Development of a novel and more holistic approach for assessing impact in health and medical research: the Research Impact Assessment Framework.

Considered investment in health and medical research (HMR) is critical for fostering a healthcare system that is sustainable, effective, responsive, and innovative. While several tools exist to measure the impact of research, few assess the research environment that nurtures and supports impactful research and the strategic alignment of research with societal needs. This perspective article discusses the limitations of existing assessment tools and presents a novel Research Impact Assessment Framework designed to enable more strategic and targeted investment towards HMR, having the potential for significant public benefit.

Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts.

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells.

Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the "glycan code") and its application (e.g., biomanufacturing high-value glycomolecules on demand).

The effect of light emitting diode phototherapy on rate of orthodontic tooth movement: a split mouth, controlled clinical trial.

OBJECTIVE: Increasing the rate of orthodontic tooth movement (OTM) can reduce risks such as periodontal disease and caries. TRIAL DESIGN: This split-mouth randomized controlled clinical trial investigated whether light emitting diode (LED) phototherapy could accelerate the rate of OTM. SETTING: The study was conducted at the Graduate Orthodontics Clinic at the University of Toronto, Faculty of Dentistry. PARTICIPANTS AND METHODS: 17 dental arches from 11 orthodontic participants with bilaterally symmetrical extraction of premolars were included. During space closure of single tooth extraction sites, LED phototherapy was applied to one side of the dental arch for a specified time and the contralateral side acted as the control. Space closure was measured immediately prior to, during and later in space closure. RESULTS: All participants were compliant with LED application. Our results revealed no significant changes in the rate of OTM with LED phototherapy over 3 months of extraction space closure. CONCLUSIONS: Our findings were contrary to previous findings of the effect of laser phototherapy on regulating the rate of OTM. Further investigations are warranted to analyse whether the duration or method of LED delivery would have an effect on the rate of OTM. Toronto. This trial was registered at clinicaltrials.gov (NCT01490385).

MiR-22 is frequently downregulated in medulloblastomas and inhibits cell proliferation via the novel target PAPST1.

Medulloblastoma is the most frequent malignant central nervous system tumor in children. MicroRNAs (miRs) are small, non-coding RNAs that target protein-coding and non-coding RNAs, and play roles in a variety of cellular processes through regulation of multiple targets. In the present study, we analyzed miR-22 expression and its effect in cell proliferation and apoptosis in medulloblastomas. Quantitative reverse transcription PCR (RT-PCR) revealed significantly lower expression of miR-22 in 19 out of 27 (70%) medulloblastomas, D341, DAOY, ONS-76 medulloblastoma cell lines, compared with normal cerebellum. Forced expression of miR-22 by lentiviral vector transfection reduced cell proliferation and induced apoptosis, while knockdown of miR-22 increased proliferative activity in DAOY and ONS-76 cells. DAOY cells with miR-22 overexpression in nude mice yielded tumors smaller than those originated from control DAOY cells. Microarray analysis in DAOY cells with forced miR-22 expression showed significant changes in expression profiles, PAPST1 being the most significantly (10 folds) downregulated gene. Quantitative RT-PCR revealed PAPST1 mRNA upregulation in 18 out of 27 (67%) medulloblastomas. In addition, a luciferase reporter assay in ONS-76 and DAOY cells suggested that miR-22 directly targets the PAPST1 gene, and lentivirus-mediated knockdown of PAPST1 suppressed proliferation of DAOY and ONS-76 medulloblastoma cells. These results suggest that frequently downregulated miR-22 expression is associated with cell proliferation in medulloblastomas, and this may be at least in part via PAPST1, which is a novel target of miR-22.

Loss of CIC and FUBP1 expressions are potential markers of shorter time to recurrence in oligodendroglial tumors.

Combined deletion of chromosomes 1p and 19q is a prognostic marker in oligodendroglial tumors. Recent studies in oligodendroglial tumors have unveiled recurrent mutations of CIC (homolog of Drosophila capicua) and FUBP1 (far upstream element binding protein 1) that are located on 19q13 and 1p31, respectively. However, the impact of CIC and FUBP1 mutations on their protein expressions has not been examined. The aims of this study were to correlate the expression patterns of CIC and FUBP1 with their mutation profiles and to evaluate the clinical relevance of these molecular markers in 55 oligodendroglial tumors diagnosed in 47 adult patients. Using direct sequencing, somatic mutations of CIC and FUBP1 were identified in 47% (22/47) and 16% (7/45) of oligodendroglial tumors, respectively. Immunohistochemical analysis revealed loss of CIC or FUBP1 protein expression in 36% (20/55) and 16% (9/55) of oligodendroglial tumors examined. Somatic mutation was significantly associated with absent protein expression for both genes (CIC, P=0.01; FUBP1, P=0.00001). Four tumors with undetectable CIC mutations exhibited absent CIC expression, suggesting that CIC inactivation could be mediated by mechanisms other than mutations and genomic loss. Univariate survival analysis revealed that 1p/19q codeletion was significantly associated with overall survival (P=0.05). Loss of CIC expression was significantly correlated with shorter progression-free survival (P=0.03), whereas CIC alteration (mutation or null expression) with worse overall survival (P=0.05). Absent FUBP1 expression was linked with unfavorable progression-free survival (P=0.02) and overall survival (P=0.01). In 16 tumors with 1p/19q codeletion, CIC mutation was associated with unfavorable survival (P=0.01). There was a correlation between lack of CIC or FUBP1 expression and poor progression-free survival (P=0.004; P=0.0003). No molecular markers showed association with survival in oligodendroglial tumors lacking 1p/19q codeletion. We conclude that absent CIC and FUBP1 expressions are potential markers of shorter time to recurrence and CIC mutation a potential marker of worse prognosis, especially in tumors carrying 1p/19q codeletion.

Mutation analysis of IDH1 in paired gliomas revealed IDH1 mutation was not associated with malignant progression but predicted longer survival.

Recurrence and progression to higher grade lesions are characteristic behaviors of gliomas. Though IDH1 mutation frequently occurs and is considered as an early event in gliomagenesis, little is known about its role in the recurrence and progression of gliomas. We therefore analysed IDH1 and IDH2 status at codon 132 of IDH1 and codon 172 of IDH2 by direct sequencing and anti-IDH1-R132H immunohistochemistry in 53 paired samples and their recurrences, including 29 low-grade gliomas, 16 anaplastic gliomas and 8 Glioblastomas. IDH1/IDH2 mutation was detected in 32 primary tumors, with 25 low-grade gliomas and 6 anaplastic gliomas harboring IDH1 mutation and 1 low-grade glioma harboring IDH2 mutation. All of the paired tumors showed consistent IDH1 and IDH2 status. Patients were analyzed according to IDH1 status and tumor-related factors. Malignant progression at recurrence was noted in 22 gliomas and was not associated with IDH1 mutation. Survival analysis revealed patients with IDH1 mutated gliomas had a significantly longer progression-free survival (PFS) and overall survival (OS). In conclusion, this study demonstrated a strong tendency of IDH1/IDH2 status being consistent during progression of glioma. IDH1 mutation was not a predictive marker for malignant progression and it was a potential prognostic marker for gliomas of Chinese patients.

MIR-137 suppresses growth and invasion, is downregulated in oligodendroglial tumors and targets CSE1L.

MicroRNA-137 (miR-137) expression has been reported to be decreased in astrocytic tumors in two expression profiling studies but its role in the pathogenesis of oligodendroglial tumors is still limited. In this study, we demonstrate that miR-137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and nine glioma cell lines compared with normal brains. Lower miR-137 expression is associated with shorter progressive-free survival and overall survival. Restoration of miR-137 expression in an oligodendroglial cells TC620, and also glioblastoma cells of U87 and U373 significantly suppressed cell growth, anchorage-independent growth, as well as invasion. Demethylation and deacetylation treatments resulted in upregulation of miR-137 expression in TC620 cells. In silico analysis showed that CSE1 chromosome segregation 1-like (yeast) (CSE1L) is a potential target gene of miR-137. Luciferase reporter assay demonstrated that miR-137 negatively regulates CSE1L by interaction between miR-137 and complementary sequences in the 3' UTR of CSE1L. Immunohistochemistry revealed that CSE1L is upregulated in oligodendroglial tumors. Knockdown of CSE1L resulted in similar outcomes as overexpressing miR-137 in oligodendroglioma cells and glioblastoma cells. Overall, our data suggest that miR-137 regulates growth of glioma cells and targets CSE1L, providing further understanding in the tumorigenesis of gliomas.

MiR-383 is downregulated in medulloblastoma and targets peroxiredoxin 3 (PRDX3).

Accumulating evidence suggests that microRNAs (miRNAs) are over- or under-expressed in tumors, and abnormalities in miRNA expression may contribute to carcinogenesis. MiR-383 was previously identified as one of the under-expressed miRNAs in medulloblastoma (MB) by miRNA expression profiling. Quantitative reverse transcription polymerase chain reaction (RT-PCR)-based miRNA assays showed an enrichment of miR-383 in normal brain. Based on these data, we speculated that miR-383 is important in MB pathogenesis. In this study, we demonstrated significant downregulation of miR-383 in 23/29 (79%) MB samples and 7/7 (100%) MB cells lines. Ectopic expression of miR-383 in MB cells led to suppression of cell growth, cell accumulation at sub-G1 phase and alteration of apoptosis-related proteins. By transcriptomic analysis and computational algorithms, we identified peroxiredoxin 3 (PRDX3) as a target gene of miR-383. Luciferase reporter assay confirmed that miR-383 negatively regulated PRDX3 by interaction between miR-383 and complementary sequences in the 3' UTR of PRDX3. MiR-383 repressed PRDX3 at transcriptional and translational levels as revealed by quantitative RT-PCR and Western blot analysis. Furthermore, depletion of PRDX3 by siRNAs resulted in similar effects as observed in miR-383-transfected cells. In conclusion, miR-383 acts as a regulator controlling cell growth of MB, at least in part, through targeting PRDX3.

Overexpression of IL-7 enhances cisplatin resistance in glioma.

Cisplatin is one of the most commonly used chemotherapeutic agents for glioma patients. In this study, array comparative genomic hybridization (aCGH) was used to identify genes associated with cisplatin resistance in a human glioma cell line. The cisplatin-resistant U251/CP2 cell line was derived by stepwise selection using cisplatin. The genetic aberrations of the U251 parental cell line and the U251/CP2 cells were analyzed using aCGH. RT-PCR was used to detect the expression of the altered genes revealed by aCGH. The sensitivity of glioma cells to cisplatin was determined by using the MTT assay. Apoptosis was detected using flow cytometry and western blot analysis. The IC 50 value of cisplatin in U251/CP2 cells was five times higher than its IC 50 in U251 cells. The U251 cells lost at least one copy each of the CFHR1 and CFHR3 genes, and both CFHR1 and CFHR3 were homozygously deleted in U251/CP2 cells. The U251/CP2 cells gained two to three copies of C8orf70 and IL-7 genes. IL-7 mRNA expression was studied in 12 glioma cell lines, and expression was positively correlated with the IC 50 of cisplatin. Furthermore, IL-7 mRNA expression was also positively correlated with the IC 50 of cisplatin in 91 clinical glioma specimens. Additionally, treatment with recombinant human IL-7 (rhIL-7) enhanced cisplatin resistance and increased the relative growth rate of the glioma cells. Moreover, the apoptosis induced by cisplatin could be inhibited by IL-7. In conclusion, our results suggest that IL-7 may play an important role in cisplatin resistance in glioma.

Immediate loading in the maxillary arch: evidence-based guidelines to improve success rates: a review.

The reliability of immediately loaded dental implants in the mandible has prompted many to investigate their application in the maxilla. Although the body of literature is growing, the long-term survivability of immediate loading in the maxilla is still pending. This review of literature investigates the status of immediate loading of dental implants in the maxilla to determine its predictability as a treatment option for partial and complete maxillary edentulism. Current terminology in the field is summarized first. Subsequently, the rationale and advantages of immediate loading in the maxilla are reviewed, and the relationships between immediate loading and osseointegration, primary stability, implant design, micromotion, immediate implant placement, and bone character are explored. The importance of a prosthodontically driven implant treatment plan emphasizing the role of splinting a high-precision and passively fitting implant restoration with reduced micromotion under function is summarized. The reliability and predictability of immediately loaded implants as a treatment option are proposed, and recommended guidelines for the successful delivery of immediately loaded implants in the maxilla are presented.

Clinical significance of vasculogenic mimicry in human gliomas.

Vasculogenic mimicry (VM) is known as non-endothelial tumor cell-lined microvascular channels in aggressive tumors. We have previously found the presence of VM in high-grade gliomas. In this study, we aimed to identify VM patterns in gliomas and to explore their clinical significance. Tumor samples as well as their detailed clinical/prognostic data were collected from 101 patients. Vasculogenic mimicry in the glioma samples was determined by dual staining for endothelial marker CD34 and periodic acid-Schiff (PAS). Tumor samples were also immunohistochemically stained for Ki-67, VEGF, COX-2 and MMP-9. The association between VM and the clinical characteristics of the patients were analyzed. A Kaplan-Meier survival analysis and log-rank tests were performed to compare survival times of the patients. Vasculogenic mimicry was present in 13 out of 101 samples. The higher grade gliomas had a higher incidence of VM than that of lower grade gliomas (P = 0.006). Vasculogenic mimicry channels were associated with the expression of COX-2 and MMP-9 (P < 0.05). While there was no association between the existence of VM and the sex, age and preoperative epilepsy of the patients, or expression of Ki-67 and VEGF. However, patients with VM-positive gliomas survived a shorter period of time than those with VM negative gliomas (P = 0.027). Interestingly, in high-grade gliomas, the level of microvascular density was lower in VM positive tumors than those VM negative tumors (P = 0.039). Our results suggest that VM channels in gliomas correlate with increasing malignancy and higher aggressiveness, and may provide a complementation to the tumor's blood supply, especially in less vascularized regions, which may aid in the identification of glioma patients with a poorer prognosis.

KIAA0495/PDAM is frequently downregulated in oligodendroglial tumors and its knockdown by siRNA induces cisplatin resistance in glioma cells.

Co-deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT-related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31-p36.32 that might be involved in OTs. A novel gene KIAA0495/p53-dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild-type p53. B-cell CCL/lymphoma 2 (BCL2)-like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM-silenced cells after cisplatin treatment, suggesting that alteration of anti-apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin-resistant phenotype. Moreover, our data suggested that PDAM might function as a non-protein-coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53-dependent anti-apoptotic genes.

Role of the RARRES1 gene in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a unique type of head and neck cancer that is most prevalent in southern China. Previous studies have suggested that genetic susceptibility, environmental carcinogens, and Epstein-Barr virus (EBV) infection contribute to the etiology of NPC. Our group has identified the retinoic acid receptor responder (tazarotene induced) 1 gene (RARRES1; alias TIG1) to be transcriptionally silenced by promoter hypermethylation in approximately 90% of NPC cases, suggesting that its inactivation may be important in NPC formation. The aim of this study was to explore the functional role of the RARRES1 protein (alias TIG1) in NPC cells with EBV infection (HK1-EBV) and without (HK1). Cellular proliferation analysis, as measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation, showed that knockdown and overexpression of TIG1 in HK1 led, respectively, to significantly increased (P = 0.005) and reduced (P = 0.027) proportions of BrdU-labeled cells, compared with control cells. In contrast, knockdown or overexpression of TIG1 had no significant effect on cellular proliferation in HK1-EBV cells. Invasion chamber assay showed that TIG1 knockdown in HK1-EBV cells resulted in significant enhancement of invasive capacity of HK1-EBV cells (P = 0.006). HK1 cells were not invasive, regardless of TIG1 status. These findings suggest that TIG1 may play a role in cellular proliferation and invasion in NPC cells and that its function may be dependent on the EBV status.

Glioblastoma stem cells resistant to temozolomide-induced autophagy.

BACKGROUND: Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance. METHODS: Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133(+) and CD133(-) glioblastoma cells was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P < 0.05. RESULTS: CD133(+) glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133(-) glioblastoma cells treated with 5 or 50 micromol/L TMZ was significantly higher than that in CD133(+) glioblastoma cells ((14.36 +/- 3.75)% vs (2.54 +/- 1.36)% or (25.95 +/- 5.25)% vs (2.72 +/- 1.84)%, respectively, P < 0.05). Atg5, LC3-II and Beclin-1 levels were significantly lower in CD133(+) glioblastoma cells than those in autologous CD133(-) cells after TMZ treatment (P < 0.05). Caspase-3 was mildly activated only in CD133(-) glioblastoma cells after exposure to TMZ (P < 0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP(+) cells following TMZ treatment. CONCLUSIONS: The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133(+) cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.

miR-124 is frequently down-regulated in medulloblastoma and is a negative regulator of SLC16A1.

Given that miR-124 is preferentially expressed in differentiating and mature neurons and external granule cells of cerebellum are thought to be cells-of-origins of medulloblastomas, we investigated if miR-124 played a role in the development of medulloblastomas. Quantitative expression analysis of 29 medulloblastomas demonstrated significant down-regulation of miR-124 in 21 (72%) tumors by at least 2-fold, with 11 of them exhibiting greater than 10-fold reduced level compared to normal cerebella (P < .01). Ectopic expression of miR-124 in medulloblastoma cell lines, ONS-76 and DAOY, inhibited cell proliferation. Using computational and expression analyses, solute carrier family 16, member 1 (SLC16A1) was identified as a candidate target of miR-124. Transfection of miR-124 resulted in down-regulation of SLC16A1 at both transcript and protein levels. Reporter assay with 3' untranslated region of SLC16A1 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of miR-124, providing strong evidence that miR-124 is a direct regulator of SLC16A1. Expression analysis further revealed that SLC16A1 transcript was elevated in 26 (90%) of 29 tumors examined. Knockdown of SLC16A1 by siRNA induced cell death in medulloblastoma cells. SLC16A1 functions to efflux lactic acid during aerobic glycolysis. We speculated that inhibition of SLC16A1 function resulted in a decrease of intracellular pH to a lethal level. In conclusion, our study demonstrates that miR-124 deregulation is common in medulloblastomas, and restoration of its function inhibits cell proliferation, suggesting that miR-124 may act as a growth suppressor. Our findings also raise the possibility that the miR-124/SLC16A1 pathway may represent a novel therapeutic target for treatment of malignant medulloblastomas.

Oncogenic role of microRNAs in brain tumors.

MicroRNAs (miRNAs) are short non-protein-coding RNAs that function as key regulators of diverse biological processes through negative control on gene expression at the post-transcriptional level. Emerging evidence indicates that miRNAs play an important role in the development of human cancers, with their deregulation resulting in altered activity of downstream tumor suppressors, oncogenes and other signaling molecules. Recent years have seen considerable progress in miRNA research in brain tumors, particularly in glioblastomas and medulloblastomas, providing novel insights into the pathogenesis of these malignant lesions. Expression profiling has unveiled miRNA signatures that not only distinguish brain tumors from normal tissues, but can also differentiate histotypes or molecular subtypes with altered genetic pathways. Moreover, specific miRNA subsets may have potential diagnostic and prognostic values in some brain tumors. Several deregulated miRNAs uncovered in glioblastomas and medulloblastomas have their gene targets and the associated genetic pathways identified. This review summarizes recent findings of miRNA study in brain tumors.

Glioblastoma stem cells resistant to temozolomide-induced autophagy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance.</p><p><b>METHODS</b>Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133(+) and CD133(-) glioblastoma cells was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P < 0.05.</p><p><b>RESULTS</b>CD133(+) glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133(-) glioblastoma cells treated with 5 or 50 micromol/L TMZ was significantly higher than that in CD133(+) glioblastoma cells ((14.36 +/- 3.75)% vs (2.54 +/- 1.36)% or (25.95 +/- 5.25)% vs (2.72 +/- 1.84)%, respectively, P < 0.05). Atg5, LC3-II and Beclin-1 levels were significantly lower in CD133(+) glioblastoma cells than those in autologous CD133(-) cells after TMZ treatment (P < 0.05). Caspase-3 was mildly activated only in CD133(-) glioblastoma cells after exposure to TMZ (P < 0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP(+) cells following TMZ treatment.</p><p><b>CONCLUSIONS</b>The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133(+) cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.</p>

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production.

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.

EMP3 overexpression is associated with oligodendroglial tumors retaining chromosome arms 1p and 19q.

The epithelial membrane protein 3 (EMP3) gene located on chromosome 19q13 has been implicated as a candidate tumor suppressor gene (TSG) in neuroblastomas and gliomas. The aim of this study was to investigate whether EMP3 is involved in oligodendroglial tumors (OTs), which frequently carry combined chromosomes 1p and 19q deletion. We first investigated the transcript level of EMP3 in a cohort of 57 OTs by quantitative real-time RT-PCR. Our results showed that 10 (18%) tumors had reduced EMP3 expression level compared to normal brains. Six of these tumors carried chromosome 19q13 deletion but no statistical correlation was found between the 2 parameters. Intriguingly, a similar proportion (11 of 57, 19%) of tumors displayed EMP3 overexpression, with 8 of them having transcript level >10-fold higher than normal brain. All 11 OTs retained chromosomes 1p36 and 19q13, and a significant association was found between EMP3 overexpression and balanced chromosomes 1p36 and 19q13 (p = 0.004). The methylation status of EMP3 was evaluated by bisulfite sequencing in 29 OTs with diverse expression levels. All tumors except 3 showed aberrant methylation of EMP3 and no correlation was observed between transcript level and methylation status, suggesting that methylation alone does not mediate transcriptional down-regulation of EMP3 in OTs. In conclusion, our study demonstrates that EMP3 overexpression is involved in OTs retaining chromosomes 1p and 19q and does not support EMP3 as the target TSG on chromosome 19q13 in OTs.