Identification and pharmacological inactivation of the MYCN gene network as a therapeutic strategy for neuroblastic tumor cells

This research was originally published in Journal of Biological Chemistry. Olesya Chayka, Cosimo Walter D’Acunto, Odette Middleton, Maryam Arab, and Arturo Sala. Identification and Pharmacological Inactivation of the MYCN Gene Network as a Therapeutic Strategy for Neuroblastic Tumor Cells. Journal of Biological Chemistry. 2015; Vol 290 (4) :pp. 2198 - 2212. © the American Society for Biochemistry and Molecular Biology.This article has been made available through the Brunel Open Access Publishing Fund.The MYC family of transcription factors consists of three well characterized members, c-MYC, L-MYC, and MYCN, deregulated in the majority of human cancers. In neuronal tumors such as neuroblastoma, MYCN is frequently activated by gene amplification, and reducing its expression by RNA interference has been shown to promote growth arrest and apoptosis of tumor cells. From a clinical perspective, RNA interference is not yet a viable option, and small molecule inhibitors of transcription factors are difficult to develop. We therefore planned to identify, at the global level, the genes interacting functionally with MYCN required to promote fitness of tumor cells facing oncogenic stress. To find genes whose inactivation is synthetically lethal to MYCN, we implemented a genome-wide approach in which we carried out a drop-out shRNA screen using a whole genome library that was delivered into isogenic neuroblastoma cell lines expressing or not expressing MYCN. After the screen, we selected for in-depth analysis four shRNAs targeting AHCY, BLM, PKMYT1, and CKS1B. These genes were chosen because they are directly regulated by MYC proteins, associated with poor prognosis of neuroblastoma patients, and inhibited by small molecule compounds. Mechanistically, we found that BLM and PKMYT1 are required to limit oncogenic stress and promote stabilization of the MYCN protein. Cocktails of small molecule inhibitors of CKS1B, AHCY, BLM, and PKMYT1 profoundly affected the growth of all neuroblastoma cell lines but selectively caused death of MYCN-amplified cells. Our findings suggest that drugging the MYCN network is a promising avenue for the treatment of high risk, neuroblastic cancers.SPARKS and the Neuroblastoma Society

Unified geometrical framework for the plastic design of reinforced concrete structures

© 2020 The Authors. Structural Concrete published by John Wiley & Sons Ltd on behalf of International Federation for Structural Concrete. Although the analysis and design of structures in static equilibrium can be intuitively carried out using simple equilibrium-based methods such as graphic statics, the application of these methods to engineering problems that take into consideration specific material properties is generally limited. Within the domain of reinforced concrete, existing geometric approaches for developing stress fields and yield lines based on the theory of plasticity are especially useful. However, these approaches usually rely on iterative constructions and are generally limited to two-dimensional cases. By taking advantage of graphic statics, this article introduces the theoretical basis for an entirely geometrical method to generate discrete stress fields and yield line patterns in two- and three-dimensional reinforced concrete structures. The proposed approach is based on the use of reciprocal stress functions and the relationship between form and force diagrams

Applications of graphic statics to analysis and design of reinforced concrete: Stress fields and yield lines

© Fédération Internationale du Béton – International Federation for Structural Concrete. Graphic statics can be effectively used for the analysis and design of structures in static equilibrium and for solving engineering problems related to specific materials, most notably, reinforced concrete. In this regard, methods for the construction of stress fields and yield lines, based on the theory of plasticity, are particularly valuable; however, their automation and generalisation to the third dimension are still open research topics. This paper introduces a unified method based on graphic statics for the generation of discrete stress fields of 2D and 3D reinforced concrete structures and the definition of admissible yield line patterns in concrete slabs. The presented approach relies on the construction of reciprocal stress functions, and the link between form and force diagrams through Minkowski sums

Free boundary problem for the role of planktonic cells in biofilm formation and development

The dynamics of biofilm lifecycle are deeply influenced by the surrounding environment and the interactions between sessile and planktonic phenotypes. Bacterial biofilms typically develop in three distinct stages: attachment of cells to a surface, growth of cells into colonies, and detachment of cells from the colony into the surrounding medium. The attachment of planktonic cells plays a prominent role in the initial phase of biofilm lifecycle as it initiates the colony formation. During the maturation stage, biofilms harbor numerous microenvironments which lead to metabolic heterogeneity. Such microniches provide conditions suitable for the growth of new species, which are present in the bulk liquid as planktonic cells and can penetrate the porous biofilm matrix. We present a 1D continuum model on the interaction of sessile and planktonic phenotypes in biofilm lifestyle which considers both the initial attachment and colonization phenomena. The model is formulated as a hyperbolic-elliptic free boundary value problem with vanishing initial value. Hyperbolic equations reproduce the transport and growth of sessile species, while elliptic equations model the diffusion and conversion of planktonic cells and dissolved substrates. The attachment is modelled as a continuous, deterministic process which depends on the concentrations of the attaching species. The growth of new species is modelled through a reaction term in the hyperbolic equations which depends on the concentration of planktonic species within the biofilm. Existence and uniqueness of solutions are discussed and proved for the attachment regime. Finally, some numerical examples show that the proposed model correctly reproduces the growth of new species within the biofilm and overcomes the ecological restrictions characterizing the Wanner-Gujer type models.Comment: 17 pages, 9 figures, preprint versio

Continuum and discrete approach in modeling biofilm development and structure: a review

The scientific community has recognized that almost 99% of the microbial life on earth is represented by biofilms. Considering the impacts of their sessile lifestyle on both natural and human activities, extensive experimental activity has been carried out to understand how biofilms grow and interact with the environment. Many mathematical models have also been developed to simulate and elucidate the main processes characterizing the biofilm growth. Two main mathematical approaches for biomass representation can be distinguished: continuum and discrete. This review is aimed at exploring the main characteristics of each approach. Continuum models can simulate the biofilm processes in a quantitative and deterministic way. However, they require a multidimensional formulation to take into account the biofilm spatial heterogeneity, which makes the models quite complicated, requiring significant computational effort. Discrete models are more recent and can represent the typical multidimensional structural heterogeneity of biofilm reflecting the experimental expectations, but they generate computational results including elements of randomness and introduce stochastic effects into the solutions

Cortico-cortical stimulation and robot-assisted therapy (CCS and RAT) for upper limb recovery after stroke: study protocol for a randomised controlled trial

Background: Since birth, during the exploration of the environment to interact with objects, we exploit both the motor and sensory components of the upper limb (UL). This ability to integrate sensory and motor information is often compromised following a stroke. However, to date, rehabilitation protocols are focused primarily on recovery of motor function through physical therapies. Therefore, we have planned a clinical trial to investigate the effect on functionality of UL after a sensorimotor transcranial stimulation (real vs sham) in add-on to robot-assisted therapy in the stroke population. Methods: A randomised double-blind controlled trial design involving 32 patients with a single chronic stroke (onset > 180 days) was planned. Each patient will undergo 15 consecutive sessions (5 days for 3 weeks) of paired associative stimulation (PAS) coupled with UL robot-assisted therapy. PAS stimulation will be administered using a bifocal transcranial magnetic stimulator (TMS) on the posterior-parietal cortex and the primary motor area (real or sham) of the lesioned hemisphere. Clinical, kinematics and neurophysiological changes will be evaluated at the end of protocol and at 1-month follow-up and compared with baseline. The Fugl-Meyer assessment scale will be the primary outcome. Secondly, kinematic variables will be recorded during the box-and-block test and reaching tasks using video analysis and inertial sensors. Single pulse TMS and electroencephalography will be used to investigate the changes in local cortical reactivity and in the interconnected areas. Discussion: The presented trial shall evaluate with a multimodal approach the effects of sensorimotor network stimulation applied before a robot-assisted therapy training on functional recovery of the upper extremity after stroke. The combination of neuromodulation and robot-assisted therapy can promote an increase of cortical plasticity of sensorimotor areas followed by a clinical benefit in the motor function of the upper limb. Trial registration: ClinicalTrials.gov NCT05478434. Registered on 28 Jul 2022

Poly(Vinyl alcohol)/gelatin scaffolds allow regeneration of nasal tissues

Need for regeneration and repair of nasal tissues occurs as a consequence of several pathologies affecting the nose, including, but not limited to infective diseases, traumas and tumor resections. A platform for nasal tissue regeneration was set up using poly(vinyl alcohol)/gelatin sponges with 20%–30% (w/w) gelatin content to be used as scaffolds, for their intrinsic hydrophilic, cell adhesive and shape recovery properties. We propose mesodermal progenitor cells (MPCs) isolated from the bone marrow as a unique stem cell source for obtaining different connective tissues of the nose, including vascular tissue. Finally, epithelial cell immune response to these scaffolds was assessed in vitro in an environment containing inflammatory molecules. The results showed that mesenchymal stromal cells (MSCs) deriving from MPCs could be used to differentiate into cartilage and fibrous tissue; whereas, in combination with endothelial cells still deriving from MPCs, into pre-vascularized bone. Finally, the scaffold did not significantly alter the epithelial cell response to inflammatory insults derived from interaction with bacterial molecules

