Aurivillius Oxides Nanosheets-Based Photocatalysts for Efficient Oxidation of Malachite Green Dye.

Aurivillius oxides ferroelectric layered materials are formed by bismuth oxide and pseu-do-perovskite layers. They have a good ionic conductivity, which is beneficial for various photo-catalyzed reactions. Here, we synthesized ultra-thin nanosheets of two different Aurivillius oxides, Bi2WO6 (BWO) and Bi2MoO6 (BMO), by using a hard-template process. All materials were characterized through XRD, TEM, FTIR, TGA/DSC, DLS/ELS, DRS, UV-Vis. Band gap material (Eg) and potential of the valence band (EVB) were calculated for BWO and BMO. In contrast to previous reports on the use of multi composite materials, a new procedure for photocatalytic efficient BMO nanosheets was developed. The procedure, with an additional step only, avoids the use of composite materials, improves crystal structure, and strongly reduces impurities. BWO and BMO were used as photocatalysts for the degradation of the water pollutant dye malachite green (MG). MG removal kinetics was fitted with Langmuir-Hinshelwood model obtaining a kinetic constant k = 7.81 × 10-2 min-1 for BWO and k = 9.27 × 10-2 min-1 for BMO. Photocatalytic dye degradation was highly effective, reaching 89% and 91% MG removal for BWO and BMO, respectively. A control experiment, carried out in the absence of light, allowed to quantify the contribution of adsorption to MG removal process. Adsorption contributed to MG removal by a 51% for BWO and only by a 19% for BMO, suggesting a different degradation mechanism for the two photocatalysts. The advanced MG degradation process due to BMO is likely caused by the high crystallinity of the material synthetized with the new procedure. Reuse tests demonstrated that both photocatalysts are highly active and stable reaching a MG removal up to 95% at the 10th reaction cycle. These results demonstrate that BMO nanosheets, synthesized with an easy additional step, achieved the best degradation performance, and can be successfully used for environmental remediation applications.

Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

Adsorption and Release of Sulfamethizole from Mesoporous Silica Nanoparticles Functionalised with Triethylenetetramine.

Mesoporous silica nanoparticles (MSN) were synthesised and functionalised with triethylenetetramine (MSN-TETA). The samples were fully characterised (transmission electron microscopy, small angle X-ray scattering, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential and nitrogen adsorption/desorption isotherms) and used as carriers for the adsorption of the antimicrobial drug sulphamethizole (SMZ). SMZ loading, quantified by UV-Vis spectroscopy, was higher on MSN-TETA (345.8 mg g-1) compared with bare MSN (215.4 mg g-1) even in the presence of a lower surface area (671 vs. 942 m2 g-1). The kinetics of SMZ adsorption on MSN and MSN-TETA followed a pseudo-second-order model. The adsorption isotherm is described better by a Langmuir model rather than a Temkin or Freundlich model. Release kinetics showed a burst release of SMZ from bare MSN samples (k1 = 136 h-1) in contrast to a slower release found with MSN-TETA (k1 = 3.04 h-1), suggesting attractive intermolecular interactions slow down SMZ release from MSN-TETA. In summary, the MSN surface area did not influence SMZ adsorption and release. On the contrary, the design of an effective drug delivery system must consider the intermolecular interactions between the adsorbent and the adsorbate.

Zinc as a Drug for Wilson's Disease, Non-Alcoholic Liver Disease and COVID-19-Related Liver Injury.

Zinc is the second most abundant trace element in the human body, and it plays a fundamental role in human physiology, being an integral component of hundreds of enzymes and transcription factors. The discovery that zinc atoms may compete with copper for their absorption in the gastrointestinal tract let to introduce zinc in the therapy of Wilson's disease, a congenital disorder of copper metabolism characterized by a systemic copper storage. Nowadays, zinc salts are considered one of the best therapeutic approach in patients affected by Wilson's disease. On the basis of the similarities, at histological level, between Wilson's disease and non-alcoholic liver disease, zinc has been successfully introduced in the therapy of non-alcoholic liver disease, with positive effects both on insulin resistance and oxidative stress. Recently, zinc deficiency has been indicated as a possible factor responsible for the susceptibility of elderly patients to undergo infection by SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic. Here, we present the data correlating zinc deficiency with the insurgence and progression of Covid-19 with low zinc levels associated with severe disease states. Finally, the relevance of zinc supplementation in aged people at risk for SARS-CoV-2 is underlined, with the aim that the zinc-based drug, classically used in the treatment of copper overload, might be recorded as one of the tools reducing the mortality of COVID-19, particularly in elderly people.

