Perceptions, intentions, and actual use of a consumer nicotine gum.

BACKGROUND: Little is known about perceptions, use intentions, and behaviors of adults regarding nicotine gum that is marketed and regulated as a consumer product rather than as a medicinal nicotine replacement therapy (NRT). METHODS: Survey data were collected from a Qualtrics online panel (N = 1000) of adults who had never used a consumer nicotine gum, recruited based on smoking behavior, and from current and former purchasers of one commercially available nicotine gum product (LUCY Chew and Park), recruited via emails to a customer database (N = 500). In addition to descriptive cross-sectional analyses, logistic regression was used to estimate the probability of intent to try and of product appeal among these different groups. RESULTS: Among online panel respondents, individuals who smoked with and without plans to quit showed high intention to try the product (odds ratios 15.6 [95% CI 9.3, 27.6] and 9.8 [95% CI 5.8, 17.3] respectively, compared to people who formerly smoked) and persons who had never smoked showed low intentions to try. These results stood regardless of flavor. Among current and former purchasers of the study product, 43.4% of persons who had smoked cigarettes regularly indicated they were motivated to try the product "to help me quit smoking." Only 0.6% of young adult consumers of the nicotine gum (aged 21-30) had not tried tobacco products previously. CONCLUSIONS: Consumer nicotine gum does not appear to attract those who have never used a tobacco product and the results for young adults suggest minimal appeal to youth. The study product was used primarily by individuals who currently smoke and/or use e-cigarettes but who wished to quit or reduce consumption. These results suggest that a consumer nicotine gum may reduce harm by substituting for higher-risk products such as combustible cigarettes.

Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity.

Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.

Necrotic reshaping of the glioma microenvironment drives disease progression.

Glioblastoma is the most common primary brain tumor and has a dismal prognosis. The development of central necrosis represents a tipping point in the evolution of these tumors that foreshadows aggressive expansion, swiftly leading to mortality. The onset of necrosis, severe hypoxia and associated radial glioma expansion correlates with dramatic tumor microenvironment (TME) alterations that accelerate tumor growth. In the past, most have concluded that hypoxia and necrosis must arise due to "cancer outgrowing its blood supply" when rapid tumor growth outpaces metabolic supply, leading to diffusion-limited hypoxia. However, growing evidence suggests that microscopic intravascular thrombosis driven by the neoplastic overexpression of pro-coagulants attenuates glioma blood supply (perfusion-limited hypoxia), leading to TME restructuring that includes breakdown of the blood-brain barrier, immunosuppressive immune cell accumulation, microvascular hyperproliferation, glioma stem cell enrichment and tumor cell migration outward. Cumulatively, these adaptations result in rapid tumor expansion, resistance to therapeutic interventions and clinical progression. To inform future translational investigations, the complex interplay among environmental cues and myriad cell types that contribute to this aggressive phenotype requires better understanding. This review focuses on contributions from intratumoral thrombosis, the effects of hypoxia and necrosis, the adaptive and innate immune responses, and the current state of targeted therapeutic interventions.

Nanocarrier-enhanced intracellular delivery of benznidazole for treatment of Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), an intracellular pathogen that causes significant morbidity and death among millions in the Americas from Canada to Argentina. Current therapy involves oral administration of the nitroimidazole benznidazole (BNZ), which has serious side effects that often necessitate cessation of treatment. To both avoid off-target side effects and reduce the necessary dosage of BNZ, we packaged the drug within poly(ethylene glycol)-block-poly(propylene sulfide) polymersomes (BNZ-PSs). We show that these vesicular nanocarriers enhanced intracellular delivery to phagocytic cells and tested this formulation in a mouse model of T. cruzi infection. BNZ-PS is not only nontoxic but also significantly more potent than free BNZ, effectively reducing parasitemia, intracellular infection, and tissue parasitosis at a 466-fold lower dose of BNZ. We conclude that BNZ-PS was superior to BNZ for treatment of T. cruzi infection in mice and that further modifications of this nanocarrier formulation could lead to a wide range of custom controlled delivery applications for improved treatment of Chagas disease in humans.

Vasoactive intestinal peptide producing pheochromocytoma and intracardiac thrombosis.