Regional Precuneus Cortical Hyperexcitability in Alzheimer's Disease Patients

Objective: Neuronal excitation/inhibition (E/I) imbalance is a potential cause of neuronal network malfunctioning in Alzheimer's disease (AD), contributing to cognitive dysfunction. Here, we used a novel approach combining transcranial magnetic stimulation (TMS) and electroencephalography (EEG) to probe cortical excitability in different brain areas known to be directly involved in AD pathology. Methods: We performed TMS-EEG recordings targeting the left dorsolateral prefrontal cortex (l-DLPFC), the left posterior parietal cortex (l-PPC), and the precuneus (PC) in a large sample of patients with mild-to-moderate AD (n = 65) that were compared with a group of age-matched healthy controls (n = 21). Results: We found that patients with AD are characterized by a regional cortical hyperexcitability in the PC and, to some extent, in the frontal lobe, as measured by TMS-evoked potentials. Notably, cortical excitability assessed over the l-PPC was comparable between the 2 groups. Furthermore, we found that the individual level of PC excitability was associated with the level of cognitive impairment, as measured with Mini-Mental State Examination, and with corticospinal fluid levels of Aβ42 . Interpretation: Our data provide novel evidence that precuneus cortical hyperexcitability is a key feature of synaptic dysfunction in patients with AD. The current results point to the combined approach of TMS and EEG as a novel promising technique to measure hyperexcitability in patients with AD. This index could represent a useful biomarker to stage disease severity and evaluate response to novel therapies. ANN NEUROL 2022

The involvement of xanthohumol in the expression of annexin in human malignant glioblastoma cells

Glioblastoma multiforme (GBM) is the most common malignant and resistant tumor of the central nervous system in humans and new therapeutic strategies are urgently required. Recently, we have shown that the potential chemotherapeutic polyphenol xanthohumol (XH), isolated from Humulus Lupulus, induces apoptosis of human T98G glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways. Then we have found, by western blotting and microscopic analysis, that XH up-regulates cytosolic levels of ANXA1 and induces translocation of the protein on the cell membrane of T98G cells in a time-dependent manner with significant effects observed after 24 h. On the basis of the above evidence, the aim of this work was to investigate the role of intracellular and cell membrane localized ANXA1 in GBM cells. RT-PCR analysis has shown that XH up-regulates mRNA levels of ANXA1 after 16 h treatment. To demonstrate the involvement of ANXA1 in apoptosis of GBM cells we down-regulated ANXA1 expression with small interfering RNA (siRNA) and then analysed apoptosis in the presence and absence of apoptotic stimuli. Importantly, apoptosis induced by XH was reduced in siRNA-ANXA1 transfected cells where western blot analysis shows a significant reduction of ANXA1 protein levels. To investigate the role of ANXA1 expression on the cell membrane of T98G cells as potential “eat-me” signal we studied phagocytosis of apoptotic cells by human macrophages. We incubated apoptotic T98G cells with human blood monocyte derived macrophages (M=). After co-incubation period we analysed the percentage of M= phagocytosing the apoptotic cells by cytofluorimetric FACS analysis and by confocal microscopy. Our results show that XH induces phagocytosis of apoptotic T98G cells by human M= in a concentration-effect manner, a processes that is dependent on caspase mediated apoptosis. ANXA1 acts as an “eat-me” signal on the cell membrane of T98G cells, and interestingly, apoptotic siRNA-ANXA1 transfected cells are not completely ingested by M=. These results were confirmed by incubating apoptotic cells with a neutralizing anti-ANXA1 antiboby and ANXA1 membrane depletion by EDTA washing. ANXA1 was also detected in supernatants of apoptotic cells and the incubation of enriched supernatants enhanced the percentage of phagocytosis by M=. These results demonstrated that ANXA1 is involved both in the apoptosis and phagocytosis of glioblastoma cells. This study shows a possible role of ANXA1 in maintenance of brain homeostasis and may lead to novel therapeutic approaches for neuro-inflammatory diseases and chemotherapy targets in the treatment of glioblastoma multiforme