AKT1, PRDM4, and BAX are the natural markers of psychological endurance threshold

Abstract Introduction Abnormal behavior can cause harm or loss to oneself, the family, and society and may be related to psychological endurance levels. With early identification and early intervention, the occurrence of harm can be prevented and the loss can be reduced. Now there is no clear definition of psychological endurance levels and no accurate measurement tools yet. Methods This study first proposes the concept of psychological endurance threshold (PET) and defines that as: “the psychological state threshold of human objective physiological characteristics and outbreaks of abnormal behavior led by subjective cognitive level difference”. The study hypothesizes that human behavior is related to it, and constructs multiple measurement method tools to measure it. Results Here we show PET exists objectively and can be measured exactly by methods such as psychological endurance threshold measurement table, experience evaluation, dopamine level detection, and genetic testing. In particular, PET is determined by AKT1, PRDM4, and BAX which are the natural markers of PET. Conclusions The significance of this study is to discover people with abnormal expression of AKT1, PRDM4, and BAX who have lower PET and tend to commit abnormal behavior more easily. Understanding PET will enable people to make self‐adjustment or to intervene by professionals as soon as possible and in a timely manner in the face of various negative stimuli in work and life, especially for people with lower PET, people should intervene as early as possible to reduce the harm to the individual, family and society.


| INTRODUC TI ON
We all know that people have a certain psychological endurance level, but what is its definition, what is the threshold, and how to measure it, have not been studied yet, there are only researches on the relationship between abnormal behavior and negative stimulation.
People often attribute abnormal behavior to constant negative stimulation. If a certain level is reached or exceeded, abnormal behaviors will occur. Some abnormal behaviors can cause great harm or loss to the individual, family, and society. Negative things are also called negative stimuli. Previous studies have conducted relevant experiments to examine the properties and performance of negative stimuli.
Negative stimuli can lead to aversive emotional changes. Disgust is a negative emotional experience caused by unpleasant and annoying stimuli (Rozin, Haidt, & McCauley, 2000). Hoefling et al. (2009), Anderson and Platten (2011, p.463-466), Markovitch, Netzer, andTamir (2016), andOlatunji, Ciesielski, Wolitzky-Taylor, Wentworth, andViar (2012) conducted experiments on people's feelings of disgust produced by feelings of deprivation and negative stimuli. The results showed that if proper intervention cannot be provided for negative events or negative stimuli, abnormal behavior will occur when these stimuli reach or exceed a certain level. Some abnormal behaviors can cause considerable harm. Hegedüs et al. (2014), Schmitz, Vierhaus, and Lohaus (2013), Ryan, Kujawa, Hammill, Le Prell, and Kil (2016), and Szelényi A (Szelényi, Journée, Herrlich, Galistu, van den Berg, van Dijk, 2013) conducted human tolerance experiments on pain, noise, and electrical currents. Black, Gabbett, Cole, and Naughton (2016), Estes (2015, p. 1,174-1,187), andNeuraz et al. (2015) conducted experiments on people's workload tolerance. The results indicated that a tolerance level exists and it varies from person to person. Some researchers have concluded (Leyro, Zvolensky, & Bernstein, 2010) that people who suffer from a low tolerance for pain not only have painful experiences but also expand the conditions that cause pain and respond to other people and the environment. If these reactions are abnormal behaviors, they can cause harm to themselves, their family, and society. Mora, Segovia, Del, de Blas, and Garrido (2012), Vaessen, Hernaus, Myin-Germeys, and van Amelsvoort (2015), and Bosker, Neuner, and Shah (2017) conducted experiments on the release of dopamine under pressure or stress responses. The results showed that neurotransmitters can affect people's stress response, and different pressures can lead to changes in dopamine levels. However, there are individual differences. Cabib, Campus, and Colelli (2012), Zannas, Wiechmann, Gassen, and Binder (2016), McEwen et al. (2015) and others have performed experiments on human genetics and stress response.
The results showed that genetics influences the human stress response. Genetic polymorphism and epigenetics participate in the stress response, resulting in significantly different human responses to stress.
In summary, given that there is no defined concept of psychological endurance levels or measurement methods and tools, this study first proposes the concept of the psychological endurance threshold (PET) for the first time and defines it as "the psychological state threshold of human objective physiological characteristics and outbreaks of abnormal behavior led by subjective cognitive level difference". We conduct experiments including the construction of a psychological endurance threshold measurement table (PETMT), experience evaluation, and dopamine and gene detection to explore the existence and different levels of PET and measurement methods and tools for PET. Understanding PET will enable people to make self-adjustment or to intervene by professionals as soon as possible and in a timely manner in the face of various negative stimuli in work and life, especially for people with lower PET, people should intervene as early as possible to reduce the harm to the individual, family, and society.

| Test subjects
The test subjects were people with normal behavior and abnormal behavior. Two groups of 9,120 volunteers between the ages of 18 and 60 (50% males and 50% females) participated in the experiment.
Consents were obtained for experimentation with human subjects.
The study materials and procedures were approved by Review Board of Chinese Academy of Sciences and Beijing Normal University. The experiment has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.
The normal behavior group was the population currently living under normal social conditions in China (provinces, municipalities, and autonomous regions), including farmers, workers, soldiers, doctors, nurses, teachers, students, scientific and technical personnel, athletes, civil servants, and company employees, as well as some foreign people who worked, studied, and lived in China (e.g., working in Chinese institutions, international students). This population may include some individuals who have encountered negative stimuli or have experienced abnormal behaviors and have caused harm due to reaching or exceeding PET but who had not yet been convicted by the court and served their sentences as well as people who had served a prison sentence and had been released from prison. Since the current technical means cannot accurately distinguish such people, but because it is a large population, such people don't affect the overall result, so the study still assigned these individuals to the normal behavior group.

| Building topics and data acquisition
Psychology research mainly uses psychological measurement, experience evaluation, biochemical indicator detection, brain wave detection, eye movement analysis, nuclear magnetic resonance detection etc. For the accuracy of the study, we used a large population to build a model, so we chose a simple and accurate PETMT. The research group collected negative stimuli content to build topics. Topics were mainly  (Table 1), a table with a total of 15 topics was finally formed (Table 2).

| Statistics
The test data were input in EXCEL and analyzed with SPSS13.0. We used critical ratios (CR values), eigenvalues >1 dimension extraction, principal component method factor analysis, reliability testing, validity testing, differential ANOVA, MANOVA (F value) and regression analysis.

| Test subjects
After thoroughly observing various social strata for many years,

| Data acquisition
Given that pregnant women during the delivery process are already under stress, our research cannot add any additional stimuli and cannot interfere with their delivery process or cause any trauma, so we choose the simple, accurate, and acceptable PETMT.
During the delivery process, the pregnant women completed the PETMT measurement orally. A nurse read the questions, the pregnant women answered the options orally, and the nurse filled out the form.

| Statistics
Same as Method 2.1.3.

| Test subjects
Same as Method 2.2.1.

| Evaluation criteria
Given that pregnant women during the delivery process are already under stress, our research cannot add any additional stimuli and cannot interfere with their delivery process or cause any trauma, so we choose the simple, accurate, and acceptable experience evaluation.
Mr. QC. Jing, a member of the research team, has participated in the rapid selection of flying students in China. Using the experience of three old flying instructors to quickly select students, the project achieved very good practical results, so the experience evaluation system began to be used. However, because it is a confidential project, it has not been published yet. Based on the method used by Mr.
QC. Jing, who used an experience evaluation system to quickly select flying students, a group of three senior doctors or midwives who had worked in the delivery room for 20-30 years was assigned. The doctors and midwives independently gave scores of high, medium, and low according to experience regarding the degree of physical and speech sound response of the pregnant women during the delivery process. The principle for the total score summary was as follows: if three people gave the same score, the total score was the score; if the score of two people was high and one was medium or low, the total score was still high; if one score was high and two people provided scores of medium, the total score was medium; if two people gave medium scores and one person gave a low score, the total score was still medium; if one person provided medium scores and two people provided low scores, the total score remained low.

| Statistics
The scoring system for the experience evaluation was input using EXCEL.

| Test subjects
Same as Method 2.2.1. The duplicate readings for each standard, control, and sample were averaged. A standard curve was constructed by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and a best fit curve was drawn through the points on the graph. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

| Data acquisition
The experiment was repeated three times with three replicates per sample. In the plates detected in the same batch, one plate was randomly taken, and six different samples were selected to calculate intra-assay %CV. In each of the three different batches of plates, one plate was randomly taken, and six different samples were selected to calculate inter-assay %CV. Intra-assay %CV was less than 10%, inter-assay %CV was less than 15%.

| Statistics
The data were processed using SPSS 13.0 and one-way ANOVA.

| Test subjects
After the subjects of Method 2.2.1 were tested for dopamine detection, experience evaluation and PETMT measurement, five individuals were randomly selected from the high PET group and five from the low PET group, for a total of 10 subjects.

