Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

The structure of the archaeal nuclease RecJ2 implicates its catalytic mechanism and inability to interact with GINS.

Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5' or 3' exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5'-exonuclease MjRecJ1 and the 3'-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3' hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins.

Theoretical study on the mechanism of alcohol photooxidation on Nb2O5 surface.

Theoretical modeling of the solid-state photocatalysis is one of the important issues as various useful photocatalysts have been developed to date. In this work, we investigated the mechanism of the alcohol photooxidation on niobium oxide (Nb2O5) which was experimentally developed, using the density functional theory (DFT)/time-dependent (TD)DFT calculations based on the cluster model. The alcohol adsorption and the first hydrogen transfer from hydroxy group to surface occur in the ground state, while the second hydrogen transfer from CH proceeds in the excited states during the photoirradiation of UV or visible light. The spin crossing was identified and the low-lying triplet states were solved for the reaction pathway. The photoabsorption in the visible light region was characterized as the charge transfer transition from O 2p of alcohol to Nb 4d of the Nb2O5 surface. The spin density and the natural population analysis indicated the generation of spin density in the moiety of carbonyl compound and its dissipation to the interface of the surface, which partly explains the electron paramagnetic resonance measurement. It was confirmed that the rate determining step is the desorption of carbonyl compound and water molecule in agreement with the experimental rate equation analysis. The present findings with the theoretical modeling will provide useful information for the further studies of the solid-state photocatalysis.

Giant Anisotropic Thermal Expansion Phase Transition of Silver Iodide Anionic Organic-Inorganic Hybrid.

Hybrid metal halide materials with charming phase transition behaviors have attracted considerable attention. In former works, much attention has been focused on the phase transition triggered by the order-disorder or displacement motions of the organic component. However, manipulating the variation of the inorganic component to achieve the phase transition has rarely been reported. Herein, two novel organic-inorganic hybrid materials, [THPM]n[AgX2]n (THPM = 3,4,5,6-tetrahydropyrimidin-1-ium, X = I for 1 and Br for 2) with the [AgX2]nn- anionic chain structure, were synthesized. At 293 K, the [AgX2]nn- chains in 1 were constructed by the tetramer units of Ag atoms, while that in 2 was assembled by the dimer structure. Upon heating to 355 K, owing to the variation of the metallophilic interaction between adjacent Ag atoms, a unique transformation process from tetramer to dimer in [AgI2]nn- chains of 1 can be detected and endow 1 with a giant anisotropic thermal expansion with linear strain of ∼7% and shear strain of ∼20%, which can be used as a mechanical actuator for switching. Alternatively, for 2, no phase transition process can be observed upon the temperature variation. This work provides an effective approach to design phase transition materials triggered by the inorganic part.

Unraveling the Mechanism of Very Initial Dendritic Growth Under Lithium Ion Transport Control in Lithium Metal Anodes.

Lithium metal deposition is strongly affected by the intrinsic properties of the solid-electrolyte interphase (SEI) and working electrolyte, but a relevant understanding is far from complete. Here, by employing multiple electrochemical techniques and the design of SEI and electrolyte, we elucidate the electrochemistry of Li deposition under mass transport control. It is discovered that SEIs with a lower Li ion transference number and/or conductivity induce a distinctive current transition even under moderate potentiostatic polarization, which is associated with the control regime transition of Li ion transport from the SEI to the electrolyte. Furthermore, our findings help reveal the creation of a space-charge layer at the electrode/SEI interface due to the involvement of the diffusion process of Li ions through the SEI, which promotes the formation of dendrite embryos that develop and eventually trigger SEI breakage and the control regime transition of Li ion transport. Our insight into the very initial dendritic growth mechanism offers a bridge toward design and control for superior SEIs.

Promoting Molecular Exchange on Rare-Earth Oxycarbonate Surfaces to Catalyze the Water-Gas Shift Reaction.

It is highly desirable to fabricate an accessible catalyst surface that can efficiently activate reactants and desorb products to promote the local surface reaction equilibrium in heterogeneous catalysis. Herein, rare-earth oxycarbonates (Ln2O2CO3, where Ln = La and Sm), which have molecular-exchangeable (H2O and CO2) surface structures according to the ordered layered arrangement of Ln2O22+ and CO32- ions, are unearthed. On this basis, a series of Ln2O2CO3-supported Cu catalysts are prepared through the deposition precipitation method, which provides excellent catalytic activity and stability for the water-gas shift (WGS) reaction. Density functional theory calculations combined with systematic experimental characterizations verify that H2O spontaneously dissociates on the surface of Ln2O2CO3 to form hydroxyl by eliminating the carbonate through the release of CO2. This interchange efficiently promotes the WGS reaction equilibrium shift on the local surface and prevents the carbonate accumulation from hindering the active sites. The discovery of the unique layered structure provides a so-called "self-cleaning" active surface for the WGS reaction and opens new perspectives about the application of rare-earth oxycarbonate nanomaterials in C1 chemistry.

Morphology-Dictated Mechanism of Efficient Reaction Sites for Li2O2 Decomposition.

In the pursuit of a highly reversible lithium-oxygen (Li-O2) battery, control of reaction sites to maintain stable conversion between O2 and Li2O2 at the cathode side is imperatively desirable. However, the mechanism involving the reaction site during charging remains elusive, which, in turn, imposes challenges in recognition of the origin of overpotential. Herein, via combined investigations by in situ atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS), we propose a universal morphology-dictated mechanism of efficient reaction sites for Li2O2 decomposition. It is found that Li2O2 deposits with different morphologies share similar localized conductivities, much higher than that reported for bulk Li2O2, enabling the reaction site not only at the electrode/Li2O2/electrolyte interface but also at the Li2O2/electrolyte interface. However, while the mass transport process is more enhanced at the former, the charge-transfer resistance at the latter is sensitively related to the surface structure and thus the reactivity of the Li2O2 deposit. Consequently, for compact disk-like deposits, the electrode/Li2O2/electrolyte interface serves as the dominant decomposition site, which causes premature departure of Li2O2 and loss of reversibility; on the contrary, for porous flower-like and film-like Li2O2 deposits bearing a larger surface area and richer surface-active structures, both the interfaces are efficient for decomposition without premature departure of the deposit so that the overpotential arises primarily from the sluggish oxidation kinetics and the decomposition is more reversible. The present work provides instructive insights into the understanding of the mechanism of reaction sites during the charge process, which offers guidance for the design of reversible Li-O2 batteries.

Antinociceptive effect of gelsenicine, principal toxic alkaloids of gelsemium, on prostaglandin E2-induced hyperalgesia in mice: Comparison with gelsemine and koumine.

Gelsemium elegans (G.elegans) is a plant of the Loganiaceae family, known for its indole alkaloids, including gelsemine, koumine, and gelsenicine. Gelsemine and koumine are well-studied active alkaloids with low toxicity, valued for their anti-anxiety and analgesic properties. However, gelsenicine, another important alkaloid, remains underexplored due to its high toxicity. This study focuses on evaluating the analgesic properties of gelsenicine and comparing them with gelsemine and koumine. The results indicate that all three alkaloids exhibit robust analgesic properties, with gelsemine, koumine, and gelsenicine showing ED50 values of 0.82 mg/kg, 0.60 mg/kg, and 8.43 µg/kg, respectively, as assessed by the hot plate method. Notably, the therapeutic dose of gelsenicine was significantly lower than its toxic dose (LD50 = 0.185 mg/kg). The study also investigated the mechanism of action by analyzing the expression levels of GlyRα3 and Gephyrin. The PGE2 model group showed decreased expression levels of GlyRα3 and Gephyrin, while groups treated with gelsemine, koumine, and gelsenicine were able to reverse this decrease. These results suggest that gelsenicine effectively alleviates PGE2-induced hyperalgesia by upregulating the expression of GlyRα3 and Gephyrin, which are key targets of the Gly receptor pathway.

Mechanistic Studies of Regiocontrolled Bisaddition of Fullerenes Driven by Oriented External Electric Fields.

The challenge of achieving regioselective multifunctionalization on highly symmetric C60 and C70 fullerenes persists as a significant hurdle. In this study, we present a novel approach involving the participation of an oriented external electric field (OEEF) to facilitate the regioselective formation of bisadducts in C60/C70 fullerenes. These products are obtained through consecutive Diels-Alder cycloaddition reactions. We constructed the field strength-barrier relationship and elucidated the OEEF-driven modulation mechanisms quantitatively. Leveraging the interplay between molecular dipoles and electric fields, the diverse reactions at distinct sites exhibit varying degrees of sensitivity to the applied electric fields, thereby leading to a pronounced regioselectivity in the bisaddition process. Our proposition suggests that the angle formed between the bonding direction (referred to as the reaction axis) and the external field can conveniently function as a predictive descriptor for the reactivity of different sites on the fullerene surface when subjected to electric fields.

Long-circulating doxorubicin and Schizandrin A liposome with drug-resistant liver cancer activity: in vitro and in vivo evaluation.

Co-loading doxorubicin (DOX) and Schizandrin A (SchA) long-circulating liposome (SchA-DOX-Lip) have been confirmed to have good antitumor activity in vitro. However, in vivo pharmacodynamics, targeting, safety, and mechanism of action of SchA-DOX-Lip still need to be further verified. We investigated the tumor inhibition effect, targeting, safety evaluation, and regulation of tumor apoptosis-related proteins of the SchA-DOX-Lip. MTT assay was used to investigate the inhibitory effect of SchA-DOX-Lip on CBRH7919 cells. The drug uptake of CBRH7919 cells was observed by inverted fluorescence microscope. The tumor-bearing nude mice models of CBRH7919 were established, and the anti-tumor effect of SchA-DOX-Lip in vivo was evaluated by tumor biological observation, H&E staining, and TUNEL staining. The distribution and targeting of SchA-DOX-Lip in nude mice models were investigated by small animal imaging and tissue distribution experiment of CBRH7919. The biosafety of SchA-DOX-Lip was evaluated by blood routine parameters, biochemical indexes, and H&E staining. The expression of tumor-associated apoptotic proteins (Bcl-2, Bax, and Caspase-3) was detected by immunohistochemistry anvd western blotting. The results showed that SchA-DOX-Lip had cytotoxicity to CBRH7919 cells which effectively inhibited the proliferation of CBRH7919 cells, improved the uptake of drugs by CBRH7919 cells and the targeting effect of drugs on tumor site. H&E staining and biochemical detection results showed that SchA-DOX-Lip had high biosafety and did not cause serious damage to normal tissues. Western-blotting and TUNEL staining results showed that SchA-DOX-Lip could improve the regulatory effect of drugs on tumor apoptosis proteins. It was demonstrated that SchA-DOX-Lip had high safety and strong tumor inhibition effects, providing a new method for the clinical treatment of hepatocellular carcinoma (HCC).

Comparison of Semen Aesculi species from different geographical origins and horse chestnut based on metabolomics and chemometrics analysis.

BACKGROUND: Semen Aesculi, a traditional Chinese herbal medicine, has a long history of use for treating chest and abdominal pain with distension. In addition, the horse chestnut (Aesculus hippocastanum L.) is another species of Aesculus in Europe and has notable clinical significance in alleviating chronic venous insufficiency, hemorrhoids, and postoperative edema. Thus, highlighting the comparative study of Semen Aesculi and horse chestnut may broaden clinical applications. OBJECTIVES: To conduct a comprehensive comparative analysis on the chemical profiling of these two varieties and determine whether they have equivalent clinical efficacy by integrating plant metabolomics and multivariate statistical methods. METHODS: Initially, a comprehensive characterisation was performed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) platform, and in total 44 active ingredients were identified. Then, untargeted metabolomics combined with principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) was applied for the discrimination of a German species and three official Chinese species. Next, 24 marker compounds responsible for the discrimination of different species were screened out and used to predict the species of unknown samples by genetic algorithm-optimised support vector machine (GA-SVM) with a high prediction accuracy. Finally, a heatmap visualisation was employed for clarifying the distribution of the identified active ingredients. RESULTS: The three species of Chinese Semen Aesculi showed distinct separation from each other, while European horse chestnut and Aesculus chinensis Bunge were similar in chemical composition. CONCLUSIONS: This work provided experimental evidence for further expanding the clinical application of Chinese Semen Aesculi and promoted the species identification and quality control of Semen Aesculi.

The Role of Water Content of Deep Eutectic Solvent Ethaline in the Anodic Process of Gold Electrode.

Traditional coupling of ligands for gold wet etching makes large-scale applications problematic. Deep eutectic solvents (DESs) are a new class of environment-friendly solvents, which could possibly overcome the shortcomings. In this work, the effect of water content on the Au anodic process in DES ethaline was investigated by combining linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS). Meanwhile, we employed atomic force microscopy (AFM) to image the evolution of the surface morphology of the Au electrode during its dissolution and passivation process. The obtained AFM data help to explain the observations about the effect of water content on the Au anodic process from the microscopic perspective. High water contents make the occurrence of anodic dissolution of gold at higher potential, but enhances the rate of the electron transfer and gold dissolution. AFM results reveal the occurrence of massive exfoliation, which confirms that the gold dissolution reaction is more violent in ethaline with higher water contents. In addition, AFM results illustrate that the passive film and its average roughness could be tailored by changing the water content of ethaline.

WITHDRAWN: Comparison of second-generation cryoballoon ablation and quantitative radiofrequency ablation guided by ablation index for atrial fibrillation.

Ahead of Print article withdrawn by publisher.

[Effect of Xiaoxuming Decoction on synaptic plasticity following acute cerebral ischemia-reperfusion in rats].

This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.

[Research advances in role of angiogenesis in diabetic ulcer and traditional Chinese medicine intervention].

Diabetic ulcer(DU) is one of the common complications of diabetes often occurring in the peripheral blood vessels of lower limbs or feet with a certain degree of damage. It has high morbidity and mortality, a long treatment cycle, and high cost. DU is often clinically manifested as skin ulcers or infections in the lower limbs or feet. In severe cases, it can ulcerate to the surface of tendons, bones or joint capsules, and even bone marrow. Without timely and correct treatment, most of the patients will have ulceration and blackening of the extremities. These patients will not be able to preserve the affected limbs through conservative treatment, and amputation must be performed. The etiology and pathogenesis of DU patients with the above condition are complex, which involves blood circulation interruption of DU wound, poor nutrition supply, and failure in discharge of metabolic waste. Relevant studies have also confirmed that promoting DU wound angiogenesis and restoring blood supply can effectively delay the occurrence and development of wound ulcers and provide nutritional support for wound healing, which is of great significance in the treatment of DU. There are many factors related to angiogenesis, including pro-angiogenic factors and anti-angiogenic factors. The dynamic balance between them plays a key role in angiogenesis. Meanwhile, previous studies have also confirmed that traditional Chinese medicine can enhance pro-angiogenic factors and down-regulate anti-angiogenic factors to promote angiogenesis. In addition, many experts and scholars have proposed that traditional Chinese medicine regulation of DU wound angiogenesis in the treatment of DU has broad prospects. Therefore, by consulting a large number of studies available, this paper expounded on the role of angiogenesis in DU wound and summarized the research advance in traditional Chinese medicine intervention in promoting the expression of angiogenic factors [vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), and angiopoietin(Ang)] which played a major role in promoting wound angiogenesis in the treatment of DU to provide ideas for further research and new methods for clinical treatment of DU.

[Effect of Xiaoxuming Decoction on activation of astrocytes in acute cerebral ischemia/reperfusion injury].

This study investigated the effect of Xiaoxuming Decoction(XXMD) on the activation of astrocytes after cerebral ischemia/reperfusion(I/R) injury. The model of cerebral IR injury was established using the middle cerebral artery occlusion method. Fluorocitrate(FC), an inhibitor of astrocyte activation, was applied to inhibit astrocyte activation. Rats were randomly divided into a sham group, a model group, a XXMD group, a XXMD+FC group, and a XXMD+Vehicle group. Neurobehavioral changes at 24 hours after cerebral IR injury, cerebral infarction, histopathological changes observed through HE staining, submicroscopic structure of astrocytes observed through transmission electron microscopy, fluorescence intensity of glial fibrillary acidic protein(GFAP) and thrombospondin 1(TSP1) measured through immunofluorescence, and expression of GFAP and TSP1 in brain tissue measured through Western blot were evaluated in rats from each group. The experimental results showed that neurobehavioral scores and cerebral infarct area significantly increased in the model group. The XXMD group, the XXMD+FC group, and the XXMD+Vehicle group all alleviated neurobehavioral changes in rats. The pathological changes in the brain were evident in the model group, while the XXMD group, the XXMD+FC group, and the XXMD+Vehicle group exhibited milder cerebral IR injury in rats. The submicroscopic structure of astrocytes in the model group showed significant swelling, whereas the XXMD group, the XXMD+FC group, and XXMD+Vehicle group protected the submicroscopic structure of astrocytes. The fluorescence intensity and protein expression of GFAP and TSP1 increased in the model group compared with those in the sham group. However, the XXMD group, the XXMD+FC group, and XXMD+Vehicle group all down-regulated the expression of GFAP and TSP1. The combination of XXMD and FC showed a more pronounced effect. These results indicate that XXMD can improve cerebral IR injury, possibly by inhibiting astrocyte activation and down-regulating the expression of GFAP and TSP1.

[Research progress in role of autophagy in diabetic wound healing and traditional Chinese medicine intervention].

Diabetic ulcer(DU) is a chronic and refractory ulcer which often occurs in the foot or lower limbs. It is a diabetic complication with high morbidity and mortality. The pathogenesis of DU is complex, and the therapies(such as debridement, flap transplantation, and application of antibiotics) are also complex and have long cycles. DU patients suffer from great economic and psychological pressure while enduring pain. Therefore, it is particularly important to promote rapid wound healing, reduce disability and mortality, protect limb function, and improve the quality of life of DU patients. By reviewing the relevant literatures, we have found that autophagy can remove DU wound pathogens, reduce wound inflammation, and accelerate ulcer wound healing and tissue repair. The main autophagy-related factors microtubule-binding light chain protein 3(LC3), autophagy-specific gene Beclin-1, and ubiquitin-binding protein p62 mediate autophagy. The traditional Chinese medicine(TCM) treatment of DU mitigates clinical symptoms, accelerates ulcer wound healing, reduces ulcer recurrence, and delays further deterioration of DU. Furthermore, under the guidance of syndrome differentiation and treatment and the overall concept, TCM treatment harmonizes yin and yang, ameliorates TCM syndrome, and treats underlying diseases, thereby curing DU from the root. Therefore, this article reviews the role of autophagy and major related factors LC3, Beclin-1, and p62 in the healing of DU wounds and the intervention of TCM, aiming to provide reference for the clinical treatment of DU wounds and subsequent in-depth studies.

Molecular landscape of pediatric type IDH wildtype, H3 wildtype hemispheric glioblastomas.

The WHO (2021) Classification classified a group of pediatric-type high-grade gliomas as IDH wildtype, H3 wildtype but as of currently, they are characterized only by negative molecular features of IDH and H3. We recruited 35 cases of pediatric IDH wildtype and H3 wildtype hemispheric glioblastomas. We evaluated them with genome-wide methylation profiling, targeted sequencing, RNAseq, TERT promoter sequencing, and FISH. The median survival of the cohort was 27.6 months. With Capper et al.'s36 methylation groups as a map, the cases were found to be epigenetically heterogeneous and were clustered in proximity or overlay of methylation groups PXA-like (n = 8), LGG-like (n = 10), GBM_MYCN (n = 9), GBM_midline (n = 5), and GBM_RTKIII (n = 3). Histology of the tumors in these groups was not different from regular glioblastomas. Methylation groups were not associated with OS. We were unable to identify groups specifically characterized by EGFR or PDGFRA amplification as proposed by other authors. EGFR, PDGFRA, and MYCN amplifications were not correlated with OS. 4/9 cases of the GBM_MYCN cluster did not show MYCN amplification; the group was also enriched for EGFR amplification (4/9 cases) and the two biomarkers overlapped in two cases. Overall, PDGFRA amplification was found in only four cases and they were not restricted to any groups. Cases in proximity to GBM_midline were all hemispheric and showed loss of H3K27me3 staining. Fusion genes ALK/NTRK/ROS1/MET characteristic of infantile glioblastomas were not identified in 17 cases successfully sequenced. BRAF V600E was only found in the PXA group but CDKN2A deletion could be found in other methylation groups. PXA-like cases did not show PXA histological features similar to findings by other authors. No case showed TERT promoter mutation. Mutations of mismatch repair (MMR) genes were poor prognosticators in single (p ≤ 0.001) but not in multivariate analyses (p = 0.229). MGMT had no survival significance in this cohort. Of the other common biomarkers, only TP53 and ATRX mutations were significant poor prognosticators and only TP53 mutation was significant after multivariate analyses (p = 0.024). We conclude that IDH wildtype, H3 wildtype pediatric hemispheric glioblastomas are molecularly heterogeneous and in routine practice, TP53, ATRX, and MMR status could profitably be screened for risk stratification in laboratories without ready access to methylation profiling.

Molecular landscape of IDH-wild-type, H3-wild-type glioblastomas of adolescents and young adults.

OBJECTIVE: We aimed to characterise glioblastomas of adolescents and young adults (AYAs) that were isocitrate dehydrogenase (IDH) wild type (wt) and H3wt. MATERIALS AND METHODS: Fifty such patients (aged 16-32) were studied by methylation profiling, targeted sequencing and targeted RNA-seq. RESULTS: Tumours predominantly clustered into three methylation classes according to the terminology of Capper et al. (2018): (anaplastic) pleomorphic xanthoastrocytoma (PXA) (21 cases), GBM_midline (15 cases) and glioblastoma RTK/mesenchymal (seven cases). Two cases clustered with ANA_PA, four cases with LGG classes and one with GBM_MYCN. Only fifteen cases reached a calibrated score >0.84 when the cases were uploaded to DKFZ Classifier. GBM_midline-clustered tumours had a poorer overall survival (OS) compared with the PXA-clustered tumours (p = 0.030). LGG-clustered cases had a significantly better survival than GBM_midline-clustered tumours and glioblastoma RTK/mesenchymal-clustered tumours. Only 13/21 (62%) of PXA-clustered cases were BRAF V600E mutated. Most GBM_midline-clustered cases were not located in the midline. GBM_midline-clustered cases were characterised by PDGFRA amplification/mutation (73.3%), mutations of mismatch repair genes (40.0%), and all showed H3K27me3 and EZH1P loss, and an unmethylated MGMT promoter. Across the whole cohort, MGMT promoter methylation and wt TERT promoter were favourable prognosticators. Mismatch repair gene mutations were poor prognosticators and together with methylation class and MGMT methylation, maintained their significance in multivariate analyses. BRAF mutation was a good prognosticator in the PXA-clustered tumours. CONCLUSION: Methylation profiling is a useful tool in the diagnosis and prognostication of AYA glioblastomas, and the methylation classes have distinct molecular characteristics. The usual molecular diagnostic criteria for adult IDHwt glioblastoma should be applied with caution within the AYA age group.