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IntroductionConsidering the dangers of adolescent tobacco use, the successful design of behavioral programs is crucial for tobacco prevention. According to preliminary research, social game interventions can improve adolescent tobacco outcomes. The current qualitative study aims to (1) uncover the gaming elements that adolescents deem important for a positive learning experience, and (2) confirm these gaming elements with adolescents who are presented with a tobacco prevention game concept that applies these elements.MethodsFindings from this study are drawn from two phases. Phase 1 involved in-person focus group discussions (n = 15) and Phase 2 included three online focus groups and a paired interview with another set of adolescents (n = 15). The study was conducted under a project that aimed to design and test a social game-based tobacco prevention program for adolescents (Storm-Heroes). With open coding and thematic analysis, two research team members identified repeated topics and relevant quotes to organize them into themes. The themes evolved as new content was identified during the process. This process was repeated until thematic saturation was reached.ResultsThematic analysis across Phase 1 and Phase 2 revealed four major themes: 1) Balance during gaming challenges, 2) Healthy social interaction, 3) Performance and creative freedom, and 4) Fictional world and game mechanics for tobacco prevention.ConclusionThis study identified specific intervention features that best fit the needs of adolescents in the context of a social game for tobacco prevention. For future research, we will use a participatory approach to allow adolescents to take part in the design process, improve Storm-Heroes, and develop health promotional messages that can be incorporated into the program. Ultimately, a board game for tobacco prevention is expected to bring adolescents together to create lasting memories that nudge them away from tobacco use and the harm it can cause.

BackgroundThe use of new and emerging tobacco products (NETPs) and conventional tobacco products (CTPs) has been linked to several alarming medical conditions among young adults (YAs). Considering that 96% of YAs own mobile phones, SMS text messaging may be an effective strategy for tobacco risk communication. ObjectiveProject Debunk is a community-based randomized trial aiming to identify specific types of messages that effectively improve perceived NETP and CTP risk among YAs in community colleges. MethodsWith YAs recruited offline from 3 campuses at the Houston Community College (September 2016 to July 2017), we conducted a 6-month randomized trial with 8 arms based on the combination of 3 message categories: framing (gain-framed vs loss-framed), depth (simple vs complex), and appeal (emotional vs rational). Participants received fully automated web-based SMS text messages in two 30-day campaigns (2 messages per day). We conducted repeated-measures mixed-effect models stratified by message type received, predicting perceived CTP and NETP risks. Owing to multiple testing with 7 models, an association was deemed significant for P

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Introduction: Communicating the risks associated with nicotine and tobacco use to adolescents can be challenging, especially with the current tobacco market's attempt to capture the attention of youths. Text message interventions have emerged to address the need to improve tobacco risk communication. This article informs the design of a message library for tobacco risk communication that is based on the transtheoretical model and addresses the risk of multiple tobacco products. Methods: We draw findings from this study from two phases. Phase 1 involved six remote focus group discussions (n = 25) and an in-depth interview, and Phase 2 involved online ideation sessions (n = 11) that led to the current version of the messages. We conducted the study within a larger project for the design and testing of a tobacco prevention program. With thematic analysis and the affinity mapping technique, two research team members identified repeated topics and relevant quotes to organize them into themes and subthemes. Results: In Phase 1, thematic analysis revealed four major themes: 1) Adolescents' gap in tobacco knowledge, 2) Social influence and popularity, 3) Attitude toward marketing, and 4) Text message framing preferences. During Phase 2, participants generated 1-to-7 iterations of the original messages. Votings and discussions resulted in a library of 306 messages under 7 sections, categorized based on the processes of change from the transtheoretical model. Conclusion: The current study presents key insights crucial for developing and evaluating a library of tobacco prevention text messages that is scientifically valid and successfully resonates with today's adolescents. Our future plan is to go beyond this initial message development and vet the message library by adolescents and expert reviewers in tobacco risk communication. Future research may consider developing messages that are tailored based on gender, ethnicity, and other factors that are predictive of tobacco use. [ABSTRACT FROM AUTHOR]