| Genetic testing
Given that pregnant women during the delivery process are already under stress, our research cannot add any additional stimuli and cannot interfere with their delivery process or cause any trauma, so we choose the simple, accurate, and acceptable genetic testing.
In order to reflect the physiological characteristics of PET more accurately, we used the method of genetic testing while detecting the dopamine level to clarify the relationship between PET and human genes at the molecular level. First, 1 ml peripheral blood F I G U R E 1 Psychological endurance threshold (PET) score curve of normal and abnormal behavior groups. PET score curve of the normal behavior group was obviously biased toward the right, and the peak was 61 points (out of 100 points). PET score curve of the abnormal behavior group was obviously biased toward the left, and the peak was 52 points (out of 100 points) following the manufacturer's instructions. Slides were scanned by GeneChip® Scanner 3,000 (Cat#00-00212, Affymetrix) and Command Console Software 4.0 (Affymetrix) with default settings. Raw data were normalized by MAS 5.0 algorithm, Affy packages in R.

| Data processing
Fold change values for genes were calculated as the ratio of the signal values of the group with low PET compared with the group with high PET. Differentially expressed genes were screened according to a gene expression changed significantly p < 0.01. Then, KEGG function enrichment analysis was performed using DAVID and KEGG database. The selection criteria were the number of differentially expressed genes on a certain term >2, and p < 0.05.

| PET can be accurately measured by PETMT
This study adopted negative stimuli of life as the content, measured PET of normal and abnormal behavior groups, explored differences in cognitive ability and effective discrimination between the two groups of people, and constructed a convenient, accurate, and effective PETMT.
The results showed that PETMT with a total of 15 topics was finally formed with three dimensions (social, economic and living) and five topics for each dimension (Table 1). The reliability and validity of PETMT were good, and there was a significant difference between the PET of normal and abnormal behavior groups (p < 0.05). PET score curve of the normal behavior group was obviously biased toward the right, and the peak was 61 points (out of 100 points). PET score curve of the abnormal behavior group was obviously biased toward the left, and the peak was 52 points (out of 100 points) ( Figure 1). The results indicate that people with a PET score higher than 61 points have a higher PET and tend to be individuals who are not susceptible to outbreaks of abnormal behavior. Those who score below 52 points have a lower PET and tend to be individuals who are susceptible to outbreaks of abnormal behavior.

| Experience evaluation is highly consistent with the measurement results of PETMT
After thoroughly observing various social strata for many years, the

| Dopamine level is highly correlated with the results of experience evaluation and PETMT
Based on the dopamine detection and experience evaluation, and PETMT in the process of maternal delivery, we explored the dopamine level released in pregnant women under stress response as well as the relationship between dopamine level and experience evaluation, and PETMT.
The results showed that the release levels of dopamine significantly changed during the delivery process of pregnant women.
The dopamine results were positively correlated with the experi-

| Gene detection first finds genetic markers related to PET
Genes are genetic material carried by human beings. The research group assumes that a low PET or a tendency toward aggressive outbreaks may be related to people's specific genes. We used whole genome detection and functional analysis during the maternal delivery of pregnant women combined with dopamine detection, experience evaluation, and PETMT to explore whether this hypothesis was established.
The results of whole genome detection, dopamine level, experience evaluation, and PETMT during maternal delivery of pregnant women were highly consistent. The gene expression levels in the group with low PET compared with the group with high PET were significantly different, and there were 66 differentially expressed genes of 38,500 genes (Figure 4a), 23 genes were upregulated and 43 genes were downregulated (Figure 4b). KEGG function enrichment analysis of differentially expressed genes using DAVID and KEGG database revealed that the differentially expressed genes were mainly involved in seven signaling pathways, of which the neurotrophin signaling pathway changed most significantly (Figure 4c).
In the neurotrophin signaling pathway, expressions of AKT1, PRDM4 and BAX were significantly changed (Figure 4d). AKT1 and PRDM4 were significantly downregulated and BAX was significantly upregulated in people with a high PET; AKT1 and PRDM4 were significantly upregulated and BAX was significantly downregulated in people with a low PET.
The expression of AKT1, PRDM4 and BAX was verified by qPCR.
It was confirmed that the expression of three genes were significantly different in groups with a high and low PET; AKT1 and PRDM4 were significantly upregulated and BAX was significantly downregulated in low PET group compared with high PET group (Figure 4e).

| D ISCUSS I ON
In this study, we found that PET exists objectively and can be measured exactly through experiments including the PETMT, experience evaluation, dopamine and gene detection which complement each other. The first PETMT can quantify the PET. We also found that PET is determined by three genes AKT1, PRDM4, and BAX.
Neurotrophic factor is a class of proteins that have a specific role in the nervous system and promote neuronal differentiation, survival, neurite outgrowth, synapse formation, and cell migration and proliferation (Hempstead, 2015, p. 9-19). Neurotrophic factors activate many complex signaling pathways by activating two different types of receptors, the Trk family receptor and the p75 receptor, and ultimately exert biological effects (Skaper, 2012, p. 1-12). The Trk receptor and the p75 receptor interact to maintain a balance between neuronal survival and death.
The Trks receptor activates the PI3K/AKT pathway, which phosphorylates a variety of molecules, especially the proapoptotic protein Bad, thereby inhibiting its apoptosis-inducing properties and promoting neuronal survival. AKT1, serine/threonine kinase 1, is a critical mediator of growth factor-induced neuronal survival in the .01 Up S1_ZY_NS S1_KXF_NS S1_SYS_NS S1_ZZY_NS S1_SMJ_NS S1_LLP_NS S1_HR_NS S1_LXL_NS S1_ZQJ_NS S1_CX_NS RAB21  1565705_x_at  MGEA5  239536_at  ZNF182  AMBRA1  FNBP1  CCP110  GOLGB1  RNF20  ZNF638  1556253_s_at  ZNF684  CIR1  MYEF2  PRDM4  AKT1  SLC25A37  PANX1  WWC3  225273_at  SIK3  UBE3B  TPST2  RHOXF1  SUSD1  CAMTA1  SERPINE2  OPN5  LRRC59  BAX  NIPSNAP3B  METTL21B  CDKN1C  P2RY12  NTPCR  CNOT1  ELAC1  ARID1B  LOC145783  TLK1  KIAA0586  RNASE2  UBE2E2  UGDH  ATP6V1F  HMGN2  RFWD3  TRMT5  In summary, this study found that AKT1 was significantly up- The significance of this study is to discover people with abnormal expression of AKT1, PRDM4, and BAX have lower PET and tend to commit abnormal behavior more easily. Understanding PET will enable people to make self-adjustment or to intervene by professionals as soon as possible and in a timely manner in the face of various negative stimuli in work and life, especially for people with lower PET, people should intervene as early as possible to reduce the harm to the individual, family, and society.

ACK N OWLED G M ENTS
Thanks to Y.R.Z. and X.L.Z. for assisting in the completion of the review of domestic and foreign literature and collecting, screening, and testing negative stimuli topics. Thanks to W.G.Y. and E.P.W. for assistance in completing the work of soliciting expert opinions. Thanks to J.M.N. and Y.Q.F. for assisting in the completion of data collection involving hospitals. Thanks to D.F. and W.S.W. for assisting in the completion of the data processing. Thanks to hundreds of individuals, such as X.S.Z. and P.L. for their hard work, support, and help in this research.

CO N FLI C T O F I NTE R E S T
The author declares no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
ZX fully presided over and conducted the entire process. QJ participated in the whole process and focused on constructing the PETMT and the experience evaluation systems as well as the planning of dopamine and gene research. HZ participated in the whole process and focused on constructing the PETMT. YL participated in the whole process and focused on dopamine and gene detection work.

I N FO R M E D CO N S E NT
Written informed consent was obtained from adults (parents or guardians of minor children and participants 18 years of age or older) and assent was obtained from minor children whose parents or guardians provided written consent.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
F I G U R E 4 Results of whole genome detection and analysis in pregnant women during maternal delivery. The gene expression levels in the group with low PET compared with the group with high PET were significantly different, and there were 66 differentially expressed genes of 38,500 genes (a), 23 genes were upregulated and 43 genes were downregulated (b). Differentially expressed genes were mainly involved in seven signaling pathways, of which the neurotrophin signaling pathway changed most significantly (c). Expressions of AKT1, PRDM4 and BAX in neurotrophin signaling pathway were significantly changed. Magenta represents upregulation of gene expression and cyan represents downregulation of gene expression. AKT1 and PRDM4 were significantly downregulated and BAX was significantly upregulated in people with a high PET; AKT1 and PRDM4 were significantly upregulated and BAX was significantly downregulated in people with a low PET (d). The results of qPCR showed the expression of three genes were significantly different in groups with a high and low PET (*<0.01), AKT1 and PRDM4 were significantly upregulated and BAX was significantly downregulated in low PET group compared with high PET group (e). [Correction added on 24 May 2019, after first online publication: in Figure 4d, the gene "SC-1" has been changed to "PRDM4".]