Gold Nanoparticles: A Powerful Tool to Visualize Proteins on Ordered Mesoporous Silica and for the Realization of Theranostic Nanobioconjugates.

Ordered mesoporous silica (OMS) is a very interesting nanostructured material for the design and engineering of new target and controlled drug-delivery systems. Particularly relevant is the interaction between OMS and proteins. Large pores (6­9 nm) micrometric particles can be used for the realization of a drug depot system where therapeutic proteins are adsorbed either inside the mesopores or on the external surface. Small pores (1­2 nm) mesoporous silica nanoparticles (MSNs), can be injected in the blood stream. In the latter case, therapeutic proteins are mainly adsorbed on the MSNs' external surface. Whenever a protein-OMS conjugate is prepared, a diagnostic method to locate the protein either on the internal or the external silica surface is of utmost importance. To visualize the fine localization of proteins adsorbed in mesoporous silica micro- and nanoparticles, we have employed specific transmission electron microscopy (TEM) analytical strategies based on the use of gold nanoparticles (GNPs) conjugates. GNPs are gaining in popularity, representing a fundamental tool to design future applications of MSNs in nanomedicine by realizing theranostic nanobioconjugates. It may be pointed out that we are at the very beginning of a new age of the nanomaterial science: the "mesoporous golden age".

Silver Enhancement for Transmission Electron Microscopy Imaging of Antibody Fragment-Gold Nanoparticles Conjugates Immobilized on Ordered Mesoporous Silica.

Ordered mesoporous silica (OMS) materials are receiving great attention as possible carriers for valuable but unstable drugs as, for example, therapeutic proteins. A key issue is to prove that the therapeutic protein is effectively able to penetrate the pores of OMS during the adsorption step. Here, we immobilized an antibody fragment [F(ab')GAMIgG] conjugated with ultrasmall gold nanoparticles (GNPs) onto amino-functionalized SBA-15 (SBA-NH2) mesoporous silica. The aim of this work is the visualization of the location of the conjugates adsorbed onto SBA-NH2 with transmission electron microscopy (TEM). Because of the ultrasmall size of GNPs (<1 nm), we use the silver enhancement procedure to amplify their size. In this procedure, ultrathin sections of conjugate-loaded SBA-NH2 particles are prepared by a ultramicrotome sectioning technique. The ultrasmall GNPs located on the top side of the 70-90 nm thick slices act as microcrystallization nucleation sites for the deposition of reduced metallic silver. Consequently, the ultrasmall GNPs increase their size. This allows for the direct imaging of the conjugates adsorbed. We clearly localize the F(ab')GAMIgG-GNPs conjugates either on the external surface of the particles or inside the mesopores of SBA-NH2 through TEM.

Loading and protection of hydrophilic molecules into liposome-templated polyelectrolyte nanocapsules.

Compartmentalized systems produced via the layer-by-layer (LbL) self-assembly method have been produced by alternatively depositing alginate and chitosan layers onto cores of liposomes. The combination of dynamic light scattering (DLS), ζ potential, and transmission electron microscopy (TEM) techniques provides detailed information on the stability, dimensions, charge, and wall thickness of these polyelectrolyte globules. TEM microphotographs demonstrate the presence of nanocapsules with an average diameter of below 300 nm and with a polyelectrolyte wall thickness of about 20 nm. The possibility of encapsulating and releasing molecules from this type of nanocapsule was demonstrated by loading FITC-dextrans of different molecular weights in the liposome system. The release of the loaded molecules from the nanocapsule was demonstrated after liposome core dissolution. Even at low molecular weight (20 kDa), the nanocapsules appear to be appropriate for prolonged molecule compartmentalization and protection. By means of the Ritger-Peppas model, non-Fickian transport behavior was detected for the diffusion of dextran through the polyelectrolyte wall. Values of the diffusion coefficient were calculated and yield useful information regarding chitosan/alginate hollow nanocapsules as drug-delivery systems. The influence of the pH on the release properties was also considered. The results indicate that vesicle-templated hollow polyelectrolyte nanocapsules show great potential as novel controllable drug-delivery devices for biomedical and biotechnological applications.

Thymosin ß4 and ß10 Expression in Human Organs during Development: A Review.

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin ß4 and thymosin ß10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin ß4 and thymosin ß10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of ß-thymosins in health and disease. The functions of ß-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "ß-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.

Astrocytes shed large membrane vesicles that contain mitochondria, lipid droplets and ATP.

Various cells types, including stem and progenitor cells, can exchange complex information via plasma membrane-derived vesicles, which can carry signals both in their limiting membrane and lumen. Astrocytes, traditionally regarded as mere supportive cells, play previously unrecognized functions in neuronal modulation and are capable of releasing signalling molecules of different functional significance. In the present study, we provide direct evidence that human fetal astrocytes in culture, expressing the same feature as immature and reactive astrocytes, release membrane vesicles larger than the microvesicles described up to now. We found that these large vesicles, ranging from 1-5 to 8 µm in diameter and expressing on their surface ß1-integrin proteins, contain mitochondria and lipid droplets together with ATP. We documented vesicle content with fluorescent-specific dyes and with the immunocytochemistry technique we confirmed that mitochondria and lipid droplets were co-localized in the same vesicle. Scanning electron microscopy and transmission electron microscopy confirmed that astrocytes shed from surface membrane vesicles of the same size as the ones detected by fluorescence microscopy. Our results report for the first time that cultured astrocytes, activated by repetitive stimulation of ATP released from neighboring cells, shed from their surface large membrane vesicles containing mitochondria and lipid droplets.

Cell starvation increases uptake of extracellular Thymosin ß4 and its complexes with calcium.

Cell metastasis is the main cause of cancer mortality. Inhibiting early events during cell metastasis and invasion could significantly improve cancer prognosis, but the initial mechanisms of cell transition and migration are barely known. Calcium regulates cell migration, whilst Thymosin ß4 is a G-actin and iron binding peptide associated with tumor metastasis and ferroptosis. Under normal cell growth conditions, intracellular free calcium ions and Thymosin ß4 concentrations are strictly regulated, and are not influenced by extracellular supplementation. However, cell starvation decreases intracellular Thymosin ß4 and increases extracellular peptide uptake above the normal range. Unexpectedly, cell starvation significantly increases internalization of extracellular Ca2+/Thymosin ß4 complexes. Elucidating the role of Ca2+/Thymosin ß4 in the early events of metastasis will likely be important in the future to develop therapies targeting metastasis.

Exploring cell surface markers and cell-cell interactions of human breast milk stem cells.

Background: Breakthrough studies have shown that pluripotent stem cells are present in human breast milk. The expression of pluripotency markers by breast milk cells is heterogeneous, relating to cellular hierarchy, from early-stage multi-lineage stem cells to fully differentiated mammary epithelial cells, as well as weeks of gestation and days of lactation. Design and methods: Here, we qualitatively analyze cell marker expression in freshly isolated human breast milk cells, without any manipulation that could influence protein expression. Moreover, we use electron microscopy to investigate cell-cell networks in breast milk for the first time, providing evidence of active intercellular communication between cells expressing different cellular markers. Results: The immunocytochemistry results of human breast milk cells showed positive staining in all samples for CD44, CD45, CD133, and Ki67 markers. Variable positivity was present with P63, Tß4 and CK14 markers. No immunostaining was detected for Wt1, nestin, Nanog, OCT4, SOX2, CK5, and CD34 markers. Cells isolated from human breast milk form intercellular connections, which together create a cell-to-cell communication network. Conclusions: Cells freshly isolated form human breast milk, without particular manipulations, show heterogeneous expression of stemness markers. The studied milk staminal cells show "pluripotency" at different stages of differentiation, and are present as single cells or grouped cells. The adjacent cell interactions are evidenced by electron microscopy, which showed the formation of intercellular connections, numerous contact regions, and thin pseudopods.

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney.

Background: A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching. Design and methods: Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination. Results: Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching. Conclusions: The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud.

Living with the enemy: from protein-misfolding pathologies we know, to those we want to know.

