Four genes (HCN1-4) encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid in situ hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula  and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of Ih-mediated “sag” in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels.Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I HCN1-4) encode HCN channels that generate a non-selective cation conductance, Ih . Tissue for Western blots was taken from one Nestin-Cre male and one Nestin-Cre x HCN4-floxed male, aged P31 and P37, respectively. For electrophysiological studies, four Nestin-HCN4 knockout mice  aged P36 – P45 were used, together with four  Nestin-Cre mice aged P29 - P35 for controls.Experiments were carried out as approved by The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee and in accordance with the guidelines of the NHMRC of Australia Code of Practice for the Care and Use of Animals for Experimental Purposes. Male C57BL/6J mice aged P40 – P42 were used for RNAscopeMice were deeply anesthetized using isoflurane, decapitated, dissected to isolate thalamus-enriched tissue, and the tissues were stored at −80°C. The isolated tissues were lysed in RIPA buffer  and made to a final protein concentration of 5 μg/μl protein, 2 M Urea, and 1xSDS reducing loading buffer . Known p2⋅2H2O, 6 mM MgCl2⋅6H2O, 2.5 mM KCl, 1.25 mM NaH2PO4 and 26 mM NaHCO3). 300 μm thick coronal slices were cut using a vibratome (Leica VT1200 S). Slices were held in a holding chamber with artificial cerebrospinal fluid  and bubbled continuously with carbogen gas at 32°C for at least 15 min and then room temperature for at least 30 min before recording. Whole-cell recordings from the medial habenula were made using borosilicate glass electrodes (3–6 MΩ) filled with an internal solution comprising 125 mM K-gluconate, 5 mM KCl, 2 mM MgCl2⋅6H2O, 10 mM HEPES, 4 mM ATP-Mg, 0.3 mM GTP-Na, 10 mM phosphocreatine, 0.1 mM EGTA, and 0.2% biocytin; with a pH of 7.2 and osmolarity of 291 mOsm. Bridge balance and capacitance neutralization were applied. Neurons were maintained at a membrane potential of −50 mV with a 5 s −70 pA current injection used to elicit Ih-mediated “sag.” The start-to-start interval was 6.5–7 s. A response over 5–10 sweeps was averaged. Sag was calculated as the difference between the most hyperpolarized point within the first second of the average trace and the final “steady state” of this trace.Mice were deeply anesthetized using isoflurane and decapitated. Brains were rapidly removed and mounted in a slice chamber containing chilled cutting solution . Coronal brain slices were cut at 15 μm thickness using a cryostat  and mounted on positively charged microscope slides. Coronal sections were cut according to the Paxinos Mouse Brain Atlas (approximately bregma −1.70 mm) (® .The fresh-frozen brain was embedded in optimum cutting temperature (OCT) embedding gel (Tissue-Tek1.70 mm) . Cryosecin situ hybridization was performed using RNAscope® (ACD) (RNA e® (ACD) . Standar2O. Sections were air-dried for 5 min at room temperature and a hydrophobic barrier was drawn around each section using an ImmEdge Hydrophobic Barrier Pen . Sections were then incubated with protease pretreatment 4  for 30 min at room temperature and then washed twice in PBS  before being incubated for 2 h at 40°C with three Mus musculus (Mm) multiplexed probe combinations: (a) Mm-HCN4 (channel 1), Mm-HCN1 (channel 2), and Mm-Slc32a1 (channel 3); (b) Mm-HCN4 (channel 1), Mm-HCN2 (channel 2), and Mm-Slc17a6 (channel 3); and (c) Mm-HCN4 (channel 1), Mm-Slc17a7 (channel 2), and Mm-Slc32a1 (channel 3). For each combination, sections from a minimum of three animals were processed in independent experiments. After hybridization, sections were washed twice in wash buffer . Signals were amplified according to the protocol using four consecutive amplification steps with the fluorescent multiplex reagent kit . For amplification step 4, the fluorescent label combination AltB was used. Sections were then washed twice using wash buffer, after which the sections were stained with DAPI for 30 s, covered with ProLong Diamond antifade reagent  and cover slipped. Company supplied positive and negative controls were both used to confirm specificity of staining.Tissue was processed according to the protocol set out in the user manual. Briefly, sections were fixed in 4% paraformaldehyde (PFA) for 15 min at 4°C and dehydrated for 5 min each at room temperature and with increasing concentrations of ethanol  diluted in (RNase-free) diHThe sections were imaged on an 8 kHz resonant scanning confocal microscope using a pixel dwell time of 72 ns  with a U Plan Apo 40× oil immersion objective with a numerical aperture of 1.3. Complete coronal brain slices were captured using the tile scanning module and Z series were collected to compensate for focal drift. Images were acquired sequentially. Excitation (ex) and emission (em) spectra were set as follows: DAPI  on Photomultiplier Tube (PMT); Alexa 488  on Hybrid Detector (HyD); Atto 550  on HyD; and Alexa 647  on PMT. For quantification analysis, images were taken as single planes. To generate single images in https://doi.org/10.26180/5cde05da74ce0. For each extracted area a minimum of three coronal slices from three different animals was analyzed.For each coronal slice, areas from a single opitcal plane were extracted according to “The Mouse Brain in stereotaxic coordinates” and “The Allen Mouse Brain Reference Atlas” . The extt test .All RNAscope data were directly extracted from the macro code and are presented as individual values and mean values. Error bars represent the standard error of the mean (SEM). For electrophysiological data, a Shapiro-Wilk test was initially applied to test for normality, and returned a value of >0.05 in all groups. Statistical significance was then determined using an unpaired two-tailed Student’s in situ hybridization was used to map the expression of HCN4, HCN1 and HCN2 mRNA in a single brain plane  of DAPI-identified cells. In the same region, HCN2 mRNA was expressed in 59 ± 3% (n = 3 slices), while HCN1 expression was only seen in 0.9 ± 0.4% (n = 4 slices) of cells (n = 3 slices) co-expressed VGlut2 (n = 3 slices) of HCN4 mRNA-positive cells also expressed HCN2 mRNA  of cells. Using VGlut2 as a marker of excitatory thalamocortical neurons (n = 4 slices) of cells were found positive for VGlut2 mRNA in the thalamus. HCN2 mRNA was seen in 67 ± 3% (n = 4 slices), with HCN1 mRNA essentially undetectable . The vast majority of VGlut2 positive cells expressed HCN4 and HCN2 mRNA (n = 4 slices) and was always co-expressed with HCN2 mRNA  (n = 4 slices) of cells positive for HCN2 mRNA co-expressed VGlut2. This indicates that HCN2 is also expressed in cell types that are VGlut2- and HCN4-negative  . In contn = 7 slices) of cells were positive for HCN4 mRNA, suggesting very low expression in the NRT. Similarly, HCN1 expression was low, with only 0.5 ± 0.3% (n = 4 slices) cells being positive (n = 3 slices) were positive for HCN2 mRNA, indicating that this is the predominant HCN channel subtype expressed in the NRT (The presence of VGat mRNA and absence of VGlut1 and VGlut2 mRNA is consistent with the predominance of GABAergic neurons in the NRT . Only 1 positive . In cont the NRT .n = 4 slices) of cells were positive for HCN4 mRNA, but in these cells, expression was very high (n = 4 slices), while HCN1 expression was very low . HCN4 mRNA-positive cells mostly co-expressed HCN2 mRNA , while co-expression of HCN1 mRNA was essentially non-existent .The striatum expressed HCN4 mRNA at very low levels . Only 2 ery high . Furtherery high . These cery high , suggestery high . In the n = 7 slices) of cells positive. 78 ± 5% (n = 4 slices) of HCN4 mRNA-positive cells co-expressed VGat mRNA, indicating that these channels are predominantly expressed in GABAergic neurons in this region. However, 29 ± 3% (n = 4 slices) of VGat mRNA-positive cells co-expressed HCN4 mRNA, suggesting that HCN4 mRNA is only present in a subset of GABAergic neurons. In the GPe, HCN1 mRNA was also expressed at low levels , while HCN2 mRNA was expressed more widely . The majority of HCN4 mRNA-positive cells also expressed HCN2 mRNA, with a smaller subset co-expressing HCN1 mRNA (HCN4 mRNA was expressed at low levels in the GPe  with 8 ±CN1 mRNA .n = 3) slices expressed HCN4 mRNA, although it should be noted that this is only a rough estimate given the low expression.In general, HCN4 mRNA expression was very low and diffuse (not clustered) in the hippocampus, making quantitative analysis unreliable. We specifically looked at co-expression using the GABAergic marker, VGat, given that HCN4 channel expression has been reported in parvalbumin-positive neurons . ConsistHCN4 mRNA expression was above background in a number of other brain regions. However, in these regions the expression pattern was generally diffuse, making quantitative analysis unreliable. We have therefore only presented the HCN4 mRNA probe results in these regions without interpreting co-expression with other HCN channels or neuron markers. These regions include the somatosensory, auditory, piriform and retrosplenial cortices , and thein situ hybridization provides a powerful method to probe mRNA expression in tissue with improved specificity and sensitivity, as well as the ability to use multiple probes simultaneously (in situ techniques (Multiplex nucleic acid aneously . Here wechniques . Importain situ hybridization technique allows high resolution mapping of HCN mRNA, the method cannot report on protein levels or post-translational aspects of function. This is especially important given that HCN channels are subject to a number of different post-translational mechanisms in both physiological and pathological settings, resulting in biophysical and trafficking changes (A workflow based on a Fiji macro enabled the quantification of mRNA expression within cell bodies identified by the nuclear marker, DAPI. This provides a powerful tool for understanding mRNA expression profiles. However, there are some important caveats to this approach. First, the method only works in regions of the brain in which cell bodies are separated sufficiently to identify single cells. In regions such as the medial habenula and piriform cortex, the high density of cell bodies precludes such analysis at the resolution imaged here. Second, the method can only provide an estimate of mRNA expression in the defined cell body. This cannot take into account mRNA in other compartments such as dendrites and axons. Similarly, mRNA in dendrites and axons that cross the cell body may “contaminate” the signal. This analytical method therefore works best for situations where high mRNA expression is clustered at the cell body and should be avoided when mRNA expression is diffuse, as seen in the cortex and hippocampus for HCN4 mRNA. Furthermore, although the multiplex  changes . This sah is a critical modulator of neuronal excitability in the lateral habenula (h has a hyperpolarized activation voltage of ∼−110 mV in this region, which is consistent with a heteromeric channel containing both HCN4 and HCN2 isoforms (h underlies spontaneous tonic firing in cholinergic neurons suggesting HCN channels play an important role in defining excitability at least in a subset of medial habenula neurons (h in these neurons.HCN4 mRNA expression is high across both the lateral and medial habenula. Ihabenula . HCN4 mRisoforms . In the isoforms . At a ce neurons . Using wThe medial habenula can be subdivided, with distinct gene expression patterns reported for neurons in the superior-inferior, ventral-central, and lateral regions . Despiteh is significantly reduced in thalamocortical relay neurons of both HCN2 and HCN4 knockout mice (HCN4 mRNA was also highly expressed in the thalamus, the main integrator and relay of sensory information to the cortex. The thalamus can be subdivided into a large number of nuclei, but given that HCN4 mRNA expression was uniform across the thalamic sub-nuclei, we combined them for analysis. Most VGlut2-positive excitatory neurons were positive for HCN4 mRNA and co-expressed HCN2 mRNA. Consistent with this, Iout mice . This sth is important for autonomous pacemaker and spiking activity in these interneurons (HCN4 mRNA expression is high in a very select subset of cells in the striatum . These crneurons . HCN4 anrneurons  althoughHCN4 mRNA expression was low and diffuse in the hippocampus. HCN4 channel expression has been reported in parvalbumin-positive neurons in the adult rodent hippocampus . The expin situ hybridization technique. HCN4 channels are known to be expressed in other central nervous system regions not investigated here, including the olfactory bulb, the cerebellum, and the spinal cord (in situ hybridization technique in these regions are warranted. Furthermore, we focused our efforts on co-expression of HCN4 mRNA with only broad markers of excitatory and inhibitory neurons. Additional markers of cell types, for example cholinergic or more specific GABAergic neuron markers as well as glia markers would refine the knowledge of expression of this channel. We also have not explored the expression of HCN3 mRNA. The functional importance of this channel is becoming evident (in situ hybridization technique to map HCN3 mRNA expression is warranted. Finally, the multiplex in situ technique could be combined with immunohistochemical approaches to identify specific neuron compartments.This study focused on high-resolution imaging of a single brain slice plane that encompassed regions known to express HCN4 channels, and established a robust workflow to measure mRNA expression using the multiplex nal cord . Future  evident  and the in situ hybridization technique to improve our knowledge about which neuron types express HCN4 mRNA. Furthermore, we have identified in several brain regions which other HCN channel subtypes co-express with HCN4 mRNA. These results provide a morphological framework on which to better investigate the functional roles of these important channels.We have used the multiplex All datasets generated for this study are included in the manuscript and/or the supplementary files.The animal study was reviewed and approved by The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee and performed in accordance with the guidelines of the NHMRC of Australia Code of Practice for the Care and Use of Animals for Experimental Purposes.JO, SP, and CR conceived the study. JO, LB, KR, and AP completed the experimental work. CN wrote the Fiji code for analysis of imaging data. JO and LB completed the analysis of data. All authors contributed to the writing of the manuscript.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.


The use of video-recording offers important advantages in observing and assessing the relationships between specific behaviors in health care settings. The purpose of this systematic review is to investigate and synthesize the methodological characteristics of studies using video-technology for measuring interactions between the older persons with dementia and staff in long-term care facilities. We searched Medline, Embase and CINHAL databases for published articles in English using a video-recording method for both staff and persons with dementia. Quantitative research design studies  were included. Among 5,605 searched papers, a total of 20 studies were selected for this review. Situations of video-recording were providing personal care (n=12), mealtime (n=6), and conversation (n=3). Concepts of measuring by video-recording were classified into two groups: 1) Staff [care practice by staff (n=13) and communication by staff (n=11)] and 2) residents . This review demonstrates that video technologies are actively used to evaluate the relationship between quality of care and health outcomes of the elderly with dementia in many international nursing studies. This study provides the foundation for a future research using video-recording technologies to examine the interactions and relationship between staff and the residents in long-term care settings.


Significant advances have been made in elucidating the potential role of p21 in promoting tumorigenesis. Here, we discuss the involvement of p21 in multiple signaling pathways, its dual role in cancer, and the importance of understanding its paradoxical functions for effectively designing therapeutic strategies that could selectively inhibit its oncogenic activities, override resistance to therapy and yet preserve its tumor suppressive functions.p21 Se. Se20]. Cdc20) control the degradation of p21 in prometaphase, and thus promote the full activation of CDK1, which is needed for the progression of mitotic events [Skp2 and CRL4Cdt2 target p21 for degradation during S phase [Ubiquitin-dependent proteasomal degradation confers an important regulatory mechanism for p21 stability, and thereby for cell cycle progression control. p21 is targeted for ubiquitylation and degradation by multiple cell cycle-related ligases at different stages of the cell cycle . For exac events . Other s S phase ,48Ubiquitin-independent degradation of p21 has been also observed in a number of studies . It has Being a tumor suppressor, p21 is usually downregulated in many types of cancer and contributes to poor prognosis of patients and decreased overall survival ,38. p21-The decreased expression of p21 correlates with mutations in the genes that activate and repress CDKN1 transcription. These genes undergo gain of function or upregulation and loss of function or downregulation, respectively . Given tTbx2, which is amplified in some breast cancers, regulates senescence and oncogenesis, at least in part, through the regulation of p21 expression . HistoneThe interaction of p21 with PCNA mediates the ubiquitination and degradation of p21 by a component of the CRL4 E3 ubiquitin ligase family, known as CRLCDT2 or DTL in UV-irradiated cells and neoplastic cell lines ,67.Loss of cell cycle control promotes tumorigenesis and reflects a crucial hallmark of cancer . In partIn contrast, the overexpression of p21, together with an observed oncogenic activity, has been detected in many human cancers associated with poor prognosis, including breast, cervical, prostate cancer, gliomas, and acute myeloid leukemia (AML). It has been shown that Ras oncogene, which is frequently mutated and activated in human cancers, induces p21 promotor partly through the transcription factor E2F1 in a p53-independent fashion . OverexpGrowth stimuli sequentially activate CDKs to promote cell cycle. One of the major events that occur early during the cell cycle is pRb phosphorylation by cyclin D-CDK4/6. Phosphorylation of pRb is indispensable for the G1/S transition as it releases the sequestered E2F transcription factors and allows for the transactivation of genes important for the exit from G1 and the entry into S phase. CyclinE/CDK2 complex is activated to further phosphorylate pRb and ensure complete separation of E2F from the hyperphosphorylated pRb . CDK3 asThe role of p21 in cell cycle control is well established. p21 can directly inhibit cyclin-dependent kinase activities by interacting with their N-terminal domains or indirectly by interfering with the phosphorylation of CDK1 and CDK2 . p21 bin161 phosphorylation [Previous work has identified the involvement of p21 in G2 checkpoint control. It is important to note that p21 has low affinity for cyclin B/CDK1 in comparison to other cyclin/CDK complexes . Howeverrylation . p21 hasrylation .Studies on p21 deficiency highlight the role of p21 in normal mitotic processes. Loss of p21 results in centriole overduplication and consequently an abnormal increase in centrosome number in tumor cells . p21 is Nonetheless, it has been suggested that p21 can promote cell growth possibly by functioning as an adaptor protein that induces the assembly, nuclear translocation, and activation of cyclin D/CDK4 in human tumor cells, resulting in cell proliferation ,84. ThesMultiple studies shed light on the potential role of p21 in modulating apoptosis and tumorigenesis. p21 is a negative regulator of p53-dependent and p53-independnet apoptosis in cancer cells . SeveralDNA damage results in two scenarios: Apoptosis or p21-dependent cell cycle arrest. In cases where p53-dependent p21 induction is repressed or p21 protein is inactivated by caspase-mediated cleavage, p53-dependent apoptosis is the end result . Followic-Jun induces p53-dependent apoptosis partly by negatively regulating the association of p53 with p21 promotor. In contrast, c-Jun-deficient cells undergo p21-mediated cell cycle arrest in response to UV . InterleGartel and Tyner  reviewed2O2) can activate two different pathways: p53-dependent apoptosis and p53-dependent and -independent induction of p21 that protects cells from apoptosis. Nitric oxide and hyperoxia are known to induce p53-dependent and -independent activation of p21 and cell cycle arrest in macrophages, vascular smooth muscle cells, and HCT116 cancer cells. Interestingly, antisense p21 impairs nitric oxide-induced G1 arrest and sensitizes cells to apoptosis. Similarly, hyperoxia induces apoptosis in HCT116 cells, in the absence of p53 or p21. p21 seems to protect cells from oxidative stress, whereby it increases survival of mice, following hyperoxic lung injury [The effects of oxidative stress on p21 was also reviewed . Hydrogeg injury .p21 has been also shown to negatively regulate p53-independent apoptosis. Signals including TGF-β, TNF-α, IFN-γ, and histone deacetylase inhibitors may simultaneously induce both p53-independent apoptosis and p53-independent upregulation of p21 . TGF-β iInterestingly, p21 has a conflicting role in promoting apoptosis. This finding is based on several reports showing that p21 overexpression enhances apoptosis response to cisplatin in glioma cells and ovarian carcinoma cells in vitro . MoreoveEvidence from multiple studies indicates that p21 has a dual role in DNA repair processes ,105,106.The role of p21 in nucleotide excision repair (NER) regulation is controversial . Defectsp21 is also involved in double-strand break (DSB) repair pathway, whereby it colocalizes with proteins involved in DNA repair, such as Rad50, MRE11, and PCNA at DNA damage sites induced by heavy ions . p21 parIt has been reported that the downregulation of the major DNA repair factor histone H2AX is mediated by Her2/NeuT oncogene and involves the p21-CDK-Rb pathway, resulting in suppression of DNA repair while promoting DNA instability in Her2-positive cancer .Taken together, the abovementioned findings show a direct role of p21 in DNA repair processes via regulating PCNA interactions with multiple DNA repair factors. Others have suggested that p21 promotes DNA repair through inducing cell cycle arrest during low levels of DNA damage, whereas high levels of DNA damage result in p21 degradation and apoptosis induction . Thus, tmyc and cdc25A genes by binding to the promotors of these genes, after being recruited together with STAT3 and E2F in response to DNA damage. The recruitment of p21 is associated with downregulation of histone H4 acetylation and inhibition of p300 histone acetylase recruitment [p21 can have positive or negative effects on transcriptional regulation in response to DNA damage ,119,120.ruitment . p21 andruitment .cyclin B1 and PLK1 [It has been reported that p21 inhibition of the polo-like kinase 1 (PLK1) is mediated in part by two promotor elements, cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR), whereas the inhibition of topoisomerase IIα (TOPO IIα) promotor by p21 is mediated through CDE and not CHR. These DNA sequences are implicated in the regulation of cell cycle-dependent transcription and have been identified in the proximity of the initiation start site in the promotors of genes inhibited by p21, such as and PLK1 . CDE wasand PLK1 .p21 is a negative transcriptional regulator of Glucocorticoid-induced tumor necrosis factor receptor (GITR), which exerts anti-apoptotic functions in T lymphocytes and keratinocytes. By downregulating GITR, p21 enhances the UVB-induced apoptosis and response to DNA damage in skin keratinocytes .p21 can also positively regulate gene transcription mediated by several transcription factors. It enhances transcriptional activation by Estrogen receptor (ERα) through two mechanisms ,120. TheWnt gene transcription by binding to Wnt4 gene promotor in association with E2F-1 and inhibiting the recruitment of p300 to the promotor, resulting in histone hypoacetylation and transcription repression [p21 regulates transcription through a third mechanism that involves interaction with co-activators, such as p300 or CBP . It negapression . The binpression . p21 indpression . p21 uprpression .p21 regulates many other genes involved in cell division, senescence, and aging . ExampleIt has been also shown that p21 mediates inflammation suppression by reducing the expression of TNF-α and IL-1β in macrophages . In contAs part of the p53-DREAM pathway, p21 has been shown to indirectly downregulate many genes involved in DNA repair , apoptosis , serine/threonine kinase 17b (STK17B), and in cell cycle regulation  . Thus, pStudies on the role of p21 in cancer therapeutics report contradictory results regarding its role as a mediator of chemosensitivity or chemoresistance. Initial studies have shown that p21 inhibits initiator apoptotic caspase cleavage by TNF-related apoptosis-inducing ligand (DR4/TRAIL) receptor, thus suppressing apoptosis and enhancing survival of human breast and colon cancer cells . It has Tripterygium wilfordii, upregulated p21 expression and caused G1 phase arrest in colon cancer cells [Some anticancer drugs function partly through their ability to induce the expression of p21. For example, histone deacetylase (HDAC) inhibitors induce the expression of p21 through Sp1 binding sites in the p21 promotor, in a p53-independent manner. Trichostatin A promotes p21 expression, and consequently cell cycle arrest and apoptosis, in human gastric carcinoma, oral carcinoma, and multiple myeloma cell lines ,147. Theer cells . Similarer cells . Senesceer cells . In addier cells . BL is aer cells . UC2288 er cells . The useThe universal CDK inhibitor p21, which was previously thought to play a tumor suppressive role is now well recognized as an important two-faced molecule with remarkable oncogenic activities. This duplicity seems to be based on its role in induction/suppression of apoptosis, its subcellular localization, and is cell type dependent. In addition to controlling cell cycle and promoting cellular senescence, p21 functions as a positive and negative regulator of transcription in response to DNA damage. Given that p21 promotes its effects through the complex interaction with multiple players involved in tumorigenesis, the challenge lies in devising specific therapeutic agents that could inhibit the oncogenic activities of p21 without interfering with its tumor suppressor functions. Therapeutic strategies targeting cytoplasmic p21 may provide a new approach for the treatment of cancer and thus alleviate the resistance to chemotherapy.


RAE was investigated from the birth trimester of the athletes, their playing position, and final ranking in the Championship. The results showed an overrepresentation of athletes born in the first two trimesters (Q1 and Q2) (χ2(3) = 32.97; p < 0.001, ω = 0.29). The analysis of the athlete’s position showed that most wings (χ2(3) = 18.37; p < 0.001, ω = 0.32) and backs (χ2(3) = 12.51; p = 0.006, ω = 0.34) were born in the first trimesters (Q1 and Q2). The ranking in the Championship presented no significant association with the date of the birth (p > 0.05). The results showed the existence of the RAE in youth handball elite athletes, especially for the back and wing positions. However, the strategy of selecting is questionable once the presence of RAE was not associated with competitive success.This study aimed to verify the occurrence of the relative age effect (RAE) in male elite young handball athletes according to the playing position and its association with team performance in a World Championship. Data from 383 handball athletes from 24 countries who participated in the 7 In a sports context related to young athletes, annual age-grouping is a common strategy widely used to divide athletes into categories and competitive levels based on their year of birth . AlthougWattie et al.  proposedRelative age presents different effects depending on the characteristics of each sport ,7. AccorThe occurrence of RAE has been widely reported in sports where physical and anthropometrics attributes are important to performance, such as soccer ,16, baskAlthough RAE is widely investigated in sport, most studies have been conducted primarily to describe their presence and associated factors. In basketball, it has been reported that having older athletes in U16 and U18 may not be decisive for the best performance of teams in championships [Therefore, the aims of the present study were to verify the existence of RAE in young elite male handball athletes and to analyze if its occurrence is specified by the playing position and whether RAE is associated with successful team performance. Due to the characteristics and inherent demands of handball, we expected to find an RAE in young elite athletes (U-19). We also hypothesized that a large RAE would be revealed for the back position because physical size and strength are important determinants of success for specific actions in that function , thus, yth Handball Men’s Youth World Championship in the under-19 category (age 18.3 ± 0.7 years). A total of 24 countries  participated in the Championship organized by the International Handball Federation in Georgia in 2017.The sample was composed of 393 athletes of the 7http://www.ihf.info/). The book contains information about the results of the 7th Handball Men’s Youth World Championship in the under-19 category, team ranking, and team roster athletes . The information was used to verify the existence of RAE between the athletes to compare playing positions and analyze its association with the final team ranking in the Championship.Data were obtained from the official results book available from International Handball Federation on its webpage (st (quarters are composed as a function of this cut-off). The variables analyzed included the birth quarter of the athletes, that is, quarters of the year the athletes were born: Q1 , Q2 , Q3  and Q4 , the specific playing position of each athlete , and the team’s ranking in the championship (first to last position).The cut-off date for youth categories of national’s teams participating in the Handball Men’s Youth World Championship was January 12) of one variable  to describe the RAE of the participants and X2 2x2 test to analyze the association between the specific playing position and team performance. For all analyses, the effect size ω of the Chi-square tests was calculated according to Cobley et al. [Data was presented in absolute and relative frequency. The birth-date distribution of the athletes was compared performing a chi-square test  = 34.78; p < 0.001, ω = 0.30), with an overrepresentation of athletes born in the first semester (Q1 and Q2).2(3) = 19.19; p < 0.001, ω = 0.33) and backs (χ2(3) = 13.04; p = 0.005, ω = 0.37) were born in the first quarter. Conversely, the distribution of birth dates for the pivots and goalkeepers were evenly distributed (p > 0.05). The observed distribution was different according to the player’s position . Specifi2(3) = 8.02; p = 0.042, ω = 0.35), octaves-finalists (χ2(3) = 20.28; p < 0.001, ω = 0.40), and bottom eight teams (χ2(3) = 11.89; p = 0.008, ω = 0.30) (2(3) = 1.73; p = 0.629, ω = 0.16).Athletes born in the Q1 and Q2 were overrepresented in the quarter-finalists (χ = 0.30) . BirthdaOR results are presented in The aim of the present study was to verify the RAE in male handball athletes in the Under-19 World Men’s Handball Championship 2017 according to the playing position and team performance. The findings showed a skewed distribution of the birth dates in Under-19 handball athletes. Specifically, more athletes were born in the first two quarters of the year. A similar pattern was observed according to the playing position for the wings and backs. On the other hand, pivots and goalkeepers were more evenly distributed. Our results also showed that despite the RAE that was observed, considering all athletes by playing position, RAE was not associated with team performance . To the best of our knowledge, this is the first study to demonstrate a lack of association between RAE and competitive success in young elite handball athletes.As hypothesized, RAE was verified for the back position, corroborating previous studies with young athletes ,14. The The position analysis also showed RAE for the athletes who played at the wings. Different from the backs, who are characterized by bigger and stronger bodies, the wings are usually the smaller athletes in the team ,29, as wRegarding performance, we found an even distribution of the birth date for the semi-finalist teams, whereas in the other teams was observed an overrepresentation of athletes born in the first months of the year. This bias was already presented at lowest level participation in handball athletes with a decreasing effect over time, indicating that relatively younger athletes still manage to achieve a higher level of competition in the sports development system . The redIn handball, young athletes perform better according to their anthropometrics and physiological characteristics ,39. RAE In the teams of the Under-19 World Men’s Handball Championship 2017, we observed that RAE was not associated with final placement in the championship. Despite the skewed birth date distribution, teams with athletes born early in the year were not likely to perform better at the championship. Additionally, previous studies showed that RAE does not predict high-performance, selection for the national team, match outcome as well as it is not presented in some Olympic sports ,41,42,43One of the primary explanations for the RAE in sport is the role of physical maturation  . For insFrom a practical perspective, the results of the present study suggest that the RAE is ineffective in explaining the competitive success of national teams with young elite handball athletes. Thus, coaches are encouraged to reduce the role of the RAE on the early stages of selection processes of their athletes. Noteworthy, the only performance indicator investigated in the present study was the final ranking in the Under-19 World Men’s Handball Championship 2017. Thus, we consider the lack of other indicators  a limitation of the present study. Future studies are warranted to investigate the RAE on these performance indicators. However, the scientific literature has not supported the efficiency of the RAE at senior and professional level team national athletes ,36. LongIn conclusion, we verified the relative age effect in young elite handball athletes. Despite the tendency of RAE disappear as age increases, our findings evidence the permanence of this phenomenon even in an age group considered advanced. The process of selecting young elite athletes in handball seems to favor those born in the first half of the year. In addition, specific demands of playing position appear to act as a task constraint and affect the magnitude of RAE. Significantly larger RAE were observed for backs and wings compared with goalkeepers and pivots. Despite the RAE occurrence in young athletes’ selection, the supposed advantage of older athletes does not seem to be a preponderant factor for competitive success in team sports. We found no association between an overrepresentation of the athletes born in the first semester and the classification of the team in championships. These findings bring the current process of athlete selection for young age groups into question. Federations, clubs, and coaches should consider that RAE might be irrelevant for high performance and reflect on alternatives to avoid the exclusion of a potentially talented handball player due to a chronological limitation.


Autism spectrum disorder (ASD) presents certain hallmark features associated with cognitive and social functions, however, the ability to estimate self-generated distance and duration in individuals with ASD are unclear. We compared the performance of 20 ASD individuals with 20 typical developments (TDs) with respect to two tasks: (1) the drawing of a line of a specified distance (10 or 20 cm) and (2) waiting for a specified time (10 or 20 s). We observed that both the line distances and waiting times were substantially shorter in the ASD group than in the TD group. Furthermore, a trait of “attention to detail,” as measured by the Autism-Spectrum Quotient, correlated with some distance and duration productions observed in individuals with ASD. We suggest that attentional functions are related to the contraction of distance and duration in ASD. Initiating communication and responding to other people are challenging for individuals with ASD, and unawareness of social situations or blindness to those around them often results in confusion2. They exhibit atypical behavior associated with space and time, such as personal space3 and time management4.The ability to understand and utilize distance and duration are vital for coordinated action control. Expressive control of space and time is a critical tool for information sharing and communication with others in social activities. Autism spectrum disorder (ASD) is a developmental disorder that begins early in childhood and lasts throughout life5, gaze7, and language8, and these problems have been explained in terms of disruption of the social brain network, including the amygdala, superior temporal sulcus, fusiform area, and prefrontal cortex10. Furthermore, cerebro-cerebellar circuits, which bidirectionally connect the neocortex and cerebellum, are damaged in some individuals with ASD12, while there is a spectrum/heterogeneity in all individuals with an ASD13, suggesting that altered cerebellar connectivity may be linked to a lack of sociality.Individuals with ASD have a marked impairment in the recognition of facial expressions15. Individuals with ASD are also less able to perceive the duration of a tone accurately in a comparison process16. However, although individuals with ASD have difficulty perceiving space and time accurately, it is not known whether they have difficulty performing within specified space and time parameters. In other words, although they show perceptual impairments, it is not known whether they show corresponding sensorimotor impairments.Moreover, the perceptual level is atypical in ASD. People with ASD show a narrowing of spatial attention and are more apt to focus on details than on the whole17. Furthermore, ASD shows a restricted range of attention/interests18. An attentional function in ASD may be related to a contraction in both space and time productions.When subjects with a typical development (TD) are asked to draw a line of a specific length, say, 10 cm, most can do this rather accurately without requiring a ruler. We might see similar results for time production . However, if the impaired perceptual mechanism reflects the sensorimotor function in ASD, the individuals may show atypical productions of space and time. A further plausible conjecture is that the level of error may be related to the level of attention to detail because visual attention dilates the perceived duration of visual stimuli21. Furthermore, to identify autism-related factors associated with this ability, we conducted standardized tests of the ASD symptoms and compared the results with errors in the production of distance and duration.To test these ideas, we conducted behavioral experiments comparing the ability of individuals with ASD and TD to generate sensorimotor functions of specified distance and duration parameters. We used durations of 10 and 20 s because this allowed us to investigate consciously available behavior in the seconds range, unlike milliseconds or circadian scales. Previous research shows differences between TDs and individuals with ASD using such a time scale23.For the selected participants (see Methods), we conducted production tasks of distance and duration in both test and feedback sessions Fig. . Furthert tests revealed that the ASD group exhibited higher total scores than the TD group  Similarly, the ASD group scored higher than the TD group in the social skills , attention switching , attention to detail , communication skills , and imagination domains .The scores on the AQ were higher for the ASD group than in the TD group Table . The t tWe attempted the tasks shown in the representative videos  and session , as well as their interaction . The post hoc tests showed that the distances produced by the ASD group (Supplementary Video p < 0.0001). In the ASD group, the distances produced in the test session were shorter than those in the feedback session (Supplementary Video p < 0.0001), while in the TD group, there was no difference between the test and feedback sessions  and session , as well as their interaction . The post hoc tests showed that the distances produced by the ASD group (Supplementary Video p < 0.0001), and in the ASD group the distances produced in the test session were shorter than those produced in the feedback session (Supplementary Video p < 0.0001), while in the TD group there was no difference between the test and feedback sessions , no effect of session , and a significant interaction . The post hoc tests showed that in the test session the duration produced by the ASD group (Supplementary Video p < 0.0001). In the ASD group, the duration produced in the test session was shorter than that in the feedback session (Supplementary Video p < 0.0001). In the TD group, the duration produced in the test session was longer than that in the feedback session (Supplementary Video p < 0.0001). Similarly, in the “T20” condition , no effect of session , and a significant interaction . The post hoc tests showed that in the test session the duration produced by the ASD group (Supplementary Video p < 0.0001). In the ASD group, the duration produced in the test session was shorter than that in the feedback session (Supplementary Video p < 0.0001). In the TD group, the duration produced in the test session was longer than that in the feedback session (Supplementary Video p < 0.0001).In the duration production task Fig. , the ASDion Fig. , the ANOt tests showed no significant differences between the S10 and S20 tasks of distance in the ASD and TD groups. Similarly, the t tests showed no significant differences between the T10 and T20 tasks in the ASD and TD groups , to test magnitude parameters as continuous variables. However, unpaired p < 0.05), and in the ASD group the velocity of the feedback session was greater than that of the test session (p < 0.0001). In the S20 condition, the tests showed that the velocity of the ASD group was greater than that of the TD group in the feedback session (p < 0.001), and in the ASD group the velocity of the feedback session was greater than that of test session (p < 0.0001).Furthermore, as a supplemental analysis of motor function, we analyzed the pen velocity in the distance tasks . In the ASD group, the “attention to detail” subscore of the AQ negatively correlated with the “S10”  and “T10” , while the score did not correlate with “S20”  and “T20”  conditions. The total scores and other subscores showed no significant correlation for any of the conditions . The results revealed significant correlations between S10 and T10  and S20 and T20  in the ASD group  was applied to the correlation analyses 20, while distance production is normal in PD21. In the case of PD, it is thought that the abnormal timing is due to the disruption of a striatum-centered neural network26; however, it has been unreported in ASD. In contrast, the cerebro-cerebellar circuits are damaged in some individuals with ASD13, and the cerebellum has been associated with sensorimotor functions including a control/modification of voluntary limb movements29. Higher brain dysfunction due to cerebellar deficits is known as cerebellar cognitive affective syndrome (CCAS)31. Individuals with ASD exhibit CCAS and also exhibit a dysmetria, which is an inability to stop a voluntary movement32. It remains possible that the contraction of distance and time duration in the current study may be caused by CCAS.Studies of spatial perception have demonstrated connections from the visual to parietal and then to the prefrontal cortices34. Applied to our results, strong attention to detail may cause compression during production of distance and duration. Another explanation relates to atypical conceptualization. The literature on spatial association during number processing is dominated by the effects of spatial-numerical associations of response codes36. People with ASD may fail to accurately perform digital-analog conversions, or more specifically, they may underestimate the distance/duration during the conversion due to enhanced attention to small numbers. Furthermore, the current study revealed the correlation between distance and duration in the ASD group  were lower than the short scale productions (S10 and T10). These results may reflect different types of processing levels. Second, another subscale of AQ  showed no relationship with the perceived distance and time duration. This may be dependent on the task demands. For instance, it appears that the subscores of the social skills, communication skills, and imagination reflect a social activity related to personal relationships. The subscore of attention switching may also be near a suppression function, and it may be correlated to a response inhibition task. Perhaps, the attention to detail subscore has a high affinity for behavioral tasks related to spatial and temporal representations. The third limitation is that we did not control for medication. Some individuals on medication were studied while medicated, because the majority of individuals with ASD suffered from restlessness and had difficulty managing the stylus pen under the drug Off condition. The number of subjects in our study was also too small to reveal a medication effect, while an additional analysis, removing 7 individuals on medication, showed that the tendency in the reduced group was similar to that of the non-reduced group. A sufficient number of samples in the On/Off states is required to determine the effects of medication because antipsychotics inhibit inappropriate behavior but can adversely affect various cognitive functions41. Finally, the current study investigated the spatial/temporal characteristics in ASD from the aspects of production not perception. Individuals with ASD show a restricted range of attention/interests18. A passive task, such as a bisection task or attentional task, may reveal further insights into the impairment of production in individuals with ASD.A limitation of our study was that we used restricted measurement ranges in terms of durations of 10 and 20 s and distances of 10 and 20 cm. With particular reference to the time scale, the processing level in the brain varies based on the time scalesWe found that individuals with ASD under-produced distances and time durations. This is a novel characteristic of ASD. Correct production of distance and duration may play a mediating role in social interactions, and deviations may participate in disrupting the communication ability in ASD. This easy-to-use experimental paradigm may be helpful in providing a diagnosis of ASD. Furthermore, a modification towards normalization of distance and duration production may contribute to an improvement of social communication ability in individuals with ASD.This study was approved by the Ethics Committees of Showa University Karasuyama Hospital  and was conducted according to the Principles of the Declaration of Helsinki. All participants provided written informed consent.42. The diagnosis of ASD was based on a consensus reached by two psychiatrists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR). Two detailed interviews were conducted independently by a clinical psychologist. Criteria included the participants’ developmental history, present illness, past history, and family history. The initial sample size included 40 individuals. From this group, 20 individuals uncomplicated with other diseases (Attention-Deficit Hyperactivity Disorder) were selected to participate in this study. Furthermore, Wechsler Adult Intelligence Scale-III43 , to ensure the high functionality of individuals with ASD. Seven were routinely prescribed antidepressant medications, and participated in behavioral experiments in the drug On condition, under which medicines were being administered. Twenty TDs were randomly selected by matching the average age, sex distribution, and schooling history of the ASDs. Therefore, we conducted the experiment on the basis that the IQs of the TDs are near average on a common distribution. There were no significant differences in education between the TD and ASD groups, and all participants were right-handed  by holding a stylus pen above an electronic tablet . At the beginning of each trial, the distance or duration to be produced was verbally stated by the experimenter, and the start of the production task was self-paced for each participant. The participants hovered the stylus over a point when moving the stylus towards the right. The distance and time duration were measured between the two points.In the production task of distance Fig. , the parAs a supplemental analysis of motor function, we analyzed the velocity of the distance task. The velocity was calculated by dividing distance (cm) by duration (s) in both the test and feedback conditions. The tasks in the feedback condition were simpler than those in the test session, so we assumed that the feedback session was appropriate for assessing sensorimotor function.t tests were performed for clinical assessments and for medication effects. The distance and duration data were analyzed separately. In addition, the 10/20 cm and 10/20 s conditions were analyzed independently. The results are shown as the mean ± standard error of mean and effect size . Furthermore, the FDR was applied to the correlation analyses.Repeated measures ANOVA and post hoc tests with Bonferroni correction were performed for the behavioral data, and unpaired Supplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7Supplementary Video 8Supplementary Video 9Supplementary Video 10Supplementary Video 11Supplementary Video 12Supplementary Video 13Supplementary Video 14Supplementary Video 15Supplementary Video 16Supplementary Info


On the cellular and tissue levels, maintaining Mg2+ within optimal levels according to the biological context, such as cell types, developmental stages, extracellular environments, and pathophysiological conditions, is crucial for development, normal functions, and diseases. Hence, Mg2+ is pathologically involved in cancers, diabetes, and neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and demyelination. In the research field regarding the roles and mechanisms of Mg2+ regulation, numerous controversies caused by its versatility and complexity still exist. As Mg2+, at least, plays critical roles in neuronal development, healthy normal functions, and diseases, appropriate Mg2+ supplementation exhibits neurotrophic effects in a majority of cases. Hence, the control of Mg2+ homeostasis can be a candidate for therapeutic targets in neuronal diseases. In this review, recent results regarding the roles of intracellular Mg2+ and its regulatory system in determining the cell phenotype, fate, and diseases in the nervous system are summarized, and an overview of the comprehensive roles of Mg2+ is provided.Magnesium (Mg) is the second most abundant cation in mammalian cells, and it is essential for numerous cellular processes including enzymatic reactions, ion channel functions, metabolic cycles, cellular signaling, and DNA/RNA stabilities. Because of the versatile and universal nature of Mg The inorganic chemistry of Mg plays a key role in the first chemical processes, which lead to the origin of life, i.e., ribozymes, and the early evolution of life ) within optimal levels according to the cell type and environment not only for normal functions and development but also prevention of diseases. In fact, disorder of Mg2+ homeostasis is involved in cancer, diabetes, and neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and demyelination  ([Mg2+]cyto) regulates the circadian rhythm of BBB permeability in Drosophila, indicating that the intracellular [Mg2+] of BBB affects the neuronal environment in the brain [2+] of CSF is greater than that of blood [2+ is actively transported from the blood into CSF [2+] of ventricular CSF is higher and more sensitive to changes in [Mg2+] of plasma than that of lumber CSF in cow [2+] of CSF correlates with the extracellular [Mg2+] around neurons, which affects neural activities. Thus, the [Mg2+] of CSF is closely related to various brain functions [2+] of CSF and cognitive functions have been reported to exhibit a positive correlation [2+] of erythrocytes significantly correlates with the [Mg2+] of CSF in the hippocampus, and further with the hippocampal synapse density and recognition and memory performance [2+]cyto of erythrocytes is a good index of recognition and memory. These facts revealed that Mg2+ homeostasis in the human body is a key factor in brain functions, especially synaptic connectivity.Mg(50–70%) . In the  tissues . The ser 1.05 mM ,12, the y person . Even unabsorbed , and mos00 hours . Even inal range . Extraceal range ,16,17, cid (CSF) . The gapnd MagT1 . Howeverd kidney ,17, furtubule 10–% and thehe brain . CSF funhe brain . The  ranges between 17 and 20 mM in mammalian cells [2+]cyto and extracellular free Mg2+ concentration ([Mg2+]ex) are maintained within less than twofold  [2+]cyto is at the electrochemical equilibrium, then its resting concentration should be 50 mM cyto is observed, within about twofold [2+] within a narrow range under resting or stimulated conditions cyto within a narrow range. In such intracellular environments, some biological stimuli induce slight but significant changes in [Mg2+]cyto [2+] is apparently slight, the contents and distributions of Mg-complexed biomolecules may change dramatically under the conditions in which Mg2+-buffering molecules are abundant. Thus, a slight fluctuation of intracellular [Mg2+] can impact on cellular processes more than expected.In the cytosol, Mgproteins ,37. In p ~78 µM) ,37,46. Tg2+]cyto ,47,48,492+] ([Mg2+]nuc) considerably varies depending on the physiological conditions nuc increases and peaks during the metaphase, and it gradually decreases during cytokinesis. The condensation of chromosomes by Mg2+ is required for cell mitosis  in the mitochondrial matrix ([Mg2+]mito). Thus, mitochondria are a key player in the regulation of intracellular Mg2+ homeostasis [2+]mito is typically 0.8 to 1.2 mM mito by the Mrs2 knockdown affects the metabolome, especially reactions involved in the TCA cycle cyto are observed in several apoptotic cells [2+]cyto via TRPM7 channels in hippocampal neurons mito [2+]cyto mito  and decr[2+]cyto  are obse the PTP ,84. Thes2+ uptake plays various roles in vital signaling processes, such as bioenergetics, cell death, and sequestration of cytosolic Ca2+ transients ) is near 100 nM in the cytosol, while [Ca2+] in the ER ranges between 100 and 800 µM  in ER/SR has not been reliably determined. ER plays