Normal-pressure hydrocephalus (NPH) is a potentially reversible syndromecharacterized by enlarged cerebral ventricles , cognitiveimpairment, gait apraxia and urinary incontinence. A critical review of theconcept, pathophysiology, diagnosis, and treatment of both idiopathic andsecondary NPH was conducted. We searched Medline and PubMed databases fromJanuary 2012 to December 2018 using the keywords “normal-pressure hydrocephalus”/ “idiopathic normal-pressure hydrocephalus” / “secondary normal-pressurehydrocephalus” / “NPH” / “ventriculoperitoneal shunt”. The initial searchproduced 341 hits. After careful selection, a total of 54 articles were chosenand additional relevant studies were included during the process of writing thisarticle. NPH is an important cause of potentially reversible dementia, frequentfalls and recurrent urinary infections in the elderly. The clinical and imagingfeatures of NPH may be incomplete or nonspecific, posing a diagnostic challengefor medical doctors and often requiring expert assessment to minimizeunsuccessful surgical treatments. Recent advances resulting from the use ofnon-invasive MRI methods for quantifying cerebral blood flow, in particulararterial spin-labeling (ASL), and the frequent association of NPH andobstructive sleep apnea (OSA), offer new avenues to understand and treatNPH. Adams.,idiopathic NPH (iNPH) andreported that in elderly patients  with iNPHmanifested by cognitive symptoms of “senile dementia,” 64% (18/28) improved aftercerebrospinal fluid (CSF) shunting.NPH is a potentially reversible syndrome characterized clinically by enlargedcerebral ventricles , cognitive impairment, gait apraxia andurinary incontinence.idiopathic (iNPH), accounting forabout 50% of cases, and secondary (sNPH), resulting fromsubarachnoid hemorrhage, meningitis, intracerebral hemorrhage, brain tumor or headtrauma,,,,The usual NPH classification into -The diagnostic criteria for NPH remain a topic of discussion. The numerouscontroversies surrounding this disease led to an interesting dichotomy: while someconsider the disorder as the most common type of hydrocephalus in adults,The aim of this review was to evaluate current knowledge on the two forms of NPH,their etiology, diagnosis, pathophysiology and treatment, highlighting novelfindings within the last 5 years; this process should ultimately help to answer thequestion on the merit of the assumption that NPH is a frequent cause of dementia,abnormal gait and falls in the elderly.We searched Medline and PubMed from January 2012 to December 2018 for relevantarticles using the keywords “normal-pressure hydrocephalus,” or “idiopathicnormal-pressure hydrocephalus,” or “secondary normal-pressure hydrocephalus,” or“NPH,” or “ventriculoperitoneal shunt (VPS).” The initial search produced 341 hitsand, after selection based on the abstracts, a total of 54 articles on etiology,diagnosis, pathophysiology or treatment were chosen and reviewed. Additionalreferences were obtained from these articles and from the authors of thisreview.rd ventricle cyst, and only2 were idiopathic NPH . In addition to symptomsof brain edema and traumatic brain injury, including akinetic mutism, they allpresented with frontal dementia plus gait disability resembling Bruns apraxia orfrontal ataxia, and frontal-type urinary and fecal incontinence. All of thesesymptoms responded dramatically to neurosurgical treatment with ventriculoatrialshuntingth ventricle - without air in the subarachnoidspace. This finding suggested communicating hydrocephalus, hence the name“normal-pressure” hydrocephalus used for the first time.,Since its description in 1965, the concept of iNPH has been a recurrent focus ofdiscussion. Of the 6 original patients, 2 were post-traumatic hydrocephalus, 1 woman (age 63) had suspected meningealcarcinomatosis, 1 man (age 62) with a III,2O, a value that, althoughnormal, is very close to the upper acceptable limit. It is also possible that180 mmH2O was not the peak intracranial pressure due to the CSFdynamics and possibility of partial obstruction.,As mentioned above, a critical evaluation of these initial descriptions revealsthat the mechanism of CSF flow obstruction that caused the hydrocephalus wasknown in all but 2 of the reported cases.,-Over the years, the term iNPH has been almost indiscriminately used for allindividuals who present with “unexplained” ventriculomegaly detected by brainimaging including computed tomography (CT) or magnetic resonance imaging (MRI),associated with the classic triad comprising cognitive impairment, gaitdisturbance, and incontinence.,According to International Guidelines the following key imaging features shouldbe employed for the diagnosis of NPH: Ventricular enlargement with Evans’s index >0.3 .Absence of macroscopic obstruction to CSF flow.At least one of these supporting features:Enlarged temporal horns of the lateral ventricles not entirely due tohippocampus atrophy;Callosal angle of 40° or greater ;Periventricular signal changes on CT and MRI due to altered brainwater content and not entirely attributable to microvascularischemic changes or demyelination ;Flow void in the Sylvian aqueduct or fourth ventricle on MRI.The Japanese Guidelines for the diagnosis of NPH did not regard periventricularchanges as relevant for the diagnosis, but included two other key imagingfeatures: narrowing of the sulci and subarachnoid spaces over the high convexityand midline surface of the brain ; and enlIn 2017, Andersson et al.-When ventriculomegaly is excluded, the NPH symptomatology becomes nonspecific.After all, dementia resulting from various causes occurs in about 35% of peopleaged over 70 years;The clinical differentiation between iNPH and the aforementioned conditions maybe very subtle, requiring specialized assessment by dementia experts.,The clinical presentation of normal pressure hydrocephalus is linked to impairedperiventricular blood flow, as well as to interstitial edema, microvascularinfarctions, neuronal injury, and gliosis,Despite being important conditions in the differential diagnosis, AD and the NPHsyndrome can differ greatly in terms of clinical presentation. In 2016,Damasceno,Brain biopsies performed on patients treated successfully with CSF derivation foriNPH, have shown various underlying brain pathologies, mainly AD, indicatingthat the 2 conditions are not mutually exclusive. In fact, about 19%-However, 32% of patients with moderate-to-severe AD pathology had worse baselinecognitive test scores and less postoperative improvement of NPH symptoms 4months after VPS.The treatment of NPH can be highly beneficial, both from the viewpoint ofprevention of frequent falls, head injury, subdural hematomas, trauma and hipfractures in the elderly, as well as for the improvement of quality of life withdisappearance of incontinence, control of the risk of repeated urinary tractinfections and sepsis, freedom of ambulation, and in some selected cases,cognitive improvement.-,Despite the existence of numerous diagnostic criteria for NPH,force exerted bythe CSF on the walls of the ventricles is equal to the product of thepressure of the fluid and the area of thewall: F = P × A. In the original example, they wrote that the CSF openingpressure is transmitted to every square centimeter of the surface of thecontainer; therefore, a pressure of 150 mmH2O exerts a force of 300mmH2O on the surrounding brain when the ventricles’ surface areais 120 cm2O) with the same opening pressure on ventricles with anormal surface of 60 cmIn the original descriptions, Hakim et al.transmantle pressure, i.e., thedifference between ventricular pressure and the pressure over the cerebralconvexity. The result is a global decrease in cerebral perfusion from thecentrifugal transmantle pressure, given that most of the arterial cerebral bloodflow (CBF) is centripetal, i.e. from the subarachnoid space towards the centerof the brain.,The force exerted on the ventricles is transmitted centrifugally, compressing thebrain and elevating the -Owler and Pickard-Enhancement of cerebral perfusion in periventricular white matter and basalganglia has been described after CSF removal,Arterial Spin-Labeling (ASL) perfusion imaging is a novel non-invasive MRItechnique that requires neither contrast media nor isotopes to measure CBF.ASL-MRI generates an endogenous contrast by using radiofrequency pulses that“label” water proton spins in blood circulating in carotid and vertebralarteries at the base of the skull. CBF-ASL-MRI images are obtained bysubtracting labeled and unlabeled spin exchanges in the brain tissue yielding amap of regional CBF (rCBF) quantified in mL/100g/min. We use 3Dpseudo-continuous ASL (pCASL) with spiral acquisition before and after the taptest for NPH. Also, CBF-ASL-MRI has been used to demonstrate regional decreaseof CBF in NPHUsing ASL-MRI, we have demonstrated a positive correlation between enhanced CBF and clinIt should be noted that the CSF is called “the third circulation” because of theconstant interaction between cerebral arterial circulation, CSF circulation, andvenous circulation. The concept of intracranial venous hypertension leading todecreased CSF absorption and hydrocephalus is critical to fully understand thepathogenesis of NPH.-Venous hypertension in intracranial circulation hinders CSF absorption throughthe arachnoid villae in the dural sinuses.,,,Also, venous hypertension alters intracranial compliance and changes CSFdynamics, affecting the intracranial Windkessel effect from brain viscoelasticproperties.Other than the effects of ventriculomegaly, in 2006, Malm and Eklund,,,They also observed that the clinical features of iNPH most likely result fromperiventricular, frontal or subcortical impairment.,In 2010, Ott et al.,,,Recently, an association between NPH and the glymphatic system has emerged,attempting to link reduced intracranial compliance and diminished arterialpulsations with inefficient glymphatic flow.The mechanisms induced by OSA cause almost total absence of REM and delta sleep,affecting glymphatic flow; also, sleep disordered breathing produces cerebralvenous hypertension due to increases in central venous pressure. The postulatednet result is a decrease in CSF outflow, leading to hydrocephalus. Nocturnalpolysomnogram is therefore indicated in the evaluation of patients withsuspected NPH.,-,-,,,,-,,-NPH is classically defined as a communicating form of hydrocephalusCSF shunting and subsequent drainage continues to be the first-line therapy forNPH, with the symptoms usually improving after the intervention. Many authorsconsider the responsiveness to shunting as the main difference between iNPH andsNPH, where clinical improvement is seen in about 50% of individuals with iNPHand in up to 70% of those with sNPH.A number of physiologically based tests have been developedAdams et al.The tap test works by temporarily decreasing intraventricular pressure, mimickingthe effect of a shunting procedure, allowing the physician to evaluate thepatient’s response to a substantial (50 ml) CSF removal. Given the diagnostic uncertainties mentioned earlier, there are major advantagesin securing consensus recommendations from a team that includes specialists inneurology, neuropsychology, physical therapy, neuroradiology and neurosurgery.Moreover, using quantifiable measurements , allows objective judgmentof each of the test components. The Román Protocol for the LVLP diagnostic testfor NPH is performed as follows:Neuropsychology. On the day prior to the LVLP, aclinical neuropsychologist evaluates the following cognitivedomains: global cognition, memory, orientation, language, praxis,and executive function. Twenty-four hours after the LVLP, the samespecialist performs the second evaluation, modified to avoidlearning and practice effects.Physical Therapy. On the day of admission for theLVLP, a pre-trained Physical Therapist examines the patient gait andbalance prior to the LVLP using the scores from the TinettitestSphincter Control. During the period of in-hospitalobservation (24 hours), pre- and post-LVLP, the patient´saccompanying relative is instructed to notify the nurse if thesubject asks to void or to evacuate, or if incontinence occurred.The number of such events in the 24 hours pre- and post-LVLP isrecorded.Non-enhanced brain MRI with CBF-ASL: The baselinenon-enhanced brain MRI test is performed on the days leading up tothe LVLP and is repeated within 24 hours after the tap test. It isusually well tolerated; does not expose the patient to X-rayradiation, requires no intravenous contrast medium, and can berepeated as often as needed. The only limitation is that MRI cannotbe performed in patients with cardiac pacemakers ordefibrillators.Large-Volume Lumbar Puncture. A neuroradiologistperforms a routine lumbar puncture under fluoroscopy with an 18- or20-gauge spinal needle;The final diagnosis and therapeutic decision should be the professionalresponsibility of the trained neurologist or neurosurgeon in charge of thepatient after considering the results of each component of the tap test.Usually, surgical treatment with insertion of a ventriculoperitoneal shunt (VPS)is recommended only for patients that present clear improvement in gait postLVLP, usually with concurrent improvement in bladder control. Few patients showimprovement in cognitive evaluation within the 24 hours post LVLP. For patientsconsidered to be non-surgical candidates, and for those that decline surgery,the use of acetazolamide (Diamox®) is recommended at relatively low doses(125-500 mg/day).Despite its novel nature and considerable advantages, the Román Protocol can beassociated with some drawbacks - predominantly related to cost-effectiveness -such as requiring a multidisciplinary team, multiple procedures, and a 24-hourstay in a hospital.Although associated with a short learning curve, the tap test should be performedby trained professionals.,Professionals must be aware that a lack of response on this test does notcontraindicate the surgical procedure,Physicians should also bear in mind that repeating the tap test at a later dateis possible. Repeated large-volume lumbar puncture is an alternative treatmentseldom used.,-External lumbar drainage and infusion testing are other predictive tests thattend to require more expertise. These tests are more frequently used in Europeancountries. An association between augmentation of CBF following glyceroladministration and a favorable response to shunt procedures has beenproposed.Adequate selection of surgical candidates leads to a 90% chance of success,according to a recent review.,In light of this, and considering that surgery may be an effective placebo,recent suggestions have been made to conduct randomized, double-blind,placebo-controlled clinical trials comparing the efficacy of shunting andplacebo procedures for iNPH,,,Also, shunting procedures are not free of complications and can be associatedwith significant adverse effects (AEs), including subdural hematomas andhygromas, shunt and CNS infection, complex partial seizures, over-drainage, andprolonged post-operative delirium.In 2018, Souza et al.,Souza et al.’s study was critically reviewed by Damasceno,Secondary normal pressure hydrocephalus encompasses all cases in which anetiology is identified.Along the same lines, a population-based MRI study,Thus, based on firm epidemiological data, it can be concluded that iNPH is afrequent disorder in the elderly, with an average age of onset of about 70years; more importantly, between 60% and 80% of patients improve with shuntsurgery, but only a minority receive the benefit of the surgical treatment withCSF shunt.Because sNPH may result from several different causes, it has become difficult tocreate practical guidelines for optimizing the treatment and diagnosis of thedisease.A recent review by Daou et al.Up to 37% of the patients with SAH developed chronic hydrocephalus,Because some studies have evaluated iNPH and sNPH as the same condition, it hasalso been questioned whether or not the sNPH concept is even valid, and to whatextent it is useful for differentiating sNPH from iNPH. We basically agree withthis concept, given that ventriculomegaly is the central axis of the NPHsyndrome. However, Daou et al. concluded that, despite differences in outcome,iNPH and sNPH should not be treated as completely separate entities.Taking everything into account, the diagnosis of NPH should always be consideredwhen facing a suggestive clinical presentation. This often occurs in theemergency room when routine brain CT in an elderly patient undergoing evaluationfor trauma surprisingly discloses ventriculomegaly, or in the evaluation ofpatients with gait disorders or cognitive decline in outpatient clinics. It isrecommended to always search for an etiology and, even when one fails to befound, the diagnosis of NPH should be properly - and rapidly - addressed.Based on a comprehensive review of the recent literature, we conclude that theclinical syndrome of NPH is the same in both iNPH and sNPH. The separation intoidiopathic and secondary NPH has resulted in a difference of approach to thesyndrome, manifested by a sense of urgent evaluation and treatment only for sNPH.The initial step in the diagnostic process is the evaluation of ventriculomegaly onbrain imaging.Both the concept and the management of normal-pressure hydrocephalus remain a topicof discussion, marked by several controversies. Nonetheless, it is clear frompopulation-based epidemiological studies that iNPH is more common than previouslythought. Therefore, each situation must be thoroughly and individually assessed inorder to prevent misdiagnosis and incorrect treatment. The confirmation of thisdisease is rather complex, requiring the involvement of dementia experts, becauseiNPH has no particular imaging, clinical or pathological features, and there are nodefinitive tests capable of accurately diagnosing the condition. It is alsoessential to keep in mind that to minimize errors the tap test should be performedby trained professionals. Overall, secondary NPH is associated with better outcomes,which is partially explained by swift intervention with adequate VPS placement,which remains the first-line of treatment. NPH may still be considered a potentiallyreversible cause of dementia; however, a randomized, placebo-controlled trial ofshunting procedures should be conducted to finally prove - or refute - the trueefficacy of surgical interventions.


TNF-α) gene polymorphisms were supposed to be involved with the interactions because TNF-α plays an important role in inflammation, a mechanism by which toxic elements cause threats to human health. This research aimed to analyze the influence of TNF-α gene polymorphisms and multi-elements on serum TNF-α level. Blood multi-elements concentrations , serum TNF-α level, and TNF-α single nucleotide polymorphisms (SNPs), including −238G > A (rs361525), −308G > A (rs1800629), −857C > T (rs1799724), −863C > A (rs1800630), and −1031T > C (rs1799964), were measured in 462 metal industrial workers. We applied mixed-effect models to analyze the interactions among multi-elements and TNF-α SNPs. Blood concentration of all elements were positively associated with serum TNF-α level, and the effects may be modified by TNF-α gene polymorphisms. Our study revealed that TNF-α −308A/A and −1031C/C may be susceptible genotypes, and thus we suggest that those workers should take preventive measures against metal toxicity.Health of the metal industrial workers should be a noteworthy issue due to the hazard of chronic exposure to metals or toxic elements. The interactions among multiple elements are sophisticated and may differ from person to person. Tumor necrosis factor-α ( Workers in metal industries are likely to be exposed to multiple metals. Lead (Pb), cadmium (Cd), and arsenic (As) were reported to be the main hazardous metals/metalloids in heavy industries. They cause severe damage to many target organs in human bodies through the mechanism of inflammation, production of oxidative stress, and interference with essential elements. The interactions among these toxic metals/metalloids are extremely complicated and some effects which have not been observed in single constituent exposure may occur ,2.On the other hand, selenium (Se), cobalt (Co), copper (Cu), and zinc (Zn) are essential trace elements which play crucial roles in maintaining normal physiological function in human bodies, but they may cause a threat to human health beyond tolerable serum concentrations ,4,5,6. UOne of the mechanisms of how toxic elements exert a negative effect on health is through inflammation. Tumor necrosis factor-α (TNF-α), possessing a wide range of biological activities, is known to be an important mediator of local and systemic inflammation, and immune response . The maiThe TNF gene cluster is located on human chromosome 6p21. Several single nucleotide polymorphisms (SNPs) have been identified in the human TNF gene promoter . The SNPMost metal industrial workers are exposed to various metals/metalloids and elements simultaneously in their working environment. The complicated interactions and impacts on health are major issues but have not been studied thoroughly. The aim of our research is to evaluate the interactive effects between multi-elements. Moreover, we also investigate how TNF-α gene polymorphisms modify the influence of multi-elements on serum TNF-α level.We collected the data of 462 metal industrial workers who received annual health examination in Kaohsiung Medical University Hospital, a medical center in southern Taiwan. All procedures were approved by the Kaohsiung Medical University Hospital Institutional Review Board -20150259), and written informed consent was obtained from all participants. The content of health examinations consisted of body mass index (BMI) and blood pressure measurement, personal history inquiry , physical examination, and blood tests. Blood sampling for biochemical analyses included white blood cell (WBC) counts, red blood cell (RBC) counts, hemoglobin, platelet counts, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglyceride, uric acid, fasting blood sugar, and creatinine. In addition, the blood samples were sent for TNF-α genotype analysis and measurement of blood multi-elements concentration  and serum TNF-α level. All blood samples were analyzed in the central laboratory in Kaohsiung Medical University Hospital.3 for dilution solution. Then, the blood samples were analyzed by Inductively Couple Plasma Mass Spectrometry (ICPMS) of Thermo Scientific XSERIES 2  3600811) . We checked whole blood concentrations of Pb, Cd, As, Se, Co, Cu, and Zn. The process of analyses was modified from the method of Ebba Barany in 2007 [Venous blood samples obtained in annual health examinations were stored at 4℃, and then were placed to room temperature prior to analysis. We set the mixed solution with 0.2 % ammonia solution, 0.1% Triton X-100, and 0.3% HNOSerum samples were collected from coagulated blood. Serum TNF-α levels were measured by Enzyme-linked immunosorbent assay (ELISA) using Human TNF-alpha Quantikine ELISA Kit from R&D Systems . Three samples of known concentration were tested twenty times on one plate to assess intraassay precision, and tested in twenty separate assays to assess interassay precision. The CVs of intraassay were all less than 5%, and CVs of interassay were all less than 8%.http://Ncbi.nlm.nih.gov/LocusLink) and HapMap database (http://hapmap.org). Genomic DNA from participants was obtained from whole peripheral blood, using standard method. TaqMan allelic discrimination assays  was used to separately analyze all the SNPs: TNF-α −238G > A (rs361525), −308G > A (rs1800629), −857C > T (rs1799724), −863C > A (rs1800630), and −1031T > C (rs1799964). The results were analyzed with a 7300 Real-time polymerase chain reaction (PCR) system (Applied Biosystems(ABI)®, Thermo Fisher Scientific, Foster City, CA, USA). Each real-time PCR was performed with a 10μL reaction volume mix fluid, containing 5μL Genotyping Master Mix, 3.87μL distilled water, 1μL DNA fluid (10 ng/μL) and 0.25μL primer-probe. Amplification reactions were performed using the following program for total 45 cycles: 50℃ for 2 minutes; 92℃ for 10 minutes; 95℃ for 15 seconds; 60℃ for 1 minute. The fluorescence level was detected by an Applied Biosystem StepOne Real-Time PCR system . The allele frequencies were determined using ABI SDS software .SNPs located within the TNF-α gene were selected from the NCBI LocusLink , respectively. We calculated the frequencies of all TNF-α genotypes, using either the Chi-square test or the Fisher’s exact test to examine deviations from Hardy–Weinberg equilibrium (HWE). The above data were analyzed using the SPSS 20 statistical package .5 = 243 kinds of combination genotypes based on five SNPs, but there were only 36 genotypes in our participants. After excluding 12 genotypes of n = 1, the result was not influenced, so we remained all 36 genotypes in our analysis for fear of deleting some genotypes. Among all the genotypes, G-G/G-G/C-C/C-C/T-T was the wild type with the most subjects, and it was set as reference. Mixed-effects models were carried out using SAS 9.4 software . Because we analyzed five SNPs at a time, we used Bonferroni’s correction for multiple testing. α level was set at 0.05, and the corrected α level would be changed to 0.01.In the Pearson correlation matrix of TNF-α and seven elements, all Pearson correlation coefficients were less than 0.5, except for Zn and Cu (γ = 0.51), so we supposed that these elements were not correlated with one another. We applied mixed-effects models for multivariate analyses, in which the blood concentrations of seven elements were set as independent variables and serum TNF-α level was set as the dependent variable. Age, gender, BMI, consumption of cigarettes and alcohol, and white blood cell (WBC) counts were incorporated as covariates. Subsequently, TNF-α SNPs were also added into independent variables in further analysis to show the modification of TNF-α gene polymorphisms. In theory, there should be 3Multivariate analyses showed that age, WBC count, and blood concentration of seven elements were positively associated with serum TNF-α level, while BMI was negatively associated with serum TNF-α level . With reIn Most researches of TNF-α gene polymorphism focus on genetic susceptibility to autoimmune diseases, inflammatory diseases, and cancers. Few studies have been reported to evaluate the correlation between TNF-α gene polymorphism and susceptibility to metal toxicity in industrial workers. It has been proposed that TNF-α gene polymorphisms may be associated with lead-induced inflammatory response , berylliTNF-α −238 and −308 SNPs are two of the most extensively studied polymorphisms in many diseases ,21,22. TOur results showed that TNF-α -857T allele might be associated with a hazardous trend. Meta-analyses exhibited that TNF-α −857T allele was associated with increased susceptibility to gastric cancer, type 1 diabetes, and psoriatic arthritis ,30,31,32In our study, age and WBC counts have a positive correlation with TNF-α, whereas BMI has a negative correlation with serum TNF-α level. Previous literature has postulated that obesity is related to low-grade inflammation, which probably results in an increased serum TNF-α level . HoweverThis is the first study using mixed-effects models to analyze the interaction of TNF-α SNPs and multi-elements in working environment. A mixed-effect model has been recommended as a powerful tool in longitudinal genome-wide association study . In our Working duration not included into the variables and relatively small sample size are two of the limitations of our research. On the other hand, the TNF-α genotype distribution in our study is in HWE except for −238G > A rs361525). Because the expected count was less than five in −238A/A genotype, we used Fisher’s exact test instead of the Chi-square test to examine deviations from HWE . On the . BecauseThe elements in our research  have positive correlation with serum TNF-α level, and the effects may be modified by TNF-α gene polymorphisms. TNF-α −238G/A and −863C/A heterozygous genotype may play protective roles, whereas −308A/A and −1031C/C genotypes and −857T allele may be susceptible genotypes. It is noteworthy that the workers with susceptible genotype combinations may predispose to metal toxicity in their working environment. Further large-scale studies are warranted to illustrate the complicated interaction of multi-elements and genetic polymorphisms.


The Journal of Physiology and British Journal of Pharmacology jointly published an editorial series in 2011 to improve standards in statistical reporting and data analysis. It is not known whether reporting practices changed in response to the editorial advice. We conducted a cross-sectional analysis of reporting practices in a random sample of research papers published in these journals before (n = 202) and after (n = 199) publication of the editorial advice. Descriptive data are presented. There was no evidence that reporting practices improved following publication of the editorial advice. Overall, 76-84% of papers with written measures that summarized data variability used standard errors of the mean, and 90-96% of papers did not report exact p-values for primary analyses and post-hoc tests. 76-84% of papers that plotted measures to summarize data variability used standard errors of the mean, and only 2-4% of papers plotted raw data used to calculate variability. Of papers that reported p-values between 0.05 and 0.1, 56-63% interpreted these as trends or statistically significant. Implied or gross spin was noted incidentally in papers before (n = 10) and after (n = 9) the editorial advice was published. Overall, poor statistical reporting, inadequate data presentation and spin were present before and after the editorial advice was published. While the scientific community continues to implement strategies for improving reporting practices, our results indicate stronger incentives or enforcements are needed. The accurate communication of scientific discovery depends on transparent reporting of methods and results. Specifically, information on data variability and results of statistical analyses are required to make accurate inferences.The quality of statistical reporting and data presentation in scientific papers is generally poor. For example, one third of clinical trials in molecular drug interventions and breast cancer selectively report outcomes , 60-95% Many reporting guidelines  have beIn response to the poor quality of statistical reporting and data presentation in physiology and pharmacology, the Journal of Physiology published an editorial series to provide authors with clear, non-technical guidance on best-practice standards for data analysis, data presentation and reporting of results. Co-authored by the Journal of Physiology’s senior statistics editor and a medical statistician, the editorial series by Drummond and Vowler was jointly published in 2011 under a non-exclusive licence in the Journal of Physiology, the British Journal of Pharmacology, as well as several other journals.  The key recommendations by Drummond and Vowler include instructions to (1) report variability of continuous outcomes using standard deviations instead of standard errors of the mean, (2) report exact p-values for primary analyses and post-hoc tests, and (3) plot raw data used to calculate variability –15. ThesWe conducted a cross-sectional analysis of research papers published in the Journal of Physiology and the British Journal of Pharmacology to assess reporting practices. Specifically, we assessed statistical reporting, data presentation and spin in a random sample of papers published in the four years before and four years after the editorial advice by Drummond and Vowler was published.All papers published in the Journal of Physiology and the British Journal of Pharmacology in the years 2007-2010 and 2012-2015 and indexed on PubMed were extracted using the search strategy: (J Physiol[TA] OR Br J Pharmacol[TA]) AND yyyy: yyyy[DP] NOT . Papers were excluded if they were editorials, reviews, erratums, comments, rebuttals, or part of the Journal of Physiology’s Crosstalk correspondence series. Of the eligible papers, a random sample of papers published in the four years before the 2011 editorial advice by Drummond and Vowler was published (2007-2010) and four years after (2012-2015) was extracted , and fulTen questions and scoring criteria were developed to assess statistical reporting, data presentation and spin in the text and figures of the extracted papers. Questions assessing statistical reporting in the text (Q1-5) determined if and how written measures that summarize variability were defined, and if exact p-values were reported for primary analyses and post-hoc tests. Questions assessing data presentation in figures (Q6-8) determined if and how plotted measures that summarize variability were defined, and if raw data used to calculate the variability were plotted. Questions assessing the presence of spin (Q9-10) determined if p-values between 0.05 and 0.1 were interpreted as trends or statistically significant.A random sample of 20 papers before and 20 papers after the editorial advice was used to assess the clarity of the scoring instructions and scoring agreement between raters. These papers were separate from those included in the full audit. All papers were independently scored by three raters . Scores that differed between raters were discussed to reach agreement by consensus. The wording of the questions, scoring criteria, and scoring instructions were refined to avoid different interpretations by raters. The questions are shown in Each rater  had to score and extract data independently from 50 papers before and 50 papers after the editorial advice was published. For each rater, papers from the 2007-2010 and 2012-2015 periods were audited in an alternating pattern to avoid an order or block effect. One rater unintentionally audited an additional 2007-2010 paper, and another rater unintentionally audited a 2007-2010 paper instead of a 2012-2015 paper. Thus, data from a random sample of 202 papers before and 199 papers after the editorial advice were analysed. When scoring was completed, papers that were difficult or ambiguous to score  were reviewed by all raters and scoring determined by consensus.It was difficult to score some papers unambiguously on some of the scoring criteria. For example in question 3, it was sometimes difficult to determine what a paper’s primary analyses, main effects and interactions were, in order to determine whether p-values for these were reported or implied. When raters could not unambiguously interpret the data, either individually or as a team, we scored papers to give authors the benefit of doubt.Counts and proportions of papers that fulfilled the scoring criteria for each question were calculated; no statistical tests were performed. Descriptive data are reported. All data processing and analysis were performed using Python (v3.5). Raw data, computer analysis code and result are available in the supporting information .The random sample of audited papers was reasonably representative of the number of papers published each year in the Journal of Physiology and the British Journal of Pharmacology in the two periods of interest .The proportions of audited papers that fulfilled the scoring criteria are presented in Of papers that reported p-values between 0.05 and 0.1, 56-63% interpreted such p-values as trends or statistically significant. Examples of such interpretations include:oes/VT  and effort–displacement ratios  were consistently greater during exercise …” (PMID 18687714)“A P < 0.05 level of significance was used for all analyses. […] As a result, further increases in the tidal P“The level of IL-15 mRNA tended to be lower in vastus lateralis than in triceps (P = 0.07) ”  but the difference was not quite significant (P = 0.05)” (PMID 18174213)“… resting activity of A5 neurons was marginally but not significantly higher in toxin-treated rats ” (PMID 22526887)“… significantly smaller than with fura-6F alone (P = 0.009), and slightly smaller than with fura-6F and EGTA (P = 0.08).” (PMID 18832426)“The correlation becomes only marginally significant if the single experiment with the largest effect is removed .” (PMID 17916607)Implied or gross spin  was noted incidentally in papers before (n = 10) and after (n = 9) the editorial advice was published. Examples of statements where implied or gross spin was present include:“However, analysis of large spontaneous events (>50 pA in four of six cells)  showed t“… whereas there was only a non-significant trend in the older group.” Post-hoc analyses revealed audit results were comparable between raters . AdditioIn 2011 the Journal of Physiology and the British Journal of Pharmacology jointly published editorial advice on best practice standards for statistical reporting and data presentation . These rOur results confirm that the quality of statistical reporting is generally poor. We found that ∼80% of papers that plotted error bars used standard error of the mean. In line with this, a systematic review of 703 papers published in key physiology journals revealed 77% of papers plotted bar graphs with standard error of the mean . SimilarOur findings also broadly agree with other observational data on the ineffectiveness of statistical reporting guidelines in biomedical and clinical research. For example, the CONSORT guidelines for the reporting of randomized controlled trials are widely supported and mandated by key medical journals, but the quality of statistical reporting and data presentation in randomized trial reports remains inadequate –20. A scOne example where editorial instructions improved reporting practices occurred in public health. In the mid-80’s the American Journal of Public Health had an influential editor who advocated and enforced the use of confidence intervals rather than p-values. An audit of papers published before and during the tenure of this editor found that the reliance on p-values to interpret findings dropped from 63% to 5% and the reporting of confidence intervals increased from 10% to 54% . HoweverWhy are reporting practices not improving? The pressure to publish may be partly to blame. Statistically significant findings that are visually and numerically clean are easier to publish. Thus, it should come as no surprise that p-values between 0.05 and 0.1 are interpreted as trends or statistically significant, and that researchers use standard errors of the mean to plot and report results. There is also a cultural component to these practices. The process of natural selection ensures that practices associated with higher publication rates are transmitted from one generation of successful researchers to the next . UnfortuThe recent reproducibility crisis has seen all levels of the scientific community implement new strategies to improve how science is conducted and reported. For example, journals have introduced article series to promote awareness , 31 and In summary, reporting practices have not improved despite published editorial advice. Journals and other members of the scientific community continue to advocate and implement strategies for change, but these have only had limited success. Stronger incentives, better education and widespread enforcement are needed for enduring improvements in reporting practices to occur.S1 FilePython code and PubMed search results used to randomly select papers for the audit. See the included README.txt file for a full description.(ZIP)Click here for additional data file.S2 FileScoring criteria and details of questions 1-10.(PDF)Click here for additional data file.S3 FileComma-separated-values (CSV) file of raw scores for questions 1-10 and the Python files used to analyse the data. See the included README.txt file for a full description.(ZIP)Click here for additional data file.S4 FileComparison of audit results across raters indicates the scoring criteria were applied uniformly across raters.(PDF)Click here for additional data file.S5 FileComparison of audit results for each year and journal. The British Journal of Pharmacology consistently had lower reporting rates of p-values for main analyses, and exact p-values for main analyses.(PDF)Click here for additional data file.


Gastroduodenal artery (GDA) pseudoaneurysm is a serious complication following pancreatic resection, associated with high morbidity and mortality rates. This review aimed to report the incidence of GDA pseudoaneurysm after pancreatic surgery, and describe clinical presentation and management.MEDLINE and Embase were searched systematically for clinical studies evaluating postoperative GDA pseudoaneurysm. Incidence was calculated by dividing total number of GDA pseudoaneurysms by the total number of pancreatic operations. Additional qualitative data related to GDA pseudoaneurysm presentation and management following pancreatic resection were extracted and reviewed from individual reports.Nine studies were selected for systematic review involving 4227 pancreatic operations with 55 GDA pseudoaneurysms, with a reported incidence of 1·3 (range 0·2–8·3) per cent. Additional data were extracted from 39 individual examples of GDA pseudoaneurysm from 14 studies. The median time for haemorrhage after surgery was at 15 (range 4–210) days. A preceding complication in the postoperative period was documented in four of 21 patients (67 per cent), and sentinel bleeding was observed in 14 of 20 patients (70 per cent). Postoperative complications after pseudoaneurysm management occurred in two‐thirds of the patients (14 of 21). The overall survival rate was 85 per cent (33 of 39).GDA pseudoaneurysm is a rare yet serious cause of haemorrhage after pancreatic surgery, with high mortality. The majority of the patients had a preceding complication. Sentinel bleeding was an important clinical indicator. The management of gastroduodenal artery (GDA) pseudoaneurysm is primarily endovascular, with the exact method dependent on vessel anatomy, clinical state of the patient, collateral hepatic blood flow and interval from index surgery.Remains an unusual but important complication Mortality from pancreatic resection has fallen significantly over the past few decades, especially in experienced centresThe aim of this systematic review was to synthesize existing evidence regarding the incidence, clinical presentation and management of GDA pseudoaneurysms after pancreatic surgery.Studies evaluating GDA pseudoaneurysm formation after pancreatic surgery were identified by means of database searches of MEDLINE and Embase. In Embase the terms used were: ‘false‐aneurysm’ AND ‘gastroduodenal artery’ AND ‘pancreaticoduodenectomy’ OR ‘pancreatectomy’ OR ‘distal pancreatectomy’ OR ‘pylorus preserving pancreaticoduodenectomy’ OR ‘pancreas surgery’. In MEDLINE the terms used were: ‘false aneurysm’ OR ‘pseudoaneurysm’ AND ‘gastroduodenal artery’ AND ‘pancreaticoduodenectomy’ OR ‘pancreas surgery’ OR ‘pancreatectomy’ OR ‘distal pancreatectomy’ OR ‘pylorus preserving pancreaticoduodenectomy’.A manual reference search was also performed to identify additional observational studies. No language restrictions were applied. Inclusion criteria were: manuscript published in a peer‐reviewed journal until 2017, investigating adult patients aged over 18 years, undergoing pancreatic surgery for any indication, developing GDA pseudoaneurysm, and reporting clinical outcomes of interest. The authors independently reviewed all relevant titles and abstracts, and all disagreements were resolved by consensus. Observational studies that reported both the number of GDA pseudoaneurysms and the total number of pancreatic operations performed were used for quantitative analysis of incidence. Qualitative information also relevant to the clinical presentation and management of GDA pseudoaneurysms was extracted from individual cases and collated. Data extracted included index surgery, sentinel bleeding defined as haemorrhage that occurred in the gastrointestinal tract  or intra‐abdominally  between 6 h and 10 days before a massive haemorrhage in the postoperative setting, day of postoperative bleeding, diagnostic method, management, other postoperative complications and mortality. GDA pseudoaneurysms were confirmed either radiologically or during surgery in all studies.Fig. A PRISMA flow diagram is shown in Five further studies were excluded as they did not provide the rate of GDA pseudoaneurysm in the postoperative periodTable Table All of the nine manuscriptsTable A total of 55 GDA pseudoaneurysms were identified in the postoperative period following 4227 pancreatic procedures, with a reported incidence of 1·3 (range 0·2–8·3) per cent  were treated using an endovascular approach. Nineteen (49 per cent) were managed using transarterial embolization (TAE) of the GDA via the common hepatic artery (CHA), and 14 (36 per cent) by selective embolization of the pseudoaneurysm. Stenting was employed in two patients (5 per cent), and only four (10 per cent) were treated by emergency laparotomy. The overall survival rate was 85 per cent (33 of 39).The GDA is the most common site for pseudoaneurysm formation after pancreatic surgeryIn this series, two‐thirds of the patients (4 of 21) had a preceding complication following pancreatic resection. Most authors favoured the hypothesis that lytic, enzyme‐rich, pancreatic fluid from a pancreatic anastomotic leak could result in autodigestion of GDA vessel wall owing to its proximity to the pancreatic anastomosis.Interestingly, a few studiesVarious techniques have been suggested to reduce the chance of pseudoaneurysm formation, including the ‘wrapping’ techniqueRecognition of a sentinel bleed may help in early management, this being a feature in most patientsSurgical intervention has been largely replaced by interventional radiologyThe endovascular management of pseudoaneurysms varied, reflecting the location and size of the pseudoaneurysm and probably institutional preferences for approach and embolization technique, and materials. TAE of the GDA was conducted via either the CHA or the superior mesenteric artery to achieve both proximal and distal occlusion, to exclude the pseudoaneurysm and prevent backflow from collateral circulationLimitations of this study include heterogeneity of the included studies, the descriptors used and study sizes. The absence of any prospective registers or clinical trials on this topic needs to be addressed.The authors declare no conflict of interest.


Binding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification.Human monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis , NF-κB activation and TNF-R1 internalization were used as biological readouts.We identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction.Our results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.The online version of this article (10.1186/s12964-019-0405-8) contains supplementary material, which is available to authorized users. Tumor necrosis factor alpha (TNF) regulates a variety of cellular processes ranging from inflammation, proliferation, to differentiation, and can induce various forms of cell death. TNF signal transduction occurs via binding of the ligand to two different receptors: TNF-R1 and TNF-R2, two members of the TNF-receptor superfamily. TNF-R1 belongs to the subgroup of death receptors (DR) and can induce cell death via its C-terminal “death domain” (DD). The selective recruitment of distinct adaptor proteins to the activated TNF-R1 determines whether cell survival or cell death signaling prevails . We and Intracellular trafficking of TNF-receptosomes and fusion with trans-Golgi vesicles facilitates maturation towards a multivesicular/lysosomal compartment. Here, a proteolytic cascade leads to the formation of ceramide via acid sphingomyelinase (aSMase) and translocation of active cathepsin D (CtsD) into the cytoplasm. CtsD cleaves and then degrades the anti-apoptotic chaperone HSP90, and activates BID by truncation to tBID. tBID is involved in mitochondrial outer membrane permeabilization and cell death –14. ManyThe reversible post-translational protein modification of cysteine residues with a palmitic acid through thioester formation  is known to modulate target protein interactions with lipids and with other proteins. The palmitoyl group is attached to a target protein by palmitoyl acyltransferases (PAT) containing the characteristic Asp-His-His-Cys (DHHC) motif. In mammals, 23 zDHHC containing proteins have been identified. Palmitoylation is fully reversible and depalmitoylation is catalyzed by palmitoyl thioesterases (PTE). The best-described PTEs are the cytosolic APT1, APT2, and the lysosomal PPT1, but the family is growing, e.g. through the identification of the ABDH17 proteins . PalmitoHere, we identify palmitoylation as a novel molecular switch that modulates TNF-R1 mediated signaling. Our initial observations that pharmacological interference with palmitoylation modulates TNF-R1 internalization prompted us to investigate how lipidation regulates TNF-R1 signaling. We focused on the palmitoylation state of TNF-R1, showing that its de-palmitoylation in response to TNF is mediated by the PTE APT2. Knock down of APT2 enhances apoptosis but blocks signaling via NF-κB. Mutagenesis of the putative palmitoylation site C248 altered TNF-R1 surface expression resulting in both, reduced cell death and NF-κB signaling.PIC: cOmplete protease inhibitor cocktail (Roche).Cell Signaling: anti-CD71 (#13113), anti-cleaved Caspase 3 (#9661S), anti-His (#2365), anti-IκBα (#4814), anti-Integrinα6 (#3750), anti-STX6 (#2869), anti-TNF-R1 (#3736), anti-PARP (#9542S).ENZO: anti-Ceramide .LSbio: anti-Lypla2/APT2 (LS-C158086).LifeTechnologies: anti-mouse Alexafluor488 (A21202).Millipore: anti-mouse light chain (AP200P), anti-rabbit light chain (MAB201P).Proteintech: anti-βActin (HRP-60008), anti-GAPDH (HRP-60004).Santa Cruz Biotechnology: anti-Rab5B (sc-598).Sigma-Aldrich: anti-PPT1 (HPA021546).ThermoFisher: anti-Lypla1/APT1 (PA5–28034).Human U937 cells  were maintained in RPMI 1640 medium  supplemented with 5% v/v FCS  and 1% v/v Pen/Strep (Merck Millipore) under standard cell culture conditions.CRISPR/Cas9 plasmids  were nucleofected (Lonza). After transfection cells were FACS sorted for GFP positive signal and single clones were isolated and further characterized.C248S was ordered from Geneart and cloned into pMOWS vector . To generate virus particles, vector was transfected to Gryphon™ cells (Allele Biotechnology) using lipofectamine 2000. After two days, virus containing supernatant was centrifuged  sterile filtrated (0.45 μm) and added to 0.5 × 106 ΔTNF-R1 U937 cells in the presence of 5 μg/ml Polybrene. Next day, medium changed to standard cell culture medium and 1.5 μg/ml puromycinwas added after 24 h.FLAG-TNF-R16 cells/sample were incubated with biotinylated TNF  coupled to streptavidin Alexafluor488 (Life Technologies) on ice for 20 min, followed by synchronized receptor internalization at 37 °C. Where inhibitors were used, these were pre-incubated for 20 min at room temperature followed by the incubation on ice. Internalization was stopped by addition of cold PBS / cell mask , incubation for 5 min on ice sedimentation and fixation of the cells in 2% PFA/PBS. At least 5000 images per experiment were acquired using the Inspire software (200.1.388.0) and analyzed using the Amnis IDEAS software (6.0.154.0).TNF-R1 internalization was analyzed by imaging flow cytometry . For this 10Cells were labeled as for internalization measurement. At least 5000 images per experiment were acquired using the Inspire software (200.1.388.0) and fluorescence intensity of Ch2 (Alexafluor488) was analyzed using Amnis IDEAS software (6.0.154.0).For apoptosis measurement by imaging flow cytometry, cells were incubated for the times indicated in the figure with TNF (100 ng/ml) under standard cell culture conditions. 30 min before end Hoechst stain (Sigma-Aldrich) was added to the culture medium finally diluted 1:10,000. Up to 10,000 images were captured per assay and quantified using Amnis IDEAS software (6.0.154.0) .6 U937 cells were pre-incubated for 10 min at room temperature followed by 10 min on ice with DPP-2 or DPP-3 fluorescent probes (provided by B.C. Dickinson)  the other part with Tris-HCl [pH 7.5], final concentration 0.5 M each. After over night incubation, beads were washed and used for SDS-PAGE.AcylRAC was performed as described by Forrester et al. , 24, wit4, 0.2 mM TBTA  and 4 mM ascorbic acid (fresh) in a total volume of 200 μl. After 2 h incubation at RT, proteins were acetone precipitated and then resuspended in 500 μl 50 mM TRIS-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1 mM EDTA, 0.25% Na-deoxycholate. 20 μl Streptavidin-microbeads  were added and incubated overnight at 4 °C. After purification via μColums (Miltenyi) and elution using SDS-Sample buffer containing β-mercaptoethanol, 15 μl were used for SDS-PAGE/WB.The labeling protocol was adapted from . In brie7 cells were washed with ice-cold PBS followed by incubation with 100 ng/ml Fc60TNF for 20 min on ice. After the temperature shift for the respective time point, cells were resuspended in 1 ml of IP lysis buffer (50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1% Triton X-100, 1 mM EDTA, Benzonase (Merck) and PIC for 45 min on ice and subsequently 10x sheared using a G21 gauge needle. The lysate was centrifuged 10 min at 10,000 x g and 50 μl protein G Microbeads were added to the supernatant and incubated for 2 h at 4 °C. Upon purification using μ Columns (Miltenyi), 10 μl eluate was analyzed by SDS-PAGE/WB.5 × 10Cells were pre-incubated for 45 min with 50 μM ML349 followed by addition of 100 ng/ml TNF for 6 h and lysis in TNE . Proteins were analyzed by SDS-PAGE/WB.Cells were cooled on ice for 15 min, followed by incubation with TNF for 15 min, and a temperature shift for the indicated times to 37 °C. Cells were then lysed in 50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1% Triton X-100, 1 mM EDTA, Benzonase (Merck) and PIC, followed by BCA assay and SDS-PAGE/WB.Where described, protein samples were labeled with lightning red (Serva) diluted 1:50 in protein sample buffer and then separated on anyKD (Biorad) or 12.5% SDS-PAGE gels. Subsequently, total protein staining was analyzed using a Typhoon trio . After transfer to PVDF membranes (Carl Roth), membranes were blocked with 5% skimmed milk/TBST, incubated with primary antibodies overnight at 4 °C, followed by washing with TBST and incubation with HRP-conjugated second step antibodies for one hour. Luminescence was detected using ECL reagent and x-ray films . Films were scanned and where mentioned, densitometric quantification was performed using ImageJ.Recombinant APT2 was generated as described . PurifieFor APT2 silencing, shRNA encoding plasmids  were nucleofected using Amaxa (Lonza), followed by puromycin treatment to generate stable pools.Internalization and trafficking of TNF-R1 is a prerequisite for the diversification of TNF signal transduction, implicating a putative role of palmitoylation to selectively recruit proteins to distinct signaling platforms in these events –7, 27. ITC) and the HA fractions, not all cellular TNF-R1 appears to be palmitoylated. This could be explained by either inefficient capturing during acylRAC or by the assumption that only a certain pool of TNF-R1 is palmitoylated (i.e. receptors at the plasma membrane). As controls, we analyzed the samples for known palmitoylated proteins  and β Actin and CoxIV as a non palmitoylated controls. Figure tc) +/− TNF contains a variety of proteins, while fewer bands are visible in the hydroxylamine (HA) fraction. The control (Tris) lanes are absent of protein, indicating the enrichment was successful.Based on the observation that perturbation of S-palmitoylation by 2BrP altered TNF signaling, we hypothesized that TNF-R1 may be palmitoylated itself, as palmitoylation of other death receptors  and implications on their downstream signaling has been reported before –30. To cTo investigate whether endogenous TNF may affect TNF-R1 palmitoylation, we incubated U937 in the presence of an anti-TNF-Fab’ for 14 days, followed by acylRAC/WB analysis. In both, untreated and TNF-Fab’ treated cells, TNF-R1 was found to be palmitoylated   and two less conserved cysteine residues C304 and 442) , the lower MW variant (filled arrowhead) is much more prominent in TNF-R1C248S expressing cells. Thus, we conclude that C248 is not the only TNF-R1 palmitoylation site but it is required for proper transport of the receptor to the plasma membrane and also for the activation of NF-κB.To analyze whether the cysteine 248 is the only putative palmitoylation site, we performed acylRAC from wt and mutant TNF-R1 expressing cells  for further functional analysis Fig. d. The upFig. e: PalmitTo investigate the in vivo role of APT2 in TNF signaling, cells were then incubated with different concentrations of the inhibitor ML349. First, we observed slightly reduced TNF-R1 internalization from ~ 70% in untreated to ~ 45–60% of the ML349 treated cells  , 46, 47.Three groups reported that TNF-R1:lipid raft association is essential for ERK and NF-κB regulation , 48, 49,We identified constitutive palmitoylation of TNF-R1 but the number, exact sites and which PAT are involved is still unclear and has to be unraveled in further studies. In an acylRAC proteome analysis of TNF-R1 CRISPR/Cas9 edited cells, which is part of a different project, we identified one TNF-R1 peptide (data not shown). Analysis of acylRAC samples by MS in this study did not result in identification of TNF-R1 peptides. The low coverage of peptides identified by mass spectrometry is in line with two earlier reports , 60, as Mutagenesis of the putative palmitoylation site C248 led to high overall cellular TNF-R1 expression but reduced TNF-R1 surface expression. IκB degradation was blocked in C248S cells. Compared to TNF-R1 knock-out cells cell death was restored but on a lower level. We assume that C248 palmitoylation is required for Golgi to PM transport, which is in line with the report by Wang et al., showing that surface TNF-R1 is shed from the cell surface in response to histamine and subsequently replenished by TNF-R1 from an intracellular Golgi pool . This isAltered subcellular distribution of TNF-R1 may also result in reduced glycosylation/sialylation of TNF-R1 , 61. ThiWe found that APT2 is involved in TNF-R1 de-palmitoylation which may be prerequisite for lipid raft translocation and NF-κB activation as reported by others , 49. ThiWe also observed APT1:TNF-R1 interaction by co-IP, while the function of APT1 in the TNF-RSC remains to be investigated. The lysosomal PTE PPT1 has recently been linked to TNF signaling by showing that PPT1/Cln-1 deficiency results in resistance to TNF induced apoptosis induction which is in line with our observations that TNF-receptosomes have to maturate into multivesicular bodies and lysosomes for full apoptosis induction . InteresEarlier reports showed that transmembrane TNF is palmitoylated which regulates its affinity to TNF-R1 , 69. FasBased on our results and reports from other groups, we propose the following model of de-palmitoylation events in the regulation of TNF-R1 signal transduction Fig. : TNF-R1 In sum, our study demonstrates that palmitoylation represents a novel layer of regulation in TNF-R1 signaling. Observation of TNF-R1 ubiquitination , 73, 74,Additional file 1:Figure S1. Endogenous TNF does not affect TNF-R1 palmitoylation. U937 cells were cultured for 14 days in the presence of 0.5 μg/ml anti-TNF-Fab’-Fragment or left untreated (ctrl). Prior to stimulation with exogenous TNF , cells were washed and acylRAC/WB was performed. One representative experiment is shown. (JPG 201 kb)Additional file 2:Figure S2. TNF-R1 sequence alignment. Alignment of TNF-R1 amino acid sequence from different species (part of the N-terminus is not shown). Red box: conserved Cys residues representing possible palmitoylation sites. Blue box: TMD. (JPG 812 kb)Additional file 3:Figure S3. Role of APT2 in TNF signaling. Loading control for Fig. Additional file 4:Table S1. Proteins identified by mass spectrometry after enrichment of palmitoylated proteins by acylRAC after 0–60 min TNF incubation. The sample processing and data analysis is described in table. (XLSX 346 kb)


I present a body of data that, I argue, cumulatively casts serious doubt on the validity of Jeanne Calment's accepted world record of human life span. First, I assess the plausibility of the record based on the life spans of other centenarians in the International Database of Longevity (IDL) and critique some arguments put forward previously in support of that plausibility, including the longevity of Calment's ancestors. Second, I review the literature dedicated to Calment and discuss multiple contradictions in her interviews, biographies, photos, and documents. I argue that the evidence from these sources motivates renewed consideration of the previously rejected hypothesis that Jeanne's daughter Yvonne acquired her mother's identity after her death to avoid financial problems and that Jeanne Calment's death was reported as Yvonne's death in 1934. Finally I discuss the importance of reconsidering the principles of validation, due to the possibility of similar problems regarding other exceptionally long-lived people and the mistaken inferences that researchers may draw from flawed datasets. The phenomenon of Jeanne Calment may prove to be an instructive example of the uncertainty of seemingly well-established facts. None of these studies, however, has identified any decline in the force of mortality with age, at least until the data become too scarce to make a determination. (This is in intriguing contrast to what has been observed in insects.) However, Calment's age at death exceeds that of the current runner-up (Sarah Knauss) by more than 3 years; thus, at a first glance she looks like an exception.8 But how extreme an exception?Much attention has recently been devoted to the dynamics of mortality in older ages (105 years and older).e.g.) the Italians born in 1904 and alive in 2009, approximately half of whom died during any year of follow-up observation through to 2016.4 Under this assumption, the probability of the event that anybody from a group consisting of N supercentenarians would be alive after t years is:We can quantify the likelihood that the last survivor of a group reaches a given age using Monte Carlo simulation. We make the conservative assumption that the force of mortality does indeed stay constant from age 110, and we use a value for that annual mortality rate of 0.5, which is what is observed in  after her 110th anniversary. All the other 48 supercentenarians in the French section of the International Database of Longevity (IDL) containing verified supercentenarians10) achieved the world record. Since IDL is free from age ascertainment bias,9 one can estimate the plausibility of the emergence of the record in IDL by 1998 by considering the group consisting of all 80 individuals in the IDL dataset born before 1876. ThenThus, the first (by date of birth) French woman to be validated as a supercentenarian by a large project searching for long-lived people  and the survival curve of the IDL group above without Calment do not differ much from the simulations .2 her validation was based on archive data, such as censuses, which may not be reliable age confirmation.11 Independent researchers from the 110 Club forum discovered that she was probably 20 years younger than her claim,12 but she is still present in the IDL and Gerontology Research Group (GRG) databases.Incidentally, the second place in this group  belongs to Lucy Hannah . Similar to the majority of the U.S. supercentenarians in the IDL,49 Similarly, the researchers who conducted the validation of Calment's age, Jean-Marie Robine and Michel Allard, attribute her exceptional life span to her extraordinary genes inherited from her presumably exceptional ancestors.13 They write: “Many ancestors of Madame Calment could have been unconsciously chosen (by the spouse or by their families) because they were carriers of genes favoring longevity. Gradually, there would have been a concentration, a unique enrichment of this type of gene, to permit such a long existence.”14 In an article13 devoted to justifying the life span of Jeanne Calment by reference to the longevity of her ancestors, Robine and Allard state that “One might hypothesize that the extraordinary longevity of Jeanne Calment is largely genetic in origin and that it is due to an exceptional genetic inheritance, randomly accumulated within the ecological niche of Arles in the 18th and 19th centuries  and 1838 . The average life span of these ancestors appears to be 72.3 years, somewhat less than the declared 80 but still higher than 58.Unfortunately, these authors do not provide the raw life span data of Calment's ancestors. Thus, with the help of French researchers Arnaud and Cyril63I thus chose to analyze this dataset in more detail. It was easy to see that representatives of the male line of the Calments, including Jeanne's father Nicolas Calment, his father, grandfather, and great-grandfather, who were all ship carpenters by profession, and their wives lived long lives. The parents of their wives also lived long. Going further back, it turned out that Vincent Calment's father was a hostler and died at the age of 68 years, and his grandfather was a worker and died at around 50 years. Other ancestors of Vincent also could not boast exceptional longevity.I thus considered three groups of Jeanne's ancestors—the Calments and their wives; the Calments, their wives, and their parents; and Jeanne's other ancestors—and drew the survival curves of these groups, as well as synthetic survival curves based on the death rates in France in 1875 and 2014 from the age of 31, when the first death was recorded .21), and high social standing. The low infant mortality among the Calments' relatives could also have been aided by the presence of a large number of midwives in this family.14 But whatever the reason, the above-average longevity of the family seems to arise mostly, if not entirely, from the absence of early-life mortality.It will be seen that the survival curves of the first two groups are almost indistinguishable, and the third group experiences much higher mortality at young ages. Apparently, the longer life span of the family members of ship carpenters was due to a more favorable environment: prosperity, quality of food, the ability to escape from an epidemic by retreating to a country house . Thanks to Cyril Depoudent,62 I obtained a list of 10 real-estate transactions conducted by Jeanne Calment between 1897 and 1955, as well as transactions made by her family members. These include a large rental deal in Arles (probably of the space of the shop which was closed in 1937) of 375,000 francs in 1938  and the sale of the farm in Saint Martin de Crau, which was formerly owned by Jeanne's father Nicolas Calment, in 1951 for 5.5 million francs, which is equivalent to around $500,000 today. One of the sellers was Jeanne and another was her grandson Frédéric Billot, who had probably bought the share of his uncle François in 1943 for 500,000 francs . This farm was probably inherited by Jeanne and François from Nicolas Calment after his death in 1931, since the only large gift by Nicolas to his children was made in 1926 and amounted to 72,000 francs . After the death of Nicolas in 1931, both Jeanne and François sold some property for 35,000 francs each, and it was stated that Jeanne had sold the apartment at Roquette, 53—the address of the parents of Jeanne Calment  in the French Wikipedia27 Interestingly, the tax laws seem to affect the timing of reported deaths.28Tax rates rose sharply in the early decades of the 20th century. “Between 1791 and 1901… the proportional tax rates were fairly small , so there was really very little incentive to cheat. The estate tax was made progressive in 1901. In the 1920s, tax rates were sharply increased for large estates. In 1901, the top marginal rate applying to child heirs was as small as 5%; by the mid 1930s, it was 35%; it is currently 40%. Throughout the 20th century, these high top rates were only applied to small segments of the population and assets.”21 in 1931. The inheritance tax for the farm in Saint Martin de Crau could amount to hundreds of thousands of dollars in modern money. It is not hard to imagine that the family had neither desire nor ability to pay the tax, especially twice in a row . Information about paid taxes must be stored in the relevant archives,27 so one can try to check the taxes paid after the death of Nicolas in 1931, given access to them. In addition, Jeanne and her husband had a lot of common property (which they had rented out after the closure of the store), the inheritance of which could also be heavily taxed.Perhaps the Calment family suffered from taxation after the death of Maria Felix  and especially after the death of Jeanne's father Nicolas Calment, the owner of land and real estate in the surrounding villages53 see section 43Another motive could be the annuity contract presumably signed before 1934,We now examine a long list of items that are (to varying degrees) surprising in the context of Jeanne Calment having died in 1997 and Yvonne in 1934 but are consistent with the reverse. No one item constitutes a “smoking gun,” but I claim that in combination they form a compelling case. Some of them were already known to previous analysts, but this is the first time that they have been systematically assembled.A remarkably large number of examples exist in which Calment or a member of her family describes someone using wording that is incompatible with the no-switch scenario but fits with the switch scenario. While the errors made by Calment may be ascribed to her age, those made by others cannot.14 Her reminiscences concerning the family shop, however, incorporate a curious confusion. In the Canadian film “Vincent and me,”29 which made Jeanne the oldest-ever movie star, the 114-year-old woman informs a young girl that she met the artist in her “father's” shop. Newspapers also mentioned this “father's” shop,30 as well as the GRG website,31 and Robert Young on the 110 Club.32 However, Jeanne's father Nicolas, unlike Yvonne's father Fernand, never had any store. There were actually no shopkeepers in Jeanne's ancestry49 contrary to what was stated in Robine et al., 199813 but shopkeepers were