Conformational diseases are caused by the aggregation of misfolded proteins. The risk for such pathologies develops years before clinical symptoms appear, and is higher in people with alpha-1 antitrypsin (AAT) polymorphisms. Thousands of people with alpha-1 antitrypsin deficiency (AATD) are underdiagnosed. Enemy-aggregating proteins may reside in these underdiagnosed AATD patients for many years before a pathology for AATD fully develops. In this perspective review, we hypothesize that the AAT protein could exert a new and previously unconsidered biological effect as an endogenous metal ion chelator that plays a significant role in essential metal ion homeostasis. In this respect, AAT polymorphism may cause an imbalance of metal ions, which could be correlated with the aggregation of amylin, tau, amyloid beta, and alpha synuclein proteins in type 2 diabetes mellitus (T2DM), Alzheimer's and Parkinson's diseases, respectively.

Undercover Toxic Ménage à Trois of Amylin, Copper (II) and Metformin in Human Embryonic Kidney Cells.

In recent decades, type 2 diabetes complications have been correlated with amylin aggregation, copper homeostasis and metformin side effects. However, each factor was analyzed separately, and only in some rare cases copper/amylin or copper/metformin complexes were considered. We demonstrate for the first time that binary metformin/amylin and tertiary copper (II)/amylin/metformin complexes of high cellular toxicity are formed and lead to the formation of aggregated multi-level lamellar structures on the cell membrane. Considering the increased concentration of amylin, copper (II) and metformin in kidneys of T2DM patients, our findings on the toxicity of amylin and its adducts may be correlated with diabetic nephropathy development.

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands.

BACKGROUND: Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. METHODS: Biopsy samples of parotid glands were excised from non-diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). RESULTS: Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. CONCLUSIONS: Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.

Ultrastructural localization of salivary mucins MUC5B and MUC7 in human labial glands.

As a result of their presence throughout the mouth in the submucosa or between muscle fibers, minor salivary glands secrete directly and continuously into the oral cavity, providing mucosal surfaces with highly glycosylated proteins that are active in bacterial aggregation and in oral tissue lubrication. In this study, we investigated the ultrastructural localization of the MUC5B and MUC7 mucins in human labial glands by means of a postembedding immunogold technique. Thin sections of normal human labial glands, obtained during surgery, were incubated with polyclonal antibodies to human salivary mucins MUC5B and MUC7, and then with gold-labeled secondary antibodies. Specific MUC5B reactivity was found in the secretory granules of mucous cells of all glands examined, and was associated with the luminal membrane of duct cells. MUC7 labeling was observed in the granules of both mucous and seromucous secretory cells of the glandular parenchyma. Quantitative analyses demonstrated that seromucous granules have higher immunogold labeling densities for MUC7 than mucous granules. Our immunohistochemical data extend the results of previous light microscopic studies of MUC5B and MUC7 localizations, pointing out the significant contribution of human labial glands in the secretion process of these two mucins.

Thymosin ß4 cytoplasmic/nuclear translocation as a new marker of cellular stress. A Caco2 case study.

Biomarkers of cell stress are important for proper diagnosis, and in studies of how cells respond to drug treatment. Biomarkers that respond early to pharmacological treatment could improve therapy by tailoring the treatment to the needs of the patient. Thymosin beta-4 (Tß4) plays a significant role in many aspects of cellular metabolism because of its actin-sequestering properties. Other physiological functions of Tß4 have been also reported. Among these, Tß4 may play a crucial role during cellular stress. We addressed the relevance of Tß4 in cellular stress conditions by using different treatments (serum starvation, DMSO, and butyrate administration) in a colon adenocarcinoma cell line (CaCo2), a cell line frequently used for in vitro experimental studies of Tß4. In this study, different stress stimuli were analyzed and the obtained results were compared using immunocytochemistry, and molecular and biochemical methods. Taken together, the data clearly indicate that the Tß4 peptide is involved in adaptive and defensive cellular mechanisms, and that different stress inducers lead to a similar Tß4 cytoplasmic/nuclear translocation. The translocation of Tß4 between the cytoplasm and the nucleus of the cell seems characteristic of a possible molecular response to cellular stress exerted by this peptide.

Assembly of Multicomponent Nano-Bioconjugates Composed of Mesoporous Silica Nanoparticles, Proteins, and Gold Nanoparticles.

