BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10−4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax–rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic  TP53 mutations, and unmutated IGHV status [Clinical outcomes of chronic lymphocytic leukemia (CLL) have significantly improved over the past 10 years with targeted therapies changing the treatment paradigm of the disease. Remarkable outcomes in first-line as well as in relapsed/therapy refractory (R/R) CLL are observed in patients with coexisting conditions, as well as in patient populations characterized by high-risk disease biology, including deletions of 11q and 17p, V status ,3,4. VenV status ,7,8. CurV status ,7,9.BCL2 diminishing venetoclax binding, overexpression of the anti-apoptotic BCL-XL, Bcl-2, MCL1, and XIAP, and the emergence of complex karyotypes [Despite the high response rates and prolonged progression-free survival, loss of efficacy and subsequent secondary venetoclax resistance occur in a subset of patients, in both continuous and fixed-duration treatment setups ,6. Over ryotypes ,15,16,17ryotypes .BCL2 gene have been demonstrated to confer resistance to venetoclax by reducing or inhibiting the binding affinity of the small molecule inhibitor for the anti-apoptotic Bcl2 protein. The glycine-to-valine substitution emerging at position G101 (G101V) was first reported by Blombery et al. in seven patients showing progressive disease during venetoclax treatment [BCL2 G101V mitigates the cellular response to venetoclax in vitro via modulation of the BH3 binding domain of Bcl2 and is associated with clinical resistance in up to 50% of the patients experiencing relapse or disease progression [BCL2 mutations, e.g., D103Y directly disrupting the BH3 binding P4 pocket, have been observed in CLL patients, unveiling various cooperating mutational patterns with coexisting mutations and overexpression of the pro-survival proteins BCL-XL and MCL1 [A handful of mutations affecting the reatment . BCL2 G1gression ,14,19. Oand MCL1 ,14,20,21and MCL1 ,13,20. Iand MCL1 ,23.BCL2 resistance mutations, G101V and D103Y, in a cohort of patients with R/R CLL outside of clinical trials receiving single-agent or venetoclax plus rituximab combination therapy. Our results provide an insight into the frequency of these variants in patients progressing on venetoclax and into the repertoire of additional, simultaneously detectable BCL2 mutations. Our data on BCL2 G101V and D103Y may also have implications for the sensitive monitoring of venetoclax therapy which may allow for the early detection of an impending relapse and the identification of patients who could potentially benefit from an alternative approach to therapeutic intervention.In this study, we have assessed the clinical value and feasibility of sensitive screening for the two most common TP53 mutation positivity screened by targeted NGS were available in 94.0% (63/67) and 83.6% (56/67) of the cases, respectively. Patients harboring del(17p) and/or TP53 mutation(s) comprised 40.3% (27/67) of the patient population. Additional cytogenetic aberrations including del(11q), del(13q), and trisomy of chromosome 12 were identified in 22.4% (15/67), 22.4% (15/67), and 11.9% (8/67) of the cases, respectively. IGHV mutation status was assessed in 52 patients, with 82.7% (43/52) of them carrying unmutated IGHV (IGHV-U). Patients with high-risk genetic features associated with adverse prognosis including TP53 aberrations, and IGHV-U status represented 83.6% (56/67) of the study cohort, which together with the failure of prior therapy lines depicted an R/R patient population typically selected for venetoclax therapy.Molecular and cytogenetic aberrations considered independent prognostic and/or predictive markers in CLL were screened as part of the standard-of-care diagnostic workflow . Del(17pBCL2 G101V and D103Y mutations was performed on samples obtained from 67 R/R patients using locus-specific ddPCR assays designed and optimized for the sensitive (10−4) interrogation of these two mutational hotspots. BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with 4 patients carrying both alterations. The two variants were detected after a median of 15 months (range: 5–48 months) of venetoclax therapy with 90.9% (10/11) of the affected patients experiencing relapse or disease progression. These ten patients represented 43.5% (10/23) of all cases developing secondary venetoclax resistance during the follow-up period. Richter’s transformation was observed in one patient who tested positive for BCL2 G101V and in another patient with wild-type G101 and D103 alleles after 15 and 25 months of venetoclax therapy, respectively. BCL2 G101V and D103Y were exclusively detected in patients with continuous venetoclax therapy, and thus cases with a time-limited treatment setup were excluded from the PFS and OS analyses. In the analyzed patient population, compared to cases with wild-type BCL2 G101 and D103 loci, significantly inferior PFS as defined by the iwCLL 2018 criteria [BCL2 G101V and/or D103Y mutations  (p = 0.6904)  and 0.28% (range: 0.018–8.94%), respectively. The median CCF value of BCL2 D103Y at the time of sample collection in four cases with wild-type G101 was 0.023% (range: 0.015–0.16%).Screening for criteria  was obse 0.0192) A, with t 0.6904) B. The TPotherapy . IntriguBCL2 G101V and/or D1013Y discontinued single-agent venetoclax after a median of 21 months (range: 10–48 months) of Bcl2 inhibition  with wild-type G101 and D103 developing secondary venetoclax resistance after a median of 15 months (range: 1–48 months) of venetoclax therapy. Targeted ultra-deep sequencing was performed covering all coding regions of the BTK, PLCG2, BCL2, and TP53 genes with a median depth of 3886× . NGS uncovered three additional BCL2 mutations, namely, D103E, A113G, and V156D in two cases. Notably, both patients harbored G101V and/or D103Y as previously assessed by ddPCR, with no BCL2 mutations unveiled in BCL2 G101 and D103 wild-type cases.Ultra-deep targeted NGS was performed on 17 samples obtained at the time of disease progression, irrespective of their TP53 variants unveiled by targeted NGS, two patients (patients #25 and #40) acquired detectable TP53 mutations during venetoclax therapy, with these aberrations being exclusively observed in samples obtained at the time of disease progression and not in diagnostic specimens. Interestingly, one patient (patient #43) was found to be TP53 wild-type at progression, while harboring a somatic TP53 variant at the time of diagnosis. Further somatic variants uncovered by ultra-deep NGS in samples obtained at the time of disease progression are listed in Considering BCL2 mutations experienced relapse or disease progression after a median of 29 months (range: 18–48 months) of continuous single-agent venetoclax therapy , 1.15% (range: 0.05–3.40%), 1.16% (range: 0.37–1.95%), 0.37% (range: 0.05–5.76%), and 2.2%, respectively. In 4 patients with samples obtained prior to progression, the emergence of BCL2 G101V and/or D103Y, as well as co-occurring BCL2 mutations preceded the first clinical signs of disease progression by a median of 11 months (range: 5–16 months)  increase in the CCF values of each mutation over time (Four patients harboring multiple  therapy . Sensitind V156D . In sampver time .BCL2 G101 and D103 as assessed by ddPCR. This subset of patients represented 56.5% of the cases developing secondary venetoclax resistance after a median of 13 months (range: 1–33 months) of Bcl2 inhibition. Interestingly, all patients experiencing relapse or disease progression received continuous single-agent venetoclax therapy. Subsequent salvage R-CHOP chemo-immunotherapy was applied in two cases. In one patient, subsequent rituximab was administered in combination with continued venetoclax and in one case, salvage ibrutinib was added to the continuous venetoclax monotherapy. Eight patients succumbed to their disease after developing venetoclax resistance without receiving any subsequent lines of treatment. As an attempt to unveil alternative molecular mechanisms underlying venetoclax resistance, targeted ultra-deep NGS was performed on samples available at the time of disease progression from nine patients carrying wild-type BCL2 G101 and D103, as previously described. No further resistance mutation affecting the BCL2 gene was identified in any of the analyzed cases, suggesting either the presence of alternative genomic lesions localized outside the coding region of BCL2 or the emergence of epigenomic/transcriptomic changes eventually leading to venetoclax resistance.Thirteen patients progressing on venetoclax carried wild-type BCL2 mutations associated with resistance in longitudinally collected samples of patients undergoing venetoclax treatment.Bcl2 inhibition by venetoclax has revolutionized the therapeutic landscape of R/R CLL with high response rates and prolonged PFS in heavily pre-treated patient populations ,6,25,26.BCL2 mutations associated with acquired venetoclax resistance in a cohort of 67 patients with R/R CLL treated with venetoclax either as monotherapy or in combination with rituximab. Using an ultra-sensitive ddPCR method, BCL2 G101V and/or D103Y mutations were detected in 16.4% of the analyzed patient population, with some 90% of the affected patients showing clinical signs of relapse during the follow-up period. This subset of BCL2 G101V and/or D103Y positive cases represented over 40% of the patients undergoing clinical disease progression. To unveil additional cooperating genetic changes driving venetoclax resistance, targeted ultra-deep NGS was performed on samples obtained at the time of disease progression, irrespective of the BCL2 G101 and D103 status previously assessed by ddPCR. In four patients, NGS uncovered three additional BCL2 mutations, such as D103E, A113G, and V156D, which were previously found to be co-occurring with G101V and associated with resistance to Bcl2 inhibition in vivo [BCL2 variants in longitudinally collected patient samples revealed a heterogeneous and dynamically changing subclonal abundance of mutations during venetoclax therapy, suggesting a complex underlying architecture of resistant CLL cells, coupled with subsequent clinical disease progression in all four cases. These patients represent the first longitudinally monitored and temporally dissected cases with venetoclax resistance to date.In the framework of a nationwide effort, we investigated the clinical value and feasibility of a sensitive ‘snapshot’ screening for the two most common  in vivo ,28. RetrBCL2 G101V in 11 cases with all patients ultimately experiencing disease progression [BCL2 mutations, including D103Y, D103E, A113G, and V156D, in all but one patient using digital and hybrid capture-based NGS approaches [BCL2 mutations, including A113G, F104L, F104S, L119V, and G101A coexisting with G101V, and the internal tandem duplication p.Arg107_Arg110, in a cohort of 24 patients with venetoclax and ibrutinib-resistant CLL [Our findings are in line with previous studies performed on patient cohorts of various clinical trials. Anderson et al. investigated 67 patients with R/R CLL receiving venetoclax in 3 early-phase trials and identified gression . Consistproaches ,29. Simitant CLL . These stant CLL ,29.BCL2 mutations, substitutions emerging at the D103 residue are especially noteworthy. D103 residue is located within the P4 pocket of Bcl2 establishing a hydrogen bond with the venetoclax [BCL2 D103Y was first reported by Tausch et al., as a cooperating mutation driving venetoclax resistance in a single patient who previously tested positive for G101V [BCL2. In addition, here we report recurrent BCL2 D103E co-occurring with other changes of BCL2 in two patients, further underlining the role of D103 residue in driving venetoclax resistance.Among the additionally unraveled netoclax . Amino anetoclax . In the or G101V . In our BCL2 variants displayed cooperating subclonal mutational patterns with allelic burdens of less than 10%, even in samples obtained at the time of relapse. Plausible explanations for this phenomenon may include the key role of mutually exclusive mutation-bearing subclones reported by Thompson et al., the permissive microenvironment as well as the ability of mutation-bearing cells to promote resistance in BCL2 unmutated CLL subclones [BCL2 mutations and experiencing clinical disease progression could have been identified with an exclusive screening for G101V and D103Y, demonstrating the potential clinical value of a standard-of-care ddPCR test targeting these two variants only for the early identification of venetoclax-resistant cases in the future. Intriguingly, no BCL2 mutation was observed among patients undergoing time-limited venetoclax therapy either as a single agent or in combination with rituximab. This finding is in line with the results of previous studies including an evaluation of the 4-year data of the phase 3 MURANO trial by Kater et al. [BIRC3, BRAF, TP53, NOTCH1, and XPO1 genes, with no variants identified in BCL2 [All cases harboring multiple ubclones ,20,29,31r et al. ,32. The BCL2 mutations and experiencing relapse or disease progression in our cohort, with 50% of these patients delivering durable responses. Consistent with our observations, a handful of studies with a small number of cases and a cohort study of 74 patients by Mato et al. concluded that the efficacy of BTKi therapy does not seem to be altered by prior venetoclax treatment [The significance of detecting resistance to Bcl2 inhibition early on in both time-limited and continuous treatment settings has been increasing with the growing number of alternative therapeutic options available for patients developing venetoclax resistance ,34,35,36reatment ,37,38,39reatment ,40,41,42reatment ,44.BCL2 mutations underlying venetoclax resistance in a real-world cohort of patients with R/R CLL. To our knowledge, this is the largest R/R CLL cohort outside of clinical trials to date in which BCL2 resistance mutations were screened during Bcl2 inhibition. The vast majority of our mutation-bearing patients underwent subsequent disease progression during the follow-up period, and based on the presented data, driver mechanisms of acquired venetoclax resistance may be uncovered in up to 45% of the cases experiencing clinical relapse using a sensitive screening method for only these two aberrations. Although longer follow-up times and prospective studies on independent patient cohorts will be required for a refined assessment of the predictive power of these ddPCR tests, the clinical value of such analyses in the standard-of-care workup algorithms of venetoclax-resistant CLL will likely be increasing in the future. Retrospective interrogation of additional loci in the anti-apoptotic gene BCL2 revealed multiple, simultaneously detectable mutations with heterogeneous and temporally changing abundance, suggesting a complex subclonal architecture characterized by minor subclones that carry distinct genomic features and cooperatively contribute to venetoclax resistance. Further genome-, epigenome-, and transcriptome-wide studies, preferably coupled with functional readout will be essential to achieve a deepened understanding of key mechanisms orchestrating the interplay between various CLL compartments driving resistance to targeted Bcl2 inhibition.In summary, our study demonstrates the feasibility and clinical value of the robust detection of the two most common Peripheral blood samples from 67 patients  with R/R CLL treated with venetoclax monotherapy or venetoclax in combination with anti-CD20 rituximab were collected from 11 Hungarian oncohematological centers. Fixed-duration venetoclax plus rituximab combination was applied in 23.9% (16/67) of the patients, while time-limited and continuous venetoclax monotherapy was administered in 7.5% (5/67) and 68.6% (46/67) of the cases, respectively. At the time of the last follow-up, 11 patients had stopped the fixed-duration venetoclax treatment after a median of 26 months (7–32 months) due to subsequent deep remission as defined by Hungarian treatment protocols. The cohort represented a pre-treated patient population with a median of two lines (range: one to six lines) of prior therapy. This cohort also comprised a subset of cases with prior ibrutinib therapy investigated within the framework of the Hungarian Ibrutinib Resistance Analysis Initiative, published previously by our research group . FollowiBCL2 mutations unveiled by ultra-deep targeted sequencing and droplet digital polymerase chain reaction (ddPCR) at the time of disease progression were collected as part of the routine diagnostic work-up and were analyzed retrospectively. The clinical characteristics of our patient cohort are summarized in Samples were collected at the time of disease progression from 23 patients experiencing relapse following a median of 14 months (1–48 months) of venetoclax therapy. In 44 cases with no clinical signs of disease progression, the median follow-up time was 23 months (range: 1–83 months) with samples obtained at the time of the last follow-up. Median 6 follow-up samples (range: 2–11) from 4 patients harboring multiple TP53 mutations and IGHV mutation status were assessed according to the most recent ERIC guidelines [Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation following the assessment of leukemic cell purity by flow cytometry using CD5/CD19/CD45 immunophenotypic markers. In cases with low whole blood counts, leukemic cell purity was assessed using CD19/CD5/CD22/CD43/CD79b/CD81/CD3/ROR1 immunophenotypic markers following the most recent European Research Initiative on CLL (ERIC) guideline on minimal residual disease monitoring in CLL . DNA isoidelines ,48. ChroBCL2 G101V and D103Y mutations, as well as additional BCL2 variants unveiled by next-generation sequencing, was performed by ddPCR using custom assays designed for the quantitative assessment of mutant and wild-type alleles in each altered genomic position . Reactions were carried out according to the manufacturer’s instructions on a QX200 Droplet Digital PCR system  with the recommended 90–100 ng of input DNA. Fractional abundance (FA) depicting the allelic burden of the selected variants was calculated from the ratio of the number of detected mutant DNA molecules (a) and the total number of detected mutant plus wild-type (b) DNA molecules (FA = a/(a + b)). FA values were normalized to the leukemic cell purity assessed by flow cytometry . The quantitative reliability of the ddPCR assays was assessed with dilution series for each variant and sensitivity was determined based on droplet counts in each sample. The median lower limit of detection proved to be 0.01% as calculated from the final droplet counts.Ultra-sensitive screening for BCL2, BTK, PLCG2, and TP53) as previously described [TP53 Database  and Seshat were used for annotating somatic TP53 mutations [Targeted next-generation sequencing (NGS) was performed on samples obtained at the time of disease progression using a QIAseq Targeted DNA Custom Panel  utilizing unique molecular identifiers and covering four genes  was used for calculating the median values and for performing the Mantel–Cox test to compare progression-free survival (PFS) and overall survival between patients harboring


Pediatric high-grade gliomas bearing either an H3G34V/R or H3K27M mutation are incurable brain tumors with unique epigenomes. Current epigenomic studies on pHGGs primarily focus on these mutations; however, the function of other important crosstalk histone posttranslational modifications must be determined to elucidate tumor mechanisms. Here, we show that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response, and thus a potential epigenetic therapeutic target for children with malignant gliomas.Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas. Brain tumors, the most common form of solid tumor in children under the age of 15, are responsible for approximately 20% of all childhood cancers. Five-year survival, following diagnosis and treatment of a primary malignant brain tumor, is ~30%. Among pediatric brain tumors, high-grade gliomas (pHGGs), including diffuse intrinsic pontine gliomas (DIPGs), are especially devastating, with an average patient survival of less than 15 months [Mutations at the histone H3 tail, which change the glycine at residue 34 to either valine or arginine (H3G34V/R) in hemisphere tumors, or the lysine to methionine substitution at residue 27 (H3K27M) in DIPGs . SETD2 iH3K4me3 levels play a role in determining the pathogenesis of various human cancers ,13, inclA human SET domain containing 1A/B protein complexes (hSETD1A/B-COMPASS) binds to DNA containing unmethylated CpG motifs and is responsible for trimethylating H3K4 . Human SIn this study, H3K4me3 levels and modifiers for this epigenetic mark were mapped using in-house pediatric glioma specimens and via in silico analysis of multiple publicly available databases. WDR82 and H3K4me3 were found to be elevated in pHGGs and associated with reduced chemotherapeutic sensitivity. A reduction in H3K4me3 by the downregulation of WDR82 decreased H3K4me3 promoter occupancy and gene expression and increased the response of pediatric glioma cells to chemotherapy. Our data suggest that H3K4me3 status is an important determinant of pediatric glioma malignancy and therapeutic response, and that WDR82, which regulates H3K4me3, is a promising target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.Formalin-fixed, paraffin-embedded (FFPE) pediatric glioma samples were used for immunohistochemical analysis. Samples were collected from patients diagnosed in the Department of Pathology, Ann & Robert H. Lurie Children’s Hospital of Chicago  under IRB# 2005–12252 . Clinicopathological information is summarized in 2.Pediatric SJ-GBM2 glioblastoma cells were obtained from the Children’s Oncology Group Cell Culture and Xenograft Repository. Pediatric KNS42 cells were purchased from the JCRB (Japanese Cancer Research Resources) cell bank. The human H3K27M DIPG cell line SF8628 was generously provided by Dr. Rintaro Hashizume . All cells were propagated as monolayers in complete medium consisting of Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  from Gibco , at 37 °C with 5% COA set of SMARTvectors inducible lentiviral shRNA plasmids with hEF1 promoter and TurboGFP reporter against human WDR82 (Entrez Gene 80335) V3SH11252-229426599 , V3SH11252-2289952521 , and non-targeting SMART vector inducible lentiviral with hEF1 promoter and TurboGFP reporter control plasmid (Cat#VSC6572) were purchased from Horizon Discovery Ltd. . These vectors were amplified and purified as per manufacturer’s instructions.Tumor cells were plated a day before transduction, in 6-well plates at 30–40% confluency, to avoid cell clumps and uneven distribution that can reduce the efficacy of