BackgroundTobacco smoking remains a public health problem among adolescents in the United States. While Web-based interventions for smoking prevention have been successful at the individual level, there is still an urgent need to understand their engagement capabilities and their effects at the social level. In the current study, we aimed to (1) learn about adolescents' subjective experience with a Web-based program called a smoking prevention interactive experience (ASPIRE), (2) obtain suggestions for improvement in ASPIRE content, (3) identify psychological outcomes of ASPIRE, and (4) explore outcomes of social interaction.Materials and methodsAfter a randomized controlled trial with 110 adolescents, 20 adolescent users of ASPIRE, aged 11-18, were randomly selected to participate in one-on-one interviews at four after-school programs in Houston, Texas. Interviews involved questions concerning adolescents' experience with the intervention. Qualitative data were coded and analyzed using a constant comparison approach for the generation of themes.ResultsDescribing their experience with ASPIRE, participants expressed comfort in material that is tailored to their demographic and preferred interactive activities over entertaining videos. Presenting suggestions for improvement, participants mainly reported the need to include gaming features into ASPIRE. Presenting psychological outcomes, they expressed emotional engagement in the program, shifts in attitudes and beliefs, and unwillingness to smoke. Finally, as outcomes of social interaction, participants reported engagement with others in discussions about tobacco and their need to hold smokers accountable for their actions.ConclusionsAdolescents' reports moved from their individual experience with ASPIRE to their active interactions with family members and friends and their attempt to persuade others to quit smoking. Future Web-based programs for adolescents may be designed with tailoring and game play in mind, in order to provide mobilization skills and foster social interactions against smoking.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined. • Native PfCRT migrates as 42 kDa polypeptide is non-phosphorylated at its C-terminal. • Post-translationally modified PfCRT variant is phosphorylated and migrates as 52 kDa polypeptide. • Antibody to PfCRT N-terminal detects both native and post-translationally modified variant polypeptides. • Antibody to PfCRT C-terminal binds only non-phosphorylated native or dephosphorylated variant PfCRT. [ABSTRACT FROM AUTHOR]

Introduction: Considering the dangers of adolescent tobacco use, the successful design of behavioral programs is crucial for tobacco prevention. According to preliminary research, social game interventions can improve adolescent tobacco outcomes. The current qualitative study aims to (1) uncover the gaming elements that adolescents deem important for a positive learning experience, and (2) confirm these gaming elements with adolescents who are presented with a tobacco prevention game concept that applies these elements. Methods: Findings from this study are drawn from two phases. Phase 1 involved in-person focus group discussions (n = 15) and Phase 2 included three online focus groups and a paired interview with another set of adolescents (n = 15). The study was conducted under a project that aimed to design and test a social game-based tobacco prevention program for adolescents (Storm-Heroes). With open coding and thematic analysis, two research team members identified repeated topics and relevant quotes to organize them into themes. The themes evolved as new content was identified during the process. This process was repeated until thematic saturation was reached. Results: Thematic analysis across Phase 1 and Phase 2 revealed four major themes: 1) Balance during gaming challenges, 2) Healthy social interaction, 3) Performance and creative freedom, and 4) Fictional world and game mechanics for tobacco prevention. Conclusion: This study identified specific intervention features that best fit the needs of adolescents in the context of a social game for tobacco prevention. For future research, we will use a participatory approach to allow adolescents to take part in the design process, improve Storm-Heroes, and develop health promotional messages that can be incorporated into the program. Ultimately, a board game for tobacco prevention is expected to bring adolescents together to create lasting memories that nudge them away from tobacco use and the harm it can cause. [ABSTRACT FROM AUTHOR]

The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent developments in the studies of drug resistance have identified compounds such as verapamil and tamoxifen that specifically target ABCB1-expressing multidrug-resistant (MDR) cells, through an ATP-dependent ROS-generating mechanism. In this report, we demonstrate that treatment of ABCB1-expressing MDR cells (CHORC5 or MDA-Doxo400) or individual clones of the latter with sub-lethal concentrations of tamoxifen or verapamil down-regulates ABCB1 protein and mRNA expression in surviving clones. Consequently, tamoxifen- and verapamil-treated cells show increased sensitivity to chemotherapeutic drugs (e.g. colchicine and doxorubicin) and decreased sensitivity to collateral sensitivity drugs (e.g. verapamil and tamoxifen). Importantly, we show for the first time that down-regulation of ABCB1 expression resulting from tamoxifen treatment and CRISPR-knockout of ABCB1 expression up-regulate α-enolase (enolase I) protein levels and activity. These findings demonstrate a possible effect of ABCB1 expression on the metabolic homeostasis of MDR cells. Moreover, given the use of tamoxifen to prevent the recurrence of oestrogen receptor-positive breast cancer, the findings of this study may be clinically important in modulating activity of other drugs. [ABSTRACT FROM AUTHOR]

Multidrug resistant tumor cells show collaterally sensitive to a range of non-toxic drugs. In this report, we describe the isolation of several P-glycoprotein-knockout cell clones, using CRISPR/Cas9, from Chinese hamster multidrug resistant model cell line and its parental cells (e.g. , CHORC5 and AuxB1, respectively). All three P-glycoprotein-knockout clones of CHORC5 cells show complete loss of resistance to anti-cancer drugs (e.g., colchicine and doxorubicin), while gaining resistance to well characterized collateral sensitivity drugs (e.g., verapamil, progesterone and NSC73306). A correlation between P-glycoprotein and Sorcin expression levels and a possible role for the latter in low grade resistance to colchicine and doxorubicin was observed. Furthermore, we show that P-glycoprotein expression is necessary for the ROS-mediated mechanism of collateral sensitivity. However, expectantly, P-glycoprotein-knockout clones of CHORC5 cells revealed a dramatic increase in the accumulation of Rhodamine 123, Mito tracker red and doxorubicin, but not Hoechst 33342. The latter findings and their significance to P-glycoprotein collateral sensitivity remain to be determined. [Display omitted] • Knockout of P-glycoprotein expression demonstrates its essential role in collateral sensitivity of multidrug resistant cells. • Collateral sensitivity of multidrug resistant cells is due to P-glycoprotein-dependent rise in reactive oxygen species. • Sorcin may mediate resistance to doxorubicin in P-glycoprotein knockout tumor cells. • Loss of P-glycoprotein expression from multidrug resistant cells unmask putative drug in-flux mechanism or transporter. [ABSTRACT FROM AUTHOR]

The P-Glycoprotein (ABCB1) is a linker domain that causes multidrug resistance in tumor cells selected with lipophilic anticancer drugs. ABCB1 helps in encoding high-affinity binding sequences to [alpha]- and [beta]-tubulins and hence can be used in various tumor cells.

P-glycoprotein, member of the B-subfamily of the ATP-binding cassette (ABC) superfamily (e.g., ABCB1), has been demonstrated to confer resistance to clinically relevant anticancer drugs. Paradoxically, ABCB1-expressing multidrug resistant (MDR) cells are hypersensitivity or collateral sensitivity to non-toxic drugs. In this report, we demonstrate the capacity of trifluoperazine (TFP), a calmodulin inhibitor, to confer a collateral sensitivity onto ABCB1-overexpressing MDR cells. We show TFP-induced collateral sensitivity to be linked to ABCB1 expression and ATPase activity, as such phenotype is abolished in ABCB1-knockout MDR cells (CHORC5ΔABCB1 clones A1-A3) or with inhibitors of ABCB1 ATPase. TFP-induced collateral sensitivity is mediated by apoptotic cell death, due to enhanced oxidative stress. The findings in this study show for first time the use TFP as a collateral sensitivity drug, at clinically relevant concentrations. Moreover, given the use of trifluoperazine in the treatment for symptoms of schizophrenia and the role of ABCB1 transporter in tissue blood barriers and other physiologic functions, the finding in this study may have implications beyond cancer chemotherapy. [Display omitted] • Trifluoperazine is selectively lethal to ABCB1-expressing multidrug resistant cells. • Trifluoperazine-induced Collateral sensitivity is mediated by oxidative cell death. • ABCB1 knockout or inhibition of ATPase activity reverses the collateral sensitivity to trifluoperazine. • Potential clinical use of trifluoperazine to prevent the rise of drug resistant tumor cells. [ABSTRACT FROM AUTHOR]

The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s). [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser33, Ser411 and Thr416) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e. , IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence (408NEDSEGE414), including Ser411. The effect of Ser411 phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser411 with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has un-phosphorylated Ser411 and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser411 in CQ-sensitive (3D7) than CQ–resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser411 phosphorylation in PfCRT structure-function. • High resolution mapping of anti-PfCRT antisera to its C-terminal sequence. • Isolation of three reactive IgG pools in PfCRT antisera against different epitopes, with IgG pool 2 specific to Ser411 phosphorylation status. • Demonstrating the presence of PfCRT homodimer, ∼90 kDa, encoding un-phosphorylated Ser411. • PfCRT phosphorylation status at Ser411 may predict P. falciparum resistance to chloroquin. [ABSTRACT FROM AUTHOR]

The study synthesized and characterized a novel photoreactive azido analogue of leucotriene (LTC4) (AALTC4). The results demonstrate a high-resolution map of LTC4 binding domains in multidrug resistance protein 1 (MRP1) and provide the first direct evidence for LTC4 binding within membrane multiple spanning domain of MRP1 (MSD0).

Tobacco smoking remains a public health problem among adolescents in the United States. While Web-based interventions for smoking prevention have been successful at the individual level, there is still an urgent need to understand their engagement capabilities and their effects at the social level. In the current study, we aimed to (1) learn about adolescents' subjective experience with a Web-based program called a smoking prevention interactive experience (ASPIRE), (2) obtain suggestions for improvement in ASPIRE content, (3) identify psychological outcomes of ASPIRE, and (4) explore outcomes of social interaction. After a randomized controlled trial with 110 adolescents, 20 adolescent users of ASPIRE, aged 11–18, were randomly selected to participate in one-on-one interviews at four after-school programs in Houston, Texas. Interviews involved questions concerning adolescents' experience with the intervention. Qualitative data were coded and analyzed using a constant comparison approach for the generation of themes. Describing their experience with ASPIRE, participants expressed comfort in material that is tailored to their demographic and preferred interactive activities over entertaining videos. Presenting suggestions for improvement, participants mainly reported the need to include gaming features into ASPIRE. Presenting psychological outcomes, they expressed emotional engagement in the program, shifts in attitudes and beliefs, and unwillingness to smoke. Finally, as outcomes of social interaction, participants reported engagement with others in discussions about tobacco and their need to hold smokers accountable for their actions. Adolescents' reports moved from their individual experience with ASPIRE to their active interactions with family members and friends and their attempt to persuade others to quit smoking. Future Web-based programs for adolescents may be designed with tailoring and game play in mind, in order to provide mobilization skills and foster social interactions against smoking. [ABSTRACT FROM AUTHOR]

The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker domains (L0 and L1) composed of highly charged sequences with multiple protein kinase A/C phosphorylation sites. In this report we made use of the scanning peptide approach to identify MRP1 linker L1 (L1 MRP1 ) interacting proteins. Scanning heptapeptides covering L1 MRP1 126 amino acids (Ile 846 - Leu 972 ) were synthesized and used in pull-down assays to isolate proteins from cell extracts (human multidrug resistant SCLC cell line; H69/AR). The results show four high affinity binding sequences in L1 MRP1 domain [ 866 FLRTYAST 867 ; 906 SAGKQLQRQLSSS 912 ; 925 ISRHHNSTA 927 and 954 AQTGQVKLSVYW 959 ] that bound ∼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and β-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and β-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1 MRP1 ), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1 MRP1 . Intriguingly, substitutions of serine residues in L1 MRP1 by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser 871, 915, 930, and 961 within L1 MRP1 may modulate its binding to tubulin. Taken together, the results of this study suggest possible interaction between MRP1 and tubulin that is modulated by phosphorylation of specific sequences in the L1 MRP1 domain. [ABSTRACT FROM AUTHOR]

P-Glycoprotein (or ABCB 1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of ∼90 amino acids, the ‘linker domain’ with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LD5617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIJVIKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of ∼80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of α- and β-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to α- and β-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells. [ABSTRACT FROM AUTHOR]

Abstract: The human ABCG2 protein, a member of ABC transporter family, was shown to transport anti-cancer drugs and normal cell metabolites. Earlier studies have demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report, we show that ABCG2 in mature human erythrocytes migrates with an apparent molecular mass of 140kDa, under reducing conditions, on Fairbanks SDS gel system. In contrast, tumor cells expressing higher levels of ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumor cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug-resistant cells but not in the parental drug-sensitive cells. Given the ability of ABCG2 to transport protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes. [Copyright &y& Elsevier]

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [125I]iodoaryl azido-rhodamine123 (IAARh123)—a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-β-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1–drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents. [Copyright &y& Elsevier]