Regulation of Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

Trained immunity induced by high-salt diet impedes stroke recovery.

A high-salt diet (HSD) elicits sustained sterile inflammation and worsens tissue injury. However, how this occurs after stroke, a leading cause of morbidity and mortality, remains unknown. Here, we report that HSD impairs long-term brain recovery after intracerebral hemorrhage, a severe form of stroke, despite salt withdrawal prior to the injury. Mechanistically, HSD induces innate immune priming and training in hematopoietic stem and progenitor cells (HSPCs) by downregulation of NR4a family and mitochondrial oxidative phosphorylation. This training compromises alternative activation of monocyte-derived macrophages (MDMs) without altering the initial inflammatory responses of the stroke brain. Healthy mice transplanted with bone marrow from HSD-fed mice retain signatures of reduced MDM reparative functions, further confirming a persistent form of innate immune memory that originates in the bone marrow. Loss of NR4a1 in macrophages recapitulates HSD-induced negative impacts on stroke outcomes while gain of NR4a1 enables stroke recovery in HSD animals. Together, we provide the first evidence that links HSD-induced innate immune memory to the acquisition of persistent dysregulated inflammatory responses and unveils NR4a1 as a potential therapeutic target.

Associations between Marketing Exposure, In-game Purchases, Problem Gaming, Simulated Gambling, and Psychological Distress among Adolescents.

This study examined the relationships between marketing exposure, in-game purchase, problem gaming, online simulated gambling game playing, and psychological distress. Data were obtained from a sample of 2,595 seventh-grade students from 30 middle schools in Taiwan. A self-administered questionnaire was conducted in 2020. The results indicated that 94% of adolescents engage in online gaming, with 38% making in-game purchases, and 9% playing online simulated gambling games. The multiple regression results showed that adolescents who are exposed to higher levels of gaming marketing, influenced by advertising effects, involved in in-game purchases, and have lower levels of active parental mediation were more likely to experience problem gaming. Adolescents who have increased exposure to gambling game marketing, are influenced by advertising effects, are involved in in-game purchases, and who are experiencing problem gaming were more likely to engage in online simulated gambling game playing and token purchasing. Involvement in in-game purchases, problem gaming, and playing online simulated gambling games were associated with higher levels of psychological distress and poor sleep quality. In conclusion, the results of this study link adolescents' exposure to marketing with their involvement in in-game purchases, problem gaming, and engaging in online simulated gambling.

Smartphone addiction and victimization predicts sleep problems and depression among children.

BACKGROUND: In this study we examined the phenomena of smartphone addiction, online harassment, and school bullying/victimization to predict the prospective influence these could have on the onset and persistence of sleep problems and depression among children. METHODS: Responses from 2155 fifth-grade children recruited from 30 primary schools in Taipei were assessed, and a follow-up was performed in the 6th grade. Self-administered questionnaires were collected for each year. FINDINGS: Children who reported smartphone addictions, online harassment, and school bullying/victimization coupled with an increase in those factors were more likely to experience the onset and persistence of sleep problems. In addition, children who reported smartphone addiction, online harassment, school bullying/victimization, and poor sleep quality were more likely to experience the onset and persistence of depression. IMPLICATIONS: School nurses or pediatric nurses should be able to assess children's Internet use and risks to understand potential influences on sleep quality and mental status and provide recommendations for children, parents and schools.

A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells.

Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with Dyn2 but inactivated in non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3ß) kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual- and triple-channel total internal reflection fluorescence microscopy, we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2 but not Dyn1 exhibits a pronounced burst of assembly, presumably into supramolecular collar-like structures that drive membrane scission and clathrin-coated vesicle (CCV) formation. Activation of Dyn1 by acute inhibition of GSK3ß results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation, and decreased CCP lifetimes but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME that occur well upstream of fission, even when present at low, substoichiometric levels relative to Dyn2. Under physiological conditions, Dyn1 is activated downstream of epidermal growth factor receptor (EGFR) signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoform-specific regulatory role for Dyn1 in CME.

Clinical outcomes of surgical resection versus radiofrequency ablation in very-early-stage hepatocellular carcinoma: a propensity score matching analysis.

BACKGROUND: The detection rate of Barcelona Clinic Liver Cancer (BCLC) very-early-stage hepatocellular carcinoma (HCC) is increasing because of advances in surveillance and improved imaging technologies for high-risk populations. Surgical resection (SR) and radiofrequency ablation (RFA) are both first-line treatments for very-early-stage HCC, but the differences in clinical outcomes between patients treated with SR and RFA remain unclear. This study investigated the prognosis of SR and RFA for very-early-stage HCC patients with long-term follow-up. METHODS: This study was retrospectively collected data on the clinicopathological characteristics, overall survival (OS), and disease-free survival (DFS) of 188 very-early-stage HCC patients (≤ 2 cm single HCC). OS and DFS were analyzed using the Kaplan-Meier method and Cox regression analysis. Propensity score matching (PSM) analysis was performed. RESULTS: Of the 188 HCC patients, 103 received SR and 85 received RFA. The median follow-up time was 56 months. The SR group had significantly higher OS than the RFA group (10-year cumulative OS: 55.2% and 31.3% in the SR and RFA groups, respectively). No statistically significant difference was observed in DFS between the SR and RFA groups (10-year cumulative DFS: 45.9% and 32.6% in the SR and RFA groups, respectively). After PSM, the OS in the SR group remained significantly higher than that in the RFA group (10-year cumulative OS: 54.7% and 42.2% in the SR and RFA groups, respectively). No significant difference was observed in DFS between the SR and RFA groups (10-year cumulative DFS: 43.0% and 35.4% in the SR and RFA groups, respectively). Furthermore, in the multivariate Cox regression analysis, treatment type (hazard ratio (HR): 0.54, 95% confidence interval (CI): 0.31-0.95; P = 0.032) and total bilirubin (HR: 1.92; 95% CI: 1.09-3.41; P = 0.025) were highly associated with OS. In addition, age (HR: 2.14, 95% CI: 1.36-3.36; P = 0.001) and cirrhosis (HR: 1.79; 95% CI: 1.11-2.89; P = 0.018) were strongly associated with DFS. CONCLUSION: For patients with very-early-stage HCC, SR was associated with significantly higher OS rates than RFA. However, no significant difference was observed in DFS between the SR and RFA groups.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

Crosstalk between Akt/GSK3ß signaling and dynamin-1 regulates clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3ß signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3ß accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3ß signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME.

A Portable Low-Power Acquisition System with a Urease Bioelectrochemical Sensor for Potentiometric Detection of Urea Concentrations.

This paper presents a portable low-power battery-driven bioelectrochemical signal acquisition system for urea detection. The proposed design has several advantages, including high performance, low cost, low-power consumption, and high portability. A LT1789-1 low-supply-voltage instrumentation amplifier (IA) was used to measure and amplify the open-circuit potential (OCP) between the working and reference electrodes. An MSP430 micro-controller was programmed to process and transduce the signals to the custom-developed software by ZigBee RF module in wireless mode and UART in able mode. The immobilized urease sensor was prepared by embedding urease into the polymer (aniline-co-o-phenylenediamine) polymeric matrix and then coating/depositing it onto a MEMS-fabricated Au working electrode. The linear correlation established between the urea concentration and the potentiometric change is in the urea concentrations range of 3.16 × 10(-4) to 3.16 × 10(-2) M with a sensitivity of 31.12 mV/log [M] and a precision of 0.995 (R² = 0.995). This portable device not only detects urea concentrations, but can also operate continuously with a 3.7 V rechargeab-le lithium-ion battery (500 mA·h) for at least four days. Accordingly, its use is feasible and even promising for home-care applications.

A dipole-assisted solid-phase extraction microchip combined with inductively coupled plasma-mass spectrometry for online determination of trace heavy metals in natural water.

We employed a polymeric material, poly(methyl methacrylate) (PMMA), for fabricating a microdevice and then implanted the chlorine (Cl)-containing solid-phase extraction (SPE) functionality into the PMMA chip to develop an innovative on-chip dipole-assisted SPE technique. Instead of the ion-ion interactions utilized in on-chip SPE techniques, the dipole-ion interactions between the highly electronegative C-Cl moieties in the channel interior and the positively charged metal ions were employed to facilitate the on-chip SPE procedures. Furthermore, to avoid labor-intensive manual manipulation, a programmable valve manifold was designed as an interface combining the dipole-assisted SPE microchip and inductively coupled plasma-mass spectrometry (ICP-MS) to achieve the fully automated operation. Under the optimized operation conditions for the established system, the detection limits for each analyte ion were obtained based on three times the standard deviation of seven measurements of the blank eluent solution. The limits ranged from 3.48 to 20.68 ng L(-1), suggesting that this technique appears uniquely suited for determining the levels of heavy metal ions in natural water. Indeed, a series of validation procedures demonstrated that the developed method could be satisfactorily applied to the determination of trace heavy metals in natural water. Remarkably, the developed device was durable enough to be reused more than 160 times without any loss in its analytical performance. To the best of our knowledge, this is the first study reporting on the combination of a dipole-assisted SPE microchip and elemental analysis instrument for the online determination of trace heavy metal ions.

The relationship between parental mediation and Internet addiction among adolescents, and the association with cyberbullying and depression.

OBJECTIVE: This study examined the relationships between parental mediation and Internet addiction, and the connections to cyberbullying, substance use, and depression among adolescents. METHOD: The study involved 1808 junior high school students who completed a questionnaire in Taiwan in 2013. RESULTS: Multiple logistic regression analysis results showed that adolescents who perceived lower levels of parental attachment were more likely to experience Internet addiction, cyberbullying, smoking, and depression, while adolescents who reported higher levels of parental restrictive mediation were less likely to experience Internet addiction or to engage in cyberbullying. Adolescent Internet addiction was associated with cyberbullying victimization/perpetration, smoking, consumption of alcohol, and depression. CONCLUSION: Internet addiction by adolescents was associated with cyberbullying, substance use and depression, while parental restrictive mediation was associated with reductions in adolescent Internet addiction and cyberbullying.

Correction: Children's mobile-gaming preferences, online risks, and mental health.

[This corrects the article DOI: 10.1371/journal.pone.0278290.].

PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins.

Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.

Adolescent Use of Dating Applications and the Associations with Online Victimization and Psychological Distress.

In this study, we examined the relationships between the use of online dating applications (apps), online victimization, and psychosocial distress among adolescents. This study was conducted in 2020. A sample of 2595 seventh-grade students from 30 Taiwanese middle schools was surveyed. We conducted a self-administered survey. Overall, 15% of the adolescents reported using online dating apps in the past year, while 78% reported having seen dating app advertisements on the internet in the past year. Multivariate analysis results indicated that adolescents' exposure to the marketing of dating apps and poor academic performance were both associated with the use of online dating apps. Adolescents who used dating apps were more likely to experience online privacy victimization, cyberbullying victimization, and online sexual harassment. The use of dating apps by adolescents, online privacy victimization, cyberbullying victimization, and online sexual harassment were associated with higher levels of depression, anxiety, and stress. In conclusion, adolescent use of dating apps is related to online victimization and psychological distress.

The Association of Influencer Marketing and Consumption of Non-Alcoholic Beer with the Purchase and Consumption of Alcohol by Adolescents.

In this study, we examined influencer marketing and consumption of non-alcoholic beer by adolescents to determine how these factors could affect the intentions of adolescents to purchase and drink alcohol. A total of 3121 high-school students recruited from 36 schools in Taiwan completed a self-administered questionnaire during the COVID-19 pandemic in 2022. The results indicate that 19% of these adolescents consumed non-alcoholic beer and 28% consumed alcohol in the past year. Multivariate analysis positively associated adolescents' exposure to influencer marketing with their purchase and consumption of non-alcoholic beer. Adolescents' exposure to influencer marketing of non-alcoholic beer combined with lower levels of parental restrictive mediation was associated with increased odds of the purchase and consumption of alcohol. For individuals who did not purchase alcohol in the past year, both the exposure to influencer marketing and the consumption of non-alcoholic beer were associated with intending to purchase alcohol in the future. Similarly, individuals who previously abstained from the consumption of alcohol, both the exposure to influencer marketing and the consumption of non-alcoholic beer were associated with intending to consume alcohol. In conclusion, when adolescents were exposed to influencer marketing of non-alcoholic beer they were more likely to consume it, which resulted in an increased likelihood that they would then purchase and consume alcohol.

Excessive Gaming and Online Energy-Drink Marketing Exposure Associated with Energy-Drink Consumption among Adolescents.

In this study, we examined excessive online gaming by adolescents and the resultant effects of their exposure to the online marketing of energy drinks and alcohol, and whether marketing literacy could serve as a mitigating factor. This cross-sectional study was conducted in 2020. Data were obtained from a sample of 2613 seventh-grade students from 30 middle schools in Taiwan. A self-administered questionnaire was conducted. The results showed that nearly 18% of the adolescent respondents had used energy drinks, while 75% reported seeing energy-drink advertisements on the internet in the past year. Multiple regression results indicated that factors such as being male, reporting excessive gaming, being exposed to higher levels of online energy-drink marketing, and reporting alcohol use were positively associated with energy-drink consumption. A higher level of online energy-drink marketing-affective literacy, however, was negatively associated with energy-drink consumption. In conclusion, factors that predicted energy-drink consumption among adolescents included excessive gaming and exposure to online energy-drink marketing, but marketing-affective literacy tended to lessen the impact of such advertising.

Children's mobile-gaming preferences, online risks, and mental health.

This study examined the relationships between children's mobile gaming preferences, online risks, and mental health. Data were obtained from a sample of 2,702 third and fourth grade students from 16 elementary schools in Taiwan and 9 schools in China. A self-administered questionnaire was used. The mental state of the children who participated in the study was assessed using the Strengths and Difficulties Questionnaire (SDQ), while mobile gaming addiction was assessed using the short form of the Internet Gaming Disorders Scale (IGDS9-SF). The results showed that about 54% of children played mobile games with others (multi-player), while 31% played mobile games alone, and 15% did not play mobile games. Multiple logistic regression results indicated that behaviors such as participating in multi-player games, playing violent games, a poor parent-child relationship, and living in a rural area were associated with a greater risk of mobile gaming addiction. Involvement in multi-player games, playing violent games, mobile gaming addiction, and exposure to mobile violence/pornography were associated with greater risks of cyber aggression/victimization. Multiple regression results showed that being a multi-player, playing violent games, mobile gaming addiction, exposure to violence/pornography, exposure to cyber aggression/victimization, and having a poor parent-child relationship were associated with emotional and behavioral problems.

Gain-of-"endocytic' function in mutant p53 cancer cells.