viral transduction. Individual wells of confluent cells were treated with packaged lentivirus (100–500 µL) and polybrene 4–8 µg/mL, followed by incubation for 6–14 h. Cell viability and protein expression were monitored. The transduction process was repeated until an optimal amount of GFP protein was expressed. GFP protein expression was checked after 48–72 h using a fluorescent microscope to visualize the fluorescent tag present in the construct. Cells were then selected with antibiotics. In cases where the construct needed a substrate such as doxycycline (Dox) for induction, Dox was added and protein expression was monitored after 24 h, 48 h, and 72 h.Total proteins were extracted with Tissue Extraction Buffer I  with proteinase , phosphatase  inhibitor cocktails, and phenylmethylsulphonyl fluoride . Total histone was extracted using histone extraction kit  as per manufacturer’s instructions. Protein concentrations were quantified with the BCA Protein Assay Kit  using Nanodrop ND-2000 . Equal amounts of cell lysate were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . Blocking was performed for 60 min with 5% nonfat dry milk in Tris-buffered saline and Tween 20, followed by blotting with primary antibodies overnight at 4 °C. Primary antibodies include polyclonal anti-rabbit WDR82 , H3K4me3 , and H3  from Cell Signaling Technology ; β-actin  from Abcam ; hSETD1A from Bethyl Laboratories ); rabbit polyclonal anti-GAPDH  from Santa Cruz Biotechnology. After washing with Tris-buffered saline and Tween 20, membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP) conjugated donkey anti-rabbit antibody , and signal was detected with enhanced chemiluminescence substrate (Bio-Rad Laboratories).http://gliovis.bioinfo.cnio.es/, accessed on 15 March 2022). Data were further analyzed with GraphPad Prism 9.0 for gene expression, gene expression correlation, and survival analysis .Two expression profiling datasets  were downloaded from the GEO database at the NCBI. Original gene expression profiles of glioma were obtained from these datasets. Expression of individual genes was identified with GEO2R. Adult low- and high-grade glioma data in TCGA were processed with the Gliovis online portal . Differential expression was determined using DESeq2. The cutoff for determining genes, which showed significant differential expression, was an FDR-adjusted p-value less than 0.05.Total RNA was prepared using the RNeasy Mini Kit  as per manufacturer’s instructions. Stranded mRNA-seq was conducted in the Northwestern University Sequence (NUseq) Core Facility. Briefly, total RNA samples were checked for quality using RINs generated from Agilent Bioanalyzer 2100 . RNA quantity was determined with the Qubit fluorometer . The Illumina TruSeq Stranded mRNA Library Preparation Kit  was used to prepare sequencing libraries from 1 mg of high-quality RNA samples (RIN = 10). The Kit procedure was performed without modification. The procedure includes mRNA purification and fragmentation, cDNA synthesis, 3′ end adenylation, Illumina adapter ligation, and library PCR amplification and validation. Illumina HiSeq 4000 sequencer  was used to sequence the libraries with the production of single-end, 50 bp reads at the depth of 30-40 M reads per sample. The quality of reads, in FASTQ format, was evaluated using FastQC. Adapters were trimmed and reads of poor quality or aligning to rRNA sequences were filtered. The cleaned reads were aligned to the H. sapiens genome using STAR . Read co® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)  per manufacturer’s instructions. ChIP-seq was conducted in the NUseq Core Facility. Briefly, ChIP and input DNA samples were checked for quality and quantity using Qubit and Agilent Bioanalyzer . After passing quality control), the Illumina TruSeq ChIP Sample Preparation Kit  was used to prepare sequencing libraries from 5-10 ng of ChIP and input DNA. This procedure includes end repair, 3-end adenylation, Illumina adapter ligation, and library PCR amplification and validation. Illumina HiSeq 4000 sequencer was used to sequence the libraries with the production of single-end, 50 bp reads at the depth of 30–40 M reads per sample. After sequencing, the quality of reads, in FASTQ format, will be evaluated using FastQC. Adapters will be trimmed; reads of poor quality will be filtered. The cleaned reads will be aligned to the H. sapiens genome (hg38) using Bowtie [http://homer.ucsd.edu/homer/index.html, accessed on 26 January 2021).Fragmental DNA for ChIP-seq was prepared using SimpleChIPg Bowtie . Peak ca4 cells/100μL were plated in 96-well plates with complete cell culture medium with or without Dox (2 µg/mL), and subjected to 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium  assay. Pediatric high-grade glioma KNS42 cells were seeded into 6-well tissue culture plates and allowed to adhere. Attached cells were treated with or without Dox  at 2 µg/mL. Cells were incubated with Dox for 2 weeks, at which time, colonies were counted following staining with methylene blue (0.66% solution in 95% ethanol). Plating efficiencies were calculated as the ratio of the number of colonies formed to the number of cells seeded. The cell lines were used for different assays due to their distinct growth patterns.SJ-GBM2 and SF8628 cells were used to determine if reducing WDR82 through inducible knockdown affected cell viability; 1 × 105) or KNS42 (2 × 105) cells, transduced with sh-WDR82 vectors, or non-transduced controls, were implanted into the right striatum. Mice were given 1% sucrose or 1% sucrose + 2 mg/mL Dox in their drinking water [2 asphyxiation followed by cervical dislocation when they became moribund . Brains were harvested and fixed with 10% paraformaldehyde in PBS overnight and switched to PBS prior to embedding. The tissue was sectioned onto slides and stained with H&E. Tissue preparation was conducted at the Mouse Histology and Phenotyping Laboratory (MHPL), Northwestern University Feinberg School of Medicine.Briefly, 6–8-week-old athymic nude mice were purchased from Taconic. All mice were housed under aseptic conditions, which included filtered air and sterilized food, water, bedding, and cages. The Institutional Animal Care and Use Committee (IACUC) approved all animal protocols. SJ-GBM2 .Cell proliferation results were read on a Synergy 2 Microplate Reader . Cell survival is presented as a percentage of viable cells compared to the viable cell number in the corresponding control, with the control set as 100%. P values were calculated using two-way ANOVA, with To investigate whether a correlation exists between clinicopathologic variables and H3K4me3, forty-six pediatric low-grade gliomas (WHO grades I and II) and ten pediatric high-grade gliomas (WHO grades III and IV) FFPE specimens were IHC-stained for H3K4me3 . H3K4me3H3K4 is reversibly modified by methyltransferases and demethylases. To identify the key modifier(s) of H3K4 in pHGGs, gene expression profiling with microarray analysis revealed that WDR82, among all H3K4 modifiers, is differentially expressed at significant levels and correlates with WHO-grade malignancy, regardless of histone mutation subtypes A,B. ThesAltered gene expression following WDR82 inducible knockdown. We have shown that WDR82 is highly expressed in pHGGs vs. pLGGs. To investigate the role of WDR82 in pHGGs, pHGG cell lines SJ-GBM2, SF8628, and KNS42 were transduced with SMARTvector inducible lentiviral shRNA targeting WDR82 (shRNA#1 and shRNA#2) or non-targeting control vector . The reshttp://geneontology.org/, accessed on 15 March 2021). The results showed that reducing WDR82 significantly decreases the expression of genes associated with mitosis, proliferation, and DNA repair  at day 5 and sequenced (RNA-seq and ChIP-seq with H3K4me3 antibody immunoprecipitation). The RNA-seq results from SJ-GBM2 cells indicate that 129 genes were differentially (cutoff: 1.5 times) expressed in cells transduced with shRNA#1 and shRNA#2, in comparison to non-significant gene expression changes with non-target shRNA (shNT) controls in the presence or absence of Dox A–C. We fA repair D. ChIP-sA repair E.To investigate if changes in gene expression link to WDR82-mediated H3K4me3 alteration at their promoters, representative genes were selected. The correlation between gene expression and WDR82 was plotted using RNA-seq results from pediatric glioma tissue specimens. Promoter H3K4me3 levels were mapped using ChIP-seq results SJ-GBM2 cells treated with the Dox inducible shRNA vector against WDR82 (shRNA#2), in the absence and presence of Dox. The representative results showed WDR82 did not correlate with the expression of NUP62 and FBOX10, genes for which levels increased in pediatric glioma tissue specimens A. AdditiTo investigate whether WDR82 levels affect pHGG cell growth, equal numbers of cells were plated in 96-well plates in the absence and presence of Dox (2 µg/mL), with relative cell numbers compared at 0~7 days by MTS assay. OD values for Dox-treated groups were normalized based on the values of corresponding untreated cells. Inducible knockdown of WDR82, following Dox treatment, significantly suppresses cell growth in SJ-GBM2 and SF8628 cells A. InduciCDK2 is associated with CCND1, which regulates cell proliferation, the cell cycle, and DNA damage repair in childhood cancer through the regulation of Rb . CDK2 deIncreased WDR82 expression is associated with tumor malignancy grade A. Recent5 cells (SJ-GBM2 transduced with shNT and WDR82 shRNA#2) were inoculated intracranially into the right striatum of 6–8-week-old athymic nude mice. Mouse housing and tissue processing as described under in vivo studies. Tumor size and mitotic cells are not significantly different in non-targeted shRNA (shNT) groups with or without Dox. In contrast, tumor size is smaller and there are fewer mitotic cells in the group treated with Dox, as indicated by comparing results from animals given Dox vs. 1% sucrose, (WDR82 contributes to tumorigenesis, malignant phenotype, and tumor proliferation of multiple human cancers ,34. To isucrose, C–E. The sucrose, F. These In a previous study, we established that H3K4me3 is associated with WHO-grade malignancy in pediatric ependymomas . In thisThe discovery of recurrent histone mutations in children with pHGGs in the last decade revealed that PTMs on histone tails are one of the milestone changes whose role must be elucidated in cancer research. In pediatric gliomas, H3G34V/R in hemisphere tumors and H3K27M in midline gliomas including DIPG are common histone mutations . H3K4me3Among the six methyltransferases for H3K4me3, SETD1A is associated with WHO malignancy in pediatric gliomas . InvestiWDR82 activity is a key factor in maintaining embryonic  and cancIn the present study, WDR82-mediated H3K4me3 alters the gene expression of NUP43 , PKMYT1 In summary, H3K4me3 and WDR82 are elevated in pHGGs vs. pLGGs. WDR82-mediated H3K4me3 is associated with the expression of genes involved in regulating stem cell features, cell mitosis and proliferation, the cell cycle, and DNA damage repair. Reduction of WDR82 increased the response of pediatric glioma cells to chemotherapy. Inducible reduction of WDR82 decreases pHGG tumor cell growth in vivo and extends animal survival. These findings suggest that WDR82-mediated H3K4me3 is a significant factor in pediatric glioma, and further investigation of WDR82 as a promising epigenetic therapeutic target for pHGG is warranted.


PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are involved in light-modulated changes in growth orientation. They act downstream of phytochromes to control hypocotyl gravitropism in the light and act early in phototropin signaling. Despite their importance for plant development, little is known about their molecular mode of action, except that they belong to a protein complex comprising phototropins at the plasma membrane (PM). Identifying evolutionary conservation is one approach to revealing biologically important protein motifs. Here, we show that PKS sequences are restricted to seed plants and that these proteins share 6 motifs (A to F from the N to the C terminus). Motifs A and D are also present in BIG GRAIN, while the remaining 4 are specific to PKSs. We provide evidence that motif C is S-acylated on highly conserved cysteines, which mediates the association of PKS proteins with the PM. Motif C is also required for PKS4-mediated phototropism and light-regulated hypocotyl gravitropism. Finally, our data suggest that the mode of PKS4 association with the PM is important for its biological activity. Our work, therefore, identifies conserved cysteines contributing to PM association of PKS proteins and strongly suggests that this is their site of action to modulate environmentally regulated organ positioning. Members of the PHYTOCHROME KINASE SUBSTRATE (PKS) family are characterized by six conserved sequence motifs, one of which is required for plasma-membrane association and biological activity of PKS4. Background: Plants can orient their leaves towards the light. This is known as phototropism and fascinated scientists since Charles Darwin. Phototropism is initiated by a blue light photoreceptor called phototropin. Light activates phototropin's protein kinase activity (an enzyme that phosphorylates proteins). This is followed by a series of poorly understood events, which lead to the redistribution of the growth hormone auxin in the stem resulting in growth towards the light. PHYTOCHROME KINASE SUBSTRATE 4 (PKS4) is phosphorylated by phototropin and is believed to act between phototropin light activation and auxin redistribution. The mechanism of PKS4 action is unknown because the protein does not contain any domains of known biochemical activity. PKS4 associates with the plasma membrane (PM), where it was postulated to act.Question: We wanted to understand how PKS4 associates with the PM and determine whether this subcellular localization is important for its biological activity.Findings: Functionally important parts of proteins are typically conserved over evolutionary timescales. We, therefore, identified genes coding for PKS proteins and found that they are present in seed plants and comprise 6 conserved sequence motifs. We showed that one of these motifs is important for efficient PKS association with the PM in Arabidopsis. Cysteine residues of this motif are modified with lipids, which presumably contribute to PM association. Moreover, in PKS4, these residues are important for its function in phototropin signaling in Arabidopsis.Next steps: Our study identified a PKS4 sequence motif that is important for biological activity and subcellular localization. However, we still don’t know what the rest of the protein does and how it may be important in linking phototropin activation and auxin redistribution. We hope that studying other conserved motives identified here will allow us to answer this question.Arabidopsis thaliana), etiolated (dark-grown) seedlings grow following the direction of the constantly present gravity stimulus: roots grow downwards while shoots grow upwards. However, in response to light, hypocotyls orient their growth following the integration of 2 main signaling pathways: gravitropism (repressed) and phototropism (activated)  . Growth tivated) . Additiotivated) . Althougtivated) .Gravitropism signaling comprises the perception of the gravity vector, which requires sedimentation of starch-filled amyloplasts for signal generation and transduction, resulting in hypocotyls growing upwards . In ArabArabidopsis has 2 phototropins, which exhibit specific and partially redundant functions: phot1 and phot2. Hypocotyl phototropism is mainly mediated by phot1 . PhototrPKSs are expressed in the hypocotyl elongation zone, consistent with their importance during hypocotyl growth regulation (PKSs are a family of basic hydrophilic proteins that do not contain domain(s) of known function. They were initially identified as phytochrome-binding proteins that regulate phytochrome signaling . Surprisgulation . PKS1, Pgulation . However2P2 sequence predictor (PKS homologs using the Orthologous matrix (OMA) browser , we identified 2 PKS genes each, which group into the PKS4 subfamily and a subfamily formed by the ancestors of PKS1, PKS2, and PKS3 . Based on the limited data available from gymnosperms and the limited bootstrap support throughout the tree, the most parsimonious interpretation of our data is that PKS was present as a single copy in the ancestral spermatophyte genome . We hypothesize that there was a duplication in the ancestral angiosperm to form 2 copies: PKS4 and PKS1/2/3. Another duplication occurred after the divergence of Amborella, P. americana, and N. colorata, giving rise to PKS1/2 and PKS3 in the ancestral Mesangiospermae. An additional Brassicaceae-specific duplication gave rise to PKS1 and PKS2. PKS4 genes are present in all analyzed monocot and dicot genomes .Using the Dredictor , we deteredictor . To defi browser  and manu browser . This annosperms . Howeverng MAFFT , removinng MAFFT , and theng MAFFT . The resng MAFFT . Howeverng MAFFT . In the  genomes . EudicotP<0.05) between motifs C and F, suggesting that parts of these motifs are related to each other  35 promoter. We first confirmed that the full-length (ABCDEF) PKS1-GFP signal was consistent with the previously reported PM localization  residues  and selected 3 independent single insertion lines accumulating PKS4 to levels comparable to a wild-type PKS4-HA control line (WT-3) (PKS4 WT-3 line showed complementation of the phototropic phenotype of the pks4 mutant as previously shown (PKS4 C* lines complemented the mutant (PKS4 C*-2) exhibits an even stronger phototropic defect than the pks4 mutant, suggesting that the expression of the PKS4 C* variant might interfere with the molecular mechanism underlying phototropism. To look at the rapid phototropin-mediated response, we analyzed the light-induced reduction of PKS4 mobility on SDS-PAGE gels triggered by phototropin-mediated PKS4 phosphorylation   (Schumace (WT-3) . The PKSly shown    with similar protein abundance as in our PKS4 WT control line  was fused to an N-terminal myristoylation (myri) sequence. We selected single insertion lines (myriPKS4 WT 1-4 and myriPKS4 C* 1-3) with comparable protein levels as in our PKS4 WT and PKS4 C* lines  to have a stronger phototropic response than PKS4 C* or Cys-12-Ser alone  displaying a slightly more robust phototropic response than the PKS4 C* construct . PKS1 and PKS2 were inferred by OMA to be in the same gene family (rooted at the Magnoliopsida level), and PKS3 and PKS4 were in 2 other families, rooted at the Pentapetalae and Mesangiospermae levels, respectively. A final, smaller gene family was found in OMA by searching for the A. trichopoda PKS4 gene, which was rooted at the Magnoliopsida level. Seventeen additional PKS sequences not present in the OMA database, selected to increase phylogenetic diversity, were added to cover the following: the basal angiosperms A. trichopoda and N. colorata; the monocots purple false brome (Brachypodium distachyon) and foxtail millet ; the magnoliid avocado (P. americana); the asterids potato (Solanum tuberosum) and tea plant (Camellia sinensis); the rosid wild strawberry (Fragaria vesca); and the Caryophyllales sugar beet (Beta vulgaris). The aforementioned sequences were obtained by reciprocal BLAST searches using Arabidopsis PKS1 and PKS4 protein sequences as query  from the January 2020 release of the OMA browser . The firas query . Two gymerms 1.0 . Thus, aerms 1.0 .The 172 sequences were aligned using MAFFT v7.313 , with thThe alignment was used to make a gene tree of all PKS sequences using IQ-TREE web server version 1.6.12  with theTo identify motifs in the highly gapped alignment, GLAM2 was utilized . Since Ghttps://meme-suite.org/meme/tools/tomtom)  . The Sanhttps://meme-suite.org/meme/tools/meme)   The BIG ls/meme)  using ArA. thaliana) Columbia-0 accession. The pks4-2 allele (GABI_312E01) was utilized in this study (35S:PKS1-GFP (pCF202), 35S:PKS4-GFP (pIS03), and PKS4pro:PKS4-3XHA (pPS09) in pks4-2 were previously described . The PKS1 cDNA from plasmid pCF173 was amplified with the following primer combinations (CF129/CF470) corresponding to amino acids (aa) 1–160 of PKS1 (AB); (CF129/CF471) corresponding to aa 1-273 (ABC); (CF472/CF473) corresponding to aa 274-439 (DEF); (CF507/CF471) corresponding to aa 161-273 (C), and (CF507/CF473) corresponding to aa 161-439 (CDEF). The PCR products were digested with Kpn1 and BamH1 and ligated into digested pCF203 to obtain pCF524 (AB), pCF525 (ABC), pCF526 (DEF), pCF534 (C), and pCF535 (CDEF). Mutations of the 3 conserved Cys residues in motif C were obtained by site-directed mutagenesis using pCF173 as a template to generate pCF546 (ABC*), pCF547 (ABC*DEF), and pCF550 (C*DEF). Mutations of the conserved Cys residues in motif F (Cys-378) were also obtained by site-directed mutagenesis using pCF173 as a template to obtain pCF393. For PKS4 C* lines, a fragment encoding PKS4 ABC (aa 1 to 186 of PKS4 with Cys-143 and Cys-145 mutated to Ser) was ordered at Eurofins and after digestion with XmaI/Bpu10I was ligated into a 35S:PKS4-GFP construct to replace the wild type with the mutant sequence to obtain pCF561. For the generation of the PKS4pro:PKS4 C*-3xHA lines (pAL10), a 517-bp DNA fragment containing PKS4 encoding the C143S and C145S variants was digested from pCF561 with the restriction enzymes EcoRV and NruI and replaced in the pPS9 construct , a synthetic 517-bp DNA fragment of the PKS4 coding sequence harboring the C143S or C145S mutation and flanked by the EcoRV and NruI sites was ordered from Eurofins and replaced in the pPS9 construct previously digested with the same restriction enzymes to replace the wild type with the mutant sequence. Similarly, for the generation of the PKS4pro:PKS4 F*-3xHA lines (pAL40), a synthetic 682-bp DNA fragment of the PKS4 coding sequence carrying the C358S mutation and flanked by the EcoRV and BamHI sites was ordered from Eurofins and replaced in the pPS9 construct previously digested with the same restriction enzymes to replace the wild type with the mutant sequence. For the generation of the PKS4pro:myriPKS4-3xHA (pAL63) and PKS4pro:myriPKS4 C*-3xHA (pAL64) and PKS4pro:myriPKS4 Cys12*-3xHA (pAL86) lines, a synthetic 171-bp DNA fragment of the PKS4 coding sequence containing the last part of the PKS4 promoter and the myristoylation signal sequence ATGGGAATTTGTATGTCTAGA followed by the beginning of the PKS4 coding sequence was ordered and digested with the restriction enzymes XhoI and NruI and replaced in the pPS9, pAL10, and pAL80 constructs previously digested with the same restriction enzymes. For the generation of the PKS4pro:PKS4-3xHAfarn (pAL70) and PKS4pro:PKS4 C*-3xHAfarn (pAL71) lines, a synthetic 225-bp DNA fragment including the last part of the PKS4 coding sequence, the 3xHA tag sequence followed by the farnesylation sequence TCT AAG GAT GGA AAG AAG AAG AAG AAG AAG TCT AAG ACT AAG TGT GTT ATT ATG, and a very short fragment of the backbone vector flanked by the unique restriction enzymes sites BamHI and PstI was replaced in the pPS9 and pAL10 previously digested with the same restriction enzymes. For the generation of PKS4pro:PKS4-GFP (pAL43) lines, a DNA fragment containing the PKS4 pro:PKS4-GFP:term in pED10 (OLE1pro:OLE1-FastRed for selection of transgenic plants) previously digested with the same enzyme. For the generation of the PKS4pro:PKS4 C*-GFP (pAL45) and PKS4pro:PKS4 F*-GFP (pAL65), PKS4pro:PKS4 Cys10*-GFP (pAL81), and PKS4pro:PKS4 Cys12*-GFP (pAL82) lines, a DNA fragment encoding PKS4 with C143S and C145S, or C358S, or C143S, or C145S mutations, respectively, was digested from the pAL10, pAL40, pAL79, and pAL80 vectors with the restriction enzymes NruI and ZraI and replaced in pAL43 previously digested with the same enzymes. For the generation of the PKS4pro:myriPKS4-GFP (pAL61), PKS4pro:myriPKS4 C*-GFP (pAL62), and PKS4pro:myriPKS4 Cys12*-GFP (pAL87) lines, the same synthetic 171-bp DNA fragment used for the generation of pAL63 and pAL64 was digested with the restriction enzymes XhoI and NruI and replaced in the pAL43, pAL45, and pAL82 previously digested with the same restriction enzymes. For the generation of the PKS4pro:PKS4-GFPfarn (pAL67) and PKS4pro:PKS4 C*-GFPfarn (pAL68) lines, the same synthetic 1,557-bp DNA fragment used for the generation of pAL70 and pAL71 was replaced in pAL43 and pAL45 previously digested with the same restriction enzymes. All constructs were verified by Sanger sequencing. Primers are provided in Agrobacterium tumefaciens)-mediated transformation with Agrobacterium GV3101  at 22 °C, and plates were placed back in the dark at 19 °C or 22 °C for 3 d before light exposure. For inhibition of gravitropism experiments, germination was induced by 1 h of RL (50 μmol m−2 s−1) at 22 °C and plates were placed back in the dark at 22 °C for 1 d before light exposure.For seed production, plants were grown on soil at 22 °C with 16 h of white light (WL) per day. For physiological experiments, seeds were surface-sterilized in 70% (v/v) ethanol and 0.05% (v/v) Triton-X for 5 min and in 100% ethanol for 5 min. Seeds were then sown on Petri plates containing half-strength MS medium, pH 5.7, 0.8% (w/v) agar. Plates were stored in the dark for 3 d at 4 °C for stratification. For experiments with dark-grown seedlings, germination was induced by 4 to 6 h of WL  of each hypocotyl with respect to the vertical after light treatment, as determined by a customized MATLAB script developed in the Fankhauser Lab. One-way ANOVA (aov) and Compute Tukey's Honest Significance Differences (HSD.test) [agricolae package] were performed in R. We considered 35S:PKS4-GFP and 35S:PKS4_C*- GFP. Excitation was accomplished using an Argon laser at 488 nm and detection used a bandpass emission between 505 and 530 nm. For some images, the PM was stained with the dye FM4-64  at a concentration of 50 μM, by soaking the seedlings for 1 min and then washing 3 times in liquid half-strength MS medium. In that case, the signal was detected with a long pass emission from 650 nm. Model LSM 880 was used for imaging PKS4pro:PKS4 F*-GFP and PKS4pro:PKS4 C*-GFPfarn. Samples were excited with an argon laser (488 nm) and detection was done between 495 and 518 nm for GFP and between 607 and 691 nm for chlorophyll and red fluorescent protein (RFP). Leica Stellaris 5 was used to image PKS4pro:PKS4-GFP, PKS4pro:PKS4 C*-GFP, PKS4pro:PKS4 Cys10*-GFP, PKS4pro:PKS4 Cys12*-GFP, PKS4pro:myriPKS4-GFP, PKS4pro:myriPKS4 C*-GFP, PKS4pro:myriPKS4 Cys12*-GFP. The objective used was HC PL APO 63×/1.40 OIL. Samples were excited with a 488-nm laser and fluorescence was detected between 496 and 520 nm for GFP, 580 to 585 nm for RFP, and 670 to 690 nm for protochlorophyllide. For each plant, between 30 and 40 images were taken at different z positions, every 1 µm starting in the epidermis and finishing in the second cortex layer. Images were taken below the apical hook, where PKS4 expression is the highest. Images were edited using ImageJ. In Confocal microscopy images were taken with a confocal microscope model LSM 510 (Zeiss), LSM 880 (Zeiss), or Stellaris 5 (Leica). Model LSM 510 was used for all PKS1 imaging and for µL of high capacity neutravidin-agarose beads (Thermo Fisher) instead of 15 µL.Palmitoylation of the PKS1 fragments was assayed as described in µL in an Eppendorf tube) were sown onto plates containing half-strength MS medium solidified with 08% (w/v) agar. After stratification for 3 to 5 d, germination was induced with 6 h of WL (80 μmol m−2 s−1). Individual plates were incubated at 22 °C for 3 d in darkness. Seedlings were collected, weighted, and ground with a mortar and pestle on ice. Two volumes of extraction buffer  were added. Samples were centrifuged for 5 min at 1600 rpm (200 g) in a benchtop centrifuge at 4 °C. The supernatant corresponds to the total fraction, S1. The supernatant was centrifuged again for 75 min at 100,000 × g at 4 °C. The supernatant corresponds to the soluble fraction, S2. The pellet was resuspended in 1 volume of extraction buffer with 0.5% (v/v) Triton X-100 and centrifuged again for 5 min at 14,000 rpm  in a benchtop centrifuge at 4 °C. The supernatant corresponds to fraction S3.One thousand seeds per replicate (100 µL of protein extract was incubated with 50 µL of magnetic beads for 90 min at 4 °C. The first 4 washes were done with extraction buffer (as defined above) + Triton X-100. Elution was done with 2× Laemmli buffer.Co-IP was done using a Miltenyi µMACS HA isolation kit (130-091-122), μ Columns (130-042-701), and a μMACS Separator (130-042-602) following the manufacturer's guidelines with a few modifications. The microsomal fraction S3 was used for the Co-IP. Briefly, 340 µL 2× Laemmli buffer for 20 mg fresh weight) and 10 µL was loaded per lane. Proteins were separated on 4% to 15% precast polyacrylamide gels except for data shown in For the biotin switch assay, proteins extracted in 2× Laemmli buffer were separated on 12% SDS/PAGE gels and transferred onto nitrocellulose membrane in CAPS buffer. Cell fractionation samples and Co-IP samples were mixed with 1 volume of 2× Laemmli buffer. For the cell fractionation assay, twice as much was loaded of S1 and S2 than S3. For all other immunoblots, total proteins were extracted in Laemmli buffer , PKS2 (At1g14280), PKS3 (At1g18810), PKS4 (At5g04190), NPH3 (At5g64330), BG1 (At5g12050), BG2 (At3g42800), BG3 (At1g54200), BG4 (At3g13980), BGL1 (At1g69160), BGL2 (At1g13670).The Arabidopsis Genome Initiative numbers for the genes mentioned in this article are as follows: koad096_Supplementary_DataClick here for additional data file.


HPV11 E6-mediated Yap activation was observed in 2D and 3D (organotypic raft) cell culture systems and HPV-infected lesions, with both NHERF1, which is a regulator of the Hippo and Wnt pathways, and E6AP, playing an important role. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways has not previously been defined. Our study suggests a model in which the preserved functions of the low and high-risk Alpha E6 proteins modulate epithelial homeostasis via E6AP activity, and lead to alteration of multiple downstream pathways, including those involving NHERF1 and YAP.Human papillomaviruses (HPV) cause persistent infections by modulating epithelial homeostasis in cells of the infected basal layer. Using FUCCI and cell-cell competition assays, we have identifed regulatory roles for E6AP and NHERF1, which are the primary HPV11 E6 cellular targets, as well as being targets of the high-risk E6 proteins, in processes governing epithelial homeostasis . Depletion of E6AP, or expression of HPV11 or 16E6 increased keratinocyte cell density and cell cycle activity, and delayed the onset of differentiation; phenotypes which were conspicuously present in HPV11 and 16 infected patient tissue. In line with proposed E6 functions, in HPV11 condyloma tissue, E6AP and NHERF1 were significantly reduced when compared to uninfected epithelium. In experimental systems, loss of HPV11 E6/E6AP binding abolished 11E6’s homeostasis regulatory functions, while loss of E6/NHERF1 binding reduced the cell density threshold at which differentiation was triggered. By contrast, a NHERF1-binding mutant of 16E6 was not compromised in its homeostasis functions, while E6AP appeared essential. RNA sequencing revealed similar transcriptional profiles in both 11 and 16E6-expressing cells and E6AP Human papillomaviruses are classified as ‘high-risk’ and ‘low-risk’ types. Both groups of viruses establish a reservoir of infection in the epithelial basal layer. To maintain persistence and support the productive lifecycle, early viral proteins such as E6 interact with cellular targets to modulate the epithelial homeostasis pathways. Here we have used a cell cycle indicator system, together with cell-cell competition assays to assess the functions of HPV E6 protein in keratinocytes. We found that both ‘low-risk’ and ‘high risk’ E6 proteins drive cell proliferation, and delay cell contact inhibition, delamination and differentiation in our model system, which are the four necessary steps to maintain the balance of basal cell expansion and loss. We identified that cellular proteins E6AP and NHERF1 both contribute to E6 functions, and discovered that E6AP itself also regulates keratinocyte differentiation. The key downstream pathways regulated by E6/E6AP include the Hippo and Wnt pathways. Its activation is known to promote cell proliferation and inhibit cell differentiation. Therefore, we propose a model in which E6 activates YAP activity by inhibiting E6AP and NHERF1 in the basal layer to support homeostasis. These findings will enhance our understanding of the nature of chronic lesions and self-limiting HPV-associated diseases. Human papillomaviruses (HPV) are non-enveloped double-stranded DNA viruses that infect multiple epithelial sites,2. So faThe epidermis is a stratified epithelium composed of morphologically distinct cellular layers that reflect the terminal differentiation process of keratinocytes . Basal kIt has been reported previously that the Alpha genus E6 proteins are the only group that preferentially interacts with E6AP rather than MAML1, with some E6 proteins further acquiring the ability to induce E6AP degradation. E6 is t-/- keratinocyte cell lines, our results further suggest that E6AP may function as a homeostasis modulator even in the absence of E6, with transcriptome analysis indicating a decline in differentiation-related gene expression in keratinocytes. Curiously, E6 expression or E6AP depletion activated a similar subset of YAP target genes involved in cell proliferation. Our results suggest that E6 regulates key homeostatic processes in the epithelial basal layer by inhibiting E6AP function, and that this is complemented in 11E6 expressing cells by the downregulation of NHERF1. We suspect that disruption of the homeostatic pathway may have detrimental effects on viral persistence and offers attractive targets for therapeutic approaches.Low-risk HPVs successfully survive and cause chronic lesions in the epithelium . Thus, lin vitro, this is possible for HPV16. Organotypic rafts expressing 16E6 and HPV16 whole genome . Both mutants were shown to be deficient for NHERF1 degradation  allele remained. Consistent with this, immunoblotting showed loss of E6AP expression   . K10 expncreased . These rns of E6  must als-/- cells that grew until post-confluence. With the standard mRNA read depth of around 20 million reads/sample, 3824 genes were differentially expressed with fold-change > = 2 and adjusted P< = 0.05 in E6AP-/- cells  downregulation is found in E6-expressing and E6AP-/- NIKS cells  in addition to NIKS, the use of organotypic raft culture, and most importantly, the evaluation of in vivo patient-derived clinical material  of The Galilee Medical Center. Approval Number NHR 0202–18 on March 12, 2018. The study was determined by the IRB to be exempt from obtaining patients’ consents forms. The mode of collection, processing, and patient data-handling of the clinical samples used in this study have been described previously .NIKS , a HPV-negative spontaneously immortalised human keratinocyte cell line, was maintained at sub-confluence on γ-Irradiated J2 3T3 feeder cells (a gift from Paul Lambert) in F medium with all supplements as previously described . 293T A.W133R, pQCXIN-Flag11E6L111Q, pQCXIN-Flag11E6L70A, pQCXIN-Flag16E6L50G, pQCXIN-Flag16E6F69A were constructed using a KOD-Plus-Mutagenesis Kit , prior to DNA sequencing to ensure that no additional base changes were present. The primer sequences used for mutagenesis are listed in pSpCas9(BB)-2A-Puro (PX459)-E6APgRNA plasmid and pSpCas9(BB)-2A-Puro (PX459)-scrambled control gRNA plasmid were kind gifts from Lawrence banks  and Dr. 5 cells were seeded in each well of a six-well plate the day before transduction. Cells were inoculated with viruses at MOI>1 in the presence of 4 μg/ml of Polybrene (Santa Cruz). Stable NIKS populations were generated following selection with puromycin (10 μg/ml), G418 (50 μg/ml) or hygromycin (10 μg/ml).The production and infection of recombinant retroviruses were accomplished as previously described ,101. To 7 cells/ml with Rat Tail Collagen Type I  to make the dermal equivalent. Dermal equivalent was allowed to contract in DMEM for four days before NIKS cells were plated on at a density of 1.5 x 106 cells/50 μl. Organotypic rafts were firstly cultured in FC media to allow attachment and expansion, followed by cornification media [Raft cultures were established as previously described ,102. EF-on media  to facilImmunofluorescence was performed as described previously . The forViral RNA in cells were detected and visualized with RNAscope in situ hybridization assay  following the manufacturer’s instructions. The probe used for low-risk E6/E7 RNA detection was RNAscope Probe-HPV6/11 (415211).4 NIKS cells of each mCherry and eGFP were seeded with 6 x 103 irradiated J2-3T3 feeder cells. Cells were cultured for up to 9 days and media was changed every other day. Cells were fixed at day 1, 3, 5, 7 and 9 by incubating in 4% PFA for 30 minutes. Bottom layer and second layer of the cells were visualised and scanned by Harmony Opera Phenix high content imaging system at MRC Institute of Metabolic Science (IMS), Cambridge. Magnification 20x, Field size 3.78 mm2.NIKS cells stably expressing both FUCCI cell cycle indicator and E6 were plated in CellCarrier-96 well ultra Microplates (Perkin Elmer) at 10 different densities, ranging from 6000 cells/well to 60,000 cells/well. FC media was changed at 24 hours after seeding and cells were fixed at 72 hours after seeding using 4% PFA. For competition assay, NIKS were seeded at high (confluent) density in CellCarrier-96 well ultra Microplates. To each well, 2.4x10Proteins were extracted from cells using RIPA buffer and quantified using the BCA protein assay kit (Pierce), before being separated on 4–12% gradient polyacrylamide-SDS-Tris-Tricine denaturing gel (Invitrogen) and transferred onto PVDF membranes . After transfer, membranes were blocked for 1 hour at room temperature in 5% milk in TBS. Blots were then incubated overnight at 4°C with appropriate primary antibody diluted in 5% milk in TBS. This is followed by incubating with appropriate IRDye 800cW fluorescent secondary antibody (Licor) for an hour at room temperature. Protein bands were detected with Odissey imaging system (Licor). Primary antibodies used in this study are listed in -/-, or NIKS transduced with E6 using the RNeasy mini kit (Qiagen). PolyA selection, reverse transcription, library construction, sequencing and bioinformatics analysis were performed by Novogene. Differentially expressed genes were selected based on a log2 (FoldChange) > = 1 & padj < = 0.05 cut-off and were analysed for enriched biological processes using the GO (Gene Ontology) enrichment analysis tool. FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) values were calculated to estimate gene expression levels from RNA-seq results.Total RNA was extracted from three independent clones of NIKS parental control cells, NIKS-E6APPTGS2, PLAU, AREG, AXL, CTGF and E6 gene and GAPDH were measured by ViiA 7 Real-Time PCR system (Life Technologies) using Fast SYBR master mix (Applied Biosystems) with 15 minutes denaturation at 95°C, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The PCR primers for qPCR are listed in Genomic DNA from NIKS for qPCR was purified using QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions. All samples were digested with DpnI to remove any residual input DNA prior to analysis. Total RNA from NIKS was purified by using the RNeasy Mini Kit (Qiagen), with genomic DNA removed by Turbo DNA-free kit (Invitrogen). cDNA was synthesised with SuperScript III Reverse Transcriptase (Thermo Fisher scientific) using 100 μM oligo dT, according to the manufacturer’s instructions. The YAP-responsive genes 2.Images of 9 fields in each well of the 96 well plate were captured by High content confocal imaging at x20 magnification (4 channels: DAPI (461nm), geminin (488nm), Cdt1 (594nm) and K10 (650nm). % geminin positive cells, number of cells and % K10 positive cells were quantified with Harmony image analysis software (Perkinelmer). Same thresholds were applied to all plates. Size of each field = 3.78 mmS1 Fig(A) NHEK cells or NIKS cells stably expressing either 11E6 or 16E6 were compared at similar densities for the expression level of MCM (green) and K10 (red). Scale bar = 200 μm (B) Graphs comparing the saturation density, cells in cycle (at saturation), and differentiation commitment at saturation among EV-transduced, 11E6 or 16E6 expressing NHEK and NIKS cell lines. Statistical significance was calculated with one-way ANOVA. *, P ≤0.05; ***, P ≤0.001; ****, P ≤0.0001.(TIF)Click here for additional data file.S2 Fig(A) NIKS rafts stably expressing 16E6 or maintaining the HPV16 full genome were stained with K10  and MCM (Abcam ab52489) and compared to NIKS-EV control raft. Scale bar = 200 μm. (B) Basal cell density, cells in cycle in the basal layer, and differentiation commitment were quantified by calculating the mean values of nuclei per mm, % MCM7-positive cells in the basal layer, and distance from the basal lamina to K10 positive cells (μm). Error bars representant standard errors. Statistical significance was calculated with one-way ANOVA. *, P ≤0.05; **, P ≤0.01; ****, P ≤0.0001.(TIF)Click here for additional data file.S3 FigK73A, 11E6L70A, 16E6K72A and 16E6F69A. Cells were cultured at 8x 106 cells/well in six-well plates and cell lysates were collected for western blotting. NHERF1  and GAPDH  primary antibodies were used to detect specific protein bands.NIKS cells were retrovirally transduced with vectors encoding 11E6, 16E6, 11E6(TIF)Click here for additional data file.S4 Fig(A) E6 mRNA expression level in NIKS cell lines stably expressing wild type E6 was compared with qRT-PCR. Results for three independent samples for each cell line were included in the analysis. Error bar shows the standard error. (B) E6 protein levels from cell extracts are shown by western blot analysis. Positive control is a flag-tagged vaccinia protein C6 with similar size to E6. GAPDH is used as house-keeping gene.(TIF)Click here for additional data file.S5 FigE6/E7 RNAScope, MCM7, K10, E6AP and NHERF1 immunofluorescence stainings were carried out on adjacent sections. Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. Lower power images are shown on the left. Enlargement images of non-infected and lesion area are presented with separated channels. The dotted lines indicate the position of the basal layer.(TIF)Click here for additional data file.S6 Fig(A) H&E staining (A), E4/MCM (B), K13 (C), and K10 (D) immunofluorescence stainings were performed on adjacent sections of high-risk HPV-infected low-grade squamous intraepithelial lesions (LSILs). Nuclei were counterstained with DAPI (blue). Scale = 200 μm. Both low power images and enlargement images of non-infected and lesion area are presented. The dotted lines indicate the position of the basal layer. (E) The graphs show quantifications of basal cell density, cells in cycle and differentiation commitment. Mean values of nuclei per mm, % MCM positive cells in the basal layer, and distance from the basal lamina to K13-positive cells were calculated and displayed with standard errors. Student t tests were performed .(TIF)Click here for additional data file.S7 Fig(A) NIKS cells expressing either 11E6 or 16E6 were lysed and subject to western blotting for E6AP (Merck). (B) NIKS cells expressing either 11E6 or 16E6 were used to establish organotypic rafts, followed by immunofluorescent staining with E6AP . and DAPI. Scale bar = 200 μm.(TIF)Click here for additional data file.S8 FigThree condyloma biopsies from different patients were stained with active YAP antibody . Nuclei were stained with DAPI. Non-infected and lesion tissue areas are presented with separated channels. Scale bar = 200 μm.(TIF)Click here for additional data file.S9 FigNIKS cells stably expressing shRNA targeting E6AP were transduced with plasmids expressing either 11E6 or 16E6. Cells were fixed at post-confluence and stained with active YAP antibody . Scale bar = 200 μm.(TIF)Click here for additional data file.S10 FigW133R, 11E6L111Q and 11E6L70A were harvested at high cell density and analysed with western blotting. Specific bands were detected using total YAP , active YAP (Abcam) and Ser127 YAP  antibodies.NIKS cells stably expressing 11E6, 16E6, 11E6(TIF)Click here for additional data file.S11 FigNIKS cells were transduced with lentiviral vectors encoding shRNA targeting either luciferase or E6AP. After selection, NIKS rafts were established, followed by H&E (left) and immunofluorescent staining (right) with K10 and DAPI. Scale bar = 200 μm.(TIF)Click here for additional data file.S12 Fig-/- expressing either 11E6 or 16E6 and NIKSE6AP-/—EV. Each dot on the graph shows the % K10 positive cells against cell density per field (3.78 mm2) for each cell line. The red arrow indicates E6AP-independent functions of E6.E6AP KO NIKS cell lines were transduced with plasmids expressing either 11E6 or 16E6 and seeded at different densities in 96 well plates. Levels of K10 were measured and compared among control NIKS cells transduced with scrambled gRNA plasmid, NIKS-E6AP(TIF)Click here for additional data file.S13 Fig(A) Total number of differentially expressed genes (DEG) (purple), number of up-regulated genes (blue) and number of down-regulated genes (red) in each experimental group. (B) Venn diagram shows the number of co-expressed genes between the samples. (C) The X-axis displays the selected gene ontology (GO) terms that are the most relevant and significant, which ranks from left to right according to degree of significance. The y-axis shows the number of genes that were up-regulated (blue) or down-regulated (red) under each GO term.(TIF)Click here for additional data file.S14 Fig-/- or control cells transfected with either HPV16 or HPV11 genome over 5 passages. The viral genome copy number per cell was measured by qPCR, using GAPDH to estimate cell number. Three biological repeats were included for each passage. Error bar indicates standard errors across three samples.Genomic DNA was extracted from NIKS E6AP(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Abnormal levels of neurotransmitters are closely associated with some mental disorders. Therefore, accurate analysis of neurotransmitters is of great clinical importance. Electrochemical sensors have shown bright application prospects in the detection of neurotransmitters. In recent years, MXene has been increasingly used to prepare electrode materials for fabricating electrochemical neurotransmitter sensors due to its excellent physicochemical properties. This paper systematically introduces the advances in MXene-based electrochemical (bio)sensors for the detection of neurotransmitters (including dopamine, serotonin, epinephrine, norepinephrine, tyrosine, NO, and H Some cations and organic molecules can be used as intercalators to weaken the interaction forces between MXene layers, resulting in single- or few-layer MXene structures. The use of intercalation agents depends on the etching methods. With acid/fluoride salt etching, cations can be directly intercalated between adjacent MXene layers, forcing them to separate by sonication. When etched with HF, MXene layers can only be delaminated by adding certain cations or organic molecules  ,48. It id Nb2CTx ,48. Surfd Nb2CTx ,50. Somen, etc.) . For biopability ,51,52. Ipability ,53,54.2S). Various analytical methods have been applied to detect the analytes, including amperometry, differential pulse voltammetry (DPV), square wave voltammetry (SWV), and cyclic voltammetry (CV). To date, a certain number of MXene-based electrochemical (bio)sensors have been constructed for the detection of neurotransmitters. The analytes detected by these (bio)sensors can be grouped into three categories: biogenic amines , amino acids (Tyr), and soluble gases  [3C2Tx promoted selective accumulation of positively charged DA. The Nafion-stabilized Ti3C2Tx/GCE-based sensor showed much better electrochemical performance than that of a reduced graphene oxide (rGO)-based sensor under similar experimental conditions. A mixed phase MXene (Ti–C–Tx) was synthsized and then deposited on GCE for DA analysis [x/GCE showed excellent electrocatalytic activity and could clearly distinguish AA and UA in DA analysis. The Ti–C–Tx/GCE-based sensor achieved a low limit of detection (LOD) for DA (0.06 µM), AA (4.6 µM), and UA (0.075 µM), and therefore can be used for the determination of DA in human urine samples. Nitrogen and sulfur co-doped Nb2C MXene (NS-Nb2C) was synthesized using thiourea as N,S resource [2C/Nafion/GCE-based sensor was satisfactory in detecting DA in simulated gastric juice. Ethylene diamine (EDA) aminated Nb2CTx MXene was prepared to modify carbon cloth (CC) for DA detection [2CTx/CC obtained an ultra-low LOD of 300 pM. A sensor for simultaneous determination of DA and Tyr was prepared based on a Ti3C2 layer modified screen-printed electrode (Ti3C2/SPE) [3C2/SPE sensor was able to simultaneously detect DA and Tyr in DA tablets and urine samples.A simple DA sensor was developed by modifying Nafion-stabilized Tide (GCE) . The neganalysis . The Ti–resource . The intetection . The sen3C2/SPE) . The DPV3C2Tx/Pt nanoparticles (PtNPs) were synthesized by reducing H2PtCl6 on the surface of accordion-like Ti3C2Tx [3C2Tx/PtNPs/GCE-based sensor achieved a good response for electrochemical analysis of DA and UA due to the intercalated PtNPs preventing aggregation of the MXene layer and facilitating electron transfer. Ti3C2Tx/DNA/Pd/Pt nanocomposites were prepared for DA detection [3C2Tx via π–π stacking. The introduction of Pd/Pt nanoparticles into Ti3C2Tx significantly improved the electrocatalytic activity. In another work, a flexible sensor based on a Au–Pd/Ti3C2Tx/LSGE (laser-scribed graphene electrode) was fabricated [3C2Tx was cross-linked with LSGE via a C–O–Ti covalent bond to form the Ti3C2Tx/LSGE, and finally Au–Pd nanoparticles were assembled on the Ti3C2Tx/LSGE through a self-reduction process , AA (3 µM) and UA (1.47 µM). In addition, the sensor was successfully applied to detect DA, AA, and UA in urine samples, and to monitor their variation in sweat samples. Single-atom catalysts (SACs) with maximum atomic utilization rate have better catalytic activity compared to metal nanoparticles. Fe-SACs/Ti3C2Tx composites were synthesized to analyze DA and its end products VMA (vanillylmandelic acid) and HVA (homovanillic acid) [3C2Tx composites exhibited excellent electrocatalytic activity for the oxidation of DA, VMA, and HVA. The sensor based on Fe-SACs/Ti3C2Tx/LSGE was feasible for analysis of DA, VMA, and HVA in urine and serum samples, as well as for real-time monitoring of in situ cellular DA productions.Introducing metals or metal oxides/sulfides/phosphides into MXene can yield MXene-based composite electrode materials with enhanced electrochemical properties. Ti Ti3C2Tx . The Ti3etection . DNA wasbricated . A 3D po process . As the ic acid) . The Fe-3C2Tx composites were synthesized by the self-assemblling of amino functionalized ZnO and Ti3C2Tx MXene. On this basis, ZnO/Ti3C2Tx/Nafion/Au electrodes were prepared for electrochemical detection of DA [2 quantum dots-functionalized Ti3C2 nanocomposites (SnO2QDs@Ti3C2 MXene) were synthesized using an in situ growth method for eletrochemical analysis of DA [2QDs could be intercalated between adjacent Ti3C2 nanosheets, effectively preventing the restacking of Ti3C2, the electroactive surface area and electron transfer mobility of the obtained composite electrode materials were significantly increased. The SnO2QDs@Ti3C2-based sensor achieved an excellent electrochemical response to DA, with a LOD of 2.0 nM. Combing metal sulfides with MXene is a feasible method for preparing high-performance composite electrode materials. Nb2C/ZnS (mass ratio 1:1) nanocomposites were synthesized for the electrochemical analysis of DA [2C/ZnS nanocomposites (Nb2C/ZnS/GCE) showed high selectivity for DA analysis. In another work, Nb2CTx@MoS2 heterostructures were synthesized using a hydrothermal method and their composition was optimized by setting different MoS2 contents. These Nb2CTx@MoS2 heterostructures were then modified on CC to detect DA [2 and Nb2CTx, the Nb2CTx@MoS2/CC-based sensor achieved ultrasensitive electrochemical detection of DA, with a wide linear range (1 fM–100 nM) and an ultra-low LOD of 0.23 fM. Bimetallic phosphides with metalloid characteristics and good electrocatalytic activity are more suitable for constructing electrochemical sensors. A highly sensitive electrochemical sensor for DA detection was fabricated based on a CC/Ti3C2Tx/NiCoP composite [3C2Tx/NiCoP composite was synthesized using a hydrothermal method and subsequent phosphating process. Combing the large specific surface area of NiCoP quantum dots, the abundant active sites of porous NiCoP nanowires, the good hydrophilicity of Ti3C2Tx nanosheets and the excellent conductivity of CC substrates, the sensor achieved a wide linear range from 0.17 μM to 784.55 μM and a low LOD of 0.18 nM.ZnO is widely used for the preparation of electrochemical sensors due to its good electronic properties and high chemical stability. ZnO/Tion of DA . SnO2 quis of DA . As the is of DA . A GCE metect DA . Due to omposite . The CC/3C2Tx/rGO heterostructures were synthesized through electrochemical reduction for electrochemical detection of DA and UA [3C2Tx and electrochemically reduced holey graphene modified glass carbon electrodes (Ti3C2Tx-ERHG/GCE) were prepared for DA detection. With the highly nanoporous ERHG hindering the agglomeration and oxidation of Ti3C2Tx, Ti3C2Tx reducing the non-specific adsorption, and their synergistic effect promoting the electron transfer rate, the Ti3C2Tx-ERHG/GCE-based sensor exhibited excellent electrochemical performance, anti-biofouling properties, and good sensitivity in the detection of DA in serum and artificial cerebrospinal fluid (aCSF) [3C2Tx/HG) nanocomposites were further prepared by the same research group for electrochemical sensing of DA [3C2 MXene, graphitized multi-walled carbon nanotubes and a ZnO nanosphere-modified glassy carbon electrode (Ti3C2/G-MWCNTs/ZnO/GCE) [Combing carbon nanomaterials such as carbon nanotubes (CNTs), graphene (Gr) and its derivatives with MXene is also a commonly used method for preparing high-performance MXene-based composite electrode materials. Bilayer TiA and UA . Ti3C2Txd (aCSF) . Later, ng of DA . A highlZnO/GCE) .3C2 MXene in order to prepare stable layered Ti3C2-PVP, Ti3C2-PVA and Ti3C2-PAM nanocomposites for electrochemical detection of DA [3C2Tx/PPy (polypyrrole) nanocomposites were synthesized for detection of DA and UA [3C2Tx composites [2-hybridized carbon were then modified with PDI-Ti3C2Tx composites for electrochemical analysis of DA. Due to the synergistic effect of MXene and PDI resulting in a highly sensitive sensing interface, and the electrostatic attraction between negatively charged PDI and positively charged DA facilitating the adsorption of DA, the PDI-Ti3C2Tx/GPE-based sensor achieved superior sensitivity and selectivity, and also exhibited a good response in analyzing DA in human serum samples. Metal organic frameworks (MOFs) have been promising materials in sensing applications because of their tunable porosity and excellent adsorption performance. 2D/2D NiCo-MOF/Ti3C2 heterojunctions were prepared and coated on a carbon cloth electrode (CCE) for the electrochemical determination of DA, UA and AP (acetaminophen) [3C2 MXene, the NiCo-MOF/Ti3C2/CCE-based sensor exhibited good performance for the oxidation of DA, UA and AP, with completely separated signal peaks. A Ti3C2 film was synthesized by hydrogen bonding self-assembly of UIO-66-NH2 and Ti3C2 [3C2 and UIO-66-NH2. Benefiting from its large specific area, excellent electronic conductivity and water dispersibility, the Ti3C2 film exhibited strong selectivity and sensitivity to DA.Conductive polymers and organic compounds can be used as effective regulators to improve the electrochemical ability and structural stability of 2D MXene. Three polymers with good biocompatibility and hydrophilicity, PVP (polyvinylpyrrolidone), PVA  and PAM (polyacrylamide), were employed to modify Tion of DA . Ti3C2TxA and UA . Poly . Integrand Ti3C2 , and its3C2Cl2 MXene was prepared using molten salt etching. Ionic liquid (IL) was then applied to stabilize the inherently unstable Ti3C2Cl2 and enhance its electrocatalytic capability. As the strong π–π interaction between IL and Ti3C2Cl2 inhibited leaching of the electrode material and promoted electron transfer at the electrode interface, the IL-Ti3C2Cl2/GPE-based sensor showed robust activity towards DA oxidation. Notably, this is the first time that MXene was prepared using molten salt etching for neurotransmitter detection.In a study , Ti3C2Cl3C2-LIG electrode was prepared by immobilizing laccase on a Ti3C2 modified laser-induced graphene electrode. The laccase-Ti3C2-LIG electrode was then encapsulated in a 3D printed microfluidic device for electroanalysis of DA. With the ideal matrix provided by the Ti3C2 modified LIG network for laccase immobilization, as well as the stable environment provided by the microfluidic device to maintain laccase sensitivity, the resulting laccase-Ti3C2-LIG-based sensor exhibited good analytical performance in real-time detection of blood serum and synthetic urine samples, and demonstrated good potential in point-of-care applications [In addition to active nanomaterials, enzymes can also be used to catalyze the oxidation of DA. A laccase-Tiications .Changing the 2D structure of MXene to other dimensional nanostructures is also an effective way to prevent the restacking or aggregation of MXene, thereby improving its electrochemical performance. In one study, the positively charged surfactant DODA (dimethyldioctadecylammonium bromide) was inserted between negatively charged MXene nanosheets through electrostatic interaction . DODA no5-HT is an inhibitory neurotransmitter in the central nervous system that plays an important role in regulating calm physiological behaviors such as learning, sleeping, and emotions . 5-HT de3C2Tx MXene to prepare Tri-AgNP/l-Cys/Ti3C2Tx for electrochemical detection of 5-HT [3C2Tx electrode material is shown in 3C2Tx MXene, with large surface area and good biocompatibility, provided a good loading platform for Tri-AgNP/l-Cys, while the intercalation of Tri-AgNP/l-Cys avoided the restacking of Ti3C2Tx nanosheets. Under optimized conditions, the Tri-AgNP/l-Cys/Ti3C2Tx/GCE-based sensor exhibited good performance for 5-HT detection, with a LOD of 0.08 μM. An electrochemical sensor based on a Ti3C2Tx MXene/single-walled carbon nanotubes (SWCNTs) nanocomposite was developed for dynamic monitoring of 5-HT secretion from cultured living cells [3C2Tx-rGO nanocomposites were synthesized by hydrazine reduction and subsequent ultrasonic treatment [3C2Tx MXene alleviated the aggregation of MXene nanosheets and exposed more effective active sites, resulting in better electrochemical performance. The Ti3C2Tx-rGO/GCE-based sensor exhibited high sensitivity for 5-HT detection, and could successfully be used to detect 5-HT in blood plasma.In one study, l-cysteine-terminated triangular silver nanoplates (Tri-AgNP/l-Cys) were loaded onto Ti of 5-HT . The preng cells . Ti3C2Txreatment . The comEP, also known as adrenaline, is an excitatory neurotransmitter synthesized in the adrenal gland and released into the blood in emergency and stressful situations ,96. EP r2CTx MXene/graphite composite paste electrode (MXene/GCPE) was fabricated for the detection of EP [3C2Tx MXene (rGO/Ti3C2Tx) was synthesized by freeze-drying and heat treatment and transferred to indium tin oxide (ITO) for electrochemical detection of EP [3C2Tx MXene/N-doped reduced graphene oxide (MXene/N-rGO) composites were synthesized via a simple hydrothermal reaction using ethylenediamine (EDA) as a GO reducing agent and nitrogen source [3C2Tx MXene and N-rGO.A Tion of EP . This elon of EP . In anotn source . The com2OH as functional monomer, and EP as template molecule [2OH-MIP/MXene/CNHs/GCE is shown in 2OH-MIP for target EP molecules, the sensor based on pEDOT-CH2OH-MIP/MXene/CNHs/GCE displayed excellent sensing performance, with a wide linear range (1.0 nM to 60.0 μM) and a low LOD (0.3 nM). The sensor also demonstrated satisfactory feasibility in urinary EP analysis.Molecularly imprinted polymers (MIP) not only have excellent selective recognition and adsorption capabilities for target molecules, but also have higher stability compared to other biological recognition components. As a result, molecularly imprinted sensors have attracted intense interest in recent years. A molecularly imprinted sensor for electrochemical determination of EP was developed using MXene/carbon nanohorns (CNHs) as electrode modification material, EDOT-CHmolecule . The preNE is a biogenic amine neurotransmitter secreted by the adrenal medulla . Abnorma2/MXene-PVA/GO) hydrogel was successfully prepared and modified on a screen-printed carbon electrode (SPCE) by repeated freezing–thawing treatment for urinary NE determination . Abnormal levels of Tyr are closely associated with various diseases, such as dementia, Parkinson’s disease, depression, anxiety, etc. ,97. As sfied GCE . The int2CTx heterostructures were prepared by partial in-situ conversion of V2CTx MXene to MOF for electrochemical NO sensing [2CTx MXene, charge redistribution at the heterojunction interface facilitating charge transfer, and enhanced NO adsorption energy at the heterojunction interface, the MOF@V2CTx heterostructures exhibited significantly improved electrochemical NO sensing performance compared to those of bare MXene and MOF.Some soluble gases have been identified as gasotransmitters. NO is a gasotransmitter involved in various neurological functions including sleep and appetite regulation, neurotransmission, learning and memory . In one  sensing . Due to 2S is another endogenous gasotransmitter that is widely distributed in the body and plays an important role in neurotransmission, antioxidation, and vascular tone regulation [2S concentrations may be related to neurodegeneractive disorders, gastrointestinal diseases, diabetes and cancer [3C2Tx MXene-modified GCE was fabricated for H2S detection [2S electrochemical sensing.Hgulation ,98. Abnod cancer . A direcetection , which e2S) is presented, focusing on the preparation strategies for electrode materials. In the past few years, research in this field has been dedicated to improving the sensitivity, selectivity, and stability of electrodes. Thus, MXene-based electrode materials with high electrical conductivity, large specific surface area, porous structure, good biocompatibility, and anti-fouling properties have been prepared by combining metal nanoparticles, metal oxides/sulfides/phosphides, carbon nanomaterials, conductive polymers, MOFs, etc., with MXene or by changing the 2D structure of MXene to other dimensional nanostructures. In view of this, significant progress has been made in MXene-based neurotransmitter electrochemical sensors. However, MXene-based neurotransmitter electrochemical sensors still face some deficiencies and need further development and improvement: (1) MXene is mainly prepared using HF etching and acid/fluoride salt etching. The residual –F on the surface of MXene affects its electrochemical performance. It is necessary to develop simple fluorine-free preparation methods to achieve large-scale production of MXene in the future. (2) The species of neurotransmitters detected by MXene-based electrochemical sensors are limited; some important species, such as histamine, acetylcholine, aspartic acid, and glutamate, have not been investigated. (3) A certain human disease or behavior is generally the result of coordinated effects of multiple neurotransmitters; therefore, it is necessary to simultaneously detect the relevant neurotransmitters. At present, simultaneous detection is still in its infancy, and thus it is of great significance to further develop electrochemical sensors for simultaneous detection of several neurotransmitters. (4) MXene-based neurotransmitter electrochemical sensors have been developed for detecting neurotransmitters in biological fluids , some of which have shown potential for POC detection. Next, it is meaningful to further implement their POC applications by developing the relevant devices. (5) MXene-based neurotransmitter electrochemical sensors have not yet achieved real-time detection in the human body; therefore, the future direction is to develop MXene-based neurotransmitter electrochemical sensors that can be used for real-time detection in the human body. It is also necessary to develop integrated, miniature, and implantable sensors for neurotransmitter detection in blood, as well as wearable flexible sensors for urinary neurotransmitter detection. These sensors can be combined with miniature electrochemical workstations and wireless sensing devices or mobile phones to ultimately enable real-time and long-term monitoring of human neurotransmitters. In summary, the research on MXene-based neurotransmitter electrochemical sensors is still at a preliminary stage, and there is still a long way to go to truly achieve their clinical applications. This paper presents the design strategies, current bottlenecks and future directions for MXene-based neurotransmitter electrochemical sensors, aiming to provide some reference for further development in this field.This paper reviews the advances in MXene-based electrochemical (bio)sensors for neurotransmitter detection. First, the composition, structure, synthesis, properties, delamination and surface modification/functionalization of MXene are briefly described. Then, a systematic summary of MXene-based electrochemical (bio)sensors for the detection of neurotransmitters including biogenic amines , amino acids (Tyr), and soluble gases (NO and H