Epitope specific antibodies to N- and C cytoplasmic domains of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) differentiate native and post-translationally modified variant.

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.

The anti-estrogen receptor drug, tamoxifen, is selectively Lethal to P-glycoprotein-expressing Multidrug resistant tumor cells.

BACKGROUND: P-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), confers resistance to clinically relevant anticancer drugs and targeted chemotherapeutics. However, paradoxically P-glycoprotein overexpressing drug resistant cells are "collaterally sensitive" to non-toxic drugs that stimulate its ATPase activity. METHODS: Cell viability assays were used to determine the effect of low concentrations of tamoxifen on the proliferation of multidrug resistant cells (CHORC5 and MDA-Doxo400), expressing P-gp, their parental cell lines (AuxB1 and MDA-MB-231) or P-gp-CRISPR knockout clones of AuxB1 and CHORC5 cells. Western blot analysis was used to estimate P-gp expression in different cell lines. Apoptosis of tamoxifen-induced cell death was estimated by flow cytometry using Annexin-V-FITC stained cells. Oxidative stress of tamoxifen treated cells was determined by measuring levels of reactive oxygen species and reduced thiols using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 5,5-dithio-bis-(2-nitrobenzoic acid) DTNB, respectively. RESULTS: In this report, we show that P-gp-expressing drug resistant cells (CHORC5 and MDA-Doxo400) are collaterally sensitive to the anti-estrogen tamoxifen or its metabolite (4-hydroxy-tamoxifen). Moreover, P-gp-knockout clones of CHORC5 cells display complete reversal of collateral sensitivity to tamoxifen. Drug resistant cells exposed to low concentrations of tamoxifen show significant rise in reactive oxygen species, drop of reduced cellular thiols and increased apoptosis. Consistent with the latter, CHORC5 cells expressing high levels of human Bcl-2 (CHORC5-Bcl-2) show significant resistance to tamoxifen. In addition, the presence of the antioxidant N-acetylcysteine or P-gp ATPase inhibitor, PSC-833, reverse the collateral sensitivity of resistant cells to tamoxifen. By contrast, the presence of rotenone (specific inhibitor of mitochondria complex I) synergizes with tamoxifen. CONCLUSION: This study demonstrates the use of tamoxifen as collateral sensitivity drug that can preferentially target multidrug resistant cells expressing P-gp at clinically achievable concentrations. Given the widespread use of tamoxifen in the treatment of estrogen receptor-positive breast cancers, this property of tamoxifen may have clinical applications in treatment of P-gp-positive drug resistant breast tumors.

Knockout of P-glycoprotein abolish the collateral sensitivity of CHORC5 multidrug resistant cells.

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.

The phenothiazine, trifluoperazine, is selectively lethal to ABCB1-expressing multidrug resistant 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.

Epitope-specific IgG pools identify PfCRT monomer and homodimer polypeptides that are differentially phosphorylated at Ser411 in Plasmodium falciparum.

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.

Malaria Detection by Third-Harmonic Generation Image Scanning Cytometry.

Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsa-stained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.

Sequences in Linker-1 domain of the multidrug resistance associated protein (MRP1 or ABCC1) bind to tubulin and their binding is modulated by phosphorylation.

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 (L1MRP1) interacting proteins. Scanning heptapeptides covering L1MRP1 126 amino acids (Ile846- Leu972) 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 L1MRP1 domain [866FLRTYAST867; 906SAGKQLQRQLSSS912; 925ISRHHNSTA927 and 954AQTGQVKLSVYW959] that bound ∼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and ß-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and ß-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1MRP1), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1MRP1. Intriguingly, substitutions of serine residues in L1MRP1 by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser871, 915, 930, and 961 within L1MRP1 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 L1MRP1 domain.

From the Experience of Interactivity and Entertainment to Lower Intention to Smoke: A Randomized Controlled Trial and Path Analysis of a Web-Based Smoking Prevention Program for Adolescents.

BACKGROUND: Web-based programs for smoking prevention are being increasingly used with some success among adolescents. However, little is known about the mechanisms that link the experience of such programs to intended nicotine or tobacco control outcomes. OBJECTIVE: Based on the experiential learning theory and extended elaboration likelihood model, this study aimed to evaluate the impact of a Web-based intervention, A Smoking Prevention Interactive Experience (ASPIRE), on adolescents' intention to smoke, while considering the experience of interactivity and entertainment as predictors of reduced intention to smoke, under a transitional user experience model. METHODS: A total of 101 adolescents were recruited from after-school programs, provided consent, screened, and randomized in a single-blinded format to 1 of 2 conditions: the full ASPIRE program as the experimental condition (n=50) or an online , text-based version of ASPIRE as the control condition (n=51). Data were collected at baseline and immediate follow-up. Repeated-measures mixed-effects models and path analyses were conducted. RESULTS: A total of 82 participants completed the study and were included in the analysis. Participants in the experimental condition were more likely to show a decrease in their intention to smoke than those in the control condition (beta=-0.18, P=.008). Perceived interactivity (beta=-0.27, P=.004) and entertainment (beta=-0.20, P=.04) were each associated with a decrease in intention to smoke independently. Results of path analyses indicated that perceived interactivity and perceived entertainment mediated the relationship between ASPIRE use and emotional involvement. Furthermore, perceived presence mediated the relationship between perceived interactivity and emotional involvement. There was a direct relationship between perceived entertainment and emotional involvement. Emotional involvement predicted a decrease in intention to smoke (beta=-0.16, P=.04). CONCLUSIONS: Adolescents' experience of interactivity and entertainment contributed to the expected outcome of lower intention to smoke. Also, emphasis needs to be placed on the emotional experience during Web-based interventions in order to maximize reductions in smoking intentions. Going beyond mere evaluation of the effectiveness of a Web-based smoking prevention program, this study contributes to the understanding of adolescents' psychological experience and its effect on their intention to smoke. With the results of this study, researchers can work to (1) enhance the experience of interactivity and entertainment and (2) amplify concepts of media effects (eg, presence and emotional involvement) in order to better reach health behavior outcomes. TRIAL REGISTRATION: Clinicaltrials.gov NCT02469779; https://clinicaltrials.gov/ct2/show/NCT02469779 (Archived by WebCite at http://www.webcitation.org/6nxyZVOf0).

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum.

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum.

P-glycoprotein mediates the collateral sensitivity of multidrug resistant cells to steroid hormones.

The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings and show that drug resistant CHO cells are also collaterally sensitive to physiologic levels of progesterone (PRO) and deoxycorticosterone (DOC). Both PRO and DOC collateral sensitivities in CH(R)C5 cells are dependent on P-gp-expression and ATPase, as knockdown of P-gp expression with siRNA or inhibition of P-gp-ATPase with PSC833 reverses PRO- and DOC-induced collateral sensitivity. Moreover, the mitochondrial complexes I and III inhibitors (antimycin-A and rotenone, respectively) synergize with PRO and DOC-induced collateral sensitivity. We also show that VRP inhibits PRO and DOC collateral sensitivity, consistent with earlier findings relating to the VRP's modulation of PRO and DOC-stimulation of P-gp ATPase. The findings of this study demonstrate a P-gp-dependent collateral sensitivity of MDR cells in the presence of physiologically achievable concentrations of progesterone and deoxycorticosterone.

Developing a text-message library for tobacco prevention among adolescents: A qualitative study.

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.

Optimization of malaria detection based on third harmonic generation imaging of hemozoin.

The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin.

Identifying adolescents' gaming preferences for a tobacco prevention social game: A qualitative study.

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.

Human ABCC1 interacts and colocalizes with ATP synthase α, revealed by interactive proteomics analysis.

Human ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily, and its overexpression has been shown to cause multidrug resistance by active efflux of a wide variety of anticancer drugs. ABCC1 has been shown to exist and possibly function as a homodimer. However, a possible heterocomplex involving ABCC1 has been indicated. In this study, we performed an interactive proteomics study to examine proteins that bind to and form heterocomplexes with ABCC1 using coimmunoprecipitation and tandem mass spectrometry (MS/MS) analyses. We found that ATP synthase α binds to ABCC1 in plasma membranes with a ratio of 2:1. The ATP synthase α binding site in ABCC1 is located in the linker domain at the carboxyl core of ABCC1, and phosphorylation of the linker domain at the protein kinase A site enhances ATP synthase α binding. The interaction between ABCC1 and ATP synthase α in a heterocomplex may indicate a novel function of ABCC1 in regulating extracellular ATP level and purinergic signaling cascade.

Down-regulation of ABCB1 by collateral sensitivity drugs reverses multidrug resistance and up-regulates enolase I.

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.

Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue.

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).

Mobile Text Messaging for Tobacco Risk Communication Among Young Adult Community College Students: Randomized Trial of Project Debunk.

BACKGROUND: The 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. OBJECTIVE: Project 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. METHODS: With 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<.007 (.05 divided by 7). RESULTS: A total of 636 participants completed the baseline survey, were randomized to 1 of 8 conditions (between 73 and 86 participants per condition), and received messages from both campaigns. By the 2-month post campaign 2 assessment point, 70.1% (446/636) completed all outcome measures. By the end of both campaigns, participants had a significant increase in perceived NETP risk over time (P<.001); however, participants had a marginal increase in perceived CTP risk (P=.008). Separately for each group, there was a significant increase in perceived NETP risk among participants who received rational messages (P=.005), those who received emotional messages (P=.006), those who received simple messages (P=.003), and those who received gain-framed messages (P=.003). CONCLUSIONS: In this trial, YAs had an increase in perceived NETP risk. However, with stratification, we observed a significant increase in perceived NETP risk upon exposure to rational, emotional, simple, and gain-framed messages. In addition, YAs generally had an increase in perceived CTP risk and presented nonsignificant but observable improvement upon exposure to emotional, complex, and loss-framed messages. With the results of this study, researchers and practitioners implementing mobile health programs may take advantage of our tailored messages through larger technology-based programs such as smartphone apps and social media campaigns. TRIAL REGISTRATION: ClinicalTrials.gov NCT03457480; https://clinicaltrials.gov/ct2/show/NCT03457480. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/10977.

P-glycoprotein (ABCB1) modulates collateral sensitivity of a multidrug resistant cell line to verapamil.

P-glycoprotein (or P-gp1, ABCB1) expression in tumor cells is causative of multidrug resistance through the active efflux of drugs across the cell membrane. However, the over-expression of P-glycoprotein in some tumor cells has been associated with increased sensitivity, or "collateral sensitivity", of multidrug resistant cells to specific drugs, including the calcium channel blocker verapamil. We previously demonstrated that collateral sensitivity to verapamil correlates with the effect of this drug on P-gp1 ATPase, and is reversed by inhibitors of P-gp1 ATPase (e.g., PSC 833 and Ivermectin). In this report, we expand on our earlier study and demonstrate that P-gp1 expression in drug-resistant cells modulates collateral sensitivity. Using P-gp1-specific siRNA, P-gp1 expression in the multidrug resistant CH(R)C5 cells was significantly down-regulated beginning on day 2 post-transfection of siRNA. Furthermore, down-regulation of P-gp1 led to increased sensitivity of CH(R)C5 cells to paclitaxel and doxorubicin, but not to cis-platinum, due to inhibition of P-gp1 drug efflux pump. Down-regulation of P-gp1 expression completely reversed collateral sensitivity to verapamil. Moreover, known inhibitors of ETC, rotenone and antimycin A which cause an increase in reactive oxygen species, synergized with verapamil-induced collateral sensitivity leading to increased cell death as determined by MTT cell survival assay. Similarly, the addition of hydrogen peroxide also synergized with verapamil. Taken together, the results of this study demonstrate a direct link between P-gp1 expression and collateral sensitivity of drug-resistant cells, possibly due to an increase in reactive oxygen species.

Autofluorescence of condensed heme aggregates in malaria pigment and its synthetic equivalent hematin anhydride (beta-hematin).

The condensed crystalline phase of iron(III) protoporphyrin IX either isolated from parasite culture as malaria pigment (hemozoin) or synthetic equivalent hematin anhydride exhibits a solid-state autofluorescence characterized by an excitation maximum of 555 nm and an emission maximum of 577 nm. The excitation spectrum maximum at 555 nm corresponds to the Q(0,0) band in the absorption spectrum which represents the lowest singlet of the material. This suggests that the fluorescent emission is due to the heme condensed phase. The photoluminescence lifetime of tau(f) = 2.7 +/- 0.8 ns as measured at four wavelengths between 550 and 600 nm is in the range of Frankel exciton in porphyrinic condensed phases. The material is shown to have an optical band gap of 2.04 eV characteristic of a semiconductor. Luminescence is markedly dependent upon the degree of hydration and the emission does not seem to be caused by presence of zinc(II) protoporphyrin IX or free-base protoporphyrin IX in the lattice. The autofluorescence can be used for in vivo tracking of hemozoin, for determination of parasitemia levels, and for infection monitoring and possibly for drug screening studies.

What is pure hemozoin? A close look at the surface of the malaria pigment.

The malaria parasite, Plasmodium spp., produces hemozoin (Hz) crystals as a by-product of hemoglobin digestion. Purification methods used to remove host or parasite products adsorbed on Hz surface lead to variable and undetermined residues. This compositional variation likely accounts for the assortment of contradictory results in studies of Hz's biomineralization, immunomodulating properties, and the mechanism of action of some antimalarials. In this work, we study the surface of Hz cleaned with two methods, both reported in the literature, one stricter than the other. We find that biomolecules are adsorbed on Hz treated with either method, they bind through carboxylate groups, and may be present within Hz structure. Their composition and amount depend on the washing protocol, which also introduces contaminants. This finding led us to question the concept of "pure" Hz, and to propose x-ray photoelectron spectroscopy (XPS) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) as characterization tools to assess surface contamination prior to further work on Hz crystals.