Re‐examination of the mechanisms regulating nuclear inositol lipid metabolism

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3‐kinase and phosphoinositide‐specific phospholipase C‐β1

Early differentiating osteoclast interactions with a well suitable bone-like composite

Osteoclasts, as well as preosteoclasts, show different adhesion features in relationship to the substrate on which cells are grown, i.e. the formation of either podosomes belt or sealing zones. Podosomes belt forms on non bone substrates, i.e. when cells interact with glass coverslips or culture plates, whereas sealing zones form when cells grow on bone-like substrates. Podosomes belt corresponds to numerous F-actin columns arranged at the cell periphery, whereas the sealing zone could be defined as a unique large band of actin [1]. In the study of bone resorption mechanisms, the employ of bone slices is not perfectly suitable to investigate actin rearrangement due to cell-extracellular matrix (ECM) interaction, since it doesn’t allow to obtain high quality preparations to be examined both by light and electronmicroscopy (TEM and SEM). In particular, TEM preparation requires demineralization which could influence the chemical properties of either bone slices or bone-like composites. Moreover, the use of bone slices as scaffold, although extensive, doesn’t allow ultrastructural details that are necessary in the study of mineral resorption by monocytes or preosteoclasts [2,3]. The aim of the present study was to set up an experimental model for the study of cell-ECM interaction between either monocytes or early differentiating osteoclasts and a mineralized ECM. RAW 264.7 cells (a monocyte-macrophage cell line that can differentiate in osteoclasts) were cultured on a composite constituted by calcium phosphate and type I collagen to investigate actin polymerization and podosome formation. This bone-like composite doesn’t present the mechanical bone properties, but it is constituted by the main bone components and exhibits the advantage that collagen glues the mineral phase in clusters that can be either added to cell cultures or applied on coverslips, as well as to the culture medium. Light and fluorescence microscopy, as well as TEM and SEM techniques were employed. Results showed that the use of this bone-like composite allowed to obtain useful morphological information about the resorption activity of RAW 264.7 cell line differentiating towards the osteoclastic phenotype

Looking for calcium phosphate composite suitable to study osteoclast endocytosis: preliminary observations

One of the issues regarding in vitro study of bone resorption is the synthesis of a bonelike biomaterial forming a thin layer onto either glass or plastic. The synthesis of a bone-like material suitable for in vitro studies can be valuable both to investigate osteoclast differentiation, that in vivo proceeds within the local microenvironment of bone and to understand how its presence triggers activation of macrophages present in situ when bone is damaged (a scenario that can occur for example in case of bone fracture). Despite the intensive studies committed to recreate synthetic bone analogues, the most used substrates for in vitro studies on bone resorption are slices of bone or dentine. Therefore morphological investigations (i.e. fluorescence analysis and phase contrast) are strongly compromised due to the thickness of the bone analogue. In the present study, with the aim to guarantee a versatile (and easy to be made) substrate, that could be suitable to study cell adhesion and morphology by epifluorescence, phase contrast and TEM, we developed a biomaterial containing a calcium phosphate salt and type I collagen. This material (made specifically for in vitro studies) forms a very thin layer that allowed to merge the morphological information derived from phase-contrast and epifluorescence observation, making possible the observation of the interface between cell and matrix. Moreover the electron microscopy evaluation of the endocytosis performed on cell differentiated could be more suitable because sample does not need the process of demineralization

Synergistic Proapoptotic Activity of Recombinant Trail Plus the AKT Inhibitor Perifosine in Acute Myelogenous Leukemia Cells

To potentiate the response of acute myelogenous leukemia (AML) cells to TNF-Related Apoptosis- Inducing Ligand (TRAIL) cytotoxicity, we have examined the efficacy of a combination with perifosine, a novel phosphatidylinositol 3-kinase (PI3K)/Akt signaling inhibitor. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease the cellular FLICE-Inhibitory Protein (cFLIP) in human lung cancer cell lines. Perifosine and TRAIL both induced cell death by apoptosis in the THP-1 AML cell line, which is characterized by constitutive PI3K/Akt activation, but lacks functional p53. Perifosine, at concentrations below IC50, dephosphorylated Akt and increased TRAIL-R2 levels, as demonstrated by western blot, RT-PCR, and flow cytometric analysis. Perifosine also decreased the long isoform of cFLIP (cFLIP-L) and the X-linked Inhibitor of Apoptosis Protein (XIAP) expression. Perifosine and TRAIL synergized to activate caspase-8 and induce apoptosis, which was blocked by a caspase- 8 selective inhibitor. Upregulation of TRAIL-R2 expression was dependent on a protein kinase Cα/ c-Jun-NH2-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine synergized with TRAIL also in primary AML cells displaying constitutive activation of the Akt pathway, by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 upregulation, cFLIP-L and XIAP downregulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients. In contrast, CD34+ cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination perifosine and TRAIL might offer a novel therapeutic strategy for AML. Originally published Cancer Research, Vol. 68, No. 22, Nov 200

Neuronal dysfunction associated with cholesterol deregulation

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith–Lemli–Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries