate of excitatory population in respective circuit model as a function of excitatory recurrence for increasing strengths of presynaptic inhibition (β).Top: Mean population models in which presynaptic inhibition not only affects excitatory recurrent synapses, but also (TIF)Click here for additional data file.S2 Figτc = 30 s) produces a narrow distribution around the target rate of 5 Hz. Slower homeostasis leads to a broad long-tailed distribution of firing rates with an inherent turnover but no temporal oscillations. A. Mean (solid line) and standard deviation (shaded area) of firing rate over time. Plasticity is switched on after 6 minutes of simulation time (asterisk). B. Distribution of single neuron firing rates. C. Firing rate of single neurons over time. Target rate is indicated by yellow colour. D. Temporal correlation of single neuron firing rates.Fast homeostasis ((TIF)Click here for additional data file.S3 FigA. Critical homeostatic time scale as a function of the presynaptic inhibition strength β in networks with (left) and without (right) heterogeneity. For simulations the vertical bars indicate the range between largest stable and smallest unstable timescale tested. B. Temporal evolution of the average firing rate for different homeostatic time constants τc with (left) and without (right) heterogeneity in the network for different strengths of presynaptic inhibition β.With heterogeneity in the initial excitatory recurrent weights, the theory holds as long as presynaptic inhibition is not too strong. Removing this source of heterogeneity from the network increases the critical timescale for strong presynaptic inhibition, and thereby provides a better match to the analytically determined critical timescales. (TIF)Click here for additional data file.S4 FigA. Exponentially decreasing (top) and sigmoid transfer functions (bottom two) linking inhibitory firing rate to release factor for different slope parameters  and 5 (bottom). Target rate of 5 Hz is indicated by gray dashed line. B. Increase in analytical critical homeostatic timescale as a function of transfer function slope at the target rate. Parametrisation in A indicated by markers of same colour. C. Release factor at target rate as a function of increase in homeostatic time constant. Markers correspond to transfer functions shown in A. D. Temporal evolution of the average firing rate in networks with presynaptic inhibition for different homeostatic time constants τc. Plasticity is switched on after 6 minutes of simulation time (asterisk). Coloured marker in top right corner specifies parametrisation of transfer function (see A).We qualitatively recover the results obtained for linear transfer functions if the release probability decreases exponentially or as a sigmoid with increasing inhibitory rate. (TIF)Click here for additional data file.S5 FigA. Eigenvalues of reduced system with compared to without presynaptic inhibition as a function of homeostatic time constant. The time constant is given in units of effective plasticity time constant τplast. The critical homeostatic timescale  is marked by a red dashed line. B. Phase plane dynamics with nullclines and example trajectories for short and long homeostatic timescales in systems with and without presynaptic inhibition.Besides increasing the critical homeostatic time constant, presynaptic inhibition preserves low firing rates beyond the point of stability. Systems without presynaptic inhibition trajectories diverge away from the fixed point  if homeostasis is close to the timescale of plasticity, whereas systems with presynaptic inhibition merely oscillate around it. (TIF)Click here for additional data file.


