Lung cancer is the leading cause of cancer-related deaths worldwide, and insights into its underlying mechanisms as well as potential therapeutic strategies are urgently needed. The microbiome plays an important role in human health, and is also responsible for the initiation and progression of lung cancer through its induction of inflammatory responses and participation in immune regulation, as well as for its role in the generation of metabolic disorders and genotoxicity. Here, the distribution of human microflora along with its biological functions, the relationship between the microbiome and clinical characteristics, and the role of the microbiome in clinical treatment of lung cancer were comprehensively reviewed. This review provides a basis for the current understanding of lung cancer mechanisms with a focus on the microbiome, and contributes to future decisions on treatment management. Actinobacteria, Proteobacteria, and Verrucomicrobia were minor components. The human gut microbiome is extremely large and scientists have not been able to determine the number of gut microbes that people may carry. It has been estimated that a 70 kg adult (3.0×1013 cells) carries approximately 3.8×1013 bacteria (0.2 kg)  , 23.Bacteroidetes to Firmicutes increases, and that of Bifidobacterium decreases. Claesson et al. reported that, compared with young people, the differences in gut microbiota composition especially in Ruminococcaceae family , among individuals was significantly higher in the elderly, and the Bifidobacterium proportions, the Clostridium cluster IV, as well as the species diversity within each individual was significantly reduced, which is likely related to diet, health status, and immune system decay . Agedness is another stage at which the gut microbiota changes dramatically. At this stage, the number of facultative bacteria increases, the ratio of em decay , 25.Staphylococcus, Streptococcus, Lactobacillus, Pseudomonas, Bifidobacterium, Corynebacterium, Enterococcus, Acinetobacter, Rothia, Cutibacterium, Veillonella and Bacteroides   , 34 (Fiori (HB) .In normal prostate, estimations concerning the microorganism number and composition are difficult since access to non-diseased prostate tissue is restricted. However, a number of previous studies have characterized the microbial composition in prostate cancer and normal surgically resected specimens, and found that no bacteria were present in normal prostate tissue –38. On t2.2The microbiome plays an important role in human development and physiology. In this context, changes in the oral microbiome may cause oral and systemic diseases  and an i2.2.1Candida is the most common. In the oral microecosystem, microbes such as bacteria and fungi attach to the surface of teeth and form a biofilm called plaque with the surrounding extracellular matrix in order to protect themselves from fluctuations in the oral environment and external drug stimuli and evade host defense mechanisms  make up the majority of the healthy oral microbiome and are mainly composed of six phyla, including Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, Bacteroides and Spirochaetas . In addichanisms .The balance of oral microecosystem not only contributes to the maintenance of oral health, but also has a potential impact on the overall health. Microorganisms in oral microecosystems achieve dynamic balance between each other and the host through complex interspecific interactions such as symbiosis, competition and confrontation . This pa2.2.1.1P. seudomonas aeruginosa invade, the oral flora inhibits their growth in saliva by producing lactic acid  can alter saliva composition or affect saliva secretion or flow rate, leading to dry mouth and poor oral hygiene . This ma2.2.2Propionibacterium, Corynebacterium, Staphylococcus and Moraxella. Prevotella, Vermicelli, Streptococcus, Haemophilus, Fusobacterium, Neisseria and Corynebacterium were predominant in oral cavity , small airways (bronchioles), and respiratory areas . Because the respiratory tract is connected to the outside world, a large number of airborne microorganisms and particles, including viruses, bacteria and fungi, continue to migrate or be removed from the respiratory tract. The bacterial burden of the upper respiratory tract is about 100-10000 times than that of the lower respiratory tract, and the nasal cavity is dominated by l cavity , 55. Precavities . As mentcavities , 57, 58.2.2.2.1The respiratory tract is the main site of continuous contact with exogenous microorganisms. Airway epithelium acts as a sensor for the presence of microorganisms, and its epithelial cells are in constant contact with the environment. This interaction is a key factor in maintaining stable homeostasis. The environmental conditions necessary for microbial growth in the respiratory tract  are heterogeneous, so considerable regional variation can be observed in a single healthy lung .2.2.2.2In health conditions, the microbiome can also regulate immune strength. Symbiotic fungi have been shown to influence the immune system and regulate the bacterial community, thus contributing to the recovery of bacterial flora after antibiotic treatment , 61.2.2.32.2.3.1Gut microorganisms can affect intestinal mucosal development and homeostasis. Comparative studies of conventional and germ-free animals have shown that the gut microbiota is essential for the formation and functional realization of the intestinal mucosal immune system during infancy . A poor 2.2.3.2Firmicutes, Bacteroidetes, and some anaerobic microorganisms can decompose complex carbohydrates in the gut to produce short-chain fatty acids (SCFAs), such as acetic acid, propionic acid, and butyric acid -α production following lipopolysaccharide (LPS) and C. albicans stimulation. Barnesiella was negatively associated with LPS-and B. fragilis-induced interferon (IFN)-γ production. This included common gut commensals, such as Dorea longicatena and Dorea formicigenerans, where higher species abundance was associated with higher IFN-γ levels in response to C. albicans hyphae. Both species of Dorea can metabolize sialic acids, which are usually found at the end of mucins; and the release of these acids is associated with mucin degradation, and may increase gut permeability. Both Streptococcus parasanguinis and Streptococcus australis were associated with IFN-γ production whereas other species, such as Streptococcus mitis/oralis/pneumoniae, were associated with IL-1β production. Also the correlation of Bifidobacterium pseudocatenulatum and IFN-γ was positive. In contrast, the correlation of Bifidobacterium adolescentis and TNF-α was negatively. In addition, P. distasonis was negatively associated with TNF-α and IL-1β after stimulation with C. albicans hyphae . SC. SCAcidorganisms  97). Le. LeAcidocificity . In addip. In AC , whereasnt in AC . In addint in AC . Last, H3.2Legionella was more abundant in patients with metastatic lung cancer , whereas Dialister is more common in patients with progressive disease, and its higher abundance is related to reduction of progression-free and overall survival (OS)  .Veillonell, Megasphaera, Actinomyces and Arthrobacter in lung adenocarcinoma without metastasis was significantly higher than that in lung squamous cell carcinoma without metastasis. The contents of Capnocytophaga and Rothia in metastatic lung adenocarcinoma were significantly lower than those in metastatic lung squamous cell carcinoma. Streptococcus content was significantly lower in lung adenocarcinoma with metastasis than in lung adenocarcinoma without metastasis. The contents of Veillonella and Rothia in lung squamous cell carcinoma with metastasis were significantly higher than those in lung squamous cell carcinoma without metastasis. Jungnickel et al.  and disease-free survival (DFS) in patients with lung cancer. On the contrary, the abundance of Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae was correlated with a decrease in RFS or DFS of lung cancer -17, PI3K, mitogen-activated protein kinase (MAPK), and ERK pathways in the airway transcriptomes of patients with lung cancer and is associated with poor prognosis - and phosphoinositide 3-kinase (PI3K)-signaling pathways of the lower airway transcriptome in patients with lung cancer are significantly upregulated, which is related to the enrichment of  airways . Recent rognosis . In humarognosis . In addirognosis .Firmicutes in the lower lobe of the lung may be a sign of increased pathogenicity and is associated with poorer prognosis  is an environment composed of various physical and chemical factors surrounding tumor cells, including neighbor tumor cells, immune cells, stromal cells, extracellular matrix, and a variety of soluble molecules, and is an important aspect of the tumor. TME plays an important role in the occurrence and development of tumors . FigureIn a mouse model of KRAS-TP53 co-mutation (KP) lung cancer, airway microbiosis disorder caused by Tiny Vibrio led to the recruitment of Th17 cells, increased IL-17 production, increased PD-1+T cell-expression, and recruitment of neutrophils, which resulted in a reduced survival and increased the burden of lung tumors . Gut micStudies have shown correlations between lung cancer cell growth and unbalances in the airway microbial community. This locally dysregulated microbiome stimulates the production of IL-1β and IL-23 in myeloid cells, which in turn induce the proliferation and adaptive activation of lung-resident Vγ6+Vδ1+γδT-immune cells. Activated γδT cells produce IL-17, which promotes neutrophil infiltration and inflammation in the TME , 108. Th5Bifidobacterium was abundant in the intestinal tract of patients with NSCLC who responded to clinical treatment. Further, when a commercial Bifidobacterium strain was used to treat mice tumors with the same genotype the tumor load could be reduced by inducing the host immune response and cooperating with immunotherapeutic or chemotherapeutic drugs (Clostridium butyricum (MIYAIRI 588 strain) to ameliorate symptoms associated with ecological disturbance caused by antibiotics (ATBs), suggesting that probiotic Clostridium butyricum therapy (CBT) has a positive effect on improving immune checkpoint blockade (ICB) in patients with cancer  also restored host homeostasis and reduced microbial-induced inflammation , 139  before or after ICI treatment significantly extended patients’ progression-free survival (PFS)non-progressive survival and overall survival (OS)  . Improve6The microbiome characteristics have significant effects in tumor development, however, how the microbiome responds to lung cancer, in particular, how lung cancer cells and TME shape the local microbial community of the lungs, is unknown. However, it has been shown that in colorectal cancer (CRC), loss of surface barrier function can cause tumor inflammation induced by symbiotic bacteria. In particular, the breakdown of tight connections between colon tumor cells allows bacterial degradation products such as LPS, to enter the tumor stroma, causing bone marrow-derived cells to be recruited to the TME. Therefore, understanding the interaction between the human microbiome and lung cancer cells, and identifying the cellular and molecular mediators involved in this interaction are relevant issues to be explored in order to find future potential targets for lung cancer treatment.In addition, when considering the influence of microbiome on the efficacy of chemotherapy, targeted therapy, and immunotherapy for lung cancer, it is necessary to distinguish between the specific roles of the local lung microbiome, the distal gut microbiome, and oral bacteria in tumor growth and related immune responses . It is pBoth authors contributed equally to the writing of the review.


The fibroblast growth factor 23 (FGF23) has been related to biological aging, but data in elderly individuals are scant. We determined the profile of serum FGF23 levels in a population of very-old individuals and studied their correlations with parameters of bone metabolism and health markers, as functional performance.This cross-sectional study was performed on 182 community dwellers aged ≥ 80 years. Serum levels of FGF23, PTH, calcium, albumin, phosphorus, creatinine, bone markers, and bone mineral density data were analyzed.  Physical performance was evaluated with the stationary march (Step), Flamingo, and functional reach tests, along with questionnaires to assess falls and fractures in the previous year, energy expenditure (MET), and the Charlson index (CI). Physical activity was evaluated with the International Physical Activity Questionnaire (IPAQ).Most participants (75%) had FGF23 levels between 30-120 RU/mL . FGF23 levels correlated with estimated glomerular filtration rate  and PTH . Individuals with FGF23 in the highest tertile had more falls in the previous year (p = 0.032), worse performance in the Flamingo (p = 0.009) and Step (p < 0.001) tests, worse CI (p = 0.009) and a trend toward sedentary lifestyle (p = 0.056). On multiple regression, FGF23 tertiles remained significant, independently of eGFR, for falls in the previous year, performance in the Flamingo and stationary march tests, lean mass index, and IPAQ classification.In a population of very elderly individuals, FGF23 levels were inversely associated with neuromuscular and functional performances. Higher concentrations were related to more falls, lower muscle strength and aerobic capacity, and poorer balance, regardless of renal function, suggesting a potentially deleterious role of high FGF23 concentrations in musculoskeletal health. Individuals over the age of 80 represent the segment of the population with the highest relative growth (3.8% per year). By 2015, the world’s population of individuals over the age of 80 was estimated at 125 million and, according to projections, will be three times higher by 2050 (2D] (Some studies have found associations between proteins regulating the metabolism of phosphorus and aging . FGF23 i2D] .klotho protein (coreceptor) , and cardiovascular diseases (To bind to its specific receptor (FGFR) located on the cell membrane, FGF23 requires the presence of klotho pathway besides renal phosphorus regulation have been studied. However, data in humans pertinent to FGF23’s non-classical effects are scarce, and virtually non-existent in the aged population.Since FGF23 receptors and/or co-receptors have been identified in extra-renal tissues like the parathyroid glands, cardiac and skeletal muscles ,5, otherWith regard to the need to elucidate the potential roles of FGF23 as a marker of aging, we measured serum levels of FGF23 in a free-living aged population of 80 years or more. We analyzed correlations between FGF23 levels and other parameters of bone and mineral metabolism as well as health markers, such as functional performance  and the presence of comorbidities.This cross-sectional study was conducted in a cohort of individuals aged 80 years or older who were participating in a Geriatrics Program at Unifesp. Inclusion criteria were age ≥ 80 years, free-living, independence in performing basic activities of daily life, ability to walk without human support, and chronic health conditions under control. Patients who had moderate or severe dementia, terminal illness, hospitalization in the last 6 months, were on dialysis or refused to participate in the study were excluded. The study included 182 individuals.Clinical evaluation was performed at the first visit, through a questionnaire that included personal history, identification of diseases, description and quantification of medications, calcium and vitamin D supplements taken, and life habits. The Charlson comorbidity index of each individual was calculated based on these data . QuestioBody mass index (BMI) was calculated from measurements of weight and height and classified according to the Lipschitz criteria, as recommended for elderly individuals .® 2010, Roche Diagnostics, Indianapolis, IN, USA; intraassay and interassay coefficients of variation 3.0% and 3.5%, respectively) and procollagen type 1 N-propeptide (P1NP) and serum C-terminal telopeptide of type 1 collagen (CTX) by electrochemiluminescence . Calcium was corrected for albumin levels  FGF23 were measured by ELISA . The concentrations of albumin, total calcium, and phosphorus were measured by an automated colorimetric method, and those of creatinine by alkaline picrate. Concentrations of 25(OH)D were quantified by chemiluminescence . PTH was measured by immunochemiluminescence  performed on the lumbar spine (L1-L4), femoral neck, total hip, and total body, and the results obtained were classified according to the World Health Organization (WHO) criteria  and body composition were measured by Dual Energy X-ray Absorptiometry (DXA) Hologiccriteria . The coecriteria , Baumgarcriteria  and the criteria . Based oThe following physical tests were used to predict the risk of falls:Flamingo balance test : the individual raises one of the lower limbs forming a 90o angle with the femur, with hands on the waist, and tries to maintain position for 30 seconds. The test is performed 3 times and the longest time the patient is able to maintain the position is recorded as the final score : the patient stands facing a wall with a tape measure attached to it, parallel to the floor, at the height of their acromion, with arms extended making a 90º angle to the trunk. The individual leans as far forward as possible and hold this position for 3 seconds, without lifting the heels off the floor. The distance reached is recorded and the best of 3 attempts is considered to be the final score : after prior training of a simulated gait, the number of times the right knee of the individual rises to at least the midpoint of the distance between the patella and the iliac crest is counted for a total duration of 2 minutes  and conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all individual participants prior to their inclusion in the study.FGF23 levels were analyzed as continuous variables and tertiles. Categorical variables are presented as absolute and relative frequencies, and continuous variables as measures of central tendency and dispersion .t- test for independent samples and ANOVA were used to compare mean values between two groups and more than two groups, respectively. When differences in mean values were observed in ANOVA, such differences were identified using Duncan’s multiple comparisons. In case of violation of data normality, the mean ranks were compared using the nonparametric Mann-Whitney and Kruskal-Wallis tests alternatively. Once differences in the Kruskal-Wallis test were detected, they were identified using Dunn-Bonferroni tests.The Kolmogorov-Smirnov test was used to evaluate the normality of the distribution of the variables and the chi-square test was used to evaluate the distribution of the participants by IPAQ, Baumgartner, and ALM/BMI classifications. Linear associations between two continuous variables were evaluated with Pearson’s correlation and nonparametric variables with Spearman’s correlation. Student’s Multiple regression models were used to evaluate the simultaneous effects of FGF23, sex, age, 25(OH)D, number of medications, number of comorbidities, and several other variables on each of the dependent variables. For the dependent variables of continuous numerical nature, linear regression models were used for continuous numerical values, and the Poisson models were used for categorical numerical values. For dependent, dichotomous variables , logistic regression models were used, and for polytomous values (IPAQ categories), a multinomial regression model was applied. A 5% significance level was adopted for all statistical tests. Statistical analyses were performed using SPSS 20.0 and Stata 12.2 for women and 18.03 ± 1.80 kg/m2 for men, and the mean Baumgartner index was 6.59 ± 0.97 kg/m2 for women and 7.49 ± 0.91 kg/m2 for men. The mean ALM/BMI index was 0.551 ± 0.077 for women and 0.775 ± 0.104 for men. According to those two last indexes, most individuals (82.4% by the Baumgartner index and 57.4% by the ALM/BMI index) had an adequate ALM.The mean age of the 182 participants was 86.5 ± 4.5 years (range 80 to 99 years); 138 (75.8%) were women (86.5 ± 4.0 years) and 44 (24.2%) were men (86.5 ± 9.0 years). An asymmetric distribution of FGF23 concentrations was verified, with 75% of the participants presenting with FGF23 values between 30 and 120 RU/mL. Concentrations above 200 RU/mL were found in 22 outliers (12%), all of whom had eGFR greater than 32 mL/min , normal phosphorus , were not current smokers, and did not take calcitriol. Hyperparathyroidism (PTH > 65 pg/mL) was observed in 11 (50%) of these individuals, including all 3 with primary hyperparathyroidism. Vitamin D deficiency (<20 ng/mL) was present in 63.6% (n = 14) of them, similar to the entire sample (57.7%).Information on falls and previous fractures were provided by 152 individuals, of whom 76 (50%) reported 160 falls  in the previous year. Eight fractures occurred after falling from a standing height in 6 individuals (2 men and 4 women); 2 occurred in the femur, 2 in the wrist, 1 in the lumbar vertebra, 1 in the shoulder, 1 in the rib, and 1 in the phalanx of the hand.There was no significant difference in mean FGF23 levels between the groups according to gender, age, smoking, use of any medication related to calcium homeostasis, or densitometric diagnosis. FGF23 levels correlated significantly with several variables, like the stationary march test scores, eGFR, and serum PTH levels, as described in The individuals were divided into tertiles based on the FGF23 levels . Those in the highest FGF23 tertile had had a greater number of falls in the previous year (p = 0.032), worse performance in the Flamingo (p = 0.009) and stationary march (p < 0.001) tests, and worse CI (p = 0.009) compared to those in the other tertiles .Multiple regression models were tested to identify independent factors that could be associated with successful aging. Differences among FGF23 tertiles remained significant in the final models for the following markers: falls in the previous year , performIn the final model for falls in the previous year , the folFor the stationary march test , the facFor the number of fractures in the previous year, only the BMI categories remained significant in the final model, and the participants with low weight presented 15 times more fractures than those with excess weight.The population evaluated in this study is singular, being all participants 80 years or older living independently in the community, and with chronic health conditions under control. As far we know, this is the first study to investigate FGF23 concentrations and their relationship with clinical data, physical performance, and falls in this population.2D, which was not measured in our cohort , and in 22 of the individuals, the FGF23 levels were above 200 RU/mL (outliers). Although there were 3 individuals with primary hyperparathyroidism among them, other factors commonly associated with very high concentrations of FGF23 were not found in this subgroup of patients, who had eGFR above 32 mL/min, normal phosphorus levels, were not current smokers, and did not use calcitriol . That sur cohort .FGF23, along with its coreceptor klotho, is currently considered an important regulator of phosphorus homeostasis and of vitamin D metabolism . FGF23 iIn the present study, the relationships observed between higher levels of FGF23 and poorer neuromuscular performance were independent of the renal function, as observed in the number of falls in the previous year, in the Flamingo and stationary gait tests, and in the lean mass index. Although the expression of FGF23 has been identified in skeletal and cardiac muscle, the actions of FGF23 in the muscular system are still contradictory and little known ,5. Some Studies have shown an inverse relationship between serum phosphate levels and muscle strength, linking hyperphosphatemia to a possible mechanism involved in sarcopenia ,29. Howe2D]. Our study not only reinforces this hypothesis through the results of stationary gait , but also shows FGF23’s correlation with static balance (Flamingo test). This finding corroborates the possibility that FGF23 exerts effects on sensory neurons  prevented further statistical analysis. However, when separated by FGF23 levels, fractures occurred in 15.8% (n = 3) individuals with FGF23 > 200 RU/mL but only in 3.8% (n = 5) of those with FGF23 < 200 RU/mL. Although this difference has not reached statistical significance, it may suggest some deleterious effect of high concentrations of FGF23 on the risk of fractures. The association between increased concentrations of FGF23 and fractures has already been described, both in populations of elderly men  and in p2D was not evaluated, and data on fractures were based on the patients’ history and were not necessarily documented. Despite these limitations, the findings of our study have great relevance in the current context, considering that life expectancy has grown exponentially in the world and data on this peculiar population of very old individuals are still scarce. Furthermore, as far as we know, these findings were the first to correlate FGF23 as an independent factor for balance and strength of lower limbs, properties that may predict falls, and separately evaluated.Our study has several limitations, including its cross-sectional design, which prevented us from evaluating associations with future outcomes. With the absence of a control group, the conclusions of this study in very old individuals comprised of independent aging subjects cannot be extrapolated to other populations of younger or fragile elderly individuals. The assay used in the study measures the pool of circulating forms of FGF23, including inactive C-terminal forms, which may not adequately represent the normal physiological state. In addition, information on dietary calcium intake was not available, 1,25(OH)In conclusion, our study demonstrated a relationship between increased levels of FGF23 and worse neuromuscular performance, demonstrated by the surrogate measures of falls during the previous year, scores in the Flamingo and stationary march tests, that especially evaluate muscle strength, aerobic capacity, and balance, independently of renal function. This may well be the first study measuring FGF23 concentrations in a community-dwelling, independent, very old individuals making correlations with these parameters. The results suggest that FGF23 could be a marker of physical performance in this population, indicating a potential role of FGF23 on musculoskeletal health.


The results show that coping style and DASS could mediate the relationship between adolescent resilience and mobile phone addiction among Chinese adolescents. A chain of coping styles and DASS mediated the relationship between adolescent resilience and mobile phone addiction in Chinese adolescents.Natural disasters cause long-term psychological problems and increase substance use in some adults. However, it is unclear whether disasters also lead to these problems in adolescents. We hypothesized the influence of adolescent resilience on mobile phone addiction during the normalization of COVID-19 and flooding. We tested the mediating role of coping style and depression, anxiety, and stress (DASS) on phone addiction among 1,751 adolescents in the Henan Province in China. The adolescents were surveyed  Due to the COVID-19 outbreak, mobile phones have become increasingly important for online teaching and learning in China. According to the 48th China Statistical Report on Internet Development, by June 2021, there were 1.07 billion mobile phone users in China, accounting for 99.7% of the total number of internet users . FurtherMany studies indicated the predictors of mobile phone addiction, such as physiological health , experieAccording to resilience framework theory, psychological resilience is the ability to cope with a crisis or quickly return to a pre-crisis status mentally or emotionally . PreviouCoping strategies are psychological models used by individuals to manage emotions, thoughts, and behaviors when they encounter various states of stress in different stages, reflecting all responses to stress that can be utilized and successfully used . Many stStress is an experience of negative emotions and behavioral changes. The environment plays an important role in generating stressors . RegardiIn July 2021, the Henan province suffered from unusually heavy rainfall and maximum continuous rainfall of 958 mm , causingChildren who had more exposure to the flood events had greater resilience than those who had less or none . ResilieMany scholars believe that adolescent resilience is also related to substance use, such as smoking and excessive drinking  . Some exGiven this, adolescents confined to their homes may be likely to overuse mobile phones and the internet due to floods in China. Thus, we hypothesized a correlation between resilience and mobile phone addiction among adolescents, and that coping style and mental health play a mediating role in that relationship.As a stressor, flooding can affect adolescent mental health and cause symptoms of anxiety and depression. Many studies show that resilience eliminates the symptoms of anxiety and depression, and increases self-esteem and mental well-being among young people . StudiesResilience is a crucial developmental stage during adolescence, as it is a transitional period characterized by significant neurobiological and psychosocial changes in amplifying environmental demands and increasing sensitivity to social contexts . During Based on the literature review, the present study constructed a chain mediation model to examine the mediating role of positive coping, stress, anxiety, and depression in the relationship between adolescent resilience and mobile phone addiction among Chinese adolescents. Furthermore, we proposed a model to test the associations among Chinese adolescent resilience, coping style, mental health, and mobile phone addiction to further clarify mobile phone addiction related to resilience .Convenience sampling was employed to recruit students from grades 7–9 in middle school and grades 1–3 in high school in Henan province of China. Inclusion criteria: (1) flood areas in Henan Province; (2) junior or senior high school students, aged 12–18; (3) floods from July to August 2021; and (4) informed consent and voluntary participation in this study. Exclusion criteria: (1) teenagers who are not in school and (2) students who have communication problems. All subjects signed informed consent online before participating in the study, which was authorized by the Human Participant Review Committee of Xinxiang Medical College. All projects are available in an easy-to-understand Chinese version. Trained graduate students in psychology handed out and recycled the scales. They are also responsible for explaining possible doubts to avoid any confusion. It takes about 20 min to fill out the questionnaire. They completed online questionnaires from July 1 to August 30, 2021. A total of 1,751 valid questionnaires were obtained, with an effective response rate of 97.28%.never to 3 = most of the time). This self-report instrument includes three subscales: (1) the stress subscale, which measures tension, agitation, difficulty relaxing, and negative affection; (2) the anxiety subscale, which assesses autonomic arousal, skeletal musculature effects, situational anxiety, and subjective experience of anxious arousal; and (3) the depression subscale, which measures hopelessness, dysphoria, lack of interest, self-deprecation, and inertia. The reliability coefficients of depression, anxiety, and stress were 0.82, 0.82, and 0.79, respectively. The Cronbach’s alpha of the total scale was 0.89.The Depression, Anxiety, and Stress Scale with 21 Items (DASS-21) was used to evaluate negative emotional states of DASS , referrinot at all; 2 = rarely; 3 = occasionally; 4 = often; and 5 = always). The scale covers four dimensions: (1) “inability to control craving,” which reflects the amount of time adolescents spend on their mobile phones, thereby leading to complaints from family and friends about their compulsive mobile phone use and causing the adolescents to lose of sleep due to excessive use; (2) “anxiety and feeling lost” assesses preoccupation, feeling lost or anxious, and having difficulty switching off the mobile phone; (3) “productivity loss” measures decreased productivity and diverted attention from pressing issues due to adolescents’ excessive use mobile phones; and (4) “withdrawal and escape” indicates that adolescents use their mobile phones to escape from isolation, loneliness, and feeling down. The Cronbach’s alpha of the scale was 0.90.Leung designed the Mobile Phone Addiction Index (MPAI) to identify addiction symptoms associated with mobile phone use among adolescents in Hong Kong . The scaThe Resilience Scale for Chinese Adolescents (RSCA) was developed by This instrument was designed by P < 0.01 and P < 0.05.All data analyses were performed using SPSS 26.0 and Amos 23 . First, descriptive data were received using SPSS 26.0, and correlations variables were calculated using Pearson’s correlations. Second, according to The demographic profiles and descriptive statistics for the final analysis of the participants are provided in As displayed in 2/df = 2.85, RMSEA = 0.047, goodness-of-fit index (GFI) = 0.982, CFI = 0.987). The hypothesis relationships between the variates are demonstrated in P < 0.01). The indirect effect of adolescent resilience on mobile phone addiction through stress, anxiety, and depression was −0.602, 95% CI , P < 0.01, excluding 0. The mediating effect was significant. The indirect effect of adolescent resilience on mobile phone addiction through positive coping was 0.122, 95% CI , P < 0.01, excluding 0, and the mediating effect was significant. In The test results revealed the goodness of fit of the proposed structural model . Written informed consent to participate in this study was provided by the participants’ or their legal guardian/next of kin.HC designed the study. XL and SZ collected the data. AM analyzed the data and wrote the manuscript. YY and SG revised the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4,622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7,659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure. Together with an ongoing genomic surveillance program, these data will contribute to a better understanding of SARS-CoV-2, as well as its evolution pattern and pandemic characteristics in Bangladesh. VOC is st May 2022 to understand the distribution of variants and mutation accumulation trends over time. We have thoroughly studied the temporal and geographical distribution of different lineages inside Bangladesh and built the transmission network to trace their back and forth circulation. To better understand the evolutionary dynamics of SARS-CoV-2 in Bangladesh over the last 2 years, we examined the genetic diversity among strains, gene-wise mutation distribution, and selection pressures.To the best of our knowledge, most of the previous studies in Bangladesh addressed lineages distribution, source determination, and potential mutations with only a few sequences from the early phase of the outbreak . Therefost May 2022 were retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) database (www.gisaid.org) (https://pangolin.cog-uk.io/)  . Prior t-uk.io/) . FurtherFirst, the selected sequences were aligned using the Mafft algorithm , followeWe have aligned each sequence with the reference sequence (NC_045512.2)  using thFirst of all, we used TASSEL software  to deterst May 2022, but many of them were incomplete and lacked quality. Therefore, we filtered the sequences based on their completeness, coverage, and gaps, resulting in 4,984 sequences for downstream analysis. In all, these sequences belonged to 93 PANGO lineages, although many of these lineages carried very few sequences. Hence, we further filtered the sequences and kept only the lineages containing at least ten sequences, resulting in 4,692 sequences from 35 lineages . The Omicron variant first emerged in Bangladesh in December 2021 and took over within a month, ultimately leading to the third wave of infections. Initially, the BA.1 sub-lineage of the Omicron variant dominated. However, BA.2 has gained a significant growth advantage over BA.1 and has taken over.We then conducted a geographical analysis in order to determine whether the variants were distributed evenly across Bangladesh’s administrative divisions. In terms of geographical distribution, Dhaka had the most diversified sequences from all thirty-five lineages, followed by Chattogram with 31. On the contrary, Mymensingh and Rangpur were less diverse areas with sequences from only 22 and 24 lineages, respectively, where most of the lineages represented only one or two sequences . InitialTo get a clearer idea of the viral circulation trend in different divisions of the country, we extensively analyzed the variants present there chronologically. We figured out that the whole country was mostly filled with a few major lineages throughout the time, but interestingly their dominance varied. We have seen that some lineages were missing from a particular area at a particular time and then reappeared, maybe due to mass people’s movement from other areas. For example, B.1.1 lineages were present in Mymensingh from the very beginning till June 2020. Then, this variant was missing there for 5 months but reappeared in the middle of December 2020. However, the variant was found present during this period in Dhaka and Chattogram. On the other hand, the sub-lineages of Beta variant B.1.351.3 were missing in Sylhet for 2 months from February to March 2021 and appeared again in April 2021, while was present in other divisions during this time. Several other back and forth circulation of strains were observed, for example, AY.100 and AY.102 in Dhaka. Detailed circulation of the variants information is provided in the supplementary file .Finally, we have built a viral transmission network using all our analysis data set sequences. Dhaka was found to be the center of viral transmission and directly connected with all other locations, while others were not. For example, we did not find any direct connection between Chattogram with Khulna and Mymensingh, Rangpur with Khulna, and Barisal did not have any connection with Sylhet . In addith February 2022, and the lowest number was only one found in a sequence from 11th May 2021. Up to the present study, we have found 7,659 unique mutations present in 4,692 sequences where 482 were extragenic mutations, and the rest were in the coding regions. In the coding region, a total of 4,103 missense, 2,865 synonymous, ten insertion, 125 deletion and 74 premature stop codon mutations were observed . MoreoveORF10 and ORF7a harbored the highest mutation density with 0.453 and 0.451 mutations per base, respectively, although only 8.05% of sequences were found to carry mutations in ORF10. On the other hand, 99.98% and 99.96% of sequences had mutations in ORF1ab and S genes, but their mutation density was lower at 0.214 and 0.252, respectively. ORF6 was found to be the most stable gene of SARS-CoV-2 in sequences from Bangladesh, with only 16.96% sequences carrying mutations, 0.301 mutations per base and 69.64% missense mutations. ORF3a was identified to harbor the highest percentage (75.69%) of missense mutations. In comparison, the least percentage of missense mutations (42.65%) with 0.223 mutations per base was found in membrane protein-encoding gene M. It was clearly evident that non-structural proteins were subjected to more missense mutations than non-synonymous mutations compared with structural proteins . In addiAmong the 7,786 mutations, 6,968 were SNP, where 4,697 and 2,271 were involved in transition and transversion events, respectively, rendering a transition transversion ratio of 2.07. Transition mutations were calculated to be more prevalent than expected if mutational events took place randomly, which clearly revealed the nucleotide substitution bias . Then, tThen, out of the ten most prevalent mutations in Bangladesh, three were extragenic, one was synonymous, and six were missense mutations, where 23403A>G (missense mutation) was the highest prevalent, followed by the second highest 14408C>T (missense mutation) which resembles the global scenario and these two mutations appeared together with 3,037>C>T (synonymous mutation). Among the top seven mutations in the coding region, three were in the spike protein , two were in the ORF1ab (P4715L and F924F), one was in membrane protein (I82T), and another was in ORF3a (S26L). In addition, these seven mutations were highly prevalent throughout the world and were found in multiple lineages of the virus. Furthermore, 1163A>T (nsp2: I120F) was a highly prevalent and unique mutation found in Bangladesh from the beginning of the pandemic, whereas it was absent from other countries during that period. There were more than 21% of sequences containing this mutation. Then, from the linkage disequilibrium analysis (LD), we found that all the mutations mentioned had a perfect correlation (R2 = 1.00) and have been present together since their occurrence . AccordiThe mutations affect viral infectivity, transmissibility, virulence, viral fitness, selection pressure, proteome structure, and evolution. Overall, the SARS-CoV-2 genomes had very high nucleotide identity with an average of 37.64 mutations and low overall nucleotide diversity (π) 0.004. Although overall nucleotide diversity was lower, it varied from gene to gene. For example, ORF8 had the highest nucleotide diversity (0.01543), while gene ORF10 was most stable with a π value of 0.00059 . Analyzip-value of 0.1, we calculated the dN/dS and found that eight of the eleven genes were under negative selection pressure, which signifies the low nucleotide diversity. Additionally, three genes  were under positive selection pressure or directional selection since the mutations present in them were advantageous to them, as a result, their frequencies were increasing. On the other hand, rest of the genes were experiencing negative evolution pressure to eliminate the deleterious mutations that they have acquired from random mutations. It is likely that eighty-two percent of the 7,659 unique mutations were present in sequences below ten, possibly due to their deleterious effects on the virus, and that these mutations were gradually purged by the purifying selection pressure. This higher number of mutations with low frequency is also indicative of a demographic process known as population expansion, which might have resulted in a reduction in the overall genetic diversity. Following that, we thoroughly analyzed the specific sites under selection pressure using the FEL, and FUBAR methods. There were only 47 sites that were under positive or divergent selection pressure compared to 190 sites that were under negative or purifying selection , was dominant. However, In April 2021 Delta variant emerged and dominated other variants until the arrival of Omicron. Omicron variants BA.1 and BA.2 are found in Bangladesh, and currently, BA.2 is the dominant variant., 20 distinct mutations in the spike protein differentiate the two sub-lineages, and BA.2 displays a marked decreased sensitivity to many neutralizing monoclonal antibodies (mAbs) when compared to previous VOCs . TherefoOn the other hand, geographical analysis depicts Dhaka and Chattogram containing a more diversified number of sequences than other parts of the country. Our analysis has limitations at this point because we had a higher number of sequences from these two areas than others. The sequences were more diversified in the first phase of the pandemic. However, with the arrival of the Delta and Omicron variants, the divergence reduced drastically, maybe due to viral adaptation following the “Survival of the fittest” theory of natural selection. Additionally, we have seen Dhaka being the viral transmission hub, which is obvious since it is the capital city of Bangladesh. From extensive analysis, we have built the SARS-CoV-2 transmission network between different administrative divisions and observed the back-and-forth circulation of the virus inside the country. This situation arose due to a lack of restrictions on mass movement; public gatherings and other socioeconomic events.From the mutational perspective, we have seen 37.64 mutations per sample where on average 24.61were coding variants, which happens to be significantly higher than the global average of 7.23, reported in July 2020 . This shMoreover, we have observed that most of the genes were under negative selection pressure while only three non-structural protein-coding genes were under Darwinian (positive) selection, indicating that most of the random mutations were deleterious for SARS-CoV-2 , which cTo sum up, considering the limitations regarding sequence number variations in different parts of the country, we have thoroughly studied the virus circulation trend and analyzed all the mutations present, which are comprehensively reported in the supplementary files. This data would further facilitate researchers from various perspectives like investigating viral transmission, the connection among isolates, evolution patterns, and dynamics of divergence of the virus.


This study aims to develop a simple, sensitive, low-cost, environmentally friendly and flexible surface-enhanced Raman scattering (SERS) platform, combined with a portable Raman spectrometer, for the rapid and on-site SERS detection of bacteria. Commercial tobacco packaging paper (TPP) with little background interference was used as a loading medium that effectively adsorbed Au nanoparticles and provided sufficient “hot spots”. This Au-tobacco packaging paper (Au-TPP) substrate used as a flexible SERS platform can maximize sample collection by wiping irregular surfaces, and was successfully applied to the on-site and rapid detection of pathogenic bacteria. Raman fingerprints of pathogenic bacteria can be obtained by SERS detection of spiked pork using wipeable Au-TPP, which verifies its value in practical applications. The results collected by SERS were further verified by polymerase chain reaction (PCR) results. It showed several advantages in on-site SERS detection, including accurate discrimination, simple preparation, easy operation, good sensitivity, accuracy and reproducibility. This study indicates that the established flexible SERS platform has good practical applications in pathogenic bacterial identification and other rapid detections. In recent years, the detection of pathogenic bacteria has been of great significance to human health ,2,3. Unt9 [Typical SERS substrates are based on rigid substrates such as silicon and glass, which are not suitable for sample collection by physical wiping ,21,22,239 . Several9 ,36,37. V9 ,41,42. V9 . Oliveir9 . Torul e9 . Importa9 . However9 ,50,51,529 ,54. Seco9 ,56. TherTobacco packaging paper (TPP) is a specially designed thin paper with high longitudinal tensile strength, air permeability, and good biocompatibility ,58. The Herein, the development of a simple, flexible, sensitive, low-cost, and environmentally friendly TPP-based enhanced substrate for rapid and on-site SERS detection is discussed. The gold nanoparticles (Au NPs) used as enhanced substrates were precipitated or centrifuged onto the surface of commercial tobacco packaging paper. The formation of “hot spots” was controlled by adjusting the amount of Au NP attachment. The prepared flexible SERS substrate was successfully applied to detect pathogenic bacterial contamination in commercial pork. The Raman characteristic fingerprints of pathogenic bacteria can be easily obtained and accurately distinguished. The established flexible SERS platform combined with a portable Raman spectrometer is suitable for large-scale and on-site market screening. This shows the feasibility of rapid and culture-free identification of pathogenic bacteria in food safety and clinical testing.Noble metal colloids are commonly used to enhance substrates for SERS detection due to their good enhancement effects and easy preparation ,62. HoweIn this study, we synthesized a Au NP colloid and then used it to prepare the Au-tobacco packaging paper-based enhanced substrate (Au-TPP) by the immersion method and the centrifuge method . As show−5 M was selected as the probe molecule to examine the SERS enhancement abilities of the Au-TPP substrates under different immersion times. As shown in The immersion method is one of the most common methods for preparing flexible substrates based on fiber paper ,66. The Following on from the immersion method, the preparation of the flexible substrate was improved, and an active SERS substrate with good uniformity and high stability was successfully prepared using the centrifugal method. In the process of centrifugal preparation, it was found that different preparation conditions would affect the concentration, aggregation and distribution of metal nanoparticles on the surface of the substrate, thereby affecting the SERS enhancement effect, as shown in The immersion method has disadvantages such as being time consuming, requiring large amounts of colloidal Au NPs, having high costs, and the inevitable coffee-ring effect between the Au NPs and the substrate. When compared with the immersion method, the centrifugation method is simpler and faster to prepare, and it only takes 10 min to prepare the Au-TPP substrate with a better enhancement effect. The preparation of SERS substrates by centrifugation also enables a controllable amount of Au NPs to be attached to the paper surface. According to the SEM image e, it is −1 of the corresponding point of CV is 6.83% . The substrates were characterized every 10 d with CV as the probe molecule, and the enhancement effect was recorded to investigate their stability. With the increase in storage time, the SERS signals of the Au-TPP substrates were stable, and the RSDs of the characteristic peaks of CV at 722 cmthan 10% e,f. ThisStaphylococcus aureus and Shigella flexneri as model pathogens, and collected bacteria by swabbing the surface of spiked pork  of two randomly selected strain samples. The three principal components PC1 (45.982%), PC2 (19.339%) and PC3 (12.425%) were obtained, and their cumulative contribution rate can reach 77.75%. Therefore, the discriminant classification model based on these three principal component factors is representative. The results are shown in nuc gene encodes a thermostable nucleotidase produced by pathogenic staphylococci, which can realize the rapid identification of S. aureus. The ipaH gene is the most important virulence pathogenic gene in Shigella. The PCR results show that the nuc gene is expressed in the S. aureus strain and absent in S. flexneri; the ipaH gene is expressed in the S. flexneri strain and absent in S. aureus . Tryptone soy broth (TSB) and nutrient broth (NB) were purchased from Beijing Land Bridge Technology Co., Ltd. . Chloroauric acid (HAuCl4) and nitric acid (HNO3) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Sodium citrate dihydrate (C6H5Na3O7) was purchased from Alfa Aesar . Hydrochloric acid (HCl) was purchased from Beijing Chemical Works. Crystal violet (CV) was purchased from Amresco . All experimental water was ultrapure water produced by the Millipore system . All glassware used in the experiment was immersed in aqua regia (HCl: HNO3 =3:1).The tobacco packaging paper was kindly gifted from the China National Pulp and Paper Research Institute . 2) was soaked in Petri dishes containing colloidal Au NPs (2 mL) for different times . For the centrifugation method, tobacco packaging paper (circular paper sheets with a diameter of 2 cm) was fixed at the bottom of centrifuge tubes and 2 mL of colloidal Au NPs was added to each centrifuge tube. The centrifuge tubes were placed in a high-speed centrifuge  at 10,000 rpm for 10 min, and then the supernatant was removed. The Au-TPPs were placed in a vacuum freeze dryer  and oven-dried for 1 h, respectively. This operation was repeated 1–2 times in parallel to prepare the Au-TPP flexible substrates under different conditions. Finally, the Au-TPP flexible substrates were placed in vacuum-sealed storage at 4 °C.The colloidal gold nanoparticles (Au NPs) used as an enhanced substrate were synthesized in our laboratory according to the method by Wei et al. . In brieThe position of the plasmon resonance peak of colloidal Au NPs can be detected by a UV-Vis spectrometer . The scanning range was set as 200–1000 nm with a scanning speed of 600 nm/min. The morphology and size of the Au NPs were observed using a transmission electron microscope , with an accelerating voltage of 75 kV and an electric current of 40 mA. The nanostructure and surface morphology of the Au-TPP substrates were characterized using a scanning electron microscope .−1 and a scanning range of 250 to 1800 cm−1, was used to collect SERS spectra from different positions on the Au-TPP substrate.A portable laser Raman spectrometer  equipped with a 785 nm laser, utilizing 250 mW laser power, a spectral resolution of 4 cmS. aureus was inoculated in TSB medium, and the bacteria were placed in a 37 °C incubator and shaken at 110 rpm/min. When the optical density value of the bacterial liquid at 600 nm wavelength was 1 (OD = 1), determined using a UV-Vis spectrophotometer, the cultivation was stopped and used for standby. After centrifuging 1 mL bacterial solution at 8000 r/min for 5 min, the supernatant was discarded, and sterilized water was added and centrifuged again. S. flexneri was inoculated in NB medium, and the same operation was repeated as with the treatment of S. aureus.S. aureus and S. flexneri were selected as the model pathogens, 200 μL of S. aureus or S. flexneri bacterial solution was applied to the surface of the pork, and the surface of the spiked pork was wiped with Au-TTP to collect bacteria. The number of wipes depended on the condition of pork contamination, generally 3–5 times, and the SERS test was required after each wipe.Pork mince samples were purchased from a local supermarket. First, 5 g samples of pork minced meat were soaked in 10 mL of a 10 mmol/L phosphate buffer. Pathogenic bacteria were collected from the Au-TPP substrate. Firstly, the Au-TPP substrate was rinsed with ultrapure water for about 3 min. Secondly, the ultrapure water containing colonies was centrifuged at 7000 rpm/min for 5 min. Finally, the bacterial pellet was suspended in 50 μL of ultrapure water to prepare the bacterial suspension used in the experiment.nuc gene and the ipaH gene. The reaction conditions of the two genes were pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 45 s, annealing at 64 °C for 45 s, extension at 72 °C for 1 min and end extension at 72 °C for 10 min. The specificity of the two genes was designed by Primer software  was used to extract the DNA of the bacterial liquid, and PCR technology was used to detect the software .In this study, a flexible paper-based Au substrate (Au-TPP) was prepared by a simple and fast method, showing the characteristics of high sensitivity, good uniformity, reproducibility and stability. The selected tobacco packaging paper itself has lower Raman peaks, which can reduce the background interference for the test object. Au NPs are regularly attached to the surface of paper fibers to significantly enhance the Raman signal of pathogenic bacteria. Due to the good biocompatibility of Au-TTP, rapid detection of pathogenic bacteria can be achieved, and an effective platform for screening and detection of microorganisms is also provided. When combined with portable Raman spectroscopy, this Au-TTP substrate is expected to be used as a next-generation portable biosensor for on-site pathogen detection in daily life.


Hepatocellular carcinoma (HCC) is one of the most deadly and common malignant cancers around the world, and the prognosis of HCC patients is not optimistic. ZNF320 belongs to Krüppel like zinc finger gene family. However, no studies have focused on the influence of ZNF320 in HCC. We first analyzed ZNF320 expression in HCC by using data from TCGA and ICGC, then conducted a joint analysis with TIMER and UALCAN, and validated by immunohistochemistry in clinical HCC samples. Then we applied UALCAN to explore the correlation between ZNF320 expression and clinicopathological characteristics. Consequently, using Kaplan-Meier Plotter analysis and the Cox regression, we can predict the prognostic value of ZNF320 for HCC patients. Next, the analysis by GO, KEGG, and GSEA revealed that ZNF320 was significantly correlated to cell cycle and immunity. Finally, TIMER and GEPIA analysis verified that ZNF320 expression is closely related to tumor infiltrating immune cells (TIIC), including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. The analysis of the TCGA and ICGC data sets revealed that ZNF320 expression was significantly correlated with m6A related genes . In conclusion, ZNF320 may be a prognostic biomarker related to immunity as a candidate for liver cancer. There is evidence that hepatocellular carcinoma is a tumor with a poor prognosis and the third primary cause of cancer death in the world. Globally, the number of deaths due to HCC is estimated to be 810,000 per year, while there exit about 854,000 new cases of HCC per year , 3. The As the largest transcription factor family in the human genome, the ZNF family is widely involved in various biological processes in the human body, and zinc finger (ZNF) transcription factors are characterized by finger-like DNA domains, which require one or more zinc ions to stabilize the structure. Previous research has established the possibility that ZNF is a dominant factor in the occurrence and development of HCC  that ultThe research data in this thesis is drawn from four main sources: TCGA, GTEx, UALCAN databases, and patients and tumor specimens from 35 patients undergoing nephrectomy in the Second Affiliated Hospital of Nanchang University from June 2017 to January 2021. In this study, we dissected the expression of ZNF320 mRNA and protein in HCC and assessed the correlation between the expression level of ZNF320 and prognosis of HCC. Besides, we also investigated the possible mechanism of high expression of ZNF320 in HCC and the relation of ZNF320 expression and cell cycle, tumor infiltrating immune cells  in HCC phttps://dcc.icgc.org/projects/LIRI-JP). [LINC-JP] Liver Cancer - NCC, JP datasets included 202 normal samples and 243 tumor samples [Hepatocellular carcinoma (HCC) clinical and mRNA expression level was collected from the TCGA Database. In terms of the gene expression profile, this study included 374 LIHC samples and 50 normal samples, and the data type of mRNA expression profile was HTSeq-FPKM. We obtained clinical information from 377 patients. In addition, RNA-seq data and clinical information were also acquired from the ICGC website  is a synthetic web used for analyzing the levels of immune invasion in diverse cancers, which includes 32 cancer types. In this work, we use “Diff-Exp module” to confirm the expression of ZNF320 in diverse tumors. Then the “gene module” was used to estimate the relationship of ZNF320 with immune infiltration. Moreover, the “SCNA module” was used to compare the level of tumor invasion among tumors with different somatic copy number changes for ZNF320. Finally, we considered the correlation between ZNF320 expression in TIICs and immune markers by using related modules. The difference between infiltration level and normal level was evaluated for each SCNA category through Wilcoxon rank-sum test. Finally, with the help of the “Correlation module”, considering the Spearman’s rho value and predicted statistical implications, we verified the relationship between ZNF320 and tumor infiltrating immune cell markers in HCC .Cut formalin-fixed, paraffin-embedded HCC tissue into 4 μm-thick sections. After deparaffinization, rehydration, and microwave heating in microwave-heated antigen unmasking solution  to repair the antigen, 30 minutes in total was used to seal the sections with goat serum. Next, incubate the sections overnight with anti-ZNF320 monoclonal antibody  at 4° C. After that, HRP-conjugated secondary antibody (Boster) was allowed to stand at room temperature for 2 hours. Subsequently, the two-step method  were used for immunostaining. Finally, three pathologists who are unaware of the clinical parameters assessed the staining intensity and the percentage of positive cells semi-quantitatively.UALCAN database is a tumor data on-line analysis website that provides comprehensive cancer transcriptome and clinical patient data (extracted from TCGA) . In our LinkedOmics, a comprehensive online site, is usually chosen to analysis multidimensional data within and across 32 kinds of cancer. Using it, we succeeded in mining the co-expressed genes linked to ZNF320 in the TCGA LIHC database through the results of analysis. Volcano plots and heat maps provided strong evidence for this. We define the top 500 related genes as co-expressed genes.Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.8 is a comprehensive, functional annotation website, which enables us to study the biological functions and signal pathways of specific gene sets. Gene annotation includes Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis .The cBioPortal database provides visualization, analysis, and download of large-scale tumor genome data for a variety of cancers. We used it to study the association between ZNF320 promoter methylation level and expression in HCC with “Plot module” .We used GSEA to conduct a group study on the RNA-sequencing data of 374 TCGA-LIHC patients downloaded from Genomic Data Commons, analyzing the pathways associated with ZNF320 expression in HCC specimen. The number of permutations: 1000; normalized enrichment score (NES) >1; false discovery rate (FDR) <0.25, p-val<0.05 .STRING database is an online tool for evaluating protein interactions in multiple ways. In our research, the top 500 genes with co-expression coefficients greater than 0.4 were collected from the STRING database. They were evaluated by Cytoscape 3.8.2 and its plug-in MCODE (Molecular Complex Detection). And the selection criteria are as follows: Max depth=100, node score cutoff=0.2, K-core=2 .Hub genes are highly connected to the nodes in the module and have been certificated to play an important role in function. The significance of the genes was measured by the absolute values of the Spearman’s correlation in our study. On the basis of the results, the top 500 genes with confidence > 0.9 were uploaded to the STRING database to construct protein-protein interaction (PPI). Then, we used Cytoscape 3.8.0 and its plug-in MCODE to evaluate the top 500 genes. Furthermore, a standard for hub genes (yellow nodes) was set with a degree cut-off = 2, node score cut-off = 0.2, k-core = 2, and max. depth= 100, which was screened with MCODE. In total, there were 6 genes scoring the highest and initially defined as hub genes. Ultimately, after digging out their backgrounds, three genes, closely related to the cell cycle, were considered to be a “real” hub gene among these genes.GEPIA , an inteAll statistical analysis in our work was done by R software (version 3.6.3). The detection of different ZNF320 expression levels between LIHC samples and normal samples was realized by using the “limma” and “beeswarm” packages of “R” and rank sum test method. We probed the relevance between ZNF320 expression and clinicopathological feature by Wilcoxon signed-rank test or Kruskal-Wallis test. Then, Kaplan-Meier survival curve using log-rank test to explore the survival distribution among patients with different levels of expression was drawn to evaluate whether the differential genes expression had a significant influence on the prognosis of patients (p<0.05). Univariate and Multivariate Cox regression analysis screened factors significantly related to prognosis (p<0.05) . Lastly, we used the ROC curve drawn by “survival ROC” to evaluate the predictive ability of ZNF320 expression level on one-year, three-year, or five-year survival. T-test was used to study the different expression of m6A interrelated genes between ZNF320 high and low expression groups.The expression profiling of genes from diabetics and diabetics nephropathy can be available from the GEO (GSE142153 and GSE26168). The other data can be obtained from the corresponding author (Bo Zhang).To compare the differential expression of ZNF320, we first studied the expression of ZNF320 in LIHC and other tumors by using TIMER database. Expression of ZNF320 in liver hepatocellular carcinoma was predicted to be vitally upregulated . Next, wAfter the expression of mRNA and protein were found to increase in HCC patients, we next used UALCAN to integrate various clinic factors of HCC samples, including patients’ individual tumor grade, cancer stages, gender nodal metastasis, age, and weight. Then we compare the expression levels of ZNF320 in each group. The result exhibited that with grade increasing, the patients had statistically higher expression of ZNF320.  AdditionTo further find the potential role of ZNF320 expression in TCGA patients with Hepatocellular Carcinoma, we used Kaplan Meier survival method for survival analysis. We found via the KM plotter that high expression of ZNF320 patients had shorter OS and DSS times than low expression  , 3B. TheSignificant increases of ZNF320 expression in HCC were observed. In consequence, we will study the reason for the overexpression of ZNF320. As methylation plays an important role in gene expression. we studied the methylation and expression of ZNF320 through the cBioPortal dataset. The finding was that ZNF320 expression was highly positively correlative with methylation  in HCC . We deteIn conclusion, the methylation level of ZNF320 was positively associated with its expression. It is supposed that the increased methylation level of ZNF320 promoter leads to the high ZNF320 expression in liver cancer, which in turn causes poor prognosis in patients with liver cancer.p-value < 0.05). The top 50 obvious gene set positive (left) and negative (right) correlation with ZNF320 were present in the heatmap.  were present by the hipplot. Analysis using the GO database showed that ZNF320 was functionally associated with G2/M transition of mitotic cells, Cell-cell adhesion, Regulation of transcription, Cell division, Cadherin binding involved in cell-cell adhesion, DNA binding, Transcription factor activity  network, and Cytoscape (MCODE plug-in) was applied to find the most vital module. What’s more these genes were highlighted in yellow . Based oIt has been confirmed that the occurrence and development of tumors and their prognosis depend on the number of immune cell infiltration. Meanwhile, KEGG and GSEA suggest that ZNF320 is associated to immune infiltration. Through analyzing whether ZNF320 expression was associated to immune infiltration levels in HCC, we discovered a positive correlation between ZNF320 expression and tumor purity, infiltrating levels of B cells, CD8+ T cells, CD4 + T cells, Macrophage, Neutrophils, and Dendritic cell . FurtherTo further explore the underlying correspondence of ZNF320 and different immunocyte, TIMER was used to analyze correlations between ZNF320 levels and multiple gene markers of immune cells, which included TAM, M1 and M2 macrophages, CD8+ T cell, T cell and B cell in HCC . BecauseMoreover, ZNF320 was also associate with the HCC-related chemokines including CCL15, CCL8, and CCL26. It could be known from the results that ZNF320 was negatively associated with CCL15 and CXCL2 while positively associated with CCL26 . TherefoThese results indicated that ZNF320 could affect immune cell infiltration partly by regulating these chemokines expression.Because ZNF320 expression was significantly correlated with immune infiltration, which is related to prognosis in HCC, we analyzed whether ZNF320 expression influences the prognosis of HCC by influencing immune infiltration. The prognosis was analyzed based on the expression level of ZNF320 in HCC related immune cell subgroup. As shown in Modification of m6A plays a significant role in HCC. By analyzing TCGA and ICGC HCC data, we detected the correlation between the expression of ZNF320 and the expression of 21 m6A related genes in HCC, and ZNF320 expression significantly positively correlated with ZNF320, RBMX, RBM15B, LRPPRC, YTHDF1, HNRNPC , P<0.01 We divided TCGA samples into two groups according to the expression of ZNF320. We tried to exam the differential expression of genes related to M6A between the high and low ZNF320 groups. As shown in Liver cancer is the fifth most common type of cancer worldwide, it is also the second leading cause of death from cancer . Due to With the purpose of detecting the expression and prognosis of ZNF320 in HCC, first through public database analysis, we noticed that ZNF320 expression in HCC tissues was highly increased compare with adjacent normal tissues, and we applied clinical samples to verify it. Secondly, we applied Kaplan-Meier survival analysis established that ZNF320 is one of the factors of poor overall survival and prognosis in HCC patients. At the same time, results showed that ZNF320 has been associated with many clinicopathological parameters, including tumor grade, tumor stage, and so on. Finally, univariate Cox analysis showed that ZNF320 was pointedly related to overall survival (OS). Variational calculation Cox analysis revealed that ZNF320 could be regarded as an independent predictor. At the same time, we explore the connection between ZNF320 methylation and ZNF320 expression through the cBioPortal dataset . ResultsWith the purpose of further exploring the function and mechanism of ZNF320 in HCC, we explored the co-expressed genes of ZNF320 in HCC through LinkedOmics, used GO and KEGG to perform functional analysis of the co-expressed genes, and performed GSEA analysis on ZNF320. The results indicated that the functional classification and KEGG pathway related to ZNF320 included: “FC epsilon pathway”, “FC gamma R mediated phagocytosis”, “cell cycle”, “WNT signaling pathway”, “pathway in cancer”, “leukocyte transendothelial migration”, “mismatch repair” and so on. In the results of GSEA, we noticed that ZNF320 can play a role by participating in DNA mismatch repair, so we deeply explored the correlation between ZNF320 and mismatch repair proteins, and the results showed that the expression of ZNF320 was associated with MSH2, MSH6, MLH1 and PMS2. All were positively correlated –1D, and The cell cycle is a highly regulated process that enables cell growth, replication of genetic material, and cell division . In cancWhat’s more, the disorder of the cell cycle is also related to the invasion, and migration of tumors. Through EdU and transwell assays, we found that interfering with the expression of ZNF320 could obviously inhibit the proliferation, invasion, and migration of HCC cells and downregulate the protein expression of CDK1, CDC20 and CCNB1. In summary, this experiment showed that down-regulation of ZNF320 can inhibit cell proliferation, invasion, and migration of HCC cells, which may be related to regulating the cell cycle signaling pathway.With the advent of immunotherapy , 37, a sN6-methyladenosine, also called m6A, refers to the methylation of the sixth N position of adenylate in RNA, which mainly regulates RNA transcription, splicing, maturation, stability, translation, etc. The effect of m6A modification on human cancer has been confirmed in different cancer types , and theIn conclusion, our research suggests that ZNF320 may be a possible biomarker for poor prognosis of hepatocellular carcinoma. ZNF320 may not only take part in cell cycle regulation and affect the proliferation of HCC, but also may assume the role in the microenvironment of HCC by regulating tumor infiltrating immune cells. In addition, the results claim that ZNF320 may be closely associated with the m6A modification of HCC, which eventually influents the progression and prognosis of HCC. These put forward that ZNF320 may serve as a target for early clinical diagnosis and treatment which may have a chance to improve diagnostic and therapeutic options, and at the same time provides a reference for further exploration of new cancer immunotherapy.Supplementary FiguresSupplementary Table 1


Saccharomyces cerevisiae. Later, possible pollutants were mined using ultrahigh-performance liquid chromatography (UPLC) coupled with a Bruker impact II Q-TOF-MS. Possible hits were identified using MetaboScape software (4000 compounds), which includes pesticide, pharmaceutical pollutant, veterinary drug, and toxic compound databases and a special library of 75 possible xenoestrogens. The presence of xenoestrogens in vegetable samples collected from two different locations was also investigated. The total estrogen equivalents according to the YES system were 2.9 ± 1.2, 9.5 ± 5, 2.5 ± 1.5, 1.4 ± 0.9 ng/L for King Talal Dam, As-Samra Wastewater Treatment Plant, King Abdullah Canal, and tap water, respectively. In Almujeb Dam and drinking water, the estrogenic activity was below the detection limit. Numbers of identified xenoestrogens were: As-Samra Wastewater Treatment Plant 27 pollutants, King Talal Dam 20 pollutants, Almujeb Dam 10 pollutants, King Abdullah Canal 16 pollutants, Irbid tap water 32 pollutants, Amman tap water 30 pollutants, drinking water 3 pollutants, and vegetables 7 pollutants. However, a large number of compounds remained unknown. Xenoestrogen pollutants were detected in all tested samples, but the total estrogenic capacities were within the acceptable range. The major source of xenoestrogen pollutants was agricultural resources. Risk evaluations for low xenoestrogen activity should be taken into account, and thorough pesticide monitoring systems and regular inspections should also be established.Sex hormone disruptors (xenoestrogens) are a global concern due to their potential toxicity. However, to date, there has been no study to investigate the presence of xenoestrogen pollutants in the Jordanian water system. Samples in triplicates were collected from six locations in Jordan, including dams, surface water, tap or faucet water, and filtered water . Xenoestrogens were then extracted and evaluated with a yeast estrogen screen utilizing  Water treatment and pollution are major concerns worldwide, i.e., heavy metals and (recently appearing) estrogen and xenoestrogen pollutants. Xenoestrogens are estrogen mimics . Many xeXenoestrogens as well as estrogen pollutants are not completely removed during the process of sewage treatment and are carried over into the general aquatic environment. After ground passage, they can eventually be found in drinking water ,7,8,9. AXenoestrogens are widely diffused in the environment, water, and in food, and thus a large portion of the human population is exposed to them worldwide . XenoestThe scarcity of water is the greatest challenge that Jordan faces. On a per capita basis, Jordan has one of the lowest levels of water resources in the world. In addition, the situation has been exacerbated by periodic massive influxes of refugees, worsening the imbalance between population and water. Despite the presence of some surface water supplies, collectible rain is the main source of water in Jordan via dams, rivers, lakes, and groundwater . To collThe pollution of estrogen and xenoestrogen in Jordan should be taken seriously, especially after it has been detected and determined in several places around Jordan, including Mediterranean coastal water , Jordan Chemicals were purchased from Sigma-Aldrich, and 17-β-estradiol stock solutions were prepared in methanol (1 µg/µL) and stored at −20°C.The water system in Jordan is complex and composed of surface water  and groundwater basins . The JorWater sample collection was conducted as described previously . All glaSamples were filtered through glass fiber or glass wool (pore sizes of 0.3–1.2 µm) before solid-phase extraction was carried out. The filtered samples were loaded with a flow rate of 5.5–6 mL/min into reactivated reversed-phase C-18 cartridges . Later, the cartridges were washed with 10 mL of methanol in water (1:1), followed by 10 mL of acetone in water (1:2).There were two methods: one for the analysis using yeast estrogen assay (YES) and the other for liquid chromatography–mass spectrometry (LC-MS) analysis.Samples for YES: 100 µL DMSO was added, and the acetone and methanol were evaporated under a gentle stream of nitrogen.Samples for LC-MS screening: samples were dried under nitrogen gas (99.99%), reconstituted in 100 μL methanol, completed to 50 mL by acetonitrile, and then centrifuged at 4000 rpm for 2.0 min. Finally, 1.0 mL of the sample was transferred to the autosampler.The sample preparation was conducted via salting-out assisted liquid–liquid extraction (SALLE) as described previously . Five miAssays were conducted as described previously ,37,38,39Data analysis was conducted as described previously . Dose–reAll samples were analyzed using LC-MS/MS. Assays were conducted as described previously . For anaThe instrument was operated using the Ion Source Apollo II Ion Funnel electrospray source. The capillary voltage was 2500 V, the nebulizer gas was 2.0 bar, the dry gas (nitrogen) flow was 8 L/min and the dry temperature was 200 °C. The mass accuracy was ˂1 ppm, the mass resolution was 50,000 FSR (full-sensitivity resolution) and the TOF repetition rate was up to 20 kHz.Standards for identification of ms/z with high-resolution Bruker TOF MS and the exact retention time of each analyst after chromatographic separation was used. Later, all possible hits were identified using MetaboScape software (more than 4000 compounds) which includes a list of potential pharmaceutical pollutants, a list of potential veterinary drug pollutants, a list of potential insecticides, pesticides, and herbicides pollutants, and an additional special library  of possible xenoestrogens. All pollutants are listed in 3 of wastewater, serving a population of 2.2 million living in the Amman and Zarqa areas [3/s and a tail discharge capacity of 6 m3/s. [Due to the governmental restriction from the Ministry of Water and Irrigation, the research was restricted to designated sites: two dams , one wastewater treatment plant (As-Samra), and one canal . King Talal Dam’s (the largest dam in Jordan) main purpose is to retain winter rainfall and treated wastewater processed at the As-Samra Wastewater Treatment Plant and is used for domestic, agricultural, and industrial uses and to control floods, improve drainage, and collect water from rivers and streams ,42, whilqa areas . King Ab 6 m3/s. . Dair Al 6 m3/s. .The estrogenic activity via yeast screening assay of each water sample measured as 17β-estradiol relative estrogenicity is summarized in Jordan’s primary sources of tap water are surface water and groundwater wells . For exaThe relative estrogenicity of tap water (1.4 ± 0.9 ng/L) is lower than that reported in other countries ,52,53,54In the LC-MS experiment, a high-resolution database included exact mass data for parent ions, adducts, fragment ions as well as isotopic pattern and retention time. The potential source of each xenoestrogen . In both circumstances, an interaction between xenoestrogens and tap water is feasible . It appeIn Jordan, water is disinfected using various disinfectants (such as chlorine), which may cause interactions with organic matter to produce disinfection by-products  . NaturalThe data show that open-source water has estrogenic activity of 2.5–9.5 ng/L and is not far from most countries in the world, including bordered countries ,46,47,48Raw wastewater has different characteristics in Jordan from most other countries. According to reports, Jordan’s wastewater is very strong, highly salinized, contains heavy metals, and contains toxic organic compounds. Furthermore, Jordan’s raw wastewater contains large organic contents resulting from low home water consumption and industrial waste .The estrogenic activity of the effluent of As-Samra treatment plant was 9.5 ± 5 ng/L. This value is not far from recorded around the world. For instance, the estrogenic activity was 0.4–6.6 ng/L in England, 5–10.6 ng/L in the USA, 5.6–11 ng/L in Germany, 2.9–7.5 ng/L in Italy, 17.9 ng/L in Switzerland and 29–65 ng/L in Brazil ,97,98. AThe distribution of detected xenoestrogens sources in the open-source water is shown in It is clear that Almujeb water has the least detected compounds and that may be explained by the location, where the dam and collected water are far from human pollution, such as heavy traffic and industrial areas . TherefoOn the other hand, King Talal Dam has easy access to people and is located in a crowded place surrounded by farms and agricultural areas. Furthermore, the dam retained the treated wastewater processed in the As-Samra Wastewater Treatment Plant. Hence, the pollutants from residents around the dam, the farms, and the treated wastewater augment the pollution in King Talal Dam. This may explain the presence of herbicides, pharmaceutical compounds, and natural products.It has previously been claimed that some chemicals, such as phenolic compounds, may be released from the pharmaceutical sector or other companies engaged and located in King Talal Dam’s catchment area . MoreoveIn case of King Abdullah Canal, residents toss waste and trash into and around it, and the presence of intermittent gatherings of migrant workers who live in the neighborhood exacerbates the problem. The continuous dumping of waste and its accumulation may explain the presence of such pollutants in the canal. Moreover, previous incidents of contamination have been reported . It has A possible explanation for the low level of xenoestrogens in all samples is that the sample collections were conducted during the winter, which may dilute the xenoestrogens. In addition, degradation, evaporation, and adsorption are potential mechanisms contributing to lowering the activity of xenoestrogens .Regarding drinking water, private stations have more purification systems with additional filters: sand filter, carbon filter, iron removal filter, and microfilter. In addition, the stations perform sterilization using ultraviolet radiation. Hence, the potential estrogenicity was low, below the detection limit of the YES assay (1 ng/L). However, several compounds  were detected by LC-MS and identified by the MetaboScape database .Despite estrogenic activity not being detected in drinking water using YES, the existence of xenoestrogen cannot be ruled out, because its concentration was below the YES detection limit. More sensitive screening methods have recently been developed, which can detect levels as low as 14.7 pg/L of estrogen equivalents . EstrogeDetected xenoestrogens are listed in Carvone is a monoterpene compound that is widely used as an insecticide, fungicide, antioxidant, and plant growth regulator . For insMercaptobenzothiazole, in addition to being utilized in pesticide manufacturing, is also employed as a sulfur vulcanization accelerator in the synthesis of rubber plumbing items such as gaskets and O-rings, which are essential parts of water networks . Mercapt8H17NO2) as pregabalin , DL-2-aminooctanoic acid has been identified in various foods, including chicken and cow milk, and has been used as a potential biomarker for the consumption of these foods. As a result, it is more likely that the pollutant came from food metabolites rather than the anticonvulsant medicine, especially as DL-2-aminooctanoic acid is not found in the MetaboScape library. Although it is unlikely to have pregabalin in drinking water as explained previously, it has been detected in several rivers around the world [The existence of pregabalin, which is used to treat neuropathic pain and convulsions, remains unknown. Pregabalin was detected in all water samples; however, its half-life has been reported to be 8 to 10 days in the aerobic environment , implyinegabalin . Hence, he world .The identification of xenoestrogen contaminants in the water does not always imply direct estrogenic activity, because the mass spectrometry detects traces of xenoestrogens and the estrogenic activity is concentration-dependent . AlthougThe presence of xenoestrogens in vegetables has been previously reported . ConsequFerula genus (Umbelliferae) contains the phytoestrogen ferutinin, which has a modest estrogenic activity [The activity  and is nactivity . TherefoButylparaben—a preservative used in cosmetic products—is not prone to photodegradation and is highly stable against sunlight; however, it is suitable for biodegradation and sorption . The expDibutyl phthalate (DBP) is a plasticizer used to improve the flexibility of plastic products, specifically polyvinyl chloride, which is used in the synthesis of packaging/greenhouse films, wires, pipes, and all flooring materials . MoreoveFusarium species) of small grain cereals and corn, and might be developed under poor storage conditions [Alpha-zearalenol (α-ZEA) is a metabolite of mycotoxin zearalenone that is widespread, particularly in pathogens  is a metabolite of chlorpyrifos , which iThe source of xenoestrogens in the vegetable samples was predominantly from direct interaction throughout the farming process , and the source of xenoestrogens discovered in water was most likely from agricultural resources rather than the other way around. Traces of pesticides have previously been detected in Jordanian fruit and vegetables ,170. FurXenoestrogens are persistent, prevalent substances in the environment that accumulate and may even be further activated by biotransformation, making them hazardous to animal and human health. Xenoestrogens were detected in all water and vegetable samples, including drinking water. However, the estrogenic activity was low and does not constitute an immediate, acute health risk to the community, though there is a necessity to limit the existence of such compounds in the water. The main source of xenoestrogens was agricultural resources . Therefore, proper training and implementation of good agricultural practices should be established, and comprehensive pesticide monitoring systems and frequent inspections should also be enforced. This research may serve as a whistleblower on the estrogenic contamination in the Jordanian water system.Six locations are insufficient to provide adequate data regarding xenoestrogen pollution in the Jordanian water system. The Ministry of Water and Irrigation restricted access to water resources and barred the collection of samples freely. As a result, the number of samples from each location was limited and restricted to certain sites. Furthermore, surface water samples were collected and prepared at the ministry labs, and the samples were subsequently analyzed in the university labs.Future studies must be more comprehensive and include more samples from various locations at each site. Even though there were more than 4000 compounds in the MetaboScape database, still there were many unidentified compounds in the samples. Hence, future work might focus on specific families of xenoestrogens, where the xenoestrogens could be quantified.