Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM.

Microglia: Immune and non-immune functions.

As resident macrophages of the central nervous system (CNS), microglia are associated with diverse functions essential to the developing and adult brain during homeostasis and disease. They are aided in their tasks by intricate bidirectional communication with other brain cells under steady-state conditions as well as with infiltrating peripheral immune cells during perturbations. Harmonious cell-cell communication involving microglia are considered crucial to maintain the healthy state of the tissue environment and to overcome pathology such as neuroinflammation. Analyses of such intercellular pathways have contributed to our understanding of the heterogeneous but context-associated microglial responses to environmental cues across neuropathology, including inflammatory conditions such as infections and autoimmunity, as well as immunosuppressive states as seen in brain tumors. Here, we summarize the latest evidence demonstrating how these interactions drive microglia immune and non-immune functions, which coordinate the transition from homeostatic to disease-related cellular states.

Current tools to interrogate microglial biology.

Microglial cells perform a plethora of functions in the central nervous system (CNS), involving them in brain development, maintenance of homeostasis in adulthood, and CNS diseases. Significant technical advancements have prompted the development of novel systems adapted to analyze microglia with increasing specificity and intricacy. The advent of single-cell technologies combined with targeted mouse models has been decisive in deciphering microglia heterogeneity and dissecting microglial functions. However sophisticated these tools have become, clear limitations remain. Understanding their pitfalls and advantages ensures their correct application. Therefore, we provide a guide to the cutting-edge methods currently available to dissect microglial biology.

Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.

Correction to 'Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma'.

[This corrects the article DOI: 10.1093/narcan/zcab009.].

mTOR-dependent translation amplifies microglia priming in aging mice.

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.

Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment.

Tumour-associated microglia/macrophages (TAM) are the most numerous non-neoplastic populations in the tumour microenvironment in glioblastoma multiforme (GBM), the most common malignant brain tumour in adulthood. The mTOR pathway, an important regulator of cell survival/proliferation, is upregulated in GBM, but little is known about the potential role of this pathway in TAM. Here, we show that GBM-initiating cells induce mTOR signalling in the microglia but not bone marrow-derived macrophages in both in vitro and in vivo GBM mouse models. mTOR-dependent regulation of STAT3 and NF-κB activity promotes an immunosuppressive microglial phenotype. This hinders effector T-cell infiltration, proliferation and immune reactivity, thereby contributing to tumour immune evasion and promoting tumour growth in mouse models. The translational value of our results is demonstrated in whole transcriptome datasets of human GBM and in a novel in vitro model, whereby expanded-potential stem cells (EPSC)-derived microglia-like cells are conditioned by syngeneic patient-derived GBM-initiating cells. These results raise the possibility that microglia could be the primary target of mTOR inhibition, rather than the intrinsic tumour cells in GBM.

Correction to: c-MYC overexpression induces choroid plexus papillomas through a T-cell mediated inflammatory mechanism.

In the original version of this article [1], there was 1 error in the affiliation of the European Institute of Oncology (affiliation 3). In this correction article the updated affiliation is shown for clarification.

Selective modification of a native protein in a patient tissue homogenate using palladium nanoparticles.

We have developed new benign palladium nanoparticles able to catalyze the Suzuki-Miyaura cross-coupling reaction on human thyroglobulin (Tg), a naturally iodinated protein produced by the thyroid gland, in homogenates from patients' tissues. This represents the first example of a chemoselective native protein modification using transition metal nanoobjects in near-organ medium.

c-MYC overexpression induces choroid plexus papillomas through a T-cell mediated inflammatory mechanism.

Choroid plexus tumours (CPTs) account for 2-5% of brain tumours in children. They can spread along the neuraxis and can recur after treatment. Little is known about the molecular mechanisms underlying their formation and only few high fidelity mouse models of p53-deficient malignant CPTs are available.We show here that c-MYC overexpression in the choroid plexus epithelium induces T-cell inflammation-dependent choroid plexus papillomas in a mouse model. We demonstrate that c-MYC is expressed in a substantial proportion of human choroid plexus tumours and that this subgroup of tumours is characterised by an inflammatory transcriptome and significant inflammatory infiltrates. In compound mutant mice, overexpression of c-MYC in an immunodeficient background led to a decreased incidence of CPP and reduced tumour bulk. Finally, reduced tumour size was also observed upon T-cell depletion in CPP-bearing mice. Our data raise the possibility that benign choroid plexus tumours expressing c-MYC could be amenable to medical therapy with anti-inflammatory drugs.

Regulatory mechanisms of incomplete huntingtin mRNA splicing.

Huntington's disease is caused by a CAG repeat expansion in exon 1 of the HTT gene. We have previously shown that exon 1 HTT does not always splice to exon 2 producing a small transcript (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The mechanisms by which this incomplete splicing occurs are unknown. Here, we have generated a minigene system that recapitulates the CAG repeat-length dependence of HTTexon1 production, and has allowed us to define the regions of intron 1 necessary for incomplete splicing. We show that manipulation of the expression levels of the splicing factor SRSF6, predicted to bind CAG repeats, modulates this aberrant splicing event and also demonstrate that RNA polymerase II transcription speed regulates the levels of HTTexon1 production. Understanding the mechanisms by which this pathogenic exon 1 HTT is generated may provide the basis for the development of strategies to prevent its production.

PLGA-PEG-supported Pd Nanoparticles as Efficient Catalysts for Suzuki-Miyaura Coupling Reactions in Water.

Chemical transformations that can be performed selectively under physiological conditions are highly desirable tools to track biomolecules and manipulate complex biological processes. Here, we report a new nanocatalyst consisting of small palladium nanoparticles stabilized on the surface of PLGA-PEG nanoparticles that show excellent catalytic activity for the modification of biological building blocks through Suzuki-Miyaura cross-coupling reactions in water. Brominated or iodinated amino acids were coupled with aryl boronic acids in phosphate buffer in good yields. Interestingly, up to 98% conversion into the coupled amino acid could be achieved in 2 h at 37 °C using the stable, water-soluble cyclic triolborate as organometallic partner in the presence of only 1 mol% of palladium. These results pave the way for the modification of biomolecules in complex biological systems such as the intracellular space.

Designing logical codon reassignment - Expanding the chemistry in biology.

Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.

Palladium: a future key player in the nanomedical field?

Metal nanostructures offer invaluable possibilities for targeted drug delivery, detection/diagnosis and imaging. Whereas iron, gold, silver and platinum nanoarchitectures have largely dominated this field to date, several hurdles impede the widespread application of those nanopharmaceuticals in a clinical context. Therefore, technologies based on alternative metals are now being evaluated for their potential in medical applications. Palladium nanostructures are characterized by remarkable catalytic and optical properties. However, until recently, very few studies have taken advantage of these unique characteristics for applications in the biomedical field. Very recently, palladium nanostructures have been reported as prodrug activator, as photothermal agents and for anti-cancer/anti-microbial therapy. With only a handful of reports available, the pharmaceutical applications of palladium nanostructures reviewed here are in their infancy. Yet their interesting performance and toxicity profiles may qualify them as future key players in the nanomedical field.

Site-specific control of N7-metal coordination in DNA by a fluorescent purine derivative.

A synthetic strategy that utilizes O6-protected 8-bromoguanosine gives broad access to C8-guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8-substituted 2'-deoxyguanosines are push-pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001-0.72) due to excited-state proton-transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal-binding affinity and specificity of 8-substituted 2'-deoxyguanosines. One derivative, 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), exhibits selective binding of Cu(II), Ni(II), Cd(II), and Zn(II) through a bidentate effect provided by the N7 position of guanine and the 2-pyridyl nitrogen atom. Upon incorporation into DNA, 2-pyridine-modified guanine residues selectively bind to Cu(II) and Ni(II) with equilibrium dissociation constants (K(d)) that range from 25 to 850 nM; the affinities depend on the folded state of the oligonucleotide (duplex>G-quadruplex) as well as the identity of the metal ion (Cu>Ni≫Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site-specific control of metal localization in alternatively folded nucleic acids. Temperature-dependent circular-dichroism studies reveal metal-dependent stabilization of duplexes, but destabilization of G-quadruplex structures upon adding Cu(II) to 2PyG-modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids.