Laser speckle contrast imaging (LSCI) has emerged as a promising tool for intraoperative cerebral blood flow (CBF) monitoring because it produces real-time full-field blood flow maps noninvasively and label free.We aim to demonstrate the ability of LSCI to continuously visualize blood flow during neurovascular procedures.LSCI hardware was attached to the surgical microscope and did not interfere with the normal operation of the microscope. To more easily visualize CBF in real time, LSCI images were registered with the built-in microscope white light camera such that LSCI images were overlaid on the white light images and displayed to the neurosurgeon continuously in real time.LSCI was performed throughout each surgery when the microscope was positioned over the patient, providing the surgeon with real-time visualization of blood flow changes before, during, and after aneurysm clipping or arteriovenous malformation (AVM) resection in humans. LSCI was also compared with indocyanine green angiography (ICGA) to assess CBF during aneurysm clipping and AVM surgery; integration of the LSCI hardware with the microscope enabled simultaneous acquisition of LSCI and ICGA.The results suggest that LSCI can provide continuous and real-time CBF visualization without affecting the surgeon workflow or requiring a contrast agent. The results also demonstrate that LSCI and ICGA provide different, yet complementary information about vessel perfusion. Both LSCI and ICGA images are restricted to the tissue surface, but their relatively simple instrumentation allows them to be incorporated into the surgical microscope. Several studies have demonstrated LSCI during neurosurgical procedures in humans and shown its promise as a CBF monitoring tool, including surgical revascularization,,Here we demonstrate an LSCI system that is integrated into the neurosurgical microscope allows real-time, continuous visualization of CBF overlayed onto the surgical field during neurosurgical procedures including simultaneous ICGA and LSCI imaging. We evaluate these capabilities during cerebral aneurysm clipping and arteriovenous malformation (AVM) resection surgeries. Although simultaneous imaging of CBF with LSCI and ICGA has been performed in animal models,22.1The LSCI hardwareBack-scattered laser light was directed to an NIR-enhanced CMOS camera  mounted on the side observer port on the same side as the craniotomy. This enabled an observer to participate during the study, which occurred for most of the surgery for patients 2 and 3 and intermittingly for patients 1, 4, and 5. The pixel area was slightly cropped during acquisition to capture only pixels over brain tissue. A filter wheel  and polarizer  were positioned between the camera adapter and camera. The filter wheel held various neutral density filters for controlling the laser power. The polarizer was integrated into a motorized rotation mount  to reduce specular reflections. A band-pass filter  was added in front of the camera to enable simultaneous LSCI acquisition during illumination of ICG, to block nonlaser light, and to avoid interference of normal white light illumination throughout each procedure.2.2–ICGA images are represented as raw fluorescence intensity images that were collected by the built-in Leica OH6 fluorescence camera. Raw LSCI images collected by the speckle camera were processed by calculating the spatial speckle contrast value . LSCI could be performed at any time when the microscope was positioned over the patient by turning on the laser illumination. LSCI did not disturb the workflow of the neurosurgeon and was performed at numerous critical times throughout the surgery. The neurosurgical co-investigator (R. A.) performed a majority of the surgeries using the surgical microscope oculars and could observe the LSCI data in real time on a monitor mounted next to the surgical microscope that displayed the overlaid blood flow images.3The preclipping LSCI images in The flow dynamics within the aneurysm shown in Video ,Despite the differences between the information obtained with LSCI and ICGA, LSCI can also be used to create images that look very similar to ICGA images. Results comparing simultaneous LSCI and ICGA images shown in 4Results of the current study show that LSCI can be used to continuously monitor CBF in real time. Results shown in ,The waveforms in One limitation of LSCI that is noticeable in The aim of this study was to demonstrate the potential of LSCI to monitor CBF continuously during neurovascular surgery. The integration of the LSCI device into the surgical microscope enabled continuous CBF visualization and allowed for simultaneous acquisition of LSCI and ICGA. The time course of the relative blood flow shown in 5Our results suggest that LSCI can provide continuous and real-time CBF visualization without affecting the surgeon workflow or requiring a contrast agent and thus is a promising tool for continuous CBF monitoring during surgery. By integrating the LSCI device into the surgical microscope, we performed LSCI at critical parts of neurovascular surgery and provided the surgeon with immediate actionable information on the success of the procedure. We also performed simultaneous acquisition of LSCI and ICGA, demonstrating that LSCI and ICGA are complementary tools for visualizing CBF to aid surgical decision making.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


In addition, an abnormally large EAT poses mechanical stress on the heart via pericardial restrain. HFpEF patients with enlarged EAT may belong to a unique phenotype that can benefit from specific EAT-targeted interventions, including life-style modifications and pharmacologically via statins and fat modifying anti-diabetics drugs; like metformin, sodium-glucose cotransporter 2 inhibitors, or glucagon-like peptide-1 receptor agonists, respectively.Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome with diverse etiologies and pathophysiological factors. Obesity and type 2 diabetes mellitus (T2DM), conditions that coexist frequently, induce a cluster of metabolic and non-metabolic signaling derangements which are in favor to induce inflammation, fibrosis, myocyte stiffness, all hallmarks of HFpEF. In contrast to other HFpEF risk factors, obesity and T2DM are often associated with the generation of enlarged epicardial adipose tissue (EAT). EAT acts as an endocrine tissue that may exacerbate myocardial inflammation and fibrosis  via para/vasocrine factors and pericardial restrain, respectively. Life-style modifications including healthy diet and regular exercise can quash the EAT expansion. Statins, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors and fat-modulating antidiabetics including metformin, sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) agonists can target EAT. FFA, free fatty acids; AGEs, advanced glycation end-products; NO, nitric oxide; ROS, reactive oxygen species; Ang-II, angiotensin II; TGF-β, Transforming growth factor beta; MCP-1, monocyte chemoattractant protein 1; IL-6, interleukin 6; TNF-α, tumor necrosis factor alpha. Figure created via Servier Medical Art and BioRender tools.Epicardial adipose tissue (EAT)-related heart failure with preserved ejection fraction (HFpEF). Obesity and type 2 diabetes mellitus (T2DM) are common triggers of HFpEF, frequently associated with EAT expansion. EAT plays metabolic and mechanical roles in HFpEF development  Commipokines .There is growing evidence that HFpEF with enlarged EAT is a clinically relevant HF phenotype that may require specific treatments , 17. We via triggering a systemic proinflammatory environment and inducing endothelial production of reactive oxygen species and reduces nitric oxide (NO) bioavailability, which especially affect the coronary microvasculature and the neighboring cardiomyocytes  mass to volume ratio compared to the non-obese phenotype which is more common in the elderly  and lower levels of glucose transporter-4 (GLUT4), leading to adverse lipid and glucose metabolic profile augmented by the proinflammatory secretome   , 67  . In linece (CMR) . The algce (CMR) . Beside ce (CMR) . Since Cce (CMR) . Recent ce (CMR) .Unlike HF with reduced EF (HFrEF), patients with HFpEF phenotype do not benefit from most standard HF therapies, but rather from lifestyle modifications , 74. SimIn addition to lifestyle modifications, the ESC 2016 guidelines have recommended the symptomatic use of diuretics in HFpEF . Owing tvia inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis  receptors on the surface of hepatocytes (Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are novel cholesterol lowering agents which act atocytes . A studyatocytes .Metformin is the first-line treatment for T2DM, particularly in obese patients. Alongside its glucose-lowering effect, metformin use is associated with weight loss independent of diabetes, primary due to reduction of VAT , 93. A rvia enhancing glucose-dependent insulin secretion, reducing glucagon secretion, and delaying gastric emptying, resulting in adequate T2DM control and weight loss  receptor agonists, indicated for the treatment T2DM. They act ght loss . Severalght loss , 99. In ght loss . In addight loss . Interesght loss . In a coght loss . Similarght loss . GLP-1 ag system –107.via prolonging GLP-1 half-life (via different mechanisms including downregulation of the receptor for AGE (RAGE) . Sitagliptin, a DPP-4 inhibitor, has recently been shown to substantially reduce EAT in subjects with obesity and T2DM alf-life . DDP-4 ialf-life –111. ThoE (RAGE) , activatE (RAGE) , reductiE (RAGE) , and dimE (RAGE) . In contignaling –118.via increasing urinary glucose excretion , for example dapagliflozin and empagliflozin are relatively new medications indicated for T2DM patients, but they are also sufficient in HFrEF patients without DM , 120. Agxcretion . In addifat burn , 123. Lifat burn . With refat burn , 125. Cafat burn . In patifat burn . Press rfat burn , all shofat burn . Similarfat burn . Weight fat burn . OverallAnterior pericardiotomy through minimally invasive percutaneous procedure is a potentially novel last-option treatment for HFpEF patients with severe LV restriction, since it eliminates the external restraint of the pericardium , 132. InO2) and ventilatory efficiency (VE/VCO2)  . WhetherVE/VCO2) . These fHFpEF is a diverse disease resulting from wide range of comorbidities. Obesity and DM are principal drivers of HFpEF. Stratifying HFpEF patients based on phenotypic data results in novel classifications including obese and diabetic HFpEF phenotypes. There is a close association between EAT volume and HFpEF. HFpEF patients can be further classified according to EAT volume using advanced imaging techniques including CMR and CT. EAT functions as endocrine tissue that contributes to myocardial inflammation. In addition, EAT expansion acts as space-occupying lesion that causes pericardial restrain, increase in ventricular filling pressures, and enhanced ventricular interaction. HFpEF patients with enlarged EAT may benefit from lifestyle modifications and symptomatic treatment with diuretics. Besides, statins, PCSK9i and fat-modulating anti-diabetic agents like metformin, SGLT2i or GLP-1 agonists can be especially effective in this subgroup of patients, being able to induce EAT regression. In addition, direct effects of SGLT2i and GLP-1 agonists on HFpEF are currently under clinical investigation.AE wrote most of the manuscript and prepared the figures. VN wrote short parts of the manuscript. SV revised the manuscript and applied changes to the text and figures. CT provided the main ideas of the manuscript and guided the other authors, revised the manuscript, and added some parts. SK provided CT has received speaker fees and/or contributions to congresses from Abbott, Abiomed, Astra Zeneca, Bayer, Böhringer-Ingelheim, Novartis Pfizer, and Servier; all outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Despite identification of numerous candidate drugs in rodent models, there has been a lack of clinical trials and there is currently no therapy that halts disease progression in NPH patients. This review covers the most important findings of therapeutic approaches in NPH model systems to date, including hypothesis-driven therapies and untargeted drug screens, approached from the pathophysiology of NPH. Importantly, most animal models used in these studies represent the cystic infantile form of NPH, which is less prevalent than the juvenile form. It appears therefore important to develop new models relevant for juvenile/adult NPH. Alternative non-orthologous animal models and developments in patient-based in vitro model systems are discussed, as well as future directions in personalized therapy for NPH.Nephronophthisis (NPH) is an autosomal recessive ciliopathy and a major cause of end-stage renal disease in children. The main forms, juvenile and adult NPH, are characterized by tubulointerstitial fibrosis whereas the infantile form is more severe and characterized by cysts. NPH is caused by mutations in over 20 different genes, most of which encode components of the primary cilium, an organelle in which important cellular signaling pathways converge. Ciliary signal transduction plays a critical role in kidney development and tissue homeostasis, and disruption of ciliary signaling has been associated with cyst formation, epithelial cell dedifferentiation and kidney function decline. Drugs have been identified that target specific signaling pathways  and rescue NPH phenotypes in  NPHP1 gene, the most frequent cause of NPH , have recently been shown to be an important cause of renal disease in adults (Nephronophthisis (NPH) is an autosomal recessive kidney disease that accounts for up to 15% of end-stage renal disease (ESRD) in children . In addin adults .Three clinical subtypes of NPH can be discerned. In juvenile NPH, the most prevalent form, ESRD develops at a median age of 13 years . JuvenilIn contrast to juvenile and adult NPH, infantile NPH leads to ESRD before the age of 5 years and typically presents with enlarged cystic kidneys, severe hypertension and a rapid disease course, making it difficult to distinguish from autosomal recessive polycystic kidney disease (ARPKD) based on renal symptoms only . InfantiPKD1 and PKD2, led to the hypothesis that cilia control oriented cell division and proliferation of tubular cells, and that ciliary defects therefore result in cystic kidneys, which became a classic sign of ciliopathies.Mutations in more than 20 different genes have been identified to cause NPH . Most ofIn accordance with the role of PC in most organ systems, 10–20% of NPH patients have extrarenal manifestations defining syndromic forms, including Senior-Løken syndrome , Bardet-Biedl syndrome , Joubert syndrome  and cranioectodermal dysplasia (narrow thorax and short stature) .Current drug therapy for NPH is merely supportive, aimed at treatment of anemia, hypertension, growth retardation and other symptoms related to chronic kidney disease (CKD) . AlthougTherapy for NPH-related ciliopathies has been an active field of research for more than 15 years. Next-generation sequencing techniques have accelerated gene discovery and uncovered numerous targetable genes and pathways. The most important drug targets for renal ciliopathies  have been reviewed by in vitro and in vivo model systems: pharmacotherapy (hypothesis-driven approaches including repurposed medication from PKD and untargeted drug screens) and gene-based therapy, in relation to the pathophysiology of different aspects of the NPH phenotype. Promising developments in personalized disease modeling and drug delivery are discussed, as well as challenges in clinical trials for NPH and orphan-drug development. Finally, we provide a rationale for future studies in this field.If juvenile NPH is detected in an early disease stage, there is a therapeutic window of a few years in which treatment could delay or prevent renal replacement therapy. Previous reviews of therapies for renal ciliopathies focused on models of PKD and cystic (infantile) NPH or did not discern between renal and extrarenal manifestations. In this review we summarize the results from two main strategies for therapeutic intervention in NPH specifically that have been investigated in 0) from the centrosome, called ‘basal body’ when engaged in ciliogenesis , basal body/centrosome  and inversin compartment  and IFT-B  sub-complexes , ZNF423 (NPHP14) and the recently discovered gene MAPKBP1 (NPHP20). However, ZNF423 has been shown to interact with CEP290 and its murine ortholog Zfp423 was shown to be involved in the transcription of ciliary genes ”] but no ciliogenesis defects , resulting in more than 90 genes associated with an ‘NPH-related ciliopathy’ . These effects could be rescued in vitro using CDK inhibitors  and upregulation of fibrosis-associated genes, further linking aberrant DDR signaling and S-phase arrest to tubulointerstitial fibrosis in NPH  stress response and mitochondrial dynamics were shown to be dysregulated in the K8/NPHP9 . In addiK8/NPHP9 . HoweverK8/NPHP9 . The relK8/NPHP9 . FinallyK8/NPHP9 . HoweverAlthough studies in animal models of NPH have provided above-mentioned insights into pathophysiology of NPH, most models do not recapitulate the juvenile NPH phenotypes.in vivo imaging, functional studies and large-scale screening of small molecules for therapeutic approaches. Most models have been based on morpholino-based knockdown of the respective nphp genes, resulting in loss of function like conditions, typically resulting in severe developmental defects including cysts in the proximal pronephros and/or defects in the distal part of the pronephros associated with ciliary defects (tmem67 (NPHP11), leading to ciliary defects and tubular cysts in adult zebrafish kidneys (Zebrafish larva (24–72 h post fertilization) is a widely used organism to study renal ciliopathies. This model is low cost, fast and efficient, allowing  defects . It is u defects , likely  defects . Interes kidneys , indicatNphp1, Nphp4, Nphp5 and Mapkbp1 do not show an obvious spontaneous renal phenotype  show enlarged cystic kidneys characteristic of infantile NPH, although pcy/Nphp3 and Anks6/Nphp16 mice have slow disease progression and (late stage) tubulointerstitial fibrosis , kidney enlargement and late-stage fibrosis PKD (Mouse models of InvsC defects (fibrosis . The pcyfibrosis . Consequ diverge . Similar (AR)PKD . These dCy rat models, with mutations in the orthologs of human NEK8/NPHP9, TMEM67/NPHP11 and ANKS6/NPHP16 genes, respectively. These all show rapidly progressive cystic kidney enlargement reminiscent of infantile NPH or ARPKD phenotypes , kidney tubuloids and organoids, could partly solve these issues  and the genetic background strain with associated genetic modifiers impact the severity of the phenotype and the utility of the model for human disease, as was demonstrated in mouse models of opathies , 2020. Pe issues .Primary kidney tubular epithelial organoids, or ‘tubuloids,’ can be efficiently established from adult stem cells derived from kidney biopsy material and from URECs. Tubuloids were employed to study infectious kidney disease in a personalized fashion and test the efficacy of cystic fibrosis treatment using a forskolin (cAMP) swelling assay, illustrating its utility in  drug screening . In contLimitations are that kidney organoids do not recapitulate the overall kidney morphology and do not take into account interactions between organs including vascularization of the glomeruli (unless they are transplanted into a host organism), an essential step required for generation of intratubular flow. In addition, only one study reported the generation of iPSCs-derived kidney organoid in the context of an NPH-related condition. The generated organoids were used as a source of tubular epithelial cells in which transciptomic and functional analyses confirmed major defects in polarity , as prevNPHP1, the most frequent genetic event in NPH. Possibilities for targeted therapies therefore include enhancing stability/function of remaining mutated NPHP protein and/or of the complex in which it functions, or targeting the signaling pathway in which the protein plays a role, downstream pathophysiological mechanisms or modifier gene products . As a result, important findings in the ADPKD field may be relevant for NPH.Of note, some therapies listed below are aimed at prevention of NPH manifestations. While this approach in animal models could provide important insights, in practice most patients are symptomatic upon diagnosis therefore precluding any preventive treatment until the identification of relevant biomarkers in asymptomatic individuals.G-protein coupled receptors are a large family of signaling receptors, many of which localize to the ciliary membrane . ExampleAVPR2. V2R is an important GPCR in renal physiology that localizes to the basolateral membrane and to cilia of renal tubular epithelial cells (AVPR2 (One of the main targetable sources of cAMP is the vasopressin receptor type 2 (V2R) encoded by al cells . Activatal cells   pathway-induced epithelial cell proliferation . Its activation was shown to stimulate cyst growth through several cAMP-mediated mechanisms including activation of PKA leading to Clferation . The useferation , has prose model . The V2Rse model . Tolvaptse model , 2018 anse model . There aere case . Such anpost hoc analysis of the above-mentioned study in ADPKD patients  and other cystic kidney diseases  oxylipins  has been shown to be elevated in diseased kidneys of rodent models of NPH  which functions as a central regulator in cellular metabolism, growth, proliferation, cell cycle and survival. It has been shown that cilia-dependent flow sensing negatively regulates mTORC1 through the kinase LKB1 . In ciliCy/Anks6/Nphp16 rats  pathway, although the role of cilia in canonical, β-catenin-dependent Wnt signaling remains controversial . ProteinDCDC2/NPHP19 has been shown to activate the canonical Wnt pathway and cause pronephric cyst formation in zebrafish, a phenotype which could be rescued