Periplaneta americana) which are solitary foragers: naive , conditioned , and mixed . The robustness of the consensus is not affected by the naive individuals' proportion, but the rate and the frequency of selection of the odorous shelter are correlated to this proportion. In mixed groups, the naive individuals act as influencers. Simulations based on the mechanisms highlighted in our experiments predict that the consensus emerges only for intermediate group sizes. The universality of these mechanisms suggests that such phenomena are widely present in social systems.Many social species are able to perform collective decisions and reach consensus. However, how the interplay between social interactions, the diversity of preferences among the group members and the group size affects these dynamics is usually overlooked. The collective choice between odourous and odorless shelters is tested for the following three groups of social cockroaches (  •Consensus is group size dependent•Consensus is not affected by an aversive group conditioning•Consensus is robust regardless the diversity of individual preferences in a group•Group choice is controlled by the preference of a minority of individuals Biological sciences; Ecology; Ethology, Social behaviour Aggregation, which results in an uneven distribution of individuals through space, is a widespread behavior occurring in living organisms, from unicellular organisms to mammals . In arthDuring decision-making, animals respond to biotic  and abiotic  cues to guide themselves through the environment . The strThe outcome of collective decisions, in response to the environmental cues and to the conspecifics, modifies the structure of the group in space  and in time  . Still, Collective decisions wherein group members cannot be coerced into their decisions by others are part of many social animals, ranging from (eu)social arthropods to mammals. In these groups, individuals influencing each other may have different behaviors or preferences. In such heterogeneous groups this influence can, lead to social learning . MoreoveIn democratic groups, the consensus is reached by choosing the most frequent preference of the group members . In nondPeriplaneta americana), we test the capacity of naive, conditioned , and mixed groups  to reach a consensus during settlement. The insects, like many others, individually explore the environment and choose to settle between one shelter without odor (hereafter CS) and one with peanut butter odor (hereafter PS) (see In a binary choice experiments with groups of 10 nymphs (L6-L7 instar) of the American cockroach ( PS) see . Cockroa PS) see , the att PS) see . Indeed, PS) see  and ther PS) see . The hyp PS) see  and theiP. americana. Conditions differed on the ratio of naive/conditioned individuals: for the naive condition is 10/0 ; for the conditioned condition is 0/10 ; and for the mixed condition is 4/6  for more details see vs mixed) and between 10 and 180 min (naive vs conditioned and conditioned vs mixed)  . After 2ps - Ncs) over time, where Nps (Nps) is the number of individuals in the PS(CS). Bimodal distributions of the difference between the sheltered populations (Nps - Ncs) are observed from particular time steps  until the end of experiments for all conditions  after 10 min for naive and mixed condition and after 30 min for conditioned condition  . In addition, a survival analysis on the values of Tw shows no significant difference between the selected shelter (PS vs CS) for the three conditions . This correlation suggests an absence of complete deconditioning of the conditioned individuals (for the conditioned and the mixed condition) during the trials.At 24 h, 97% of the experiments end with 90% or more of the total sheltered population in the same shelter . This shows that the level of consensus  is independent of the shelter type. These collective choices can actually be predicted at 180 min, as the population inside the selected shelter at 180 min is positively correlated to the one at 24 h . Nonetheless, the proportion in the PS between conditions is significantly different . A nonlinear least squares fit of At 24 h, the proportion of trials that ended with 90% or more of the total sheltered population within the PS(CS) is 0.8(0.2) for the naive, 0.37(0.63) for the conditioned, and 0.64(0.36) for the mixed . In thisχ2 test shows that during the experiment, the fraction of naive sheltered individuals is larger than the fraction of conditioned ones, from 30 to 170 min of the trials. Moreover, at each time step (except at t = 20 min), the fraction of naive sheltered individuals is linearly correlated to the fraction of conditioned sheltered individuals .In the mixed condition, groups are composed of 4 naive and 6 conditioned individuals marked with red and black dots, respectively, on their pronotum , it is the relative individual response to the quality of the shelter PS (CS). As for the leaving probability at every time step, it depends on the shelter quality as well as on its population. Like many other gregarious animals, cockroaches are retained by conspecifics and the probability of leaving decreases with the sheltered population (ρps(ρcs) is the leaving probability when individuals are isolated by unit of time , ςps(ςcs) is the social strength, and Xps (Xcs) is the number of individuals present in the shelter.The sheltering dynamics result from the entries to the shelters and the exits from them, which depend on the individual probabilities of joining and leaving the shelters. Usually, these probabilities depend on the qualities of this shelter and its housed individuals. Based on previous works , the propulation . Here, wpulation :. The fitting of the total sheltered population with such simplification gives the minimal value of μ, ρ, and ς) and identify their extremes values  are in the same ranges of values, but not μ. Although this analysis is compatible with the experimental results at 24 h, it is only partially consistent with the full dynamics.A first approach to approximate the values of of θ see . Moreoves values  that ares values  of our mμ, ρ, and ς), we performed dynamical stochastic simulations of the model . We summarize hereafter the different steps of the simulations:•t = 0, all individuals are outside.At •Jps or Jcs (At each time step (second), individuals outside the shelters have a probability s or Jcs  to find •Qps or Qcs  and for 10,000 realizations.To further approximate the parameters of the model  and CS (μcs) were kept equal, regardless of the type of individual, the simulated data were significantly different from the experimental data . Finally, Keeping the experimental proportions of the three groups 100% naive (naive), 40% of naive (mixed), and 0% naive (conditioned), we perform stochastic simulations  based on the mechanisms described previously, to study the effect of the population size on the consensus at 24 h. P. americana). In these cockroaches, like in many other arthropods, the interactions between individuals are linked to specifics activities, in particular, the collective choice of a resting site  ended up with the selection of a particular shelter (consensus for the PS or the CS) at the third h which is stabilized or is increased further until the 24th h. This suggests that no memory loss occurs during the duration of the experiments, which is in agreement with previous works showing that cockroaches can retain acquired memories up to one week . HoweverThe selection of the winning shelter appears early in time, indicating that the process of decision-making is fast and irreversible. The relatively small area of our setup partially contributes to this high rate . Nonetheς) and the number of sheltered individuals is not large enough to retain them and to lead to a consensus. Reaching a consensus at large population sizes is also less frequent but for a different reason. In this case, the probability to find numerous conspecifics inside both shelters is large  A. The mois large  leading As seen in multiple studies, a leader can either be an individual with a low threshold  or an experienced one with accurate information   King an. The colFrom an adaptive point of view, the fact that individuals in mixed groups may choose an option which does not fit their preference or their needs, strongly suggests that benefits associated with settling with many other individuals offset the costs of a nonoptimal choice , a largeHere, we tackled an important phenomenon in social organisms: groups dynamics and decision-making when individuals possess unshared preferences. We highlighted how group dynamics are linked to the interindividual variability . FinallyThe results described in this article and the examples provided previously clearly suggest that the collective behaviors generated by the conflict between individual preferences and cooperative feedbacks are a widespread phenomenon. In this context, we proposed a generic script that can be at work whatever the origin of diversity be it within the same species  or in heterospecific situations.All animals came from ten-year-old rearing cages, thus, consanguinity could play an important role in the cohesiveness observed between individuals. For this reason, it could be interesting to use cockroaches from other strains in the experiments. Furthermore, in this study, the controlled individual variability is induced adversely (electroshocks), and it is therefore not known if the conditioned cockroaches are less stress outside thus, their displacement is slower or if they are physiologically affected by the electricity. Among the important issues, that are not addressed here, are the ones related to the roles of starvation on social strength and of the loss of the conditioned memory during the experiments. Indeed, although there is no indication that a memory loss occurs in the short term , in the mcalvoma@ulb.ac.be).Information and requests for resources should be directed to and will be fulfilled by the lead contact, Mariano Calvo Martín  ad libitum. Groups of 10 nymphs (L6-L7 instar of both sexes) were kept in a box (15.5 × 11 × 6 cm) with water and food for 24 h before being conditioned or tested. Cockroaches did not have access to water or food during the experiments. The sex of L6-L7 instar of this species is not recognizable at naked eye, and therefore, the ratio male/female is unknown. However, there is no behavioral differences between the sexes at these life stage cycle.Cockroaches of the species Rosco E+ # 019 - fire). This dark shelter contains a cup filled with peanut butter (Boni) covered with a perforated plastic layer so that the individuals can only scent the peanut butter. The bottom of the shelter is an electric plate (silicate plate with copper strips) linked to a power source. A group of individuals is placed in the illuminated part and when an individual enters the shelter, an electric shock (7.5 V and 2 A) is manually delivered after 2 s. This delay allows the animal to associate the odor and the shelter. The shocked cockroach always runs out of the shelter. The procedure is applied for at least 30 min and is then continued until no individual enters the shelter for 5 consecutive min. At the end of the procedure, cockroaches were aggregated outside the shelter, this suggest that sociality is not affected by the conditioning. Thus, they are doubly conditioned: to lose their inherent preference for the odor and to spend more time outside. For example, for the conditioned condition, the group received a mean ± sd of electric shocks of 55.26 ± 14.4, with a mean ± sd duration of a conditioning trial of 2469 ± 376 s. Finally, the number of shocks administrated decreased over time  and are disposed symmetrically in a rectangular arena , whose bottom is covered with a white paper layer covering the floor as well inside the shelters, this is changed after every trial. A light source (8 fluorescent tubes lamps – 80 W/T5 SYLVANIA), placed 190 cm above the arena, provides a homogeneous illumination intensity at the ground level . The shelters are as described in the conditioning procedure section. Under each shelter, there is a cup (diameter: 0.16 cm) covered with a plastic film perforated with a needle, to prevent cockroaches to have direct contact with the interior of the cups. One of the shelters' cups is filled with peanut butter (Boni), this being the odorous shelter (PS). For the other, the control shelter (CS), the cup is empty. The group is released into the center of the arena all at once and left to explore and seek shelter for 24 hr.For the experiments, we used 69 groups of 10 cockroach nymphs without any external damage. The groups are divided into 3 conditions: 15 groups of individuals without training, the naive condition (naive); 19 groups of conditioned individuals, the conditioned condition (conditioned); and 35 groups composed of 4 naive individuals and 6 