The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Intervention to Improve Diarrhea-Related Knowledge and Practices Among Informal Healthcare Providers in Slums of Kolkata.

BACKGROUND: In the densely populated slums of Kolkata, informal healthcare providers' (IHP) diarrhea-related knowledge and rationality of practices should be improved to reduce risk of adverse outcome, expenditure, and antimicrobial resistance. METHODS: A multicomponent intervention was conducted among 140 representative IHPs in the slums of 8 wards in Kolkata to assess its impact on their diarrhea-related knowledge and practice. Six intervention modules in local languages were provided (1 per month) with baseline (N = 140) and postintervention (N = 124) evaluation. RESULTS: Mean overall (61.1 to 69.3; P < .0001) and domain-specific knowledge scores for etiology/spread (5.4 to 8.1; P < .0001), management (6.4 to 7.2; P < .0001), and oral rehydration solution ([ORS] 5.7 to 6.5; P < .0001) increased significantly (at α = 0.05) after intervention and were well retained. Impact on knowledge regarding etiology/spread (adjusted odds ratio [aOR] = 5.6; P < .0001), cholera (aOR = 2.0; P = .0041), management (aOR = 3.1; P < .0001), ORS (aOR = 2.3; P = .0008), and overall (aOR = 4.3; P < .0001) were significant. Intervention worked better for IHPs who practiced for ≥10 years (aOR = 3.2; P < .0001), untrained IHPs (aOR = 4.8; P < .0001), and pharmacists (aOR = 8.3; P < .0001). Irrational practices like empirical antibiotic use for every cholera case (aOR = 0.3; P < .0001) and investigation for every diarrhea case (aOR = 0.4; P = .0003) were reduced. Rationality of testing (aOR = 4.2; P < .0001) and antibiotic use (aOR = 1.8; P = .0487) improved. CONCLUSIONS: Multicomponent educational intervention resulted in sustainable improvement in diarrhea-related knowledge and practices among IHPs in slums of Kolkata. Policy implications should be advocated along with implementation and scale-up.

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Evaluation of intra-operative imaging during scarf osteotomy - an unnecessary cost or an essential prerequisite?

Scarf osteotomy is an effective surgical treatment option for hallux valgus. It can manipulate alignment in three planes, allowing accurate anatomical correction. The potential benefit of intra-operative image intensification (II) to gauge deformity correction during surgery however, has not been quantitatively reported. This study aims to compare the correction of hallux valgus by scarf osteotomy with and without intra-operative imaging. Retrospective analysis of a consecutive series of scarf osteotomy in 2 groups. Group A had intra-operative radiographic assessment and group B did not. Patient and surgical data was collected with a mean follow-up of 14 months. Of 99 scarf osteotomies there was no significant difference in age, gender or pre-operative deformity between the groups (p<0.05). No statistical difference was found between the radiographic corrections of the two groups (p<0.05), although operating time was less in group B. This series shows that intra-operative imaging does not improve accuracy of deformity correction, or implant position in scarf osteotomy. We suggest it is not required routinely during scarf osteotomy.

Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content.

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here, we report that the oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell-derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases can be detected in EVs purified from plasma of tumor-bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804 but not with anti-EGFR antibody (Cetuximab) or etoposide, triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR, and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Tyr-845, Tyr-1068, and Tyr-1173), ERK, and AKT varies between cells and their corresponding EVs and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.

Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma.

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%-60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.

Differential transformation capacity of neuro-glial progenitors during development.

Gliomas represent the most common type of brain tumor, but show considerable variability in histologic appearance and clinical outcome. The phenotypic differences between types and grades of gliomas have not been explained solely on the grounds of differing oncogenic stimuli. Several studies have demonstrated that some phenotypic differences may be attributed to regional differences in the neural stem cells from which tumors arise. We hypothesized that temporal differences may also play a role, with tumor phenotypic variability reflecting intrinsic differences in neural stem cells at distinct developmental stages. To determine how the tumorigenic potential of lineally related stem cells changes over time, we used a conditional transgenic system that integrates Cre-Lox-mediated and Tet-regulated expression to drive K-ras(G12D) expression in neuro-glial progenitor populations at different developmental time points. Using this model, we demonstrate that K-ras(G12D)-induced transformation is dependent on the developmental stage at which it is introduced. Diffuse malignant brain tumors develop during early embryogenesis but not when K-ras(G12D) expression is induced during late embryogenesis or early postnatal life. We show that differential expression of cell-cycle regulators during development may be responsible for this differing susceptibility to malignant transformation and that loss of p53 can overcome the transformation resistance seen at later developmental stages. These results highlight the interplay between genetic alterations and the molecular changes that accompany specific developmental stages; early progenitors may lack the regulatory mechanisms present at later, more lineage-restrictive, developmental time points, making them more susceptible to transformation.

Identification and selected reaction monitoring (SRM) quantification of endocytosis factors associated with Numb.

Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.

Extracellular vesicles--vehicles that spread cancer genes.

Once regarded as cellular 'debris' extracellular vesicles (EVs) emerge as one of the most intriguing entities in cancer pathogenesis. Intercellular trafficking of EVs challenges the notion of cancer cell autonomy, and highlights the multicellular nature of such fundamental processes as stem cell niche formation, tumour stroma generation, angiogenesis, inflammation or immunity. Recent studies reveal that intercellular exchange mediated by EVs runs deeper than expected, and includes molecules causative for cancer progression, such as oncogenes (epidermal growth factor receptor, Ras), and tumour suppressors (PTEN). The uptake of oncogenic EVs (oncosomes) by various cells may profoundly change their biology, signalling patterns and gene expression, and in some cases cause their overt tumorigenic conversion. Moreover, EVs circulating in blood and present in body fluids provide an unprecedented access to the molecular circuitry driving cancer cells, and new technologies are being developed to exploit this property as a source of unique cancer biomarkers.

Endoscopic Endonasal Trans-Sphenoidal Minimally Invasive Pituitary Surgery with Image Guided Navigation System (Igns): Learning Experience of Ent Surgeon: First Author.

Introduction- Endoscopic minimally invasive pituitary surgery (MIPS) is advantageous over microscopic technique, as it provides superior close up, wide angle view of surgical target area. Image guided navigation system (IGNS) guides the surgeon to localize the lesion. In the present study we analyzed the Image Guided Surgical procedure and outcome of Endoscopic minimally invasive pituitary surgery and shared our experiences regarding disease clearance. MATERIALS AND METHODS: During the period of April 2015 to August 2022 a total 104 patients, diagnosed with pituitary adenoma underwent surgery and further followed up in a multidisciplinary team approach in a tertiary care hospital of Kolkata, India. The data obtained were reviewed statistically to satisfy the study objectives. RESULTS: Total 104 operations were done on 98 patients and total cases taken for calculation and analysis was 98, which consist of 11 microadenomas, 81 macroadenomas. Among 35 patients with normal preoperative hormonal assay, one patient developed postoperative hypopituitarism. Among 6 patients with preoperative hypopituitarism 4 patients (66.6%) recovered after surgery. Overall, 85 cases had total disease clearance as detected on post-operative MRI. In functioning pituitary adenoma (FPA) clinical and endocrinological improvement occurred after primary surgery in 85.36% (n = 35) and after revision surgery it was 84.44% (n = 38). Macroadenomas, giant adenomas were found to have statistically significant higher risk of incomplete disease clearance but large adenomas do not have statistically higher risk of incomplete clearance. CONCLUSION: IGNS requires extra time for setup, but with proper registration of tracker instruments it adds precision to the surgery. IGNS supplements endoscopic visualization with localization of target lesion by real time stereotactic feedback using preset preoperative imaging data, thus increasing accuracy, safety and effectiveness of minimally invasive surgery.

Loss of p53 cooperates with K-ras activation to induce glioma formation in a region-independent manner.

Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morphological features. These differences, between and within varying grades of gliomas, have not been explained solely on the grounds of an oncogenic stimulus. Interactions with the tumor microenvironment as well as inherent characteristics of the cell of origin are likely a source of this heterogeneity. There is an ongoing debate over the cell of origin of gliomas, where some suggest a progenitor, while others argue for a stem cell origin. Thus, it is presumed that neurogenic regions of the brain such as the subventricular zone (SVZ) containing large numbers of neural stem and progenitor populations are more susceptible to transformation. Our studies demonstrate that K-ras(G12D) cooperates with the loss of p53 to induce gliomas from both the SVZ and cortical region, suggesting that cells in the SVZ are not uniquely gliomagenic. Using combinations of doxycycline-inducible K-ras(G12D) and p53 loss, we show that tumors induced by the cooperative actions of these genes remain dependent on active K-ras expression, as deinduction of K-ras(G12D) leads to complete tumor regression despite absence of p53. These results suggest that the interplay between specific combinations of genetic alterations and susceptible cell types, rather than the site of origin, are important determinates of gliomagenesis. Additionally, this model supports the view that, although several genetic events may be necessary to confer traits associated with oncogenic transformation, inactivation of a single oncogenic partner can undermine tumor maintenance, leading to regression and disease remission.

Role of moesin in hyaluronan induced cell migration in glioblastoma multiforme.

BACKGROUND: A major barrier to effective treatment of glioblastoma multiforme (GBM) is the invasion of glioma cells into the brain parenchyma rendering local therapies such as surgery and radiation therapy ineffective. GBM patients with such highly invasive and infiltrative tumors have poor prognosis with a median survival time of only about a year. However, the mechanisms leading to increased cell migration, invasion and diffused behavior of glioma cells are still poorly understood. METHODS: In the current study, we applied quantitative proteomics for the identification of differentially expressed proteins in GBMs as compared to non-malignant brain tissues. RESULTS: Our study led to the identification of 23 proteins showing overexpression in GBM; these include membrane proteins, moesin and CD44. The results were verified using Western blotting and immunohistochemistry in independent set of GBM and non-malignant brain tissues. Both GBM tissues and glioma cell lines (U87 / U373) demonstrated membranous expression of moesin and CD44, as revealed by immunohistochemistry and immunofluorescence, respectively. Notably, glioma cells transfected with moesin siRNA displayed reduced migration and invasion on treatment with hyaluronan (HA), an important component of the extracellular matrix in GBM. CD44, a transmembrane glycoprotein, acts as a major receptor for hyaluronan (HA). Using co-immunoprecipitation assays, we further demonstrated that moesin interacts with CD44 in glioma cells only after treatment with HA; this implicates a novel role of moesin in HA-CD44 signaling in gliomas. CONCLUSIONS: Our results suggest that development of inhibitors which interfere with CD44-moesin interactions may open a new avenue in the future to mitigate cellular migration in gliomas.

A novel neurofibromin (NF1) interaction with the leucine-rich pentatricopeptide repeat motif-containing protein links neurofibromatosis type 1 and the French Canadian variant of Leigh's syndrome in a common molecular complex.

Loss-of-function mutations and deletions in the neurofibromin tumor suppressor gene (NF1) cause neurofibromatosis type 1 (NF-1), the most common inherited syndrome of the nervous system in humans, with a birth incidence of 1:3,000. The most visible features of NF-1 are the neoplastic manifestations caused by the loss of Ras-GTPase-activating protein (Ras-GAP) activity mediated through the GAP-related domain (GRD) of neurofibromin (NF1), the protein encoded by NF1. However, the syndrome is also characterized by cognitive dysfunction and a number of developmental abnormalities. The molecular etiology of many of these nonneoplastic phenotypes remains unknown. Here we show that the tubulin-binding domain (TBD) of NF1 is a binding partner of the leucine-rich pentatricopeptide repeat motif-containing (LRPPRC) protein. These two proteins complex with Kinesin 5B, hnRNP A2, Staufen1, and Myelin Basic Protein (MBP) mRNA, likely in RNA granules. This interaction is of interest in that it links NF-1 with Leigh's syndrome, French Canadian variant (LSFC), an autosomal recessive neurodegenerative disorder that arises from mutations in the LRPPRC gene. Our findings provide clues to how loss or mutation of NF1 and LRPPRC may contribute to the manifestations of NF-1 and LSFC.

Radial glia cells are candidate stem cells of ependymoma.

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.

Comparison of three different fixation methods of calcaneal osteotomies.

BACKGROUND: There are various methods available to fix a calcaneal osteotomy, ranging from screws to plates and staples. It is not clear if one method is superior to the other. In this series we compare the complications and union rates of 3 different methods of fixation. METHODS: A retrospective review of the records of a consecutive series of patients who had a calcaneal osteotomy was undertaken. All patients had their osteotomy by the same technique, however the subsequent fixation was performed using 3 different methods: a lateral locking plate, a headless, or a headed screw. The screws were placed through a separate stab incision inserted from the infero-posterior heel. Records were kept of subsequent symptoms from the hardware and need for hardware removal as well as any complications. When screws were inserted, the entry point in relation to the weight-bearing surface of the calcaneus was also recorded. Sixty-seven osteotomies were investigated, of which 17 were fixed using a headed screw, 18 using a headless screw, and the remaining 32 were fixed using a lateral plate. RESULTS: There was an overall 97% union rate. The only 2 cases of delayed union were both fixed using a lateral plate. Overall, 47% of the headed screws, 11% of the headless screws, and 6% of the lateral plates were removed to address symptoms that were suspected to arise from the hardware. There was a 10% rate of wound complication in the lateral plate cohort. There were no cases of sural nerve injury or neuroma. No correlation was found between entry position of screw and subsequent hardware symptoms. CONCLUSIONS: Calcaneal osteotomies have high union rates regardless of fixation method. Fixation using a headed screw is associated with a high rate of secondary screw removal. This was unrelated to the position of the screw in relation to the weight-bearing surface of the calcaneus in our series. Hardware problems were less frequent in the headless screw or the lateral plate groups; however, the incidence of local wound complications and radiological delayed union was higher in the group fixed with a lateral plate. This may be related to the greater soft tissue dissection and lesser compression achieved at the osteotomy site. LEVEL OF EVIDENCE: Level III, retrospective case control study.

Microarray-based copy number analysis of neurofibromatosis type-1 (NF1)-associated malignant peripheral nerve sheath tumors reveals a role for Rho-GTPase pathway genes in NF1 tumorigenesis.

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumors. Approximately 15% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-Wide Human single-nucleotide polymorphism (SNP) Array 6.0 was used to perform SNP genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH), and copy number neutral-LOH (CNN-LOH) analyses of DNA isolated from 15 MPNSTs, five benign plexiform neurofibromas (PNFs), and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level CNN-LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN-LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA, Ras-related C3 botulinum toxin substrate 1 (RAC1) (7p21-p22), PDGFRL (8p22-p21.3), and matrix metallopeptidase 12 (MMP12) (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho-GTPase pathway genes and several cytoskeletal remodeling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1, ROCK2, PTK2, and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines, whereas wound healing, cell migration, and invasiveness were reduced, consistent with a role for these Rho-GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs.

Comparison of aprotinin and tranexamic acid in adult scoliosis correction surgery.

PURPOSE: A retrospective review of consecutive adult patients undergoing scoliosis correction surgery was performed to compare the effects of aprotinin and tranexamic acid in blood conservation and to define a comprehensive blood conservation strategy for such surgery. METHODS: Medical records of all patients who underwent scoliosis correction surgery in this unit between January 2003 and December 2008 were reviewed. The patients were divided into three cohorts: group 1 receiving no antifibrinolytics, group 2 aprotinin and group 3 tranexamic acid. Information was collected regarding number of vertebral levels fused, pre- and post-operative haemoglobin, intra-operative blood loss and peri-operative autologous and allogenic blood transfusion performed. RESULTS: Aprotinin was used in 28 patients (38%), tranexamic acid in 26 (36%), while 19 (26%) received no antifibrinolytics. 21 patients had anterior surgery, 34 patients had posterior surgery and 18 had combined anterior and posterior procedures. Mean blood loss in the patients who received aprotinin and tranexamic acid was 710 and 738 ml, respectively. This was significantly less than the patients receiving no antifibrinolytics (972 ml, p = 0.037). Blood transfusion was required in only two patients undergoing anterior correction surgery. CONCLUSION: Aprotinin and tranexamic acid reduce blood loss in adult spinal deformity correction surgery. With aprotinin being unavailable for clinical use, we recommend the use of tranexamic acid along with other blood conservation measures for adult spinal deformity correction surgery.

Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme.

Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is metabolized to lactate rather than by oxidative metabolism, even in the presence of oxygen. This metabolic adaptation provides a survival advantage and facilitates synthesis of biosynthetic precursors required for continued cellular proliferation. An important mediator of aerobic glycolysis is our demonstration that in malignant gliomas there is over-expression of the glycolytic enzyme hexokinase 2 (HK2), phosphorylating glucose as the first step of the glycolytic pathway. In contrast, normal brain preferentially expresses HK1 and undergoes oxidative glucose metabolism. In this study, we examine whether this switch in HK isoform also occurs in the developing embryo and central nervous system (CNS). Bioinformatic analysis of available microarray data, including that of The Cancer Genome Atlas, demonstrated a ~17% overlap in metabolic-related genes in blastocyst stage embryo and human GBM tissue, including upregulation of HK2 and downregulation of HK1. Quantitative RT-PCR on mouse brains isolated at different embryonic and postnatal development time-points demonstrated HK2 expression was highest in the early embryo, while HK1 expression increased with CNS maturation. The downstream glycolytic enzymes PKM2 and LDHA had similar temporal profiles as HK2. Expression of the HK2 isoform was due in part to epigenetic regulation of HK2. In support, adult normal human brain and the few human GBM cell lines with low HK2 expression had methylation of CpG islands within intron 1 of HK2. In contrast, developing human fetal brain and GBM tissue expressing HK2 demonstrated significantly lower percent methylation. Furthermore, treatment of GBM cells lacking HK2 with 5-aza-2-deoxycytidine restored HK2 transcript expression. Overall, our results demonstrate that proliferative states including the developing embryo and malignant gliomas, which rely on aerobic glycolysis, preferentially express the HK2 isoform, found to be regulated in part epigenetically.

Lipoma compressing the brachial plexus in a patient with sarcoidosis: case report.

Lipomas associated with distal peripheral nerves are well recognized; however, those impinging on the brachial plexus are rare. We document a unique case of a sarcoidosis patient with an extraneural lipoma compressing the brachial plexus, and present a review of the current literature.

Preclinical in vivo evaluation of rapamycin in human malignant peripheral nerve sheath explant xenograft.

Neurofibromatosis type 1 (NF1) patients are prone to the development of malignant tumors, the most common being Malignant Peripheral Nerve Sheath Tumor (MPNST). NF1-MPNST patients have an overall poor survival due to systemic metastasis. Currently, the management of MPNSTs includes surgery and radiation; however, conventional chemotherapy is not very effective, underscoring the need for effective biologically-targeted therapies. Recently, the NF1 gene product, neurofibromin, was shown to negatively regulate the phosphoinositide-3-kinase (PI3K)/Protein Kinase-B (Akt)/mammalian Target Of Rapamycin (mTOR) pathway, with loss of neurofibromin expression in established human MPNST cell lines associated with high levels of mTOR activity. We developed and characterized a human NF1-MPNST explant grown subcutaneously in NOD-SCID mice, to evaluate the effect of the mTOR inhibitor rapamycin. We demonstrate that rapamycin significantly inhibited human NF1-MPNST mTOR pathway activation and explant growth in vivo at doses as low as 1.0 mg/kg/day, without systemic toxicities. While rapamycin was effective at reducing NF1-MPNST proliferation and angiogenesis, with decreased CyclinD1 and VEGF respectively, there was no increase in tumor apoptosis. Rapamycin effectively decreased activation of S6 downstream of mTOR, but there was accompanied increased Akt activation. This study demonstrates the therapeutic potential and limitations of rapamycin in NF1-associated, and likely sporadic, MPNSTs.