Beyond its well-known canonical function as a tumor suppressor, p53 is also involved in numerous cellular processes through altered transcription under both normal and pathological conditions. The functional diversity of p53 outputs is complex and dependent on cell context. However, the underlying mechanisms responsible for this diversity remain largely unclear. The emerging evidence of p53 mutations involved in regulating endocytic trafficking and signaling, in tandem to promote malignancy (invasion, exosome biogenesis and immune evasion), sheds light on possible mechanisms behind the p53-driven complexity. The interrelated nature of endocytic trafficking and receptor signaling that form dynamic and adaptable feedback loops - either positive or negative - functions to modulate multiple cellular outputs. Biasing the tunable endocytic trafficking and receptor signaling network by mutant p53 expands the purview of p53, allowing its contribution to diverse and aggressive phenotypes. In this review, we explore recent studies in which the novel role of mutant p53 in altering endocytic trafficking to bias receptor signaling and drive transforming phenotypes is revealed. Understanding the complex crosstalk of mutant p53, endocytic trafficking and receptor signaling will allow the development of therapies to selectively target p53-altered endocytic processes.

Parent-Child Discrepancies in Reports of Exposure to Violence/Pornography on Mobile Devices and the Impact on Children's Psychosocial Adjustment.

This study assessed the discrepancies between reports from parents and children concerning children's exposure to violence/pornography on mobile devices and the impact on the psychosocial adjustment of children. Data were obtained from a sample of 2,230 parent-child dyads recruited from 16 elementary schools (1,140 dyads) in Taiwan and 9 schools (1,090 dyads) in China. A self-administered questionnaire was used. The results showed that about 30 percent of children reported being exposed to violence on mobile devices. Approximately 70 percent of their parents were unaware of their child's exposure to violence on mobile devices. About 16 percent of children reported exposure to pornography on mobile devices, and 80 percent of their parents were unaware of this exposure. Multinomial logistic regression results showed that after controlling for parent and child sociodemographic variables, factors related to parental unawareness of child exposure to violence on mobile devices included a child's ownership of mobile devices, smartphone/tablet use time, a lower level of parental understanding, and a residence in China or in a rural area, whereas the parent-child relationship and a child's smartphone/tablet use time were associated with parents who were unaware of their child's exposure to pornography. Multiple regression results showed that children who were living with household poverty, had a poor parent-child relationship, spent much time using a smartphone/tablet, and with parents who were unaware of their exposure to violence/pornography on mobile devices were more likely to have emotional and behavioral problems.

Rapid identification of bloodstream bacterial and fungal pathogens and their antibiotic resistance determinants from positively flagged blood cultures using the BioFire FilmArray blood culture identification panel.

BACKGROUND/PURPOSE: Rapid and accurate identification of pathogens and their antibiotic resistance directly from flagged blood cultures can aid clinicians in optimizing early antibiotic treatment and improve the clinical outcomes, especially in settings associated with high rates of bloodstream infection caused by vancomycin-resistant Enterococci (VRE) and carbapenem-resistant Enterobacteriaceae (CRE). We compared the results of the BioFire FilmArray Blood Culture Identification (BCID) panel with those of conventional methods for identifying the pathogens and their antibiotic susceptibility status. METHODS: In total, 100 randomly selected positive blood cultures (BACTEC Plus Aerobic/F bottles or BACTEC Anaerobic Lytic/10 bottles) were analyzed. The pathogen detection efficiency of FilmArray BCID panel was compared with that of conventional method using MALDI-TOF MS system (Bruker MALDI Biotyper) and susceptibility testing by the Vitek 2 system. The sequencing analysis of antibiotic resistance genes was performed for discrepant results obtained from MALDI Biotyper and Vitek 2. RESULTS: Among the 100 positively flagged blood cultures, 94% of FilmArray BCID panel results were consistent with the MALDI Biotyper results. All five VRE isolates positive for vanA/vanB genes, 10 of 12 Staphylococcus species positive for mecA gene, and only one Klebsiella pneumoniae isolate positive for K. pneumoniae carbapenemase gene (blaKPC) detected in the FilmArray BCID panel were also concordant with results by the results by conventional susceptibility testing/molecular confirmation. CONCLUSIONS: The FilmArray BCID panel results not only demonstrated good correlation with conventional blood culture identification and susceptibility results but also provided results rapidly, especially for the early detection of MRSA, VRE and blaKPC-mediated CRE.