A case of pheochromocytoma producing vasoactive intestinal peptide (VIP) and left ventricular thrombus in the absence of cardiomyopathy or wall motion abnormalities on echocardiogram is presented along with a review of the relevant literature. A 30-year-old female of Afghani descent with past medical history of panic attacks presented with fever, cough, sore throat, vomiting, and was found to have an 11 cm adrenal mass consistent with primary adrenocortical adenoma versus carcinoma. Her tumor elicited catechols and vasoactive intestinal peptide. Her hospitalization was complicated by left ventricular thrombosis leading to an embolic injury to her right kidney, respiratory failure, need for transient dialysis and urinary tract infections. She developed a profuse secretory diarrhea and decision was made to treat with empiric octreotide infusion and imodium with improvement in symptoms. She underwent coil and particle embolization followed by resection. Followup PET gallium scan showed no evidence of residual disease or metastasis. VIP producing pheochromocytoma associated with intracardiac thrombosis is rare. Outcomes depend on prompt diagnosis of the pheochromocytoma and multidisciplinary approach to management.

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need.

Widespread tumor cell invasion is a fundamental property of diffuse gliomas and is ultimately responsible for their poor prognosis. A greater understanding of basic mechanisms underlying glioma invasion is needed to provide insights into therapies that could potentially counteract them. While none of the currently available in vitro models can fully recapitulate the complex interactions of glioma cells within the brain tumor microenvironment, if chosen and developed appropriately, these models can provide controlled experimental settings to study molecular and cellular phenomena that are challenging or impossible to model in vivo. Therefore, selecting the most appropriate in vitro model, together with its inherent advantages and limitations, for specific hypotheses and experimental questions achieves primary significance. In this review, we describe and discuss commonly used methods for modeling and studying glioma invasion in vitro, including platforms, matrices, cell culture, and visualization techniques, so that choices for experimental approach are informed and optimal.

Cell Cycle Inhibition To Treat Sleeping Sickness.

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.

Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts.

Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-ß-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.

The Lipid Raft Proteome of African Trypanosomes Contains Many Flagellar Proteins.

Lipid rafts are liquid-ordered membrane microdomains that form by preferential association of 3-ß-hydroxysterols, sphingolipids and raft-associated proteins often having acyl modifications. We isolated lipid rafts of the protozoan parasite Trypanosoma brucei and determined the protein composition of lipid rafts in the cell. This analysis revealed a striking enrichment of flagellar proteins and several putative signaling proteins in the lipid raft proteome. Calpains and intraflagellar transport proteins, in particular, were found to be abundant in the lipid raft proteome. These findings provide additional evidence supporting the notion that the eukaryotic cilium/flagellum is a lipid raft-enriched specialized structure with high concentrations of sterols, sphingolipids and palmitoylated proteins involved in environmental sensing and cell signaling.

Sphingosine Kinase Regulates Microtubule Dynamics and Organelle Positioning Necessary for Proper G1/S Cell Cycle Transition in Trypanosoma brucei.

UNLABELLED: Sphingolipids are important constituents of cell membranes and also serve as mediators of cell signaling and cell recognition. Sphingolipid metabolites such as sphingosine-1-phosphate and ceramide regulate signaling cascades involved in cell proliferation and differentiation, autophagy, inflammation, and apoptosis. Little is known about how sphingolipids and their metabolites function in single-celled eukaryotes. In the present study, we investigated the role of sphingosine kinase (SPHK) in the biology of the protozoan parasite Trypanosoma brucei, the agent of African sleeping sickness. T. brucei SPHK (TbSPHK) is constitutively but differentially expressed during the life cycle of T. brucei. Depletion of TbSPHK in procyclic-form T. brucei causes impaired growth and attenuation in the G1/S phase of the cell cycle. TbSPHK-depleted cells also develop organelle positioning defects and an accumulation of tyrosinated α-tubulin at the elongated posterior end of the cell, known as the "nozzle" phenotype, caused by other molecular perturbations in this organism. Our studies indicate that TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology. Cytotoxicity assays using TbSPHK inhibitors revealed a favorable therapeutic index between T. brucei and human cells, suggesting TbSPHK to be a novel drug target. IMPORTANCE: Trypanosoma brucei is a single-celled parasite that is transmitted between humans and other animals by the tsetse fly. T. brucei is endemic in sub-Saharan Africa, where over 70 million people and countless livestock are at risk of developing T. brucei infection, called African sleeping sickness, resulting in economic losses of ~$35 million from the loss of cattle alone. New drugs for this infection are sorely needed and scientists are trying to identify essential enzymes in the parasite that can be targets for new therapies. One possible enzyme target is sphingosine kinase, an enzyme involved in the synthesis of lipids important for cell surface integrity and regulation of cell functions. In this study, we found that sphingosine kinase is essential for normal growth and structure of the parasite, raising the possibility that it could be a good target for new chemotherapy for sleeping sickness.

