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The development of drug addiction is characterized by molecular changes in brain reward regions that lead to the transition from recreational to compulsive drug use. These neurobiological processes in brain reward regions, such as the nucleus accumbens (NAc), are orchestrated in large part by transcriptional regulation. Our group recently identified the transcription factor E2F3a as a novel regulator of cocaine's rewarding effects and gene expression regulation in the NAc of male mice. Despite this progress, no information is available about the role of E2F3a in regulating cocaine reward at the sex- and cell-specific levels. Here, we used male and female mice expressing Cre-recombinase in either D1- or D2-type medium spiny neurons (MSNs) combined with viral-mediated gene transfer to bidirectionally control levels of E2F3a in a cell-type-specific manner in the NAc during conditioned place preference (CPP) to cocaine. Our findings show that selective overexpression of E2F3a in D1-MSNs increased cocaine CPP in both male and female mice, whereas opposite effects were observed under knockdown conditions. In contrast, equivalent E2F3a manipulations in D2-MSNs had no significant effects. To further explore the role of E2F3a in sophisticated operant and motivated behaviors, we performed viral manipulations of all NAc neurons in combination with cocaine self-administration and behavioral economics procedures in rats and demonstrated that E2F3a regulates sensitivity aspects of cocaine seeking and taking. These results confirm E2F3a as a central substrate of cocaine reward and demonstrate that this effect is mediated in D1-MSNs, thereby providing increased knowledge of cocaine action at the transcriptional level.

Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b ( Pde1b ), a Ca 2+ /calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Introduction: Recalling personal memories on the loss and deriving new meanings from them is deemed necessary for adapting to bereavement. Recent studies suggest that games can afford players meaningful experiences that can support players through stressful life events, but its potential on the meaning-making of loss has not been much explored. To address this gap, we investigated the bereaved players' experiences of playing commercial video games that elicited their personal memories of loss and what meanings they derived from those experiences., Method: Twelve adult players with bereavement experiences (six male, six female, age range: 20-31) played two video games (Bear's Restaurant and Spiritfarer). Their experiences during and after gameplay were tracked via play diaries and in-depth interviews. Data was analyzed using thematic analysis method., Results: We discovered seven themes on the meanings that players made from their gameplay experience: "Recalling memories", "Avoiding engagement with the pain", "Recognizing positive emotions", "Acknowledging the deceased's perspective", "Reviewing the meaning of loss", "Planning a better future", and "Fulfilling a wish". Our findings indicate that bereaved players recalled and related their autobiographical memories to their in-game experiences. Furthermore, they derived new meanings on both the loss and their post-loss life after playing video games., Discussion: We discuss how video games can provide a unique meaning-making experience to bereaved players by affording them an agency to actively reconstruct their narrative of loss and facilitating the sharing of grief., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Eum and Doh.)

In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly through the use of more powerful neuroproteomic approaches. However, mass spectrometry (MS)-based neuroproteomic studies of synapses still require cell-type, spatial, and temporal proteome information. With the advancement of sample preparation and MS techniques, we have just begun to identify and understand proteomes within a given cell type, subcellular compartment, and cell-type-specific synapse. Here, we review the progress and limitations of MS-based neuroproteomics of synapses in the mammalian CNS and highlight the recent applications of these approaches in studying neuropsychiatric disorders such as major depressive disorder and substance use disorders. Combining neuroproteomic findings with other omics studies can generate an in-depth, comprehensive map of synaptic proteomes and possibly identify new therapeutic targets and biomarkers for several central nervous system disorders.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This study tested an ecological model of resilience that illustrated the influence of COVID-19-related stressors (i.e., social and health stressors) and various socio-ecological factors at microsystem (i.e., parent-child conflicts and couple relationship) and exo-system levels (i.e., the utilization of community resources) on family functioning among Chinese families during COVID-19. An anonymous telephone survey was conducted using random sampling method. The sample contained 322 respondents who were co-habiting with their child(ren) and their partner. Hierarchical regression analysis and structural equation modelling were used to examine the differential impacts of various levels of factors and the model that were proposed. Results showed that 13.2% of the households were categorized as at-risk of poorer family functioning. Couple relationship and stressors significantly accounted for much of the variance in family functioning. While stressors had a significant direct effect on family functioning, couple relationship, but not parent-child conflicts or utilization of community resources, significantly mediated and moderated the impact of stressors on family functioning. The findings highlighted the impacts of both individual and ecological factors on family functioning under COVID-19. Importantly, cultural and contextual factors should be considered when adopting ecological model of resilience to examine family functioning in diverse cultural groups., Competing Interests: We have no known conflicts of interests to disclose., (© 2022 John Wiley & Sons Ltd.)

Ligand binding to G protein–coupled receptors (GPCRs), such as the α 2a -adrenergic receptor (α 2a AR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gβγ dimers. α 2a AR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gβγ effector. To understand how the Gβγ-SNARE complex underlies the α 2a AR-dependent inhibition of synaptic transmission, we examined the specificity of Gβγ subunits for the SNARE complex in adrenergic neurons, in which auto-α 2a ARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α 2a ARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gβ and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α 2a ARs, Gβ 1 and Gγ 3 interacted with the SNARE complex. However, Gβ 1 , Gβ 2 , and Gγ 3 were found in the complex when auto-α 2a ARs were activated by epinephrine. Further understanding of the specific usage of distinct Gβγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

Purpose: Hong Kong has experienced four waves of COVID-19 since the first case was confirmed in January 2020. Several studies have highlighted the psychological impacts of the outbreak in Hong Kong but have largely ignored the protective factors that contribute to resilience among vulnerable families. This study adopted an ecological resilience framework to explore the impact of this epidemic on members of families with youth with a delinquent tendency/mental health concerns and the ecological protective factors for these vulnerable families., Methods: Random sampling based on a sampling frame provided by one of the largest local social service organizations in Hong Kong led to the recruitment of 407 respondents who were interviewed using a battery of standardized questionnaires., Results: The results showed that 30.6% and 11.5% of respondents reported a moderate and a severe level of psychological distress, respectively, almost double the percentages reported in a previous study conducted in Hong Kong before the COVID-19 outbreak. Around 36.6% of respondents indicated they had encountered financial problems and almost 40% indicated aggravated financial circumstances since the outbreak. Hierarchical regression analysis revealed that financial stress was the strongest predictor of psychological distress. Structural equation modeling indicated that family support, indoor leisure activities and community resources significantly mediated the negative influence of COVID-19-related stressors on psychological distress of family members., Conclusion: Family leisure activities, family support, community spirit and mutual help within the context of social-distancing restrictions may need to be promoted to benefit vulnerable families in Hong Kong under the COVID-19 epidemic., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

While player characters (PCs) are the key element in engaging players in narrative games, the experience and relationship of the player with the PC have received scarce attention from the perspective of the subjective player experience. The diversity of players and the importance of the PC in the game suggests meaningful connections between how players relate to their PC and the resulting satisfaction with the game. We, therefore, investigated in this study how the player-character relationship influences satisfaction of the player with the game. We performed semi-structured in-depth interviews with 12 players of The Last of Us Part II , a game that has elicited highly polarized reactions in relation to how players responded to a switch of the PC in the game. Through thematic analysis, three themes were found, illustrating the connection between aspects of the player-character relationship and the overall game satisfaction. The themes are "Tolerance of forced character switch", "Malleability of character image" and "Flexibility of character attachment". We discuss how those findings should be taken into consideration when designing diverse and meaningful gaming experiences., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Erb, Lee and Doh.)

Background: The number of older adult gamers who play mobile games is growing worldwide. Earlier studies have reported that digital games provide cognitive, physical, and socioemotional benefits for older adults. However, current mobile games that understand older adults' gameplay experience and reflect their needs are very scarce. Furthermore, studies that have analyzed older adults' game experience in a holistic manner are rare., Objective: The purpose of this study was to suggest mobile game design guidelines for adults older than 50 years from a holistic gaming experience perspective. Adopting a human-centric approach, this study analyzes middle-aged and older adults' gameplay experience and suggests practical design guides to increase accessibility and satisfaction., Methods: We organized a living laboratory project called the "Intergenerational Play Workshop." In this workshop, 40 middle-aged and older adults (mean age 66.75 years, age range 50-85 years) played commercial mobile games of various genres with young adult partners for 1 month (8 sessions). Using a convergent parallel mixed-method design, we conducted a qualitative analysis of dialogue, game diaries, and behavioral observations during the workshop and a quantitative analysis of the satisfaction level of the game elements for the mobile games that they played., Results: This project was active from April 2019 to December 2021, and the data were collected at the workshops from July 1 to August 28, 2019. Based on the identified themes of positive and negative experiences from the qualitative data, we proposed 45 design guides under 3 categories: (1) cognitive and physical elements, (2) psychological and socioemotional elements, and (3) consumption contextual elements. Our empirical research could reaffirm the proposals from previous studies and provide new guidelines for improving the game design. In addition, we demonstrate how existing commercial games can be evaluated quantitatively by using the satisfaction level of each game's elements and overall satisfaction level., Conclusions: The final guidelines were presented to game designers to easily find related information and enhance the overall understanding of the game experience of middle-aged and older adults., (©Seyeon Lee, Hyunyoung Oh, Chung-Kon Shi, Young Yim Doh. Originally published in JMIR Serious Games (https://games.jmir.org), 20.05.2021.)

Studies over the past several decades have identified numerous epigenetic mechanisms associated with pathological states in psychiatric and neurological disease. Until recently, studies investigating chromatin-regulatory proteins, using overexpression or knockdown approaches, did not establish causal roles for epigenetic modifications at specific genes because these techniques typically affect hundreds or thousands of genomic loci. In this Review, we describe recent efforts in using locus-specific neuroepigenome editing in vivo to, for the first time, define causal relationships between a single chromatin modification at a specific gene in a defined cell population and downstream measures at the molecular, cellular, circuit and behavioural levels. We briefly introduce three epigenome-editing platforms: zinc-finger proteins, transcriptional activator-like effectors and clustered regularly interspaced short palindromic repeats (CRISPR). We then explore the development of in vivo neuroepigenome-editing tools and their applications to resolve epigenetic contributions to the pathophysiology of brain diseases. We also discuss technical considerations for in vivo neuroepigenome-editing experiments and ongoing innovations in the field, including new tools to investigate chromatin marks, manipulate chromatin topology and induce epigenetic modifications at multiple genes in the same cell. Lastly, we explore the potential clinical applications of in vivo neuroepigenome editing for treating brain pathology.

Background: Research on problematic internet use has focused on devising diagnostic criteria or describing the factors that influence internet overuse. However, a paradigm shift is necessary in studying the phenomenon of increased internet use not just from a pathological point of view but also from a developmental point of view that considers children's behavior of adapting to a technology-oriented society., Objective: In this paper, we propose the Cyclic Value-Context Reinforcement Model (CVCRM) to understand problematic internet use behavior. The purpose of our study was to construct a developmental process model that provides a holistic understanding of problematic internet use behavior of children and to empirically validate the proposed model by conducting a thematic analysis on actual counseling data., Methods: To validate the CVCRM, we conducted thematic analysis using the counseling data from 312 Korean children aged 7-18 years. For the coding process, 7 master's and doctoral student researchers participated as coders, and 2 professors supervised the coding process and results., Results: This project was funded from October 2015 to September 2019 to analyze counseling data from 312 children who participated in counseling sessions during January 2012 to May 2014. Based on the data analysis, we present the CVCRM, which integrates existing theoretical approaches and encompasses the 3 interacting aspects that induce and reinforce problematic internet use in children: psychosocial value, environmental context, and internet utility. Specifically, using counseling data, we empirically ascertained that problematic internet use behavior feeds into children's psychosocial values and environmental contexts, which in turn facilitates problematic internet use in a cyclical manner., Conclusions: Through this empirical validation, the CVCRM can provide a theoretical framework and an integrated perspective on the developmental mechanism of problematic internet use behavior of children., (©Young Yim Doh, Bugeun Kim, Seul Lee, Gahgene Gweon. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 14.07.2020.)

Although Korea has achieved successful economic, social, cultural, and technological development over the past decades, Korean people do not seem to be particularly happy. To enhance an individual's happiness, we need to be aware of what situations and environmental conditions are conducive for happiness and explore the values of happiness we pursue. This study investigated the types of happiness expressed by Korean people using a mixed-method approach. Personal in-depth ( n = 15) and focus group ( n = 16) interviews were conducted with people who reported feeling a high level of happiness. Happiness categorization was conducted using Q methodology ( n = 63). Subsequently, we surveyed 999 nationally representative samples of Korean adults to generalize the results of the Q analysis. The findings revealed seven types of adult happiness in Korea: (1) Self-actualization, (2) Belongingness, (3) Mission, (4) Social recognition, (5) Enjoyment, (6) Material success, and (7) Parenting. The combined results of the qualitative and quantitative analyses showed that in Korea, people pursuing money or social success feel the unhappiest, whereas people pursuing a mission or sense of belonging feel the happiest. In conclusion, we discussed the need for happiness literacy education, to provide each adult an opportunity to understand the type of happiness they pursue., Competing Interests: The authors declare no conflict of interest.

G protein-coupled receptors (GPCRs) that couple to G i/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca 2+ channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca 2+ entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABA B receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT 1b receptors and adrenergic α 2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by G i/o -coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α 2a -adrenergic receptors (α 2a ARs) in both adrenergic (auto-α 2a ARs) and non-adrenergic neurons (hetero-α 2a ARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α 2a ARs from mouse models including transgenic FLAG-α 2a ARs and knock-in HA-α 2a ARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α 2a ARs and hetero-α 2a ARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ 2 , Gγ 2 , Gγ 3 , and Gγ 4 with activated auto α 2a ARs, whereas we found Gβ 4 and Gγ 12 preferentially interacted with activated hetero-α 2a ARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.

Throughout the past five decades, tremendous advancements have been made in our understanding of G protein signaling and presynaptic inhibition, many of which were published in the Journal of Biological Chemistry under the tenure of Herb Tabor as Editor-in-Chief. Here, we identify these critical advances, including the formulation of the ternary complex model of G protein-coupled receptor signaling and the discovery of Gβγ as a critical signaling component of the heterotrimeric G protein, along with the nature of presynaptic inhibition and its physiological role. We provide an overview for the discovery and physiological relevance of the two known Gβγ-mediated mechanisms for presynaptic inhibition: first, the action of Gβγ on voltage-gated calcium channels to inhibit calcium influx to the presynaptic active zone and, second, the direct binding of Gβγ to the SNARE complex to displace synaptotagmin downstream of calcium entry, which has been demonstrated to be important in neurons and secretory cells. These two mechanisms act in tandem with each other in a synergistic manner to provide more complete spatiotemporal control over neurotransmitter release., (© 2019 Zurawski et al.)

Modulation of neurotransmitter exocytosis by activated G i/o coupled G-protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is the interaction between Gβγ and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAREs). There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR and the specificity to effectors, such as SNARE, in vivo are not known. Although less studied, Gβγ binding to the exocytic fusion machinery (i.e. SNARE) provides a more direct regulatory mechanism for neurotransmitter release. Here, we review some recent insights in the architecture of the synaptic terminal, modulation of synaptic transmission, and implications of G protein modulation of synaptic transmission in diseases. Numerous presynaptic proteins are involved in the architecture of synaptic terminals, particularly the active zone, and their importance in the regulation of exocytosis is still not completely understood. Further understanding of the Gβγ-SNARE interaction and the architecture and mechanisms of exocytosis may lead to the discovery of novel therapeutic targets to help patients with various disorders such as hypertension, attention-deficit/hyperactivity disorder, post-traumatic stress disorder, and acute/chronic pain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background and Aims: Past research on the classification of problematic Internet use (PIU) has focused on symptom-based severity and usage motive in order to understand its mechanism. Recently, usage context, such as family or social relationships, has been identified as a key influencing factor of PIU. Therefore, we extended the classification of PIU to include usage context in addition to symptom-based severity and usage motive., Methods: To classify PIU types, we conducted two studies. First, we performed a clustering analysis, where 265 counseling cases were clustered into usage types in terms of motive and context. After characterizing each usage type, we examined their hierarchical relationships by considering symptoms. Second, we performed a focus group interview with six counselors to increase the transferability of usage types. This transferability was established by matching counselors' quotations to the usage types. When usage types showed consistency between quotations and the clustering analysis, we identified the progression patterns between hierarchical relationships., Results: The clustering analysis of motive and context yielded six usage types with three hierarchical relationships. The focus group interview results verified the transferability of these six types and identified two progression patterns between the hierarchical relationships., Discussion and Conclusion: Although usage motive and context were given equal weight before the clustering analysis, the resulting types revealed that usage context played a greater role in the classification process.

