Kaizer 2 Initiating β-blocker therapy, possibly combined with mexiletine depending on genotype, has yielded remarkable outcomes in reducing morbidity and mortality in LQTS patients, and the imperative of early diagnosis and management in this context cannot be overstated.3 LQTS is a major cause of cardiac arrest in children and young adults5 and is causally implicated in 8–10% of sudden infant deaths7 and unexplained stillbirth.8 This understanding presents pre-natal diagnosis of LQTS as a pivotal window of opportunity to proactively mitigate the risk of life-threatening arrhythmias and sudden cardiac events in affected neonates and children.In this nascent era of precision medicine, the unfolding tale of long QT syndrome (LQTS) shines as a luminous chapter and a prominent testament to what the future holds for genetic cardiovascular diseases.9 On the other hand, many—but not all10—experts believe that there is sufficient evidence to recommend routine ECG screening for LQTS in infancy.11 However, slight QT prolongation unrelated to LQTS might be seen in the early days of life, and there is a need for comprehensive assessment of ECG screening performance in this population.10Diagnosing LQTS in a foetus proves to be an arduous task as a reliable foetal ECG is not available, and magnetocardiography is only offered in a few specialized centres globally. While pre-implantation genetic testing or pre-natal genetic testing by way of chorionic villus sampling and amniocentesis can be performed to ascertain foetal LQTS genetic status, these procedures come with their own challenges and risks and many families elect to defer diagnosis to the post-natal period. In addition, these options are only available in babies where there is pre-existing knowledge of familial LQTS and a clear pathogenic variant.12 underscoring the need for better FHR-based prediction models.Another diagnostic avenue being explored is foetal heart rate (FHR), a biomarker widely used in multiple domains, that could raise suspicion for foetal LQTS. In early studies, FHR of <110 bpm at any gestational age (GA)—the obstetric definition of foetal bradycardia—was employed as an indicator warranting consideration of LQTS. It was later demonstrated in a Swedish LQT1 founder cohort that FHR ≤ 3rd percentile for GA offers a better performance, boasting a specificity of 97% while exhibiting sensitivity of 50% for detecting foetal LQTS,13 The authors used a multi-source identification to recruit an extensive cohort of 267 offspring from 164 LQTS families across eight countries, of which 60% inherited the familial LQTS-causative variant . Since some of the earlier data regarding the FHR/GA relationship relied on two Swedish LQT1 Founder populations,12 the authors included both the Swedish Founder LQT1 cases and other inherited LQT1 foetuses to assess potential differences. Their analysis, utilizing a linear mixed effects regression model, controlled for GA, maternal β-blocker use, and the interaction between β-blocker use and foetal LQT1 status, revealed no significant differences in the mean FHRs between the founder cohort and other LQT1 foetuses. Taking precision to the next level, the authors unveiled for the first time that in LQT1 foetuses, FHR is affected by both the KCNQ1 variant type and its functional impact on current density and β-adrenergic response, with lowest FHRs observed in foetuses hosting non-nonsense variants and variants leading to severe functional loss (i.e. current density ≤ 25% of wild type or impaired β-adrenergic response). Exposure to β-blockers in utero potentiated the intrinsically low FHRs in foetuses with LQTS without notable effect on those without LQTS, further supporting the safety profile of β-blocker treatment.In this issue of the journal, the Fetal LQTS Consortium presents an elegant study that informs the natural history of LQTS by expanding and refining the current understanding of FHR/GA dynamic in familial LQTS pregnancies, taking cohort, genotype, variant type/effect, and maternal β-blocker therapy into consideration.12 indicating the low reproducibility of this scheme. Finally, the authors went on develop a novel prediction model using GA as a continuous predictor to estimate the probability that a foetus is LQTS genotype-positive from a logistic regression model, assuming 50% prevalence. Compared to the FHR < 3rd percentile for GA model, this strategy demonstrated similar accuracy with a better balance between sensitivity (71.1%) and specificity (80.6%) for identifying LQT1 and LQT2 foetuses.Leveraging this extensive dataset, existing prediction schemes for foetal LQTS were put to the test. The obstetric definition of foetal bradycardia exhibited an accuracy of only 45%, failing to identify 92% of LQT1 and LQT2 cases. As expected, employing the previously proposed definition of foetal bradycardia as FHR < 3rd percentile for GA showed improved accuracy at 74%, with a decent sensitivity of 84.9%, albeit with an unacceptably low specificity of 59.2%. This contrasts with the performance of this approach in the Swedish Founder LQT1 cohort, exhibiting an inversely lower sensitivity but higher specificity,PRKAG2-associated glycogen storage disorders, Holt–Oram syndrome, short QT syndrome, tuberous sclerosis, and sick sinus syndrome associated with HCN4 or SCN5A mutations), and other systemic diseases.14 There are limited data on the expected heart rates of foetuses with the aforementioned bradycardic conditions, and this may significantly impact the specificity of this model in an unselected population. Discerning the specific type of bradyarrhythmia, which is critical to ruling out other more common causes of foetal bradycardia, would require evaluation of heart rate and AV relationship using more costly modalities to limit false positive screenings for LQTS. This, in turn, may limit the cost-effectiveness and the accessibility of such a screening. Further investigation can also help identify unique characteristics of foetuses with missed LQTS diagnoses using this model, thereby informing further refinements. For instance, it would be interesting to see whether missed cases could be effectively identified through pulsed Doppler-based measurements of left ventricular isovolumetric relaxation—another marker with diagnostic potential.15 Lastly, data remain limited on prediction of foetal LQT3 and de novo LQTS , which tend to present with frequent and lethal post-natal cardiac arrhythmias.16 Conducting longitudinal screening and implementation studies in large populations, combining the proposed FHR/GA threshold model with pre-/post-natal genetic testing in foetuses with complex and novel rhythms, can be pivotal in advancing diagnostic protocols to a higher level of accuracy for both inherited and de novo forms of LQTS.The authors of this study should be commended for compiling such a large and rare cohort of unique cases, delivering more insights into the intricate links between foetal bradycardia and congenital LQTS, and offering an improved screening model for prediction of foetal LQT1 and LQT2. A few limitations would benefit from further research and validation. While the investigation shed light on the effects of β-blocker therapy on FHR, a deeper exploration of maternal β-blocker therapy would benefit from investigating dose–response relationships, β-blocker type, treatment duration, and the impact of timing and compliance on FHR in the context of different LQTS genotypes. Interestingly, only 46% (83/179) of LQTS mothers in the study were treated with β-blockers during pregnancy, mostly due to five-fold lower treatment rates in the Swedish Founder cohort (16%) as compared to LQT1 and LQT2 cases from international centres (83%); this is explained by the historical nature of the Swedish Founder cohort, recruited in 1980s. This indicates the need for physician reassurance on β-blocker safety (and importance) during pregnancy in women with LQTS to increase treatment rates and improve outcomes. Additionally, while promising, the novel LQTS prediction model requires rigorous external validation and cross-validation to assess its applicability beyond the study cohort and ensure its reliability in real-world clinical practice for perinatal LQTS prediction. Additionally, the isolated detection of FHR using Doppler or cardiotocography, without evaluation of atrioventricular relationship with foetal echo/m-mode or magnetocardiograpy, may yield false positive concerns for foetal LQTS in the general population where other causes of foetal bradycardia are much more prevalent. Numerous other conditions have been associated with primary and secondary foetal bradycardia including complete and high-grade atrioventricular (AV) block caused by maternal autoantibodies, structural heart disease, or unknown aetiologies, foetal distress, non-LQTS monogenetic disorders (e.g.


GBA1 are a genetic risk factor for rapid eye movement sleep behavior disorder (RBD), which is a known early feature of synucleinopathies. Recently, a population-specific non-coding variant (rs3115534) was found to be associated with PD risk and earlier disease onset in individuals of African ancestry.Damaging coding variants in GBA1 rs3115534 PD risk variant is associated with RBD.To investigate whether the GBA1 rs3115534 risk variant status was imputed from previous genotyping for all. Symptoms of RBD were assessed with the RBD screening questionnaire (RBDSQ).We studied 709 persons with PD and 776 neurologically healthy controls from Nigeria. The GBA1 rs3115534 risk variant is associated with possible RBD in individuals of Nigerian origin , as well as after adjusting for PD status  suggesting that this variant may have the same downstream consequences as GBA1 coding variants.The non-coding GBA1 rs3115534 risk variant is associated with increased RBD symptomatology in Nigerians with PD. Further research is required to assess association with polysomnography-defined RBD.We show that the non-coding  GBA1) variants have been documented as the most significant genetic risk for Parkinson’s disease (PD) globally.1GBA1-associated PD has an earlier age of disease onset, faster motor progression and more frequent non-motor symptoms specifically cognitive decline/dementia, visual hallucinations, hyposmia, autonomic features and rapid eye movement (REM) sleep behavior disorder (RBD). 3Glucocerebrosidase , or in rare cases, multiple system atrophy (MSA) within 10–15 year.GBA1 locus, including p.Glu365Lys (rs2230288) and p.Asn409Ser (rs76763715), as linked to RBD risk in the European ancestry population .9 To the best of our knowledge, no research aimed at exploring genetic risk factors linked to RBD in the African population has been performed.The first genome-wide association study (GWAS) in RBD recently nominated two coding variants at the GBA1 locus as the major genetic contributor to PD in this population .10 This variant exerts substantial risk of PD as compared with common variation identified through GWAS and it was found to be present in approximately 55% of the PD cases of Nigerian ancestry and 39% of the PD cases from African admixed ancestry.10 Importantly, all current GBA1 knowledge, ranging from clinical to functional, is based on GBA1 coding variants and not much is known about the impact of non-coding variants on disease etiology. Here, we aimed to investigate the role of GBA1 rs3115534 and the risk of RBD in the African population.The first GWAS of PD in the African and African admixed populations has identified a novel population-specific intronic variant linked to PD risk and age at onset at the 11 Research ethics approval for the study was obtained from the institutional health research ethics committees, the National Health Research Ethics Committee (NHREC) in Nigeria and the University College London (UCL) Institutional Review Board. All participants provided written informed consent. Individuals with PD fulfilled the United Kingdom Parkinson’s Disease Society Brain Bank (UKPDSBB) criteria .12 Controls were healthy volunteers of Nigerian origin, with no known family history of PD, no clinically evident neurological condition, and matched for age.This is a secondary analysis of data obtained from study participants recruited by neurologists from the Nigeria Parkinson’s Disease Research (NPDR) network.13 It is a 10-item, patient self-rating instrument with short questions to be answered by either ‘yes’ or ‘no’. The validity of the questionnaire has been shown to perform with high sensitivity and reasonable specificity in the diagnosis of possible RBD (pRBD).14 Briefly, items 1 to 4 address the frequency and content of dreams and their relationship to nocturnal movements and behavior. Item 5 asks about self-injuries and injuries of the bed partner. Item 6 consists of four sub items assessing nocturnal motor behavior more specifically, e.g., questions about nocturnal vocalization, sudden limb movements, complex movements, or bedding items that fell down. Items 7 and 8 deal with nocturnal awakenings. Item 9 focuses on disturbed sleep in general and item 10 on the presence of any neurological disorder. The maximum total score of the RBDSQ is 13 points.The RBD screening questionnaire (RBDSQ), a specific questionnaire for RBD, was used to assess the most prominent clinical features of RBD.https://github.com/GP2code/Neuro_Booster_Array).10DNA extraction as previously described was done either from saliva samples collected using DNA Genotek saliva kits or from venous whole blood samples using standard protocols while genotyping was performed at the National Institutes of Health/Laboratory of Neurogenetics using the NeuroBooster Array (https://github.com/GP2code/). As previously reported, the GBA1 rs3115534 was reliably imputed in all samples.10Genetic data were QCed and imputed using standard GP2 pipelines and protocols  and disease duration as co-variates.All samples with complete data were included in the analyses. Two initial screening models were built to test the association between the quantitative RBD score and a dichotomized pRBD outcome with the outcome positive at RBD score six or higher. For the linear regression model testing RBD score as the outcome and the logistic regression model testing pRBD status as the outcome, female sex and enrollment age were used as covariates with the dosage of the GBA1 non-coding rs3115534 risk variant had an effect on pRBD status. Similar to previous observations, the GBA1 non-coding rs3115534 risk variant was overrepresented in the PD cases vs the neurologically healthy controls (MAF_cases = 0.34 vs MAF_controls = 0.21). A detailed description of demographics and clinical characteristics of the participants under study can be seen in GBA1 non-coding rs3115534-G allele and RBD status  as well as the continuous RBD score . The association between RBD score and GBA1 non-coding rs3115534-G allele persisted after adjusting for PD case status , showing that the association with GBA1 rs3115534-G and severity of pRBD is at least partially independent of PD case status in this dataset. The association with RBD score persists when data is subset to only cases . There are no associations between either RBD score or RBD status among neurologically normal controls (P > 0.05). For a detailed description of all statistical tests performed see In total we included 709 PD cases and 776 neurologically healthy controls of Nigerian descent and assessed whether the novel Identifying RBD symptoms in the general population becomes especially important when considering its robust connection to alpha-synucleinopathies including PD, DLB and MSA. Because RBD can manifest many years before the onset of obvious motor symptoms, exploring genetic risk factors for RBD are crucial for prompt detection and intervention or disease modification.GBA1 rs3115534 PD risk variant and an increased risk of RBD symptoms, and show that risk carriers are ~1.43 times more likely to have RBD symptoms than non-carriers. Our results are in line with those from previous PD studies, including other ethnicities, which clearly demonstrated similar relationships between GBA1 coding variants and the risk for RBD. 15 The fact that RBD serves as a potent predictor for the onset of dementia suggests that this variant may also be implicated in PD-dementia and DLB risk. In the current study, we were unable to explore whether this variant is associated with dementia risk in the African population given that we did not have access to dementia data. Additionally, the most commonly used cognitive screening tests such as MoCA and MMSE do not seem to be well suited for this population. 16 .Here we perform the first genetic assessment of RBD risk in the Nigerian population. Our results demonstrate the association between the novel GBA1 rs3115534 affects the rate of phenoconversion from RBD to PD and other synucleinopathies such as DLB and MSA.Our study has some limitations. First, questionnaires are subject to recall bias and inaccuracies. Validation of current scales are necessary to ensure reproducibility, reliability and accuracy as polysomnography-defined RBD data becomes available. In the present study, we were not able to confirm that RBD symptoms preceded PD diagnosis due to the lack of longitudinal data in this cohort. Further studies are needed to explore whether GBA1 rs3115534 is associated with symptoms of RBD defined by a commonly used screening questionnaire. Identifying individuals at risk of RBD represent an appealing target for clinical trials focused on preventing neurodegeneration in conditions such as PD and DLB. As neuroprotective treatments emerge in the future, an early identification of RBD will be key to treat these conditions.In summary, we show here that the non-coding Supplement 1https://1drv.ms/x/s!Aj-0YTgBXcwfhJ9orGkbfhHqocIZEASupplemental Table 1 – Nigeria Parkinson Disease Research (NPDR) network members Supplement 2https://docs.google.com/spreadsheets/d/1dum-N2jBjW8DUzRjg2z_VGgJ0WPsSOZ8/edit#gid=1769939610Supplemental Table 2 - Global Parkinson’s Genetics Program (GP2) members


RELN gene encoding reelin.In a recent study, Lopera and colleagues investigate a person with extreme resilience to autosomal-dominant familial Alzheimer’s disease, which they attribute to a rare variant in the  RELN gene encoding reelin.In a recent study, Lopera and colleagues investigate a person with extreme resilience to autosomal-dominant familial Alzheimer’s disease, which they attribute to a rare variant in the  Alzheimer’s disease (AD) is the most common cause of dementia, constituting a rapidly growing financial and personal burden as populations of the world age. The pathological hallmarks of AD include accumulation of amyloid-β in amyloid plaques, tau aggregation in neurofibrillary tangles, and brain atrophy resulting from neuron and synapse loss. Understanding factors that confer brain resilience to AD is at the forefront of research. Around 30% of older adults exhibit neuropathological features of AD without cognitive impairment,PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Lopera and team have characterized more than 1,200 people with the PSEN1 E280A mutation from the world’s largest autosomal-dominant AD kindred and used this powerful cohort to look for genetic factors that can delay the age of onset of dementia in the hopes of finding pathways that confer resilience.APOE3 “Christchurch” variant was discovered by this group in 2019. In a woman in the Columbian kindred, this variant was associated with an approximate 30-year delay in developing AD.Nature Medicine, the same group report another gene variant associated with AD resilience. The team described a Colombian brother and sister carrying a rare variant in the RELN gene (H3347R coined as RELN-COLBOS after the Columbia-Boston biomarker research study) who showed delayed onset of autosomal-dominant Alzheimer disease despite carrying the PSEN1 E280A mutation known to cause mild cognitive impairment (MCI) by around age 44 and AD type dementia by age 49.RELN-COLBOS variant was functionally independent and cognitively intact until age 67 when he was diagnosed with MCI. This corresponds to an approximate 20-year delay in symptom onset compared to the rest of the cohort. He progressed to mild dementia by age 72 and died at age 74.Based on the age of onset and genetic predisposition, AD is classified into sporadic (late-onset) AD or familial (early-onset) AD. Even though sporadic AD accounts for 95% of all cases, our understanding of AD has been greatly influenced by the study of families with AD-causing autosomal-dominant mutations in presenilin 1 , so although the RELN-COLBOS variant substantially delayed onset, it did not prevent typical neuropathological progression by end stage. Added to that, there were only three mice per group in the tau pathology study so the effects RELN-COLBOS on tau accumulation require further substantiation.One potential mechanism underlying the observed resistance to AD in the PSEN1 E280A mutation and RELN-COLBOS variant was less protected than her brother (delayed onset by 12 years). However, her earlier dementia onset could be attributed to various comorbidities including brain trauma, hypertension, and depression. Potential sexually dimorphic effects of RELN-COLBOS variant were further investigated in the new mouse model in this study. In the RELN-COLBOS mice, the males had a small but statistically significant increase in phosphorylated Dab1 levels compared to the females . Also, male human tau transgenic mice expressing the RELN-COLBOS allele had rescued abnormal limb-clasping phenotype. However, like the human study, this mouse study had caveats—small sample sizes and behavior only in male animals—so more work will be needed to confirm whether the RELN-COLBOS variant has meaningful sexually dimorphic effects on resilience to AD.This report also highlights potential sex differences in resilience with this variant. The woman with the same ,APOE4 and clusterin and synaptic pathology accumulation,RELN-COLBOS carriers who have amyloid pathology in their brains.One interesting potential mechanism linking reelin variants to AD resilience that was not explored in the Lopera paper is potential synaptic protection. The activation of Dab1 by reelin is known to influence synaptic function by modulating post-synaptic receptor activity.APOE2 (apolipoprotein E epsilon 2) reducing risk while APOE4 is associated with a large increase in risk of developing AD. Recent genetic studies in sporadic AD have reported multiple gene variants and pathways associated with resilience.RELN-DAB1 pathway in AD was published last year by Bracher-Smith and colleagues who found a novel genome-wide significant locus associated with AD mapping to DAB1 in APOE4 homozygots.Genetic contributors to sporadic AD  have also informed understanding about mechanisms of brain resilience and vulnerability to AD with variants like RELN-COLBOS variant. Together, this study and the Bracher-Smith paper provide strong genetic support for an important role of reelin-Dab1 signaling in resilience to AD pathology. Further exploration of mechanisms using the important mouse model developed by Lopera and team will hopefully lead to new avenues to prevent, delay, or treat Alzheimer’s disease by enhancing the brain’s brilliant powers of resilience.This study by Lopera and colleagues shows remarkable resilience to familial Alzheimer’s disease in two people with the newly described


DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor ( Accurate diagnosis is crucial for an optimal management of children with these diseases. During the last several years, remarkable advances in our understanding of the molecular underpinnings of these tumors have occurred as a result of comprehensive (epi-)genetic profiling and led to substantial progress in the classification and therapy of pediatric CNS tumors2. In addition, several novel and extremely rare tumor types have been identified using state-of-the-art molecular methods such as DNA methylation arrays and genetic profiling3. More recently, a wide range of different oncogenic gene fusions outside the mitogen-activated protein kinase (MAPK) pathway have shown to play an important role in driving tumorigenesis of pediatric CNS tumors6, some of them with the potential to provide novel therapeutic options.Pediatric neoplasms in the central nervous system (CNS) are extremely heterogeneous and diagnostically challenging tumors. According to data from the Central Brain Tumor Registry of the United States (CBTRUS), CNS tumors have become the leading cause of cancer-related death in childhood9. Such an approach is particularly valuable for the discovery and characterization of rare and novel tumor types that show a wide variety of clinicopathological appearances5.DNA methylation profiling of CNS tumors has been demonstrated to be a powerful tool for molecular tumor classification with the additional evaluation of copy number profiles being extremely useful for the identification of oncogenic gene fusionsHere, we describe a novel molecular CNS tumor type, primarily occurring in children, identified through unsupervised visualization of a large cohort of genome-wide DNA methylation profiling data, together with targeted next-generation DNA sequencing, and RNA transcriptome sequencing.n = 16), that did not match any known DNA methylation class. This group was comprised of tumors with a wide spectrum of original histological diagnoses, including predominantly high-grade glioma , with many tumors considered as not classifiable or with a descriptive diagnosis (Supplementary Table CIC::LEUTX fusion (see below) that have already been published were included into subsequent molecular profiling10. A more selected visualization (t-SNE) of DNA methylation patterns of tumors in this novel cluster compared with well-characterized reference samples  and leucine twenty homeobox (LEUTX) in all samples  as the 3′ partner, both located on chromosome 19q13.2  were fused in frame to exon 3 of LEUTX (NM_001143832.2), retaining the DNA-binding high-mobility group (HMG) box of CIC and the suggested 9aaTAD domain of LEUTX12. These findings are in line with the breakpoints detected in a previously reported pediatric embryonal tumor of the CNS13  exons 2–7 (NM_001284293.1) was observed, very similar to the CIC::LEUTX rearrangement  demonstrated gene fusions between 3.2 Fig. . In all X NM_00113832.2, rX NM_00113832.2, rn = 9) that revealed a morphologically heterogeneous group. Histologically, all reviewed tumors shared a high cellular density with most neoplasms showing slightly pleomorphic neoplastic cells often with remarkably condensed chromatin . All evaluated tumors had absent immunostaining for NeuN (n = 5). CD34 (n = 6) expression was restricted to the vessels  and the sex distribution was not significantly biased when considering the small number of patients. Clinical outcome data were available for only six patients. Median PFS was 13.5 months (range 6–16 months) with all of the patients experiencing a relapse during the follow-up period. Only one of the patients died of the disease during the follow-up period at 15 months after diagnosis. Together, these initial data suggest an intermediate malignancy grade  as an additional unifying feature.Here, we describe a previously uncharacterized group of rare, pediatric CNS tumors that was discovered through unsupervised visualization of genome-wide DNA methylation profiles. This novel group of tumors, epigenetically distinct from all known CNS neoplasms, shows recurrent gene fusions involving the transcriptional repressor CIC, a human homolog of capicua in Drosophila, acts as a transcriptional repressor with a DNA-binding high-mobility group (HMG) box domain that normally inhibits ETV1/4/5 expression and counteracts activation of genes downstream of receptor tyrosine kinase (RTK) signaling14. Aberrations in CIC have been identified in various types of cancer, with loss-of-function mutations frequently observed in oligodendroglioma16, leading to activation of downstream RTK signaling. Intriguingly, rearrangements involving CIC and the double homeobox 4 (DUX4) commonly found in high-grade round cell undifferentiated sarcoma18, have been shown to enhance the transcriptional activity of CIC downstream targets, including the member of the ETS family of transcription factors, such as ETV1/4/520. Consistent with that, an upregulation of members of the ETS transcription factor family have been reported in CIC-rearranged sarcoma  harboring oncogenic fusions between CIC and NUTM111.LEUTX is a member of the paired (PRD)-like homeobox gene family of transcription factors and is expressed almost exclusively in early embryos where it is thought to play a role during preimplantation development22. Although rearrangements between CIC and LEUTX have been reported recently23, the exact role in tumorigenesis of LEUTX remains poorly defined. However, since LEUTX and DUX4 belong to the same class of paired (PRD)-like homeobox genes an oncogenic mechanism very similar to that of the CIC::DUX4 chimeric transcript seems very likely. Downstream functional consequences of this novel fusion protein and potential therapeutic implications need to be further investigated.In contrast, CIC::LEUTX-fused CNS tumor samples10, that were now included into the present series, also clustered with this novel epigenetic group, it seems still too early at this point to say whether this alteration is specific enough to use it as an “essential diagnostic criterion” according to the recently published fifth edition of the World Health Organization (WHO) classification of CNS tumors24. The same considerations also apply to the histomorphological and