Formation Of Membrane Ruffles Research Articles

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

Manipulation of microvillar proteins during Salmonella enterica invasion results in brush border effacement and actin remodeling.

Enterocyte invasion by the gastrointestinal pathogen Salmonella enterica is accompanied by loss of brush border and massive remodeling of the actin cytoskeleton, leading to microvilli effacement and formation of membrane ruffles. These manipulations are mediated by effector proteins translocated by the Salmonella Pathogenicity Island 1-encoded type III secretion system (SPI1-T3SS). To unravel the mechanisms of microvilli effacement and contribution of SPI1-T3SS effector proteins, the dynamics of host-pathogen interactions was analyzed using live cell imaging (LCI) of polarized epithelial cells (PEC) expressing LifeAct-GFP. PEC were infected with S. enterica wild-type and mutant strains with defined defects in SPI1-T3SS effector proteins, and pharmacological inhibition of actin assembly were applied. We identified that microvilli effacement involves two distinct mechanisms: i) F-actin depolymerization mediated by villin and ii), the consumption of cytoplasmic G-actin by formation of membrane ruffles. By analyzing the contribution of individual SPI1-T3SS effector proteins, we demonstrate that SopE dominantly triggers microvilli effacement and formation of membrane ruffles. Furthermore, SopE via Rac1 indirectly manipulates villin, which culminates in F-actin depolymerization. Collectively, these results indicate that SopE has dual functions during F-actin remodeling in PEC. While SopE-Rac1 triggers F-actin polymerization and ruffle formation, activation of PLCγ and villin by SopE depolymerizes F-actin in PEC. These results demonstrate the key role of SopE in destruction of the intestinal barrier during intestinal infection by Salmonella.

Functional significance of ion channels during macropinosome resolution in immune cells.

Macropinocytosis is a unique type of endocytosis accompanied by membrane ruffle formation. Closure of membrane ruffles leads to the uptake of large volumes of fluid phase and, subsequently, the formation of large vacuoles termed macropinosomes. Immune cells, such as dendritic cells, T cells, and macrophages, endocytose the surrounding amino acids and pathogens via macropinocytosis either constitutively or in a stimulus-dependent fashion. This process is critical for cell migration, mammalian target of rapamycin complex 1 (mTORC1) activation, and antigen presentation. Large vacuoles are fragmented into tubules and smaller vesicles during the progression and maturation of macropinosomes in immune cells. This process is called "macropinosome resolution" and requires osmotically driven shrinkage of macropinosomes, which is controlled by ion channels present in them. The crenation of membranes on shrunken macropinosomes is recognized by curvature-sensing proteins and results in intracellular membrane trafficking. In this mini review, we highlight the recent progress in research on macropinosome resolution in macrophages, with a focus on ion channels (TPC1/2 for Na+ and TMEM206 for Cl-) that is required for macropinosome resolution. We also discuss the potential contribution of membrane lipids to this process.

The Effect of the Nanoparticle Shape on T Cell Activation.

The mechanism of extracellular ligand nano-geometry in ex vivo T cell activation for immunotherapy remains elusive. Herein, the authors demonstrate large aspect ratio (AR) of gold nanorods (AuNRs) conjugated on cell culture substrate enhancing both murine and human T cell activation through the nanoscale anisotropic presentation of stimulatory ligands (anti-CD3(αCD3) and anti-CD28(αCD28) antibodies). AuNRs with large AR bearing αCD3 and αCD28 antibodies significantly promote T cell expansion and key cytokine secretion including interleukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). High membrane tension observed in large AR AuNRs regulates actin filament and focal adhesion assembly and develops maturation-related morphological features in T cells such as membrane ruffle formation, cell spreading, and large T cell receptor (TCR) cluster formation. Anisotropic stimulatory ligand presentation promotes differentiation of naïve CD8+ T cells toward the effector phenotype inducing CD137 expression upon co-culture with human cervical carcinoma. The findings suggest the importance of manipulating extracellular ligand nano-geometry in optimizing T cell behaviors to enhance therapeutic outcomes.

SeV C Protein Plays a Role in Restricting Macrophage Phagocytosis by Limiting the Generation of Intracellular Double-Stranded RNA.

Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-β stimulation and neutralizing antibodies against IFN-β suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-β-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells.

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.

Discovery of 5,6-Bis(4-methoxy-3-methylphenyl)pyridin-2-amine as a WSB1 Degrader to Inhibit Cancer Cell Metastasis.

The gain of cell motility is an essential prerequisite for cancer metastasis. The ubiquitin ligase subunit WD repeat and SOCS box-containing 1 (WSB1) has been demonstrated to regulate hypoxia-driven tumor cell migration. However, there is still a lack of methods for discovering inhibitors targeting the WSB1 axis. Here, we employed phenotypic screening models and identified compound 4 that displayed migration inhibitory activity against WSB1-overexpressing cells. Further studies indicated that it may function as a WSB1 degrader, thus leading to the accumulation of the Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2) protein, reversing the expression of downstream F-actin and formation of membrane ruffles, and disturbing the migration capacity of cancer cells. Moreover, compound 4 exhibited a promising in vivo anticancer metastatic effects. Our findings show the discovery of a new WSB1 degrader, providing a unique solution for the treatment of cancer metastasis.

Oncogenic RAS drives the CRAF-dependent extracellular vesicle uptake mechanism coupled with metastasis.

Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non‐transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium‐hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS‐transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS‐driven EV uptake, along with adhesion‐independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS‐driven cancers.

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

The Role of ATP8A1‐CDC50a on Learning Deficits

ATP8A1-CDC50a is a Type 4 P-type ATPase protein composed of two subunit proteins, the ATP8A1 section and the CDC50a section, connected by heterodimerization. Originally found in red blood cells, the ATP8A1 section was the first identified member of the P4-ATPase family and works as a flippase that uses the energy from ATP hydrolysis to translocate phospholipids from the outer to inner leaflet of eukaryotic cellular membranes in order to maintain phospholipid asymmetry necessary for membrane trafficking, membrane biogenesis, apoptosis, and signaling pathways. CDC50 proteins are common binding partners of the human class-1 P-ATPases to form heteromeric complexes which, in the case of ATP8A1-CDC50a, associate with and recruit P4-ATPase ATP8A1 to the plasma membrane. The CDC50a section functions as a chaperone protein to ATP8A1 and facilitates the proper folding and flippase activity. Mutations in some P4-ATPases cause inherited genetic diseases such as with ATP8A1-CDC50a and ATP8A2-CDC50, two closely related proteins in the P4-ATPase family, which can cause neurological disorders and learning deficits; however, only ATP8A2-CDC50 mutations are linked to severe neurological defects, whereas there is no clear disease associations linked to ATP8A1-CDC50a. Despite this, overexpression of CDC50a leads to extensive cell spreading and enhanced cell migration while a deficiency of either CDC50A or ATP8A1 may lead to severe defects in the formation of membrane ruffles, in turn inhibiting cell migration. The Walton SMART team has designed a 3D model of ATP8A1-CDC50a to investigate the relationship between its structure and function.

\u03b1-Linolenic acid induces clearance of Tau seeds via Actin-remodeling in Microglia

Alzheimer’s disease (AD) is known by characteristic features, extracellular burden of amyloid-β and intracellular neuronal Tau. Microglia, the innate immune cell of the brain has the ability to clear the burden of accumulated proteins via phagocytosis. But the excessive proinflammatory cytokine production, altered cellular signaling and actin remodeling hampers the process of migration and phagocytosis by microglia. Actin remodeling is necessary to initiate the chemotactic migration of microglia towards the target and engulf it. The formation of lamellipodia, filopodia, membrane ruffling and rapid turnover of F-actin is necessary to sense the extracellular target by the cells. Omega-3 fatty acids, are known to impose anti-inflammatory phenotype of microglia by enhancing its ability for migration and phagocytosis. But the role of omega-3 fatty acids in cellular actin remodeling, which is the basis of cellular functions such as migration and phagocytosis, is not well understood. Here, we have focused on the effect of dietary supplement of α-linolenic acid (ALA) on extracellular Tau internalization and assisted actin polymerization for the process. ALA is found to induce membrane ruffling and phagocytic cup formation along with cytoskeletal rearrangement. ALA also enhances the localization of Arp2/3 complex at the leading edge and its colocalization with F-actin to induce the actin polymerization. The excessive actin polymerization might help the cell to protrude forward and perform its migration. The results suggest that dietary supplement of ALA could play a neuroprotective role and slow down the AD pathology.

Computational and in vitro Pharmacodynamics Characterization of 1A-116 Rac1 Inhibitor: Relevance of Trp56 in Its Biological Activity.

In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.

The metabolic signature of T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prototypic autoimmune disease manifesting as chronic inflammation of the synovium and leading to acceleration of cardiovascular disease and shortening of life expectancy. The basic defect causing autoimmunity has remained elusive, but recent insights have challenged the notion that autoantigen is the core driver. Emerging data have added metabolic cues involved in the proper maintenance and activation of immune cells as pathogenic regulators. Specifically, studies have unveiled metabolic pathways that enforce T cell fate decisions promoting tissue inflammation; including T cell tissue invasiveness, T cell cytokine release, T cell-dependent macrophage activation and inflammatory T cell death. At the center of the metabolic abnormalities lies the mitochondria, which is consistently underperforming in RA T cells. The mitochondrial defect results at least partially from insufficient DNA repair and leads to lipid droplet accumulation, formation of invasive membrane ruffles, inflammasome activation and pyroptotic T cell death. T cells in patients with RA, even naïve T cells never having been involved in inflammatory lesions, have a unique metabolic signature and the changes in intracellular metabolites drive pathogenic T cell behavior. Recognizing the role of metabolic signals in cell fate decisions opens the possibility for immunomodulation long before the end stage synovial inflammation encountered in clinical practice.

Long noncoding RNA AURKAPS1 potentiates malignant hepatocellular carcinoma progression by regulating miR-142, miR-155 and miR-182

The mitotic serine/threonine kinase aurora kinase-A (AURKA) has been identified as carcinogenic in hepatocellular carcinoma (HCC). AURKAPS1, a long non-coding RNA (lncRNA), is the pseudo-gene of AURKA, which play important roles in the cancer. Its underlying functions and mechanisms in liver cancer progression remain largely unknown. The mRNA expression of AURKAPS1 in HCC tumor tissues was significantly higher, which is associated with tumor size and TNM stage. The high expression of AURKAPS1 promotes cell movement, migration and invasion. AURKAPS1 can increases the protein expression of RAC1, promotes the activation of ERK, and enhance the formation of membrane ruffles by binding with miR-182, miR-155 and miR-142 competively. Thus, AURKAPS1 could be a useful marker, and the combination of AURKAPS1/miRNAs (miR-142, miR-155 and miR-182) may be a new theoretical basis for the treatment of HCC.

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis

ObjectiveHighly elevated plasma levels of interleukin-10 (IL-10) are causally associated with “Disappearing HDL Syndrome” and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. Methods and resultsMacrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting.Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01). ConclusionsInterleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to “Disappearing HDL Syndrome”.

Human Immunodeficiency Virus (HIV) Infection and Use of Illicit Substances Promote Secretion of Semen Exosomes that Enhance Monocyte Adhesion and Induce Actin Reorganization and Chemotactic Migration.

Semen exosomes (SE) from HIV-uninfected (HIV−) individuals potently inhibit HIV infection in vitro. However, morphological changes in target cells in response to SE have not been characterized or have the effect of HIV infection or the use of illicit substances, specifically psychostimulants, on the function of SE been elucidated. The objective of this study was to evaluate the effect of HIV infection, psychostimulant use, and both together on SE-mediated regulation of monocyte function. SE were isolated from semen of HIV− and HIV-infected (HIV+) antiretroviral therapy (ART)-naive participants who reported either using or not using psychostimulants. The SE samples were thus designated as HIV−Drug−, HIV−Drug+, HIV+Drug−, and HIV+Drug+. U937 monocytes were treated with different SEs and analyzed for changes in transcriptome, morphometrics, actin reorganization, adhesion, and chemotaxis. HIV infection and/or use of psychostimulants had minimal effects on the physical characteristics of SE. However, different SEs had diverse effects on the messenger RNA signature of monocytes and rapidly induced monocyte adhesion and spreading. SE from HIV infected or psychostimulants users but not HIV−Drug− SE, stimulated actin reorganization, leading to the formation of filopodia-like structures and membrane ruffles containing F-actin and vinculin that in some cases were colocalized. All SE stimulated monocyte chemotaxis to HIV secretome and activated the secretion of matrix metalloproteinases, a phenotype exacerbated by HIV infection and psychostimulant use. SE-directed regulation of cellular morphometrics and chemotaxis depended on the donor clinical status because HIV infection and psychostimulant use altered SE function. Although our inclusion criteria specified the use of cocaine, humans are poly-drug and alcohol users and our study participants used psychostimulants, marijuana, opiates, and alcohol. Thus, it is possible that the effects observed in this study may be due to one of these other substances or due to an interaction between different substances.