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Increased de novo synthesis, mobilization and uptake of fatty acids are required to provide sufficient lipids for membrane biogenesis in support of rapid tumor cell division and growth. In addition to their structural roles as components of the plasma membrane, fatty acid-derived lipids regulate ferroptotic cell death, a type of programmed cell death, when oxidized by iron-dependent lipoxygenase enzymes. De novo lipogenesis and the defense against oxidative lipid damage require large amounts of cytosolic NADPH. Our group has recently found that HGG up-regulate wild-type Isocitrate dehydrogenase 1 (referred to hereafter as ‘wt-IDH1high HGG’) to generate large quantities of cytosolic NADPH. RNAi-mediated knockdown of wt-IDH1, alone and in combination with radiation therapy (RT), slows the growth of patient-derived HGG xenografts, while overexpression of wt-IDH1 promotes intracranial HGG growth. Isotope tracer and liquid chromatography-based lipidomic studies indicated that wt-IDH1 supports the de novo biosynthesis of mono-unsaturated fatty acids (MUFAs) and promotes the incorporation of monounsaturated phospholipids into the plasma membrane, while displacing polyunsaturated fatty acid (PUFA) phospholipids. In addition, enhanced NADPH production in wt-IDH1high HGG increases glutathione (GSH) level, reduces reactive oxygen species (ROS), activates the phospholipid peroxidase glutathione peroxidase 4 (GPX4)-driven lipid repair pathway, and dampens the accumulation of PUFA-containing lipid peroxides, known executioners of ferroptosis. To pharmacologically target wt-IDH1,we have used and characterized wt-IDH1i-13, a first-in-class competitive α,β-unsaturated enone (AbbVie). wt-IDH1i-13 potently inhibits wt-IDH1 enzymatic activity, by covalently binding to the NADP+ binding pocket. Our data indicate that wt-IDH1i-13 promotes ferroptosis, which can be rescued by pre-treatment of cells with the peroxyl scavenger and ferroptosis inhibitor ferrostatin. wt-IDH1i-13 is brain-penetrant, and similar to genetic ablation, reduces progression and extends the survival of wt-IDH1high HGG bearing mice, alone and in combination with RT. These studies credential to wt-IDH1i-13 as a novel therapeutic modality for the treatment of wt-IDH1 gliomas.

PURPOSE Growing evidence indicates that the neurotransmitters dysregulated in psychiatric disorders are similarly dysregulated in glioblastoma (GBM) biology. GBM cells are dependent on bountiful neuronal glutamate, utilize elevated dopamine receptor expression to augment progression, and catabolize serotonin to drive proliferation and inhibit anti-tumor immunity. The clinical induction of seizure, known as electroconvulsive therapy (ECT), has been used by psychiatrists since the 1930s to correct these dysregulations and can additionally improve medication blood-brain barrier (BBB) penetrance. We hypothesized that seizure-induced changes in the glioma microenvironment occur with ECT, slowing tumor progression, increasing BBB permeability, and prolonging overall survival in glioma-bearing mice. METHODS C57BL6 mice were orthotopically injected with CT-2A-Luc mouse glioma cells. Mice were randomized to receive ECT via ear-clip electrodes or sham treatment daily up to five times per week. Intracranial progression was monitored via bioluminescent signal from CT-2A-Luc xenografts. BBB permeability was assessed by subjecting mice to ECT or sham treatment immediately following intravenous injection of sodium fluorescein. RESULTS Intracranial progression was maximally reduced in ECT-treated mice relative to sham-treated mice after 17 ECT treatments (ECT radiance 2.6 x 109 photons/second versus sham 4.7 x 109 photons/second, p=0.013), which was further confirmed by both decreased tumor weight and tumor size on histologic evaluation. This translated into an improvement in overall survival from median 29 days in sham-treated mice to 38 days in ECT-treated mice (p=0.0018). Mean seizure duration was 41.8 seconds and positively correlated with overall survival (Pearson coefficient r=0.63, p=0.028). Brain parenchymal uptake of sodium fluorescein was significantly higher in ECT-treated mice (mean relative increase in ECS to sham radiance of 1.47, p< 0.05). CONCLUSION Repeated ECT slows tumor progression and prolongs overall survival in C57BL6 mice bearing CT-2A-Luc xenografts. The BBB is compromised immediately following ECT. ECT merits further oncologic investigation as a potential therapeutic in GBM.