present in the ancestry of Yvonne.According to Robine and Allard, “Madame Calment didn't really become famous until 1988, when the centennial of Vincent van Gogh's stay in Arles was celebrated. Without the link to a famous artist, perhaps she would have remained unknown. In 1994, the second-oldest French person died at the age of 113 years without either having been studied or having received national recognition. But Jeanne Calment quickly passed from local fame in Arles to worldwide fame.”Gilles, whose name was that? “It's my grandmother's name, my grandparents on my father's side.”14 After some discussion, she corrected herself and said that it was her mother's side; but in fact “Gilles” was the name of Jeanne's maternal grandfather and was thus both the married name of her maternal grandmother and also the maiden name of her mother. Regardless of whether maiden or married names are more often used in such situations, it is somewhat surprising that she would preferentially mention her grandmother rather than her mother.Here is another example of confusion: 33 Jeanne was an unusual mother-in-law; she got along perfectly with Joseph, with whom they previously co-raised Frédéric.14 Moreover, in the 1962 census, the marital status of Joseph Billot was corrected: it shows “M” (for “Married”) and “V”  typed over each other.33According to the 1954 and 1962 censuses, Madame Calment lived together with her son-in-law colonel Joseph Billot, and Frédéric lived with his wife Renée in a nearby apartment.33 the validators write that Jeanne mentioned the servant Marthe Fousson, and they cite the census of 1911 in which she is counted together with the Calment family. However, the only mention of a servant with the name Marthe that I found in the biographies of Calment was the following: Did you go to school with your friends, or did you go alone? “My father or the maid accompanied me…” What was the maid called? “Marthe, Marthe Touchon.”34In their report,14 On the basis of almost identical-sounding surnames and the coincidence of the name, and taking into account that there was nobody named Marthe living with Jeanne or nearby when she was a child according to censuses, and the only servant found in the census of 1886 was called Marguerite Minaud , I conclude that Marthe Touchon is highly likely to be the same maid as Marthe Fousson from the validation report. However, according to the archives of the commune of Fontvieille, she was born on March 8th, 1885 and thus was 10 years younger than Jeanne, so she couldn't accompany Jeanne to school. By contrast, she could well accompany her daughter Yvonne.In the last years of Calment's life when these validation talks took place, she had difficulty with speaking precisely.14 and François Hureau discovered that most probably she was in fact Césarie Gachon . But Gachon was only 14 in 1982 when Jeanne was 7. By contrast, when Yvonne was 7 (in 1905), Gachon was 37, and indeed she was listed as a piano teacher in the 1896 census59 and a music teacher in the 1911 census.60Although Jeanne did not recall the names of her early school teachers, she remembered her first piano teacher (when she was seven) very well. She identified this teacher as “Cesari Gaston,”35 when Frédéric was 1 year old, his parents went for a vacation to Italy, leaving the child to his grandparents. The child persistently tried to call his grandma “mama,” but they reached a compromise. After Yvonne's death, Jeanne treated Frédéric as her child.35After Yvonne's official death, her son Frédéric was brought up by his grandmother Jeanne Calment. He called her “Manzane,” meaning “mama Jeanne.” According to Cavalié, 1995,14In another interview, Calment was asked: “Did you ever meet Frédéric Mistral?”  She replied: “Yes! Yes, he was a friend of my father… um, he was a friend of my husband.”33 The validators explain the absence of Yvonne by a recopying error.33 However, an alternative interpretation is that Jeanne's health was by then in decline and the family was already in the early stages of devising an ID-switch plan, leading them to provide obfuscating information.According to the 1931 census, the Calments' house on Gambetta Street in Arles was inhabited by homeowner Fernand Calment (Jeanne's husband) along with his mother Maria, his wife “Maria” , Yvonne's husband Joseph Billot, Yvonne's son Frédéric Billot, and two maids.14 Soviet validators of centenarians from Azerbaijan have frequently used questions about past cholera epidemics for validation purposes11 because they have a good track record of being remembered. It is thus surprising that Jeanne initially omitted it, even though the family retreated to Saint Martin de Crau during that period.21In her numerous interviews, Madame Calment discusses a lot of hard-to-check details of her childhood, starting from the age of three, but does not mention the epidemic of cholera that devastated Arles in 1884 when Jeanne was 9 years old.63 and confirmed by patent,57 mortgage62 and census58 documents. This change of home was so thoroughly absent from Jeanne's reminiscences that the validators said she had only one address before marriage;33 moreover, Jeanne stated explicitly in 1988 that she was born at Roquette 53,54 where in fact neither Jeanne nor Yvonne was born.33 Later, illustrating similar confusion, she stated that she was born at “Du Roure 53” .35Another important event of Jeanne's childhood was the moving of her family from Du Roure 5 to Roquette 53 in 1888 when she was 13, as uncovered recently by François Robin-Champigneul35 just before the start of World War I, when Jeanne was almost 40 years old while her daughter Yvonne was around 15, she said that she was very excited to go hunting with Fernand and not being afraid of anything, she acquired the masculine nickname “Jean Calment” and became a member of the hitherto male-only hunting club. After that, she went on to master skis on the glaciers of Grenoble.35According to the biography of Calment,Do you remember how were you dressed at the time… when you were 20 years old for example? Oh, you ask too much of me. You go too far! I led a man's life. I was a huntress, you understand. That will surprise you.”14However, in another biography, Jeanne states that she was already a huntress when she was 20 years old: “36 Thus, I only briefly summarize that aspect of the evidence here.This work was inspired by my conversations with Dr. Valery Novoselov, who argued that Madame Calment was highly suspicious from a point of view of clinical geriatrician. Novoselov is going to discuss her clinical phenotype in a separate study.37). A similar fibroma can be seen in one of the photos of the old Calment .35In addition, there appears to be inconsistency with regard to Jeanne's eye color in youth versus her later years. In the color version of Finally, the old Jeanne looked almost unbelievably young for her age. While it is to be expected that those who reach exceptional ages will look similar to considerably younger people (because they “stayed younger longer”), the magnitude of that disparity in this case may be viewed as so extreme as to raise suspicion.38 where there is a short paragraph in the chapter on insurance fraud devoted to Madame Calment. This paragraph was based on an article52 (kindly sent to me by Benoit Martin) by Jacques de Baudus, who wrote that during the dinner a very respectable guest told nine respectable persons that a state insurance company , which paid a lifetime annuity to Jeanne Calment for a long time, discovered that it was a daughter of Jeanne Calment who received the rent, but, by agreement with the authorities, it kept the matter secret given how much the character of the “doyenne of the French” had by then become legendary. The details of the story are not clear. According to it, Jeanne Calment signed this contract before her death in 1934  Fernand Calment, so Jeanne should have been constantly seen by the commune. This belief can be traced back to Robine and Allard's validation report on Jeanne Calment's life span,However, the commune of Arles is actually the largest commune in France, with a surface almost nine times the surface of Paris. It incorporates several nearby villages where its wealthy inhabitants  used to have country houses. The population of the commune of Arles was around 24,000 people in 1875 and almost doubled by 1997, while there are around 380 inhabitants in a median-sized French commune.35 There is now only a renewed historical sign on the façade of the Maison Calment, the house where the store was formerly located. Contrary to what is asserted in the official validation,33 the drapery store itself has been permanently closed since 1937,39 and now there is a supermarket, Casino, in that location.According to her biographies and mortgage documents, Calment had many other places in several communes to live in, and her favorite property was La Miquelette, a villa in the village of Paradou near Arles which Jeanne and Fernand bought in the early 1930s for their daughter Yvonne and her son Frédéric Billot (1926–1963).33 Her husband, Captain Joseph Billot, travelled a lot . In fact, Calment told her validators that she followed her husband and enjoyed travelling (and hunting) with him.14 In the early 1930s, Yvonne may have spent a lot of time outside of Arles, residing in her villas where she could be known under the pseudonym Jeanne. In addition, most people knew her just as Madame Calment, no matter what name she used.Similarly, the trail of Yvonne Calment went cold after her wedding in 1926. As noted earlier, she was not listed in the 1931 census. She was also not counted with her parents in 1926.39 The fact that she is mentioned in many censuses at the address of the store does not mean that she lived there all the time. According to Robine and Allard, Jeanne is counted twice in the 1901 census: at St Martin de Crau and in Gambetta Street in Arles.33 Similarly, in the 1901 census her brother François was also counted at St Martin de Crau33 while also being counted with his wife and daughter in Toulon.62 Of note, Robine and Allard write that François had no family and children.14 Might this be another case of deliberate obfuscation of census data by the family? Of course it would not be related to the ID switch, since it occurred decades earlier, but it might indicate a pattern of behavior.Pierre Maxence is the son of Marius Maxence, the oldest employee of the store Maison Calment, who worked there for about 30 years until its closure in 1937. According to his testimony, Madame Calment almost never appeared there.41 cites a newspaper report64 a week later that an “especially big crowd followed Madame Billot-Calment for her last journey.” However, the cause of death may provide an answer. Yvonne may have suffered from a chronic condition,35 specifically tuberculosis, eventually degenerating into pleurisy, for several years before her death55—but where? Wealthy people were more often treated at home,41 and Le Pomellec failed41 to find confirmatory notes at the hospital in Arles, in spite of the following testimony of Madame Calment: “When she came back from hospital, it was Advent and she came to die at home.”14 This contradiction would be resolved if she was sent to another location, and indeed rural sanatoria were a popular option for tuberculosis sufferers at that time.65 Clearly, that would have helped keep a planned ID-switch secret; indeed, maybe it was Jeanne who had tuberculosis . In line with this, she told her validators “we took her to… Oh! I don't recall any more.”14 Death from such a severe infection could also have been an excuse to keep the dead woman's casket closed and to justify the living woman wearing a heavy veil. Camille also notes that unlike her mother's, son's and husband's names, Yvonne's name is not present on the tombstone of the familial grave.41Even if not many people knew Jeanne and Yvonne in the relevant period, there was one event at which it should have been particularly difficult to maintain secrecy: Yvonne's funeral. It was a very public event: Camille Le Pomellec35 and after the war it may have been relatively easy to promulgate the fiction that Madame Calment was always Madame Jeanne Calment.The late 1930s were turbulent times in Europe. World War II brought chaos with it,Moreover, one should also take into account the specifics of Provence, which was a region with its own language and mentality where (as everywhere in France) the idea of paying huge taxes to the French Treasury could be not very popular and corruption at the local level may not have been uncommon.21 Being in the nursing home and not being able to destroy the documents herself, Jeanne resorted to the help of a distant relative.42  Robine and Allard address this point but downplay its significance, as follows: “While she was still in the Maison du Lac, Madame Calment destroyed all her personal archives, all her family photographs. That's a bit surprising; yet, this attitude is not so unusual. Many people in her situation, without direct descendants or close friends, decide, in the twilight of their lives, to leave nothing ‘dragging’ behind them–nothing that affects them, no trace of their existence… She would say: ‘I don't want my image scattered.’ ”14 However, in the context of the pattern of behavior documented here, it becomes plausible that this destruction was a result of cold calculation and acute necessity instead of an emotional act.It is also somewhat suggestive that Jeanne decided to destroy the photos and other documents when she was requested to send them to the archives of Arles.41 Such a character would help her under the ID-switch scenario by minimizing contact with those who might not be trusted to keep the secret if they discovered it. When one journalist asked Madame Calment: “You were a charming woman; you must have had a lot of gallant proposals?” she answered him: “My husband was a marvelous man. When one has an angel, one keeps him, and the rest don't see you.”14Finally, there is evidence that the family was not socially active after the war. Claudine Serena, who nursed Madame Calment during her last years, told Camille Le Pomellec that Madame Calment despised ordinary people and people disliked her.62 Doubtless a number of other friends and colleagues knew the truth but viewed it as not their business  to whistle-blow on a family with substantial wealth and influence in the region. But there was one individual for whom these arguments may initially appear insufficient.Under the ID-switch hypothesis, a manageably small inner circle knew about the substitution but did not tell anyone. The circle consisted of the family themselves, who obviously had a financial interest in perpetuating the deception, and also a number of people who were close friends of the family and thus would be expected to have kept the secret merely out of loyalty. For example, a 71-year-old widow, Joséphine Audibert (born in Tarascon), claimed to have seen Yvonne's corpse, and her testimony was signed by the aide of the mayor of Arles, Justin Valle, who claimed to have ascertained the fact that Yvonne Calment died at 2:00 a.m. on January 19, 1934, at the age of exactly 36 years at her home on Gambetta Street.51 and he apparently had no doubts about her age.34One of the most famous stories about Jeanne is that she settled a life annuity deal with the notary André-François Raffray,51 but many other sources state that it was first signed when Jeanne was 90, i.e in 1965).34 Thus, the notary  had to pay 10 times more than expected.51 Huguette informed me by phone that the apartment was sold immediately after the death of Jeanne, and she does not know who the buyer was.Raffray agreed to pay 2500 francs monthly in exchange for acquiring her apartment in the center of Arles, where she had been counted in censuses since her marriage in 1896. The annuity was calculated according to the life expectancy of 90-year-old French women in 1965, which was around 3 years.  about the switch at the time he entered into the agreement (since then the expected time until he received the property would be many times longer). However, we cannot ignore the possibility that he discovered it later. At that point, he could presumably have terminated his liability in an instant by revealing the switch to the authorities. Were the social pressures noted above so strong as to be worth losing so much money? It seems unlikely.This initially seems to be a big challenge to the ID-switch hypothesis. Since Raffray moved to Arles only in 1959,53 Thus, she had both a motive and the ability to repay Raffray “under the table” in order to buy his silence.But two explanations are easy to posit that are fully compatible with the ID-switch scenario. Firstly, at the time of Raffray's (putative) discovery of the switch, Yvonne's reliance on perpetuating the secret would have been enormous: probably the threat of a large tax bill would have reached a statute of limitations, but there might have been the prospect of a criminal trial, or at very least a huge embarrassment for Yvonne personally. Moreover, as noted in the Alternatively, it is equally reasonable that Raffray was indeed aware of the switch before signing the deal, but was friendly with Yvonne, so they signed the deal  precisely to make the ID switch seem less plausible to the wider world, and less likely to be investigated by those who would have suffered financially from it.42 and maybe some other projects (there was even some information on her HLA group in Wikipedia). Curiously, Jeanne and Fernand were double second cousins while there was no consanguinity detected in at least four generations of Jeanne's ancestors34 and thus Yvonne is expected to have significantly higher level of homozygosity50 than Jeanne if their official genealogy34 is correct. Thus one can test the validation of Jeanne Calment's age by analyzing the DNA from cells taken from Madame Calment late in life. It is unfortunate that Calment's medical card is not available for study21 and her last geriatrician, Dr. Catherine Levraud, refused to provide me with any medical information; political will may be needed to clarify these matters.Forensic comparison of the bodies and DNA of family members buried in the familial grave could potentially be performed to check whether there was an identity switch. In fact, it is also possible that biological material from the person who died in 1997 is still in storage: cells belonging to her were probably taken for analysis by the project ChronosIn addition, there is a case for expert analysis of surviving photos of Jeanne and Yvonne to arrive at a firm determination of who is pictured, since various commentators have suggested that the old Jeanne looks more like the young Yvonne than the young Jeanne and found multiple confusions in naming of photos of Yvonne and Jeanne in newspaper articles published while Jeanne was a recognized supercentenarian.43More broadly, however, we must ask what can be learned from the fact that more than 20 years passed with (as I have argued here) severely inadequate scrutiny of what is probably the single most famous data point in the entire field of gerontology. According to Norris McWhirter, the editor of the Guinness Book of World Records, “No single subject is more obscured by vanity, deceit, falsehood and deliberate fraud than the extremes of human longevity.”44 Robert Young, the director of the GRG, a consultant for the Guinness Book of Records and the administrator of the 110 Club, writes that in cases when the declared age is approaching 120 years, the validation should be very thorough, as was done in the case of Jeanne Calment. To stress this point, a list of false examples of long-lived people is provided in Young et al., 2010.43 But there is still room for improvement, as illustrated not only by the Calment case but also by the following examples.It should first be noted that the science of extreme longevity is becoming more rigorous. In the past, oral testimony was enough to recognize the age of a person, but now multiple pieces of documentary evidence are required for validation, and numerous myths have been debunked.24 who died in 1814 at the age of 113, was not questioned until the end of the 20th century, when the demographer Hubert Charbonneau showed that Pierre Joubert had in fact died in 1766 and it was his son, born in 1732, who has been mistakenly considered as the oldest Canadian for a long time. It is important to note that in this and other false examples of long-lived people,44 the substitution was not pre-planned, so it was quite easy to expose . In the case of Jeanne Calment, on the other hand, we are talking about a well-planned identity switch in the distant past of 1934.One of the well-known examples of disproved supercentenarians, Pierre Joubert,For obvious reasons, among claimed supercentenarians, there should be a disproportionately large number of people whose documents contain intentional or accidental errors that overstate their age. On February 21st, 1986, when Jeanne Calment turned 111 years old, the previously universally recognized record holder, Shigechiyo Izumi, died at the claimed age of 120. It was eventually discovered, however, that Izumi acquired the birth certificate of his deceased older brother, and, in fact, he lived “only” until 105.Two other universally recognized rivals of Jeanne Calment were also recently “devalidated”: 117-year-olds Lucy Hannah, for whom researchers at the 110 Club showed that the census data referred to another person, and Carrie White .9The significance of such errors is disproportionate to their number. Jean-Marie Robine, one of the validators of Jeanne Calment, and his colleagues from the IDL note that “Studying extreme ages necessarily involves small numbers, and therefore, a single age error, especially at the highest ages, may well have strong implications for predicting the trajectory of mortality. It is thus of great importance to check the reliability and accuracy of each reported age from such a dataset.”46 This is in line with ideas expressed by Brouard.47There are also quantitative ways to see that errors in a small fraction of birth and death records can significantly distort the observed dynamics of mortality. Even if errors are distributed normally, survivor bias means that the observed force of mortality can plateau or even decrease in old age, even if in reality, it is increasing exponentially.3 as was explained in Zak, 2018),7 and truly verified list of semisupercentenarians . If, in addition to participating in validation, geriatricians become involved in the study of these long-lived people and collect longitudinal data on their health, the analysis of such data will help in understanding the process of individual aging. It is also necessary to avoid any conflict of interest. Selective revalidation of existing supercentenarians will allow estimation of the frequency of fake cases. It would be highly valuable to develop and implement objective methods of age estimation with narrow confidence intervals using molecules with negligible turnover rate in the human body .48However, the mortality plateau in humans may still exist. To ascertain this more rigorously, it is necessary to revisit validation principles and try to create a small, representative (the probability of being recorded should not depend on age—this principle was not taken into account in Gavrilova et al., 2017In summary: the cumulative weight of evidence presented in this study leads me to the conclusion that Jeanne Calment probably never became a centenarian, let alone a “double supercentenarian.” I conclude that the scientific community should cease to consider her as the validated oldest human until additional tests are done to reject the identity-switch hypothesis.The name Calment is probably one of the most frequently mentioned in books and articles on the topic of aging. The study of this story can be useful not only for gerontologists but also for scientists in general, as it shows the potential fragility of common knowledge.