Comprehensive analysis of MYB/MYBL1-altered pediatric-type diffuse low-grade glioma.

BACKGROUND: Pediatric-type diffuse low-grade gliomas (pLGG) harboring recurrent genetic alterations involving MYB or MYBL1 are closely related tumors. Detailed treatment and outcome data of large cohorts are still limited. This study aimed to comprehensively evaluate pLGG with these alterations to define optimal therapeutic strategies. METHODS: We retrospectively reviewed details of pLGG with MYB or MYBL1 alterations from patients treated or referred for pathologic review at St. Jude Children's Research Hospital. Tumor specimens were centrally reviewed, and clinical data were collated. RESULTS: Thirty-three patients (18 male; median age, 5 years) were identified. Two tumors had MYBL1 alterations; 31 had MYB alterations, MYB::QKI fusion being the most common (n = 10, 30%). Most tumors were in the cerebral hemispheres (n = 22, 67%). Two patients (6%) had metastasis at diagnosis. The median follow-up was 6.1 years. The 5-year event-free survival (EFS) rate was 81.3% ±â€…8.3%; the 5-year overall survival (OS) rate was 96.4% ±â€…4.1%. Patients receiving a near-total or gross-total resection had a 5-year EFS of 100%; those receiving a biopsy or subtotal resection had a 5-year EFS rate of 56.6% ±â€…15.2% (P < .01). No difference in EFS was observed based on location, histology, or molecular alterations. However, the tumors that progressed or metastasized may have distinct methylation profiles with evidence of activation of the MAPK and PI3K/AKT/mTOR pathways. CONCLUSIONS: pLGG with MYB/MYBL1 alterations have good outcomes. Our findings suggest that surgical resectability is a crucial determinant of EFS. Further characterization is required to identify optimal treatment strategies for progressive tumors.

Retrospective analysis of outcomes for pediatric acute lymphoblastic leukemia in South American centers.

Introduction: Acute lymphoblastic Leukemia (ALL) is the most common pediatric malignancy. While the survival rate for childhood ALL exceeds 90% in high-income countries, the estimated survival in low-and middle-income countries ranges from 22-79%, depending on the region and local resources. Methods: This study retrospectively reviewed demographic, biological, and clinical parameters of children under 18 years of age with newly diagnosed ALL presenting between 2013-2017 across five pediatric centers in 4 countries in South America. Survival analyses were estimated using the Kaplan-Meier method. Results: Across the five centers, 752 patients were analyzed (Bolivia [N=9], Ecuador [N=221], Paraguay [N=197], Peru [N=325]) and 92.1% (n=690) patients were diagnosed with B-cell and 7.5% (n= 56) with T-cell ALL. The median age was 5.5 years old (IQR 7.29). At diagnosis, 47.8% of patients were categorized as standard and 51.9% as high risk per their institutional regimen. Advanced diagnostics availability varied between modalities. MRD was evaluated in 69.1% of patients; molecular testing was available for ETV6-RUNX, BCR-ABL1, TCF3-PBX1, and KMT2A-rearranged ALL in 75-81% of patients; however, karyotyping and evaluation for iAMP21 were only performed in 42-61% of patients. Central nervous system (CNS) involvement was evaluated at diagnosis in 57.3% (n=429) patients; of these, 93.7% (n=402) were CNS 1, 1.6% (n=7) were CNS 2, 0.7% (n=11) were CNS3, 1.9% (n=8) had cranial nerve palsy, and 2.1% (n=9) results unavailable. Chemotherapy delays >2 weeks were reported in 56.0% (n=421) patients during treatment. Delays were attributed to infection in 63.2% (n=265), drug-related toxicities in 47.3% (n=198), and resource constraints, including lack of bed availability in 23.2% (n=97) of patients. The 3-year Abandonment-sensitive EFS and OS were 61.0±1.9% and 67.2±1.8%, respectively. The 3-year EFS and OS were 71.0±1.8% and 79.6±1.7%, respectively. Discussion: This work reveals opportunities to improve survival, including addressing severe infections, treatment interruptions, and modifications due to drug shortages. In 2018, healthcare professionals across South America established the Pediatric Oncology Latin America (POLA) group in collaboration with St. Jude Children's Research Hospital. POLA collaborators developed an evidence-based, consensus-derived, adapted treatment guideline, informed by preliminary results of this evaluation, to serve as the new standard of care for pediatric ALL in participating institutions.

Evaluation of factors leading to poor outcomes for pediatric acute lymphoblastic leukemia in Mexico: a multi-institutional report of 2,116 patients.

Background and aims: Pediatric acute lymphoblastic leukemia (ALL) survival rates in low- and middle-income countries are lower due to deficiencies in multilevel factors, including access to timely diagnosis, risk-stratified therapy, and comprehensive supportive care. This retrospective study aimed to analyze outcomes for pediatric ALL at 16 centers in Mexico. Methods: Patients <18 years of age with newly diagnosed B- and T-cell ALL treated between January 2011 and December 2019 were included. Clinical and biological characteristics and their association with outcomes were examined. Results: Overall, 2,116 patients with a median age of 6.3 years were included. B-cell immunophenotype was identified in 1,889 (89.3%) patients. The median white blood cells at diagnosis were 11.2.5 × 103/mm3. CNS-1 status was reported in 1,810 (85.5%), CNS-2 in 67 (3.2%), and CNS-3 in 61 (2.9%). A total of 1,488 patients (70.4%) were classified as high-risk at diagnosis. However, in 52.5% (991/1,889) of patients with B-cell ALL, the reported risk group did not match the calculated risk group allocation based on National Cancer Institute (NCI) criteria. Fluorescence in situ hybridization (FISH) and PCR tests were performed for 407 (19.2%) and 736 (34.8%) patients, respectively. Minimal residual disease (MRD) during induction was performed in 1,158 patients (54.7%). The median follow-up was 3.7 years. During induction, 191 patients died (9.1%), and 45 patients (2.1%) experienced induction failure. A total of 365 deaths (17.3%) occurred, including 174 deaths after remission. Six percent (176) of patients abandoned treatment. The 5-year event-free survival (EFS) was 58.9% ± 1.7% for B-cell ALL and 47.4% ± 5.9% for T-cell ALL, while the 5-year overall survival (OS) was 67.5% ± 1.6% for B-cell ALL and 54.3% ± 0.6% for T-cell ALL. The 5-year cumulative incidence of central nervous system (CNS) relapse was 5.5% ± 0.6%. For the whole cohort, significantly higher outcomes were seen for patients aged 1-10 years, with DNA index >0.9, with hyperdiploid ALL, and without substantial treatment modifications. In multivariable analyses, age and Day 15 MRD continued to have a significant effect on EFS. Conclusion: Outcomes in this multi-institutional cohort describe poor outcomes, influenced by incomplete and inconsistent risk stratification, early toxic death, high on-treatment mortality, and high CNS relapse rate. Adopting comprehensive risk-stratification strategies, evidence-informed de-intensification for favorable-risk patients and optimized supportive care could improve outcomes.

Physician Perceptions of and Barriers to Pediatric Palliative Care for Children With Cancer in Brazil.

PURPOSE: Early integration of pediatric palliative care (PPC) for children with cancer is critical to improving the quality of life of both the patient and family. Understanding physician perceptions of palliative care and perceived barriers to early integration is necessary to develop PPC in Brazil. METHODS: The Assessing Doctors' Attitudes on Palliative Treatment survey was modified for use in Brazil. The survey was open from January 2022 to June 2022 and distributed to physicians of all specialties from participating institutions who treat children with cancer. Statistical analysis was complemented by qualitative analysis of open-ended responses. RESULTS: A total of 272 respondents participated. Most respondents reported access to PPC experts for consultation (77.2%) and 34.5% indicated previous palliative care training. Physician knowledge of PPC was generally aligned with WHO guidance (median alignment, 93.0%; range, 80.5%-98.2%). However, about half (53.3%) felt comfortable addressing physical needs of patients receiving PPC, 35.3% addressing emotional needs, 25.8% addressing spiritual needs, and 33.5% addressing grief and bereavement needs. Most respondents (65.4%) felt palliative care should be involved from diagnosis, but only 10.3% stated that this occurred in their setting. The most important barriers identified were physician discomfort (89.0%), limited physician knowledge (88.6%), and lack of home-based services (83.8%). CONCLUSION: Despite a strong understanding of the role of palliative care, physicians in Brazil reported low confidence delivering PPC to children with cancer. Additionally, physicians generally believed that PPC should be integrated earlier in the disease trajectory of children with cancer. This work will direct educational and capacity building initiatives to ensure greater access to high-quality PPC for children with cancer in Brazil to address patient and family suffering.

SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules.

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.

The Mi-2 homolog Mit1 actively positions nucleosomes within heterochromatin to suppress transcription.

Mit1 is the putative chromatin remodeling subunit of the fission yeast Snf2/histone deacetylase (HDAC) repressor complex (SHREC) and is known to repress transcription at regions of heterochromatin. However, how Mit1 modifies chromatin to silence transcription is largely unknown. Here we report that Mit1 mobilizes histone octamers in vitro and requires ATP hydrolysis and conserved chromatin tethering domains, including a previously unrecognized chromodomain, to remodel nucleosomes and silence transcription. Loss of Mit1 remodeling activity results in nucleosome depletion at specific DNA sequences that display low intrinsic affinity for the histone octamer, but its contribution to antagonizing RNA polymerase II (Pol II) access and transcription is not restricted to these sites. Genetic epistasis analyses demonstrate that SHREC subunits and the transcription-coupled Set2 histone methyltransferase, which is involved in suppression of cryptic transcription at actively transcribed regions, cooperate to silence heterochromatic transcripts. In addition, we have demonstrated that Mit1's remodeling activity contributes to SHREC function independently of Clr3's histone deacetylase activity on histone H3 K14. We propose that Mit1 is a chromatin remodeling factor that cooperates with the Clr3 histone deacetylase of SHREC and other chromatin modifiers to stabilize heterochromatin structure and to prevent access to the transcriptional machinery.

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast.

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.

The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS.

RNA interference (RNAi) is critical for the assembly of heterochromatin at Schizosaccharomyces pombe centromeres. Central to this process is the RNA-induced initiation of transcriptional gene silencing (RITS) complex, which physically anchors small noncoding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which forms a bridge between Chp1 and Ago1. Chp1 is a large protein with no recognizable domains, apart from its chromodomain. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing the tight association of Chp1 and Tas3. The structure also shows a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice.

OBJECTIVE: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. METHODS: We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. RESULTS: Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. CONCLUSION: Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex.

Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent "priRNAs." The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.

High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

Chp1-Tas3 interaction is required to recruit RITS to fission yeast centromeres and for maintenance of centromeric heterochromatin.

The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3(Delta)(10-24)) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies.

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.

Chlamydia pneumoniae infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells.

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca2+ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca2+ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca2+ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic.

Elicitation of reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a Ca2+-dependent process involving simultaneous activation of NADPH oxidase and cytochrome oxidase genes.

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals.

Induction of lipoprotein lipase gene expression in Chlamydia pneumoniae-infected macrophages is dependent on Ca2+ signaling events.

Unregulated uptake of low density lipoprotein (LDL) in macrophages is the hallmark of early atherogenic lesions, and Chlamydia pneumoniae infection of macrophages induces this process by an unknown mechanism. It was therefore aimed in this study to investigate (i) the role of C. pneumoniae in macrophage expression of the lipoprotein lipase (LpL) gene, (ii) the probable role of Ca2+ influx signals and (iii) the effect of the process on LDL uptake. Lipoprotein lipase mRNA expression and LpL activity in infected RAW-264.7 cells were significantly upregulated. A biphasic Ca2+ influx signal was observed in infected cells with a moderate influx (303 nM Ca2+) favoring optimal LpL gene expression. Also, the antagonists of L-type Ca2+ channel in macrophages significantly down-regulated LpL gene expression and the biomolecular content of C. pneumoniae responsible for the observed events was in part found to be Chlamydia lipopolysaccharide (cLPS). Investigations aimed at determining the specific relevance of Ca(2+)-dependent lipoprotein lipase gene expression in C. pneumoniae-infected macrophages showed that the condition caused enhanced uptake of LDL which was abrogated by Calphostin-C-mediated down-regulation of LpL. This discovery of a specialized Ca2+ influx signal-mediated LpL upregulation in C. pneumoniae-infected macrophages provides a mechanistic insight into early events involving C. pneumoniae in macrophage foam cell formation resulting from LDL uptake.