Purpose/Objective(s) Growing evidence indicates that the neurotransmitters dysregulated in psychiatric disorders are similarly dysregulated in glioblastoma (GBM) biology. Patients with psychiatric disorders are classically thought of having excessive glutamate (major depression, bipolar depression), excessive dopamine (mania, psychosis), and/or insufficient serotonin (major depression, anxiety). Analogously, GBM cells are dependent on bountiful neuronal glutamate, utilize elevated dopamine receptor expression to activate the hypoxic response and augment progression, and catabolize serotonin to drive proliferation. The clinical induction of seizure, known as electroconvulsive therapy (ECT), has been used by psychiatrists since the 1930s to treat severe cases of depression, mania, psychosis, and catatonia. ECT has been shown to decrease extracellular glutamate and glutamate receptor expression in vivo and increase blood-brain barrier (BBB) permeability. ECT, then, may have previously unexplored oncologic value. We hypothesized that seizure-induced changes in the glioma microenvironment occur with ECT, increasing permeability of the BBB, slowing tumor progression, and prolonging overall survival in glioma-bearing mice. Materials/Methods C57BL6 mice were orthotopically injected with CT-2A-Luc mouse glioma cells. Mice were randomized to receive ECT via ear-clip electrodes or sham treatment daily up to five times per week until survival endpoints were reached. Seizure duration and rates of tonic hindlimb extension were recorded. Intracranial progression was monitored via bioluminescent signal from CT-2A-Luc xenografts. BBB permeability was assessed by subjecting mice to ECT or sham treatment immediately following tail vein injection of sodium fluorescein. Mice were saline perfused and brains were harvested for fluorescent spectroscopy. Results Intracranial progression was maximally reduced in ECT-treated mice relative to sham-treated mice after 17 treatments (mean sham radiance 4.7 × 109 photons/s versus mean ECT radiance 2.6 × 109 photon/s, P = 0.013). This translated into an improvement in overall survival from median 29 days in sham-treated mice to 34.5 days in ECT-treated mice (P = 0.0018). Mean seizure duration was 41.8 seconds and positively correlated with overall survival (Pearson coefficient r = 0.63, P = 0.028). Tonic hindlimb extension occurred in 68% of seizures and positively correlated with overall survival (Pearson coefficient r = 0.72, P = 0.0078). Brain parenchymal uptake of sodium fluorescein was significantly higher in ECT-treated mice, with a mean relative increase in ECS to sham radiance of 1.47 (P Conclusion Repeated ECT slows tumor progression and prolongs overall survival in C57BL6 mice bearing CT-2A-Luc xenografts. The BBB is compromised immediately following ECT. ECT merits further oncologic investigation as a potential therapeutic in GBM. Author Disclosure T. Sita: None. L. Hurley: None. C. Dussold: None. S. Sachdev: None. R. Stupp: Research Grant; CarThera, Paris. Consultant/ad hoc advisor for research protocol development; GT Medical Technologies, Insightec; Northwest Biotherapeutics. Scientific Advisory Board; CranioVation, TriAct, Hemispherian. IDMC of an ongoing clinical trial; Celularity. A. Stegh: None.

The lack of precision therapies combined with limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain/blood-tumor barriers have contributed to glioblastoma (GBM) being one of the most difficult cancers to effectively treat. We have developed a novel precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference (RNAi)-based spherical nucleic acids (SNAs). In this study, we used SNAs consisting of gold nanoparticle cores covalently conjugated with radially oriented and densely packed siRNA oligonucleotides. Based upon previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in non-human primates, and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event-level (NOAEL), followed by tumor resection. Safety assessment revealed no significant treatment-related toxicities. Inductively coupled plasma mass spectrometry, X-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold (Au) enrichment observed in the tumor-associated endothelium, macrophages and tumor cells. NU-0129 uptake into glioma cells correlated with significant reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of NU-0129-treated recurrent vs. matched primary (i.e., untreated) tumor. Our results establish SNA nanoconjugates as a brain-penetrant precision medicine approach for the systemic treatment of GBM.

Significance Abnormal EGFR signaling is frequently observed in glioblastoma multiforme (GBM). However, the clinical benefit of EGFR inhibitors has been limited. Here we show that the cell surface molecule CD44s splice isoform acts as a signaling modulator that attenuates EGFR degradation and sustains EGFR’s downstream AKT signaling. CD44s internalizes in cells and inhibits Rab7A-mediated EGFR trafficking for degradation, resulting in sustained EGFR protein levels. CD44 depletion combined with EGFR inhibitor results in a robust and synergistic GBM cancer cell killing. Because CD44s-mediated inhibition on Rab7A also affects other receptor tyrosine kinases (RTKs), inhibiting CD44s may be an exciting approach for perturbation of multiple RTKs in GBMs.

IDH3α promotes glioblastoma progression and links mitochondrial metabolism to cSHMT-controlled one-carbon metabolism., Mutation or transcriptional up-regulation of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) promotes cancer progression through metabolic reprogramming and epigenetic deregulation of gene expression. Here, we demonstrate that IDH3α, a subunit of the IDH3 heterotetramer, is elevated in glioblastoma (GBM) patient samples compared to normal brain tissue and promotes GBM progression in orthotopic glioma mouse models. IDH3α loss of function reduces tricarboxylic acid (TCA) cycle turnover and inhibits oxidative phosphorylation. In addition to its impact on mitochondrial energy metabolism, IDH3α binds to cytosolic serine hydroxymethyltransferase (cSHMT). This interaction enhances nucleotide availability during DNA replication, while the absence of IDH3α promotes methionine cycle activity, S-adenosyl methionine generation, and DNA methylation. Thus, the regulation of one-carbon metabolism via an IDH3α-cSHMT signaling axis represents a novel mechanism of metabolic adaptation in GBM.

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood–brain/blood–tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.

Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

A standard operating process for assessment, design, and follow-through of simulation-based education was developed to improve simulation experiences and optimize customer and staff satisfaction in a large multidisciplinary simulation center. Information is provided to describe development and implementation of a standard operating process for a large multidisciplinary simulation center. The Model for Improvement, a continuous improvement method, is the framework used to systematically describe the design, implementation, and monitoring of the standard operating process. A standard operating process is essential for success when high volumes and multiple disciplines are doing simulation-based education in a large simulation center. Utilization of a process for helping new and recurrent customers develop simulation-based education within a continuous improvement framework can increase both efficiency and the satisfaction of customers and simulation faculty and staff.

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P

Previous work on the electrochemistry of palladium in aqueous acid solution demonstrated the existence of two multilayer hydrous oxide reduction peaks, one at ca. 0.24 V and another at ca. 0.55 V vs. RHE, plus the presence of a reversible active surface state transition at ca. 0.24 V. In the present work with thermally activated palladium it was observed that, in agreement with the hydrous oxide reduction behaviour of the system, there is a second active state transition at E≥ca. 0.45 V. In most of its reactions in aqueous acid solution, apart from its unusual capacity to absorb hydrogen, palladium exhibits properties very similar to those of platinum; however, palladium seems to be more prone to dissolution and subsurface oxygen formation. Also the premonolayer oxidation responses of these two metals are often different as the more active state of the palladium surface is not as readily generated as that of platinum. The electrocatalytic properties of palladium, as reported earlier, correlate quite well with the hydrous oxide and premonolayer oxidation behaviour of this electrode system.

It is now well established that gold, in the form of oxide-supported microparticles or even as conventional macroelectrodes, displays inexplicably high catalytic activity for some reactions. In the present work, gold surfaces were superactivated by a combination of thermal and cathodic pretreatment and such electrodes in base yielded up to five distinct, and quite marked, premonolayer oxidation responses within the double layer region, over the range 0.0–1.0 V (RHE). As outlined in earlier publications, such unusual behaviour is important from an electrocatalytic (and heterogeneous catalysis) viewpoint. A new mode of active site adsorption, involving highly localized electron transfer from active surface atoms to either the external circuit (in electrocatalysis) or an adsorbing reactant (in heterogeneous catalysis), is proposed. Such localized (active site) adsorption, which is based on surface quantum confinement effects, is virtually independent of (or only indirectly related to) the electronic properties of the bulk metal.

Severe thermal pretreatment of gold wire electrodes in an inert gas atmosphere resulted in the appearance of dramatic premonolayer oxidation responses, which in some instances commenced at 0.25 V (RHE), for the resulting active gold electrodes in aqueous acid media. Similar behaviour was reported earlier for platinum and gold activated by cathodic pretreatment in acid solution; these active noble metal surfaces are evidently more susceptible to oxidation than bulk copper. Such behaviour was attributed to the effect of surface disorder; many of the metal atoms at the interface are assumed to be in a very active, metastable state possessing quite low lattice stabilization energy. Premonolayer oxidation responses are again correlated with electrocatalytic behaviour and the existence of unusual chemisorption behaviour for molecules reacting at highly active states of metals is outlined.

The inhibiting effect of COads on platinum-based anodes is a major problem in the development of ambient temperature, polyelectrolyte membrane-type fuel cells. One of the unusual features of the response for the oxidative removal of the species in question is that the response observed for this reaction in the positive sweep is highly dependent on the CO admission potential, for example, when the COads is formed in the Hads region it undergoes oxidation at unusually low potentials. Such behaviour is attributed here to hydrogen activation of the platinum surface, with the result that oxide mediators (and COads oxidation) occurs at an earlier stage of the positive sweep. It is also demonstrated, for both platinum and gold in acid solution, that dramatic premonolayer oxidation responses may be observed following suitable preactivation of the electrode surfaces. It is suggested that the defect state of a solid electrode surface is an important variable whose investigation may yield improved fuel cell anode performance.

There is increasing independent evidence, e.g. from the application of surface enhanced Raman spectroscopy (SERS) and contact electrical resistance (CER) techniques that the behaviour of metal/aqueous solution interfaces is more complicated than is generally realised. It is demonstrated in the present work, for three different electrode systems (copper in base and platinum and gold in acid), that metal surfaces may be activated, i.e. the lattice energy in the outer layers of the metal may be reduced, by severe cathodic or thermal pretreatment. The redox behaviour of the resulting metastable, or non-equilibrium, surface states were investigated using cyclic voltammetry. It is suggested, on the basis of both the present and earlier work in this laboratory, that such surface states are of importance in electrocatalysis as they reflect the behaviour of active sites present at conventional electrode surfaces.

The present work is a continuation of an earlier investigation of the effect of severe thermal pretreatment on the redox properties of platinum electrodes in aqueous media. The most interesting observation was that raising the solution temperature to ca. 75 °C resulted in a dramatic anodic response in the positive sweep at ca. 0.25 V (RHE). Such behaviour was noted earlier at room temperature but only when the thermally pretreated sample was further activated by cathodic polarization. This transition at 0.25 V was partially reversed on the negative sweep, but the cathodic process involved was subject to severe inhibition. There is independent evidence for similar behaviour for gold in aqueous acid solution. The relevance of the present results to the operation of fuel cell anodes is outlined.

Subjecting platinum wire electrodes to short periods (10 to 25 s) of intense heating ( T >ca. 1200°C) in an inert gas atmosphere leads to considerable changes in the surface microstructure of the metal; metastable active states become frozen-in as the temperature of the thermally excited sample drops rapidly to ambient values. Unusual redox responses and, in particular, enhanced premonolayer oxidation peaks in the positive sweep, were observed when such wires were subsequently immersed in aqueous media and investigated using cyclic voltammetry. These unusual responses, which are similar to those reported earlier following prolonged electrochemical activation of platinum (the thermal procedure is much shorter and the behaviour more consistent), were further enhanced by subsequently polarizing the thermally treated samples at low potentials. For a number of reactions and, in particular, methanol oxidation which is important from a fuel cell viewpoint, the electrocatalytic activity of platinum at low potentials was greatly enhanced. However, for other processes, e.g. hydrazine oxidation, thermal pretreatment resulted in a decrease in electrocatalytic activity; it appears that in some cases the active sites become overoxidised. A synopsis is given of the active site behaviour of platinum surfaces in aqueous media and a comparison is made between the present results and those reported elsewhere for platinum single crystal electrodes where similar thermal pretreatment is frequently employed.

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro . In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) α but not ERβ knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERα. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERα signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERα (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERα-null (−/−) background, we examine the ability of ERE-independent ERα signaling pathways to convey estrogen feedback regulation of the female hypothalamic–pituitary axis in vivo . ERα −/AA exhibited 69.9% lower serum LH levels compared with ERα −/− mice. Additionally, like wild type, ERα −/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERα −/AA mice. However, unlike wild type, both ERα −/AA and ERα −/− mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERα signaling is sufficient to convey a major portion of estrogen's negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERα signaling.

A subset of transcription factors function as pivotal regulators of cell differentiation pathways. Pituitary transcription factor-1 (Pit-1) is a tissue-specific homeodomain protein that specifies the development of pituitary somatotropes and lactotropes. In this study, adenovirus was used to deliver rat Pit-1 to mouse liver. Pit-1 expression was detected in the majority (50–80%) of hepatocyte nuclei after tail vein injection (2 × 109 plaque forming units). Pit-1 activated hepatic expression of the endogenous prolactin (PRL), GH, and TSHβ genes along with several other markers of lactotrope progenitor cells. Focal clusters (0.2–0.5% of liver cells per tissue section) of periportal cells were positive for PRL by immunofluorescent staining. The PRL-producing cells also expressed proliferating cell nuclear antigen as well as the hepatic stem cell markers (c-Kit, Thy1, and cytokeratin 14). These data indicate that Pit-1 induces the transient differentiation of hepatic progenitor cells into PRL-producing cells, providing additional evidence that transcription factors can specify the differentiation pathway of adult stem cells.

Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (-/-) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (-/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

Sox3, a member of the high mobility group (HMG) family of transcription factors, is expressed in neural progenitor cells and in the gonads. Targeted deletion of Sox3 in mice causes abnormal development of the diencephalon and Rathke's pouch, the progenitor of the anterior pituitary gland. Male and female mice are also infertile and exhibit a primary defect in gametogenesis. In this study, we examined the expression and function of Sox3 in C57BL/6 mice to better understand its role in spermatogenesis. Testis development was normal during embryogenesis. However, spermatogenesis failed to progress during the postnatal period, with germ cell loss beginning at postnatal day 10 (P10). By P14, Sox3 null mice were nearly agametic, retaining only Sertoli cells and undifferentiated spermatogonia. Pituitary gonadotropin and testosterone levels were normal, suggesting a defect in Sertoli cell and/or germ cell function. Immunostaining revealed that Sox3 was expressed in a subpopulation of germ cells localized at the base of the seminiferous tubules. Sox3 expression was restricted to proliferating germ cells and colocalized with neurogenin 3 (Ngn3), a helix–loop–helix transcription factor implicated in spermatogonial differentiation. The absence of Sox3 decreased Ngn3 and increased expression of Oct4, a marker of undifferentiated spermatogonia. We conclude that Sox3 is expressed in As, Apr and Aal spermatogonia and is required for spermatogenesis through a pathway that involves Ngn3.