conditioned individuals (mixed). For the mixed condition, conditioned individuals were marked on their pronotum with a black color dot and naive individuals with red color dot, after they were taken from their breeding box. Each group is tested in only one trial, which consists of a binary choice set-up . This inhttps://www.r-project.org/) and Fortran90 (https://www.fortran90.org/).Simulations are performed using R studio , located over the set-up, at 17 frames per s for the first 3 h of the trial. The red filter covering the shelter, allow the video camera to detect the cockroaches inside the shelters. The distribution of individuals among the shelters is recorded every time-step (10 min) during the first 3 h and 24 h after the beginning of the trial by counting the number of individuals in each shelter.Trials are recorded by a video camera (https://www.r-project.org/). The significance of the statistical tests is fixed to α = 0.05. As the dynamics of sheltering process of gregarious and social insects are usually nonlinear and/or their variances vary over time, we used permutation and resampling tests . A survival analysis (log rank test) is performed on this variable. Indeed, the inverse of Tw can be viewed as the mean probability per unit time that a shelter becomes the most populated one until the end. Moreover, we use the Spearman correlation tests to quantify the correlation between the population of a winning shelter of times 180 and 1440 min and for the mixed condition, the correlation between the numbers of naive and conditioned individuals present in the same shelter. We use permutation tests  to compare the sheltered population between the PS and CS within each condition and at each time step : at eachps + Ncs) per unit of time is proportional to θ and to the number of individuals outside the shelters. The inferior limit of θ can be approximated if we neglect the exits from the shelters (ρ = 0). Thus, the mean total sheltered population as a function of time based on Our model assumes that the total number of entering individuals in both shelters (Nbased on N(1−e−θt)equation  and alloequation .P which at time t, i is the number of individuals sheltered in PS and j in CS . Each differential equation P that the system occupies the state . The equation counts the transitions leading the system to the state and those removing it from this state. In our case, the transitions depend on both the probabilities of joining the shelters PS or CS (The system of differential equations This system of equations is particularly effective for quickly obtaining the theoretical distribution of the different states. Indeed, by numerically integrating the master equation In the case of ɩ = 0, Probability to be out of the shelters: Probability to be within the CS: Probability to be within the PS: Further approximation of the parameter values, from


Fibrosarcomas are rare, malignant neoplasms of mesenchymal origin. Fibrosarcomas appear to be sporadic, but cases of fibrosarcomas secondary to radiation of nasopharyngeal carcinomas have been reported. Paranasal sinus fibrosarcomas (PNFS) are even rarer with few cases being reported since the 1950s. There have been several retrospective cohort studies examining PNFS; however, to our knowledge, no comprehensive review exists. This review aims to summarize the findings of all published cases of PNFS from the 1950s to the 2020s. We hope that a comprehensive review will assist in accurate and early diagnoses of PNFS, and help guide treatment as early treatment is associated with a favorable prognosis.This systematic review reports results following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A search was conducted on PubMed, Embase, and Cochrane Library. Studies were screened using established inclusion/exclusion criteria. A total of 26 studies were included for data extraction, and relevant data were collected and analyzed.In our study, the most common study type was case reports (n = 19). The most common presentation for PNFS included male gender (n = 17) with maxillary sinus (n = 57) involvement. Patients commonly presented with complaints of nasal obstruction (n = 15), epistaxis (n = 11), and facial fullness/pain (n = 9). Surgical resection was the mainstay treatment, with the use of chemotherapy or radiation depending on surgical margins and resectability. The diagnosis was commonly made with histological analysis. This review of the literature provides a summary and reference of important presenting factors, elements of diagnosis, and treatment options regarding PNFS to help bring awareness and guide the treatment of such a rare disease. Moving forward, there is a greater need for larger standardized studies that can further complement our findings, as well as more consistent reporting of cases. Fibrosarcomas are rare, malignant neoplasms of mesenchymal origin that comprise 7-10% of all head and neck sarcomas . ParanasAs with other nasal cavity and paranasal sinus pathologies, PNFS often presents with unilateral nasal obstruction and epistaxis, sometimes being mistaken as a papilloma . PreviouPNFS is associated with a high risk of local recurrence and a low risk of distant metastasis . Due to This review aims to summarize the findings of all published cases of PNFS from the 1950s to the 2020s. A total of 109 cases from 26 articles were collected from PubMed, Embase, and Cochrane. This review covers study characteristics, presentation of symptoms, location of the tumor, pathological findings, diagnosis, treatment, and complications. We hope that a comprehensive review will assist in accurate and early diagnoses of PNFS, as early treatment is associated with a favorable prognosis.MethodsThis systematic review reports results following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . A searcStudy SelectionTitles and abstracts of studies retrieved were screened for inclusion by two independent reviewers, and a separate third reviewer resolved all conflicts. The full text of included studies was subsequently reviewed. Studies were screened for inclusion using the Medical Subject Heading (MeSH) terms related to fibrosarcoma of the paranasal sinuses. Inclusion criteria included studies of patients with fibrosarcomas in the paranasal regions. Articles that were non-English, non-human studies, review articles, books, and studies unrelated to fibrosarcoma of the paranasal sinuses were excluded. Twenty-six studies were included for data extraction.Data ExtractionData extraction was conducted using a Microsoft Excel spreadsheet . Patient demographics were collected, such as age and gender. Study characteristics, including study type, year of publication, and authorship, and the number of patients in the study were collected. Clinical data regarding presenting symptoms, location of the fibrosarcoma, use of chemotherapy and/or radiation, surgical interventions, and histopathology characteristics were gathered. Comments regarding patient outcomes were collected including survival and mortality. The collected data were then organized into graphical figures and tables.ResultsGeneral OverviewA preliminary search yielded 632 articles after duplicates were removed, with 26 ultimately meeting inclusion criteria and were included in our study. No previous systematic reviews related to fibrosarcomas of the paranasal sinuses were identified. Articles ranged in publication date from 1952 to 2019. A total of 109 patients were derived from 19 case reports, four case series, and three retrospective cohort studies. All included studies are listed in Table Clinical Characteristics AnalysisThe described data for clinical characteristics can be found in Table Type of Treatment AnalysisThe aggregate data for treatment and outcome of patients with fibrosarcoma can be found in Table DiscussionTo the best of our knowledge, no comprehensive review of PNFS exists. Consequently, a consensus on PNFS demographics, presentation, diagnosis, treatment, and prognosis has not been found. This is significant because only a few cases of PNFS have been reported since the 1950s and an early diagnosis is associated with a more favorable outcome. We include our recommendations below.Demographics and SymptomsCancer of the paranasal sinuses is a rare condition alone, with one case occurring in every 100,000 people. Studies have shown that paranasal sinus tumors tend to occur at an average age between 50 and 60 years . ParanasIn our review, the most common presenting symptom was nasal obstruction followed by epistaxis and facial fullness/pain. These symptoms are consistent with other cancers of the paranasal sinus. These presenting symptoms are common with many other conditions and can oftentimes be overlooked. The presence of unilateral symptoms that do not improve with treatment should raise the suspicion of a possible mass such as a fibrosarcoma and should warrant further workup .Diagnostic MethodsIn routine clinical practice, paranasal fibrosarcoma is commonly misdiagnosed as other neoplasms due to its rarity and non-specific symptomatology. Therefore, it is critical to be familiar with the imaging features that differentiate paranasal fibrosarcoma from other malignancies . AdditioWhen suspecting PNFS, there are particular findings found on certain modalities that may rule in or out other diagnoses on the differential . One stuTreatment and MortalityAn accepted mainstay treatment of fibrosarcoma of the sinuses currently does not exist. It is important to recognize that local recurrence is common for PNFS, and distant metastasis rarely occurs as well. According to analyzed studies, the most commonly used type of treatment was surgical management, as shown in Table Additional research is required to give a definitive statement on indications and contraindications in the use of endoscopic surgeries in fibrosarcoma removal. According to the analyzed studies, it is suggested that surgeons are opting to mainly treat with surgical management as opposed to a multimodal type of management. We recommend that surgeons approach fibrosarcomas with local excision and large surgical border, followed by chemotherapy and radiotherapy, especially if the tumor cannot be fully removed or if surgical border involvement on pathology report is revealed. In addition, for tumors that are unresectable, it is suggested that preoperative chemotherapy should be used to decrease tumor size followed by resection.Our results suggest that PNFS is associated with a high rate of mortality, with death occurring within one year of treatment in 35% n = 18) of cases. However, mortality information was only available for 47% (n = 52) of patient cases. Studies that include long-term patient outcomes are needed to better assess PNFS prognosis. Published data have suggested that prognosis is associated with the degree of histological differentiation [ of casesLimitationsThere are several notable limitations worth mentioning. Since there were no prospective studies found, all data are based on retrospective research. Additionally, the majority of studies were case studies and series, and there were no randomized controlled trials comparing various treatment approaches, making it difficult to compare the efficacies of treatment approaches. The use of solely one primary treatment was not explicitly in the articles, which may skew the results. The mortality data were gathered from sources that mentioned any mortality information within the article. Since timeframes varied greatly for reporting the death and postoperative prognosis of patients, the mortality results may be skewed. Furthermore, the reporting of larger studies oftentimes grouped varying tumors as well as locations other than the paranasal sinuses in analysis, making some data difficult to extract. Across studies, there was not a uniform way in which data were presented, causing some information to be unavailable. To address these limitations, it would be important for larger and more standardized studies to further support our findings. Furthermore, more consistent reporting of patient progress following treatment will help with the assessment of optimal treatment options.Fibrosarcoma of the paranasal sinuses is a rare but dangerous disease. By conducting this study, we aim to provide physicians with a comprehensive review to assist in the management of PNFS. Physicians should maintain a high index of suspicion when presented with a patient with non-specific symptoms, such as nasal obstruction and facial fullness and pain, which are unresolved. Nasal endoscopy can be utilized to visualize the mass, and the use of CT and MRI can aid in further diagnosis. Ultimately, histology can confirm the final diagnosis. The mainstay of treatment is surgical excision with the use of radiation or chemotherapy depending on resectability and surgical borders. With a high rate of mortality, early identification and treatment are essential.


Ketoprofen exhibits different binding-site preferences when interacting with mammalian albumins from different species. The drug-binding properties of albumins cannot easily be predicted based only on a complex of albumin from another species even when the drug-binding sites exhibit a high degree of conservation between species. Serum albumin is a circulatory transport protein that has a highly conserved sequence and structure across mammalian organisms. Its ligand-binding properties are of importance as albumin regulates the pharmacokinetics of many drugs. Due to the high degree of structural conservation between mammalian albumins, nonhuman albumins such as bovine serum albumin or animal models are often used to understand human albumin–drug interactions. Ketoprofen is a popular nonsteroidal anti-inflammatory drug that is transported by albumin. Here, it is revealed that ketoprofen exhibits different binding-site preferences when interacting with human serum albumin compared with other mammalian albumins, despite the conservation of binding sites across species. The reasons for the observed differences were explored, including identifying ketoprofen binding determinants at specific sites and the influence of fatty acids and other ligands on drug binding. The presented results reveal that the drug-binding properties of albumins cannot easily be predicted based only on a complex of albumin from another organism and the conservation of drug sites between species. This work shows that understanding organism-dependent differences is essential for assessing the suitability of particular albumins for structural or biochemical studies. S1), the drug-binding properties of albumin are typically expected to be similar across mammals. Nevertheless, significant differences in the effects of some drugs in humans compared with other animals have been reported, and it has been suggested that this may be due to differences in albumin binding . The crystallization plate was incubated at room temperature for three months and then at 37°C for several days until the first crystals were observed. Harvested crystals were flash-cooled without any additional cryoprotectant.Crystallization was performed in 96-well plates  that were set up using a Mosquito crystallization robot (TTP Labtech). Prior to crystallization, HSA solution at concentration of 162 mg ml2.4.HKL-3000 - or (R)-enantiomers of ketoprofen were chosen by careful evaluation of the fit of each candidate to the 2mFo − DFc and mFo − DFc omit maps . Each choice was supported by comparing the fit to the maps after refinement, the resulting ADP values and the interactions with the protein . Partial occupancy was evaluated for the (R)-ketoprofen molecule in drug site 9, which resulted in the appearance of positive electron density and comparatively low ADP values; therefore, the occupancy was kept at 100%. The ACHESYM server  and ChemSketch were used for figure generation. The DALI server , for which electron density was not observed. The electron density revealed the binding of one (S)-ketoprofen molecule to drug site 2, two (S)-ketoprofen molecules to drug site 3 and one (R)-ketoprofen molecule to drug site 9 . However, it is noticeably different from a ligand-free HSA structure (PDB entry 4k2c), as can be concluded from the r.m.s.d. values between the aligned Cα atoms . Fatty acids were not added during crystallization and are most likely remnants from purification. Free cysteine was added by the manufacturer to the protein during purification to block the sole free cysteine residue in HSA and prevent albumin dimerization. Based on the observed electron density, Cys34 forms a disulfide bond with another molecule of cysteine. The quality of electron density observed for ligands in the determined structure can be inspected interactively at https://molstack.bioreproducibility.org/project/view/VW8s7hb1Z9mnCLbg3NBU/. As a control, we also determined a 2.70 Å resolution structure of HSA obtained from the same crystallization conditions but not containing ketoprofen . In the control structure, all ketoprofen binding sites  remain unoccupied, Cys34 also forms a disulfide bond with another molecule of cysteine, and fatty acids bind to the same sites as in the HSA–ketoprofen complex.The crystal of the complex of HSA with ketoprofen grew in space group  9 Fig. 2. All thr3.2.et al., 2020et al., 1975S)-ketoprofen molecule occupying this site is stabilized by strong hydrophobic interactions with surrounding residues  and by hydrogen bonds between its carboxylate group and the hydroxyl groups of Tyr411 and Ser489. Arg410 may also contribute a remote charge–charge interaction with the carboxylate group. The residues involved in the binding of (S)-ketoprofen to HSA at drug site 2 are listed in Table 2S)-ketoprofen molecule bound to drug site 2 overlaps with the fatty acid previously reported to bind in FA4 -ketoprofen molecules bound and thus can be considered as two subsites. Subsite A overlaps with the previously characterized FA1 site -ketoprofen bound. (S)-Ketoprofen in subsite A is stabilized by strong hydrophobic interactions with residues forming a narrow binding pocket , by a salt bridge between its carboxylate group and the guanidino group of Arg117, and by a hydrogen bond from its carboxylate group to the hydroxyl group of Tyr161 (Table 2S)-ketoprofen molecule surrounded by sparse hydrophobic residues, mainly Ile142, Phe149, Leu154, Phe157, Tyr161 and the aliphatic part of the side chain of Lys190. At this subsite, the carboxylate group of (S)-ketoprofen forms a hydrogen bond to the His146 side chain (NE2 atom) and a remote charge–charge interaction with Arg145. In comparison to drug site 2 and subsite A, subsite B offers a significantly smaller hydrophobicity (as can be seen by the significantly lower number of hydrophobic residues taking part in the interaction) and weaker hydrophilic interactions , which may suggest weaker binding of (S)-ketoprofen. Indeed, the high atomic ADP values observed for this ligand (Table 1S)-ketoprofen bound to the subsites within site 3 have their phenyl rings located within 4 Å of each other -ketoprofen molecules and may possibly result in cooperative binding.Drug site 3, which is also called the oncological drug site and FA1 -ketoprofen molecule in the reported structure. The (R)-ketoprofen molecule is stabilized by several hydrophobic interactions ; a hydrogen bond is formed between its carboxylate group and the hydroxyl group of Tyr452 and a salt bridge between the carboxylate group and the nitrogen group of Lys436. The (R)-ketoprofen molecule has relatively high ADP values, suggesting partial occupancy or positional variability, which is also correlated with a significantly smaller hydrophobicity of this site, which is likely to result in a lower binding affinity.Drug site 9, which is located near FA8 and FA9, is a much less common drug-binding site in SA -ketoprofen at drug sites 4, 6 and 10 -ketoprofen in sites 4 and 6 may be attributed to these differences. However, all of the residues involved in the binding of (S)-ketoprofen at drug site 10 in ESA are the same in HSA, except for Ile7, which is a Val in HSA. Moreover, Ile7 in ESA only contributes to hydrophobic interactions with the drug molecule, further suggesting conservation of the site and leading to the expectation that drug site 10 in HSA may also bind (S)-ketoprofen, even though it was not observed in the structure reported here. Drug site 2, where (S)-ketoprofen binds to HSA, is occupied by a myristate molecule in the structure of the ESA–ketoprofen complex, which potentially prevents drug binding. Drug sites 3 and 9 remain unoccupied in this structure.ESA (pairwise sequence identity of 76.1% to HSA) has recently been reported to bind -enantiomer modeled at this site -ketoprofen at drug site 1 are conserved between BSA and HSA  has been reported to be the only ketoprofen binding site in BSA (pairwise sequence identity of 75.6% to HSA), with the -ketoprofen at drug sites 2 and 6 -ketoprofen in both HSA and LSA, but the binding modes in these structures differ -ketoprofen molecule at drug site 2 in LSA is stabilized by hydrophobic interactions with surrounding residues and forms a salt bridge and a hydrogen bond between its carboxylate group and Lys414 and Tyr411, respectively. Most of the residues involved in these interactions are conserved (89%). In addition, the two residues that differ between LSA and HSA  do not change the character of the binding site -ketoprofen. Previously, (S)-ibuprofen was reported to bind to drug site 2 in HSA and ESA via two different respective binding modes that resemble those of (S)-ketoprofen te Fig. 5. The carS)-ketoprofen molecule binds to drug site 6 in LSA, where it is stabilized by hydrophobic interactions and by hydrogen bonds between its carboxylate group and the side chain of Asn397 (NE2 atom) and between its carbonyl group and the side chains of Asn402 (ND2 atom) and Lys545. This binding site is 82% conserved between LSA and HSA. Two residues that differ in HSA (Asn402 to Lys and Asn541 to Lys) change the overall charge of the cavity, and due to the larger size of the Lys side chains in HSA may affect the conformation of the drug or even prevent binding completely. Drug site 6 is unoccupied in the HSA–ketoprofen structure. In the LSA–ketoprofen structure, drug site 3 is occupied by a polyethylene glycol molecule, which may prevent drug binding to this site. An acetate ion is present in drug site 9.Another (4.S)-enantiomers at drug sites 2 and 3 (two molecules) and an (R)-enantiomer at drug site 9. We compared the HSA–ketoprofen complex with previously reported ketoprofen–albumin complexes from other mammals. Ketoprofen was shown to bind to drug sites 4, 6 and 10 in ESA , well conserved binding sites . The calculated r.m.s.d. values are higher in the structural comparison of complexes with fatty acids and ligand-free albumins than when comparing albumin complexes with ketoprofen and ligand-free albumins (Supplementary Table S1). These results indicate that fatty-acid binding alters the conformation of albumin more significantly than the binding of ketoprofen. Plasma fatty-acid levels are dynamic and are chronically elevated in some disease states -ketoprofen molecules (drug sites 2 and 3) and only one (R)-ketoprofen molecule (drug site 9). The shape of drug sites 2, 3 and 9 clearly supports the binding of the specific enantiomer of ketoprofen, suggesting differences in the binding affinity of HSA to the respective ketoprofen enantiomers. This observation agrees with previous reports stating that HSA can bind (R)- and (S)-enantiomers of ketoprofen with different affinities depending on the experimental conditions -‘profens’, but its stereoselectivity is ultimately dictated by the structure of the drug and its fit to a particular binding site. The ten binding sites available on albumins have differing affinities for different stereoisomers, and some of them may go against the general trend, as is in the case of site 9, which binds (R)-ketoprofen. We believe that different stereoisomers of drugs have distinct binding affinities to various SA drug-binding sites simply because they differ in spatial structure and their binding is affected by shape complementarity.HSA is known to bind chiral drugs stereoselectively (Shen The results reported here provide insight into the molecular basis of ketoprofen transport across species and indicate a need for similar studies for other drugs. Structural determination and biochemical characterization of drug complexes relating to HSA in parallel with those involving albumin from animals used as model organisms, are necessary to evaluate the appropriateness of such models and contribute to our understanding of drug transport. In cases of high sequence similarity, the conservation of a particular ligand–protein interaction across organisms is traditionally expected. However, this study shows that this assumption is not necessarily valid, at least for ketoprofen–albumin complex studies. It remains to be seen whether these differences in drug binding to albumin from different species will be discovered for other drugs.human serum albumin, complex with ketoprofen, 7jwnPDB reference: 10.1107/S2052252522006820/lz5056sup1.pdfSupplementary Figures and Tables. DOI: https://doi.org/10.18430/m37jwnDiffraction images.: https://doi.org/10.1101/2021.04.03.438117A preprint of this article is available from bioRxiv.:


We aim to investigate its value for target volume definition, as a prognosticator, and associations with whole-blood transcriptome liquid biopsy (WBT lbx) for which we recently reported feasibility to mirror tumor characteristics and response to particle irradiation in recurrent HGG (rHGG).Selective uptake of (18)F-fluoro-ethyl-tyrosine (18F-FET-PET data from n = 43 patients with primary glioblastoma (pGBM) and n = 33 patients with rHGG were assessed. pGBM patients were irradiated with photons and sequential proton/carbon boost, and rHGG patients were treated with carbon re-irradiation (CIR). WBT (Illumina HumanHT-12 Expression BeadChips) lbx was available for n = 9 patients from the rHGG cohort. PET isocontours  and MRI-based treatment volumes (MRIvol) were compared using the conformity index (CI) . Associations with WBT lbx data were tested on gene expression level and inferred pathways activity scores (PROGENy) and from transcriptome estimated cell fractions .vs. during radiotherapy  and in non-resected  vs. resected tumors . In rHGG, a trend toward higher SUVmax values in grade IV tumors was observed (p = 0.13). Median MRIvol was 32.34 (R 8.75–108.77) cm3 in pGBM (n = 16) and 20.77 (R 0.63–128.44) cm3 in rHGG patients (n = 27). The highest median CI was observed for 40%  and 50%  isodose, with 70% (40%) isodose in grade III (IV) rHGG tumors . High SUVmax was linked to shorter survival in pGBM  and rHGG . SUVmax showed associations with inferred monocyte fractions, hypoxia, and TGFbeta pathway activity and links to immune checkpoint gene expression from WBT lbx.In pGBM, median SUVmax was higher in PET acquired pre-radiotherapy  1.5–7.8; n = 20) 18F-FET-PET imaging on gross tumor volume (GTV) definition for particle radiotherapy warrant further evaluation. SUVmax might assist in prognostic stratification of HGG patients for particle radiotherapy, highlights heterogeneity in rHGG, and is positively associated with unfavorable signatures in peripheral whole-blood transcriptomes.The benefits of  Despite intensive research and a multimodal treatment approach, the prognosis for glioblastoma (GBM) and recurrent high-grade glioma (rHGG) remains poor , 2. TherParticle therapy might be a promising treatment strategy for glioma patients. It allows for better normal tissue sparing due to higher physical dose conformity  and deliMetabolic imaging with 18)F-fluoro-ethyl-tyrosine positron emission tomography (18F-FET-PET) has been proposed to help delineate target volumes in glioma , which iF-fluoro-Whole-blood transcriptome (WBT) liquid biopsy (lbx) data have been used as a surrogate to monitor disease states , 22. DifWith this study, we aimed to investigate the value of 18F-FET-PET for tumor delineation in treatment planning and outcome prediction in primary glioblastoma (pGBM) and rHGG treated with particle radiotherapy and to assess a link between WBT lbx readouts and 18F-FET-PET-based tumor metabolic activity quantification.All patients consented to participate in this study, and ethical approval was obtained by the IRB-Ethics Committee of the Medical Faculty of Heidelberg University .n = 76 patients with pGBM (n = 43) or rHGG (n = 33) were included in this study. All patients underwent high-precision charged particle beam radiotherapy (RT) at the Heidelberg Ion Beam Therapy Center (HIT) between 2010 and 2017. Clinical data were retrospectively recorded in the institute’s own database.Patients with pGBM were treated in or analogous to the CLEOPATRA trial  for incoPatients with rHGG (n = 33) were treated in or according to the CINDERELLA trial  and receFor pGBM, 18F-FET-PET information was not considered for photon RT treatment planning but could be used for particle boost delineation. In the rHGG cohort, 18F-FET-PET information was not considered for GTV delineation but was included in clinical target volume (CTV) delineation; i.e., areas with high tracer uptake were effectively irradiated with CIR. No patient received particle RT in both the primary and recurrent settings.A static amino acid 18F-FET positron emission tomography (PET) CT scan was available before the start of particle RT for every patient. Tracer uptake and survival analyses were performed on the entire cohort (n = 76). For analyses on the impact of 18F-FET-PET on tumor delineation  in pGBM patients, 18F-FET-PET-derived isocontours were compared to MRI-based GTV for photon RT. Analyses were carried out for n = 16 (37%) pGBM patients. N = 27 patients could not be included due to one or more of the following reasons: photon RT was delivered at another institution, and RT plans were not available for further analyses (n = 14); photon RT planning MRI was not extractable from the institution’s picture archiving and communication system (PACS), i.e., imaging performed at another institution (n = 8); PET showed unspecific tracer uptake without significant evidence of residual tumor (n = 6); PET was carried out >50 days prior to photon RT start (n = 2).N = 27 (82%) patients with rHGG were included in analyses on the impact of 18F-FET-PET on tumor delineation. 18F-FET-PET-derived isocontours were compared to MRI-based GTV. N = 6 patients could not be included because CIR planning MRI was not extractable from the institution’s PACS (n = 3), because PET was carried out >50 days prior to CIR start (n = 1), which showed uncharacteristic low tracer uptake (n = 1), or imaging was performed at another institution and without a corresponding CT dataset needed for further data procession (n = 1).Since a relevant proportion of patients could not be included in the analyses of PET vs. MRI-based treatment volumes, it was evaluated whether the characteristics of the enrolled patients were different from those excluded (MRI showing tumor progression after particle (re)RT was available for n = 34 (79%) pGBM patients and n = 25 (76%) rHGG patients. Recurrence patterns were analyzed in a semiquantitative approach classifying tumors into one or more of the following categories: 1) recurrence in the area of initial contrast uptake in T1-MRI/MRI-based treatment volume, 2) recurrence in the area of increased PET signal, and 3) distant recurrence.Whole-blood transcriptome data (Illumina HumanHT-12 Expression BeadChips) were available for n = 9 patients of the rHGG cohort participating in a liquid biopsy study for the effect of CIR in rHGG . Blood samples were collected before the start of CIR and at different time points after CIR , between two surgeries , between surgery and start of photon RT (n = 16), and during photon RT (n = 23). In rHGG patients, PET images were acquired before CIR in all cases (n = 33), with one patient having had a partial tumor resection between PET acquisition and RT start. Most PET/CT scans were performed with Biograph 6 (n = 61). Other PET/CT devices used were Biograph 64 (n = 5), Biograph mCT Flow (n = 4), Biograph HiRes Model 1080 (n = 2), Biograph TruePoint Model 1094 (n = 2), and Biograph 40  and Gemini TF 16 .For analyses of PET imaging data, the standardized uptake value (SUV) was determined. The required parameters  were extracted from the static PET DICOM files with Loni Inspector  software. Median injected activity was 180 (R 124–314) MBq and 2.2 (R 1.39–4.24) MBq/kg body weight for pGBM patients and 198 (R 130–270) MBq and 2.49 (R 1.69–4.15) MBq/kg body weight for rHGG patients. The median time from injection to image acquisition was 12 min (R 5–42) in pGBM patients and 11 min (R 2–41) in rHGG patients.www.mitk.org)  , 25. In itk.org) .18F-FET-PET-based tumor volumes were generated as isocontours from SUVmax in increments of 10 from 40% to 70% within the ROI encompassing the tumor. PET images were registered to the treatment planning MRI (rigid registration). To assess differences in PET-derived isocontours (PETvol) and MRI-derived RT treatment volumes (MRIvol), different 3D structures were created and analyzed (see RNA was extracted from whole blood (PAX gene blood RNA tubes), using the PAXgene Blood RNA Kit . Expression was quantified from 200 ng of quality-controlled RNA  on Illumina HumanHT-12 Expression BeadChip array in the genomics Core Facility DKFZ . xCell  and PROGStatistical analyses were conducted with IBM SPSS Statistics Version 27  and R v 4.0.5 R . Time-toAn overview of the cohort, treatment, and patients’ characteristics is shown in The median age at diagnosis of pGBM was 58 years (R 21–75), with a median Karnofsky Performance Score (KPS) of 90% (R 60–100). Prior to RT, 33 (77%) patients underwent resection ; subtotal resection, 20 (47%)), and 10 patients (23%) underwent biopsy only. The sequential particle boost was delivered with protons (5 × 2 Gy) in 26 cases (60%) and CIR (6 × 3 GyE) in 17 cases (40%). Tumor tissue was available from all pGBM patients, and the diagnosis of glioblastoma was based on the 2007 WHO Classification of Tumors of the Central Nervous System  in most In the rHGG cohort, the median age at the time of CIR was 54 (R 22–71) years, and the median KPS was 90% (R 60–100). For rHGG patients, tumor tissue was available for n = 15 (45%) patients at the time point of recurrence. Tumor classification was mostly based on the 2007 WHO Classification  with one case being classified according to the 2016 WHO Classification. In n = 18 (55%) patients, where no biopsy or resection was performed at recurrence, the diagnosis was based on imaging criteria suggestive of rHGG (contrast medium enhancement) and an interdisciplinary consensus (tumor board). At disease recurrence, 16 (48%) patients had a WHO grade III, and 17 (52%) patients had a WHO grade IV tumor. The initial diagnosis was determined histologically in all rHGG patients. The underlying primary tumor was low-grade glioma (LGG) in n = 10 (30%) and HGG in n = 23 (70%) cases, with n = 8 (24%) grade III vs. n = 15 (45%) grade IV gliomas. In the primary setting, classification was according to the 2007 WHO classification in n = 23 (70%) and according to previous versions in the remaining n = 10 (30%) cases. The median time from the first course of photon RT to CIR was 22 months (R 6–208). Initially, n = 29 (88%) patients underwent tumor resection vs. n = 4 (12%) biopsy only. Re-resection had been performed in 15 cases (45%). The median time between the last resection and 18F-FET-PET was 14 months (R 1–289). CIR was delivered at a median dose of 39 GyE (R 30–45) in 3-GyE fractions. Median OS in rHGG was 17.5 months (95% CI 11.3–27.2) with the date of death known for 23 (70%) patients. PFS was 5.0 months (95% CI 3.4–7.6).Tracer uptake, as quantified by SUVmax and SURmax, is shown for both cohorts in In rHGG, PET images yielded a median SUVmax of 3.09 (R 1.1–8.39). A trend toward higher SUVmax values in grade IV tumors was observed . SUVmax median cutoff and SUVmax optimal cutoff were also prognostic factors for OS . Other prognostic variables on the univariate analysis included age  and degree of resection, whereby partial resection  and subtotal resection  were associated with improved OS. Multivariate analysis confirmed SUVmax optimal  and age ≥ 65 years  as independent adverse prognostic markers for OS in pGBM Figure 3In rHGG, SUVmax as a continuous variable was not prognostic of OS on univariate analysis Figure 3SUVmax optimal cutoff was also prognostic for PFS in pGBM  and rHGG . The data are shown in Comparison of 18F-FET-PET-defined isocontours with MRI-based treatment volumes was carried out for a subset of patients due to the limited availability of data  and CTV in 3 (11%) cases, where GTV was not defined but where areas of high tracer uptake morphologically corresponded to contrast enhancement in T1-MRI. Concordance between 18F-FET-PET isocontours and MRIvol was assessed with CI 5 and DiThe highest conformity was observed for isocontour 40% in pGBM and isocontour 50% in rHGG /24.35  cm3. Its union with GTV resulted in a median 26% (R 3–328)/37% (R 0.2–4836) increase in volume, with the median absolute volume added by “best matching isocontour” being 8.35 (1.37–159.04)/6.3 (R 0.13–73.03) cm3. Absolute volumes for each patient are visualized in Next, volumetric parameters in rHGG were evaluated (intersections of isocontour volumes and MRI-based target volumes) as well as tracer uptake metrics , where 18F-FET-PET was only utilized for boost delineation, showed n = 21 (62%) local recurrences, n = 5 (15%) distant recurrences, and simultaneous local and distant recurrences for n = 8 (24%) patients at first progression. In four cases, recurrences occurred in areas with increased tracer uptake , one of which did not show contrast enhancement on MRI and the remaining three being partially included in photon-RT MRIvol.In rHGG (subcohort of n = 25 patients with follow-up imaging showing progress), where 18F-FET-PET was used for CTV delineation, no local recurrence was observed in 18F-FET-PET-positive/MRI T1ce-negative areas. N = 3 (12%) patients showed distant recurrences, n = 17 (68%) local recurrences, and n = 5 (20%) simultaneous local and distant recurrences at first progression.We assessed if SUVmax correlates with whole-blood transcriptomes. Analysis of the most variable 10% of genes adjusted for initial tumor grade identified 38 genes as being associated with SUVmax  and 936 genes with an FDR < 0.05  MRI , 40, 41.To further examine the hypothesis that 18F-FET-PET delineates highly aggressive tumor regions, we simultaneously assessed a number of tracer uptake metrics and volumetric features derived from isocontour/target volume intersections in rHGG tumors. We observed a separation of tumors by grade, and random forest analysis identified I40 features as being able to discriminate between tumor grades. The added volume based on I40 is higher in grade III tumors vs. grade IV tumors, and accordingly, the median CI for I40 is lower for grade III vs. IV. Thus, the overlap of 18F-FET-PET and target volumes is higher in grade IV rHGG, thus supporting the hypothesis that 18F-FET-PET may be better suited for more aggressive tumors.In this line, we found significantly increased tracer uptake for non-irradiated (18F-FET-PET acquisition before RT) as well as non-resected GBM and a trend toward higher SUVmax in GBM. It seems plausible that higher metabolic activity resulting in a higher tracer uptake is observed in untreated and more aggressive tumors.Tracer uptake as a prognostic factor has been described previously. Gempt et al. reported that a tumor-to-normal ratio (SUVmean tumor/SUVmean background) of 1.88 was best in discriminating OS in patients with primary WHO grade II to IV glioma , whereasWe confirmed that tracer uptake is associated with prognosis both in the primary (pGBM) and in the recurrent setting (rHGG). Our identified thresholds/threshold ranges leading to prognostic separation are in line with the reported data. Thus, tracer uptake may allow for prognostic stratification independent of the therapeutic situation (primary vs. recurrent tumor). However, determination of a fixed tracer uptake cutoff for prognostication is difficult, as tracer uptake not only is dependent on the tumor itself but also reflects the imaging protocol used. It is also biased by previous therapies, as shown in our analyses, and the prognosis is obviously dependent on antitumor treatment and patient-related factors as well.In our study, analyses of tracer uptake were based on SUVmax. We also calculated SURmax values, which are the SUVmax values corrected for the appropriate SUV background signal , and we Finally, using WBT profiling of a subset of the rHGG cohort, we found gene expression differences associated with SUVmax, adjusted for initial tumor grade . Among The main limitation of the present study is the limited number of patients, especially for the whole-blood transcriptome analyses. Here, further independent studies including larger cohorts should be performed to validate our findings. Technical differences (different scanners), missing data, and (molecular) heterogeneity within the group of recurrent high-grade glioma might affect the present results. In addition, dynamic 18F-FET PET might reveal additional insights into tumor metabolism.18F-FET PET has a prognostic value in both treatment-naïve glioblastoma and recurrent high-grade glioma. Its value on target volume definition remains less clear, with overall low concordance between 18F-FET-PET and target volumes. High metabolic activity, as quantified by SUVmax, is linked to worse prognosis and unfavorable whole-blood transcriptome liquid biopsy readouts. Our results warrant confirmation in larger, prospective studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by IRB-Ethics Committee of the Medical Faculty of Heidelberg University . The patients/participants provided their written informed consent to participate in this study.Conceptualization and methodology: MW, MR, JF, PS, JD, UH, MK, LK, CD, AG, AK, MD, UW, CH-M, SC, AA. Formal analysis: MW, MK. Investigation: MW, MK, CS, UW. Writing - original draft: MW, BT, AG, MK, AA. All authors contributed to the article and approved the submitted version.This research was funded by the German Research Foundation Collaborative Research Center , EU Predict, and intramural funds of the National Center for Tumor Diseases (NCT).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Editorial on the Research TopicThe Fontan Circulation: Problems and SolutionsThe Fontan circulation has been developed as a strategy for multiple, unrepairable complex cardiac malformations in which there is only one effective ventricle. Almost 50 years later, the Fontan circulation is seen as an important achievement, resulting in good hemodynamic outcome for an otherwise untreatable heart disease. At the same time, complications occur due to challenges associated with a failing circulation due to the physiological features of chronically elevated systemic pressure and decreased cardiac output, obligatory features in the absence of a sub-pulmonary ventricle. As a consequence, patients are at risk for various complications including ventricular failure, organ failure, arrhythmias, protein losing enteropathy and plastic bronchitis. Despite recent improvements in tackling these complications, patients still face a high morbidity and mortality.This special issue focuses on analyzing these challenges in a problem and potential solution framework. Fifteen manuscripts addressed various aspects of the Fontan circulation in children and adults. The contributions in this special issue articulate for practitioners and scientists what we can do today, and what we need to develop going forward.The Fontan operation is the palliative procedure of choice for single ventricle patients. It is achieved by connecting both caval veins directly to the pulmonary arteries without the interposition of a sub-pulmonary ventricle. This approach separates the systemic and pulmonary venous returns to the heart and markedly diminishes mixing of deoxygenated and oxygenated blood as well as reduces volume overload of the single ventricle. However, the resulting Fontan circulation, in which the systemic and pulmonary circulation are connected in series, is characterized by a distinct physiology with chronically elevated central venous pressure. Chronic preload deprivation of the single ventricle reduces cardiac output.Currently, we are still facing a lack of comprehensive knowledge regarding Fontan circulatory physiology which translates into a lack of effective treatment options.Van de Bruaene et al. (2022) shed light on underlying factors of Fontan failure and contributed with a comprehensive review drawing the focus on its most limiting factor: the pulmonary circulation. The systemic ventricle in a Fontan circulation lacks preload at rest and during exercise. Therefore, systemic venous return through the pulmonary circulation becomes the main determinant in the regulation of pulmonary blood flow and hence exercise capacity. Since there is no sub-pulmonary ventricle, even minimal changes in pulmonary vascular resistance cause significant changes in cardiac output. The authors suggest optimizing the Fontan conduit early in life to allow for sufficient pulmonary artery growth and later in life by stenting of the conduit to adult size. In particular, stenting of a hypoplastic left pulmonary artery if present is important. The value of physical activity-which introduces some degree of pulsatility to the pulmonary vasculature and improves filling of the ventricle with stiffness-reducing stretching- as well preventing the accumulation of risk factors for heart failure with a preserved ejection fraction phenotype  cannot be understated. In practice, pediatric cardiologists and congenital cardiac surgeons should always aim for the “perfect” Fontan circulation. The patient should maintain an active, healthy lifestyle avoiding weight gain and the adult congenital cardiologist should not accept suboptimal hemodynamics .Kramer et al. (2022) developed an uncomplex yet remarkably accurate score to classify Fontan failure and late mortality in adult Fontan patients to allow a timely risk stratification. The score is based on hemodynamic, clinical and laboratory findings and is composed through analysis of a cohort of 198 adult Fontan patients with a median follow-up of about 20 years. Fifteen parameters were identified associated with Fontan failure and/or mortality. The accuracy to discriminate between patients with and without late Fontan failure as well as late mortality and survival was assessed in their cohort and patients with Fontan failure had a significantly higher median Fontan Failure Score compared to non-failing Fontan patients. Mortality associated with Fontan failure was substantial (48.1%). A prospective validation and likely refinement and calibration of the score in larger and preferably multi-institutional cohorts is still required.Clinical presentation and hemodynamic phenotypes of Fontan failure are considerably variable. Laohachai et al. (2022) reviewed the impact of impaired lung function which is characterized by restrictive ventilatory patterns and a reduced lung volume. In addition, the authors report on the reduced skeletal and respiratory muscle strength in Fontan patients. Respiratory muscle training has shown potential promise to improve exercise capacity.Respiratory function plays a crucial role in Fontan circulation by having the systemic and pulmonary circulations in series. Ventilation strongly determines pulmonary blood flow and cardiac output at rest and with exercise. van der Woude et al. (2021) discussed the influence of respiration on blood flow based on insights gained from imaging-based clinical evaluations. In contrast to the healthy circulation, respiration is the main source of blood flow pulsatility in the Fontan circulation, whereas cardiac contraction mostly drives the effective forward flow rate. In particular hepatic venous flow, which contributes approximately 38% to the total Fontan tunnel flow, is strongly influenced by respiration. In essence, the higher blood flow during inspiration is countered by depressed flow rates during expiration. Since MRI examination is recommended every 2 years in Fontan patients, clinicians should be aware that most conventional MRI flow sequences do not capture the pulsatility of the blood flow as a result of respiration. Therefore, authors state that conventional ECG-gated PC-MRI acquisitions can be used for the measurement of clinical parameters based on net forward flow. Inclusion of respiratory phasic pulsatility in state-of-the-art patient-specific CFD models are recommended for evaluation of detailed, time-resolved hemodynamic metrics , continuing to provide important insights for clinicians in the function of the Fontan circulation.While respiration influences phasic pulsatility, it has a limited effect on the effective forward flow in the Fontan circulation. Interestingly, Ohuchi et al. (2022) clarified the incidence, clinical characteristics and its influence on mortality. PAVF were present in 9.2% of 391 Fontan patients investigated by pulmonary artery angiography and/or contrast echocardiography during catheterization. Most patients (83%) showed a diffuse type of PAVF, associated with a significant decrease in mean arterial blood oxygen saturation compared to the non-PAVF group. Oxygen values in these patients decreased further corresponding to the postoperative stage from 90% at 1 year to 88% in the long-term follow-up of >25 years postoperatively. Authors highlighted that discrete-PAVF had no influence on SaO2 or mortality, whereas the presence of diffuse-PAVF caused hypoxia and had an adverse impact on all-cause mortality. The incidence of PAVF increases with patient age.Pulmonary arteriovenous fistulae (PAVF) are one of the major complications after Fontan operation. Dori et al. (2022) report on the management of patients with PLE and PB, potential life-threatening diseases affecting approximately 5%–15% of single ventricle patients. Conservative management of PLE involves use of diuretics including high-dose aldactone and sildenafil. The role of low-fat high-protein diet is less clear. Cardiac catheterization must be performed to determine hemodynamics and any reversible Fontan pathway obstruction. Fenestration creation or recreation can also be attempted. Patients that remain symptomatic can be started on enteric steroids, however this treatment strategy should be finite and limited due to the risk of serious side-effects and complications. Interventions for protein-losing enteropathy include embolization of hepatoduodenal and periduodenal lymphatic networks and procedures to lower pressure in the lymphatic system such as thoracic duct decompression.Patients with single ventricle palliation are susceptible to a variety of lymphatic abnormalities of both the thorax and abdomen, such as protein-losing enteropathy (PLE) and plastic bronchitis (PB). Imaging the central lymphatic system by dynamic contrast MR lymphangiography allows visualization of the lumbar lymphatic networks, the cisterna chyli and the thoracic duct. Utilizing these imaging tools, Conservative management of PB involves inhaled bronchodilators, inhaled steroids and pulmonary vasodilators. If symptoms persist, medications aimed at breaking down the casts are used including inhaled tissue plasminogen activator. However, if active lymph leaking into the airway is present, lymphatic imaging and selective lymphatic duct embolization are needed. If symptoms in both entities, PLE and PB, persist despite lymphatic intervention, then thoracic duct decompression  or orthotopic heart transplant should be considered.Luo et al. (2022) identified an increased central venous pressure and intraoperative aortic cross-clamping as risk factors for postoperative and persistent liver dysfunction after day 7 postoperatively. Special attention to this patient group is suggested by the authors to prevent liver impairment.Hepatic dysfunction after Fontan operation can occur as a postoperative complication and might also develop long term. In a retrospective case control study of 409 patients after TCPC, Schleiger et al. (2021) reported on Fontan-associated liver disease in the adult population. They investigated the metabolic liver function with the liver maximum function capacity test (LiMAx®) in 39 patients and compared it to laboratory testing, elastography and ultrasound. The authors found preserved metabolic hepatic function in about 80% of the patients. Results of metabolic testing did not correlate to the severity of abnormal hepatic findings evaluated by sonography or laboratory analysis, indicating that while abnormalities are present, hepatic functionality remains relatively intact.Both studies indicate that the development and progression of liver dysfunction in Fontan patients is not a linear or uniform process. Therefore, Schleiger and colleagues suggest that the diagnostic approach during follow-up should encompass a variety of modalities in order to obtain the most comprehensive picture. More work is necessary to determine the most valuable surveillance strategy in this domain.Ritmeester et al. (2022) present an interesting review on this subject and report on abnormalities in the nervous system, pituitary, kidneys, and musculoskeletal system. The thyroid axis may be affected by pituitary edema which is related to its unique vasculature. Therefore, awareness for potential hypothyroidism is important. Renal dysfunction is frequent and might be underestimated by creatinine based renal function testing due to myopenia as both lean muscle mass and bone mineral density are decreased in most of the Fontan patients. The assessment of cystatin C for renal function is recommended by the authors.Organ dysfunction occurs beyond the heart, lungs, liver and gut in those with Fontan circulation. Rubenis et al. (2021) assessed the sexual function of men with a Fontan circulation by performing a prospective, cross-sectional study based on the data from the Australian and New Zealand Fontan registry. Self-reported erectile function of 54 men with Fontan circulation was not significantly impaired when compared with historical controls, however sexual desire and overall satisfaction were reduced. The presence of erectile dysfunction was not correlated to the Fontan type or the New York Heart classification and the proportion of the cohort who had a prior pregnancy was congruent with population data.Little is known about the sexual health in patients after Fontan operation. Calderon et al. (2022) reviewed the findings that many Fontan patients present with impaired neurodevelopmental and mental health outcomes. Patients experience difficulties in areas of cognition related to attention and executive functioning, visual spatial reasoning and psychosocial development. A high risk for mental health morbidities, particularly anxiety disorders and depression, is present. Underlying alterations in brain processes may occur during fetal development which may be further influenced by hemodynamic parameters and perioperative factors. Variables such as multiple interventions requiring a prolonged hospital stay, is shown to be associated with adverse long-term neurodevelopmental outcome. The authors emphasize the benefit of early screening for neurodevelopmental deficits to initiate adequate support, thus allowing for early interventions and support to achieve the best potential outcomes.Van Den Helm et al. (2022) addressed this topic and reviewed the nature of thromboembolism post Fontan surgery and the evidence for thromboprophylaxis management using anti-platelet and anti-coagulant agents. The authors highlight that the complex pathophysiology of the highly thrombogenic environment in a Fontan circulation is based on all 3 elements of Virchow's triad: endothelial cell dysfunction, abnormal blood flow and a hypercoagulable state caused by coagulation factor abnormalities. Further dysregulation of hemostasis can be caused by a tendency towards liver function abnormalities and associated serum protein imbalances. Subclinical cerebrovascular thromboembolic events are still underdiagnosed. Importantly, authors stated that the risk of thromboembolic events, mainly occurring in the venous system, is the highest in the first year post Fontan operation with a second peak 10 years post Fontan. Furthermore, the authors focus on the needs for an emerging consensus on prophylaxis. Fontan patients with no clinical complexities should receive life-long aspirin as thromboprophylaxis, whereas further anti-coagulation with vitamin K antagonists can be reserved for patients with special risk factors like previous thrombotic events or in the older Fontan patient. A comprehensive follow-up including monitoring and attention for bone mineral density should be provided. Currently, the use of new classes of oral anti-coagulants may hold promise but is not yet recommended as primary prevention due to the lack of evidence. Despite prophylaxis, a significant risk of thromboembolic disease post Fontan surgery remains.The Fontan circulation is associated with an increased risk of thromboembolism. To prevent patients from pulmonary embolism or ischemic stroke Fontan patients are commonly treated with anti-platelet agents and/or anti-coagulants. A markedly reduced exercise performance affects most Fontan patients. Reduced exercise capacity influences long-term quality of life and is associated with worse prognosis, although a subset of patients have high physical performance (“Super-Fontan”), which may represent a unique low-risk phenotype. Typical cardiopulmonary exercise testing response in Fontan patients includes a depressed peak heart rate (HR), elevated minute ventilation /carbon dioxide production slope (ventilatory inefficiency), reduced peak work rate and an increased breathing frequency.Tran et al. (2021) studied 60 patients from the Australian and New Zealand registry with the “Super Fontan” phenotype defined as having a normal exercise tolerance. When compared to a Fontan group with impaired exercise capacity, the “Super-Fontan” phenotype is associated with a healthy weight, lower age at Fontan completion, better exercise self-efficacy and higher overall levels of sport and physical activity participation during physical development. This said, exercise capacity has significant implications on clinical outcome and survival. As the mechanisms underlying improvements in aerobic exercise capacity and the effects of exercise