immunohistochemical findings that demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin and CD99. High expression of CD44, EMP3 and VIM may be useful to distinguish the tumors from CIC-rearranged sarcoma. However, this will need further validation. Thus, we suggest the specific epigenetic signature by DNA methylation profiling as so far the only method to accurately identify these tumors. Although there are strong arguments for consideration of these tumors as a specific type of glioma , the lack of a clear indication of a particular lineage and the small number of cases with sufficient material for a comprehensive histopathological evaluation suggest the provisionally use of the term “neuroepithelial” to describe these neoplasms. CIC fusion-positive neuroepithelial tumors may be considered for inclusion into upcoming classifications of CNS tumors to help diagnose these tumors more precisely. This would be either possible as a provisional tumor type in the category “gliomas, glioneuronal tumors, and neuronal tumors” or within a new “molecular” category of tumors with currently unclear lineage. In addition, more clinical workup in terms of patient outcome data is urgently needed to characterize these neoplasms in more detail. Our initial follow-up data suggest an intermediate malignancy grade with all tumors showing early relapses. However, it seems like maybe whatever second-line therapies were applied are more effective, with only one death and one longer survivor despite initial quick relapse.Although both previously described CIC. These findings also imply a growing biological understanding of the genomics underlying rare pediatric CNS tumors. Given their broad morphological spectrum and recurrent gene fusions involving CIC, we suggest the term ‘high-grade neuroepithelial tumor CIC fusion-positive’ to describe this novel tumor type.In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusion involving CIC::LEUTX fusion that have already been published were included into subsequent molecular profiling10. Furthermore, DNA methylation array data of numerous well-characterized reference samples included in the most recent classifier version (v12.5) representing CNS tumors were used for comparative analyses. Detailed descriptions of the reference DNA methylation classes are outlined under (https://www.molecularneuropathology.org). This study is covered by the ethical approval of the University of Heidelberg medical faculty  which confirmed that written consent specific for this study can be waived since it is based on archival material that was remaining after regular diagnostic workup. Clinical details of the patients are listed in Supplementary Table Patient tumor samples and retrospective clinical information were provided by multiple national and international collaborating centers and collected at the Department of Neuropathology of the University Hospital Heidelberg  and German Cancer Research Center . Sample selection was based on unsupervised visualization (t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP)) of genome-wide DNA methylation array data that revealed a molecularly distinct group of tumors forming a cluster separate from all established tumor types. In addition, two further CNS tumor samples harboring a n = 9), a histopathological review was retrospectively performed to investigate the morphological and immunohistochemical features of tumors within the novel group. Due to the aspect of a multicenter cohort, the availability of tissue was restricted for some of the cases. Hematoxylin and eosin (H&E) and immunohistochemical staining was either performed at the Department of Neuropathology of the University Hospital Heidelberg or received from the respective collaborator institutes. Immunohistochemical staining was performed on a Ventana BenchMark ULTRA Immunostainer using the ultraView Universal DAB Detection Kit . Antibodies were directed against: glial fibrillary acid protein , oligodendrocyte lineage transcription factor 2 , MAP2 , Synaptophysin , NeuN , CD56 , CD34 , CD99 , Ki-67 , CD44 , vimentin  and EMP3 .For a subset of samples . Genomic DNA was extracted from fresh frozen or formalin-fixed and paraffin-embedded (FFPE) tissue samples with the Maxwell 16 Tissue DNA Purification Kit or the Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega), according to the manufacturer’s instructions. RNA was extracted from FFPE tissue samples by following the Maxwell 16 LEV RNA FFPE Kit protocol (Promega). Nucleic acid concentrations were determined using the Invitrogen Qubit dsDNA BR Assay Kit  on a FLUOstar Omega Microplate Reader . For single cases, DNA was extracted using either the AllPrep DNA/RNA FFPE Kit , QIAamp DNA Micro Kit (Qiagen) or FormaPure Kit , according to the manufacturer’s instructions.9. Raw data were generated at the Department of Neuropathology of the University Hospital Heidelberg, the Genomics and Proteomics Core Facility of the DKFZ or at respective international collaborator institutes, using both fresh–frozen and FFPE tissue samples. All computational analyses were performed in R version 4.6.1 . Copy number variation analysis from 450k and EPIC methylation array data was performed using the conumee Bioconductor package version 1.12.0. Summary copy number profiles to display rates of copy number gains and losses per DNA methylation class were generated using an in-house R script (https://github.com/dstichel/CNsummaryplots). Focal copy number alterations were called based on manual review of the log2 ratio plots for each sample. Raw signal intensities were obtained from IDAT-files using the minfi Bioconductor package version 1.21.426. Illumina EPIC and 450k samples were merged to a combined data set by selecting the intersection of probes present on both arrays . Each sample was individually normalized by performing a background correction (shifting of the 5% percentile of negative control probe intensities to 0) and a dye-bias correction  for both color channels. Subsequently, a correction for the array type (450k/EPIC) was performed by fitting univariable, linear models to the log2-transformed intensity values . The methylated and unmethylated signals were corrected individually. Beta-values were calculated from the retransformed intensities using an offset of 100 (as recommended by Illumina). All samples were checked for duplicates by pairwise correlation of the genotyping probes on the 450k/EPIC array. To perform unsupervised nonlinear dimension reduction, the remaining probes after standard filtering9 were used to calculate the 1-variance weighted Pearson correlation between samples. The resulting distance matrix was used as input for t-SNE analysis (Rtsne package version 0.13). The following non-default parameters were applied: is_distance = T, theta = 0, pca = F, max_iter = 10,000, perplexity = 30. Estimation of differential methylated positions (DMP) was done in R by using the function “dmpFinder” from the minfi package (v1.43). The Illumina EPIC platform was used to annotate CpGs by their position in the genome and associated genes. The tests were carried out on the M-values of all promoter-associated genes as well as the top 100k CpGs according to mean average deviance. CpGs with an FDR q-value smaller than 0.05 were considered as significant differential methylated. Volcano plots of the DMPs were generated by the R-package ggplot2 (v3.3.6). CpGs are distributed according to their −log10 Q values and fold change (intersect). Further significant different methylated CpGs are depicted in red while the associated gene top100 DMP is shown.Genome-wide DNA methylation profiling of all samples was performed using the Infinium MethylationEPIC (EPIC) BeadChip  or Infinium HumanMethylation450 (450k) BeadChip array (Illumina) according to the manufacturer’s instructions and as previously describedn = 9), DNA sequencing using a customized enrichment/hybrid-capture-based next-generation sequencing (NGS) gene panel were performed on a NextSeq 500 or NovaSeq 6000 instrument (Illumina) at the Department of Neuropathology of the University Hospital Heidelberg 27. The NGS panel comprised the entire coding  and selected intronic and promoter regions of 170 genes of particular relevance in CNS tumors, and was designed to detect single nucleotide variants (SNV), small insertions/deletions (InDel), exonic rearrangements, and recurrent fusion events. Paired-end sequencing was applied to increase the detection sensitivity of duplicates and possible gene fusions. Sequence reads were mapped to the reference human genome build GRCh37 (hg19) using the Burrows–Wheeler aligner (BWA).For a subset of samples with DNA available (n = 8) was performed as previously described28. In brief, RNA sequencing libraries were prepared using the TruSeq RNA Library Prep for Enrichment kit (Illumina) and paired-end reads were sequenced on a NextSeq 500 or NovaSeq 6000 instrument (Illumina). After adapter trimming, reads were aligned to the human genome (GRCh37) with the STAR aligner29 and counted using RSEM30. Fastq files from transcriptome sequencing were used for de novo annotation of fusion transcripts using the Arriba (v1.2.0) algorithm31 with standard parameters, which removes recurrent alignment artifacts, transcript variants also observed in normal tissue, reads with low sequence complexity, and events with short anchors or breakpoints in close proximity or a low number of supporting reads relative to the overall number of predicted events in a gene.RNA sequencing for the purpose of gene fusion detection of samples for which RNA of sufficient quality and quantity was available . Data on survival could be retrospectively retrieved for six patients. Overall survival (OS) and progression-free survival (PFS) probabilities were displayed using the Kaplan–Meier method.Further information on research design is available in the Supplementary InformationSupplementary TablesReporting Summary


The strong intertwinement of the short-range polarization switching and long-range ionic movement in conjunction with the van der Waals layered structure gives rise to unique morphological and polarization evolutions under repetitive electric cycles. With the help of concerted chemical, structural, lattice vibrational and dielectric analyses, we unravel the critical role of the synergy of ionic migration and surface oxidation on the anomalous polarization enhancement and the eventual polarization degradation. This work provides a general insight into the polarization fatigue characteristics in ionically-active van der Waals ferroelectrics and delivers potential solutions for the realization of fatigue-free capacitors.Recent progress in two-dimensional ferroelectrics greatly expands the versatility and tunability in van der Waals heterostructure based electronics. However, the switching endurance issue that widely plagues conventional ferroelectrics in practical applications is hitherto unexplored for van der Waals layered ferroelectrics. Herein, we report the observation of unusual polarization fatigue behaviors in van der Waals layered CuInP 2S6, due to a synergic effect of ionic conduction and surface oxidation.The authors find unconventional fatigue characteristics, involving dramatic morphological changes and anomalous polarization enhancement in van der Waals layered ferroelectric CuInP Ever since the first report of ferroelectric fatigue phenomenon in BaTiO3 by Merz and Anderson3, numerous efforts were devoted to understanding its microscopic mechanisms6, and thereupon exploring potential remedies9. Most existing models were established on oxide ferroelectrics, in which near-electrode charge injections and oxygen vacancy redistributions are extensively involved13. Recent research fevor in two-dimensional (2D) materials rejuvenated considerable interest in van der Waals (vdW) layered ferroelectrics, primarily composed of chalcogenides and halides18. The reduced lattice dimensionality and unique stacking degree of freedom bring about unconventional properties and physics in 2D vdW ferroelectrics, such as negative longitudinal piezoelectricity and sliding/moiré ferroelectricity20. Additionally, lower electronegativities of the chalcogens and halogens, compared to oxygen, usually result in weaker chemical bonds and potentially higher ionic conductivity in vdW layered ferroelectrics22. This results in anomalous switching characteristics, including multi-well polarization states25, ionic-defect-coupled polarization switching27, and potentially quantum polarization switching29. The successful integration of 2D vdW ferroelectrics into innovative device paradigm pivots on the reliable and enduring switching of ferroelectric polarization. However, polarization fatigue characteristics in vdW layered ferroelectrics are hitherto unreported, and the role of finite ionic conduction in the fatigue process remains unknown. In a bid to throw light into these issues, we systematically investigated the polarization degradation behaviors of a prototypical vdW layered ferroelectric ion conductor, CuInP2S6 (CIPS), under repetitive electrical stress. Through field strength-, cycling frequency-, and temperature-dependent tests, we discovered unconventional fatigue characteristics of CIPS capacitors, such as protrusion formation, strong morphological rippling, and unconventional enhancement of the polarization. These unique features are closely linked to the layered structure and sizable Cu+ ion activities of CIPS, as revealed by our correlative and comparative chemical, structural and local piezoelectric response measurements. This work delivers in-depth understanding of the microscopic mechanism of ferroelectric degradation process in vdW layered ferroelectrics, enabling future quests for fatigue-free vdW ferroelectrics.Ferroelectric fatigue, manifested by the loss of switchable polarization under repetitive bipolar electric cycles, is a long-standing issue that critically impedes the practical applications of ferroelectric materials in reliable logic and memory devicesC) of ≈25–30 kV cm−1 (See Table C), the hysteresis loop shows a progressive increase of the switched polarization with cumulative cycles , the switched polarization value also increases initially, but drops after certain cycles, accompanied by the enhancement of the leakage current as indicated by the vertical blown-up of the loops. Meanwhile, all samples show some increments in the coercive field. To accurately determine the switchable remanent polarization (ΔPr), positive-up-negative-down (PUND) method was employed to subtract the contributions from non-remanent polarization and leakage charges. As shown in Fig. 6 cycles. Such polarization enhancement is further verified by standard and remanent hysteresis loops as well as PUND measurements . Lastly, surface ripples begin to form in the surrounding area, resulting in Newton’s rings due to the delamination of the vdW layers (yellow arrow). With incremental cycles, the aforementioned three features grow larger and denser. Besides, there is a general trend that the surface ripples produced by low-field stressing are smaller and denser, while high field promotes large-scale layer delamination  was performed to reveal possible changes of surface chemistry associated with the morphology modifications. By a direct comparison of elemental mapping between pristine and fatigued capacitors, large amount of oxygen is found to accumulate at the electrode edge of the fatigued sample Fig. . EDS spe8 rings at the electrode edge  was used to probe the conductivity changes caused by the potential oxidation in the rippled area and electrode edge. As the measurement on top of the Au electrode would result in huge currents that might damage the tip, we chose another dark region  was conducted on both pristine and fatigued capacitors. The sample details are shown in Fig. SNext, we traced the depth profiles for each element from an etching depth of 25 nm onwards as shown in Fig. p, In 3d and S 2p  that can be assigned to the rocking motions of the P2S6 unit, P2 (~240 cm−1) and P3 (~305 cm−1) modes involving symmetric and asymmetric bending of the PS3 bonds respectively, and P4 (~375 cm−1) mode driven by the symmetric P-P dimer stretching  may provide more clues to the structural changes. To this end, the incident X-ray was focused to a beam size of 50 μm to detect the structural changes due to fatigue test within an electrode. As shown in Fig. S33, and consequently opposite phase signal23. With only the PFM results, it is not possible for us to distinguish these two scenarios. However, our micro-XRD  of ~315 K, whereas the rise of the low-frequency imaginary part above ~ 250–260 K corresponds to the onset of thermally-activated ionic conduction33. With increasing fatigue cycles, additional contribution starts to appear and gradually intensifies in the real dielectric permittivity as indicated by the arrows. Meanwhile, the dielectric peak at TC is strongly suppressed. This behavior is somewhat analogous to the case of relaxor ferroelectrics, which demonstrate similar frequency-dependent dielectric enhancement due to the release of the frozen electric dipoles34. This finding is consistent with the inhomogeneous piezoresponse and pinned domains revealed in PFM images 36 and/or electric polarization gradient (converse flexoelectric effect)39 may strongly disturb the crystal lattice order perpendicular to the vdW layers due to random Cu+ hopping motions, which is confirmed by the cross-sectional Raman and μXRD results. Dipole-glass like relaxation process and enhanced DC ionic conductivity as derived from the dielectric spectra further corroborates the microscopic ionic dynamics during the fatigue process.By bringing together the aforementioned results and evidence, we can conclude that the polarization fatigue characteristics of CIPS is closely related to its ionically mobile nature at room temperature. The displacive instability of the monovalent CuXPS Fig. . This ef41.The anomalous morphological evolution, such as bubble-like protrusion and surface wrinkling, is closely related to the cooperation of oxidation and switching-induced piezostrain during electric cycling. Our time-lapse video clearly demonstrated that the darkening of the electrode edge (an indication of the oxidation) and the protrusion outgrowth initiate concurrently  were also performed to check the oxidation effect  for a reaction time of several days. High-quality and regular-shape single crystals were carefully picked out from the raw reaction products by optical microscopy inspection. The composition stoichiometry and phase purity were checked by energy dispersive X-ray spectroscopy (EDS) and Raman spectroscopy. To fabricate the parallel-plate ferroelectric capacitors, the bottom face of the crystals first coated with Au electrode by sputtering. The top electrodes were defined by shadow masks to form circular or square pads with a dimension of 200–300 μm. The detailed samples/devices information and involved experiments are included in the Supplementary Information . The fatigue tests were performed using square bipolar pulse with different amplitudes and frequencies, and the switchable remanent polarization was measured using PUND method. Complex dielectric data were collected by a commercial LCR meter . Temperature dependent electrical measurements were carried out in a cryogenic micromanipulator probe station equipped with a heating stage .The EDS spectra and spatial mapping were collected by the energy dispersive spectrometer  equipped on a commercial field-emission scanning electron microscope . A 20 kV accelerating voltage was used to ensure the excitation of the sufficient X-ray lines for the elements. Standardless quantitative analysis of the sample composition is achieved by calculating the ratio of peak intensities to determine the relative abundance of the element. For the elemental mapping, a long acquisition time of at least 5 min were used to ensure the measurement accuracy and reproducibility.−1, producing a spectral resolution better than 1 cm−1 . In spatial Raman mapping, the fiber-coupled laser was focused by a long working distance objective  into a Gaussian beam with a diameter less than 1 μm. The raw data was processed and analyzed by LabSpec6 software.Raman spectra were measured by a confocal Raman microscope with a 532 nm laser and a grating of 2400 gr mmα source (1486.6 eV) and a beam diameter of 50 µm. Depth profiling was performed by etching the surface using Ar+ ion beam for different durations. The nominal etching thickness is estimated based on the etching rate of SiO2.Depth-profiling XPS was carried out on a micro-focus X-ray photoelectron spectrometer  with monochromated Al Kβ radiation. A pinhole collimator was used to focus the Cu Kα beam down to a diameter of 50 µm onto the sample surface.μXRD was performed on a commercial X-ray diffractometer  with an area detector. A Gobel mirror parallel optics system was used to remove the Cu KAll scanning probe microscopic imaging were performed on a commercial atomic force microscopy . The ambient PFM studies were carried out using both dual AC resonance tracking (DART) method with a Ti/Pt-coated tip  and under non-resonant mode with a hard Pt/Ir-coated tip . The inverse optical lever sensitivity (InvOLS) was calibrated using combined Sader method and thermal noise method to obtain quantitative piezoelectric displacements in PFM images. For DART-PFM, the PFM data are fitted by simple harmonic oscillator (SHO) model to obtain effective piezoelectric response off resonance. SKPM was carried out using a two-path method, where the topographic image was acquired in the first path and in the second path, surface potential can be extracted by nulling the electrostatically induced oscillation of the cantilever. In CAFM, a DC bias of 5 V was applied between the probe and bottom electrode to measure the spatial distribution of the current. In both SKPM and CAFM, the Ti/Pt-coated tip (OMCL-AC240TM) was used.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