Polyspecific ligand recognition by P-glycoprotein includes compensatory mechanisms in the form of ligand-binding shifts when ligand-interacting residues are challenged by mutagenesis. Point mutations of ligand-interacting phenylalanine residues to alanines resulted in binding shifts of an environmental pollutant, yet its inhibitory function on ATPase activity was preserved. The multidrug transporter P-glycoprotein (Pgp)/ABCB1/MDR1 plays an important role in multidrug resistance (MDR) and detoxification owing to its ability to efflux an unusually large and chemically diverse set of substrates. Previous phenylalanine-to-alanine scanning mutagenesis of Pgp revealed that nearly all mutations retained full MDR function and still permitted substrate transport. This suggests that either the loss of any single aromatic side chain did not affect the ligand-binding modes or that highly adaptive and compensatory drug recognition is an intrinsic property including ligand-binding shifts that preserve function. To explore this hypothesis, the ATPase function and crystallographic localization of five single-site mutations in which the native aromatic residue directly interacted with the environmental pollutant BDE-100, as shown in previous crystal structures, were tested. Two mutants, Y303A and Y306A, showed strong BDE-100 occupancy at the original site (site 1), but also revealed a novel site 2 located on the opposing pseudo-symmetric half of the drug-binding pocket (DBP). Surprisingly, the F724A mutant structure had no detectable binding in site 1 but exhibited a novel site shifted 11 Å from site 1. ATPase studies revealed shifts in ATPase kinetics for the five mutants, but otherwise indicated a catalytically active transporter that was inhibited by BDE-100, similar to wild-type Pgp. These results emphasize a high degree of compensatory drug recognition in Pgp that is made possible by aromatic amino-acid side chains concentrated in the DBP. Compensatory recognition forms the underpinning of polyspecific drug transport, but also highlights the challenges associated with the design of therapeutics that evade efflux altogether. Thus, it appears that aromaticity is likely to play a role in ligand/substrate recognition, i.e. specificity, but could also permit many different substrates to be recognized and transported, i.e. polyspecificity.Several structures of Pgp determined by X-ray crystallography (XRC) and cryo-EM revealed small molecules bound to the DPB , we selected five aromatic residues  that appeared to form direct contacts with BDE-100 for mutagenesis to test the hypothesis of polyspecificity compensation in the form of shifts in ligand-binding modes. Individual aromatic residues were mutated to alanines on a background wild-type mouse Pgp mimetic mutation, C952A, that had also previously been shown to form well diffracting crystals . Following overnight methanol induction (0.5 ml min−1), the cells were harvested and lysed at 276 MPa by a single pass through a cell disrupter . Cell debris was separated by centrifugation at 15 900g at 4°C. Membranes were isolated by centrifugation at 38 400g at 4°C for 90 min and resuspended in 750 ml 75 mM NaCl, 15% glycerol, 20 mM Tris–HCl pH 8.0. The Pgp extracted from solubilized membranes with 9% Triton X-100 was purified using nickel–nitrilotriacetic acid (Ni–NTA) Superflow resin (Qiagen) on an ÄKTA FPLC. The Ni–NTA eluate was concentrated to 1 ml  and ultracentrifuged for 1.5 h, and the supernatant was loaded onto a size-exclusion column  that had been pre-equilibrated in SEC buffer . Elution peaks from 55 to 65 min were pooled and the concentration was determined by the Coomassie Plus Protein Assay (Pierce) at 595 nm using a standard curve of bovine serum albumin.P-glycoprotein and mutant constructs on a C952A template were overexpressed in 2.2.et al. . The ATPase activity was calculated using the equation ΔOD/(∊ × [protein] × time), where ΔOD is the change in absorbance and ∊ is the molar extinction coefficient for NADH. EC50 values were calculated using GraphPad Prism over the entire concentration range.The ATPase activity of purified Pgp was measured using an ATP-regenerating system at 37°C as described by Vogel & Steinhart 1976 and modi al. 1995. Briefly2.3.et al.  and 16% formaldehyde  were added to 1–3 mg mouse Pgp. The mixture was incubated for 2 h at 4°C and further dimethylamine–borane and formaldehyde were added after 2 h. After incubation for 16 h at 4°C, the reaction was quenched with cold glycine on ice for 2 h and flushed with SEC buffer . The eluted protein was concentrated to 2–3 mg ml−1 and incubated with 0.5 mM BDE-100 (AccuStandard) dissolved in DMSO in the dark for 16 h at 4°C. The precipitate was centrifuged and the extracted sample was diluted 1:10 in SEC buffer to give a final concentration of 10–13 mg ml−1. Crystallization was set up using 3 µl sitting drops in 24-well Cryschem plates (Hampton