by a β-catenin inhibitor  mouse models of infantile NPH and PKD, respectively, with long-lasting effects. Analysis of the targets of roscovitine revealed induction of a G1/S cell-cycle block, transcriptional regulation and inhibition of apoptosis as mechanisms of action   effectively arrested kidney volume expansion, cyst progression and renal insufficiency in f action . In addif action . Anotherormation . In conthibition . Finallyargement . In summ8/Nphp9) . InteresCep290/Nphp6 mouse model characterized by slowly progressive kidney disease. Treatment of kidney cells from Cep290-deficient mice with CDK1/2 inhibitor rescued DNA-damage signaling, supernumerary centrioles and ciliation defects  is an important mediator of the endoplasmic reticulum stress response, oxidative stress and the mitochondrial apoptotic pathway, which were shown to be upregulated in kidneys from jck/Nek8/Nphp9 mice. Pharmacological inhibition of CaMKII restored these pathways and significantly reduced kidney volume and cystogenesis , directly modulates the calmodulin/NPHP5 interaction therefore increasing the amount of NPHP5 at the transition zone in the absence of CEP90 , is involved in regulation of cell cycle and proliferation and was shown to be activated as a consequence of increased cAMP in ADPKD . Inhibithp3 mice . It alsose model . The samnvs mice .Cy/Anks6/Nphp16 rats and pcy/nphp3 mice) treated with the anti-inflammatory drug methylprednisolone showed reduced fibrosis and preserved kidney function compared to untreated animals . This suggests differential microtubule involvement between NPH and other renal disorders.Interestingly, expression of HDAC6 was found to be increased in various kidney conditions including fibrotic, ADPKD and CKD models, and HDAC6 inhibitors showed a positive effect on disease evolution . HDAC6 iPharmacological screens for ciliopathies comprise small molecule compound screens and high-throughput testing of chemical libraries such as United States Food and Drug Administration (FDA)-approved drugs with the aim of drug repurposing. The latter is an attractive option because it is cheaper, faster, and has a higher success rate than development of novel drugs . Thus faCEP290-invalidated human RPE1 cells and identified 22 compounds that restored ciliogenesis, of which the flavonoid eupatilin showed the strongest effect. Eupatilin was demonstrated to restore IQCB1/NPHP5 levels at the ciliary transition zone in the absence of CEP290 (see above). Eupatilin treatment did not significantly rescue ciliogenesis defects associated with the knockdown of other NPH-associated genes, including NPHP4, CEP164/NPHP15 and CEP83/NPHP18, suggesting a CEP290-specific effect. Treatment of rd16 mice harboring a Cep290 in-frame deletion with eupatilin improved cone photoreceptor function, showing its efficacy in vivo  does not present a kidney phenotype  was tested in zebrafish and did not have a significant effect on the kidney phenotype, consistent with the results from the screen  screen to identify genes that modulate ciliogenesis and can be targeted pharmacologically. For example, an siRNA screen in RPE1 cells targeting 7,784 therapeutically relevant genes identified 36 positive modulators of ciliogenesis including proteins involved in the regulation of actin cytoskeleton dynamics. Pharmacological inhibition of actin polymerization using cytochalasin D rescued ciliogenesis in roblasts . Howevered cells . One of e screen . High-the screen .CEP290/NPHP6 mutation present in 10–15% of LCA cases, which introduces a cryptic splice site leading to inclusion of a pseudo exon with a premature stop codon  to modulate splicing. This approach was first employed to correct a recurrent intronic op codon . ASO tarop codon  and led op codon . ASO wasted mice . While Ced genes . Althougkd1 mice . An advakd1 mice . Researckd1 mice .in vitro systems. In contrast to classical gene therapy in which a wild-type copy of the gene is integrated into the host genome, CRISPR/Cas9 is a precise genome-editing technique that allows use of a repair template. This correction approach is widely used to obtain isogenic control iPSCs and it was shown to efficiently restore the cilia phenotype in iPSC-derived kidney organoids from a patient with IFT140-related Mainzer-Saldino syndrome. In addition, spheroids from epithelial cells sorted from uncorrected organoids were less polarized than spheroids from gene-corrected cells  or dysregulated cell cycle (for example CDK inhibitors) as well as the use of immunomodulatory drugs (for example senolytic drugs) have improved cystic phenotypes in zebrafish and rodent models of NPH . In addisyndrome . ASO thesyndrome . Howevernile NPH .Glis2/Nphp7 knockout mice, and GLIS2 mutations are an atypical and rare cause of NPH , encoding a regulator of p53 during DDR, also leads to phenotypes similar to juvenile NPH including tubular atrophy, interstitial fibrosis and cysts at the corticomedullary junction  the NPH phenotype, there is a striking lack of clinical trials for NPH. To exclude possible publication bias, ClinicalTrials.gov was queried for registered ongoing and future clinical trials in ciliopathies. This yielded trials for ADPKD, ARPKD, primary ciliary dyskinesia, Von Hippel Lindau disease and various extrarenal manifestations of ciliopathies, including obesity in Bardet-Biedl and Alström syndromes . There are several possible reasons for the absence of clinical trials in NPH, including challenges related to the genetic and phenotypic heterogeneity in NPH, for example small sample sizes and variability within cohorts, the relatively short therapeutic window compared to ADPKD, challenges of clinical trials in a pediatric population and financial aspects of orphan drug development.Despite NPHP3-related infantile NPH isosorbide dinitrate was used to treat hypertension by restoring nitric oxide generation renal symptoms and fluctuation in renal phenotype, for example statins which can be prescribed to treat hypercholesterolemia in patients with Bardet-Biedl or Alström syndrome have shown promising but inconsistent results in ADPKD . In addineration . Nitric neration .in vitro modeling, for example the use of URECs or the generation of kidney organoids from iPSCs, enable testing of medication directly in NPH patient-derived cells that harbor the causal mutation and the genetic background of the patient including modifier gene variants. This should facilitate translation of findings back to therapy in individual patients. Despite current limitations, improvement of NPH-kidney organoids can aid drug development in NPH, as has been successfully illustrated in a kidney organoid model of cystinosis (Another consequence of the low prevalence and phenotypic heterogeneity in NPH is the need for personalized therapy. Developments in stinosis .in vivo therapeutic application of CRISPR/Cas9, genome editing can be applied ex vivo, for example to populate bioartificial kidneys with corrected patient-derived renal epithelial cells as a future alternative for donor kidneys if safety issues of cell-based therapies have been sufficiently resolved (Patient-derived organoids can be combined with CRISPR/Cas9 technology to correct the NPH phenotype for various applications . To overresolved , b. Bioeex vivo applications, developments in targeted delivery methods bring gene therapy in NPH patients one step closer (Bbs4 and Cep290 mouse models, respectively, delivery of viral vector to the kidney via systemic injection is limited because AAV particles do not pass the glomerular filtration barrier (Besides p closer . Whereas barrier . Targete barrier . This sy barrier .Many potential therapies for NPH have been explored in model systems, from targeted treatments aimed at restoring ciliary signaling, CDK inhibition, immune system suppression and ASO therapy to untargeted drug screens. Translation of results is limited by the lack of animal models that recapitulate the juvenile NPH phenotype and in the case of infantile NPH, for which models exist, by the short therapeutic window. Employment of non-orthologous animal models and developments in organoid technology can potentially fill this gap and provide new opportunities for personalized treatments. Ultimately, safety and efficacy of potential NPH therapies will have to be tested in clinical trials. Given obstacles of genetic and phenotypic heterogeneity in renal ciliopathies and clinical trials in children, repurposing of medication used in other cystic or chronic kidney diseases could be the most viable approach. In addition to identification of effective drugs, successful treatment or prevention of NPH depends on the discovery of biomarkers that identify NPH patients in an early disease stage.MS and AB conceived the idea and wrote the manuscript. AB and SS provided critical feedback. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Preeclampsia (PE) involves inadequate placental function. This can occur due to elevated pro-inflammatory tumor necrosis factor-α (TNF-α). In other tissues, TNF-α signals via sphingosine kinase 1 (SphK1). SphK1 hinders syncytial formation. Whether this occurs downstream of TNF-α signaling is unclear. We hypothesized that placental SphK1 levels are higher in PE and elevated TNF-α decreases syncytial function, increases syncytial shedding, and increases cytokine/factor release via SphK1 activity. Term placental biopsies were analyzed for SphK1 using immunofluorescence and qRT-PCR. Term placental explants were treated after 4 days of culture, at the start of syncytial regeneration, with TNF-α and/or SphK1 inhibitors, PF-543. Syncytialization was assessed by measuring fusion and chorionic gonadotropin release. Cell death and shedding were measured by lactate dehydrogenase release and placental alkaline phosphatase-positive shed particles. Forty-two cytokines were measured using multiplex assays. Placental SphK1 was increased in PE. Increased cell death, shedding, interferon-α2, IFN-γ-induced protein 10, fibroblast growth factor 2, and platelet-derived growth factor-AA release induced by TNF-α were reversed upon SphK1 inhibition. TNF-α increased the release of 26 cytokines independently of SphK1. TNF-α decreased IL-10 release and inhibiting SphK1 reversed this effect. Inhibiting SphK1 alone decreased TNF-α release. Hence, SphK1 partially mediates the TNF-α-induced PE placental phenotype, primarily through cell damage, shedding, and specific cytokine release. Preeclampsia (PE) is a pregnancy disorder characterized by high blood pressure and end organ damage. PE is the cause of 76,000 maternal and 500,000 annual deaths globally and increases the rate of preterm and low birthweight neonates ,2. The cThe human placenta is a complex organ composed of multiple cell types performing different functions and interacting with each other’s signaling pathways. Placental tissue includes cells found in other organs such as endothelial cells, vascular smooth muscle cells, fibroblasts, and immune cells. However, the unique cell type of the placenta is the pluripotent cytotrophoblast (CT) that can differentiate into an extravillous trophoblast lineage, responsible for anchoring the placenta into the maternal decidua, or into the villous trophoblast lineage .The villous CT proliferates, differentiates, and then fuses into the basal side of the overlying syncytiotrophoblast (ST), a multinucleated epithelial layer that regulates feto-maternal exchanges. The ST also acts as an immune and pathogen barrier between mother and fetus. This placental layer is in direct contact with maternal blood and releases hormones, such as human chorionic gonadotropin (hCG), essential for maintaining the pregnancy .Similar to other epithelial cells, the ST is in constant turnover throughout pregnancy, due to a balance between formation of the ST through differentiation and fusion of CTs and loss of the ST through shedding. The mature ST aggregates into syncytial knots and sheds into the maternal bloodstream. These syncytial aggregates are characterized by high levels of placental alkaline phosphatase (PLAP) enzyme activity .The placenta in PE is characterized by poor development of the syncytium, high levels of shedding and cell damage, and high levels of inflammatory cytokines . In PE, Pro-inflammatory cytokines are notorious for disrupting placental function, notably tumor necrosis factor-α (TNF-α) . ElevatePhysiological levels of TNF-α, however, have pregnancy enhancing roles such as inducing mucin 1 shedding from uterine epithelial cells, a major feature of embryo implantation . Normal One of the mediators of TNF-α signaling in non-placental endothelial and epithelial cells is sphingosine 1-phosphate (S1P) ,25,26,27S1P is synthesized by sphingosine kinases 1/2  and is degraded by S1P lyase (SPL) or dephosphorylated by S1P phosphatases (SPP) and lipid phosphate phosphatases (LPPs) . SphK1 aS1P is also associated with inflammatory pathologies like atherosclerosis , cancer p < 0.0001) and diastolic  blood pressures compared to those in the healthy control group. All participants in the PE group were positive for proteinuria. Infants born to mothers in the PE group had lower gestational ages (37.2 ± 0.3) compared to those from healthy control mothers . There were no significant differences in infant birth weights between groups.Seventeen women with healthy pregnancies and 12 women with PE were enrolled in the study and their clinical characteristics are summarized in n = 12) and the healthy control (n = 17) groups were observed in the trophoblastic layers . In Quantitative analysis of SphK1 mRNA expression demonstrated increased SphK1 in placental biopsies from women with PE (PE develops after the second trimester of pregnancy . Even thn = 3). E-cadherin is expressed at epithelial adherens junctions, which are found in both CTs and the ST in the placenta, since trophoblasts are epithelial cells. High E-cadherin expression was saturated at the basal ST membrane where the apical side of the CT fuses into the ST. Since ST differentiation involves downregulating E-cadherin expression, the apical side of the ST expresses E-cadherin at low levels and is often not visualized with the microscope parameters optimal for the signal on the basal membrane of the ST. Any nuclei present on the apical side of the heavy E-cadherin demarcation belong to the multinucleated ST layer. Explants incubated for 0, 1, 6, or 8 days after initiation of culture showed multiple nuclear clusters on the apical side of the strong E-cadherin demarcation that define areas of multinucleated ST  up to 8 days and then assessed for the presence of ST using immunohistochemistry ; n = 3. eated ST B.n = 3). As shown in Since SphK1 expression and activity is increased in response to proinflammatory stimuli ,44 and Sp = 0.003, n = 4, p = 0.02, n = 4, n = 4). We analyzed samples after 24 h of treatment as well, but there were no significant differences among the groups. Levels of β-hCG released into the supernatant were measured in explants treated with 1 ng/mL TNF-α, 1 µM PF-543, or a combined treatment of 1 ng/mL TNF-α and 1 μM PF-543 after 24 or 48 h of treatment. None of these treatments affected β-hCG (n = 6) or LDH (n = 8) levels released by the explants (TNF-α treatment (1 ng/mL) decreased the formation of multinucleated ST area after 48 h (explants . n = 6). No significant changes were observed in β-hCG levels compared to controls in any of the groups at 24 h post-treatment (p = 0.008). However, inhibiting SphK1 (1 μM PF-543) did not counteract this decrease (p = 0.05). This is likely due to the inhibitory effects of TNF-α as the treatment interaction was not significant. As well, β-hCG levels after co-treatment with PF-543 and TNF-α compared to TNF-α alone were not significant. This indicates that SphK1 activity does not mediate TNF-α effects on this ST endocrine function. Since 1 ng/mL TNF-α had no significant effects on β-hCG and LDH release, we tested whether a higher TNF-α concentration could hinder explant resyncytialization and increase cell death through SphK1. Explants were treated with 10 ng/mL TNF-α, 1 μM PF-543, or the combination of 10 ng/mL TNF-α and 1 µM PF-543  and inhibiting SphK1 in the presence of TNF-α decreased LDH levels from TNF-α-induced levels (p = 0.02). Since the 24-h treatment showed no differences in LDH release, we measured ST shedding only at 48 h. ST shedding, demarcated by positive PLAP activity in the culture supernatant, was elevated in response to TNF-α treatment alone . This effect was diminished upon Sphk1 inhibition (1 μM) with a treatment interaction of p = 0.02.No significant changes were observed in LDH levels compared to controls in any of the groups at 24 h post-treatment . Howeverp = 0.03) and MIP-1β (p = 0.001) after 48 h (n = 5). The treatment also increased the release of sCD40L (p = 0.01), RANTES (p < 0.0001), GM-CSF (p = 0.06), IL-18 (p = 0.002), MCP-3 (p = 0.0006), MIP-1α (p = 0.009), IL-6 (p = 0.055), IL-12p40 (p = 0.02), IL-12p70 (p = 0.002), IL-2 (p = 0.007), IL-15 (p = 0.004), and IL-1RA (p = 0.006), cytokines whose circulating levels are elevated in PE (p = 0.0006) and IFN-gamma-associated protein 10 or IP-10 (p = 0.02). The increase in these cytokines was reversed by SphK1 inhibition with a treatment interaction of p = 0.06 and p = 0.03, respectively : fractalkine (p = 0.009), IL-7 (p = 0.01), EGF (p < 0.0001), and Flt3L (p < 0.0001). Two interleukins that are decreased in PE, IL-4 (p = 0.002) and IL-9 (p = 0.02), were also increased by this treatment. The increased release of these factors was independent of SphK1 activation. TNF-α increased the release of FGF2 (p = 0.02) and PDGF-AA (p = 0.01)  with treatment interactions of p = 0.06 and p = 0.05, respectively. Post-hoc tests further confirmed this interaction as TNF-α increased FGF2 (p = 0.004) and PDGF-AA (p = 0.003) and inhibiting SphK1 in the presence of TNF-α decreased FGF2 (p = 0.02) and PDGF-AA (p = 0.01) levels from TNF-α-induced levels. PF-543 treatment showed an overall change in FGF2 levels (p = 0.05). This change, however, was most likely due to its interaction with TNF-α, since post-hoc tests showed no difference between PF-543 treated groups and the no-treatment control. The only cytokine tested that was decreased by TNF-α treatment alone was IL-10 (p = 0.05). PF-543 treatment in combination with TNF-α not only reversed this decrease but further increased IL-10 release to 44.7 ± 20.6% higher than the no-treatment control with a treatment interaction of p = 0.003. This highlights the PF-543 treatment effect (p = 0.02). Inhibiting SphK1 with PF-543 increased GRO-α release   which wan = 4) on day 4 of explant culture. β-hCG and LDH levels were measured. With respect to ST endocrine function, no significant changes were observed in β-hCG at any PF-543 concentration after 24 h of treatment  to inhibit SphK1 . One expIn contrast to the important role SphK1 may play in CT fusion, inhibiting SphK1 activity alone increased β-hCG release from cultured placental explants. The seemingly contradictory effects of SphK1 inhibition on syncytial function versus CT fusion in our findings might point to differential effects of SphK1 inhibition depending on the cell type, CT versus ST. This, however, remains to be investigated. While CT fusion and β-hCG release are both used as markers for syncytial differentiation, it is important to remember that these processes occur by separate signaling pathways and in different cell types. A differential effect on fusion versus endocrine function was also seen by Singh et al. when using the non-specific DMS to inhibit SphK; however, the results were opposite to ours, with increased CT fusion and decreased β-hCG release.Interestingly, in our study, the effects on β-hCG release were only observed with 10 ng/mL of TNF-α, but not with 1 ng/mL of TNF-α. TNF-α was previously established to have pro-apoptotic effects in the syncytium and to decrease β-hCG release at 10 ng/mL in villous explants , whereasThe syncytium is maintained by a balance of trophoblast fusion/differentiation and syncytial shedding. Since cell damage and syncytial shedding are increased in PE, we assessed the levels of LDH release and PLAP-positive shed particles, respectively. Inhibiting SphK1 alone had no effect on placental cell membrane damage or syncytial shedding. However, these measures were increased in explants treated with TNF-α alone, reflecting increased cell death. Inhibiting SphK1 while treating with TNF-α decreased cell death and syncytial shedding induced by TNF-α alone, indicating that TNF-α exerts its effects on cell death and shedding through SphK1. LDH is a cytosolic enzyme that can induce autophagy in tumor cells . TNF-α , as wellStudies in epithelial and endothelial cells show TNF-α administration increases activity and expression of SphK1 levels ,25,26,27In our study, the only factor that TNF-α treatment of placental explants decreased was IL-10 and inhibiting SphK1 activation not only reversed this effect but also led to increased levels above the control. Siwetz et al. show that IL-10 levels are unchanged in response to TNF-α in first-trimester villous explants . HoweverTNF-α induced the release of a wide variety of inflammatory and growth cytokines but the majority of these were independent of SphK1 activation. This could be because TNF-α acts on specific cell types in the placenta that release some cytokines and not others. Hence, TNF-α could be dependent on SphK1 activation only in specific cell types. Alternatively, TNF-α could be inducing certain cytokines through TNFR1, and some through TNFR2 whose signaling and interaction with SphK1 have yet to be investigated. Interestingly, inhibiting SphK1 led to a decrease in TNF-α release, implying that SphK1 activity induces TNF-α release from the placenta. This release could be a normal physiological response when SphK1 activation levels are within normal ranges and could lead to the release of TNF-α to increase normal processes like angiogenesis and proliferation . HoweverFinally, only GRO-α increased in response to SphK1 inhibition. While the role of GRO-α in placental and trophoblast function throughout pregnancy has not been extensively investigated, GRO-α is commonly secreted from trophoblasts in response to cell damage . In our Explants are a complex three-dimensional culture with multiple cell types, matrices, and cell-cell interactions. Hence, potent inhibitors like PF-543 can potentially bind proteins, be metabolized, or sequester within the tissue, thereby losing some potency. To investigate if increasing concentrations of PF-543 could have distinguishable effects on explant syncytialization, we exposed explants to increasing PF-543 concentrations that were still within the specific range . PF-543 Certain studies show a difference in function for S1P produced by SphK1 or SphK2 . SphK1 iCurrently, there are no definitive treatments for PE except for delivery of the fetus, which often leads to preterm birth . This stWomen were recruited and provided written informed consent. The study was approved by the University of Alberta Health Research Ethics Board  and conformed to the standards set by the Declaration of Helsinki. Term placentae were collected immediately after elective caesarean section deliveries at the Royal Alexandra Hospital, Edmonton, AB, Canada, from women with no health complications and from women with PE. Women with PE were defined as those who had blood pressures above 140/90 mmHg on two occasions measured 6 h apart and at least 30 mg/dL proteinuria in a 24 h collection. Seventeen women with healthy pregnancies and 12 women with PE were enrolled in the study. Placental biopsies from all women were collected for staining. Biopsies from 13 placentas from the healthy control group and nine placentas from the PE group were collected and processed for qRT-PCR. 2 biopsies from each patient were embedded into one mold with Tissue-TekⓇ O.C.T. . Three 8 μm sections from varying depths of one mold were cut on a cryostat and these sections were placed on a Superfrost slide . The sections were fixed with 100% ice-cold methanol for 20 min in −20 °C and blocked for 1 h with 10% normal goat serum. The sections were then incubated in a moist chamber simultaneously with a rabbit anti-SphK1 primary antibody  and a mouse anti-E-cadherin primary antibody  in 1× PBS overnight. Slides were washed 3 times with 1× PBS for two minutes per wash. AlexaFluor goat anti-rabbit 488 and goat anti-mouse 594  secondary antibodies were added simultaneously and incubated in the dark for 1 h at room temperature. After washing 3 times with 1× PBS, the sections were incubated for 10 min in the dark with DAPI . The slides were washed again and mounted with PermaFluor Aqueous Mounting Medium . Three randomly selected areas from each section were imaged using the 20× objective on Zeiss LSM 700 microscope and Zen Black software . Images were analyzed using ImageJ software . Briefly, images were set to a consistent threshold value, and mean integrated density was calculated. Integrated density values were normalized to the number of nuclei in the image, which were calculated through the “Analyze particles” function on ImageJ. Normalized values for each sample were averaged and the standard error of the mean was determined.Placental biopsies were collected from placentas within 2 h of delivery from the area between the umbilical cord and the placental edge. Three evenly spaced 6 mm2 placental biopsies were obtained from term placentas in the area between the umbilical cord and the edge of the placenta. Samples were collected within 2 h of delivery and biopsies from each placenta were placed together in a single tube. Placental biopsy tissues were placed into liquid nitrogen. The frozen samples were wrapped with aluminum foil, re-submerged in liquid nitrogen, and crushed using a mortar and pestle until the tissues were in powder form. Next, each sample was transferred into a 2 mL PCR tube containing a single steel bead . Each tube was then filled with 1 mL of TRIzol reagent . The samples were lysed using a TissueLyser II  at a setting of 25.00 frequency/s for 2 min, repeated 3 times. 200 μL of chloroform was then added to each sample before vigorously shaking for 1 min, followed by a 5 min incubation at room temperature. Samples were then centrifuged for 15 min at 15,000 rcf at 4 °C. Following this step, the RNA layer (aqueous top layer) was extracted and transferred to a new 2 mL tube and the Invitrogen RNA isolation kit  was used to purify the isolated RNA. Finally, quantification of the RNA in these samples was carried out using the Nanodrop Spectrophotometer . Samples that were below a 260/280 ratio of 1.7 were discarded, but this was very uncommon with the isolation kit that was used. Three evenly spaced 6 mm® Green MasterMix , 0.6 μL of primers (10 μM), 6.3 μL of UltraPure TM Distilled Water, and 2.5 μL of cDNA from each sample. The ΔΔCT method of relative quantification was used to analyze the results [Purified RNA was reverse transcribed into cDNA using 500 ng of total RNA, 4 μL of ABM 5× all-in-one RT MasterMix , and UltraPure TMDistilled Water  as needed to reach a final volume of 20 μL. The primer sequences for SphK1 and the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT1), were designed using OligoPerfect Designer . SphK1 forward: 5′-CTG GCA GCT TCC TTG AAC CAT3′; SphK1 reverse: 5′-TGT GCA GAG ACA GCA GGT TCA-3′; HPRT1 forward: 5′-GAC CAG TCA ACA GGG GAC ATA A-3′; HPRT1 reverse: 5′-AAG CTT GCG ACC TTG ACC-3′. The PCR reaction was carried out using Applied Biosystems Power Up TM SYBR results .2 and the media was changed every 48 h. Placental chorionic villi were dissected within 1–2 h following delivery and washed 3 times with gentle shaking using Hanks’ Balanced Salt Solution . A self-regenerating explant culture method was adapted from Siman et al. and Trowell ,81 by cuExplants were incubated for 4 days to allow the shedding of the original syncytium  and all Cultured explants from duplicate wells (3 explants/well) were fixed in 1% paraformaldehyde in PBS, embedded and sectioned as described above. The 3 villi from each well were placed in 1 mold that was sectioned at 3 varying depths and these sections were placed onto 1 slide. Similar blocking and washing procedures were followed as described above. Samples were then incubated with the E-cadherin primary antibody (1:200) overnight. AlexaFluor-488 goat anti-mouse secondary antibody (1:200) and DAPI (1:100) were used as described above. Slides were similarly mounted as before and 20 Z-stacks of each image were captured with a confocal Zeiss LSM 700 microscope and Zen Black software . Three randomly selected fluorescent images were captured per each section with the 20× objective and analyzed using ImageJ software Version 1.52a. E-cadherin localizes at the apical membrane of the CT layer, which fuses into the basal membrane of the syncytial region. Syncytialized portions were defined as nuclei external to the E-cadherin demarcation. The total area of the syncytialized portions of the explants were measured and normalized against the total surface area of the villi. 4P2O7, 100 mM NaF, 1% NP-40) with protease inhibitor cocktail  mixed in a 100:1 ratio in a tube with a metal bead. Samples were lysed using the Qiagen Tissue Lyser III  through three cycles of 25 shakes/s, with 2 min per cycle. Total protein mass was quantified using the Pierce BCA Protein Assay Kit  with an albumin standard following manufacturer recommendations. Samples were run in duplicates at a 1:10 dilution. Absorbance was measured with a Synergy HTX spectrophotometer  at a 562 nm wavelength.Explant tissues were placed in lysis buffer . If LDH were present, it would result in a color change to red due to the formation of Formazan, and the absorbance was read at 562 nm using a Synergy HTX spectrophotometer. Samples were run at a 1:2 dilution in duplicates. Culture media alone was used as a negative control and subtracted from the experimental sample absorbance values. Values were normalized to total protein mass and calculated as a ratio of the no-treatment control. Duplicate wells for each treatment were averaged.Lactate dehydrogenase (LDH) is released from all cell types as a result of cell membrane damage and is a marker for cell death. Experimental supernatants were centrifuged at 15,000 rpm for 15 min, and supernatant LDH levels were measured with the CytoTox 96β-hCG is secreted by the ST and is a marker of a functional ST. β-hCG from experimental supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) with the ELISA 1911 kit  following the manufacturer’s instructions. After centrifuging the samples at 15,000 rpm for 15 min, the supernatant was collected and run at a 1:5 dilution with the manufacturer’s supplied buffer. Absorbance was measured with the Synergy HTX spectrophotometer at a wavelength of 450 nm. The values from each assay obtained from culture medium alone were subtracted from experimental sample values. β-hCG concentrations were extrapolated using spectrophotometer measurements compared to those from a standard curve. Measured concentrations were normalized to total protein mass per well and calculated as a ratio of the no-treatment control. Duplicate wells for each treatment were averaged.PLAP activity was measured from villous particles shed into the bottom wells of the insert cultures to estimate the amount of ST shedding in response to various treatments. All particles reaching the bottom well were smaller than the 8 μM pore size. Experimental supernatants were diluted 10 times in manufacturer supplied buffer and analyzed following the manufacturer’s protocol . Briefly, phosphorylated p-Nitrophenol was added to the samples as a substrate. The alkaline phosphatase in the samples converts the substrate into the non-phosphorylated p-Nitrophenol, ultimately leading to a change in color. Absorbance was measured with a Synergy HTX spectrophotometer at a 405 nm wavelength. The values from each assay obtained from culture medium alone were subtracted from experimental sample values. PLAP activity of samples was extrapolated using spectrophotometer measurements compared to those from a standard curve. Measured concentrations were normalized to total protein mass per well and calculated as a ratio of the no-treatment control. Duplicate wells for each treatment were averaged.Supernatants from cultured explants were tested for 42 cytokines/chemokines in duplicate: EGF, Eotaxin-1, FGF-2, Flt-3L, CX3CL1, G-CSF, GM-CSF, GRO-α, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, TGF-α, TNF-β, VEGF-A. Tests were performed by Eve Technologies  using a Luminex array with a Bio-Plex™ 200 system  and a (HD42) kit .p value less than 0.05 was considered significant. All groups were subjected to post-hoc analyses. Only significant post-hoc p-values are shown in the figures.Results were analyzed with GraphPad Prism . Results from imaging were analyzed through Students’ t-test. Normalized fusion, β-hCG, LDH, PLAP, and cytokine/chemokine results from cultured explant experiments were analyzed by two-way ANOVA and two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-hoc test to control for the false discovery rate. Dose-response experiments were analyzed by non-parametric Kruskal–Wallis test and the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-hoc. A


The measurement of pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α), which is an important indicator of the inflammatory process, has received increasing attention recently because it is easy to extract from body fluid and serves as an early sign of a serious systemic inflammatory disease. Developing fast and simple detection methods to quantify the concentration of TNF-α is essential. Saliva, tears, and urine, which can easily be sampled in a non-invasive way, are considered to be important matrices for monitoring and assessing the physiological status of humans; importantly, they also provide an ideal window for monitoring the concentration of TNF-α. As a fast, accurate, inexpensive, portable, and scalable method, electrochemical biosensors are very promising for biomarker detection in matrices obtained in a non-invasive manner. This review summarises and compares the electrochemical biosensors for the detection of TNF-α in a non-invasive manner and highlights recent advances and future prospects in developing high-performance electrochemical platforms for noninvasive measurement of TNF-α. Tumour necrosis factor alpha (TNF-α), a pleiotropic cytokine produced by activating monocytes/macrophages in the inflammatory process, is considered an important biomarker of autoimmune diseases . It has −1 and increases to about 30 pg ml−1 in the saliva of patients with severe autoimmune diseases   . Its cone fluid) . TherefoFor this purpose, intensive research on TNF-α determination has been conducted, using techniques such as enzyme-linked immunosorbent assay (ELISA), radio immunoassays, chemiluminescence imaging, mass spectrometry, fluorescence immunoassay, and electrochemical immunosensors . Among tAs a promising tool for non-invasive detection, the saliva matrix can comprise more than 25% of the proteins in the human serum with strong correlations. Benefiting from the characteristics of easy collection and storage as well as good stability, saliva detection can effectively decrease the demand for cost, sophisticated instruments, and professional operators. In this section, recent studies focused on the detection of salivary TNF-α are summarised, and their advantages and disadvantages are discussed.−1 with a fast response in 5 s to the Ag-TNF-α cytokine (−1 with a limit of detection (LOD) of 0.85 pg ml−1 and 0.5–100 pg ml−1 with an LOD of 0.38 pg ml−1 for TNF-α and IL-1β, respectively, which was sufficient for application in clinical testing. In addition, the cross-talk between the adjacent SPCEs that were modified with anti-IL or anti-TNF in the dual immunosensor was non-perceptible, and no obvious differences were detected in the amperometric responses of immunosensors when faced with the non-target antigen or without the corresponding target antigen  transducer  undecanal) (TEUSD) was further modified for anti-TNF-α bonding, and the bonded condition was checked using the micro-contact printing technique. The sandwich antibody-antigen bio-recognition was chosen for TNF-α determination, in which the anti-TNF-α antibody was labelled with rhodamine. Mott-Schottky analyses of this capacitance electrochemical biosensor were used for TNF-α quantification. The result showed that this biosensor had a sensitivity of 4.4 mV pM−1 with an LOD of 1 pg ml−1 in artificial saliva .In addition to saliva, electrochemical biosensors for TNF-α in other non-invasively obtained body fluids, such as tears, were also studied. −1 in artificial human saliva against other cytokines, such as IL-1 and IL-8. They further applied the same system for TNF-α determination in human saliva with a linear range of 1–100 pg ml−1 and an LOD of 3.1 pg ml−1  and IL-4. This can be attributed to the introduction of SPDP monolayer which is considered to be effective for Ab-TNF-α immobilization.This review summarises the progress in electrochemical biosensors for non-invasive detection of TNF-α cytokines, including immunosensors for detection of TNF-α in saliva, electrochemical biosensors based on other principles or other non-invasive fluids (such as tears), and smart POC biosensors. Among these sensors, saliva testing plays an important role in non-invasive TNF-α cytokine determination because of its ability to be easily collected, good stability, and suitable TNF-α concentration within the detection capability of electrochemical biosensors. These electrochemical biosensors for non-invasive detection of TNF-α cytokines have promising prospects for continuous, fast, real-time, and portable monitoring of cardiovascular disease. Moreover, smart POC biomarkers provide a great solution for chronic disease self-management, such as chronic heart failure. To make a clearer comparison, the sensing performance, such as sensitivity and specificity, of different non-invasive biosensors that discussed in this review are listed in 2α, C-reactive protein, and IL-1β from saliva can also act as biomarkers for cardiovascular disease (Although research attention has been rapidly gained in the area of non-invasive TNF-α cytokine detection via electrochemical biosensors, further optimisation of sensor reliability should be critically investigated, and biosensors that can reliably and accurately determine TNF-α in clinical settings are still in their infancy. Besides the TNF-α cytokine, lactate, 8-isoprostalandin F disease . Designi


Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the Acinetobacterdrug efflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ–TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics.Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism  The tetracycline class of antibiotics is composed of powerful antimicrobials that are widely used in modern medicine . Tetracy1–Tetracyclines typically function by binding directly to the bacterial ribosome . The rib7–Because of its complex and coordinated functions, the bacterial ribosome contains many sites that can be targeted by antibacterial agents. Studies have shown that different antibiotic compounds can bind to specific regions of this complex biomacromolecular machine , 11. ForWhile currently effective, the potency of many antibiotics is expected to diminish with time, likely due to both misuse and bacterial adaptation that leads to enhanced antimicrobial resistance (AMR) . MultidrAcinetobacter baumannii, commonly utilize the upregulation of the resistance-nodulation-cell division (RND) superfamily of transporters to mediate resistance to multiple antimicrobial agents . The World Health Organization (WHO) also classifies carbapenem-resistant A. baumannii as a “first-priority pathogen” for the research and development of new antibiotics.A. baumannii is AdeIJK -bound structures, we show that modifications to the tetracycline core in positions 4 through 8 of TP-6076 can significantly alter binding to AdeJ with little to no change in its ribosome-bound position  was used to solve the structures of each complex. Here, we present the first structures of TP-6076 bound separately to the position . TogetheA. baumannii AdeJ recognizes the TP-6076 substrate, we utilized single-particle cryo-EM to determine its structure to high resolution. A. baumannii AdeJ was expressed in Escherichia coli, purified, and reconstituted into lipidic nanodiscs. The sample was incubated with TP-6076 to form the AdeJ–TP-6076 complex, and the structure of the complex was solved  Data processing workflow of apo-AdeJ and AdeJ–TP-6076. (B) Representative 2D classes of AdeJ–TP-6076. (C). Gold-standard Fourier shell correlation (GS-FSC) curve of AdeJ–TP-6076. (D and E). Side and top views of AdeJ–TP-6076 density maps. Chains in AdeJ–TP-6076 are labeled in tomato, gold, and royal blue. Download Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03732-21.5TABLE S1Table S1, DOCX file, 0.02 MB.AdeJ cryo-EM data collection and refinement statistics. Download Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the AdeJ assembles as a homotrimer and adopts the typical fold of a hydrophobe-amphiphile efflux (HAE)-RND pump . In agreA. baumannii AdeB multidrug efflux pump (Escherichia coli AcrB (residue R717) and Pseudomonas aeruginosa MexB (residue R716) have previously been determined to be important for drug recognition , which shuttles the ligand from the proximal to distal binding sites prior to extrusion, is formed by residues 615 to 624 in AdeJ. In AdeB, the G-loop has been shown to be flexible and occupy different conformations in apo and bound protomers . In addiIn AcrB, the distal binding site is made up of more than 20 residues , 33. SixCampylobacter jejuni CmeB  are shown as cyan sticks and surface density. Hydrogen bonds between K952 and D407, D408, K953, and T989 are labeled with yellow dashes. (B) AdeJ binding state. (C) AdeJ extrusion state. Download FIG S2, JPG file, 2.2 MB.AdeJ structural state assignment.  Distance between DN and DC domains (exit tunnel) in the AdeJ access state. (Bottom) The proton relay network of the AdeJ access state. DN and DC domain distances of the three apo-AdeJ protomers are measured between the CCopyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03732-21.6TABLE S2Table S2, DOCX file, 0.01 MB.Classification of AdeJ protomer states. Download Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The entrance site of the access protomer of AdeJ is open, whereas its extrusion site is closed  and C. WUnlike the access protomer, the conformation of the extrusion protomer depicts that the periplasmic cleft between subdomains PC1 and PC2 is closed. Therefore, the entrance site of this protomer is also closed. An extrusion tunnel can be traced from the distal binding site out through the extrusion site, which is open in this conformational state. Presumably, this tunnel allows the ligand to be extruded from the distal binding pocket.In the binding protomer, the entrance site between PC1 and PC2 is open  and C. A2+ is known to coordinate with tetracyclines in the ribosome, it is somewhat surprising that no Mg2+ ions were found to bind the TP-6076 drug in the AdeJ–TP-6076 structure. However, this observation agrees with the X-ray structure of AcrB in complex with minocycline and the cryo-EM structure of AdeB in complex with eravacycline  to extrude ligands from the periplasmic domain , 28, 35.A. baumannii ribosome to better understand the mode of the tetracycline-scaffold-ribosome interaction. The A. baumannii 70S ribosome was incubated with TP-6076 for 1 h, cryo-EM grids were prepared, and cryo-EM data were collected for this ribosome–TP-6076 complex. Using extensive classification, we were able to solve three separate cryo-EM structures of the A. baumannii 70S ribosome bound to TP-6076. These three 70S–TP-6076 structures are designated P-site tRNA, E-site tRNA, and empty 70S ribosome structures  A modified build-and-retrieve protocol was used to separate the data set and increase particle counts. (C) Only 70S ribosomal particles were taken for further processing. (D) 3D variability analysis was used to separate 70S particles based on the tRNA population. (E and F) Homogeneous refinement followed by local refinement, CTF refinement, and density modification resulted in final maps. Download FIG S3, JPG file, 2.1 MB.Cryo-EM workflow of ribosome–TP-6076 processing. (A) Initial processing of 2,922 micrographs resulted in initial maps of assembled Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The cryo-EM maps of these three ribosome structures were resolved to resolutions of between 2.38 Å and 3.05 Å , allowin10.1128/mbio.03732-21.4FIG S4A. baumannii ribosome–TP-6076 complexes. (A) Representative 2D classes (top left), final locally refined maps (bottom left), and GS-FSC resolutions (right) of the 70S P-site tRNA bound with TP-6076. (B) Representative 2D classes (top left), final locally refined maps (bottom left), and GS-FSC resolutions (right) of the 70S E-site tRNA bound with TP-6076. (C) Representative 2D classes (top left), final locally refined maps (bottom left), and GS-FSC resolutions (right) of empty 70S bound with TP-6076. For all maps, 50S is in blue, while the 30S core is in yellow, and the 30S head is in red. Download FIG S4, JPG file, 2.1 MB.Final refinements of Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03732-21.7TABLE S3Table S3, DOCX file, 0.02 MB.Ribosome cryo-EM data collection and refinement statistics. Download Copyright © 2022 Morgan et al.2022Morgan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ ions and a number of electrostatic interactions from nucleotides rU962, r2MG963, rG1050, rC1051, rC1192, rA1193, rA1194, and rG1195. This site overlaps the A-site tRNA pocket of the 30S subunit, posing the possibility that the drug is responsible for blocking the attachment of tRNA to the A site and halting translation elongation, a trademark characteristic of tetracycline antibiotics  of the 16S rRNA, which is vital for translational fidelity and initiation  were solved  ΔacrB cells and grown in 6 L of lysogeny broth (LB) medium with 100 μg/mL of ampicillin at 37°C. At an optical density at 600 nm (OD600) of 0.5, the expression of AdeJ was induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Bacteria were harvested after 4 h of induction. Cells were resuspended in low-salt buffer  and disrupted with a French pressure cell. The collected membrane fraction was washed twice with high-salt buffer  and once with final buffer (20 mM Na-HEPES [pH 7.5] and 1 mM PMSF). Membrane protein was solubilized using 2% (wt/vol) n-dodecyl-β-d-maltoside (DDM) for 3 h. Insoluble material was removed by centrifugation at 100,000 × g, and the extracted protein was purified using a Ni-nitrilotriacetic acid (NTA) column. The purity of AdeJ (>95%) was determined via SDS-PAGE gels stained with Coomassie brilliant blue. The purified protein was then dialyzed against 20 mM Na-HEPES (pH 7.5) and concentrated to 7 mg/mL (60 μM) in a solution containing 20 mM Na-HEPES (pH 7.5) and 0.05% DDM.AdeJ from escribed . In shorE. coli total lipid extract. The mixture was incubated at room temperature for 15 min. To remove the detergent, 0.8 mg/mL prewashed Bio-Beads (Bio-Rad) was added. The mixture was incubated on ice for 1 h, followed by incubation at 4°C overnight. The sample was filtered using a 0.22-μm nitrocellulose filter to remove Bio-Beads and purified from empty nanodiscs using a Superose 6 column  in a solution containing 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. Fractions containing the trimeric AdeJ-nanodisc complex were collected for cryo-EM sample preparation.AdeJ was assembled into nanodiscs using a mixture of 20 μM AdeJ, 45 μM membrane scaffold protein (MSP) (1E3D1), and 930 μM A. baumannii AB0057 as previously described (A. baumannii cells were lysed in a solution containing 20 mM Tris (pH 7.5), 50 mM magnesium acetate (MgOAc), 100 mM NH4Cl, 1 mM dithiothreitol (DTT), and 0.5 mM EDTA  and centrifuged for 16 h at 100,000 × g. Ribosome-containing pellets were stored at −80°C until further use.Ribosomes were purified directly from escribed . In brie mM EDTA  using a g was used to increase 70S purity. Fractions containing 70S particles were collected, concentrated, exchanged into ribosome buffer , aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until further use.Sucrose gradient purification using a sucrose gradient buffer  and 16 h of centrifugation at 100,000 × A. baumannii ribosome, 100 nM ribosome sample was incubated with 50 μM TP-6076 for 2 h prior to grid preparation to create the ribosome–TP-6076 complex. Grids were plunge-frozen with a Vitrobot using a 15-s blot time and graphene oxide-coated Quantifoil R1.2/1.3 grids. All grids were transferred to cartridges prior to data collection.A total of 10 μM AdeJ-nanodisc was incubated with 20 μM TP-6076 for 1 h prior to grid preparation to create the AdeJ–TP-6076 complex. The sample was applied to glow-discharged holey carbon grids , blotted for 5 s, and then plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher). For the −/Å2 over 2 s using SerialEM (−/Å2 using EPU (Thermo Fisher Scientific).For the AdeJ–TP-6076 complex, data were collected on a Titan Krios system equipped with a K3 direct electron detector (Gatan). Data were collected in superresolution mode at a magnification of ×81,000, resulting in a physical pixel size of 1.08 Å/pix (superresolution of 0.54 Å/pix). Each micrograph was collected over 40 frames with a total dose of 36 eSerialEM . For theab initio and heterogeneous three-dimensional (3D) classifications were used to remove false picks and contamination from ice and carbon. 3D classification using 3D variability analysis on the remaining 595,961 particles resulted in a single high-resolution class for refinement. Nonuniform refinement followed by masked local refinement with nonuniform sampling resulted in 2.91-Å-resolution maps of the AdeJ–TP-6076 complex based on the gold-standard Fourier shell correlation (GS-FSC)  was estimated using patch CTF in cryoSPARC v3 . Blob pi(GS-FSC) . The finab initio and heterogeneous 3D classifications were used to generate initial templates. Using a modified “build-and-retrieve” approach  were based on the cryo-EM maps. Previously determined structures for AdeJ  and the ribosome  were useribosome . The widribosome . For the in Coot . Models  in Coot  and refi in Coot  from the in Coot , with fi7RY3 and the Electron Microscopy Data Bank (EMDB) under accession number EMD-24732. Atomic coordinates for TP-6076 in complex with A. baumannii P-site tRNA 70S, E-site tRNA 70S, and empty 70S have been deposited in the PDB under accession numbers 7RYF, 7RYG, and 7RYH and in the EMDB under accession numbers EMD-24738, EMD-24739, and EMD-24740, respectively.Atomic coordinates and the EM map for the AdeJ–TP-6076 complex have been deposited in the RCSB Protein Data Bank (PDB) under accession number


RSC Advances.1 These retractions are on the basis of what we believe to be the systematic production of falsified research. Such manipulation of the publication system has been covered widely in the media during 2020,2,3 and, while we are not the only journal or publisher to have been affected, this has prompted us to stringently review our processes to ensure that, as far as possible, such papers cannot make it through to publication in the future. We have chosen to publish this editorial in order to be as transparent as possible about the situation, and hope that this will encourage other publishers to take the same approach.As a journal it is our responsibility to uphold the integrity of the scientific record, and this is something we take very seriously. Over the next few weeks, we will be retracting 68 articles published in Over the course of 2020 we carried out an extensive investigation into a number of published papers. These papers came to our attention through an update to the ScholarOne system that generated an alert linking many papers from different authors. We began investigating papers that triggered this alert, working with independent image integrity and scientific experts, and consulting with other publishers who are also affected. We identified common features across these papers, as well as instances of image duplication and manipulation to varying degrees. The papers cover similar topics, usually related to manipulation of proteins or other biomolecules to target specific genes or cellular processes for beneficial medicinal effects. Many of these papers are written with very similar structures or templates, despite having no common authors. These papers often appear to be legitimate when viewed in isolation, and many of the concerning features only come to light when comparing features across many papers, making them very difficult for individual editors or reviewers to detect.RSC Advances has always stood for high levels of ethical publishing behaviour, and we are therefore disappointed that our values have been systematically attacked. The size of our journal and breadth of our scope make us a particular target for this type of manipulation, but that does not excuse us from taking responsibility. This is a serious breach of our ethical policies, and as members of the Committee on Publication Ethics (COPE), we have followed their guidelines during our investigation. The thoroughness of our investigation highlights our determination to uphold the highest ethical standards in our journal and we will continue to leave no stone unturned, to ensure there are no more papers linked to this investigation published in our journal. Further information on the individual circumstances for each paper will be included in the retraction notice for that paper.As a journal committed to upholding the highest standards of peer review and ethical publishing we are taking a number of actions in order to strengthen our defences against organised and systematic attempts to subvert the publication process.We have already implemented enhanced screening of papers at the initial stages of assessment, using knowledge gained from this investigation to identify submissions of concern and to reject them. We are also trialling image manipulation software at the initial screening stages that will help to identify instances where images have been altered.We have implemented stricter data requirements for papers featuring western blots and other types of electrophoresis, requiring authors to provide more complete data to support their conclusions. We are also reviewing the data requirements across the rest of the journal’s scope and making updates where necessary. We will be asking reviewers to specifically comment on whether the data provided with a manuscript meets our new stricter guidelines.We will continue to work with our associate editors to ensure they are equipped with the knowledge to be able to identify concerning submissions and take necessary action. We have learned a lot about how to identify suspect papers and what steps we can take to ensure the validity of a