training on circulatory and end-organ function remain incompletely understood, Tran et al. (2022) focused on the need for developing adapted exercise programs. In this second manuscript, the authors present the planning of a large well designed, multi-center randomized controlled trial: The Fontan Fitness Intervention Trial which will investigate as a primary outcome the change in aerobic exercise capacity after a 4-month supervised aerobic and resistance exercise training program of moderate-to-vigorous intensity followed by an 8-month maintenance phase in children and adults. Results of this study are pending and have the potential to strongly impact clinical practice.Stating that patients with the best clinical outcome might provide important insights, Dirks et al. (2022) report on their innovative prospective study of home-based bicycle-ergometer and inspiratory muscle training for children and adults with Fontan circulation. After performing 90 min of endurance training per week in addition to inspiratory muscle training (30 breaths per day) for 10 months the authors observed significant increases in maximum relative workload and in maximum inspiratory and expiratory pressures. Peak VO2 values did increase significantly as compared to baseline in a subgroup analysis of teenage/adult patients while the subjective quality of life remained unchanged under a potential influence of COVID times. This study confirms that an individually adapted home-based training program is safe and is associated with improvements in exercise test variables.In line with this therapeutic strategy, Van de Bruaene et al. (2022) the areas in need of innovation to improve the Fontan circulation include:- retraining of the pulmonary vasculature and ventricle- using right-sided assist devices prior to transplant- performing further research on implantable right-sided assist devices- reducing the risk of AV valve surgery-  determining novel pathways to improve the thrombo-inflammatory state in Fontan patients.In this issue, perspectives are highlighted to improve long-term risk stratification and the implementation of new tools for future care to effectively improve the quality and duration of life of individuals with a Fontan circulation.The contributions of research efforts and reviews in this special issue on the Fontan circulation teach us to focus on principles of management and the identification of risk factors to improve patient outcomes. There is a great need for advancing multidisciplinary health care for this unique group. Special focus should be given to optimization of cardiovascular parameters pre-Fontan e.g., the pulmonary vasculature and the lymphatic vasculature. A comprehensive follow-up surveillance program post Fontan needs to pay attention to early discovery of organ dysfunctions. An important measure to prevent or reverse negative outcome and the development of complications appears to be the maintenance of a good exercise capacity with encouragement of physical activity and regular exercise training. According to


Using analytical Size-Exclusion Chromatography (SEC), Isothermal Titration Calorimetry (ITC), and SAXS we were able to unravel the mechanism of inhibition of Ru4POM. Ru4POM binds to the positively-charged substrate binding region of the enzyme through electrostatic interactions, triggering the dimerization of the enzyme which consequently is inactivated. Ru4POM is the first non-peptide molecule showing a substrate-competitive mechanism of inhibition for CK2. On the basis of SAXS data, a structural model of the inactivated (CK2α)2(Ru4POM)2 complex is presented.CK2 is a Ser/Thr protein kinase involved in many cellular processes such as gene expression, cell cycle progression, cell growth and differentiation, embryogenesis, and apoptosis. Aberrantly high CK2 activity is widely documented in cancer, but the enzyme is also involved in several other pathologies, such as diabetes, inflammation, neurodegeneration, and viral infections, including COVID-19. Over the last years, a large number of small-molecules able to inhibit the CK2 activity have been reported, mostly acting with an ATP-competitive mechanism. Polyoxometalates (POMs), are metal-oxide polyanionic clusters of various structures and dimensions, with unique chemical and physical properties. POMs were identified as nanomolar CK2 inhibitors, but their mechanism of inhibition and CK2 binding site remained elusive. Here, we present the biochemical and biophysical characterizing of the interaction of CK2α with a ruthenium-based polyoxometalate, [Ru Protein kinase CK2 (previously known as casein kinase 2 or CK2) is a eukaryotic Ser/Thr protein kinase, ubiquitous and extremely conserved throughout evolution . Despiteα catalytic subunit (or its isoform α’) and the β “regulatory” subunit are associated to form the α2β2 tetrameric holoenzyme, the prevailing form of CK2 in cells . Yet, many of these compounds show unfavorable side effects and are convenient only when conventional treatments of cancers have been ineffective  appears to be a promising approach particularly regarding the selectivity issue. In fact, unlike most protein kinases, CK2 exclusively phosphorylates acidic substrates, with a minimum consensus sequence S/T-X-X-E/D/pX, often with acidic residues also in position n + 1 and/or n + 2  are other interesting compounds explored as CK2 inhibitors. POMs are a class of polynuclear oxo-bridged transition metal complexes, which have received extensive attention due to rich topology and tunable chemical/physical properties. In addition to the application in material design  and cataOwing to their nano-size, POMs are able to establish various types of interactions with peptides and proteins, ranging from electrostatic interaction and hydrogen bonds, also mediated by water or cations, to other weaker types of interactions . These i4(H2O)4(μ-O)4(μ-OH)2(γ-SiW10O36)2]10− (Ru4POM)  , revealium atoms , and it um atoms .10[Ru4(H2O)4(μ-O)4(μ-OH)2(γ-SiW10O36)2], Ru4POM (CH2)4CONH(CH2)3Si}2O]4−, Biotin-SiW10 (10O36{(C16H9)SO2NH(CH2)3Si}2O]4−, Trp-SiW10 12[Mo36(NO)4O108(H2O)16], Mo36POM, was also prepared . Concerning the hybrid polyoxotungstates, we observed a moderately higher activity for the lipophilic TBA salts. In addition, both the nature and the configuration of the organic ligands had an impact on the inhibitory effect. The larger polyoxomolybdate showed very weak activity on CK2. Owing to these results, we focused the rest of our study on Ru4POM.First, we analyzed the efficacy of different POMs in decreasing the catalytic activity of recombinant CK2 . We foun4POM . We then checked whether Ru4POM competes with the substrates for the CK2 binding-site. As shown by the kinetics analysis reported in 4POM exerts a competitive inhibition towards both model substrates, since it reduced the affinity , without decreasing the Vmax values.We then evaluated if the Ruin vitro conditions, namely at pH 7.5 and in the absence of NaCl. Given the high negative charge of Ru4POM, we evaluated if the inhibitory efficacy was affected by changes in pH and ionic strength. Deviation from the optimal conditions reduced the activity of the controls . Therefore, we tried to treat cells with Ru4POM in combination with increasing amounts of two different cationic transfection reagents, the stable cationic polymer polyethylenimine (PEI) and the cationic liposome JetOptimus  and the effect was potentiated by increasing the amount of the transfecting reagents, likely due to higher amounts of Ru4POM delivered into the cells. These results indicate that Ru4POM is suitable for CK2 inhibition in cell, as it can be transferred inside intact cells, and that it is able to target endogenous CK2.Next, we wanted to assess whether RutOptimus . EndogentOptimus . We obse4POM and CK2α in vitro. SEC elution profiles , but also at 500 nm, where only the signal of Ru4POM is present (red curve). The lack of absorption of CK2α at 500 nm (green curve) allows to unequivocally recognize the elution of Ru4POM. In 25 mM Tris, 500 mM NaCl, 1 mM DTT, pH = 8.5, free CK2α elutes at 12.6 ml retention volume, in the monomeric form 2(Ru4POM)2 is formed.The elution profile of samples with Ru4POM, we analysed samples with increasing molar ratios in a Superdex 200 column, which has a higher MWs resolution range. The elution profiles with the unchanged buffer system, 25 mM Tris, 500 mM NaCl, 1 mM DTT, pH = 8.5, of samples with various Ru4POM/CK2α ratios, namely 1:1, 2:1, 5:1, and 10:1, are shown in 4POM induce the formation of species with larger hydrodynamic dimensions, that is, high-order oligomeric forms of CK2α/Ru4POM complexes. Very similar elution profiles for ratios 5:1 and 10:1 indicate that the saturation is reached for such values. The large variations in the elution volumes are explained by the formation of oligomeric forms of CK2α/Ru4POM complexes rather than by the formation of species where monomeric CK2α (MW 40 kDa) is interacting with multiple POMs units (MW 5 kDa). Overall, the SEC experiments indicate that Ru4POM induces the oligomerization of CK2α, with the formation of dimers when the ratio is 1:1 and of larger assemblies when the ratio is higher, with the saturation reached around 5:1 ratio.To further investigate the interaction between CK2α and Ru4POM, the thermodynamic parameters of the binding were determined by isothermal titration calorimetry (ITC). In the ITC measurements, CK2α was titrated with Ru4POM; both species were in 25 mM Tris, 500 mM NaCl, pH = 8.5. A representative experiment is shown in 4POM titration produces a typical single transition, saturation-shaped thermogram, which can be fitted by the sigmoidal binding isotherm of the one binding-site model. From two independent experiments, we derived an apparent mean KD of 2.09 ± 0.36 μM for the single macroscopic dissociation constant. The derived stoichiometry for the complex is 1:1 (1.04 ± 0.02), in accordance with the chromatographic data, thus supporting the formation of a stable (CK2α)2(Ru4POM)2 complex. The analysis of the thermodynamic signature for the binding of Ru4POM to CK2α reveals the dominance of the enthalpic contribution (ΔH = −32.99 ± 1.42 kJ/mol) over a negligible entropic term (−TΔS = 0.50 ± 2.43 kJ/mol), and a final ΔG = −32.48 ± 0.46 kJ/mol (2W12O40]6− (H2W12) interacting with human serum albumin (HSA) were also characterized by enthalpically driven exothermic process, while bigger W-based POMs as [NaP5W30O110]14− (P5W30), showed an important endothermic component, mainly attributed to the unfolding of the protein  traces as functions of the SEC elution profile frames showed that the protein was monodispersed in presence of 0.5 M NaCl , on a SEC GE Superdex Increase 200 (3.2/300) column, equilibrated with buffer 25 mM Tris pH 8.5. The Rg value of 2.3 nm, corresponding to an estimated molecular weight of about 39 kDa, as expected from monomeric CK2α, in line with the MW estimation from the previous SEC experiments. The comparison between the CK2α crystal structure and the SAXS data showed good fitting value (χ2 1.29)   and the χ2 1.29) , further4POM at 1:1 M ratio in presence of 0.5 M NaCl, and the scattering data on the single monodisperse species displayed significant structural differences compared to the free form of CK2α 2(Ru4POM)2 complex. Importantly, in accordance with the lack of a significative entropic contribution for the CK2α/Ru4POM interaction seen with ITC, SAXS data do not show any evidence of protein denaturation.We then analyzed a mixture of CK2α:Ru of CK2α . Notablyprofiles . The Rf 3.1 nm  and a mo4POM at lower ionic strengths, i.e., 0.3 and 0.2 M NaCl, caused protein precipitation of the sample, as in the case of free CK2α, hampering the possibility of reliable SAXS analyses  traces showed the formation of soluble complexes but in a multi-component system, with different size and molecular weight species co-existing in solution (2(Ru4POM)2 complex from the available SAXS data. Starting from the evidence that Ru4POM actively induces the dimerization of CK2α, the most reasonable assumption is that Ru4POM structurally mediate the interaction between two molecules of the enzyme. This is the simpler and more plausible explanation that globally takes into account our SEC, ITC and SAXS data.Next, we monitored the effect of 2 and 10 M equivalent of Rusolution . We then4POM and CK2α, trying to identify the binding site 2(Ru4POM)2 model direct contacts between the two molecules of CK2α are established. The predicted structural model overlays well with the DAMMIF elongated envelope obtained from CK2α-Ru4POM SAXS data analysis 2(μ-O)4(H2O)4 (γ-SiW10O36)2]10−. Some hybrid organic-inorganic POMs belonging to the Keggin decatungstosilicate family, which share the same SiW10O36 unit of Ru4POM, were also shown to be promising. The variation of the inhibitory activity with the organic domains will thus deserve attention, for the possibility to anchor suitable recognition motifs and develop, for example, bi-specific inhibitors , indicating that the full molecule and not some decomposition fragments are active on the enzyme, as instead reported for other POMs 2(Ru4POM)2 complex, with no residual presence of the single components, indicating the formation of a strong and stable complex. SAXS experiments confirm the presence of such a complex, with dimeric CK2α when Ru4POM is present in equimolar amount. According to SEC data, larger assemblies are formed at higher molar ratios, with the saturation reached at around 5:1 for Ru4POM:CK2α.To possibly decipher the mechanism of inhibition of Ru4POM and CK2α with the formation of a stable complex is confirmed by ITC measurements, which indicates a macroscopic apparent mean KD of 2.09 ± 0.36 μM and a 1:1 stoichiometry, in accordance with SEC and SAXS data. The observed thermodynamic parameters agree with literature data on the interaction between inorganic POMs and proteins, which are characterized by KD in the μM range 2(Ru4POM)2 complex, to see whether the structural information derived by SAXS are valid also for the tetrameric holoenzyme  and the positively charged P + 1 loop of the α-subunit of the other tetramer (Arg191–Lys198). It is plausible that Ru4POM is able to interfere with the normal regulation of the enzyme activity by affecting the oligomerization process of the tetrameric form of CK2. It is also possible that Ru4POM affect the interaction of CK2 with at least some of the interacting partners of this kinase.As Ruloenzyme . Indeed,4POM binds to CK2 and induces the formation of a (CK2α)2(Ru4POM)2 complex, which is inactive because the substrate-binding site and the ATP-binding site are inaccessible to substrates and ATP, respectively. This inhibition mechanism is entirely different from the common ATP-competitive one typical for most of the known protein kinase inhibitors. Ru4POM has physical and chemical properties very different from that of conventional kinase inhibitors , and this, coupled to its ability of inhibition in cell-based assays, make this compound particularly interesting for further developments. Notably, Ru4POM is the first non-peptide molecule showing a substrate-competitive mechanism of inhibition. Atomic details of the interaction between CK2 and Ru4POM can be unravelled by the crystallographic analysis of the (CK2α)2(Ru4POM)2 complex, which would be useful to obtain information regarding the most relevant interactions that stabilize the complex. While many crystal structures of CK2 are available, alone or in complex with inhibitors (mainly ATP-competitive), complexes with substrate peptides have never been crystallized, suggesting that also the crystallization of CK2 in complex with POMs acting as substrate-competitive inhibitors could not be straightforward.In summary, we could unravel the mechanism of inhibition of a Ru-based POM, a compound stable in aqueous solution and able to inhibit CK2 in the very low nanomolar range. Cell internalization has also been addressed by using suitable transfecting agents. Ru4POM against a protein kinase panel is beyond the purpose of this study, our findings suggest that a remarkable specificity for CK2 is highly conceivable, being this compound able to function as a substrate-competitive inhibitor of a kinase, CK2, with the uncommon property of targeting negatively charged substrates.While a selectivity profile of Ru2β2)  were kied as in . Briefly10[Ru4(H2O)4(μ-O)4(μ-OH)2(γ-SiW10O36)2], Ru4POM, was prepared following a published procedure , in buffered aqueous environment  over 24 h (Narocedure . Ru4POM ver 24 h .nBu4N)3H[γ-SiW10O36{(C5H7N2OS)(CH2)4CONH(CH2)3Si}2O], Biotin-SiW10 -(nBu4N)4[γ-SiW10O36{(C16H9)SO2NH(CH2)3Si}2O], Trp-SiW10  enantiomer, were prepared following previously reported procedures. The corresponding sodium salts were prepared by counterion exchange as described in (4)12[Mo36(NO)4O108(H2O)16], Mo36POM, was prepared as reported in - or a 10/30 Superdex 200 GL column  equilibrated with 25 mM Tris, 1 mM DTT, pH = 8.5 and NaCl at various concentrations, namely 0.5, 0.4, 0.3, and 0.2 M. For the preparation of CK2α/POM mixtures in different ratios, a concentrated stock solution of Ru4POM (5 mM) obtained by solubilizing dry powder in 25 mM Tris, 0.5 M NaCl, 1 mM DTT, pH = 8.5 was used. CK2α concentration varied from 200 to 300 μM, in accordance with SEC-SAXS experiments (see below).Analytical SEC analyses on isolated CK2α and on CK2α/Ru4POM (200–400 μM) samples were in 25 mM Tris, 500 mM NaCl, pH = 8.5. After baseline stabilization, a further delay of 60 s was used before the first injection. In each individual titration, a starting Ru4POM injection of 0.4 μl in 0.8 s was followed by other 12 injections of 3 μl with a duration of 6 s each. A delay of 150 s was applied between each Ru4POM injection. Wiseman plot of integrated data was automatically obtained by software analysis (excluding the heat referred to the first 0.4 μl injection) and then it was fitted by theoretical binding curve using the one site model.Isothermal titration calorimetry (ITC) experiments were performed using a Malvern PEAQ-ITC microcalorimeter, at 25°C, with a 270 μl sample cell and a computer controlled microsyringe for titrant injections. CK2α (20–30 μM) and Ru4POM mixtures were measured by SEC-SAXS approaches  column. For samples measured at 0.3 and 0.2 M NaCl  CK2α concentration was 130 μM. Samples with 200 μM CK2α with CK2α:Ru4POM at 1:2 and at 1:10 M ratio in buffer 25 mM Tris, 1 mM DTT, pH 8.5 and 0.5 M NaCl were loaded on a Agilent AdvanceBio SEC (4.6/300) and on a GE Superose 6 Increase (3.2/300) columns, respectively. The samples were centrifuged  prior the measurements and 50 μl of sample were injected in the columns. The purifications were carried-out via a high-performance liquid chromatography device  attached directly to the sample-inlet valve of the BM29 sample changer. The columns were equilibrated with 3 CV to obtain a stable background signal before measurement. A flow rate of 0.3 ml/min was used for all sample measurements. All SAXS data were collected at a wavelength of 0.99 Å using a sample-to-detector  distance of 2.867 m. Data reduction and preliminary data processing were performed automatically using the Dahu/FreeSAS pipeline implemented at BM29. In the SEC-SAXS chromatograms, frames in regions of stable Rg were selected with CHROMIXS and averaged using PRIMUS to yield a single averaged frame for each protein sample. Analysis of the overall parameters was carried out by PRIMUS from ATSAS 3.0.4 package , were used to calculate ab initio models in P1 and P2 symmetries, respectively, with DAMMIF  as receptor. The docking resulted in 19 plausible models of a complex formed by CK2α monomer binding a single molecule of Ru4POM bound in different positions along the entire positively charged cavity of CK2α. Symmdock . Our data showed that CK2α never forms dimers or multimers in solution while Ru4POM induces CK2α dimerization at 1:1 M ratio. Based on this experimental observation, only the Symmdock-generated CK2α:Ru4POM dimers, where Ru4POM mediates intermolecular contacts between two CK2α monomer, were selected. The structures of models were ranked according to the lower χ2 value obtained by structure comparison between the models and the SAXS data of the CK2α-POM complex using CRYSOL (The molecular docking of Ruatchdock  using thSymmdock  was theng CRYSOL . The besg CRYSOL .50 (concentrations inducing 50% inhibition) analysis, recombinant tetrameric CK2 (α2β2), or C-terminal-deleted CK2α (CK2α1–336), or full length CK2α (7.5 ng) were incubated with 0.1 mM synthetic peptide substrate RRRADDSDDDDD (CK2-tide), in a phosphorylation buffer containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20 μM [γ-33P]ATP , in a final volume of 20 μl, with increasing concentrations of the inhibitor. In the case of tetrameric CK2, 0.1 M NaCl was also present in the phosphorylation mixture. Controls were performed in the absence of any inhibitor, but with equal volume of vehicle (H2O). Reactions were performed at 30°C for 12 min and stopped by sample absorption on phospho-cellulose papers. Papers were washed three times with 75 mM phosphoric acid and counted in a scintillation counter (PerkinElmer). In the case of casein substrate (used at 0.05 mg/ml concentration), reactions were stopped by the addition of Laemmli loading buffer, samples were analyzed by 11% SDS-PAGE, and radioactive bands were detected and quantified by digital autoradiography . IC50 calculation was obtained by analysis of the results with GraphPad Prism 7.0a software.For ICFor kinetics analysis, the assays were performed with increasing concentrations of CK2-tide or casein, in the phosphorylation buffer described above, but with 100 μM ATP concentration.2, maintained in DMEM medium (Sigma), supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell treatments with Ru4POM were performed in the culture medium. When used, the cationic transfection reagents PEI (Thermo Scientific) or JetOpitmus (Polyplus-transfection) were mixed with Ru4POM and incubated for 1 h at room temperature, before adding the mix to the cells. After 24 h cells were lysed as previously described (HEK-293T cells (human embryo kidney fibroblasts) were cultured in an atmosphere containing 5% COescribed . ProteinEndo-cellular CK2 activity was evaluated by assessing the phosphorylation state of the CK2 substrate Akt phospho-Ser129 (Abcam) . For thiQuantitation of the signal was obtained by chemiluminescence detection on ImageQuant LAS 500  and analysis with Carestream Molecular Imaging software (Carestream).p < 0.05.Statistical significance was evaluated by One-way Anova analysis using GraphPad Prism 7 program. All values are expressed as means ± SEM. Comparisons of more than two groups were made with a one-way ANOVA using post-hoc Bonferroni’s test. Comparison of two groups was obtained by the Student’s t-test for unpaired data when appropriate. Differences were considered statistically significant at values of