Research) with a 1:1 ratio of protein solution and reservoir solution . Crystals could be visualized after ∼3 days at 4°C and reached full size in ∼4 weeks.To reproduce the binding of BDE-100 to Pgp and maintain crystal isomorphism, we utilized the reductive-methylation protocol of Ward  al. 2013 with somThe crystals were cryoprotected using an identical pH and identical lithium sulfate and EDTA concentrations as the growth conditions but with 29–30% PEG.2.4.K edge to optimize for the strongest anomalous signal at the beginning of each data-collection session and whenever data were collected from a new mutant. The data were processed, integrated and scaled with HKL-2000  calculated via CNS  mFo − DFc electron density surrounding the ligands, which was still present after additional refinement steps using a fixed occupancy of 1.0. We conclude there is a sufficient data-to-parameter ratio to refine both B factors and occupancy for the BDE-100 ligand in PDB entry 4xwk as well as in all of our structures. Our own refinement of PDB entry 4xwk yielded a ligand occupancy of 0.66 and our control mutant C952A agreed very well, with a refined occupancy of 0.67 . Atomic coordinates of the co-crystal structures of BDE-100 with the C952A Pgp control mutant, as well as of BDE-100 with the Pgp double mutants, were deposited in the Protein Data Bank with the following codes: 6ujn (C952A), 6ujt (C952A/Y303A), 6ujw (C952A/Y306A), 6ujr (C952A/F724A), 6ujs (C952A/F728A) and 6ujp (C952A/F979A).X-ray diffraction data were collected at 100 K on beamline 23-ID-D at the Advanced Photon Source (APS). MAD scans were obtained from Pgp–BDE-100 co-crystals near the Br 3.et al., 2013125I]-iodoarylazidoprazosin  , the BDE-100 ligand adopted a well defined single binding site in the DBP comprising the same transmembrane regions, i.e. TM5, TM6, TM7 and TM12, and appeared to make significant contact with the side chains of the aromatic residues Tyr303, Tyr306, Phe724, Phe728 and Phe979 . Accurate BDE-100 localization and modeling was accomplished by inspecting the anomalous difference Fourier electron density of the Br atoms as well as difference Fourier (mFo − DFc and 2mFo − DFc) electron density  was preserved in our C952A control  and was formed predominantly by the aromatic residues Tyr303, Tyr306, Phe310, Phe331, Phe724, Phe728, Phe755 and Phe979. Interestingly, the F979A mutant did not perturb BDE-100 binding to site 1 [Fig. 1b)]. A close inspection shows that the Phe979 side chain in the other structures appears to be just outside van der Waals contact distance with the ligand (not shown), and therefore it does not appear to contribute much to the binding energy. Surprisingly, the F724A mutant exhibited no detectable BDE-100 occupancy in site 1, but revealed a novel site 3 that was shifted ∼11 Å from site 1 and was located directly on the axis of pseudosymmetry of the DBP formed by residues Met68, Phe71, Phe332, Phe728, Tyr949, Phe974 and Val978 [Fig. 1c)]. The F728A, Y303A and Y306A mutants showed dual-occupancy BDE-100 binding in sites 1 and 2  and the ligands were in contact with the following residues: Met68, Phe71, Tyr303, Tyr306, Phe310, Phe331, Phe332, Gln721, Phe724, Ser725, Phe728, Val731, Phe755, Tyr949, Leu971, Phe974 and Phe979. The site 1 residues present in the Y303A and Y306A mutants but not the F728A mutant were Tyr303 or Tyr306 or Gln721, Ser725, Phe728 and Val731 owing to a rotation of the ligand to form a site that we designate site 1A in the F728A mutant, as discussed below.Site 1 binding of BDE-100  using the deposited structure factors (Table 273% (8/11) of the residues that form site 1 are aromatic in character. For sites 2 and 3, the percentages of aromatic residues contributing to ligand binding are 67% (4/6) and 56% (5/9), respectively Fig. 2. The abiet al., 2016Supplementary Fig. S5). These results suggested that BDE-100 itself is a poorly transported substrate of Pgp. In contrast, BDE-100 exhibited a significant inhibitory effect on verapamil-stimulated ATPase activity for all five mutants at the verapamil EC50 determined for each of the five mutants and the control C952A , revealed that the Phe974 side chain is highly disordered when BDE-100 is not present in any other site besides the canonical BDE-100 site 1  is challenged by mutagenesis.Phe974 in particular appears to make important contributions to the compensatory ligand-recognition mechanism. More specifically, close inspection of the 2 1 Fig. 1. In thesd) and Fig. 5Phe724 and Phe728 (both on TM7) appear to be important for maintaining the integrity of the canonical BDE-100 site 1, since the mutation of either residue resulted in ligand shifts away from this site. The F728A mutation resulted in an ∼75° rotation of the ligand around TM7 at the pivot point of Ala728 to site 1A that is closer to the pseudosymmetric axis and maintains van der Waals contact between the ligand and TM7 [Figs. 1B factors in refinement and some smearing of the anomalous electron density. Nevertheless, the pattern of anomalous density was highly consistent between multiple crystals of each mutant . It is tempting to speculate that some disorder of the ligand is tolerated in the alternate sites as a form of polyspecificity. In other words, very tight, ordered binding may not be essential for recognition by Pgp (and presumably transport), and may slow transport rates if the Gibbs binding free energy becomes highly favorable for certain substrates, particularly if hydrogen bonding is involved , yet Tyr303 and Tyr306 are involved in site 1 formation and are as far from site 2 as any site 1 residue. Furthermore, we detected no significant differences in the binding pose of BDE-100 in site 1 between the Y303A, Y306A, C952A or F979A mutants. One possibility is that long-range communication, or potentially quantum considerations, between site 1 binding and site 2 occurs but is not detectable given the limits of our data.4.Compensatory ligand recognition appears to contribute an important component to the mechanism of polyspecificity in MDR1/Pgp. In essence, polyspecificity is the opposite of ‘lock-and-key’ recognition in which, in this case, the lock (Pgp DBP) appears to recognize different keys (substrates) even when the shape of the lock is changed slightly at specific locations. For Pgp, the loss of one of several individual critical aromatic contacts in the DBP by mutagenesis to alanine allowed a shift in the binding of the BDE-100 ligand while still preserving the ligand’s known function of ATPase inhibition. Since aromatic residues are a dominant component of both the canonical and the novel ligand sites, we conclude that aromaticity itself is fundamental to the mechanism of polyspecificity. A slight perturbation of the canonical site allowed a second copy of BDE-100 to bind to the DBP in some cases, and therefore stoichiometry does not appear to be a stringent requirement for polyspecificity. In our structures, the transporter is essentially in the same conformation regardless of the amino-acid mutation, allowing us to conclude that the binding shifts were not owing to differences in the overall protein conformation. It would thus seem that the DBP of Pgp in the conformation that we characterized by XRC must have a relatively flat energy landscape, at least with respect to BDE-100, that allows the ligand to move to new sites if the primary binding site is perturbed. The compensatory nature of polyspecific ligand recognition would also seem to make it very challenging to modify any chemotherapeutic drug substrate that would allow it to evade efflux by Pgp altogether.10.1107/S2052252520005709/jt5047sup1.pdfSupporting figures and legends. DOI: P-glycoprotein, C952A mutant, complex with BDE-100, 6unjPDB reference: C952A/F979A mutant, complex with BDE-100, 6ujpPDB reference: C952A/F724A mutant, complex with BDE-100, 6ujrPDB reference: C952A/F728A mutant, complex with BDE-100, 6ujsPDB reference: C952A/Y303A mutant, complex with BDE-100, 6ujtPDB reference: C952A/Y306A mutant, complex with BDE-100, 6ujwPDB reference:


Tau protein, a neuronal microtubule-associated protein, becomes hyperphosphorylated in several neurodegenerative diseases called tauopathies. Hyperphosphorylation of tau is correlated to its redistribution from the axon to the somato-dendritic compartment at early stages of tauopathies. Interestingly, tau hyperphosphorylation begins in different regions of the brain in each tauopathy. In some regions, both neurons and glial cells develop tau hyperphosphorylation. Tau hyperphosphorylation is also observed in physiological conditions such as hibernation and brain development. In the first section of present article, we will review the spatiotemporal and cellular distribution of hyperphosphorylated tau in the most frequent tauopathies. In the second section, we will compare the pattern of tau hyperphosphorylation in physiological and pathological conditions and discuss the sites that could play a pivotal role in the conversion of non-toxic to toxic forms of hyperphosphorylated tau. Furthermore, we will discuss the role of hyperphosphorylated tau in physiological and pathological conditions and the fact that tau hyperphosphorylation is reversible in physiological conditions but not in a pathological ones. In the third section, we will speculate how the differences and similarities between hyperphosphorylated tau in physiological and pathological conditions could impact the elaboration of therapies to prevent tau pathology. In the fourth section, the different therapeutic approaches using tau as a direct or indirect therapeutic target will be presented. MAPT gene on human chromosome 17q21 , giving rise to the six isoforms in human adult brain . Alt. AltMAPTlt brain , 6. Its lt brain .Tau undergoes several post-translational modifications in physiological