Calcium-dependent membrane association of a flagellar calcium sensor does not require calcium binding.

Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca(2+) sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca(2+) binding. It is similar to neuronal Ca(2+) sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca(2+)-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca(2+), permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca(2+), disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca(2+)-binding mutant FCaBP(E151Q/E188Q) and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP(E151Q/E188Q) were found to associate with lipid rafts, indicating the Ca(2+)-independence of this association. To our initial surprise, FCaBP(E151Q/E188Q), like wildtype FCaBP, exhibited Ca(2+)-dependent flagellar membrane association, even though this protein does not bind Ca(2+) itself [16]. One possible explanation for this is that FCaBP(E151Q/E188Q), like some other Ca(2+) sensors, may form dimers and that dimerization of FCaBP(E151Q/E188Q) with endogenous wildtype FCaBP might explain its Ca(2+)-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca(2+). These results suggest that FCaBP possesses two distinct Ca(2+)-dependent interactions-one involving a Ca(2+)-induced change in conformation and another perhaps involving binding partner association.

Video game violence use among "vulnerable" populations: the impact of violent games on delinquency and bullying among children with clinically elevated depression or attention deficit symptoms.

The issue of children's exposure to violent video games has been a source of considerable debate for several decades. Questions persist whether children with pre-existing mental health problems may be influenced adversely by exposure to violent games, even if other children are not. We explored this issue with 377 children (62 % female, mixed ethnicity, mean age = 12.93) displaying clinically elevated attention deficit or depressive symptoms on the Pediatric Symptom Checklist. Results from our study found no evidence for increased bullying or delinquent behaviors among youth with clinically elevated mental health symptoms who also played violent video games. Our results did not support the hypothesis that children with elevated mental health symptoms constitute a "vulnerable" population for video game violence effects. Implications and suggestions for further research are provided.

Endothelial transmigration by Trypanosoma cruzi.

Chagas heart disease, the leading cause of heart failure in Latin America, results from infection with the parasite Trypanosoma cruzi. Although T. cruzi disseminates intravascularly, how the parasite contends with the endothelial barrier to escape the bloodstream and infect tissues has not been described. Understanding the interaction between T. cruzi and the vascular endothelium, likely a key step in parasite dissemination, could inform future therapies to interrupt disease pathogenesis. We adapted systems useful in the study of leukocyte transmigration to investigate both the occurrence of parasite transmigration and its determinants in vitro. Here we provide the first evidence that T. cruzi can rapidly migrate across endothelial cells by a mechanism that is distinct from productive infection and does not disrupt monolayer integrity or alter permeability. Our results show that this process is facilitated by a known modulator of cellular infection and vascular permeability, bradykinin, and can be augmented by the chemokine CCL2. These represent novel findings in our understanding of parasite dissemination, and may help identify new therapeutic strategies to limit the dissemination of the parasite.

(1)H, (15)N, and (13)C chemical shift assignments of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei.

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of Trypanosoma brucei. (BMRB no. 18011).

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei.

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF-hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF-hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N-terminal myristoyl group may be important for membrane binding. Non-conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein.

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.

Determination of the internal exposure hazard from plutonium work in an open front hood.

The particle size and quantity of a plutonium dioxide surrogate escaping an open front hood during routine work was measured. Aerosols were collected using a cascade impactor, and the amount of surrogate on each stage of the impactor was measured using neutron activation analysis. The aerodynamic mass median diameter of particles in the breathing zone of a worker was found to be 0.35 ± 5.1 µm. During a 2-h period when normal work activities were mimicked, an average of 3% of the material or 11.13 ± 0.54 µg escaped the hood. This was calculated to be equivalent to 10,000 ± 487 Bq (²³9Pu). The activity in the breathing zone of the worker is estimated, based on other research, to be 36 times less than this, or approximately 280 Bq. Worker occupancy factors were considered and a committed effective dose equivalent of 5 mSv was calculated. The cost-benefit analysis showed the use of the open front hood as a reasonable protective measure. Although worker exposure may approach the International Commission on Radiological Protection limit, the cost of the ergonomic injuries caused by work in a glove box is 5,000 times greater than the dose received by the worker. Protective measures such as respiratory protection should be evaluated on a case by case basis to keep worker exposure as low as reasonably achievable.

Trypanosoma cruzi GP63 proteins undergo stage-specific differential posttranslational modification and are important for host cell infection.

The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane.

Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms.

Flagellar membrane localization via association with lipid rafts.

The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca(2+) sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains.