mTORC2 Drives ZFX-mediated Ganglioside Biosynthesis to Promote Breast Cancer Progression.

Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside synthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in Luminal breast cancer-specific MCF-7 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of the EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian Luminal breast cancer patient samples, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.

(–)-Epicatechin Alters Reactive Oxygen and Nitrogen Species Production Independent of Mitochondrial Respiration in Human Vascular Endothelial Cells

Introduction. Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction, and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPI’s effects remain unclear. Objective(s). Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. Methods. HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. Results. Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL ( P < 0.001 ). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P < 0.001 ). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL ( P = 0.010 ). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL ( P = 0.015 and P = 0.001 , respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P = 0.035 and P = 0.011 ). Conclusion(s). EPI dose-dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates

We present a multiomic cell atlas of human lung development that combines single cell RNA and ATAC sequencing, high throughput spatial transcriptomics and single cell imaging. Coupling single cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial and erythrocyte/leukocyte compartments from 5-22 post conception weeks. We identify new cell states in all compartments. These include developmental-specific secretory progenitors that resemble cells in adult fibrotic lungs and a new subtype of neuroendocrine cell related to human small cell lung cancer; observations which strengthen the connections between development and disease/regeneration. Our datasets are available for the community to download and interact with through our web interface (https://fetal-lung.cellgeni.sanger.ac.uk). Finally, to illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signalling and transcription factor hierarchies which we test using organoid models.

Lithium Chloride Sensitivity Connects the Activity of PEX11 and RIM20 to PGM2 translation

Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signalling and signalling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5′-untranslated region (5′-UTR) of the PGM2 mRNA.

PhysiBoSS 2.0: a sustainable integration of stochastic Boolean and agent-based modelling frameworks

Motivation: Cancer progression is a complex phenomenon that spans multiple scales from molecular to cellular and intercellular. Simulations can be used to perturb the underlying mechanisms of those systems and to generate hypotheses on novel therapies. We present a new version of PhysiBoSS, a multiscale modelling framework designed to cover multiple temporal and spatial scales, that improves its integration with PhysiCell, decoupling the cell agent simulations with the internal Boolean model in an easy-to-maintain computational framework. Results: PhysiBoSS 2.0 is a redesign and reimplementation of PhysiBoSS, conceived as an add-on that expands the PhysiCell agent-based functionalities with intracellular cell signalling using MaBoSS having a decoupled, maintainable and model-agnostic design. PhysiBoSS 2.0 successfully reproduces simulations reported in the former PhysiBoSS and expands its functionalities such as using user-defined models and cells' specifications, having mechanistic submodels of substrate internalisation with ODEs and enabling the study of drug synergies. Availability and implementation: PhysiBoSS 2.0 is open-source and publicly available on GitHub (https://github.com/PhysiBoSS/PhysiBoSS) under the BSD 3-clause license. Additionally, a nanoHUB tool has been set up to ease the use of PhysiBoSS 2.0 (https://nanohub.org/tools/pba4tnf/).

Positive ROS (Reactive Oxygen Species) Modulator Engineered Device Support Skin Treatment in Locally Advanced Breast Cancer (LABC) Enhancing Patient Quality of Life

The development of research in genetic and biochemical fields has made it possible to investigate certain metabolic aspects of the microenvironment of chronic skin lesions, including altered cell signalling, highlighting its importance in determining the blockage of repair processes. The purpose of this prospective observational study is to evaluate the efficacy of a medical device consisting of a polyester scaffold enriched with an oleic matrix with controlled release of ROS in the management of LABC skin lesions. During the period from October 2018 to March 2020, 20 patients with locally advanced breast cancer were enrolled and ten were treated with the devices abovementioned. After 30 days of treatment all patients treated reported a general improvement in local conditions with reduction in ulceration area, exudate and odour. The results suggest that the application of these devices even in particular conditions (healthy and neoplastic tissue) does not lead to the onset of negative effects due to the release of ROS, though their role in tissue repair requires further study to fully understand their potential and increase the fields of application of the device by exploiting its modulation capabilities.

A Role of Phosphatidylserine in the Function of Recycling Endosomes

Cells internalize proteins and lipids in the plasma membrane (PM) and solutes in the extracellular space by endocytosis. The removal of PM by endocytosis is constantly balanced by the replenishment of proteins and lipids to PM through recycling pathway. Recycling endosomes (REs) are specific subsets of endosomes. Besides the established role of REs in recycling pathway, recent studies have revealed unanticipated roles of REs in membrane traffic and cell signalling. In this review, we highlight these emerging issues, with a particular focus on phosphatidylserine (PS), a phospholipid that is highly enriched in the cytosolic leaflet of RE membranes. We also discuss the pathogenesis of Hermansky Pudlak syndrome type 2 (HPS2) that arises from mutations in the AP3B1 gene, from the point of view of dysregulated RE functions.

In Situ Cell Signalling of the Hippo-YAP/TAZ Pathway in Reaction to Complex Dynamic Loading in an Intervertebral Disc Organ Culture

Recently, a dysregulation of the Hippo-YAP/TAZ pathway has been correlated with intervertebral disc (IVD) degeneration (IDD), as it plays a key role in cell survival, tissue regeneration, and mechanical stress. We aimed to investigate the influence of different mechanical loading regimes, i.e., under compression and torsion, on the induction and progression of IDD and its association with the Hippo-YAP/TAZ pathway. Therefore, bovine IVDs were assigned to one of four different static or complex dynamic loading regimes: (i) static, (ii) “low-stress”, (iii) “intermediate-stress”, and (iv) “high-stress” regime using a bioreactor. After one week of loading, a significant loss of relative IVD height was observed in the intermediate- and high-stress regimes. Furthermore, the high-stress regime showed a significantly lower cell viability and a significant decrease in glycosaminoglycan content in the tissue. Finally, the mechanosensitive gene CILP was significantly downregulated overall, and the Hippo-pathway gene MST1 was significantly upregulated in the high-stress regime. This study demonstrates that excessive torsion combined with compression leads to key features of IDD. However, the results indicated no clear correlation between the degree of IDD and a subsequent inactivation of the Hippo-YAP/TAZ pathway as a means of regenerating the IVD.

Minimizing IL-6 and IL-10 signalling pathway elements using lumping species and parameters

There are many cell signalling pathways that include a higher set of elements. Understanding the dynamics of such systems becomes a difficult issue in systems biology. Mathematical approaches with computational simulations provide a wide range to simplify such complex models and to predicate their dynamics. A powerful technique for reducing the complexity of cell signalling pathways is lumping variables and parameters. In this work, we suggest this technique to reduce the number of elements of IL-6 and IL-10 signalling pathways. The reduced model given in this work provides one a better understanding and predicting some model dynamics, and gives accurate approximate solutions. Computational results show that there is a good agreement between the model dynamics for the original and the simplified models.

Oxyamine Linkers for the Protecting Group Free Synthesis of Glycoconjugates

<p>Glycoconjugates, such as glycolipids and glycoproteins, are involved in a variety of cellular functions including cell-to-cell signalling and carbohydrate-protein recognition. Accordingly, glycoconjugates play important roles in health and disease and are promising new leads as carbohydrate-based therapeutics. However, for the development of glycoconjugates to study biological processes, or for the use of these adducts as therapeutics, the glycan needs to be conjugated to the carrier molecule or scaffold of choice. Many procedures for the conjugation of glycans involve lengthy protecting group strategies that install the aglycone at the start of glycan total synthesis and are therefore unsuitable for naturally derived sugars. Other glycan conjugation strategies can affect the integrity of the reducing end sugar or lead to adducts where the reducing end sugar adopts the ring-opened rather than the ring-closed form. N,O-Dialkyl oxyamine linkers, however, can be attached to the free reducing end of sugars in a single step without the need for protecting groups. This thesis therefore explores the synthesis and application of oxyamine linkers for the synthesis of glycoconjugates. First, the synthesis of an O-alkyl-N-methyl oxyamine linker (“Type A”) containing an amine at its terminus was improved by reducing the number of synthetic steps from six to four and by increasing the overall yield from 8% to 38%. This oxyamine linker was then conjugated to GlcNAc in 83% yield. The hydrolytic stability of this glycosyloxyamine was then compared to that of the analogous N-alkyl-O-methyl glycosyloxyamine (“Type B”). The stability of the two types of glycosyloxyamines has never been directly compared. Accordingly, it was not known whether the difference in substitution pattern between the two linkers affects their hydrolytic stability. To this end, the hydrolysis rates of the GlcNAc conjugated linkers were assessed at various pH values, glycoconjugate concentrations and buffer concentrations. In all instances, the “Type B” glycoside was found to have marginally better kinetic stability, while the “Type A” glycoside had marginally better thermodynamic stability, but overall, these differences were negligible. The pKa of the conjugate acid of these glycosyloxyamines was also determined to provide insight into the mechanism of hydrolysis. By considering this data, along with the observation that the rate of hydrolysis of these glycoconjugates increases with increasing buffer concentration, it was proposed that the hydrolysis of the oxyamines occurs via general acid catalysis at pH 4-6. A novel dithiol functionalised oxyamine linker was also designed and synthesised for the multivalent display of glycans on gold nanoparticles. With the successful attachment of this thiol linker to GlcNAc, the monomer unit of chitin, this work has paved the way for the future syntheses of chitin-functionalised gold nanoparticles. Such chitinfunctionalised AuNPs can be used to assess chitin’s ability to invoke the asthma allergic immune response, thereby bringing the possibility of an anti-asthma vaccine a step closer to fruition.</p>