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Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand–receptor interactions, which may have important implications in cancer.

PurposeInfluencers market products for tobacco companies on social media. This is the first study to systematically examine leading cigar brands’ use of influencers on their brand Instagram pages.MethodsWe identified 24 leading cigar brands, using July 2017–June 2018 US retail data. We identified cigar brands that had official appearing Instagram pages, with at least one influencer in the past 20 posts. We coded characteristics of the past three posts from each of five brand pages that contained influencers, such as setting and what the influencer was doing. Finally, we described influencer characteristics.ResultsApproximately one-third of the 24 brands had official Instagram accounts with at least one influencer in the past 20 posts. We identified 28 influencers, typically people of colour from the hip-hop music industry, some with millions of followers. Influencers included Bella Thorne (@bellathorne), Shaquille O’Neal (@shaq) and T.I. (@troubleman31). Brands’ posts that contained influencers showed the influencer using/holding a product, wearing branded merchandise or appearing in photos with a brand watermark. Three brands’ pages posted sponsored event photos (ie, concerts and events using branded backgrounds).DiscussionCigar brands commonly use influencers to market their products on brand Instagram pages. Results are consistent with previous findings that cigar companies’ marketing may target younger African Americans and highlight the potential utility of education campaigns that similarly engage influencers.

Shape recognition in images represents one of the complex and hard-solving problems in computer vision due to its nonlinear, stochastic and incomplete nature. Classical image processing techniques have been normally used to solve this problem. Alternatively, shape recognition has also been conducted through metaheuristic algorithms. They have demonstrated to have a competitive performance in terms of robustness and accuracy. However, all of these schemes use old metaheuristic algorithms as the basis to identify geometrical structures in images. Original metaheuristic approaches experiment several limitations such as premature convergence and low diversity. Through the introduction of new models and evolutionary operators, recent metaheuristic methods have addressed these difficulties providing in general better results. This paper presents a comparative analysis on the application of five recent metaheuristic schemes to the shape recognition problem such as the Grey Wolf Optimizer (GWO), Whale Optimizer Algorithm (WOA), Crow Search Algorithm (CSA), Gravitational Search Algorithm (GSA) and Cuckoo Search (CS). Since such approaches have been successful in several new applications, the objective is to determine their efficiency when they face a complex problem such as shape detection. Numerical simulations, performed on a set of experiments composed of images with different difficulty levels, demonstrates the capacities of each approach.

BackgroundYouth and young adults frequently use social media and are susceptible to tobacco use. This study is the first to provide a systematic overview of how leading tobacco product brands use popular social media platforms.MethodsWe identified 112 leading brands of e-cigarettes, hookah, cigars, cigarettes and smokeless tobacco based on sales and self-report user data. We searched for each brand on six platforms: Instagram, Facebook, Twitter, YouTube, Pinterest and Tumblr. In early 2019, we conducted a content analysis of each page, coding: age restrictions, warning display, page characteristics and post characteristics.ResultsCigarette brands were generally not present. Most e-cigarettes, hookah and cigar brands had pages on at least two platforms. One-third of smokeless brands had pages on at least one platform. Few brands had pages on Pinterest and Tumblr. Most pages had existed for 3–5 years. Overall, brand pages rarely used age gating, did not display health warnings, generally posted images of a product alone and often used hashtags unrelated to tobacco. Brands commonly used special features like ephemeral posts on Instagram and pop-up chat windows on Facebook. Many pages displayed images of young people and mentioned flavour. Median followers per brand ranged from about 1 000–10 000, and total followers summed across brands reached over 5 million on Facebook and Instagram alone.ConclusionsLeading brands of most tobacco product types use social media extensively. Several findings identify issues related to youth exposure to and appeal of tobacco social media marketing. Findings can inform tobacco education efforts and regulation.

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes—ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

SignificanceMost US adults use smartphones for internet access. Understanding what they see when they view smartphone-optimised (mobile) tobacco websites is important, as it can inform tobacco education and cessation strategies. This study describes mobile tobacco websites for leading brands of cigarettes, cigars, smokeless tobacco (smokeless), e-cigarettes and hookah.MethodsWe identified 130 leading tobacco brands based on sales, advertising spending and self-report data. Of these, 62 brands had mobile websites. We conducted an inductive content analysis (ie, where we derived the coding scheme from what we observed) of website characteristics by dual-coding: age requirements, warning display, brand engagement methods (eg, social features) and sales strategies (eg, coupons).ResultsAll cigarette and most smokeless websites required age-verified accounts for entry, while 76% of e-cigarette websites required accounts only for making purchases. All cigarette and smokeless websites showed warnings, but a minority of e-cigarette and cigar websites did, and no hookah websites did. Many websites required users to scroll up to view warnings. Most e-cigarette websites, most hookah websites, and half of cigar websites linked to multiple social media platforms; however, most cigarette and smokeless websites facilitated socialisation internally. All cigarette, most smokeless and no hookah websites offered coupons. Many cigarette and smokeless coupons were time-sensitive and location-based.ConclusionsWe highlight issues in how tobacco brand websites address youth access, display warnings, engage consumers and facilitate purchase. Results can help public health educators and practitioners better understand tobacco marketing as a context for designing tobacco interventions.

PurposeTwo previous studies indicate that prosmoking apps might encourage smoking behaviour via smoking cues. The current paper seeks to build on these studies and provide an updated overview of the characteristics of tobacco industry-sponsored apps.MethodsIn November 2017, we identified 19 unique top-selling cigarette brands, 20 smokeless tobacco brands, 30 e-cigarette brands and 43 cigar brands based on Nielsen sales from 2016 Nielsen Scantrack data and 2016 Kantar advertising data from the Kantar Media Stradegy database. We searched for these brand-sponsored apps in the Google Play and Apple iTunes US online stores.ResultsWe identified four cigarette and one smokeless tobacco brand-sponsored apps on the Google Play store, but none in the Apple store. The apps sponsored by Grizzly, Newport, Skoal, Camel and Winston used the last four digits of the users’ social security number to verify age. The Marlboro app offered another option in addition to providing a partial social security number—providing a valid home address. The main feature of all apps was location-based, time-sensitive coupons. Some apps had additional functions such as additional detailed product information, interactive help menus and games.DiscussionThis paper provides an up-to-date description of apps that are sponsored by tobacco companies. Cessation interventions could consider reminding their target audience to delete these apps to support quit attempts.

•Machine learning and qualitative coding provide context to social media analysis.•Predicted Reddit user age groups allows nuanced comparisons on thematic topics by age group.•Opposition to flavor restrictions was prominent for both age groups.•Emergent themes by the age group 13–20 years were opposition to minimum age laws and flavored ENDS discussions.•Posts by the age group 21–54 years commonly mentioned general vaping use behavior.

Context: Approximately 22 people die each day in the United States as a result of the shortage of transplantable organs. This is particularly problematic among Spanish-dominant Hispanics. Increasing the number of registered organ donors can reduce this deficit.Objective: The goal of the current set of studies was to conceptually replicate a prior study indicating the lack of utility of a lone, immediate and complete registration opportunity (ICRO).Design and Setting: The study, a quasi-experimental design involving a total of 4 waves of data collection, was conducted in 2 different Mexican consulates in the United States. Guided by the IIFF Model (ie, an ICRO, information, focused engagement, and favorable activation), each wave compared a lone ICRO to a condition that likewise included an ICRO but also included the 3 additional intervention components recommended by the model (ie, information, focused engagement, and favorable activation).Participants: Visitors to the Mexican consulates in Tucson, Arizona, and Albuquerque, New Mexico, constituted the participant pool.Main Outcome Measure: New organ donor registrations represented the dependent variable.Results: When all 4 components of the IIFF Model were present, approximately 4 registrations per day were recorded; the lone ICRO resulted in approximately 1 registration every 15 days.Conclusion: An ICRO, without the other components of the IIFF Model, is of minimal use in regard to garnering organ donor registrations. Future studies should use the IIFF Model to consider how the utility of ICROs can be maximized.

Substrate stiffness, biochemical composition, and matrix topography deeply influence cell behavior, guiding motility, proliferation, and differentiation responses. The aim of this work was to determine the effect that the stiffness and protein composition of the underlying substrate has on the differentiation of induced pluripotent stem (iPS) cells and the potential synergy with specific soluble cues. With that purpose, murine iPS-derived embryoid bodies (iPS-EBs) were seeded on fibronectin- or collagen I-coated polyacrylamide (pAA) gels of tunable stiffness (0.6, 14, and 50 kPa) in the presence of basal medium; tissue culture polystyrene plates were employed as control. Specification of iPS cells toward the three germ layers was analyzed, detecting an increase of tissue-specific gene markers in the pAA matrices. Interestingly, soft matrix (0.6 kPa) coated with fibronectin favored differentiation toward cardiac and neural lineages and, in the case of neural differentiation, the effect was potentiated by the addition of specific soluble factors. The generation of mature astrocytes, neural cells, and cardiomyocytes was further proven by immunofluorescence and transmission electron microscopy. In summary, this work emphasizes the importance of using biomimetic matrices to accomplish a more specific and mature differentiation of stem cells for future therapeutic applications.

Burkholderia pseudomalleiis the etiological agent of melioidosis, a severe endemic disease in South-East Asia, causing septicemia and organ failure with high mortality rates. Current treatments and diagnostic approaches are largely ineffective. The development of new diagnostic tools and vaccines toward effective therapeutic opportunities against B. pseudomalleiis therefore an urgent priority. In the framework of a multidisciplinary project tackling melioidosis through reverse and structural vaccinology, BPSL1050 was identified as a candidate for immunodiagnostic and vaccine development based on its reactivity against the sera of melioidosis patients. We determined its NMR solution structure and dynamics, and by novel computational methods we predicted immunogenic epitopes that once synthesized were able to elicit the production of antibodies inducing the agglutination of the bacterium and recognizing both BPSL1050 and B. pseudomalleicrude extracts. Overall, these results hold promise for novel chemical biology approaches in the discovery of new diagnostic and prophylactic tools against melioidosis.

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1 in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the vein's integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin–expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.

The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the vein's integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin–expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprint (PMF) are one of the most powerful combined tool for protein identification. Frequently it is the method of choice as it is faster and less expensive than protein sequencing. However, sometimes PMF only allows the identification of a subset of peptides, requiring further MS/MS or Edman degradation to attain unequivocal protein identification. This work describes an approach that combines several matrices to improve the fidelity of the protein identification by MALDI-TOF MS. The matrices used for sample preparation are 2,5-dihydroxybenzoic acid (DHB), 2,6- dihydroxyacetophenone (DHAP), CHCA and 2,4,6-Trihydroxy acetophenone (THAP). We have also developed an algorithm called Matrix Assisted Protein Identification (MAPI) that processes Mascot datasets derived from each one of the four matrix preparations, integrating them and achieving a significant improvement in protein identification.

A proteome map of Mycoplasma penetrans has been constructed using two-dimensional gel electrophoresis in combination with mass spectrometry (MS). Mycoplasma penetrans infects the urogenital and respiratory tracts of humans. A total of 207 spots were characterized with MS and, in comparing the experimental data with the DNA sequence-derived predictions, it was possible to assign these 207 spots to 153 genes. The Pro-Q Diamond phosphoprotein dye technology was used for the fluorescent detection of 26 phosphoproteins in the 4−7 pH range. Keywords: proteomics • mass spectrometry • two-dimensional gel electrophoresis • phosphoproteome • genome annotation