conditions; phosphorylation being the predominant modification. Most of these modifications impact tau function and contribute to the heterogeneity of tau forms found in developing and adult brain . In neurIn Alzheimer's disease (AD), both tau and amyloid-beta (Aβ) pathologies are landmarks of the disease whereas in primary tauopathies, tau pathology is the main neuropathological landmark –17. FronTau contains 85 phosphorylation sites. Ten sites are phosphorylated in human control brain whereas in AD brain, 55 sites become phosphorylated , 20. ProMAPT gene. Forrest and al found that 59% of their MAPT cases could be pathologically subclassified as other sporadic tauopathies including PSP, CBD, and PiD , 55 phosphorylated sites were identified in two cohorts of AD . The higSixteen sites phosphorylated in AD were detected in PSP . The patIn both brain development and hibernation, hyperphosphorylated tau is present. Three important points will be compared between these physiological conditions and tauopathies in the present section (1) the pattern of tau hyperphosphorylation; (2) the role of hyperphosphorylated tau; and (3) the reversibility of tau hyperphosphorylation during brain development and hibernation but not in tauopathies.Several groups reported that tau is hyperphosphorylated during fetal and post-natal brain development of rodents at sites similar to those observed in AD and primary tauopathies –54. Thisper se that is toxic but rather the phosphorylation of specific residues and/or the cellular context (development vs. aging) that seem to dictate the toxicity of tau. Further studies are needed to confirm this using more accurate methods such as MS to obtain the full picture of tau phosphorylation sites in fetal brain.The above studies revealed that in fetal brain, human tau is hyperphosphorylated at multiple epitopes, some of them overlapping with those seen in tauopathies. Human fetal tau appears to be able to form aggregates that are not seed competent. The function of such aggregates remains to be determined. All together, the above observations indicate that it is not hyperphosphorylation None of the studies mentioned above examined the distinct phosphorylation pattern of each tau isoform. This is an important point to consider since the pattern of tau isoforms is differently regulated in fetal and adult human brains. Only the smallest tau isoform is present in fetal brain whereas all 3R and 4R tau isoforms are found in the adult brain at a ratio of 1:1 , 58–60. in vivo in mouse brain  studies have detected hypometabolic states in isocortical brain areas of patients at preclinical stages of AD and patients with probable AD (subjects at risk) , 100. ThIt was proposed that tau would play a role in regulating neuronal activity during the hypometabolic state. Indeed, the localization of tau at the synapse and its interaction with post-synaptic proteins modulating the insertion of receptors, in particular AMPA and NMDA receptors, at the plasma membrane make it a plausible player in regulating neuronal activity . HyperexIn humans, the hyperphosphorylation and aggregation of tau were frequently detected under the age of 30 in a systematic survey of more than 2 300 non-selected autoptic cases aged between 1 and 100 years , 125. ThDuring brain development and hibernation, tau hyperphosphorylation is transient and reversible. The highly phosphorylated tau is dephosphorylated around birth and during post-natal brain development to remain less phosphorylated in normal adult brain , 56. TheAs mentioned above, during hibernation, multiple periods of torpor lasting few days are periodically interrupted by short arousal periods of activity lasting hours in which the animals return to euthermia and normal metabolism, blood flow and body temperature , 131. ThThree main observations on hyperphosphorylation of tau during brain development and hibernation could impact the elaboration of therapies to prevent tau pathology and/or slow down its propagation in the brain. Firstly, the comparison of tau phosphorylation sites between physiological conditions and tauopathies indicates that the combination of phosphorylation sites is most likely a determinant factor in the toxicity mediated by hyperphosphorylated tau and therefore this should be taking into consideration when designing tools to neutralize phosphorylated forms at specific sites. Secondly, tau hyperphosphorylation is not found in glial cells in physiological conditions as noted in primary tauopathies indicating that they could play important role in the process of neurodegeneration. Thirdly, all tauopathies are linked to aging.in vitro in presence of heparin (Examining tau hyperphosphorylation in brain development and hibernation revealed that some sites could be protective preventing tau from forming toxic seed-competent aggregates. However, it is important to mention that a full characterization of tau phosphorylation sites using an approach such as MS is needed to identify the key sites involved in the shift from non-toxic to toxic tau species. Based on what it is known so far, it is clear that the combination of phosphorylation sites is a determinant factor in tau aggregation propensity. For example, in a recent study, it was reported that the amount of phosphorylation on doubly phosphorylated T231 and S235 and on singly phosphorylated S262, positively correlated with seeding capacity of tau whereas some phosphorylation sites negatively correlate with tau seeding such as the single phosphorylation within the clusters of sites contained in peptides S198/S199/S202 or S400/T403/S404 . This in heparin . Lastly, heparin . From thIn some tauopathies, both neurons and glial cells are affected . It is sThe fact that glial cells are affected earlier than neurons in some brain regions could indicate that the insults triggering the disease altered the functioning of glial cells more than that of neurons in these regions. The mechanisms leading to tau pathology in glial cells remain poorly characterized. As shown for neurons, tau is believed to be attached to microtubules and to regulate their assembly in glial cells . This isThe fact that tau hyperphosphorylation does not exert any toxic effects during brain development and hibernation could indicate that the cellular context plays a pivotal role in determining whether hyperphosphorylated tau is part of either a response supporting neuronal survival or a response compromising neuronal survival. Four of the main changes occurring in aging could contribute to tau hyperphosphorylation. Firstly, it is well-documented that the efficacy of the two systems involved in tau degradation, the proteasome and the autophagy, is significantly decreased in aging favoring the accumulation of phosphorylated tau . SecondlSeveral clinical trials that directly target tau are currently active. The main ones are testing tau immunotherapy for sequestration of pathological tau species . Such anPhosphorylation epitopes that are shared between the tauopathies could be used to design antibodies that should work for all tauopathies unless these epitopes are rendered inaccessible to antibodies because of tau conformation. Indeed, the analysis of tau atomic structure using Cryo-EM has revealed that tau filaments are distinct in AD, PiID and CBD . These aIn several clinical trials, hyperphosphorylated tau is an indirect target. In most of these trials, either the inhibition of tau kinases or the activation of PP2A, the main tau phosphatase is tested . Tau is d I2PP2A , 164. PPd I2PP2A , 166. Cod I2PP2A . This coTau phosphorylation can be regulated by other post-translational modifications. O-GlcNAcylation was shown to regulate tau phosphorylation . In AD bAt early stages of the disease, tau detachment from microtubules in both neurons and glial cells could be reversible and therefore therapy aiming at increasing tau binding to microtubules could be efficient in stopping the disease. On one hand, if tau first detaches from microtubules because of its hyperphosphorylation, an effective therapy would be to prevent this hyperphosphorylation. On the other hand, if changes in microtubule dynamics trigger the detachment of tau from them, the therapy should act on microtubules instead of tau. This avenue was explored by using drugs that stabilize microtubules. Promising results were obtained in tau mouse models but no beneficial effect was observed in humans –173. ThiThe interactome of hyperphosphorylated tau was recently characterized . In partLastly, a recent study revealed that excitatory neurons having a selective vulnerability to tau pathology present an elevated expression of tau aggregation-prone proteins and a decreased expression of tau aggregation protector proteins . This inFrom the above observations, one can speculate that therapies targeting mechanisms involved in tau hyperphosphorylation such as the inhibition of tau kinases would be efficient in all tauopathies but a lot more complex to elaborate because of their secondary effects on substrates other than tau. Using tau as a direct target appears to be the most achievable approach although the diversity of pathological tau forms implies the adaptation of a therapy for a each tauopathy. The success of the direct approach relies on a full characterization of tau phosphorylation sites and its other post-translational modifications in both physiological and pathological conditions to identify the pivotal sites in the conversion from non-toxic to toxic species. Furthermore, as revealed in a recent study, tau phosphorylation varies during the progression of AD indicating that the strategy applied at early stages of the disease will be different from that applied at later stages.AV, CP, AD, and NL wrote the manuscript. AV prepared 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.