submission. We will make sure that all of our associate editors continue to stay informed of any new developments in this area and continue to share learning.RSC Advances reviewer panel, ensuring that it fully represents our scope and the diversity of our scientific community, and provides opportunities for early career researchers to gain valuable skills by acting as reviewers. We will continue to recruit new reviewers during 2021 and aim to specifically appoint reviewers with expertise in data and image manipulation, further supporting our associate editors in identifying suspect submissions. We stress that individual reviewers could not be expected to spot individual cases of this type of fraud, but working collaboratively we believe that editors, referees, and journal staff can achieve this.During 2020 we have been working to refresh the RSC Advances is a broad open access journal and these papers were not explicitly outside of our scientific scope, we have taken this opportunity to fully review and refine that scope, noting that, first and foremost, the journal exists to represent the chemistry community. Our clarified scope states that “RSC Advances papers should provide an insight that advances the chemistry field. Papers that contain little or no chemistry and are not considered to be of interest or relevance to the chemistry community are not within the scope of the journal.” This clarification will benefit the chemistry community, ensuring that RSC Advances only publishes the most relevant content.While Finally, we would like to apologise to our community for the original publication of these papers. While it appears that there has been a systematic effort to dishonestly subvert the publication process, our procedures should have been tighter and we are sorry that we did not reject these papers before publication. We believe the actions we have already taken, along with those planned during 2021, will demonstrate our commitment to remaining a trusted, high quality, open access chemistry journal.We wish all of our readers and authors a happy and healthy 2021.Laura FisherRSC AdvancesExecutive Editor, Russell CoxRSC AdvancesEditor-in-Chief,


Cuculus canorus exploit the parental abilities of their hosts, hosts avoid brood parasitism and predation by showing specific behavior such as loss of feathers, emission of fear screams and contact calls, displaying wriggle behavior to avoid hosts or potential prey, pecking at hosts and prey, and expressing tonic immobility (showing behavior like feigning death or rapid escape from predators and brood parasites). These aspects of escape behavior are consistent for individuals but also among sites, seasons, and years. Escape behavior expressed in response to a broad range of cuckoo hosts and prey are consistently used against capture by humans, but also hosts and brood parasites and predators and their prey. An interspecific comparative phylogenetic analysis of escape behavior by hosts and their brood parasites and prey and their predators revealed evidence of consistent behavior when encountering potential parasites or predators. We hypothesize that personality axes such as those ranging from fearfulness to being bold, and from neophobic to curiosity response in brood parasites constitute important components of defense against brood parasitism that reduces the overall risk of parasitism.Brood parasites such as the common cuckoo  Predators impose strong selection pressures on their prey. Therefore, it is not surprising that a large number of different kinds of escape behavior has evolved . In particular, not all captured animals are dead, because a non-negligible fraction of individuals escape predation events and even escape successful capture. Such escape behavior shows diversity ranging from autotomy and feather loss  to fightEscape behavior from predators and parasites can broadly be arranged along a personality-like axis from attempts to escape to physiological changes that may allow an individual to increase the probability of escape. That would be the case if behavior would relate to anti-predation and anti-parasitism behaviors. Aggression toward a predator by prey may result in escape , includiThe behavior of animals when escaping from a predator does not only reflect means by which specific individuals may be able to escape, but may also reflect the risks that specific categories of individuals may encounter. Such integral differences in suites of behavior that show strong covariation can be considered to reflect personalities . TherefoThe objectives of this study were to quantify behavior during escape attempts or during attempts by parasites to exploit their hosts, assess the reliability of estimates of such escape behavior, and identify the potential function of escape behavior. Our working hypothesis suggested that escape behavior represents evasive behavior in a predation context because escaped individuals generally can be considered to be dead unless they somehow manage to escape. Individual animals may differ in the risk of encounters with predators, with individuals having bold and neophilic personalities being more likely to encounter such situations than cautious and neophobic individuals. If that were the case, we should expect escape behavior to reflect personality traits with specific personalities being associated with specific kinds of anti-predator and anti-parasitism behavior. Escape behavior is likely to converge between parasitic brood parasites, hawk mimicry in brood parasites, and convergence in phenotype between brood parasites and their predators .To analyze these questions we studied the escape behavior of birds at Kraghede , Denmark (since 1971), as part of a long-term project . The stuWe recorded information on all 98 species of hosts of the common cuckoo. A total of 1.24% of 72 host species for which 1.24% were on average parasitized with a range from 0% to 21.89%. We recorded the prevalence of common cuckoo among host species. We also recorded the total number of cuckoo eggs in museum and private collections 1850–1940 and between 1941 and 1990.We used intra- and inter-clutch variation in egg appearance based on data reported by Host eggs vary within and among clutches. Hosts also vary in rejection rate among populations. We made an exhaustive compilation of data for the common cuckoo by making a literature search on ResearchGate, Google Scholar, and Web of Science.Birds were captured weekly during 2010–2012 from arrival in early spring until the end of the breeding season using mist nets. All recaptures were subsequently recorded, with this variable taking on values of either 0 (not recaptured again) or 1 (recaptured as least once).F = 1.99, df = 1, 97, P = 0.012, repeatability R = 0.28 (SE = 0.13) to a maximum repeatability of F = 90.70, df = 1, 97, P < 0.0001, R = 0.97 (SE = 0.01).Repeated measurements of the same individuals in the same season revealed that phenotypic characters had repeatabilities above 28% , 1994. HWe used information on the size of the uropygial gland as an estimate of the ability of hosts to provide secretions from the gland to the plumage and hence the ability to maintain a functional plumage. We hypothesize that a large uropygial gland that produces ample amounts of secretions will allow for efficient escape from brood parasites . Such seWe quantified 7 aspects of behavior during and immediately following escape. (1) Wriggle score. The measure quantifies how much a bird budges while held in a hand . (2) Biting. Holding an index finger in front of the beak and giving a score of 0, if the bird does not peck, or 1 if it does. (3) Breath rate. The number of inhalations recorded during 30 s with the fingers on the breast muscles. (4) Escape sequence. The relative ranking from 0 to 1 of the sequence of all escapes in a given year. (5) Fear scream. Whether the bird gives a fear scream when held in the hand. (6) Tonic immobility. Just before a bird is released, we placed it with my right hand on its back on my flat left hand. When the bird was lying still, we removed the right hand and recorded time until the individual righted itself and flew away. If the bird had not flown away after 30 s, we terminated the trial. (7) Alarm call. Whether the bird gave an alarm call when departing from your hand. (8) Feather loss. Whether the individual lost feathers when removed from the mist net or while held during measurements and scoring of behavior.r. We adopted the guidelines of r = 0.10 explaining 1% of the variance is a small effect, r = 0.30 explaining 9% of the variance is an intermediate effect, and r = 0.50 explaining 25% of the variance is a large effect. We estimated effect size as Pearson’s product-moment correlation coefficients, using the equations in r = 0.22–0.26 explaining 5–7% of the variance. We weighted statistical tests by sample size to weight results. Such weighting can be more important than adjustment of phylogenetic analyses by similarity due to common phylogenetic descent  models for the comparative analyses using the function “pgls” in R package “caper” . Phyloge10 number of cuckoo eggs  until 26 September 2014. Parasitism rate was positively related to the log10 number of cuckoo eggs in museum collections across the globe 1850–1940 . There was also a positive relationship between the number of cuckoo eggs during 1941–1990 expressed as the log10 number of cuckoo eggs . There was also a positive relationship for the log10 number of cuckoo eggs recorded during 1990–2013 . We assumed that the first estimate was an estimate of the number of cuckoo eggs in the following analyses.We found 98 species of hosts with information on escape behavior, and the parasitism rate was on average 1.24% among 72 species of potential hosts ranged from 0% to 21.89% of nests were parasitized. Parasitism rate was positively related to the logPGLS models showed an intermediate effect of −0.23 for fear screams implying that fear screams were less common at higher levels of intra-clutch variation . There wA PGLS showed an intermediate effect of tonic immobility of +0.23 suggesting that species with greater inter-clutch variation also had higher level of tonic immobility . There wA PGLS revealed that rejection rate was larger in species with higher level of tonic immobility . This inPersonality has in recent years been related to many aspects of animal behavior. It is only recently that personality has been related to brood parasitism . For exaLife history is central to studies of personality because life history trade-offs constitute one reason why no single personality prevails in heterogeneous environments. Some personalities do better in one environment, while others do well elsewhere, and none do well everywhere . Here, wIn conclusion, we have shown that different aspects of behavior displayed by host birds during escape and subsequent handling as reflected by tonic immobility were related to intra- and inter-clutch variation in egg appearance with small to intermediate effect sizes and rejection rate of cuckoo eggs.