In a total of 181 photographic spectrograms, obtained at the Manuel Foster Observatory of the Catholic University in Santiago (Chile), the equivalent widths of the absorption lines HeI 4471 and MgII 4481 have been determined for 10 selected southern Be stars and 4 normal B-type stars. The mean equivalent widths of each star are compared to published mean values from largers samples of B stars. On the average, Be stars coincide with normal B-type stars in their equivalent width. However, a striking difference between rapidly and slowly rotating stars in their line ratios (HeI/MgII) was found, which is probably an artifact due to a systematic misclassification of spectral types of rapidly rotating B stars (v sini>200 km s-1). Six of the ten Be stars show evidence for variability in HeI and/or MgII with time-scales shorter than a few days and amplitudes up to a factor 2 in equivalent width. Line variability occurs in the entire range of projected rotation velocity (70 km s-1-1), but seems to be restricted to spectral types B2-B4. In addition, variations at longer time-scales (years) were observed in two cases. Three of the variable stars (HR 4074, HR 4537, and a Ara) present correlated variations in both lines, one (µ Cen) anti-correlated variability. We interpret our results in terms of nonradial pulsations and stress the importance to study the HeI/MgII lines and their variability in a larger sample of Be, Bn, and normal B stars, including archival material in order to follow-up line variability for several decades in the past.

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma.

Amyloid β-protein (Aβ) toxicity is hypothesized to play a seminal role in Alzheimer’s disease (AD) pathogenesis. However, it remains unclear how Aβ causes synaptic dysfunction and synapse loss. We hypothesize that one mechanism of Aβ-induced synaptic injury is related to the cleavage of amyloid β precursor protein (APP) at position D664 by caspases that release the putatively cytotoxic C31 peptide. In organotypic slice cultures derived from mice with a knock-in mutation in the APP gene (APP D664A) to inhibit caspase cleavage, Aβ-induced synaptic injury is markedly reduced in two models of Aβ toxicity. Loss of dendritic spines is also attenuated in mice treated with caspase inhibitors. Importantly, the time-dependent dendritic spine loss is correlated with localized activation of caspase-3 but is absent in APP D664A cultures. We propose that the APP cytosolic domain plays an essential role in Aβ-induced synaptic damage in the injury pathway mediated by localized caspase activation.

The combination of a SMAC mimetic and a caspase inhibitor kills AML cells by inducing necroptosis.

ABSTRACTIn this study, we have characterized the functional properties of a novel Escherichia coliantigen named EsiB (E. colisecretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. colipathotypes revealed that esiBis preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site.IMPORTANCEPathogenic Escherichia coliinfections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. colihave not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. colito subvert the host immune response and avoid neutrophil-dependent clearance.

Neural stem cells persist after embryonic development in the subventricular zone (SVZ) niche and produce new neural cells during postnatal life; ependymal cells are a key component associated with this neurogenic niche. In the animal model of human hydrocephalus, the hyh mouse, the ependyma of the lateral ventricles is progressively lost during late embryonic and early postnatal life and disappears from most of the ventricular surface throughout its life span. To determine the potential consequences of this loss on the SVZ, we characterized the abnormalities in this neurogenic niche in hyh mice. There was overall disorganization and a marked reduction of proliferative cells in the SVZ of both newborn and adult hyh hydrocephalic mice in vivo; neuroblasts were displaced to the ventricular surface, and their migration through the rostral migratory stream was reduced. The numbers of resident neural progenitor cells in hyh mice were also markedly reduced, but they were capable of proliferating, forming neurospheres, and differentiating into neurons and glia in vitro in a manner indistinguishable from that of wild-type progenitor cells. These findings suggest that the reduction of proliferative activity observed in vivo is not caused by a cell autonomous defect of SVZ progenitors but is a consequence of a reduced number of these cells. Furthermore, the overall tissue disorganization of the SVZ and displacement of neuroblasts imply alterations in the neurogenic niche of postnatal hyh mice.