The purpose of this work was the assembly of multicomponent nano-bioconjugates based on mesoporous silica nanoparticles (MSNs), proteins (bovine serum albumin, BSA, or lysozyme, LYZ), and gold nanoparticles (GNPs). These nano-bioconjugates may find applications in nanomedicine as theranostic devices. Indeed, MSNs can act as drug carriers, proteins stabilize MSNs within the bloodstream, or may have therapeutic or targeting functions. Finally, GNPs can either be used as contrast agents for imaging or for photothermal therapy. Here, amino-functionalized MSNs (MSN-NH2) were synthesized and characterized through various techniques (small angle X-rays scattering TEM, N2 adsorption/desorption isotherms, and thermogravimetric analysis (TGA)). BSA or lysozyme were then grafted on the external surface of MSN-NH2 to obtain MSN-BSA and MSN-LYZ bioconjugates, respectively. Protein immobilization on MSNs surface was confirmed by Fourier transform infrared spectroscopy, ζ-potential measurements, and TGA, which also allowed the estimation of protein loading. The MSN-protein samples were then dispersed in a GNP solution to obtain MSN-protein-GNPs nano-bioconjugates. Transmission electron microscopy (TEM) analysis showed the occurrence of GNPs on the MSN-protein surface, whereas almost no GNPs occurred in the protein-free control samples. Fluorescence and Raman spectroscopies suggested that proteins-GNP interactions involve tryptophan residues.

Mesoporous silica nanoparticles functionalized with hyaluronic acid. Effect of the biopolymer chain length on cell internalization.

Mesoporous silica nanoparticles (MSNs) were functionalized with amino groups (MSN-NH2) and then with hyaluronic acid, a biocompatible biopolymer which can be recognized by CD44 receptors in tumor cells, to obtain a targeting drug delivery system. To this purpose, three hyaluronic acid samples differing for the molecular weight, namely HAS (8-15 kDa), HAM (30-50 kDa) and HAL (90-130 kDa), were used. The MSN-HAS, MSN-HAM, and MSN-HAL materials were characterized through zeta potential and dynamic light scattering measurements at pH = 7.4 and T = 37 °C to simulate physiological conditions. While zeta potential showed an increasing negative value with the increase of the HA chain length, an anomalous value of the hydrodynamic diameter was observed for MSN-HAL, which was smaller than that of MSN-HAS and MSN-HAM samples. The cellular uptake of MSN-HA samples on HeLa cells at 37 °C was studied by optical and electron microscopy. HA chain length affected significantly the cellular uptake that occurred at a higher extent for MSN-NH2 and MSN-HAS than for MSN-HAM and MSN-HAL samples. Cellular uptake experiments carried out at 4 °C showed that the internalization process was inhibited for MSN-HA samples but not for MSN-NH2. This suggests the occurrence of two different mechanisms of internalization. For MSN-NH2 the uptake is mainly driven by the attractive electrostatic interaction with membrane phospholipids, while MSN-HA internalization involves CD44 receptors overexpressed in HeLa cells.

Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands.

One of the main functions of the translocator protein (18 kDa) or TSPO, previously known as peripheral-type benzodiazepine receptor, is the regulation of cholesterol import into mitochondria for steroid biosynthesis. In this paper we show that TSPO ligands induce changes in the distribution of intracellular cholesterol in astrocytes and fibroblasts. NBD-cholesterol, a fluorescent analog of cholesterol, was rapidly removed from membranes and accumulated into lipid droplets. This change was followed by a block of cholesterol esterification, but not by modification of intracellular cholesterol synthesis. NBD-cholesterol droplets were in part released in the medium, and increased cholesterol efflux was observed in [(3)H]cholesterol-prelabeled cells. TSPO ligands also induced a prominent shrinkage and depolarization of mitochondria and depletion of acidic vesicles with cytoplasmic acidification. Consistent with NBD-cholesterol changes, MTT assay showed enhanced accumulation of formazan into lipid droplets and inhibition of formazan exocytosis after treatment with TSPO ligands. The effects of specific TSPO ligands PK 11195 and Ro5-4864 were reproduced by diazepam, which binds with high affinity both TSPO and central benzodiazepine receptors, but not by clonazepam, which binds exclusively to GABA receptor, and other amphiphilic substances such as DIDS and propranolol. All these effects and the parallel immunocytochemical detection of TSPO in potentially steroidogenic cells (astrocytes) and non-steroidogenic cells (fibroblasts) suggest that TSPO is involved in the regulation and trafficking of intracellular cholesterol by means of mechanisms not necessarily related to steroid biosynthesis.