Problems related to high levels of gaming and Internet usage are increasingly recognized as a potential public health burden across the developed world. The aim of this review was to present an international perspective on prevention strategies for Internet gaming disorder and related health conditions (e.g., Internet addiction), as well as hazardous gaming and Internet use. A systematic review of quantitative research evidence was conducted, followed by a search of governmental reports, policy and position statements, and health guidelines in the last decade. The regional scope included the USA, UK, Australia, China, Germany, Japan, and South Korea. Prevention studies have mainly involved school-based programs to train healthier Internet use habits in adolescents. The efficacy of selective prevention is promising but warrants further empirical attention. On an international scale, the formal recognition of gaming or Internet use as a disorder or as having quantifiable harms at certain levels of usage has been foundational to developing structured prevention responses. The South Korean model, in particular, is an exemplar of a coordinated response to a public health threat, with extensive government initiatives and long-term strategic plans at all three levels of prevention (i.e., universal, selective, and indicated). Western regions, by comparison, are dominated by prevention approaches led by non-profit organizations and private enterprise. The future of prevention of gaming and Internet problems ultimately relies upon all stakeholders working collaboratively in the public interest, confronting the reality of the evidence base and developing practical, ethical, and sustainable countermeasures.

Gβγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gβ and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gβ and Gγ subunits selectively interact to form unique Gβγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gβγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gβ and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gβ and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gβ and Gγ subunits and their subcellular localizations. For example, Gβ 1 was mostly localized at the membrane while Gβ 2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gβ and Gγ subunits may affect the Gβγ dimerization and Gβγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gβ and Gγ subunits but also new insights into the in vivo distribution of Gβ and Gγ subunits, and Gβγ dimer assembly in normal brain function.

Treatment services for Internet gaming disorder are becoming increasingly prevalent worldwide, particularly in East Asia. This international systematic review was designed to appraise the quality standards of the gaming disorder treatment literature, a task previously undertaken by King et al. (2011) prior to the inclusion of Internet gaming disorder in Section III of the DSM-5 and 'Gaming disorder' in the draft ICD-11. The reporting quality of 30 treatment studies conducted from 2007 to 2016 was assessed. Reporting quality was defined according to the 2010 Consolidating Standards of Reporting Trials (CONSORT) statement. The results reaffirmed previous criticisms of these trials, namely: (a) inconsistencies in the definition, diagnosis, and measurement of disordered use; (b) lack of randomization and blinding; (c) lack of controls; and (d) insufficient information on recruitment dates, sample characteristics, and effect sizes. Although cognitive-behavioral therapy has a larger evidence base than other therapies, it remains difficult to make definitive statements on its benefits. Study design quality has not improved over the last decade, indicating a need for greater consistency and standardization in this area. Continuing international efforts to understand the core psychopathology of gaming disorder are vital to developing a model of best practice in treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

G-protein βγ subunits (Gβγ) interact with presynaptic proteins and regulate neurotransmitter release downstream of Ca 2+ influx. To accomplish their roles in sensory signaling, photoreceptor synapses use specialized presynaptic proteins that support neurotransmission at active zone structures known as ribbons. While several G-protein coupled receptors (GPCRs) influence synaptic transmission at ribbon synapses of cones and other retinal neurons, it is unknown whether Gβγ contributes to these effects. We tested whether activation of one particular GPCR, a metabotropic glutamate receptor (mGluR), can reduce cone synaptic transmission via Gβγ in tiger salamander retinas. In recordings from horizontal cells, we found that an mGluR agonist (L-AP4) reduced cone-driven light responses and mEPSC frequency. In paired recordings of cones and horizontal cells, L-AP4 slightly reduced cone I Ca (∼10%) and caused a larger reduction in cone-driven EPSCs (∼30%). Proximity ligation assay revealed direct interactions between SNAP-25 and Gβγ subunits in retinal synaptic layers. Pretreatment with the SNAP-25 cleaving protease BoNT/A inhibited L-AP4 effects on synaptic transmission, as did introduction of a peptide derived from the SNAP-25 C terminus. Introducing Gβγ subunits directly into cones reduced EPSC amplitude. This effect was inhibited by BoNT/A, supporting a role for Gβγ/SNAP-25 interactions. However, the mGluR-dependent reduction in I Ca was not mimicked by Gβγ, indicating that this effect was independent of Gβγ. The finding that synaptic transmission at cone ribbon synapses is regulated by Gβγ/SNAP-25 interactions indicates that these mechanisms are shared by conventional and ribbon-type synapses. Gβγ liberated from other photoreceptor GPCRs is also likely to regulate synaptic transmission. SIGNIFICANCE STATEMENT Dynamic regulation of synaptic transmission by presynaptic G-protein coupled receptors shapes information flow through neural circuits. At the first synapse in the visual system, presynaptic metabotropic glutamate receptors (mGluRs) regulate cone photoreceptor synaptic transmission, although the mechanisms and functional impact of this are unclear. We show that mGluRs regulate light response encoding across the cone synapse, accomplished in part by triggering G-protein βγ subunits (Gβγ) interactions with SNAP-25, a core component of the synaptic vesicle fusion machinery. In addition to revealing a role in visual processing, this provides the first demonstration that Gβγ/SNAP-25 interactions regulate synaptic function at a ribbon-type synapse, contributing to an emerging picture of the ubiquity of Gβγ/SNARE interactions in regulating synaptic transmission throughout the nervous system., (Copyright © 2017 the authors 0270-6474/17/374619-17$15.00/0.)

Symptomatic laryngeal chondrometaplasia is rare. To the best of our knowledge, there are only few case reports on laryngeal chondrometaplasia. The imaging appearance of this uncommon disease is even more rarely described. There are only two case reports describing its appearances in computed tomography (CT) and magnetic resonance imaging (MRI). Ultrasound (US) features have not been reported so far. This case report is to show the US, CT and MRI features of this disease entity to stress the role of imaging in this disease.

Evaluating sites of protein-protein interactions can be an arduous task involving extensive mutagenesis work and attempts to express and purify individual proteins in sufficient quantities. Peptide mapping is a useful alternative to traditional methods as it allows rapid detection of regions and/or individual residues important for binding, and it can be readily applied to numerous proteins at once. Here we describe the use of the ResPep SL SPOT method to evaluate protein-protein binding interactions such as that between G-protein βγ subunits and SNARE proteins, identifying both regions of interest and subsequently individual residues which can then be manipulated in further biochemical assays to confirm their validity.

Object: Moyamoya disease (MMD) is a cerebrovascular occlusive disease affecting bilateral internal carotid termini. Smooth-muscle cells are one of the major cell types involved in this disease process. The characteristics of circulating smooth-muscle progenitor cells (SPCs) in MMD are poorly understood. The authors purified SPCs from the peripheral blood of patients with MMD and sought to identify differentially expressed genes (DEGs) in SPCs from these patients., Methods: The authors cultured and isolated SPCs from the peripheral blood of patients with MMD (n = 25) and healthy control volunteers (n = 22). After confirmation of the cellular phenotype, RNA was extracted from the cells and DEGs were identified using a commercially available gene chip. Real-time quantitative reverse transcription polymerase chain reaction was performed to confirm the putative pathogenetic DEGs., Results: The SPC-type outgrowth cells in patients with MMD invariably showed a hill-and-valley appearance under microscopic examination, and demonstrated high α-smooth muscle actin, myosin heavy chain, and calponin expression (96.5% ± 2.1%, 42.8% ± 18.6%, and 87.1% ± 8.2%, respectively), and minimal CD31 expression (less than 1%) on fluorescence-activated cell sorter analysis. The SPCs in the MMD group tended to make more irregularly arranged and thickened tubules on the tube formation assay. In the SPCs from patients with MMD, 286 genes (124 upregulated and 162 downregulated) were differentially expressed; they were related to cell adhesion, cell migration, immune response, and vascular development., Conclusions: With adequate culture conditions, SPCs could be established from the peripheral blood of patients with MMD. These cells showed specific DEGs compared with healthy control volunteers. This study provides a novel experimental cell model for further research of MMD.

Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ₁γ₁. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca²⁺ -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.

Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R(2):C(2)). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ(2):C(2) is different from its closest homolog, RIα(2):C(2). The full-length RIβ(2):C(2) crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R(1):C(1) heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling "foci" paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.

Object: Supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma are highly malignant embryonal brain tumors. They share morphological similarities, but differ in their differentiation patterns and global gene expression. The authors compared the expression of specific genes involved in neuroglial differentiation in supratentorial PNETs and medulloblastomas to define the distinct characters of these tumors., Methods: The mRNA expression of 8 genes (SOX2, NOTCH1, ID1, ASCL-1, NEUROD1, NEUROG1, NEUROG2, and NRG1) was evaluated in 25 embryonal tumors (12 supratentorial PNETs and 13 medulloblastomas) by quantitative real-time polymerase chain reaction. The expression levels of the transcripts of these genes were compared between the tumor groups. Activation of the JAK/STAT3 pathway was assessed by immunoblotting. Relative expression levels of STAT3 and phosphorylated STAT3 proteins were compared., Results: Supratentorial PNETs expressed significantly higher levels of SOX2, NOTCH1, ID1, and ASCL-1 transcripts, whereas the transcription of proneural basic helix-loop-helix factors, NEUROD1, NEUROG1 (significantly), and NEUROG2 (not significantly) was upregulated in medulloblastomas. The proportion of phosphorylated STAT3alpha relative to STAT3alpha was significantly greater in supratentorial PNETs than in medulloblastomas, indicating activation of the JAK/STAT3 pathway in supratentorial PNETs., Conclusions: These results indicate that supratentorial PNET predominantly has glial features and medulloblastoma largely follows a neuronal differentiation pattern. These divergent differentiation patterns may be related to the location and origin of each tumor.

Objective: To document the expression patterns of various matrixins, cytokines and angiogenic factors in plasma to assess their involvement in the pathogenesis of moyamoya disease (MMD)., Methods: This study included plasma samples from 20 MMD patients and nine healthy individuals. The plasma concentration of five matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-12), monocyte chemoattractant protein-1 (MCP-1), resistin, three interleukins (IL-1beta, IL-6, IL-8), tumour necrosis factor-alpha, vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor was determined using multianalyte profiling systems. The concentration of the tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) was measured using ELISA. Gelatin zymography for MMP-2 and MMP-9 was also performed., Results: MMD patients exhibited significantly higher plasma concentrations of MMP-9, MCP-1, IL-1beta, VEGF and PDGF-BB, and lower plasma concentrations of MMP-3, TIMP-1 and TIMP-2 compared with healthy controls. Significant correlations were found among MMP-9, MCP-1, VEGF, PDGF-BB and TIMP-2 in MMD patients., Conclusion: There were distinctive expression patterns of matrixins, cytokines and angiogenic factors in MMD patients, which seemed to correlate with disease pathogenesis. The balance between MMPs and TIMPs was disrupted in MMD and correlated with disease pathogenesis. Increased plasma levels of MCP-1 and VEGF in MMD patients may play a role in the recruitment of vascular progenitor cells and in the formation of collateral vessels.

Circulating endothelial progenitor cells (EPCs) play an important role in physiological and pathological neovascularization and may be involved in attenuating ischemic diseases. This study aimed to characterize circulating EPCs in moyamoya disease (MMD), one of the most common pediatric cerebrovascular diseases. Twenty-eight children with MMD prior to any surgical treatment and 12 healthy volunteers were recruited. Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured in endothelial cell growth medium. Temporal change of phenotype of cells was analyzed on days 0 and 7. The formation of EPC clusters was evaluated on day 7. The CD34(+), CD133(+), and KDR(+) cells, and the number of EPC clusters was significantly reduced in children with MMD. In controls, CD34(+) cells were significantly decreased on day 7 compared with day 0, but in MMD they were only slightly decreased. The change in KDR(+) cells on day 7 compared with day 0 was the reverse of that for CD34(+) cells. Functional assay of EPC demonstrated less tube formation and increased senescent-like phenotype in children with MMD. Analysis of the circulating EPCs of MMD children reveals decreased level and defective function. This study suggests that circulating EPCs may be associated with MMD pathogenesis., (Copyright 2009 Wiley-Liss, Inc.)

CKD-602 7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, belotecan) is a synthetic water-soluble camptothecin derivative and topoisomerase inhibitor that has been shown to have clinical anticancer effect against ovarian and lung cancer. We studied its anticancer effects on four human glioma cell lines, U87 MG, U343 MG, U251 MG and LN229. Cell viability was quantified by a modified 2-(2-methoxy-4-nitropheyl)-3-(4-nitropheyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and significant time- and dose-dependent cytotoxicity was observed in all cell lines. Susceptibility to CKD-602 at 48 h after treatment varied among the four cell lines and their IC50 value was as follows: 9.07 nM (95% CI 0.18-37.42) for LN229, 14.57 nM (95% CI 0.86-47.33) for U251 MG, 29.13 nM (95% CI 0.35-101.23) for U343 MG, and 84.66 nM (95% CI 34.63-148.25) for U87 MG. CKD-602 induced cell cycle arrest at G2 phase and produced antiproliferative activity and apoptosis in all cell lines. Thus, CKD-602 showed a significant anticancer effect on glioma cells in vitro and is a promising candidate for further studies on malignant gliomas.

Background: Characterization of a rare subgroup of glioblastoma patients who survive for more than 3 years (long-term survival glioblastoma, LTSGBL, patients) may be helpful to identify prognostic factors., Materials and Methods: A molecular-profiling proteomic approach using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify prognostic factors associated with glioblastoma by comparing frozen tumor tissue from LTSGBL patients with matched samples from short-term survival glioblastoma (STSGBL) patients. Western blot (WB) analysis, reverse-transcriptase polymerase chain reaction (RT-PCR) and immmunohistochemical (IHC) staining were used for confirmation., Results: Among most candidate spots identified by 2-DE, lack of overexpression of manganese superoxide dismutase (MnSOD) in LTSGBL samples was consistently observed using WB and RT-PCR., Conclusion: These results suggest that MnSOD expression level in tumor tissue is a candidate marker for the prognosis of glioblastoma patients., (Copyright 2009 S. Karger AG, Basel.)

We studied the expression of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) in microcystic meningiomas to investigate a possible underlying mechanism for the development of microcysts and of peritumoral edema, which are frequent characteristics of this rare subtype. Between October 1995 and June 2004, 10 of 19 patients who had histologically confirmed pure microcystic meningiomas were enrolled in the study. Six patients with meningothelial meningiomas, three with atypical meningiomas, and one with a transitional meningioma were included as a control group. Immunohistochemistry with paraffin blocks and real-time RT-PCR analysis for MMP-2, MMP-9, TIMP-1, TIMP-2 and vascular endothelial growth factor (VEGF) were performed using stored frozen tissues. Compared with the control group, MMP-9 was invariably and highly expressed in immunohistochemical staining of microcystic meningiomas. MMP-2, TIMP-1, TIMP-2 and VEGF were weakly expressed or not expressed in both microcystic and non-microcystic meningiomas. Real-time RT-PCR showed increased ratios of MMP-9 to TIMP-1 in microcystic meningiomas compared with the control group (55.855 +/- 106.353 vs. 1.858 +/- 2.575, respectively; p = 0.00). The expression of MMP-2 (0.72 +/- 1.20 vs. 2.54 +/- 3.01, p = 0.01) and TIMP-2 (1.22 +/- 1.67 vs. 1.61 +/- 1.82, p = 0.02) was higher in the control group. The results suggested that the increased ratio of MMP-9 to TIMP-1 might be associated with the formation of microcysts and peritumoral edema in microcystic meningioma.

Objective: The genes encoding tissue inhibitor of metalloproteinase (TIMP) 4 and TIMP2 span chromosomes 3p24.2-p26 and 17q25, respectively, which are the locations of familial moyamoya disease (FMMD) genes. We investigated single nucleotide polymorphisms of the TIMP2 and TIMP4 genes in FMMD patients to determine genetic predispositions., Methods: Eleven blood samples from FMMD patients were recruited. Controls included 50 blood samples from patients with nonfamilial moyamoya disease (MMD) and another 50 blood samples from non-MMD persons. We evaluated the promoter regions, exon-intron junctions, and the exons of the TIMP2 and TIMP4 genes by direct sequencing, and compared single nucleotide polymorphisms frequencies among the study groups., Results: A significantly higher frequency of a heterozygous genotype was found in the TIMP2 promoter region at position -418 in FMMD; that is, the G/C heterozygous genotype at position -418 was observed in nine of 11 patients with FMMD, in 16 out of 50 nonfamilial MMD control participants, and in 14 out of 50 non-MMD control participants (FMMD versus nonfamilial MMD: odds ratio, 9.56; 95% confidence interval, 1.85-49.48; P = 0.005; and FMMD versus non-MMD: odds ratio, 10.50; 95% confidence interval, 2.02-54.55; P = 0.001). This base at position -418 corresponds to the third base of the GAGGCTGGG sequence, an Sp1 binding site. Thus, changes in this position may influence Sp1 binding and subsequent transcription of the gene., Conclusion: Our findings suggest that the presence of a G/C heterozygous genotype at position -418 in TIMP2 promoter could be a genetic predisposing factor for FMMD.

The roles of IL-1alpha and TNF-alpha in early pulp inflammation were investigated by determining the alkaline phosphatase (ALP) activity, the osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) expression using an immunoblot method. Primary cultured dental pulp cells were treated with IL-1alpha, TNF-alpha, or both for 3, 7, and 14 days. The pulp cells treated with IL-1alpha for 3 days showed elevated ALP activity and increased ON, OC, and HO-1 expression, whereas TNF-alpha treatment did not increase the ALP activity and no BSP was expressed until day 14. The pulp cells treated with both IL-1alpha and TNF-alpha for 3 days showed increased HO-1 expression compared with that of the control. These data suggest that IL-1alpha and TNF-alpha produced in the early inflammatory reaction have different functions in human pulp cells. IL-1alpha induces ALP, ON, and OC in tooth mineralization and it may play a role in the cytoprotection of pulp cells via HO-1 expression, while long-term treatment of TNF-alpha may inhibit the tooth mineralization.

Radiation-induced glioblastoma multiforme (GBM) is a rare complication of radiotherapy. The authors report such a case occurring 10 years after treatment of cerebellar medulloblastoma. The patient was a 15-year-old boy who had undergone a gross-total removal of a medulloblastoma and received radiation therapy at the age of 5 years. He had experienced no tumor recurrences for 10 years until a new enhancing mass was found at the original site of the medulloblastoma. Following its resection the new lesion was found to be a GBM and there was no evidence of a medulloblastoma. The second tumor developed at the same site as the previous one after a sufficient latent period and fulfilled the criteria for a radiation-induced neoplasm. The original tumor cells expressed synaptophysin without p53 overexpression, a characteristic feature of medulloblastomas. In contrast, cells from the later tumor expressed glial fibrillary acidic protein and p53 but not synaptophysin. A sequence analysis of the p53 gene showed deletion at codon 233 and a C to G transition at codon 278 in the GBM but no mutation in the medulloblastoma. A GBM specimen revealed no amplification of the epidermal growth factor receptor compared with a normal control specimen. In conclusion, the clinical features of a radiation-induced GBM are similar to that of the primary GBM, whereas its genetic alterations render it a secondary GBM. These findings indicate that radiation-induced GBM should be considered a distinct clinical entity.

The management of ependymomas remains one of the most frustrating issues in pediatric neuro-oncology. Gross total resection is not always possible, and intensive chemotherapy and craniospinal radiotherapy have made no clear advances. Moreover, the chemoresistance of ependymomas may be explained by the expression of membrane transport molecule P-glycoprotein (P-gp). In this study the expression of cyclooxygenase 2 (COX-2) and the role of the specific COX-2 inhibitor NS-398 in growth and multi-drug resistance of ependymomas were investigated. COX-2 protein expression was assessed in 19 ependymomas immunohistochemically, and the effect of NS-398 on growth and multi-drug resistance was investigated using two primary cultured ependymoma cell lines. COX-2 protein expression was observed in 15 (79%) of the 19 ependymomas. NS-398 was found to reduce the proliferation of monolayer cell cultures in a dose- and time-dependent manner and to induce apoptosis and lower bcl-2 protein levels. After NS-398 treatment, Western blotting showed reduced P-gp expression and a rhodamine 123 efflux assay demonstrated a significant decrease in P-gp activity. Our findings demonstrate that COX-2 is overexpressed in ependymomas and that NS-398 is able to induce apoptosis and suppress P-gp expression and activity.

Cortical dysplasia (CD) is a well-recognized cause of intractable epilepsy, especially in children and is characterized histologically by derangements in cortical development and organization. The objective of this study was to expand the current knowledge of altered gene expression in CD as a first step towards in the identification of additional genes operative in the evolution of CD. Surgical specimens were obtained from eight patients (4 males and 4 females; age range 2-38 years; mean 15 years) with a pathologic diagnosis of CD. Nondysplastic temporal neocortex was obtained from a 2-year-old boy with intractable epilepsy and medial temporal lobe ganglioglioma. After total RNA isolation from frozen brain tissues, we carried out gene expression profiling using a cDNA expression array. Differences in gene expressions between CD and the nondysplastic neocortex were confirmed by semi-quantitative conventional reverse transcription-PCR. Three genes (recombination activating gene 1 (RAG1), heat shock 60 kDa protein 1 (HSP-60), and transforming growth factor beta1 (TGF beta1)) were found to be up-regulated more than two-fold in CD, whereas four genes (phosphoinositide-3-kinase regulatory subunit polypeptide 1 [p85 alpha] (PI3K), frizzled homolog 2 [Drosophila], Bcl-2/adenovirus E1B 19 kDa interacting protein (NIP3), and glia maturation factor beta (GMF beta)) were down-regulated to less than 50% of their normal levels. Interestingly, the majority of genes showing altered expression were associated with apoptosis. Our study demonstrates diverse changes in gene expression in CD. However, it remains to be shown which of these are causally related to the evolution of CD.

In gliomas, a high frequency of homozygous p16 gene deletions have been demonstrated, which are believed to be linked with malignant progression. The aim of this study was to assess the role of p16 in growth, invasion, and senescence. The human glioma cell lines U87 MG and U373 MG were transduced with Ad-p16, and cell viability was assessed by trypan blue staining. To examine the mechanism of cell growth inhibition, cell cycle analyses and annexin assays were performed. The invasive potential of Ad-p16 transduced cells was evaluated using a Matrigel invasion assay, and trimolecular complex (MMP-2/MT1-MMP/TIMP-2) synthesis was proven by zymography and Western blotting. To establish the link between p16 and cell senescence, we stained for Senescence-Associated beta-galactosidase activity. A cell proliferation assay demonstrated that Ad-p16 treatment significantly inhibits cell growth. Moreover, this cell growth inhibition was induced by cell cycle arrest, not by apoptosis. In vitro treatment of malignant glioma cells with Ad-p16 significantly decreased their invasive potential by Matrigel invasion assay. However, we were unable to demonstrate any differences in the constitutive productions and secretions of MMP-2, MT1-MMP, and TIMP-2, among the mock-treated, Ad-lacZ-transduced, and Ad-p16-transduced cells. p16 expression caused an enlargement of all cells, and these were morphologically similar to senescent cells. Staining for Senescence-Associated beta-galactosidase activity showed that the enlarged cells stained positively. Taken together these data strongly suggest that the anti-cancer effect of p16 is modulated by p16-mediated cell cycle arrest and by the induction of senescence.

Peritumoral edema (PTE) in meningioma occurs variably and can adversely affect the clinical course. Moreover, the etiology of PTE in meningioma is not well documented. To examine possible correlations with PTE, the authors investigated the clinical parameters and the expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in 20 meningiomas. The estimation of tumor volume (V(T)) and edema volume (V(E)) was done using Osiris software with magnetic resonance images and the edema index (EI) was calculated. The expression of VEGF, MMP, and TIMP were estimated in all 20 meningiomas by immunohistochemical staining, Western blotting, zymography, and laser densitometry. Tumor location was closely related with PTE. Meningiomas of the frontal lobe or the frontotemporal base had large PTEs, whereas those of the occipitoparietal lobe, posterior fossa or petroclivus were small. The level of VEGF expression bore correlation with the extent of PTE but not with histologic malignancy. MMP-2 and -9 were detected in 100% of meningiomas by zymography. The levels of MMP-9 were significantly elevated in moderate to severe edema (EI > 1.0) group (p < 0.05) whereas those of MMP-2 were elevated in minimal to mild edema (EI < 1.0) group. TIMP-1 and -2 were detected in 19 (95%) and 12 (60%) of meningiomas respectively and their presence had no significant correlations statistically with PTE between two groups (p = 0.190 and 0.089, respectively). Meningiomas with severe PTE expressed high levels of VEGF and MMP-9 and low levels of MMP-2. The expressions of MMP-2, -9, and TIMPs as well as VEGF in meningioma suggests that they are strongly related with the presence of PTE in meiningiomas, and that they might play the important role in the formation of PTE in meningiomas.

Object: Multiple gene replacements have been examined as a potential treatment modality for malignant gliomas. Nevertheless, no reports are available that detail the synergy, additivity, or antagonism of multiple genes. The aim of this study was to assess the interaction between p53 and p16 genes in the growth of glioma cell lines., Methods: The human glioma cell lines U87MG and U373MG were transduced using an adenoviral vector with Ad-p53, Ad-p16, or both. Western blotting was performed to determine the expression of the protein products of the transduced p53 and p16 genes. To establish whether the combination of Ad-p53 and Ad-p16 would be beneficial, the effects of gene combinations at the median inhibitory concentration level were analyzed using the isobologram method. Annexin assays and cell cycle analyses were performed on the transduced cells. Western blotting demonstrated the expression of p53 and p16 in transduced cells. Simultaneous exposure to Ad-p53 and Ad-p16 produced additive effects in both glioma cell lines. Experimental data points in U373MG lay near the Mode I line, indicating that the vectors had a different mode of action. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis, whereas in the wild-type p53 cell lines, the overexpression of wild-type p53 resulted in a moderate degree of apoptosis and G1 arrest. Furthermore, Ad-p16 induced more marked G1 arrest than Ad-p53 in cells with wild-type p53., Conclusions: The results show that interaction between Ad-p53 and Ad-p16 is additive, regardless of p53 gene status.