Introduction: Revision surgery of a previous lumbosacral non-union is highly challenging, especially in case of complications, such as a broken screw at the first sacral level (S1). Here, we propose the implementation of a new method based on the CT scan of a clinical case using 3D reconstruction, combined with finite element analysis (FEA), computer-assisted design (CAD), and 3D-printing technology to provide accurate surgical navigation to aid the surgeon in performing the optimal surgical technique by inserting a pedicle screw at the S1 level.Materials and Methods: A step-by-step approach was developed and performed as follows: (1) Quantitative CT based patient-specific FE model of the sacrum was created. (2) The CAD model of the pedicle screw was inserted into the sacrum model in a bicortical convergent and a monocortical divergent position, by overcoming the geometrical difficulty caused by the broken screw. (3) Static FEAs  were performed using 500 N tensile load applied to the screw head. (4) A template with two screw guiding structures for the sacrum was designed and manufactured using CAD design and 3D-printing technologies, and investment casting. (5) The proposed surgical technique was performed on the patient-specific physical model created with the FDM printing technology. The patient-specific model was CT scanned and a comparison with the virtual plan was performed to evaluate the template accuracyResults: FEA results proved that the modified bicortical convergent insertion is stiffer  compared to monocortical divergent placement . The final template was created via investment casting from cobalt-chrome. The template design concept was shown to be accurate  based on the comparison of the simulated surgery using the patient-specific physical model and the 3D virtual surgical plan.Conclusion: Compared to the conventional surgical navigation techniques, the presented method allows the consideration of the patient-specific biomechanical parameters; is more affordable, and the intraoperative X-ray exposure can be reduced. This new patient- and condition-specific approach may be widely used in revision spine surgeries or in challenging primary cases after its further clinical validation. Spinal fixation is a routine procedure for the treatment of unstable spine due to trauma, congenital malformations, degenerative diseases, and tumors . The accThe revision surgery of a lumbosacral non-union can be complicated by an implant related failure, with a broken pedicle screw. In the S1 segment, the convergent bicortical screw trajectory provides superior anchoring compared to any other directions, but the proper insertion of the new screws in a revision surgery due to the broken screw is extremely difficult without surgical navigation. Here, we present a complex clinical case in which the accurate surgical technique required the development of a computer-aided design (CAD) and finite-element analysis (FEA) combined method for affordable spine surgical navigation with a 3D-printed customized navigation template.The study was approved by the National Ethics Committee of Hungary and the National Institute of Pharmacy and Nutrition (reference number: OGYÉI/163-4/2019). Informed consent was obtained from the patient. A 38-years-old patient underwent multiple spine surgeries at the L5–S1 level over a 5-years period with transforaminal interbody fusion (TLIF). During the latest surgery, implant removal and S1 left side nerve root decompression were performed. Six months later, the patient was referred to our institution due to manifestation of mechanical low back pain, with no sign of sensorimotor deficit. Medical imaging at admission  demonstr3) with an intensity of 225 mA and voltage of 120 kV. The imaging protocol was previously defined in the MySpine project   (For the study Quantitative Computed Tomography (QCT) scans were used, performed with a Hitachi Presto CT machine  using an in-line calibration phantom with five cylindrical insertions of known mean equivalent bone mineral density (BMD) values  , 20, andtp.aspx) . The thrtp.aspx)  to definThe resulting masks (group of voxels) were homogenously filled by preserving the outer contour of the geometrical border in 2D. From the mask, a triangulated surface mesh was automatically generated. On the 3D geometries