bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro and in vivo synergistic effects of tigecycline combined with aminoglycosides on carbapenem-resistant Klebsiella pneumoniae
Authors: Ni Wentao , Yang Deqing , Guan Jie , Xi Wen , Zhou Dexun , Zhao Lili , Cui Junchang , Xu Yu , Gao Zhancheng , Liu Youning
Published: March 2021

Objectives

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies.

Methods

Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time–kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened.

Results

The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time–kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants.

Conclusions

Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice.
ZmMs25 encoding a plastid-localized fatty acyl reductase is critical for anther and pollen development in maize
Authors: Zhang Simiao , Wu Suowei , Niu Canfang , Liu Dongcheng , Yan Tingwei , Tian Youhui , Liu Shuangshuang , Xie Ke , Li Ziwen , Wang Yanbo , Zhao Wei , Dong Zhenying , Zhu Taotao , Hou Quancan , Ma Biao , An Xueli , Li Jinping , Wan Xiangyuan
Published: March 2021

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.
Investigating the theory of propagating fluctuations with numerical models of stochastic accretion discs
Authors: Turner Samuel G D , Reynolds Christopher S
Published: March 2021

Across a large range of scales, accreting sources show remarkably similar patterns of variability, most notably the log-normality of the luminosity distribution and the linear root-mean square (rms)–flux relationship. These results are often explained using the theory of propagating fluctuations in which fluctuations in the viscosity create perturbations in the accretion rate at all radii, propagate inwards, and combine multiplicatively. While this idea has been extensively studied analytically in a linear regime, there has been relatively little numerical work investigating the non-linear behaviour. In this paper, we present a suite of stochastically driven 1D α-disc simulations, exploring the behaviour of these discs. We find that the eponymous propagating fluctuations are present in all simulations across a wide range of model parameters, in contradiction to previous work. Of the model parameters, we find by far the most important to be the time-scale on which the viscosity fluctuations occur. Physically, this time-scale will depend on the underlying physical mechanism, thought to be the magnetorotational instability (MRI). We find a close relationship between this fluctuation time-scale and the break frequency in the power spectral density of the luminosity, a fact which could allow observational probes of the behaviour of the MRI dynamo. We report a fitting formula for the break frequency as a function of the fluctuation time-scale, the disc thickness, and the mass of the central object.
ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data
Authors: Dhariwal Achal , Junges Roger , Chen Tsute , Petersen Fernanda C
Published: March 2021

The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules—the ‘Antimicrobial Resistance Gene Table’ module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the ‘Integration’ module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the ‘Antimicrobial Resistance Gene List’ module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.
BIMG-11. PHARMACODYNAMIC EVALUATION OF IDH AND EGFR INHIBITION IN HUMAN GLIOMAS USING MOLECULAR MRI
Authors: Ellingson Benjamin , Yao Jingwen , Hagiwara Akifumi , Nathanson David , Oughourlian Talia , Everson Richard , Salamon Noriko , Pope Whitney , Nghiemphu Phioanh , Lai Albert , Liau Linda , Cloughesy Timothy
Published: March 2021

Metabolic differences are inherent to specific glioma subtypes and can be altered using targeted treatments, including IDH and EGFR inhibition. Using a large cohort of patients scanned at UCLA and other centers over the last 5 years, we demonstrate that IDH, 1p19q, and EGFR alterations uniquely contribute to alterations in glycolysis and oxygen utilization using a clinically available molecular MRI technique termed amine chemical exchange saturation transfer spin-and-gradient-echo echoplanar imaging (CEST-SAGE-EPI). Our data shows that CEST-SAGE-EPI estimates of tumor acidity are strongly associated with the degree of glycolysis as evaluated with direct pH measurements, quantitative IHC, bioenergetics experiments, and correlations with 18F-FDG PET images. Data further reveals that IDH wild type gliomas have higher acidity and oxygen utilization compared with IDH mutant gliomas, 1p19q non-codeleted gliomas (astrocytomas) have higher tumor acidity compared to 1p19q codeleted gliomas (oligodendrogliomas), and EGFR amplified gliomas have higher oxygen utilization compared with non-amplified gliomas. Additionally, phase II clinical trial data suggests successful IDH inhibition results in an early and measurable increase in tumor acidity and further reduction in oxygen utilization, signifying suppression of oxidative phosphorylation and/or glutaminolysis in favor of glycolysis. Alternatively, phase II clinical trial data suggests successful EGFR inhibition with brain penetrant agents results in early reductions in tumor acidity and 18F-FDG PET uptake, consistent with a reduction in glycolysis. Data also indicates that continual increases in tumor acidity during routine follow-up after initial therapeutic changes results in uniformly worse outcomes in all tumor subtypes under all mentioned treatment scenarios.
BIMG-19. 18F-FLUCICLOVINE PET/CT TO DISTINGUISH RADIATION NECROSIS FROM TUMOR PROGRESSION IN BRAIN METASTASES TREATED WITH STEREOTACTIC RADIOSURGERY: A PROSPECTIVE PILOT STUDY
Authors: Tom Martin
Published: March 2021

PURPOSE

To estimate the accuracy of 18F-Fluciclovine PET/CT in distinguishing radiation necrosis (RN) from tumor progression (TP) among patients with brain metastases (BM) having undergone prior stereotactic radiosurgery (SRS) who presented with a follow-up MRI brain (with DSC-MR perfusion) which was equivocal for RN versus TP.

METHODS

Within 30 days of equivocal MRI brain, subjects underwent 18F-Fluciclovine PET/CT on a Siemens Biograph mCT scanner with a 10 mCi bolus dose immediately prior to PET. Data were collected in list mode for 25 minutes post-injection and were reconstructed as a static image of data 10–25 minutes post-injection, and as a dynamic series of four 5-minute frames between 5–25 minutes post-injection. Quantitative metrics for each lesion were documented including SUVmax, SUVmean, SUVpeak, and normal brain SUVmean. Lesion to normal brain ratios were calculated. The reference standard was clinical follow-up with MRI brain (with DSC-MR perfusion) every 2–4 months until multidisciplinary consensus (or tissue confirmation) for diagnosis of RN versus TP.

RESULTS

From 7/2019-11/2020, 16 of 16 planned subjects enrolled and underwent 18F-Fluciclovine PET/CT for evaluation of 21 brain lesions. Primary histology included NSCLC (n=7), breast (n=5), melanoma (n=3), and endometrial (n=1). Ranges of quantitative metrics were: SUVmax, 2.18–12.1; SUVmean, 1.16–7.37; SUVpeak, 1.06–5.14; normal brain SUVmean, 0.19–0.50; SUVmax/normal ratio, 7.5–45.4; SUVmean/normal ratio, 4.2–26.3; and SUVpeak/normal ratio, 3.9–26.4. Among the patients 10 patients with 12 lesions who completed follow up, estimates of the area under the receiver operating characteristic curve for SUVmax, SUVmean, and SUVpeak were: 0.93, 0.93, and 0.82, respectively.

CONCLUSION

In this population, 18F-Fluciclovine PET/CT favorably produces a wide range of lesion quantitative metric values, low uptake in the normal brain, and promising accuracy to distinguish RN from TP. Completion of follow-up for all patients is required. Phase II and III studies are under development.
DDRE-14. DE-NOVO PURINE BIOSYNTHESIS IS A MAJOR DRIVER OF CHEMORESISTANCE IN GLIOBLASTOMA
Authors: Ahmed Atique
Published: March 2021

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumor is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is glioblastoma cells’ ability to adapt to complex changes within the tumor microenvironment. To elucidate this adaptation’s molecular mechanisms, specifically during chemotherapy temozolomide, we employed chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ALR13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with Liquid Chromatography-Mass Spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5’-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction function as a negative regulator for purine salvaging. Inhibition of ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using a Food and Drug Administration-approved drug, Mycophenolate Mofetil, that can block the IMPDH2 activity and enhance the therapeutic efficacy of TMZ. Our results suggest and support clinical evaluation of MMF in combination with TMZ treatment in glioma patients.
DDRE-15. THE EVOLUTIONARY ENIGMA OF FATTY ACID DESATURATION IN GLIOBLASTOMA
Authors: Oatman Nicole , Dasgupta Biplab
Published: March 2021

Fatty acid desaturation is an enzymatic reaction in which a double bond is introduced into an acyl chain. Of the four functionally distinct desaturase subfamilies, the First Desaturase Family of enzymes introduce the first double bond into a saturated fatty acid, resulting in the synthesis of monounsaturated fatty acids (MUFA). MUFA are essential components of membrane and storage lipids and exert a profound influence on the fluidity of biological membranes. A disequilibrium in saturated to unsaturated fatty acid ratio alters cell growth, differentiation and response to external stimuli, and thus affects a range of pathologies including cancer. The most abundant and key First Desaturase Family enzyme is the delta 9 desaturate called Stearoyl Co-A Desaturase (SCD and SCD5 in humans, and SCD1-4 in mice). SCD desaturates Stearoyl-CoA (C18) and palmitoyl-CoA (C16) to oleoyl-CoA (C18:1) and palmitoyl-CoA (C16:1), respectively. Besides SCD, the only known First Desaturase in mammals with dual function is FADS2 which desaturates palmitate to Sapienate (C16:1, a positional isomer of palmitoleate) in skin cells. A recent study showed that some cancer cells can use FADS2 to bypass the SCD reaction. SCD and SCD5 are by far the most abundant desaturases expressed in the human brain. We made an unexpected discovery that SCD undergoes monoallelic codeletion with PTEN on chromosome 10, and is also highly methylated in glioblastoma (GBM). More surprisingly, all GBM cell lines with SCD codeletion/methylation (that expressed very little SCD protein) are completely resistant to SCD/SCD5 inhibition, yet their phospholipids contained abundant oleic acid. It is unknown if GBMs bypassed SCD, but retained the delta 9 desaturation reaction through a novel enzymatic activity. Our targeted and untargeted metabolomics studies revealed unexpected findings that cannot be explained by conventional wisdom, and may lead to identification of novel lipogenic targets in GBM.
DDRE-22. TARGETING SERINE SYNTHESIS IN BRAIN METASTASIS
Authors: Ngo Bryan , Kim Eugenie , Osorio-Vasquez Victoria , Doll Sophia , Bustraan Sophia , Liang Roger , Luengo Alba , Davidson Shawn , Ali Ahmed , Ferraro Gino , Fischer Grant , Plasger Ariana , Rajasekhar Vinagolu , Kastenhuber Edward , Eskandari Roozbeh , Bacha Sarah , Sriram Roshan , Bakhoum Samuel , Snuderl Matija , Cotzia Paolo , Healey John , Sabatini David , Jones Drew , Zhao Jean , Yu Min , Jain Rakesh , Keshari Kayvan , Davies Michael , Heiden Matthew Vander , Hernando Eva , Mann Matthias , Cantley Lewis , Pacold Michael
Published: March 2021

The brain environment is low in amino acids, including serine and glycine, both of which are important for tumor growth as they are precursors of proteins and nucleotide bases. How tumor cells overcome these conditions to proliferate and survive in the brain is incompletely understood. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first and rate-limiting step of glucose-derived serine synthesis, enables brain metastasis in multiple human types and in preclinical models. Genetic suppression and small molecule inhibition of PHGDH attenuated brain metastasis, but not extra cranial tumors, and improved the overall survival of mice bearing brain metastasis. These results demonstrate that the tumor nutrient microenvironment determines tumor cell sensitivity to loss of serine synthesis pathway activity and raise the possibility that serine synthesis inhibitors may be useful in the treatment of brain metastases.
DDRE-27. IDH MUTATED GLIOMAS PROMOTE EPILEPTOGENESIS VIA D-2-HYDROXYGLUTARATE DEPENDENT MTOR HYPERACTIVATION
Authors: Mortazavi Armin , Fayed Islam , Bachani Muzna , Dowdy Tyrone , Steiner Joseph , Maric Dragan , Yang Chun Zhang , Larion Mioara , Ksendzovsky Alexander , Zaghloul Kareem
Published: March 2021

INTRODUCTION

Epileptic seizures in patients with low-grade, isocitrate dehydrogenase (IDH) mutated gliomas reach 90%, a major source of morbidity for these patients. Albeit there are multiple features that contribute to tumor related epileptogenesis, IDH mutations are determined to be an independent factor, although the pathogenesis remains poorly understood. We demonstrate IDH-mutated tumors promote epileptogenesis through D-2-hydroxyglutarate (D-2-HG) dependent mTOR hyperactivation and metabolic reprogramming.

METHODS

Human epileptic and nonepileptic cortex were identified via subdural electrodes in patients with IDH-mutated gliomas (n=5). An in vitro rat cortical neuronal model on microelectrode arrays were utilized to investigate the role of D-2-HG on neuronal excitability. mTOR and lysine demethylase (KDM) modulators were applied to elucidate the epileptogenic mechanism. Tetrodotoxin was utilized to evaluate the contribution of neuronal activity to mTOR signaling and metabolism. mTOR signaling was evaluated through western blot analysis and multiplex immunofluorescence. Metabolic function were analyzed via Seahorse assays and metabolomic analysis.

RESULTS

D-2-HG increased normalized bursting rate in the neuronal cultures (p<0.0001). Inhibition of mTOR with rapamycin corrected bursting levels to control levels. Furthermore, D-2-HG induced mTOR hyperactivation, independent of bursting activity, which correlated with upregulation of mTOR signaling in human epileptic tissue. KDM inhibition resulted in mTOR hyperactivation and neuronal hyperexcitability, which we demonstrated with D-2-HG, succinate, and PFI-90, a small molecule KDM inhibitor. Epileptic cortex and D-2-HG-treated neurons, have distinct metabolisms independent of neuronal activity compared to peritumoral nonepileptic cortex and control, respectively.

CONCLUSION

We demonstrate IDH-mutated gliomas promote epileptogenesis through a D-2-HG dependent mTOR hyperactivation via KDM inhibition, a putative mechanism and potential therapeutic targets. Furthermore, we argue mTOR hyperactivation results in metabolic reprogramming, independent of neuronal firing, which may contribute to epileptogenesis, a heretofore unrecognized aspect of pathologic mTOR signaling in neurological diseases.
DDRE-28. MECHANISTIC AND THERAPEUTIC LINKS BETWEEN PURINE BIOSYNTHESIS AND DNA DAMAGE IN GLIOBLASTOMA
Authors: Scott Andrew , Zhou Weihua , Wilder-Romans Kari , Feng Jiane , Wu Zhe , Andren Anthony , Zhang Li , Sajjakulnukit Peter , Kachman Maureen , Umemura Yoshie , Schmitt Melanie , Qi Nathan , Lawrence Theodore , Lyssiotis Costas , Wahl Daniel
Published: March 2021

Glioblastoma (GBM) is the most common and aggressive adult brain cancer. Radiation therapy (RT) is a critical treatment modality, and development of RT resistance is the predominant cause of recurrence and mortality in GBM patients. Using cell line models as well as patient-derived xenografts and neurospheres in orthotopic brain tumor models, we have identified increased rates and dependence upon de novo purine biosynthesis as a hallmark of GBM RT resistance. More recently, we have discovered that radiation treatment acutely stimulates flux through de novo purine synthesis in cell line and neurosphere models of GBM. This RT-induced increase in de novo purine synthesis is dependent on signaling through the DNA damage response and thus appears to be an adaptive mechanism to supply purines to repair radiation-induced DNA damage. To determine whether this regulatory mechanism also exists in vivo, we have used advanced metabolomic and metabolic tracing techniques with 13C-labeled glucose and 15N-labeled glutamine in mice bearing RT-resistant GBM patient-derived orthotopic brain tumors. We found that that orthotopic GBM PDXs had elevated activity of de novo purine synthesis that increased further after RT, while normal cortex had little activity even after RT. These observations have therapeutic relevance, as targeting this metabolic pathway with the FDA-approved purine biosynthesis inhibitor mycophenolate mofetil (MMF) overcomes GBM radiation resistance in mouse models in vivo. The lack of de novo purine synthesis in normal cortex suggests that targeting this pathway may be tumor specific. Collectively our data suggest that de novo synthesis of purines mediates RT resistance in GBM and that treatment of brain tumors with MMF in combination with RT may be a promising therapeutic strategy in patients.
ETMM-02. PRECLINICAL MODELS REVEAL BRAIN-MICROENVIRONMENT SPECIFIC METABOLIC DEPENDENCIES IN GLIOBLASTOMA
Authors: Minami Jenna , Bayley Nicholas , Tse Christopher , Zhu Henan , Morrow Danielle , Yong William , Liau Linda , Cloughesy Timothy F , Graeber Thomas , Nathanson David A
Published: March 2021

Metabolic reprogramming is a hallmark of cancer, and malignant cells must acquire metabolic adaptations in response to a multitude of intrinsic and extrinsic factors to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Conventional cell lines do not recapitulate the metabolic heterogeneity of glioblastoma (GBM), while primary cultured cells do not account for the influences of the microenvironment and the blood brain barrier on tumor biology. Additionally, these systems are under strong selective pressure divergent from that in vivo, leading to reduced heterogeneity between cultured tumor cells. Here, we describe a biobank of direct-from-patient derived orthotopic xenografts (GliomaPDOX) and gliomaspheres that reveal a subset of gliomas that, while able to form in vivo, cannot survive in vitro. RNA sequencing of tumors that can form both in vivo and in vitro (termed “TME-Indifferent”) compared to that of tumors that can only form in vivo (termed “TME-Dependent”) revealed transcriptional changes associated with altered nutrient availability, emphasizing the unique metabolic programs impacted by the tumor microenvironment. Furthermore, TME-dependent tumors lack metabolic signatures associated with nutrient biosynthesis, thus indicating a potential dependency of these tumors on scavenging specific nutrients from the extracellular milieu. Collectively, these data emphasize the metabolic heterogeneity within GBM and reveal a subset of gliomas that lack metabolic plasticity, indicating a potential brain-microenvironment specific metabolic dependency that can be targeted for therapy.
ETMM-03. CANCER CELLS DEPLOY LIPOCALIN- 2 TO COLLECT LIMITING IRON IN LEPTOMENINGEAL METASTASIS
Authors: Chi Yudan , Remsik Jan , Kiseliovas Vaidotas , Derderian Camille , Sener Ugur , Alghader Majdi , Saadeh Fadi , Nikishina Katie , Bale Tejus , Iacobuzio-Donahue Christin , Thomas Tiffany , Pe’er Dana , Mazutis Linas , Boire Adrienne
Published: March 2021

The tumor microenvironment plays a critical regulatory role in cancer progression, especially in central nervous system metastases. Cancer cells within the cerebrospinal fluid (CSF)-filled leptomeninges face substantial microenvironmental challenges, including inflammation and sparse micronutrients. To investigate the mechanism by which cancer cells in these leptomeningeal metastases (LM) overcome these constraints, we subjected CSF from five patients with LM to single-cell RNA sequencing. We found that cancer cells, but not macrophages, within the CSF express the iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17. These macrophages generate inflammatory cytokines that induce cancer cell LCN2 expression but do not generate LCN2 themselves. In mouse models of LM, cancer cell growth is supported by the LCN2/SLC22A17 system and is inhibited by iron chelation therapy. A Phase Ia/1b clinical trial focused on this novel treatment approach is underway.
FSMP-02. CHANGES IN GLUTAMINE METABOLISM INDUCED BY OXALOACETATE IN GLIOBLASTOMA
Authors: Ijare Omkar , Conway David , Cash Alan , Baskin David , Pichumani Kumar
Published: March 2021

Anhydrous Enol-Oxaloacetate (AEO) has been shown to significantly increase survival and decrease tumor growth rates in animal models of glioblastoma and hepatocellular carcinoma. In the body, AEO is metabolized to “oxaloacetate” (OAA). Earlier, we demonstrated that AEO drastically reduced Warburg glycolysis in glioblastoma cells which was determined by the increase in pyruvate to lactate ratio and a 48.8% decrease in lactate production in 13C-labeled glucose metabolism studies. We have expanded this previous work to examine 13C-labeled glutamine metabolism. Cultured solid tumor cancer cells strongly rely on both glucose and glutamine to synthesize carbon intermediates for anaplerotic reactions. With treatment of OAA, we hypothesize that glutamine-derived OAA may be reduced which can be tracked through the use GC-MS based 13C-labeled glutamine isotopomer experiments. Patient-derived glioblastoma cells were grown in 15 mM glucose and 2 mM glutamine containing DMEM medium supplemented with 2 mM OAA for 10 days. 24 hours prior to harvesting the cells, 4 mM of [U-13C]glutamine was introduced to the medium. OAA treated cells showed significant decrease in the protein levels of lactate dehydrogenase A and C which indicates the switching off of glycolysis to support the utilization of elevated OAA levels during glutamine metabolism in the TCA cycle. 13C mass distribution analysis showed significant decrease in malate, aspartate and citrate pools (malate: 8.8%, p = 0.0098; aspartate: 9.2%, p = 0.0064; citrate: 9.5%, p = 0.0036) in their M+4 isotopomer labeling in OAA treated group compared to the control group. Decrease in lactate generation may reduce cancer proliferation, migration and invasion. Together, these data provide alternative way to modulate energy metabolism in glioblastoma using AEO treatment. Similarly, AEO treatment in other solid tumors (e.g. pancreatic ductal adenocarcinoma) may produce altered glutamine metabolism which may be of therapeutic value.
FSMP-03. INVESTIGATING CO-OPTED ASTROCYTIC METABOLISM IN MELANOMA BRAIN METASTASIS
Authors: Farnan Julia , Jackson Joshua , Hartsough Edward
Published: March 2021

Melanoma, an aggressive form of skin cancer, frequently metastasizes to the brain. While peripheral melanoma is largely treatable, MBM fail to respond to current therapeutics and is a clear unmet clinical need. Initial clinical symptoms of Melanoma Brain Metastases (MBM) typically include headaches, seizures and other neurological deficits, suggesting that MBM disrupt normal brain functions. One of the major cell types that melanoma encounter and interact with during brain metastasis are astrocytes. Astrocytes, the most abundant cell in the brain, interact with neurons and the vasculature, provide trophic and energetic support to neurons, and regulate local blood flow. Metabolic pathways in astrocytes, particularly the glutamate-glutamine cycle, are essential for the recycling and resupply of neurotransmitters needed to maintain the excitation/inhibition balance. We propose that MBM co-opt astrocytic metabolism, fueling MBM growth, and deplete metabolic intermediates crucial for neuronal activity leading to altered neurologic function. We begin to unravel the metabolic interactions between astrocytes and MBM using novel modeling platforms with genetic and pharmacological tools to manipulate the tumor microenvironment. This project investigates the contribution of astrocytic metabolism to MBM growth. We intend on dissecting the distinct metabolic needs of metastatic brain melanoma in the CNS microenvironment and the subsequent neurological consequences. Completion of this project will provide a platform to study MBM and interaction with the local brain microenvironment. Inhibiting metabolic interactions between melanoma and glial cells may provide new avenue for therapeutic targeting of MBM.
FSMP-10. CYSTEINE INDUCES CYTOTOXICITY IN GLIOBLASTOMA THROUGH MITOCHONDRIAL HYDROGEN PEROXIDE PRODUCTION
Authors: Noch Evan , Palma Laura , Yim Isaiah , Binder Bhavneet , Benedetti Elisa , Krumsiek Jan , Elemento Olivier , Cantley Lewis
Published: March 2021

Glioblastoma (GBM) is a poorly treatable disease with high mortality. Tumor metabolism in GBM is a critical mechanism responsible for growth because of upregulation of glucose, amino acid, and fatty acid utilization. However, little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate metabolic signatures unique to GBM, we interrogated the TCGA and a cancer metabolite database for alterations in glucose and amino acid signatures in GBM relative to other human cancers and relative to low-grade glioma. From these analyses, we found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers and that GBM exhibits high levels of cysteine metabolites compared to low-grade gliomas. To study the role of cysteine in GBM pathogenesis, we treated patient-derived GBM cells with FDA-approved cyst(e)ine-promoting compounds in vitro, including N-acetylcysteine (NAC) and the cephalosporin antibiotic, Ceftriaxone (CTX), which induces cystine import through system Xc transporter upregulation. Cysteine-promoting compounds, including NAC and CTX, inhibit growth of GBM cells, which is exacerbated by glucose deprivation. This growth inhibition is associated with reduced mitochondrial metabolism, manifest by reduction in ATP, NADPH/NADP+ ratio, mitochondrial membrane potential, and oxygen consumption rate. Mechanistic experiments revealed that cysteine compounds induce a rapid increase in the rate of H2O2 production in isolated GBM mitochondria, an effect blocked by the H2O2 scavenger, catalase. Such findings are consistent with reductive stress, a ROS-producing process whereby excess mitochondrial reducing equivalents prevent electron transfer to oxidized electron acceptors, inducing O2 reduction to H2O2. We show that cysteine-promoting compounds reduce cell growth and induce rapid mitochondrial toxicity in GBM, which may be due to reductive stress. This pathway is targetable with FDA-approved cysteine-promoting compounds and could synergize with glucose-lowering treatments, including the ketogenic diet, for GBM.
The Liability of Internet Intermediaries
Authors: Riordan Jaani
Published: March 2021

Internet intermediaries play a central role in modern commerce and society. Although their economic and social importance is well-recognised, their legal liability remains poorly understood, and, until now, no work has specifically addressed their legal responsibility for wrongdoing carried out by third parties using their facilities or platforms. This work fills that gap by providing comprehensive coverage of the legal duties owed by intermediaries and the increasingly complex schemes that regulate their activities. The first part of the work introduces the concept of an internet intermediary, general doctrines of primary and secondary liability, and the European enforcement regime. The second part examines the liability of intermediaries in specific areas of law, with a detailed analysis of the applicable liability rules, and the major English case law, and decisions of the Court of Justice that interpret and apply them. The final part of the work provides guidance on remedies and limitations. Written by an expert author from the intellectual property chambers at 8 New Square, Lincoln's Inn, this is an essential guide for lawyers advising on liability, privacy, and online regulation.
Written, interim and oral procedures
Authors: von Falck Andreas , Dorn Stephan
Published: March 2021

The main proceedings before the UPC consist of three procedural steps: the written procedure, the interim procedure, and the oral hearing. According to Rule 10 UPCARoP, all inter partes procedures before the Court of First Instance are divided into those three parts. The same applies for the appeal procedure before the Court of Appeal. The same structure, according to Rule 85 UPCARoP, is applicable for the proceedings in actions against a decision of the EPO concerning the EPO’s functions assigned to it pursuant to Art 9 EPUE Reg.
Application to amend the patent
Authors: Plassmann Clemens , Dorn Stephan
Published: March 2021

Rule 13 UPCARoP contains the requirements for an Application by the proprietor of the patent to amend the patent in pending infringement proceedings before the UPC and regarding the material and formal aspects of such application by the patent proprietor.
Powers of the Court as regards representatives
Authors: Tilmann Winfried
Published: March 2021

Persons vested with power of representation, being admitted to the Court, are designated by the German legal expression of ‘Organe der Rechtspflege’ (officers for the administration of justice). The willingness to act in such capacity is a condition for national authorization to practise as a lawyer. For that reason, the UPC may exercise disciplinary power over representatives authorized to act before the Court. The Presidents, the presiding judges, and judges rapporteur or single judges or standing judges also exercise domiciliary authority within the premises of the UPC. The measures they may take, on one basis or another, range from formal warnings to revoking a representative’s authorization to act in particular proceedings (Rule 291) or even in all proceedings, as well as a limited or permanent ban from the premises of the Court.
Examination and decision
Authors: Gundt Miriam
Published: March 2021

The Registry first examines the formal admissibility of the application, ie observance of the requirements of Rule 378.
Article 34 TFEU
Authors: Klamert Marcus , Moustakali Maria , Tomkin Jonathan
Published: March 2021

Article 28 EC Quantitative restrictions on imports and all measures having equivalent effect shall be prohibited between Member States.
Article 122 TFEU
Authors: Flynn Leo
Published: March 2021

Article 100 EC Without prejudice to any other procedures provided for in the Treaties, the Council, on a proposal from the Commission, may decide, in a spirit of solidarity between Member States, upon the measures appropriate to the economic situation, in particular if severe difficulties arise in the supply of certain products, notably in the area of energy.
Article 19 TEU
Authors: Klamert Marcus , Schima Bernhard
Published: March 2021

Member States shall provide remedies sufficient to ensure effective legal protection in the fields covered by Union law.
Judicial Conduct
Authors: Hamer Kenneth
Published: March 2021

The Judicial Discipline (Prescribed Procedures) Regulations 2014 set out the process for dealing with complaints about judicial conduct. The Judicial Conduct Investigations Office (JCIO) is responsible for considering an allegation of judicial misconduct and for assessing whether the behaviour alleged could amount to misconduct. The Regulations provide for the making of rules about the investigation process to be applied in respect of an allegation of misconduct and the appointment of a ‘nominated judge’, whose role is to make a recommendation to the Lord Chancellor and the Lord Chief Justice as to whether the matter complained of amounts to misconduct and, if so, what disciplinary sanction may be appropriate. Where the case is particularly complex or serious, or requires further investigation, an ‘investigating judge’ may be appointed to carry out further inquiries, and a disciplinary panel may be convened at the request of the subject of the complaint when the nominated judge has made a finding of judicial misconduct and has recommended that the subject of the complaint be removed or suspended from office. Regulation 17 provides for interim suspension and regulation 18 provides for publication of decisions.
Article 4(1). Personal data
Authors: Bygrave Lee A , Tosoni Luca
Published: March 2021

The principles of data protection should apply to any information concerning an identified or identifiable natural person. Personal data which have undergone pseudonymisation, which could be attributed to a natural person by the use of additional information should be considered to be information on an identifiable natural person. To determine whether a natural person is identifiable, account should be taken of all the means reasonably likely to be used, such as singling out, either by the controller or by another person to identify the natural person directly or indirectly.
Article 4(7). Controller
Authors: Bygrave Lee A , Tosoni Luca
Published: March 2021

Controllers are key actors in the operationalisation of data protection law as they are the primary bearers of the obligations set by such law towards data subjects. Thus, definition of the criteria for attaining the role of controller is essential for understanding the law’s application. As detailed below, the tendency under EU data protection law is to apply these criteria in an expansive and flexible way, thus rendering the threshold for attaining controller status quite low.
Article 8 Conditions applicable to child’s consent in relation to information society services
Authors: Kosta Eleni
Published: March 2021

Article 7 (Conditions for consent); Article 12 (Transparent information, communication and modalities of exercise of the rights of the data subject) (see also recital 58); Article 40(2)(g) (Codes of conduct); Article 22 (Automated individual decision-making) (see also recital 71); Article 35 (Data Protection Impact Assessments).
Committal for Sentence
Authors: Sprack John , Engelhardt–Sprack Michael
Published: March 2021

Where a defendant is convicted or pleads guilty in the magistrates’ court, the magistrates will usually proceed to sentence, in accordance with the procedure detailed in Chapter 22. In doing so, however, they are limited by the restrictions on their powers of punishment. Notwithstanding these restrictions, they may be able to commit the offender to the Crown Court for sentence, and this Chapter deals with such committals.
The Course of the Trial
Authors: Sprack John , Engelhardt–Sprack Michael
Published: March 2021

The judge at a trial on indictment must be a professional judge (i.e., a High Court judge, circuit judge or deputy circuit judge, or recorder). The distribution of work between these various types of judge is described at 14.14. The prosecution must be legally represented; the accused usually chooses to be so, and will probably be able to obtain representation from the Legal Aid Agency. Although some solicitors now have rights of audience in the Crown Court, legal representation for trial on indictment still usually involves instructing solicitors who in turn brief counsel (see 14.21 to 14.23). The following Paragraphs deal with some aspects of the role of prosecuting and defence counsel, and their relationship with the judge. The discussion is based upon decided cases and also upon the Code of Conduct for the Bar. The Code does not have the force of law, but is of great persuasive value, and a barrister who conforms with its spirit cannot be guilty of a breach of professional discipline.
The Council
Authors: Dann Philipp
Published: March 2021

The Council is the indispensable institution of the European Union (EU). Its importance is based on its ubiquity in the EU’s exercises of public authority, which in turn rests on its functional and institutional complexity and hybridity. For the Council is neither only a legislature nor an executive; neither simply a meeting of state parties nor a regular second chamber of parliament; neither just political nor administrative, neither clearly national nor supranational—but it is all of these aspects at different points. It has fittingly been called a chameleon.
Remedies Before National Courts
Authors: Arnull Anthony
Published: March 2021

This chapter is concerned with the decentralized enforcement of European Union law, that is, the way in which national courts uphold the rights it confers on litigants. When the Court of Justice established in Van Gend en Loos that Union law was capable of conferring on litigants rights which the national courts were bound to protect, it had written only the first sentence (albeit a very striking one) of a long and complex story that remains unfinished. For while the Court in Van Gend en Loos had enlisted the help of the national courts in ensuring that Union law was observed, it had not made clear how exactly it expected them to discharge that responsibility. The Union legislature might have attempted to resolve the issue systematically and today there is a significant body of Union legislation dealing with the remedies national courts must provide in discrete areas. However, such legislation was slow to develop and remains piecemeal in nature. This has meant that much of the responsibility for fleshing out the remedial obligations of national courts when seeking to uphold rights granted by Union law has fallen to the Court of Justice.
Copyright, Moral Rights, and the Right to One’s Image
Authors: Bate Stephen , Wilde Gervase de
Published: March 2021

UK copyright is a right created by statute and is now contained in the Copyright, Designs and Patents Act 1988 (CDPA). Copyright is mainly regarded as a means of protecting the economic interests of creators of original works. However, it has a significant role to play in protecting privacy interests. Private correspondence and diaries are obvious examples of material that may attract copyright protection in the domestic sphere. In the commercial context, examples include corporate memoranda, other documents, and recordings containing confidential information. Copyright subsists in various descriptions of ‘works’, such as ‘literary works’, ‘films’ and ‘sound recordings’ and there may be more than one copyright work in any article. For example, a recording may include a ‘literary work’ as well as a ‘sound recording’ for copyright purposes. A copyright work may contain private information or the work may be unpublished and therefore private in that sense. Copyright is apt to protect privacy interests, because it gives the copyright owner the legal right to prohibit reproductions, ie copying, as well as other means of dissemination.
Interest Groups’ Recruitment of Incumbent Parliamentarians to Their Boards
Authors: Huwyler Oliver
Published: March 2021

Interest groups (IGs) recruit incumbent parliamentarians to their boards to influence policy, improve their resources and signal political connectedness. To detect parliamentarians’ characteristics that drive recruitment, this study analyses three decades of annual data (1985–2016) of 903 Swiss parliamentarians and their board seats. It compares 5249 cases of parliamentarians’ successful recruitment by 3291 different organisations to counterfactual cases where no recruitment took place. The results show that IGs recruit parliamentarians for both knowledge and networks (professions, other board seats) and influence (committee seats) in IGs’ policy areas. Moreover, recruited parliamentarians are more likely newcomers, ideologically proximate to IGs, moderate and from the same district as them.
Long-term persistence of rituximab in patients with rheumatoid arthritis: an evaluation of the UCL cohort from 1998 to 2020
Authors: Norris-Grey Caitlin , Cambridge Geraldine , Moore Samantha , Reddy Venkat , Leandro Maria
Published: March 2021

Objectives

B cell depletion therapy based on rituximab in patients with RA was pioneered at University College London Hospitals/University College London in 1998. The objective of this study was to evaluate long-term persistence of rituximab and identify factors associated with discontinuation of treatment.

Methods

Retrospective review of medical records from all rituximab-treated RA patients followed up in a dedicated clinic (1998–2020). Data collected included gender, disease duration, previous DMARDs, autoantibody status, age and concomitant therapy at first cycle, length of follow-up, and number of cycles. Drug survival and factors associated with drug discontinuation were analysed using Kaplan–Meier survival curves, log-rank test and Cox regression analysis.

Results

A total of 404 patients were included. Median disease duration and age at time of first rituximab cycle were 10 and 57 years, respectively. Median total follow-up was 55 months and median number of cycles five. 93.1% of patients were seropositive. Overall, 31.2% of patients stopped rituximab, with the largest reason for discontinuing being primary inefficacy (42.1%). Comparison of Kaplan–Meier curves showed that rituximab drug survival was lower in seronegative patients and in patients who had previously failed at least one biologic DMARD (bDMARD). Cox regression analysis revealed that rituximab discontinuation was associated with a greater number of previous bDMARDs.

Conclusion

Many patients with RA achieve good control of their disease with repeated cycles of rituximab treatment. The most common reasons for treatment discontinuation were either primary or secondary inefficacy. Patients who were seronegative and who had previously failed other bDMARDs were more at risk of drug discontinuation.
Reduction of Smad2 caused by oxidative stress leads to necrotic death of hypertrophic chondrocytes associated with an endemic osteoarthritis
Authors: He Ying , Fan Lihong , Aaron Nicole , Feng Yiping , Fang Qian , Zhang Ying , Zhang Dan , Wang Hui , Ma Tianyou , Sun Jian , Chen Jinghong
Published: March 2021

Objective

The occurrence and development of an endemic OA, Kashin–Beck disease (KBD), is closely related to oxidative stress induced by free radicals. The aim of the study was to find the key signalling molecules or pathogenic factors as a potential treatment strategy for KBD.

Methods

Real-time PCR and western blotting were performed to detect the mRNA and protein expression levels in cells and tissues. Immunohistochemical staining was assayed in rat models and human samples obtained from children. The type of cell death was identified by annexin V and propidium iodide staining with flow cytometry.

Results

Oxidative stress decreased levels of Smad2 and Smad3 in hypertrophic chondrocytes both in vitro and in vivo. In the cartilage of KBD patients, the expression of Smad2 and Smad3 proteins in the middle and deep zone was significantly decreased with an observed full deletion in the deep zone of some samples. Reduction of Smad2 protein induced necrotic death of hypertrophic chondrocytes, while reduction of Smad3 protein induced apoptosis. The reduction of Smad2 protein was not accompanied by Smad3 protein reduction in hypertrophic chondrocyte necrosis. Furthermore, the reduction of Smad2 also impaired the construction of tissue-engineered cartilage in vitro.

Conclusion

These studies reveal that oxidative stress causes necrosis of hypertrophic chondrocytes by downregulating Smad2 protein, which increases the pathogenesis of KBD cartilage. The importance of Smad2 in the development of KBD provides a new potential target for the treatment of KBD.