Chromosome-scale genome assembly of oil-tea tree Camellia crapnelliana.

Camellia crapnelliana Tutch., belonging to the Theaceae family, is an excellent landscape tree species with high ornamental values. It is particularly an important woody oil-bearing plant species with high ecological, economic, and medicinal values. Here, we first report the chromosome-scale reference genome of C. crapnelliana with integrated technologies of SMRT, Hi-C and Illumina sequencing platforms. The genome assembly had a total length of ~2.94 Gb with contig N50 of ~67.5 Mb, and ~96.34% of contigs were assigned to 15 chromosomes. In total, we predicted 37,390 protein-coding genes, ~99.00% of which could be functionally annotated. The chromosome-scale genome of C. crapnelliana will become valuable resources for understanding the genetic basis of the fatty acid biosynthesis, and greatly facilitate the exploration and conservation of C. crapnelliana.

Novel PIKfyve/Tubulin Dual-target Inhibitor as a Promising Therapeutic Strategy for B-cell Acute Lymphoblastic Leukemia.

OBJECTIVE: In B-cell acute lymphoblastic leukemia (B-ALL), current intensive chemotherapies for adult patients fail to achieve durable responses in more than 50% of cases, underscoring the urgent need for new therapeutic regimens for this patient population. The present study aimed to determine whether HZX-02-059, a novel dual-target inhibitor targeting both phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) and tubulin, is lethal to B-ALL cells and is a potential therapeutic for B-ALL patients. METHODS: Cell proliferation, vacuolization, apoptosis, cell cycle, and in-vivo tumor growth were evaluated. In addition, Genome-wide RNA-sequencing studies were conducted to elucidate the mechanisms of action underlying the anti-leukemia activity of HZX-02-059 in B-ALL. RESULTS: HZX-02-059 was found to inhibit cell proliferation, induce vacuolization, promote apoptosis, block the cell cycle, and reduce in-vivo tumor growth. Downregulation of the p53 pathway and suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway and the downstream transcription factors c-Myc and NF-κB were responsible for these observations. CONCLUSION: Overall, these findings suggest that HZX-02-059 is a promising agent for the treatment of B-ALL patients resistant to conventional therapies.

Evaluation the Effect of Sonodynamic Therapy with 5-Aminolevulinic Acid and Sodium Fluorescein by Preclinical Animal Study.

Sonodynamic therapy (SDT) is a novel tumor treatment that combines biosafe sonosensitizers and noninvasive focused ultrasound to eradicate solid tumors. Sonosensitizers such as 5-aminolevulinic acid and fluorescein have great potential in tumor treatment. Here, rodent subcutaneous and brain tumor models were used to evaluate the treatment effect of both 5-ALA- and fluorescein-mediated SDT. The subcutaneous tumor growth rates of both SDT groups were significantly inhibited compared with that of the control groups. For intracranial tumors, 5-ALA-SDT treatment significantly inhibited brain tumor growth, while fluorescein-SDT exerted no therapeutic effect in animals. The distribution of fluorescein in the brain tumor region underwent further assessment. Seven days post tumor implantation, experimental animals received fluorescein and were sacrificed for brain specimen collection. Analysis of the dissected brains revealed no fluorescence signals, indicating an absence of fluorescein accumulation in the early-stage glioma tissue. These data suggest that the fluorescein-SDT treatment response is closely related to the amount of accumulated fluorescein. This study reports the equivalent effects of 5-ALA and fluorescein on the treatment of somatic tumors. For orthotopic brain tumor models, tumor vascular permeability should be considered when choosing fluorescein as a sonosensitizer. In conclusion, both fluorescein and 5-ALA are safe and effective SDT sonosensitizers, and the tumor microenvironment and pathologic type should be considered in the selection of adequate sonosensitizers.

Prognostic value of an APOBEC3 deletion polymorphism for glioma patients in Taiwan.

OBJECTIVE: The molecular pathogenesis of malignant gliomas, characterized by diverse tumor histology with differential prognosis, remains largely unelucidated. An APOBEC3 deletion polymorphism, with a deletion in APOBEC3B, has been correlated to risk and prognosis in several cancers, but its role in glioma is unclear. The authors aimed to examine the clinical relevance of the APOBEC3 deletion polymorphism to glioma risk and survival in a glioma patient cohort in Taiwan. METHODS: The authors detected deletion genotypes in 403 glioma patients and 1365 healthy individuals in Taiwan and correlated the genotypes with glioma risk, clinicopathological factors, patient survival, and patient sex. APOBEC3 gene family expression was measured and correlated to the germline deletion. A nomogram model was constructed to predict patient survival in glioma. RESULTS: The proportion of APOBEC3B-/- and APOBEC3B+/- genotypes was higher in glioblastoma (GBM) patients than healthy individuals and correlated with higher GBM risk in males. A higher percentage of cases with APOBEC3B- was observed in male than female glioma patients. The presence of APOBEC3B-/- was correlated with better overall survival (OS) in male astrocytic glioma patients. No significant correlation of the genotypes to glioma risk and survival was observed in the female patient cohort. Lower APOBEC3B expression was observed in astrocytic glioma patients with APOBEC3B-/- and was positively correlated with better OS. A 5-factor nomogram model was constructed based on male patients with astrocytic gliomas in the study cohort and worked efficiently for predicting patient OS. CONCLUSIONS: The germline APOBEC3 deletion was associated with increased GBM risk and better OS in astrocytic glioma patients in the Taiwan male population. The APOBEC3B deletion homozygote was a potential independent prognostic factor predicting better survival in male astrocytic glioma patients.

Glioblastoma Primary Cells Retain the Most Copy Number Alterations That Predict Poor Survival in Glioma Patients.

Gliomas are solid tumors that originate from glial cells in the brain or spine and account for 74.6% of malignant primary central nervous system tumors worldwide. As patient-derived primary cells are important tools for drug screening and new therapy development in glioma, we aim to understand the genomic similarity of the primary cells to their parental tumors by comparing their whole-genome copy number variations and expression profile of glioma clinicopathologic factors. We found that the primary cells from grade II/III gliomas lost most of the gene copy number alterations (CNAs), which were mainly located on chromosome 1p and 19q in their parental tumors. The glioblastoma (GBM) primary cells preserved 83.7% of the gene CNAs in the parental GBM tumors, including chromosome 7 gain and 10q loss. The CNA gains of LINC00226 and ADAM6 and the chromosome 16p11 loss were reconstituted in primary cells from both grade II/III gliomas and GBMs. Interestingly, we found these CNAs were correlated to overall survival (OS) in glioma patients using the Merged Cohort LGG and GBM dataset from cBioPortal. The gene CNAs preserved in glioma primary cells often predicted poor survival, whereas the gene CNAs lost in grade II/III primary cells were mainly associated to better prognosis in glioma patients. Glioma prognostic factors that predict better survival, such as IDH mutations and 1p/19q codeletion in grade II/III gliomas, were lost in their primary cells, whereas methylated MGMT promoters as well as TERT promoter mutations were preserved in GBM primary cells while lost in grade II/III primary cells. Our results suggest that GBM primary cells tend to preserve CNAs in their parental tumors, and these CNAs are correlated to poor OS and predict worse prognosis in glioma patients.

CCL5 of glioma-associated microglia/macrophages regulates glioma migration and invasion via calcium-dependent matrix metalloproteinase 2.

BACKGROUND: Glioma-associated microglia/macrophages (GAMs) comprise macrophages of peripheral origin and brain-intrinsic microglia, which support tumor progression. Chemokine C-C ligand 5 (CCL5) is an inflammatory mediator produced by immune cells and is involved in tumor growth and migration in several cancers, including glioma. However, the mechanisms detailing how CCL5 facilitates glioma invasion remain largely unresolved. METHODS: Glioma migration and invasion were determined by wound healing, transwell assay, and 3D µ-slide chemotaxis assay. The expression levels of CCL5, CD68, matrix metalloproteinase 2 (MMP2), phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII), p-Akt, and phosphorylated proline-rich tyrosine kinase 2 were determined by cytokine array, quantitative PCR, western blot, or immunohistochemistry. Zymography and intracellular calcium assays were used to analyze MMP2 activity and intracellular calcium levels, respectively. RESULTS: CCL5 modulated the migratory and invasive activities of human glioma cells in association with MMP2 expression. In response to CCL5, glioma cells underwent a synchronized increase in intracellular calcium levels and p-CaMKII and p-Akt expression levels. CCL5-directed glioma invasion and increases in MMP2 were suppressed after inhibition of p-CaMKII. Glioma cells tended to migrate toward GAM-conditioned media activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in which CCL5 was abundant. This homing effect was associated with MMP2 upregulation, and could be ameliorated either by controlling intracellular and extracellular calcium levels or by CCL5 antagonism. Clinical results also revealed the associations between CCL5 and GAM activation. CONCLUSION: Our results suggest that modulation of glioma CaMKII may restrict the effect of CCL5 on glioma invasion and could be a potential therapeutic target for alleviating glioma growth.

Functional Change of Effector Tumor-Infiltrating CCR5+CD38+HLA-DR+CD8+ T Cells in Glioma Microenvironment.

Human glioma facilitates an impaired anti-tumor immunity response, including defects in circulation of T lymphocytes. The level of CD8+ T-cell activation acts as an immune regulator associated with disease progression. However, little is known about the characteristics of peripheral and tumor-infiltrating CD8+ T cells in patients with glioma. In this study, we examined the level of CD8+ T-cell activation in a group of 143 patients with glioma and determined that peripheral CD3+ T cells decreased in accordance with disease severity. The patients' peripheral CD8+ T-cell populations were similar to that of healthy donors, and a small amount of CD8+ tumor-infiltrating lymphocytes was identified in glioma tissues. An increase in activated CD8+ T cells, characterized as CD38+HLA-DR+, and their association with disease progression were identified in the patients' peripheral blood and glioma, and shown to display enriched CCR5+ and TNFR2+ expression levels. Ex vivo examination of CD38+HLA-DR+CD8+ T cells indicated that this subset of cells displayed stronger secretion of IFN-γ and IL-2 before and after a 6-h stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) relative to healthy CD38+HLA-DR+CD8+ T cells, indicating the functional feasibility of CD38+HLA-DR+CD8+ T cells. Higher CCL5 protein and mRNA levels were identified in glioma tissues, which was consistent with the immunohistochemistry results revealing both CCL5 and CD38+HLA-DR+CD8+ T cell expression. Patients' CCR5+CD38+HLA-DR+CD8+ T cells were further validated and shown to display increases in CD45RA+CCR7- and T-bet+ accompanied by substantial CD107-a, IFN-γ, and Granzyme B levels in response to glioma cells.

Suppression of tumor growth via IGFBP3 depletion as a potential treatment in glioma.

OBJECTIVE: Despite intensive medical treatment, patients with glioblastoma (grade IV glioma [GBM]) have a low 5-year survival rate of 5.5%. In this study, the authors tried to improve currently used therapies by identification of a therapeutic target, IGFBP3, for glioma treatment. METHODS: IGFBP3 RNA expression in 135 patients newly diagnosed with glioma was correlated with clinicopathological factors. Immunohistochemical analysis was performed to determine IGFBP3 protein expression in glioma specimens. The effect of IGFBP3 depletion on cell proliferation was examined using IGFBP3 knockdown glioma cells. Intracranial infusion of IGFBP3 siRNAs was performed to evaluate the effect of IGFBP3 depletion in mouse intracranial xenograft models. RESULTS: We demonstrated higher IGFBP3 expression in GBM than in tumor margin and grade II glioma. IGFBP3 expression was not only positively correlated with tumor grades but also associated with tumor histology and IDH1/2 mutation status. Additionally, higher IGFBP3 expression predicted shorter overall survival in glioma and GBM proneural subgroup patients. In vitro cell culture studies suggested IGFBP3 knockdown suppressed cell proliferation and induced cell cycle G2/M arrest as well as apoptosis in glioma cells. Also, accumulation of DNA double-strand breaks and γH2AX was observed in IGFBP3 knockdown cells. IGFBP3 knockdown delayed in vivo tumor growth in mouse subcutaneous xenograft models. Furthermore, convection-enhanced delivery of IGFBP3 siRNA to mouse brain suppressed intracranial tumor growth and prolonged survival of tumor-bearing mice. CONCLUSIONS: Our findings suggest IGFBP3 predicts poor outcome of glioma patients and is a potential therapeutic target for which depletion of its expression suppresses tumor growth through inducing apoptosis and accumulation of DNA damage in glioma cells.

The rs16906252:C>T SNP is not associated with increased overall survival or temozolomide response in a Han-Chinese glioma cohort.

The methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) is associated with the prognosis in gliomas and in other cancers. Recent studies showed that rs16906252, an SNP in the MGMT promoter, is associated with promoter methylation and is a predictor of the overall survival time (OST) and the response to temozolomide (TMZ) treatment. However, these findings haven't been systematically investigated in the Han-Chinese population. We analyzed the relevance between rs16906252 polymorphisms, the MGMT methylation status, and the OST in 72 Han-Chinese gliomas patients. The MGMT promoter methylation was measured by bisulfite conversion followed by pyro-sequencing, while rs16906252 was measured by restriction endonuclease digestion. Contrary to the previous findings, we found no association between rs16906252 genotypes and promoter methylation on MGMT. The lower-grade glioma (LGGs) patients carrying the C allele with rs16906252 showed a surprisingly better OST (P = 0.04). Furthermore, the LGG patients carrying hypo-methylated MGMT promoter and rs16906252 T allele showed significantly poorer prognosis. The prognostic benefit of MGMT promoter methylation and genotypes on gliomas patients is marginal. A new molecular stratified patient grouping of LGGs is potentially associated with poorer OST. Active MGMT might have a protective role in LGG tumors, enabling evolution to severe malignancy.

1,3-Bis(2-chloroethyl)-1-nitrosourea-loaded bovine serum albumin nanoparticles with dual magnetic resonance-fluorescence imaging for tracking of chemotherapeutic agents.

To date, knowing how to identify the location of chemotherapeutic agents in the human body after injection is still a challenge. Therefore, it is urgent to develop a drug delivery system with molecular imaging tracking ability to accurately understand the distribution, location, and concentration of a drug in living organisms. In this study, we developed bovine serum albumin (BSA)-based nanoparticles (NPs) with dual magnetic resonance (MR) and fluorescence imaging modalities (fluorescein isothiocyanate [FITC]-BSA-Gd/1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU] NPs) to deliver BCNU for inhibition of brain tumor cells (MBR 261-2). These BSA-based NPs are water dispersible, stable, and biocompatible as confirmed by XTT cell viability assay. In vitro phantoms and in vivo MR and fluorescence imaging experiments show that the developed FITC-BSA-Gd/BCNU NPs enable dual MR and fluorescence imaging for monitoring cellular uptake and distribution in tumors. The T1 relaxivity (R1) of FITC-BSA-Gd/BCNU NPs was 3.25 mM(-1) s(-1), which was similar to that of the commercial T1 contrast agent (R1 =3.36 mM(-1) s(-1)). The results indicate that this multifunctional drug delivery system has potential bioimaging tracking of chemotherapeutic agents ability in vitro and in vivo for cancer therapy.

Macrophage mannose receptor 1 and S100A9 were identified as serum diagnostic biomarkers for colorectal cancer through a label-free quantitative proteomic analysis.

BACKGROUND: Simple blood tests that could be used for early detection are crucial for the ultimate control and prevention of colorectal cancer (CRC). In this study, we performed a serum proteomic analysis of CRC and health volunteers to identify the novel biomarkers involved in CRC. METHOD: A shotgun proteomic method was applied to identify serum proteins in the serum samples of three CRC and three health volunteers using a combination of high-performance liquid chromatography and mass spectrometry. Label-free protein profiling was conducted to quantify the proteins and compare the profiles of the CRC and health volunteers. Two differentially expressed proteins were further validated by western blot analysis. Quantity analysis was performed through enzyme linked immunosorbent assay (ELISA) in serum from 96 healthy and 118 CRC volunteers. RESULTS: Among of the 373 identified proteins, 69 were linked to CRC (33 upregulated and 36 downregulated). The Gene Ontology and DAVID databases were used to identify the location and function of the different proteins. Among the 69 proteins linked to CRC, two proteins, namely, macrophage mannose receptor 1 (MRC1) and S100A9, were verified to be upregulated in CRC by western blot analysis and could be used to identify CRC from healthy volunteers with high accuracy through ELISA analysis. CONCLUSION: MRC1 and S100A9 may contribute to the determination of the mechanisms and screening involved in CRC.

Non-invasive synergistic treatment of brain tumors by targeted chemotherapeutic delivery and amplified focused ultrasound-hyperthermia using magnetic nanographene oxide.

The combination of chemo-thermal therapy is the best strategy to ablate tumors, but how to heat deep tumor tissues effectively without side-damage is a challenge. Here, a systemically delivered nanocarrier is designed with multiple advantages, including superior heat absorption, highly efficient hyperthermia, high drug capacity, specific targeting ability, and molecular imaging, to achieve both high antitumor efficacy and effective amplification of hyperthermia with minimal side effects.

Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study.

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

Novel magnetic/ultrasound focusing system enhances nanoparticle drug delivery for glioma treatment.

Malignant glioma is a common and severe primary brain tumor with a high recurrence rate and an extremely high mortality rate within 2 years of diagnosis, even when surgical, radiological, and chemotherapeutic interventions are applied. Intravenously administered drugs have limited use because of their adverse systemic effects and poor blood-brain barrier penetration. Here, we combine 2 methods to increase drug delivery to brain tumors. Focused ultrasound transiently permeabilizes the blood-brain barrier, increasing passive diffusion. Subsequent application of an external magnetic field then actively enhances localization of a chemotherapeutic agent immobilized on a novel magnetic nanoparticle. Combining these techniques significantly improved the delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea to rodent gliomas. Furthermore, the physicochemical properties of the nanoparticles allowed their delivery to be monitored by magnetic resonance imaging (MRI). The resulting suppression of tumor progression without damaging the normal regions of the brain was verified by MRI and histological examination. This noninvasive, reversible technique promises to provide a more effective and tolerable means of tumor treatment, with lower therapeutic doses and concurrent clinical monitoring.

Evaluation of the prognostic value of CD44 in glioblastoma multiforme.

BACKGROUND/AIM: Glioblastoma and astrocytoma are the most common brain tumors affecting adults 45-60 years of age. The poor prognosis for glioblastoma patients results from recurrence after treatment. There is therefore an urgent need to develop diagnostic and prognostic markers as well as new therapies. PATIENTS AND METHODS: Microarray analyses of clinical specimens from glioblastoma patients were used to identify potential tumor markers. Expression of candidate genes was analyzed by real-time reverse transcription-polymerase chain reaction and by immunoblotting and immunohistochemistry. RESULTS: Five potential markers (CD44 antigen (CD44), growth arrest and DNA-damage-inducible, alpha (GADD45A), fibronectin 1 (FN1), CD63 antigen (CD63) and secreted phosphoprotein 1 (SPP1)) showed expression patterns that correlated significantly with malignant glioma. In particular, expression of the CD44 antigen was elevated in more severe tumor types, and higher in tumor cores than in peripheral regions. However, lower levels of CD44 expression surprisingly correlated with lower survival. CONCLUSION: The CD44 antigen is a promising candidate for further development as a prognostic and therapeutic tool.