We previously identified 16,772 colorectal cancer-associated hypermethylated DNA regions that were also detectable in precancerous colorectal lesions (preCRCs) and unrelated to normal mucosal aging. We have now conducted a study to validate 990 of these differentially methylated DNA regions (DMRs) in a new series of preCRCs.We used targeted bisulfite sequencing to validate these 990 potential biomarkers in 59 preCRC tissue samples , each with a patient-matched normal mucosal sample. Based on differential DNA methylation tests, a panel of candidate DMRs was chosen on a subset of our cohort and then validated on the remaining part of our cohort and two publicly available datasets with respect to their stratifying potential between preCRCs and normal mucosa.Strong statistical significance for the difference in methylation levels was observed across the full set of 990 investigated DMRs. From these, a selected candidate panel of 30 DMRs correctly identified 58/59 tumors (area under the receiver operating curve: 0.998).These validated DNA hypermethylation markers can be exploited to develop more accurate noninvasive colorectal tumor screening assays.The online version contains supplementary material available at 10.1186/s12885-023-11487-w. The past 40 years have witnessed considerable reductions in both the mortality and incidence of colorectal cancer (CRC). These trends are largely attributable to the increasing use of population-based colonoscopy screening, which allows the identification and removal of early-stage CRCs as well as neoplastic lesions regarded as precancerous (preCRCs), such as conventional adenomas (cADNs) and sessile serrated lesions (SSLs) . The preDue to its high diagnostic efficacy, colonoscopy is now the primary tool for CRC screening, but it has several well-known shortcomings. Studies based on same-day tandem colonoscopy findings indicate that endoscopists overlook around 26% of adenomas , and misInterest is growing in the development of DNA methylation-based markers for CRC screening. Compared with genetic mutations, methylation abnormalities are far more frequent in colorectal tumor genomes of all stages . AlthougOur group recently demonstrated that the abnormal methylation phenotype of specific DNA regions described in CRCs  is alreatumorigenesis-specific” [Some regions that were hypermethylated in preCRCs, however, also displayed mildly increased methylation in NM samples from older (vs. younger) tumor-free women. We therefore conducted a follow-up study to explore the overlap between the ~ 20,000 tumor-associated DMRs and genomic regions that appeared to undergo methylation as an effect of aging (aging-associated DMRs) . The vaspecific” . In the DMR validation was performed on a series of colorectal tissue samples collected during colonoscopy. 59 preCRCs , each with a patient-matched NM sample collected > 2 cm from the tumor were included in the study ; and 20 other DMRs we had classified as “aging-specific” (based on their absence in cADNs and SSLs and the presence in NM samples from tumor-free women aged 40–70 but not in NM samples from tumor-free women aged < 40)  ; forty-sTarget enrichment of the selected genomic regions was achieved by solution-based hybrid capture performed with a synthetic library ultimately consisting of 96,456 RNA probes . Capture oligonucleotides  were designed to target approximately 0.5 Mb of the genome and contained 44,177 CpG sites derived from the Hg19 genome build. For each DMR, we designed nine probes for the detection the following methylation patterns: (1) genomic reference (genomic sequence before bisulfite conversion); (2) all CpG sites methylated in both cis and trans sequences; (3) no methylation in either cis or trans sequences, (4) a random half of the CpG sites methylated in both cis and trans sequences, and (5) the other half of the CpG sites methylated for both cis and trans sequences. . DNA concentrations were quantified with a Qubit fluorometer , and quality was assessed with a NanoDrop spectrophotometer . Genomic DNA (100ng) was bisulfite-converted with the EZ-DNA Methylation-Gold kit  prior to preparation of the library with the Accel-NGS Methyl-Seq DNA Library Kit . Equimolar amounts of eight indexed libraries were pooled for hybridization with customized RNA baits, which was performed twice at 63 °C for 16 to 24 h. AMPure Xp beads  were used to isolate biotinylated DNA, and the bead-bound enriched libraries were amplified with KAPA HiFi HotStart Ready Mix  and adaptor-specific primers. The concentrations of the enriched libraries were measured with a Qubit fluorometer. Library size was determined with a Tapestation  for subsequent library titration and next generation sequencing. Each pooled library was sequenced on an Illumina NovaSeq 6000 instrument  using one lane to generate an average of 25 million (2 × 150 bp) reads per sample.training set consisting of 70 samples  and a test set comprising 48 samples . Per-CpG differential methylation (DM) tests were conducted in a per-patient paired design using the limma R package. Per-region p-values were obtained by aggregating all per-CpG p-values using the Simes Method [top 30 candidate biomarker panel) was then further validated in the test set.The patient sample database was split by batch  to obtain a s Method . A candiPaired-end sequencing reads were trimmed using Trim Galore (0.6.5) software  and subsFor the selection of a top 30 DMRs, we split the patient population (n = 59) by batch  in a stratified manner, considering the ratio of SSL and cADN samples per-batch using the function stratified from the splitstackshape package .The training dataset was subjected to differential methylation (DM) testing to select the regions characterized by consistent methylation differences between preCRCsand NM. The DM-strategy chosen was based on functionalities of the limma R package –19, whicTo test the stratifying potential of the top 30 candidate biomarker DMRs, we calculated the mean methylation level of the regions across the panel for each sample. In addition to the test set described above, we used two published datasets, each comprising a sufficiently high number of colorectal tissue samples and NM samples. The first (referred to hereafter as the GSE131013 dataset)  was geneFor the GSE131013 and GSE48684 datasets from the literature, we considered the Beta-values of all CpGs overlapping the region coordinates of this study (as downloaded May 2022) for the subsequent tasks. Receiver operating characteristics (ROC) curves were obtained, and the area under the curve (AUC) for each dataset was calculated using the auc function from the pROC package . Tissue The 100 randomly selected sets of 30 DMRs each shown in the ROC plot were taken from the 990 candidate biomarker DMRs, and the mean methylation level was used to calculate the ROC curves.The F1-score was applied using the cutpointr  R packagAll MDS plots were generated using asin-transformed per-region methylation levels. Subsets of the regions  were used to determine the MDS dimensions with the plotMDS function .The heatmaps showing methylation differences reflect the difference between the methylation level per region  as obtained by the getMeth function of the bsseq library.Methods) and reduced the amount of DNA needed per sample from 1000ng to 100ng—a substantial advantage for future studies, especially those conducted on low-volume tissue samples. We comparatively analyzed two preCRC lesions and their matched NM samples with the custom myBaits protocol and the previously used SeqCap Epi CpGiant protocol. The strong correlation (r = 0.97) observed between methylation values obtained with the two methods : As shown in Fig. VIM, NDRG4/BMP3, and SEPT9; see Discussion).We then validated the top 30 candidate biomarker DMRs using our own test dataset and two published GSE131013  and GSE4KRAS or BRAF, which are identified in only ~ 40% and ~ 10% of colorectal neoplasms, respectively).With this validation study conducted on a new prospectively collected series of colorectal tissues, we have demonstrated that numerous hypermethylated DMRs are robust indicators of the precancerous stages of colorectal tumorigenesis. Because these same DNA regions are also highly hypermethylated in CRC , 10, theVIM (ColoSure assay) or NDRG4 and BMP3 (Cologuard assay) [SEPT9 [Pre-colonoscopic screening assays based on the methylation analysis of fecal DNA might thus complement (or even replace) the immunochemical stool tests widely used in today’s clinics for the identification of individuals who might need colonoscopy. Indeed, some currently available fecal DNA assays include one or two methylation markers, such as hypermethylation of d assay) , and the) [SEPT9 . At pres) [SEPT9 , 30. OurResults) owing to their very low levels of DNA hypermethylation. A more promising strategy for enhancing assay sensitivity might therefore involve adding methylation-based markers to panels containing immunochemical and genetic markers, an approach similar to the one used in the Cologuard assay.Our findings also indicate that a screening assay based exclusively on DNA hypermethylation markers is likely to miss a few preCRCs (~ 2% in our validation study, see We have validated an extensive set of DMRs as markers capable of accurately predicting the presence of a colorectal tumor regardless of the patient’s age. The list of DMRs validated in this study can thus be productively mined to establish promising biomarker sets for fecal DNA based screening assays.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Crop diseases cause enormous yield losses and threaten global food security. Deployment of resistant cultivars can effectively control the disease and to minimize crop losses. However, high level of genetic immunity to disease was often accompanied by an undesired reduction in crop growth and yield. Recently, literatures have been rapidly emerged in understanding the mechanism of disease resistance and development genes in crop plants. To determine how and why the costs and the likely benefit of resistance genes caused in crop varieties, we re-summarized the present knowledge about the crosstalk between plant development and disease resistance caused by those genes that function as plasma membrane residents, MAPK cassette, nuclear envelope (NE) channels components and pleiotropic regulators. Considering the growth-defense tradeoffs on the basis of current advances, finally, we try to understand and suggest that a reasonable balancing strategies based on the interplay between immunity with growth should be considered to enhance immunity capacity without yield penalty in future crop breeding. Triticum aestivum) streak mosaic virus R gene Wsm1 (transferred from Thinopyrum intermedium) and stem rust R gene Sr26 from Agropyron elongatum are associated with a mean yield reduction of 21% and 9%, respectively (Brown Hordeum vulgare) mlo has a 5.2% TGW (thousand-grain-weight) reduce and 4.2% yield penalty  and wheat–Th1Js(1D) (Thinopyrum ponticum) confer resistance to powdery mildew and stripe rust in wheat but causing a significant spike length shorten and tiller inhibition  occurs during development, defense response or when exposed to adverse conditions. It is no doubt that CD, especially in programmed cell death (PCD) and/or regulated cell death (RCD), is happened at a fitness cost of energy, which sometimes is associated with yield costs’ (Nejat and Mantri Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov) though interacting with GhWAK7A  by the activated MEKK1–MKK1/2–MPK4 module after bacterial pathogen attack  encodes the mitogen-activated protein kinase phosphatase OsMKP1, and directly interacts with and inactivates the mitogen-activated protein kinase OsMPK6 to regulate the role of OsMKKK10-OsMKK4-OsMPK6 cascade in determining panicle architecture and grain size  cascades that play essential roles in signal transduction and amplification for many distinct cellular responses  enhancing resistance to powdery mildew in wheat , also known as SUN–KASH (INM Sad1/UNC-84 and Klarsicht/ANC-1/Syne-1 homology) nuclear envelope (NE) bridges are responsible for connecting the nucleoplasm and cytoplasm in eukaryotes (Graumann and Evans Brassinazole-resistant (BZR), a key bHLH-type transcription factor (TF) in BR signaling, plays dual roles in plant development and defense. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight -mediated rice blast resistance  resistance tests using OsNPR1 knockdown and overexpressing rice lines demonstrated the essential role of OsNPR1 in benzothiadiazole (BTH)-induced blast resistance  encodes a transcript factor Taken these aforementioned results together, it is not difficult to infer that pleiotropic functional genes, including TFs and other functional genes, could also affect the balance of plant immunity and development.PigmR confers broad-spectrum resistance in rice with yield penalty, while PigmS is associated with a relative increase in grain yield but attenuates resistance. This means that PigmS and PigmR could be used to balance high disease resistance and yield through epigenetic regulation of paired antagonistic in hybrid varieties as a tool to develop elite crop varieties , but to select for reasonable moderate resistance lines with excellent agronomy performances. It’s time to put the pieces back together and to study how to balance responses to disease and crop yield as an integrated part of the crop plant biology.


When people enjoy salty snacks at a bar, their thirst can be quenched with an extra drink. The plant in the window, however, is unable to place an order. To cope with osmotic stress induced by drought conditions, plants rapidly increase their intracellular calcium and produce abscisic acid, a plant hormone involved in many stress responses. These signaling processes cause plants to close their stomata to prevent water loss and shape their root architecture to find more water—strategies needed for short- and long-term osmotic stress .Wei Fan and colleagues  deepens our understanding of the mechanisms underlying plant osmotic stress responses. Plant stress-induced signaling is often linked to increases in intracellular Ca2+ and the activation of calcium-dependent protein kinases (CPKs). CPK4 and CPK11 are activated under osmotic stress and promote abscisic acid signaling , showing that they are hypersensitive to the effects of osmotic stress on root elongation, whereas pub44 mutants showed the opposite phenotype (longer roots under osmotic stress). The pub44 phenotype was not apparent in cpk4 cpk11 pub44 triple mutants. Therefore, PUB44 negatively affects osmotic stress–induced root growth inhibition, dependent on the presence of CPKs. However, these observations raise the question of how osmotic stress leads to an increase in CPK4 protein levels in wild-type plants. Interestingly, a kinase-dead variant of CPK4 was preferentially degraded by PUB44. The authors hypothesize that activated CPK4 is resistant to PUB44-mediated degradation due to a conformational change following CPK4 activation that shields the lysine residues targeted by PUB44 for ubiquitination. Thus, osmotic stress induces CPK4 activation and makes the protein resistant to PUB44-mediated degradation. Future structural biology work would shed light on this mechanism.The authors found that regulation of CPK4 stability by PUB44 plays a role in osmotic stress responses. Plants that overexpressed This work identifies the CPK4/PUB44 signaling module and how it allows plants to rapidly adjust CPK4 protein levels in response to osmotic stress. The authors demonstrate that PUB44 is capable of targeting CPK4 for degradation and that osmotic stress could shield CPK4 from interacting with PUB44 to promote downstream signaling.


The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions  to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins  and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor β (TGFβ), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV ( Olfactory dysfunction (OD) manifests itself primarily in the partial (hyposmia) or total (anosmia) inability to smell odors, including the diminished perception of aromas in food. The prevalence of OD in the general adult population is about 20% in Europe and the United States . This coSniffing an odorant induces a nasal airflow, bringing odorants toward the olfactory epithelium located at the roof of the nose . The olfA particularity of olfactory sensory neurons (OSNs) is their life-long, perpetual renewal . The actOlfactory axons are particularly susceptible to injury, especially after head trauma, one of the most frequent causes of smell loss . For insThere is currently no cure for this debilitating and often permanent condition. To restore smell function after a traumatic brain injury, it is necessary to (1) create an implantable scaffold allowing axonal regrowth through the scar tissues on the cribriform plate and (2) incorporate growth factors and extracellular proteins into it. However, there are several limitations and knowledge gaps that prevent us from readily determining the ideal growth factors or extracellular proteins, which motivated the design of the present study.First, many studies about in vitro regrowth of olfactory axons use fetal bovine serum , containSecond, regarding the type of olfactory tissues used to test these conditions, many studies utilize embryonic olfactory epithelium  carryingFinally, many methods exist to measure axonal length but none of them are “high-throughput” enough to be used in our study. For example, Neuron J requires the investigators to manually trace each single axon, which is highly time-consuming.The aim of this study is to (1) create an automated quantification tool to measure axonal length and (2) determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model.To identify the growth factors and extracellular proteins that are essential for olfactory neuron axonal elongation, we developed an automated quantification tool. We developed a MATLAB framework, which operates on pictures of β-III-tubulin stained OE explants A. First,We compared our automated quantification tool with another quantification tool based on Image J 2.0 Java 1.8 (using Neuron J)  that req2  = 8.79, p = 0.032, Four regions were used as the primary donor sites for olfactory epithelium, anterior and posterior septum , and anterior and posterior turbinate  regions. For 14 days, all these primary olfactory biopsies were cultured in 2D as organotypic cultures A,B and sp = 0.0012 and p = 0.0060, respectively) . Attachment rates were not influenced by the coating. Although the presence of cellular outgrowth was higher for fibronectin (32%) when compared to collagen IV (21%), laminin (14%), and the uncoated plastic coverslip (Ctrl), (5%), this association was not significant A. Regardctively) C.Together, optimal axonal elongation was observed in the presence of fibronectin, which was selected for subsequent GF screening .In order to assess the influence of growth factors on our organotypic explant model, we used a two-step approach. On fibronectin-coated wells, we first used FGF2 to promote progenitor’s proliferation, and then, we used factors known to promote neuronal maturation . We monitored proliferation by observing the presence of growing cells from olfactory explants, which we referred to as cellular outgrowth. In the context of organotypic models, the timing of the first appearance of cellular outgrowth can vary between specimens. To establish a normalized time-point for most biopsies to exhibit cellular outgrowth, we closely monitored the proliferation rates in OE biopsies. This allowed us to identify a specific time frame in which most of the biopsies demonstrated cellular outgrowth, thus enabling us to switch from proliferation media (containing FGF2) to differentiation media. The earliest signs of cellular outgrowth among the explants were observed after three days, and this process continued up to the 12th day . We obse2 (9) = 39.97; p = 0.0001)  or FGF2 from day 0 to 7 followed by FGF2 and NGF from day 7 to 15  led to a higher cellular outgrowth rate. Similarly, the rate of neuronal outgrowth within these explants, assessed by observing the presence or absence of cells positive for the βIII-tubulin marker, was significantly increased in the presence of growth factors vs. control (χ2 (9) =23.7; p = 0.005). Our results indicate that FGF2 administration throughout the entire culture duration (0–15 days) led to a neuronal outgrowth rate of 12%. While NGF alone (from days 7 to 15) achieved only 7%, the combination of FGF2 and NGF during the same period yielded the highest neuronal outgrowth rate of 33%  (We observed a significantly enhanced cellular outgrowth rate upon growth factor supplementation (χ 0.0001) D. In par 0.0025) E.In summary, our findings demonstrate that applying FGF2, followed by either GDNF or FGF2 and NGF, significantly affects the cellular outgrowth rate in olfactory explants. However, only the latter condition, involving the combination of FGF2 and NGF, resulted in the highest rate of neuronal outgrowth. This highlights the considerable potential of this specific growth factor combination for promoting neuronal regrowth.Using our automated quantification tool , we deteWhile several combinations of growth factors were able to promote OSN axonal elongation in our setting, the condition exhibiting the highest rate of neuronal outgrowth was FGF2 and NGF.The present technical study allowed us to improve the culture methods of murine olfactory neurons. The anatomical donor site for harvesting primary olfactory epithelium in mice was restricted to the posterior septum and posterior turbinate regions, thereby allowing a higher yield of olfactory neuronal outgrowth in 2D cultures. Fibronectin and collagen IV scaffold also improved axonal elongation over untreated and laminin-coated surfaces. Finally, FGF2 alone or in combination with BDNF, GDNF, or NGF exhibited a similar effect on the neuronal outgrowth and axonal elongation and outperformed single growth factor supplementation , summaryThis quantification tool measures βIII-tubulin positive cells and axons surrounding the olfactory explant. It delivers an automated and comprehensive measurement of the total axonal length, which correlated highly with the measures found manually. Although it does not discriminate between axons that originate from neurons in the explants and new axons from neurons that migrated out of the explant, we believe that this tool gives valuable insight into the overall dynamics of neuronal growth.The definition of an optimal scaffold composed of both ECM proteins and soluble factors to regrow olfactory neurons is of the utmost importance to initiating a novel therapeutic path toward regenerating the olfactory system in patients suffering from traumatic brain injury. Based on our results, it seems to be of particular interest to treat olfactory explant cultures with a combination of growth factors containing FGF2. Earlier studies claimed that FGF2 may induce differentiation in murine and human olfactory epithelium cultures ,36; howeOur study has also shed light on the remarkable ability of FGF2 to enhance axonal elongation in vitro. However, it is crucial to consider the potential confounding factors in this observed effect. The exceptional performance of FGF2 in promoting axonal elongation may be attributed to the stimulation of progenitor and ensheathing cell proliferation. The continuous proliferation of progenitors may increase the likelihood of subsequent differentiation into neurons. Meanwhile, the ongoing proliferation and migration of ensheathing cells may facilitate the migration of these young neurons toward the edges of the quantification field, increasing the chances of βIII-tubulin-positive neurons being detected far away from the analysis starting point.Nevertheless, this observation only partially aligns with the high expectations regarding neuronal outgrowth. A combination with a growth factor that explicitly stimulates neuronal differentiation may be necessary to achieve optimal results. While FGF2 demonstrates remarkable potential as an in vitro option for boosting progenitor and ensheathing cell proliferation, additional factors that promote neuronal differentiation are crucial for achieving the desired level of neuronal outgrowth.NTR [Also, NGF exerts an impact on ensheathing cells, which are the glial cells located within the OE; those cells express both the low-affinity NGF receptor p75NTR  and the NTR . Thus, tNTR ; thus, tNGF’s role in promoting axonal elongation may extend beyond its influence on OECs. Specifically, this growth factor might play a specific role in the regeneration of OSNs following injury. In support of this, Miwa et al.  demonstrWhen combined with FGF2, NGF elicited the highest percentages of neuronal outgrowth among all tested conditions (33%), surpassing even the effects of FGF2 alone (12%). However, the axonal elongation observed  was not significantly different from that induced by FGF2 alone . Despite the crucial role of NGF in OE regeneration, our study revealed that the singular administration of NGF alone is insufficient to stimulate axonal elongation effectively. This could be attributed to the fact that NGF has been shown to primarily facilitate clustering rather than actual outgrowth in organized bundles of olfactory neurons, potentially limiting its ability to promote extensive axonal elongation .These observations support our hypothesis that a coordinated interplay of multiple growth factors and soluble proteins orchestrates the regeneration of OSNs. This concerted action might serve to optimize the regenerative processes involved in OSNs recovery after trauma. However, additional investigations are warranted to unravel the specific molecular mechanisms implicated when these promising combinations of soluble proteins are employed.When considering the application of these elicitor proteins to functionalize implants for olfactory neuronal regeneration in vivo, it is crucial to account for the fibrogenic effect of FGF2, meaning the role it has in recruiting fibroblasts to the site of injury. While FGF2 may exhibit optimal activity in vitro, its use in vivo may increase scarring due to its fibrogenic properties. Further investigations are needed to elucidate how FGF2 can be replaced or its fibrogenic effect minimized.This study introduces an optimized organotypic model specifically tailored to address regeneration of murine olfactory neurons while mitigating the potential biases associated with using poorly characterized growth factors and extracellular protein cocktails in OE cultures. Thanks to this tailored in vitro model, we demonstrated that OSN regeneration can be effectively induced, controlled, and accurately quantified using our automated quantification tool. These results hold promising implications for advancing the therapeutic paradigm of olfactory regeneration toward preclinical investigations.Basal media (BM) was a combination of DMEM-F12 media  supplemented with Hepes (15 Mm) and L-Glut (15 mM), 1× N-2 , B-27 (ref. Gibco 17504044), and Penicillin streptomycin . Proliferation media (PM) corresponds to BM supplemented with fibroblast growth factor 2 (FGF2) 40 ng/mL  and was used to culture olfactory explants from days 0–7. Differentiation media (DM) was a combination of BM with various growth factors, and was used from culture days 7–15.Harvesting techniques for OE in humans ,30,43,44Coverslips (Thermofisher 12 mm diameter) were cleaned with 70% alcohol before being placed in sterile 24-well plate for cell culture . Coating solutions for the different matrices were prepared in DPBS (Dulbecco’s Phosphate Buffer from Sigma) using the following concentrations: laminin 50 μg per mL (Sigma-Aldrich L2020 from Human Placenta); fibronectin 50 μg × mL (Sigma-Aldrich FC010 from human plasma ); collagThe growth factors (GFs) used in the study, with reference numbers, supplier information, and concentrations are shown in To harmonize the cellular outgrowth frequency among explants, we monitored the biopsies; proliferation rate along the 14 days of culture: 30% of biopsies were giving visible outgrowth already after day 5, more than a half of explants after day 7 (63%), 74% after day 9, and 81% after day 12  for 20 min at room temperature and treated with a blocking solution  for another 20 min. Primary antibodies  equipped with a Hamamatsu Orca Flash 4 camera and a Zeiss 20× objective lens (Plan-Apochromat 20×/0.8 M27) with a resolution of 0.325 microns/pixel. Slides containing three specimens each were loaded onto the scanner to enable simultaneous image acquisition, leveraging its high capacity to accommodate up to 100 slides. Before initiating the acquisition, the region of interest was determined for each specimen. The acquisition process adhered to the predefined parameters outlined in the Scanning profile, encompassing the acquisition of 9 sections per specimen (z-stack) with intervals of 2.50 µm. The Extended Deep Focus technique used the “Variance” method to capture detailed images. The primary focus during image acquisition was directed toward βIII-tubuline-positive cells, with the secondary antibody conjugated to Alexa Fluor 555 Donkey . To achieve this, a filter with excitation at 540 nm and emission at 561 nm was used. The light source intensity was 58.32%, and the exposure time was fixed at 100 ms A. ImagesAs outcome measures, we evaluated the attachment rate, cellular outgrowth rate, neuronal outgrowth rate, and axonal elongation (in mm). Cellular outgrowth refers to the presence or absence within the culture of a general cellular outgrowth and can be evaluated using a simple bright field microscope  associated with explant attachment and cellular or neuronal outgrowth. For post hoc analysis of Pearson’s chi-square, we calculated squared adjusted residuals (or z-square) and transformed them into pothesis .Since the axonal elongation data were not parametrically distributed, we performed a logarithmic transformation on the axonal elongation and then analyzed it ,49 with