central roles in Ca2+ signaling in cells, including neurons cyto by caffeine as a RyR activator through an as yet unidentified mechanism in myocytes [2+ signals. The accurate determination of [Mg2+] in the ER/SR without Ca2+ interference has been eagerly awaited.ER/SR accumulates Mgholipids . The cond 800 µM . As high neurons ,91. Ca2+ of IP3R  and RyR  of IP3R ,94,95. Aasticity ,91,96, tmyocytes  suggesteEscherichia coli comprises more than 170 Mg2+ atoms cyto is closely associated with its ribosome content [2+]cyto is highly correlated with the rate of protein synthesis [2+ deficits cause the loss of peptidyl transferase activity and subsequent ribosome disassembly [2+]cyto promotes protein expressions for Mg2+ uptake and ATP reduction, consequently making Mg2+ available for translation to satisfy the cellular demands of Mg2+ [2+] are suggested to be evolutionarily conserved in eukaryotic cells [2+ physiologically enhances mTOR signaling activities [2+ apparently regulates protein synthesis through the effect on ribosomal functions via the mTOR pathway [2+]cyto affects the formation of a neural network via ribosome biogenesis.The ribosome is a complex molecular machine that serves as the site of protein synthesis, i.e., translation. Ribosomes comprise ribosomal RNA (rRNA) molecules and ribosomal proteins. A single 70S ribosome of 2+ atoms , meaninggle cell ,100. Sin cations ,103,104, content ,106. Sinynthesis . Furtherassembly  and leadassembly . Studies of Mg2+ ,112,113.ic cells . Intracetivities ,115. Thu pathway . In neur pathway ,118. The pathway . The def pathway ,120 and  pathway ,122,123.+, Na+, Ca2+, and Mg2+, exceed the number of anions ).).2+ affes to 6.0 , leading+]), indicating that Mg2+ impacts cellular biochemical reactions in an organelle-specific manner. Indeed, intracellular [Mg2+] rhythms dynamically tune cellular biochemistry in response to the metabolic demands throughout the daily cycle [Each intracellular organelle has a characteristic concentration of protons  in response to cellular events [2+] is maintained within the narrow and sub-millimolar ranges, which is considerably greater than that in the case of [Ca2+] cyto-dependent manner within the physiological dynamic range cyto regulation of mTOR activities exhibits sigmoidal curves, indicating that Mg2+ functions as a cooperative signal amplifier -dependent properties of such intracellular signaling are apparently more primitive and fundamental than are other intracellular signals considering the involvement of Mg2+ in the early emergence of life.The free energies for the binding of ATP to protein kinase in the presence of Mg of Mg2+ . Mg2+ po decades ,132. In lization ,47,49,73r events ,36. In ct manner ,133. Thiic range . CREB siic range ,135. mTOmplifier ,136. In 2−), are toxic to the living body , the membrane potential of neurons is weakly depolarized because of the relaxation of the Mg2+ block of the NMDA receptor, leading to hyperexcitability. As the NMDA receptor is involved in excitatory neurotransmission, neuroplasticity, and neuroexcitotoxicity, it plays an important role in developmental plasticity  in multiple steps. Mg2+ contributes to the accurate transfer of genome information and resultant translation of functional protein.Mg+ to DNA . The poss of DNA . Mg(H2O)from ROS . In addid in DNA . The gend repair . DNA temfidelity ,151. DNAfidelity . To main2+ is crucial for the growth and differentiation at the cellular and tissue levels [2+]cyto mobilized from mitochondria stimulates mTOR activities in a [Mg2+]-dependent manner and facilitates the maturation of neural networks cyto controls the strength of the electrical gap junctions [2+]cyto-dependent long-term plasticity through the regulation of electrical gap junctions  around NSCs affect differentiation to oligodendrocytes via the Mg2+ modulation of NMDA receptor activities. Furthermore, TRPM7 channels may play a critical role in the proliferation and migration of astrocytes via the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways cyto cyto . Moreoveproteins . A 6-hydproteins  and Mg2+eins  in the CSF [2+] in the serum are likely to show more severe symptoms [2+ deficiency causes emotional memory dysfunction [2+ administration improves learning and memory in dementia patients [2+] conditions, the accumulation of the C terminal fragment β (CTFβ) and Aβ occur under low extracellular [Mg2+] conditions [2+] prevents the Aβ-induced reduction of the synaptic NMDA receptors via the suppression of the calcineurin overactivation in hippocampal slices mito level should increase the risk of mtDNA damage. In addition, according to the endosymbiosis theory, mitochondria are endosymbiotic bacteria [2+ plays a key role in the regulation of protein synthesis [2+ may regulate protein synthesis in mitochondria. In fact, the separated regulation of mitochondrial and cytosolic protein synthesis in the process of the central dogma plays a central role in adaptation in the cellular nutrient environment [In demyelination mutant  ,240,241.bilities , a low [bacteria ,243. In ynthesis . Thus, mironment .2+ is a versatile divalent cation because of its unique physicochemical properties. In the brain, the extracellular and intracellular Mg2+ levels dynamically change according to the biological context. Mg2+ plays crucial roles in cell proliferation, differentiation, survival, and neural network formation via the regulation of cellular metabolism, intracellular signaling, channel opening, protein synthesis, and ROS toxicity. Typically, the mobilization of intracellular Mg2+ stimulates catabolism and protein synthesis, consequently activating the cellular processes that determine the fate and phenotype. Although Mg2+ usually protects neuronal cells against cellular stress, excess levels of Mg2+ are sometimes deleterious to healthy neuronal functions. Therefore, the appropriate regulation of cellular Mg2+ homeostasis is essential for neuronal functions in the brain, and the dysregulation of Mg2+ homeostasis potentially causes and aggravates neurodegenerative diseases, such as Parkinson’s diseases, Alzheimer’s disease, and demyelination. The recovery of healthy Mg2+ homeostasis through chemotherapy targeting Mg2+-transporting system can improve cellular functions under pathological conditions. In summary, the regulation of Mg2+ homeostasis can be a candidate for a therapeutic target in neurodegenerative diseases. However, the roles of Mg2+ and its regulatory mechanisms have not been investigated much. Thus, further study is crucial for developing future therapies and deepening the understanding of its neuro(patho)physiology.Mg


Arteriovenous malformations (AVM) developed in the small intestine are rare, and it is sometimes difficult to identify and treat bleeding from small intestinal AVMs endoscopically because of their localization. We present a case of a jejunal AVM successfully treated with the combination of metallic coil embolization and laparoscopic surgery.A 50-year-old woman with a history of repetitive gastrointestinal bleeding was admitted to the hospital. Selective angiography revealed a jejunal AVM that was treated with metallic coil embolization. However, the lesion rebled 3 months later, and it was embolized again with metallic coils. Considering the risk of rebleeding, we performed laparoscopic resection of the jejunal AVM. Under laparoscopy alone, it was impossible to detect the lesion of the AVM. We used X-ray fluoroscopy intraoperatively to detect the metallic coils at the AVM. Partial resection of the jejunum with the AVM was performed followed by functional end-to-end anastomosis. The patient was discharged from the hospital without any complications after the surgery.The combination of metallic coil embolization by angiography and laparoscopic surgery with X-ray fluoroscopy can be effective for patients with repetitive bleeding from jejunal AVM. Gastrointestinal arteriovenous malformations (AVM) often cause gastrointestinal bleeding, but sometimes they are difficult to diagnose by endoscopy in the absence of active bleeding. In particular, a small intestinal AVM cannot be diagnosed by either upper or lower endoscopy because of its location, even if it is actively bleeding. The diagnosis of small intestinal AVMs is usually made by a selective mesenteric arteriography, demonstrating characteristic vascular tufts and very early venous phase . In 1986A 50-year-old woman presented to our hospital with hematochezia and anemia. 1 year earlier, she experienced severe anemia (hemoglobin 4.0 g/dL) that was treated with a blood transfusion at another hospital. The diagnosis at that time was a hemorrhagic gastric ulcer, and she was treated with a proton pump inhibitor. Contrast-enhanced abdominal computed tomography (CT) done just before the first administration to our hospital showed multiple liver lesions of arterioportal and portal venous shunts, hemangiomas, and a large focal nodular hyperplasia. She had hematochezia and anemia (hemoglobin 7.0 g/dL) again and was referred to our hospital for further examination. Upper and lower gastrointestinal endoscopies including double-balloon enteroscopy did not reveal any bleeding lesions in her esophagus, stomach, duodenum, proximal jejunum, colon, or rectum, although she had grade 1 esophageal varices. Angiographic examination revealed an AVM, with signs of extravasation, at the jejunal branch of the superior mesenteric artery  of small intestinal AVM resected under laparoscopic surgery (Table We report a rare case of jejunal AVM treated with metallic coil embolization followed by laparoscopic resection. This case suggests the effectiveness of the combination of metallic coil embolization and laparoscopic surgery for patients with repetitive bleeding from a jejunal AVM.


Long intergenic non-coding RNA p21 (lincRNA-p21) is down-regulated in some solid tumors. Glutamine catabolism plays an important role in cancer development. However, the role of lincRNA-p21 and its association with glutamine catabolism remain unknown in bladder cancer (BC). In the present study, we investigated the involvement of lincRNA-p21 and glutamine catabolism in BC cell growth and found that ectopic linRNA-p21 expression reduced the proliferation and growth of BIU87 and 5637 cells. Opposite results were observed in lincRNA-p21 silenced J82 and T24 cells. The expression of glutaminase (GLS), intracellular level of glutamate and α-Ketoglutarate (α-KG) were negatively regulated by lincRNA-p21. GLS overexpression reversed the suppressive function of lincRNA-p21 on BC cell growth and proliferation. In contrast, GLS reduction by siRNA blunted the viability of lincRNA-p21 lowly expressed BC cells. Furthermore, lincRNA-p21 and GLS abundance dictated the sensitivity of BC cells to bis-2-ethyl sulfide (BPTES) treatment. Importantly, reduced lincRNA-p21 expression and increased GLS mRNA level were observed in BC tissues compared with the normal tissues. Our results demonstrate that lincRNA-p21 suppresses the BC cell growth through inhibiting GLS and glutamine catabolism. Targeting this cascade may be a promising treatment strategy for BC patients. Bladder cancer (BC) is one of the most common malignancies, which represents the ninth of all cancer types throughout the world . It causLong intergenic non-coding RNA p21 (lincRNA-p21) is initially identified as the direct transcriptional target of P53 . It contGlutamine is the most abundant amino acid in humans and an important metabolite for cell proliferation and growth . It is fIn the present study, we investigated the role of lincRNA-p21 and glutamine catabolism in BC cell growth. The results showed that lincRNA-p21 knockdown promoted BC cell proliferation and growth. GLS expression, glutamate, and α-KG production were enhanced in lincRNA-p21 silenced BC cells. Blockage of GLS with siRNA or bis-2-ethyl sulfide (BPTES) suppressed the viability of lincRNA-p21 lowly expressed BC cells. Human BC tissues exhibited reduced lincRNA-p21 and enhanced GLS expression as compared with the normal tissues.Human BC and normal specimens were collected after patients treated with radical cystectomy. All of the patients were diagnosed as transitional cell carcinomas by three independent pathologists. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. A written informed consent was obtained from each patient and all the experiments were approved by the Ethics Committee of Shenzhen Second People’s Hospital. The relative expression of lincRNA-p21 and GLS was detected by quantitative real-time PCR (qRT-PCR) assay.2.The human BC cells J82, T24, BIU87, and 5637 were obtained from the American Type Culture Collection  and Cell Bank of the Chinese Academy of Sciences . All the cells were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% fetal bovine serum , and 1% penicillin and streptomycin solution , in a 37°C incubator with 5% COFor lincRNA-p21 knockdown experiments, control shRNA (shCtrl) and shlincRNA-p21, lentivirus particles were designed and purchased from GenePharma company. The target sequences of lincRNA-p21 were GGAGGACACAGGAGAGGCA. siRNA oligos against GLS were purchased from GenePharma company. The BC cells were transfected with 100 nM siGLS or siCtrl using Lipofectamine 2000 , according to the manufacturer’s instructions. The target sequences of GLS were GCTCTCCTTCGGAGATCTT.pSLIK lentivirus system was used for lincRNA-p21 overexpression in BIU87 and 5637 cells. Briefly, human lincRNA-p21 cDNA was subcloned into the pSLIK particles. Virus packaging was performed using 293T cells. Seventy-two hours later, the viruses were harvested and infected the BIU87 and 5637 cells with polybrene. Stable cell lines were seleted by puromycin. GLS was overexpressed in BC cells using pCDH lentivirus vectors. The coding sequence (CDS) of GLS was synthesized and cloned into the pCDH vectors. PSPAX2 and PDM2G served as packaging vectors. These three factors were co-transfected into the 293T cells. Seventy-two hours later, the virus supernatants were collected, filtered, and subjected to infection of BC cells.Total RNA was extracted from human BC and normal specimens or the cancer cells using TRIzol reagent . A total of 0.5–1 μg of the total RNA was subjected to reverse transcription using M-MLV reverse transcriptase . Subsequent qRT-PCR was performed using TB Green qPCR Mix  on the Bio-Rad machine. The qPCR primer sequences were listed as follows: lincRNA-p21 forward: 5′-CCTGTCCCACTCGCTTTC-3′, and reverse: 5′-GGAACTGGAGACGGAATGTC-3′; GLS forward: 5′-TCTACAGGATTGCGAACGTCT-3′, and reverse: 5′-CTTTGTCTAGCATGACACCATCT-3′; β-actin forward: 5′-GACCTGACTGACTACCTCATGAAG-3′, and reverse: 5′-GTCACACTTCATGATGGAGTTGAAGG-3′. β-actin served as the internal control.The proteins were harvested from indicated cells using TNTE buffer containing protease inhibitor cocktail . The proteins were boiled, centrifuged, and separated on SDS/PAGE gels and transferred on to PVDF membranes. Then the membranes were blocked by 5% skim milk and incubated with primary antibodies at 4°C overnight. After washing by TBST for three times, the membranes were incubated with the secondary antibodies and the protein abundance was detected by enhanced chemiluminescence . Antibody against GLS was purchased from Abcam . Antibody against β-actin and all the secondary antibodies were obtained from Santa Cruz. Quantitation was analyzed by ImageJ software.The viability of J82, T24, BIU87, and 5637 cells transfected indicated lentivirus was determined using cholecystokinin (CCK) kit . In brief, a total of 2000 BC cells containing 200 μl medium was seeded in 96-well plates. Eight hours after seeding (0 h shown in the figure), 20 μl CCK reagent was added into each well and the plates were maintained at 37°C. The optical density (OD) value at 450 nm was measured on a microplate reader. The cell viability of each time was normalized to 0 h.A total of 3000 indicated cells were seeded in 96-well plates and were immediately treated with different concentrations of BPTES  . Two days later, culture medium was removed and the 100 μl medium containing 10 μl CCK reagent was added into each well and the plates were maintained at 37°C. Then the OD value at 450 nm was measured on a microplate reader.Equal number of BIU87 and 5637 cells expressing Ctrl and lincRNA-p21 lentivirus, or 5637 cells expressing Ctrl, lincRNA-p21, and GLS lentivirus were seeded. Ten days after incubating at 37°C, the plates were washed by TBS and fixed with methanol for 30 min. Then the plates were stained with Crystal Violet solution for 20 min. The colonies were photographed using a camera.The concentration of glutamate and α-KG were measured by Glutamate Assay Kit  and α-Ketoglutarate Assay Kit , respectively. All the experiments were conducted following the manufacturers’ instructions. The intracellular glutamate and α-KG abundance was normalized to the cell number.t test. One-way ANOVA was used for analyzing the difference when more than two groups. Difference was considered significant when *P<0.05; **P<0.01; ***P<0.001.All the statistical data were presented as mean ± S.E.M. of at least three independent repeats. The difference between two groups was analyzed by unpaired Student’s The function of lincRNA-p21 has been described in other cancers, whereas little is known about its role in BC. First, we analyzed lincRNA-p21 abundance in different BC cells. LincRNA-p21 was highly expressed in J82 and T24 cells comparing with BIU87 and 5637 cells A. TherefTo determine the function of lincRNA-p21 in BC, we subjected control and lincRNA-p21 overexpressed BIU87 and 5637 cells to CCK and colony formation assays. CCK results showed that lincRNA-p21 ectopic expression reduced the viability of BIU87 cells A. In addGlutamine transition to glutamate and α-KG plays an important role in cell proliferation. We initially checked the intracellular glutamate and α-KG level after lincRNA-p21 overexpression and knockdown. We found that intracellular glutamate and α-KG abundance were suppressed by lincRNA-p21 overexpression, while they were stimulated by lincRNA-p21 knockdown A,B. We pTo explore whether lincRNA-p21 regulation of GLS and glutamine catabolism participates in BC cell growth and proliferation, we overexpressed GLS in 5637 cells with ectopic lincRNA-p21 expression. GLS was efficiently overexpressed using lentivirus-mediated strategy A. GLS upTo determine the abundance of lincRNA-p21 in BC patients, we collected BC and normal tissues and analyzed the mRNA expression of lincRNA-p21 using qRT-PCR assay. The results showed that lincRNA-p21 was down-regulated in BC tissues compared with normal tissues A. LikewiThe high incidence and lethality makes BC a serious threat to society health. Once diagnosed, the prognosis is gloomy in most of the patients and effective therapies are limited. The development of BC involves multiple changes, including genetic, epigenetic, and metabolism . TherefoLncRNAs are regulatory non-coding RNA molecules that play an important role in cell activity. Dysregulation of lncRNAs participates in cancer development. Among them, lincRNA-p21 is a well-characterized one. It was identified as a repressive factor in p53 transcriptional regulation network . LikewisRecent studies have shown that lincRNA-p21 regulates signaling transduction in tumor growth. Down-regulated lincRNA-p21 promotes gastric cancer development through activation of YAP independent of Hippo . LincRNAEven though GLS expression and glutamine catabolism was mediated by lincRNA-p21, this does not imply that it contributes to the suppressive function of lincRNA-p21 in BC cell proliferation and growth. Thus, we performed rescue experiments. GLS was overexpressed and its ectopic expression reversed the growth and proliferation suppressed by lincRNA-p21. By contrast, GLS knockdown inhibited the enhanced viability of lincRNA-p21 silenced BC cells. The GLS inhibitor BPTES significantly blunted the growth of lincRNA-p21 knockdown, while exhibited minimal effect on lincRNA-p21 overexpressed 5637 cells. Additionally, GLS re-stimulation in the 5637 cells enhanced the inhibitory effect of BPTES and its knockdown reduced the effect. Thus, lincRNA-p21 suppression of GLS and the glutamine catabolism indeed participates in BC cell growth and proliferation.In summary, we provided for the first time that lincRNA-p21 was involved in glutamine catabolism and BC cell viability. LincRNA-p21 was reduced in BC specimens. Down-regulation of lincRNA-p21 resulted in up-regulated GLS, enhanced glutamine catabolism, and accelerated growth and proliferation of BC cells. Blockage of GLS reversed the viability of lincRNA-p21 silenced BC cells. Thus, lincRNA-p21 is a potential tumor suppressor in BC through regulation of glutamine catabolism which depends on GLS.


To assess the moderated mediation effect of normative beliefs about aggression and family environment on exposure to violent video games and adolescent aggression, the subjects self-reported their exposure to violent video games, family environment, normative beliefs about aggression, and aggressive behavior. The results showed that there was a significant positive correlation between exposure to violent video games and adolescent aggression; normative beliefs about aggression had a mediation effect on exposure to violent video games and adolescent aggression, while family environment moderated the first part of the mediation process. For individuals with a good family environment, exposure to violent video games had only a direct effect on aggression; however, for those with poor family environment, it had both direct and indirect effects mediated by normative beliefs about aggression. This moderated mediation model includes some notions of General Aggression Model (GAM) and Catalyst Model (CM), which helps shed light on the complex mechanism of violent video games influencing adolescent aggression. The relationship between violent video games and adolescent aggression has become a hot issue in psychological research . Based oAlthough Anderson et al. insisted on using the GAM to explain the effect of violent video games on aggression, other researchers have proposed alternative points of view. For example, a meta-analysis by Anderson and his collaborators  suggesteIn fact, the relation of violent video games to aggression is complicated. Besides the controversy between the above two models about whether there is an influence, other studies explored the role of internal factors such as normative belief about aggression and external factors such as family environment in the relationship between violent video games and aggression.Normative beliefs about aggression are one of the most important cognitive factors influencing adolescent aggression; they refer to an assessment of aggression acceptability by an individual . They caStudies found that normative beliefs about aggression are directly related to aggression. First, self-reported aggression is significantly correlated to normative beliefs about aggression . GeneralAccording to Bandura’s social cognitive theory , violentFamily violence, parenting style, and other family factors have major effects on adolescent aggression. On the one hand, family environment can influence directly on aggression by shaping adolescents’ cognition and setting up behavioral models. Many studies have found that family violence and other negative factors are positively related to adolescent aggression , while aOn the other hand, family environment can act on adolescent aggression together with other factors, such as exposure to violent video games. Analysis of the interaction between family conflict and media violence (including violence on TV and in video games) to adolescent aggression showed that teenagers living in higher conflict families with more media violence exposure show more aggressive behavior . ParentaDespite GAM and CM hold opposite views on the relationship between violent video games and aggression, both of the two models imply the same idea that aggression cannot be separated from internal and external factors. While emphasizing on negative effects of violent video games on adolescents’ behavior, the GAM uses internal factors to explain the influencing mechanism, including aggressive beliefs, aggressive behavior scripts, and aggressive personality . AlthougAlthough previous studies have investigated the roles of normative beliefs about aggression and family factors in the relationship between violent video games and adolescent aggression separately, the combined effect of these two factors remains unstudied. The purpose of this study was to analyze the combined effect of normative beliefs about aggression and family environment. This can not only confirm the effects of violent video games on adolescent aggression further but also can clarify the influencing mechanism from the integration of GAM and CM to a certain extent. Based on the above, the following three hypotheses were proposed:Hypothesis 1: There is a significant positive correlation between exposure to violent video games and adolescent aggression.Hypothesis 2: Normative beliefs about aggression are the mediator of exposure to violent video games and adolescent aggression.Hypothesis 3: The family environment can moderate the mediation effects of normative beliefs about aggression in exposure to violent video games and adolescent aggression; exposure to violent video games, family environment, normative beliefs about aggression, and aggression constitute a moderated mediation model.SD = 1.60).All subjects gave informed written consent for participation in this investigation, and their parents signed parental written informed consent. The study was reviewed and approved by the Professor Committee of School of Psychology, Nanjing Normal University, which is the committee responsible for providing ethics approvals. A total of 648 Chinese middle school students participated in this study, including 339 boys and 309 girls; 419 students were from cities and towns, and 229 from the countryside. There were 277 and 371 junior and high school students, respectively. Ages ranged from 12 to 19 years, averaging 14.73  and normative beliefs about aggression  were significant, which boys scored significantly higher than girls.As described in c = 0.28, t = 6.96, p < 0.001), the regression of normative beliefs about aggression to violent video games , and the regression of aggression to violent video games  and normative beliefs about aggression  were all significant. Thus, normative beliefs about aggression played a partial mediating role in exposure to violent video games and aggression. The mediation effect value was 0.06, accounting for 21.43% (0.06/0.28) of the total effect.To examine the mediation effect of normative beliefs about aggression on the relationship between exposure to violent video games and aggression, gender factor was controlled firstly. Stepwise regression analysis showed that the regression of aggression to violent video games , while the effect of family environment × exposure to violent video games on aggression was not significant , indicating that the relationship between exposure to violent video games and aggression was not moderated by family environment.In the first step, a simple moderated model (Model 1) between exposure to violent video games and aggression was established. The result showed that exposure to violent video games had a significant effect on aggression , and the effect of family environment × exposure to violent video games on normative beliefs about aggression was significant .Next, a moderated model (Model 2) between exposure to violent video games and normative beliefs about aggression was established. The results showed that exposure to violent video games had a significant effect on normative beliefs about aggression , and the effect of family environment × exposure to violent video games on normative beliefs about aggression was not significant . Because both a3 and b1 were significant, exposure to violent video games, family environment, normative beliefs about aggression, and aggression constituted a moderated mediation model. Normative beliefs about aggression played a mediating role between exposure to violent video games and aggression, while family environment was a moderator between exposure to violent video games and normative beliefs about aggression. Mplus analysis proved that the moderated mediation model had good model fitting .In the third step, a moderated mediation model (Model 3) between exposure to violent video games and aggression was established. As shown in b = 0.08, SE = 0.08, p = 0.37). For individuals with low score of family environment, exposure to violent video games could significantly predict normative beliefs about aggression . Based on the overall findings, individuals with high scores of family environment showed a nonsignificant mediating effect of normative beliefs about aggression on the relation of exposure to violent video games and aggression; however, for individuals with low scores of family environment, normative beliefs about aggression played a partial mediating role in the effect of exposure to violent video games on aggression.To further analyze the moderating effect of the family environment and exposure to violent video games on normative beliefs about aggression, the family environment was divided into the high and low groups, according to the principle of standard deviation, and a simple slope test was performed . The resR2 for exposure to violent video games in predicting adolescent aggression was 0.05, indicating that 5% of variation in adolescent aggression could be explained by exposure to violent media. These consistent findings confirm the effect of exposure to violent video games on adolescent aggression and can be explained by the GAM. According to the GAM  cohesion reflecting the degree of mutual commitment, assistance, and support among family members; (2) conflict reflecting the extent of anger, aggression, and conflict among family members; and (3) moral-religious emphasis reflecting the degree of emphasis on ethics, religion, and values. Individuals with high scores of family environment often help each other; seldom show anger, attack, and contradiction openly; and pay more attention to morality and values. These positive aspects would help them understand violence in video games from the right perspective, reduce recognition and acceptance of violence or aggression, and diminish the effect of violent video games on normative beliefs about aggression. Hence, exposure to violent video games could not predict normative beliefs about aggression of these individuals. By contrast, individuals with low scores of family environment are less likely to help each other; they often openly show anger, attack, and contradiction and do not pay much attention to morality and values. These negative aspects would not decrease but increase their acceptance of violence and aggression. For these individuals, because of the lack of mitigation mechanisms, exposure to violent video games could predict normative beliefs about aggression significantly.The moderated mediation model of the relationship between exposure to violent video games and aggression could not only help reveal that exposure to violent video games can affect aggression but also provide an elaboration of the influencing mechanism. According to this model, for individuals with high scores of family environment, exposure to violent video games had only direct effect on aggression. However, for those with low scores of family environment, there was not only a direct effect of exposure to violent video games on aggression but also an indirect effect mediated by normative beliefs about aggression. In short, exposure to violence video games affecting aggression through normative beliefs about aggression is more likely to happen to adolescents with poor family environment than those with good family environment. That is, generation of adolescent aggression is not only related to internal cognitive factors but also to external situations. As Piotrowski and Valkenburg  pointed Compared with the simple mediation or moderation model, the present moderated mediation model provided deeper insights into the internal mechanism of the effect of violent video games on aggression, providing inspirations for preventing adolescent aggression. First, in view of the close relationship between exposure to violent video games and adolescent aggression, relevant government departments should continue to improve the grading system of video games; meanwhile, parents should appropriately monitor the types of video games used by teenagers as well as the time spent and reduce the degree of exposure to violent video games. Second, by allowing teenagers to objectively distinguish between violence in games and reality, the mediating role of normative beliefs about aggression could inspire people to identify rational ways to solve violence problems and to experience the hurtful consequences of aggression. This would help adolescents change normative beliefs about aggression, establish a correct view of right and wrong, and reduce the occurrence of aggression. Finally, the moderating effect of family environment on the mediation process suggests that more attention should be paid to the important role of family environment. On the one hand, family education is closely related to adolescent aggression. Then, parents should create a good family atmosphere, publicly show anger and aggression as little as possible, and advocate and practice positive moral values. Parents should adopt authoritative styles, abandoning autocratic and indulgent parenting styles  to minimLimitations of the current study should be mentioned. First, only Chinese school students were assessed, in a relatively small number, which could affect sample representativeness. A large sample of teenagers from different countries and in different ages, also including juvenile offenders, would be more accurate in revealing the effect of violent video games on adolescent aggression. Second, this study only focused on violent video games, not involving violent media such as internet and television, daily life events, wars, and other major social events. Indeed, these factors also have important effects on adolescent aggression, and their influencing mechanisms and combined effect are worth investigating further. Third, this study mainly adopted the self-report method. Use of peer, parent, or teacher reports to assess exposure to violent video games and aggression would help improve the effectiveness of the study. Fourth, there might be other mediators, moderating variables and relational models. In addition to normative beliefs about aggression and family environment, individual emotions, personality characteristics, school climate, and companions may play mediating or moderating roles in the relationship between violent video games and aggression. This study developed a moderated mediation model between family environment and normative beliefs about aggression, but the possibility of multiple mediation and mediated moderation models cannot be ruled out.The current study showed that exposure to violent video games is positively related to adolescent aggression; normative beliefs about aggression have a mediating effect on exposure to violent video games and adolescent aggression, while the family environment regulates the first part of the mediation process. For individuals with good family environment, exposure to violent video games only has a direct effect on aggression; however, for those with poor family environment, there is an indirect effect mediated by normative beliefs about aggression alongside a direct effect. This moderated mediation model incorporates some perspectives of GAM and CM, enriching studies of generative mechanism of adolescent aggression.YW and RS conceived the idea of the study. RS analyzed the data. YW and RS interpreted the results and wrote the paper. YW discussed the results and revised the manuscript.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.