surface smoothing  and uniform remeshing was applied .A CD Horizon Legacy (Medtronic) polyaxial pedicle screw, 45 mm long and 6.5 mm in diameter, was scanned with the ScanBox 3D scanner . The model of the screw was reconstructed and modified  in 3-matic  software. The triangulated surface mesh of the screw model was uniformly re-meshed  . The scrThe assembly was exported to the 3-matic software where nine FE meshes were generated for each of the implantation scenarios . The broken screw, inserted implant, and sacrum-implant interface had a triangle set with an edge length of 0.6 mm. The outer surface of the sacrum mesh was changed in the nine models by defining the uniform triangle mesh edge length as 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 mm. Adaptive meshing protocol was used for the volume mesh creation with 10-node tetrahedral elements. The maximum edge length of the meshing process corresponded with the initial edge length of the sacrum surface mesh , for theHounsfield Unit) values to BMD values based on the in-line phantom was performed, the conversion curve was assumed to be linear according to studies (app = −0.0829 + 0.0026 HU (ρapp [g/cm3]). Then, the bone tissue was assumed to be isotropic and linearly elastic with a Poisson's ratio of 0.3 . The FE models were exported to the Abaqus/CAEv11 . For the broken and the inserted pedicle screws the material properties were defined as follows: Poisson's ratio of 0.3 . The contact surface and the axes were exported to the Autodesk Fusion 360  CAD software which was used for the finalization of the design . The vir-chrome . The acc-chrome .The accuracy of the template was tested on a patient-specific sacrum physical model, 3D-printed with a Fused Deposition Modeling (FDM) printer . The drill template was placed on the FDM sacrum model; then, a cylinder inlet was connected to the template to support the drill bit, and the drilling of the model was performed according to the S1 and ALA positions .The template was removed and two CT scans were performed of the sacrum model with drill bits inserted in the S1 and ALA positions. The CT scan images were imported into the Mimics software where the segmentation (thresholding) and 3D reconstruction of the patient-specific FDM sacrum model geometry and drill bits were performed. The models were registered to the initial sacrum geometry derived from the QCT via point based rigid registration by selecting anatomical landmarks in the caudal part of the sacrum . This stThe investment casted cobalt-chrome drill template retains the geometrical properties of the pattern (3D-printed drill template model created with MSLA technology) based on the 3D scanning evaluation N = 9, S1 and N = 9, ALA) with increasing element numbers based on the virtual surgical plan. The FE simulation results converged above 2*105 elements for both screw insertion scenarios at ~5 min solve times on two cores. The solve time at two cores for the S1 orientation was higher compared to the ALA  with a larger sample size would be preferred to demonstrate the clinical efficacy and cost-effectiveness of the developed methodology.A patient-specific template for pedicle screw insertion allows the surgeon to insert the screw into its optimal position. The advantages of our technique compared to the conventional surgical navigation tools are the affordability, the potential to reduce intraoperative X-ray exposure, and the possibility for the consideration of patient-specific bone geometry and biomechanics. This new patient- and condition-specific approach can be widely used in revision spine surgeries or in challenging primary cases after its further clinical validations.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by National Ethics Committee of Hungary and the National Institute of Pharmacy and Nutrition  for the publication of any potentially identifiable images or data included in this article.PEE, AL, PPV, MB, and DL: research design. PEE, AL, MB, BH, AJP, and LK: acquisition of data. PEE, AL, MB, BH, AJP, and DL: analysis and/or interpretation of data. All authors: drafting the paper or revising it critically and approval of the submitted and final versions.MB was employed by the company DO3D Innovations Ltd. The remaining 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.