CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

BACKGROUND: Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. METHODS: Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. RESULTS: pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. CONCLUSION: We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

KCNA1 promotes the growth and invasion of glioblastoma cells through ferroptosis inhibition via upregulating SLC7A11.

BACKGROUND: The high invasiveness and infiltrative nature of Glioblastoma (GBM) pose significant challenges for surgical removal. This study aimed to investigate the role of KCNA1 in GBM progression. METHODS: CCK8, colony formation assay, scratch assay, transwell assay, and 3D tumor spheroid invasion assays were to determine how KCNA1 affects the growth and invasion of GBM cells. Subsequently, to confirm the impact of KCNA1 in ferroptosis, western blot, transmission electron microscopy and flow cytometry were conducted. To ascertain the impact of KCNA1 in vivo, patient-derived orthotopic xenograft models were established. RESULTS: In functional assays, KCNA1 promotes the growth and invasion of GBM cells. Besides, KCNA1 can increase the expression of SLC7A11 and protect cells from ferroptosis. The vivo experiments demonstrated that knocking down KCNA1 inhibited the growth and infiltration of primary tumors in mice and extended survival time. CONCLUSION: Therefore, our research suggests that KCNA1 may promote tumor growth and invasion by upregulating the expression of SLC7A11 and inhibiting ferroptosis, making it a promising therapeutic target for GBM.

TEM1/endosialin/CD248 promotes pathologic scarring and TGF-ß activity through its receptor stability in dermal fibroblasts.

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

WISE: whole-scenario embryo identification using self-supervised learning encoder in IVF.

PURPOSE: To study the effectiveness of whole-scenario embryo identification using a self-supervised learning encoder (WISE) in in vitro fertilization (IVF) on time-lapse, cross-device, and cryo-thawed scenarios. METHODS: WISE was based on the vision transformer (ViT) architecture and masked autoencoders (MAE), a self-supervised learning (SSL) method. To train WISE, we prepared three datasets including the SSL pre-training dataset, the time-lapse identification dataset, and the cross-device identification dataset. To identify whether pairs of images were from the same embryos in different scenarios in the downstream identification tasks, embryo images including time-lapse and microscope images were first pre-processed through object detection, cropping, padding, and resizing, and then fed into WISE to get predictions. RESULTS: WISE could accurately identify embryos in the three scenarios. The accuracy was 99.89% on the time-lapse identification dataset, and 83.55% on the cross-device identification dataset. Besides, we subdivided a cryo-thawed evaluation set from the cross-device test set to have a better estimation of how WISE performs in the real-world, and it reached an accuracy of 82.22%. There were approximately 10% improvements in cross-device and cryo-thawed identification tasks after the SSL method was applied. Besides, WISE demonstrated improvements in the accuracy of 9.5%, 12%, and 18% over embryologists in the three scenarios. CONCLUSION: SSL methods can improve embryo identification accuracy even when dealing with cross-device and cryo-thawed paired images. The study is the first to apply SSL in embryo identification, and the results show the promise of WISE for future application in embryo witnessing.

Leveraging federated learning for boosting data privacy and performance in IVF embryo selection.

PURPOSE: To study the effectiveness of federated learning in in vitro fertilization on embryo evaluation tasks. METHODS: This is a retrospective cohort analysis. Two datasets were used in this study. The ploidy status dataset consisted of 10,065 embryo records, 3760 treatments, and 2479 infertile couples from 5 hospitals. The clinical pregnancy dataset consisted of 4495 embryo records, 4495 treatments, and 3704 infertile couples from 4 hospitals. Federated learning and the gradient boosting decision tree algorithm were utilized for modeling. RESULTS: On the ploidy status dataset, the areas under the receiver operating characteristic curves of our model trained with federated learning were 71.78%, 73.10%, 69.39%, 69.72%, and 73.46% for 5 hospitals respectively, showing an average increase of 2.5% compared to those of our model trained without federated learning. On the clinical pregnancy dataset, the areas under the receiver operating characteristic curves of our model trained with federated learning were 72.03%, 56.77%, 61.63%, and 58.58% for 4 hospitals respectively, showing an average increase of 3.08%. CONCLUSIONS: Federated learning can improve data privacy and data security and meanwhile improve the performance of embryo selection tasks by leveraging data from multiple sources. This study demonstrates the effectiveness of federated learning in embryo evaluation, and the results show the promise for future application.

Differential influence of quadriceps rate of torque development on single- and double-leg landing mechanics in anterior cruciate ligament reconstructed and control females.

PURPOSE: The capacity to explosively contract quadriceps within the critical timeframe associated with anterior cruciate ligament (ACL) injury, quantified by the rate of torque development, is potentially essential for safe landing mechanics. This study aimed to investigate the influence of explosive quadriceps strength on ACL-related sagittal-plane landing mechanics in females with and without ACL reconstruction (ACLR). METHODS: Quadriceps explosive strength and landing mechanics were assessed in 19 ACLR and 19 control females during isometric contractions and double- and single-leg jump landings. A stepwise multiple linear regression model determined the variance in each of the landing biomechanics variables for the ACLR limb and nondominant limb of controls that could be explained by the group, rate of torque development and/or their interaction. If peak kinetic variables could be predicted by the rate of torque development or interaction, additional analyses were conducted, accounting for knee flexion as a covariate in the regression model. RESULTS: During single-leg landings, ACLR females exhibited greater knee flexion at initial contact than controls (p = 0.04). Greater quadriceps rate of torque development predicted higher peak posterior ground reaction force and anterior tibial shear force in both groups (p = 0.04). However, after controlling for knee flexion angle at those peak forces, quadriceps rate of torque development was not predictive. In double-leg landings, greater explosive quadriceps strength was associated with quicker attainment of peak knee extension moment and posterior ground reaction force in the ACLR limb (p = 0.03). CONCLUSION: Regardless of ACL injury status, females with greater explosive quadriceps strength adopted safer single-leg landings through increased knee flexion, potentially mitigating ACL loading despite encountering higher peak forces. During double-leg landings, a greater explosive quadriceps strength of the ACLR limb is associated with faster achievement of peak force upon landing. Incorporating explosive quadriceps strengthening into post-ACLR rehabilitation and injury prevention programmes may enhance landing mechanics for reducing primary and subsequent ACL injury risks. LEVEL OF EVIDENCE: Level II.

Targeting GBM with an Oncolytic Picornavirus SVV-001 alone and in combination with fractionated Radiation in a Novel Panel of Orthotopic PDX models.

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.

Does prior concussion lead to biomechanical alterations associated with lateral ankle sprain and anterior cruciate ligament injury? A systematic review and meta-analysis.

OBJECTIVE: To determine whether individuals with a prior concussion exhibit biomechanical alterations in balance, gait and jump-landing tasks with and without cognitive demands that are associated with risk of lateral ankle sprain (LAS) and anterior cruciate ligament (ACL) injury. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Five electronic databases (Web of Science, Scopus, PubMed, SPORTDiscus and CiNAHL) were searched in April 2023. ELIGIBILITY CRITERIA: Included studies involved (1) concussed participants, (2) outcome measures of spatiotemporal, kinematic or kinetic data and (3) a comparison or the data necessary to compare biomechanical variables between individuals with and without concussion history or before and after a concussion. RESULTS: Twenty-seven studies were included involving 1544 participants (concussion group (n=757); non-concussion group (n=787)). Individuals with a recent concussion history (within 2 months) had decreased postural stability (g=0.34, 95% CI 0.20 to 0.49, p<0.001) and slower locomotion-related performance (g=0.26, 95% CI 0.11 to 0.41, p<0.001), both of which are associated with LAS injury risk. Furthermore, alterations in frontal plane kinetics (g=0.41, 95% CI 0.03 to 0.79, p=0.033) and sagittal plane kinematics (g=0.30, 95% CI 0.11 to 0.50, p=0.002) were observed in individuals approximately 2 years following concussion, both of which are associated with ACL injury risk. The moderator analyses indicated cognitive demands (ie, working memory, inhibitory control tasks) affected frontal plane kinematics (p=0.009), but not sagittal plane kinematics and locomotion-related performance, between the concussion and non-concussion groups. CONCLUSION: Following a recent concussion, individuals display decreased postural stability and slower locomotion-related performance, both of which are associated with LAS injury risk. Moreover, individuals within 2 years following a concussion also adopt a more erect landing posture with greater knee internal adduction moment, both of which are associated with ACL injury risk. While adding cognitive demands to jump-landing tasks affected frontal plane kinematics during landing, the altered movement patterns in locomotion and sagittal plane kinematics postconcussion persisted regardless of additional cognitive demands. PROSPERO REGISTRATION NUMBER: CRD42021248916.

ASC-J9 Blocks Cell Proliferation and Extracellular Matrix Production of Keloid Fibroblasts through Inhibiting STAT3 Signaling.

Keloids are a fibrotic skin disorder caused by abnormal wound healing and featuring the activation and expansion of fibroblasts beyond the original wound margin. Signal transducer and activator of transcription 3 (STAT3) has been found to mediate the biological functions of keloid fibroblasts (KFs). Therefore, we aimed to demonstrate whether ASC-J9, an inhibitor of STAT3 phosphorylation, can suppress the activation of KFs. Western blotting results showed that ASC-J9 inhibited the levels of COL1A1 and FN1 proteins, which were upregulated in KFs, by decreasing the expression of pSTAT3 and STAT3. RNA sequencing and in vitro studies further demonstrated that ASC-J9 treatment of KFs reduced cell division, inflammation, and ROS generation, as well as extracellular matrix (ECM) synthesis. ELISA assays verified that ASC-J9 treatment significantly mitigated IL-6 protein secretion in KFs. Transmission electron microscopy images revealed that ASC-J9 induced the formation of multilamellar bodies in KFs, which is associated with autophagy-related signaling. These results suggested that inhibiting a vicious cycle of the ROS/STAT3/IL-6 axis by ASC-J9 may represent a potential therapeutic approach to suppress cell proliferation and ECM production in KFs.

USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4.

BACKGROUND: Glioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM. METHODS: Immunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7. RESULTS: Immunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes. CONCLUSIONS: Targeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

Bilateral Comparisons of Quadriceps Thickness after Anterior Cruciate Ligament Reconstruction.

Background and objectives: Anterior cruciate ligament reconstruction (ACLR) often results in quadricep atrophy. The purpose of this study was to compare the bilateral thickness of each quadricep component before and after ACLR. Materials and Methods: Cross-sectional study design. In 14 patients who underwent ACLR, bilateral quadricep muscle thicknesses were measured using a portable ultrasound device, 1 h before and 48-72 h after ACLR. Two-way analysis of variance (ANOVA) was used to compare muscle thickness pre- and post-ACLR between the limbs. Results: The primary finding was that the vastus intermedius (VI) muscle was significantly smaller in the reconstructed limb after ACLR compared to that in the healthy limb (Reconstructed limb; RCL = Pre-operated (PRE): 19.89 ± 6.91 mm, Post-operated(POST): 16.04 ± 6.13 mm, Healthy limb; HL = PRE: 22.88 ± 6.07, POST: 20.90 ± 5.78 mm, F = 9.325, p = 0.009, η2p = 0.418). Conclusions: The results represent a selective surgical influence on the quadricep muscle thickness. These findings highlight the need of advanced strengthening exercises in order to restore VI thickness after ACLR.

Impact of 5'-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription.

Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.

Conductive films based on sandwich structures of carbon nanotubes/silver nanowires for stretchable interconnects.

A variety of conductive films made of a hybrid of two conductive nanomaterials have been used as stretchable electrodes or interconnectors, desirable for stretchable electronic devices. Their intrinsic stretchability of electrical conductivity allows for accommodating mechanical strain to a certain extent under various deformations. However, few efforts have been made to enhance the interactions between two conductive components in a hybrid system. Herein, we reported new conductive films with tri-layer sandwich structures based on carbon nanotubes (CNTs) and Ag nanowires (NWs), encapsulated in silicone rubber, exhibited high stretchability along with insignificant piezoresistivity. They are suitable to be stretchable interconnectors. A successive vacuum filtration method was used to stack the conductive components layer by layer. The effects of the stacking sequence and the interactions between layers on the stretchability and stability of the electrical properties under mechanical deformations were studied. In the case of a tri-layer conductive film comprising two CNT outer layers and one AgNW central layer in the presence of enhanced interfacial interactions, it showed exceptionally durability in withstanding repetitive deformations.

Knockdown of long non-coding RNA NEAT1 inhibits glioma cell migration and invasion via modulation of SOX2 targeted by miR-132.

BACKGROUND: A better understanding of the molecular mechanism involving lncRNA-miRNA-mRNA network underlying glioma genesis is beneficial to the treatment of glioma. This study was designed to investigate the role of lncRNA NEAT1, miR-132 and SOX2 interaction in glioma. METHODS: Microarray analysis was conducted to identify the differentially expressed lncRNAs in glioma tissues. The expression levels of NEAT1, miR-132 and SOX2 were determined by qRT-PCR and western blot. Proliferation of glioma cells was detected by MTT assay, while migration and invasion were determined by transwell assay. The target relationships were predicted by miRcode algorithm, and confirmed by dual luciferase reporter gene assay. RESULTS: NEAT1 was up-regulated in glioma. Knockdown of NEAT1 inhibited glioma cells' viability, migration and invasion. MiR-132 was down-regulated while SOX2 was up-regulated in glioma cells. NEAT1 negatively regulated the expression of miR-132 in glioma while miR-132 targeted SOX2 to down-regulate its expression. CONCLUSION: NEAT1 promoted glioma development by promoting SOX2 expression through suppressing miR-132.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE: Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.

EGF/EGFR upregulates and cooperates with Netrin-4 to protect glioblastoma cells from DNA damage-induced senescence.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. METHODS: Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. RESULTS: Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. CONCLUSIONS: This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

MiR-338-5p Promotes Glioma Cell Invasion by Regulating TSHZ3 and MMP2.

This study was designed to examine differential expression of miR-338-5p in gliomas and the role of miR-338-5p in glioma cell invasion via its potential target gene TSHZ3 encoding Teashirt zinc finger homobox 3, predicted by bioinformatics, and matrix metallopeptidase 2 (MMP2), the key pro-invasive protease overexpressed in gliomas. Quantitative real-time reverse transcription PCR (qRT-PCR) and Spearman correlation analysis were used to determine differential expressions of miR-338-5p and TSHZ3 in astrocytic gliomas of different grades (n = 35) and glioblastoma cell lines (U87 and U251) in comparison to non-neoplastic brain (NNB) tissues (n = 6). Western blotting was used to determine the protein levels of TSHZ3 and MMP2 in glioblastoma cell lines and Matrigel invasion assay to examine the role of miR-338-5p in cell invasiveness. The results showed that the expression of miR-338-5p, normalized to hsnRNA U6, was significantly higher in grade III and IV gliomas and glioblastoma cell lines compared to that in NNB and grade II gliomas, whereas TSHZ3 expression, normalized to GAPDH, was inversely related to miR-338-5p (R = -0.636, P < 0.01). Luciferase assays showed TSHZ3 to be a target gene of miR-338-5p. In both U87 and U251 cells, miR-338-5p mimics increased MMP2 and invasiveness of the cells. Overexpression of ectopic TSHZ3 suppressed the cell invasiveness and attenuated the pro-invasive effect of miR-338-5p mimics. Overall, our results showed that miR-338-5p has a function in promoting glioma cell invasion by targeting TSHZ3 suppression on MMP2. In conclusion, miR-338-5p is a possible potential biomarker for the diagnosis and target for therapy of high-grade glioma.

Low pressure hydrocephalus: clinical manifestations, radiological characteristics, and treatment.

Low pressure hydrocephalus (LPH) is a rare type of hydrocephalus with low intracranial pressure and ventriculomegaly. The recognition of LPH is important, and the treatment is difficult and very complicated. An understanding of how to drain cerebrospinal fluid when the intracranial pressure is lower than the opening pressure of the value represents a critical issue. Seven patients who suffered from the pain of hydrocephalus syndrome and were diagnosed with LPH were retrospectively reviewed. A ventricle peritoneal shunt was applied to all patients, and the valve system was adjusted to the lowest pressure; however, the clinical manifestations of hydrocephalus in five of seven patients did not improve over one week. Intermittent pressing of the valve in combination with the maintenance of a semi-reclined position were subsequently implemented. The symptoms of hydrocephalus began to improve from three days to two months following the initiation of intermittent valve pressing in combination with the maintenance of a semi-reclined position. At the twelve months follow up, six of seven patients (85.7%) showed good recovery to minimal disability. Intermittent valve pressing in combination with a semi-reclined position is an effective and easy method to drain cerebrospinal fluid when the intracranial pressure (ICP) is lower than the opening pressure of the value and improve hydrocephalus symptoms.

Downregulation of lncRNA-MALAT1 Affects Proliferation and the Expression of Stemness Markers in Glioma Stem Cell Line SHG139S.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is among the most abundant and highly conserved lncRNAs, which has been detected in a wide variety of human tumors, including gastric cancer, gallbladder cancer, and so on. Previous research has showed that MALAT1 can activate LTBP3 gene in mesenchymal stem cells. However, the specific roles of MALAT1 in glioma stem cells (GSCs) remain unclear. In this study, we aimed to identify the effects of MALAT1 on proliferation and the expression of stemness markers on glioma stem cell line SHG139S. Our results showed that downregulation of MALAT1 suppressed the expression of Sox2 and Nestin which are related to stemness, while downregulation of MALAT1 promoted the proliferation in SHG139S. Further research on the underlying mechanism showed that the effects of MALAT1 downregulation on SHG139S were through regulating ERK/MAPk signaling activity. And we also found that downregulation of MALAT1 could activate ERK/MAPK signaling and promoted proliferation in SHG139 cells. These findings show that MALAT1 plays an important role in regulating the expression of stemness markers and proliferation of SHG139S, and provide a new research direction to target the progression of GSCs.

Preservation of KIT genotype in a novel pair of patient-derived orthotopic xenograft mouse models of metastatic pediatric CNS germinoma.

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of ß-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.