Neuroinflammation signaling has been identified as an important hallmark of Alzheimer’s disease (AD) in addition to amyloid β plaques (Aβ) and neurofibrillary tangles (NFTs). However, the molecular mechanisms and biological processes of neuroinflammation remain unclear and have not well delineated using transcriptomics data available. Our objectives are to uncover the core neuroinflammation signaling pathways in AD using integrative network analysis on the transcriptomics data.From a novel perspective, i.e., investigating weakly activated molecular signals , we developed integrative and systems biology network analysis to uncover potential core neuroinflammation signaling targets and pathways in AD using the two large-scale transcriptomics datasets, i.e., Mayo Clinic (77 controls and 81 AD samples) and ROSMAP (97 controls and 260 AD samples).Our analysis identified interesting core neuroinflammation signaling pathways, which are not systematically reported in the previous studies of AD. Specifically, we identified 7 categories of signaling pathways implicated on AD and related to virus infection: immune response, x-core signaling, apoptosis, lipid dysfunctional, biosynthesis and metabolism, and mineral absorption signaling pathways. More interestingly, most of the genes in the virus infection, immune response, and x-core signaling pathways are associated with inflammation molecular functions. The x-core signaling pathways were defined as a group of 9 signaling proteins: MAPK, Rap1, NF-kappa B, HIF-1, PI3K-Akt, Wnt, TGF-beta, Hippo, and TNF, which indicated the core neuroinflammation signaling pathways responding to the low-level and weakly activated inflammation and hypoxia and leading to the chronic neurodegeneration. It is interesting to investigate the detailed signaling cascades of these weakly activated neuroinflammation signaling pathways causing neurodegeneration in a chronic process, and consequently uncover novel therapeutic targets for effective AD treatment and prevention.The potential core neuroinflammation and associated signaling targets and pathways were identified using integrative network analysis on two large-scale transcriptomics datasets of AD. CD33  is the complexity of AD. More than 42 genes/loci have been associated with AD . UnfortuCD33 , TREM2  and had no any of the dementia-related diseases. The clinical and neuropathological data available from the ROSMAP cohort were used to classify controls and AD cases in our analyses. Specifically, subjects that exhibit no decline in cognition and scored Low or No AD on the Reagan scale, and with a BraakTau score of ≤3, were identified as controls. The AD cases were identified as patients who exhibit clear cognition decline as well as scored high or intermediate on the Reagan scale  values of 16,132 common protein-coding genes were then obtained in the two datasets by applying the Salmon quantification tool  . Subsequ6 The Stage 1 p-Value Data (updated by 26 February 2019) and Stage 2 p-Value Data (updated by 27 February 2019) were downloaded. The 553 candidate GWAS genes and also available in the KEGG signaling pathways were obtained by a filter with p-value ≤ 1.0 × 10–5.The GWAS data of AD was obtained from NIAGADS database .6 The StThe KEGG signaling pathways were extracted using the graphite R package , which cijP ≤ i, gk1, gk2g,…, jg >, i belongs to V0where g, and j belongs to V1g. Third, all of the genes on the signaling path Pij and belong to V1 were selected and added to V0, and removed from V1. This process was repeated until all the genes were added to V0. The data analysis codes are available upon request.To infer the signaling cascades among a set of genes of interest, we developed a network inference approach. First, we divided the genes into two groups: signaling sources  and signaling targets . Second, a signaling network was constructed by linking the signaling source genes to the signaling target genes iteratively. Specifically, the signaling source genes were used as the initial signaling source nodes set: V0. The signaling target genes were used as the target nodes set: V1. In the iterative process, the shortest signaling cascades/paths between the nodes in V0 and V1 were calculated and identified: There were 77 control subjects and 81 AD cases in Mayo dataset; and 97 control samples and 260 AD cases in ROSMAP dataset. p-value thresholds. As seen in p-value ≤ 0.05. With the fold-change threshold ≥1.25, only about 230 upregulated and about 60 downregulated genes were identified , in both studies. When relaxing both thresholds to fold change ≥1.1 and p-value ≤ 0.1, 1,120 upregulated genes and 689 downregulated genes were identified . Based on these observations, we hypothesized that the AD-associated signaling pathways are weakly activated or inhibited. The “weakly” term is defined as that the differentially expressed genes have small fold-change values.We further conducted a widely used differential expression analysis method to identify differentially expressed genes (DEGs) between the AD and control samples. To identify the common set of DEGs between the two datasets, we applied a number of fold-change and p-value ≤ 0.15. We then clustered these activated signaling pathways empirically into 7 categories. Using these 417 upregulated genes, the first principal component values in the MDS analysis of the AD and control samples were used to compare the difference between AD and control samples. The OR, absolute beta values, and p-values of logistic regression analysis  and p-value = 4.2 × 10–6 (ROSMAP)] can separate the AD and control samples much better than using all protein genes [p-value = 0.036 (Mayo) and p-value = 0.027 (ROSMAP)] in the two datasets, respectively. The box plots are also provided in As was noted previously, we believe it is important to identify AD-associated weakly activated signaling pathways and understand their roles in AD disease progression, as well as their potential roles as targets for AD therapeutics. Among the 1,120 common upregulated genes (identified from Mayo and ROSMAP datasets), 417 genes were included in the 311 KEGG signaling pathways, and the rest genes were not available in the KEGG signaling pathways. To this end, we first conducted an enrichment analysis of KEGG signaling pathways using Fisher’s exact test applied to the 417 upregulated genes. ysis see  indicateEscherichia coli infection, Staphylococcus aureus infection, Yersinia infection, Human cytomegalovirus infection, Human papillomavirus infection, Malaria, Human immunodeficiency virus 1 infection, Rheumatoid arthritis, and Inflammatory bowel disease (IBD)]. Out of the 417 upregulated genes, 111 genes were in common across these pathways highlighting a set of core genes implicated on these processes. These results indicated that weakly activated inflammation-related signaling pathways, such as inflammation, cytokine, and immune response, may be represent activated signaling pathways in the AD brain microenvironment.As seen in our results see , a set oMAPK, PI3K-Akt, and mTOR signaling pathways, and then activated the HIF-1 signaling pathway. However, the activation of HIF-1 may fail to bring enough oxygen to protect against hypoxic injury to the involved neurons. The dysfunction of blood vessel functions, leading to hypoxia, might be partially indicated by the recent study showing that blood and cerebrospinal fluid flow cleaning the brain during sleeping  were identified. sleeping .p-value of observing the 66 upregulated inflammation signaling targets from 417 upregulated genes identified in the AD vs. control samples was 8.34E-05 , and the odds ratio (OR) = 1.77, which indicates that the activation of inflammation signaling is concomitant with AD progression. Furthermore, there are 66 overlapping upregulated genes spanning the virus infection (from 111 upregulated genes) and x-core signaling pathways (from 136 upregulated genes), which indicate that the x-core signaling pathways are the likely pathways being activated in response to this inflammation. In addition, the activation of HIF-1 signaling pathway indicates the presence of hypoxia in the AD brain environment.As was introduced above, many genes that are activated as a function of virus infection, immune response, and the x-core signaling pathways are inflammation-related genes. It is well known that virus infection and immune response signaling pathways respond to inflammation. Our analyses identified 1,043 inflammation response genes in the gene ontology (GO) database (GO:0006954), which includes 492 genes in the KEGG signaling pathways. Interestingly, among the 417 upregulated genes, 66 genes were inflammation related. The p-value ≤ 1.0 × 10–5): PIK3CB, AKT3, RAF1, MAPK10, PPP2R2B, ERBB4, MECOM, IL1R1, MYD88, CAMK2D, GNB4, VAV3, PRKD3, PRKCE, THRB, FN1, LTBP1, and WWTR1, which were reported in the GWAS analysis  receptors  were upregulated. We reconstructed these signaling pathways linking the TNF receptors to the upregulated genes in TNF and apoptosis signaling pathways see in  using thTREM2 (In addition to Aβ and NFTs, neuroinflammation has been identified as another important AD pathogenesis . A set oTREM2 , TNF (DeTREM2 , IL-1betTREM2 , IL-6 (NTREM2 , and FkBTREM2  have beeTREM2 . The neuTREM2 . Also, tTREM2 . The netTREM2 . HoweverThere are some limitations in the analysis. Through integrative and network analysis, we uncovered a set of core signaling pathways of neuroinflammation. However, first, the detailed signaling cascades among these neuroinflammation signaling genes in AD brain niche remain unclear. The general known signaling pathways in KEGG are not specifically designed for AD. For example, though the activation of T and NK cell signaling pathways can indicate the activation of some immune signaling–related proteins, the roles of these proteins in neuroinflammation remain unclear because the T and NK cells are not involved in the brain. In addition to the identified inflammation signaling pathways, it is interesting to further investigate the specific and detailed inflammation signaling cascades among the identified signaling targets that are potentially caused by the abnormal β-amyloid and tau protein aggregations. The other limitation is that the identified inflammation-related targets were not experimentally validated at the RNA or/and protein levels, such as qPCR or/and Western blotting/IHC. We will look for wet-lab collaborators for the potential experimental validations. Also, different brain regions might have different dysfunctional signaling pathways or different levels of neuroinflammation. Moreover, the current RNA-seq datasets are bulk tissue based, including multiple cell types, which confounds the gene expression change. Therefore, cell type composition correctness might affect the differential expression analysis. In addition, the neuroinflammation signaling pathways are reported to be affected by multiple cell types, such as microglia and astrocytes, in addition to neuron cells. Thus, it is important to analyze brain-region specific, single nuclear or cell RNA-seq data  to uncovhttps://adknowledgeportal.synapse.org/Explore/Studies?Study=syn3219045, https://www.synapse.org/#!Synapse:syn23277389, and https://www.synapse.org/#!Synapse:syn3191087.Publicly available datasets were analyzed in this study. This data can be found here: Project was conceived by FL and CC. Transcriptomics datasets of AD samples were collected and pre-analyzed by AE, CS, OH, and CC. Methodology was developed and data were analyzed by FL. Results were discussed by FL, CC, AE, OH, EM, CK, and PP. The manuscript was prepared by FL and revised by CC, AE, WB, GW, CX, PP, EM, CK, and OH. All authors contributed to the article and approved the submitted version.


Periodontitis is one of the most prevalent diseases in oral cavity, which could not merely lead to the destruction of supporting or surrounding tooth structures but also affect the whole-body health such as the digestive and nervous systems. Epidemiological investigations suggested that in some developed countries, more than 45% or even 50% population were suffering from periodontitis. However, the prevalence increases with age remarkably and it is investigated that a high prevalence (>50%) is affecting the elderly who is over 65 years old. There is an increasing interest in the direct and indirect relationships between periodontitis and hyperuricemia. Currently, hyperuricemia has become the second major metabolic disease in modern society and the prevalence of hyperuricemia among adult males and females was 21.7% and 14.4% respectively. As an inflammatory disease associated with various systemic diseases, periodontitis may have certain connections with hyperuricemia. Partial existing research announced that hyperuricemia could act as an inhibitory factor for periodontitis, while other scholars presented that a high uric acid (UA) level was more likely to aggravate inflammatory immune response and lead to more serious tissue destruction. This article provides a detailed and comprehensive overview of the relationship underlying hyperuricemia and periodontitis in the molecular mechanisms. Given the impact of hyperuricemia, this review could provide insight into its comorbidities. Periodontitis is a chronic multifactorial inflammatory disease caused by pathogenic biofilm and results in progressive destruction of the tooth-supporting apparatus including alveolar bone resorption, which could lead to tooth loss . Althougvia crossing inflammatory signaling pathway.Hyperuricemia is a common disorder impacting patients of all ages and genders that could lead to the formation of tophus in the peripheral joint which is clinically manifesting as gouty arthritis. At present, approximately 120 million people in China and 38 million individuals in the United States are suffering from such torturing disorders . In addiHowever, it has not been as widely discussed in the medical literature about the influence of hyperuricemia in periodontitis as diabetes mellitus. Thus, this review will discuss the concrete evidence of fundamental experiments and epidemiological surveys to explain the possible comorbidity between periodontitis and hyperuricemia. The inadequate parameters will be dissected as well. This review covered the pathogenic mechanism of periodontitis, the systemic effect of elevating soluble UA levels, and the facilitation of hyperuricemia in periodontal immune-inflammatory response (the interaction relationship between hyperuricemia and periodontitis). Among the above, the former two parts consist of the foundation of the last but most significant part. Meanwhile, this review will also hint to physicians and periodontists about the potential effect and therapy based on the premise of their interaction refer to Figure 1Tannerella forsythia and Porphyromonas gingivalis  were linked to early periodontitis  by the pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) is critical for the host-microbe interaction, as well as the subsequent innate and adaptive immune responses . Usuallyvia the phosphorylation of interferon (IFN)-regulatory factor (IRF3) during the late phase of NF-κB activation  in innate immunity . Mature via TLR4 in the canonical pathway and promote the transcription of NLRP3 and pro-IL-1β, then resulting in the assembly of NLRP3 inflammasome with K+ efflux and ATP , the degradation product of bacterial peptidoglycans, could trigger the assembly of the NLRP3 inflammasome complex . This increasing pro-inflammatory mediator expression is associated with the phosphorylation of MAPKs within 15–30 min after LPS stimulation –48. MAPKDamage-associated molecular pattern (DAMP), such as UA, is produced or released by damaged or dying cells. Although momentous for tissue repair and regeneration, DAMPs could bring about sterile inflammation . In stervia TLR2/4 compared with other crystals like allopurinol crystals and basic calcium phosphate. Whereas soluble UA does not act as stated above . Monosodium urate crystals have a specific ability to stimulate DCs maturation and promote the priming of T cells ed above , 53, Lu ed above . Wang eted above . TherefoSoluble urate could specifically downregulate the IL-1Ra (IL-1 receptor antagonist) secretion even at a lower dose and enhance the IL-1β production dose-dependently when TLR ligands are presented . Howevervia the p38-PI3K-Akt signaling pathway and thereby increasing the neutrophil cytokine synthesis  soluble UA could facilitate immune responses via the PI3K-Akt-mTOR signaling pathway.The rising level of phosphorylated proline-rich AKT substrate 40kD (PRAS40) in monocytes was revealed when stimulated by soluble urate. Phosphorylated PRAS40 could strongly activate mTOR which is also known as the PRAS40-Akt-mTOR signal pathway . The aboynthesis . Crisan ynthesis . AlthougThe researchers also concluded that MSU crystals could boost inflammation by their capacity to initiate and accelerate the assembly of NLRP3 inflammasome . In periUA excretion usually depends on the kidneys, and the reabsorbing part accounts for 90% of the gross body output . The evoOut of anticipated, some scholars verified that soluble UA could only play an antioxidative role in neurodegenerative diseases . MultiplVeronese et al. concluded that hyperuricemia had a protective effect on bone metabolic disorder independently of physiological bone metabolism . Yan et Based on these results, we made the following daring hypothesis-there may be more or less association between periodontitis and hyperuricemia. In fact, the interrelationship between periodontitis and other common systemic diseases has been verified in succession these years. Holm et al. discovered a higher incidence of undiagnosed diabetes in the patients who sought dental treatment, and higher HbA1c above the threshold for pre-diabetes could also be observed . PeriodoAs we could learn from the previous and present research, the signal pathway crossing between hyperuricemia and periodontitis included the following 4 points refer to Figure 41) As PAMPs, periodontal pathogens identify TLRs to stimulate the NF-κB signal pathway, while MSU crystals could also activate NF-κB as DAMP with the synergy of TLRs. Periodontal pathogens and MSU crystals initiate similar signaling pathways, which could enable the levels of inflammatory cytokines to get strikingly higher than when a single factor activates NF-κB.2) On the whole, the NLRP3 inflammasome could be activated by endogenous DAMPs such as MSU and PAMPs like bacterial toxins. Derived from the stimulus described above, the activated NLRP3 inflammasome could stimulate caspase-1, which will process the pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 to induce pyroptotic cell death as inflammatory factors. Both MSU crystals and MDP could stimulate a variety of immune cells like macrophages, and initiate the assembly of NLRP3 inflammasome to accelerate the production and release of active IL-1β. The synergies may be gained with the presence of the two aforementioned stimuli virtually.3) MSU could enable the phosphorylation of p38 and MAPK at a low level, which may amplify the effect of phosphorylation of MAPK stimulated by LPS to induce pro-inflammatory cytokines. Consequently, the collective effect could lead to a more violent inflammatory response.4) Soluble UA could restrain autophagy through the Akt-PRAS40 signaling pathway, which results in the inhibition of inflammatory cytokines inactivation induced by periodontal pathogens. In addition, soluble UA could also cause the persistence of elevated levels of inflammatory cytokines either locally or systemically, which cannot be reversed by effective UA-lowering drugs at least for a short time.Both salivary and plasma inflammatory IL-18 levels could rise significantly in the chronic periodontitis patients compared with the control individuals, which is consistent with the alteration in patients with high soluble UA levels . Crisan Research related to the interaction between high soluble UA levels and periodontitis is still deficient and not profound relatively. While with the help of research about the interplay between high soluble UA levels and inflammation in other tissue, this review attempts to explore the mechanism of interplay between high soluble UA levels and periodontitis indirectly. Due to the capacity to increase the production of soluble UA, alum has been applied to medical practice as a vaccine adjuvant for a long time to enhance the immune response . Yan et Liu et al. demonstrated that the Type 1 CD8+ T cells were the main participants in the inflammatory cytotoxic responses when exposed to LPS . A signiP. gingivalis and Prevotella intermedia are both closely related to the initiation and development of periodontitis, while Serratia marcescens is a conditional pathogen. In the in vitro experiments, applying P. gingivalis (MOI (multiplicity of infection) of 100) to stimulate THP-1 macrophages for 24h could lead to the significantly increased release of soluble UA , Clinical Medicine +X Research Project of Affiliated Hospital of Qingdao University (QDFY+X2021055).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.


Using a global warming audience segmentation tool (Six Americas Super Short Survey (SASSY)) as a case study, we consider how public health can use consumer panels and online crowdsourcing markets (OCMs) in research. Through a secondary analysis, we aim to understand how consumer panels and OCMs are similar to or different from each other on demographics and global warming beliefs through SASSY, and how they compare to US Census estimates. With this information, researchers will understand public opinion of global warming in their sample, which is useful for many climate change initiatives. Neither the consumer panel (Ipsos) or OCM sample (MTurk) matched US estimates of population demographics. Both panels achieved similar SASSY segments, showing that even with diverse sampling frames, SASSY is a useful tool for understanding global warming sentiment. Compared to Ipsos, MTurk was younger , had higher educational attainment, and lower income. Both panels were majority White, but Ipsos was more diverse than the unweighted MTurk. Ipsos had more respondents from the South whereas MTurk had more respondents from the West. Across the MTurk SASSY segment, there were no significant differences for the majority of demographic characteristics except for age; younger generations were more Alarmed or Concerned, and older generations were more Doubtful and Dismissive. Researchers interested in understanding their sample’s opinions of global warming should use SASSY and consider oversampling in key demographic variables if they intend to achieve a nationally representative and diverse sample. The Yale Project on Climate Change Communication and the George Mason University (GMU) Center for Climate Change Communication have, since 2008, provided reports and publications on how American’s perceive and react to global warming. American public opinion about climate change and the understanding that global warming is happening and is human-caused reached an all-time high in 2020 . The effA representative sample is a subset of a population that seeks to reflect the characteristics of a larger group. Because it is largely impossible to survey every person in a larger population, samples are useful because they contain smaller, more manageable data on a larger group. However, representative samples can be difficult to achieve in public health research . SamplinMany non-probability-based survey methods are available to public health researchers, including convenience sampling through web-based survey platforms, such as consumer panels, or online crowdsourcing markets (OCMs). We aim to understand how consumer panels and OCMs are similar to or different from each other on demographics and global warming beliefs, and how they compare to US Census estimates. In this secondary analysis, we start by reviewing the similarities and differences between consumer panels and OCMs. Second, we demonstrate through a case study how an audience segmentation tool can be used in an OCM to understand a sample’s opinions of global warming. Finally, we compare demographics across both samples to US Census demographic estimates to understand differences between sampling frames and provide recommendations for future research. With this information, researchers will understand their sample’s opinion on global warming, which they can use for a range of climate change initiatives, including communications, messaging, and research efforts.Online survey companies, such as Qualtrics and Ipsos, are market research companies that offer access to consumer panels for research and marketing activities. Consumer panels provide researchers with an able and willing pool of respondents for research, with little effort on the researchers to collect each response. However, they can be costly, and consumer panels may differ demographically from the underlying population unless sampling weights are used. OCMs such as Amazon’s Mechanical Turk (MTurk)  provide MTurk participants are slightly more demographically diverse than standard internet samples, and the data obtained is as reliable as other traditional methods . US-baseNon-probability-based sampling methods, such as consumer panels and OCMs, risk having selection bias and non-response bias . In the In 2020, we sought to understand how consumer panel and OCM sampling frames differed by using a pre-validated questionnaire, called the Six Americas Super Short Survey (SASSY) ,14, whicThe survey we developed for use in MTurk included 67 survey questions from SASSY, demographics, and questions regarding the health impacts of global warming . Demogran = 1029 in Ipsos), Leiserowitz et al. [We formatted the survey flow to include attention check questions in each block as described in Smith et al.  to encoun = 200 and from 15 July to 14 August 2020 for target recruitment of n = 300. Participants consented to participate in the survey and confirmed that they were at least 18 years of age. Because respondents in non-US OCM samples can significantly vary in their demographics compared to US OCM respondents [The MTurk survey was distributed from 14 to 15 July 2020 for target recruitment of pondents , and to p-values when cells had less than 10 respondents in order to limit generalizations about the results. All data analysis was completed in Stata 16 [Demographics from our survey (MTurk) and the US Census demographics were mapped to match Ipsos’ demographic variables in order to make comparisons across samples see . We exclStata 16 .We collected 508 responses from 14 to 15 July 2020 and from 15 July to 14 August 2020. The sensitivity analysis between the first and second waves, as well as between the first 90% and last 10%, did not show any differences. We excluded 4 responses due to failure to answer attention check questions, for a total of 504 responses for the MTurk survey. Compared to the US Census, Ipsos and MTurk were significantly different on the majority of demographic levels .p < 0.001), whereas Ipsos was older (more Baby Boomers and Silent Generation p < 0.001) (p < 0.001). Although they had higher educational attainment, MTurk had a higher proportion of respondents with lower income levels , and Ipsos had more respondents with income levels greater than $125,000 (p < 0.001). Both panels were majority White, but Ipsos had more Hispanic, multiple race categories, and other race categories than MTurk (p < 0.001). There were no significant differences among the samples for categories of gender. Compared to MTurk, Ipsos had more respondents in the South whereas MTurk had more respondents from the West. For the SASSY Segment, MTurk had more respondents in the Alarmed category, whereas Ipsos had more respondents in the Disengaged category  . Ipsos hcategory .p = 0.005); younger generations were more Alarmed or Concerned, and older generations were more Doubtful and Dismissive.Across mTurk SASSY segments, there were no significant differences for the majority of demographic characteristics . There wThis study provided an overview of the similarities and differences between US-based OCM and consumer panels in order to understand public opinion on global warming using SASSY. One of the main motivations for this work is to show how SASSY can be used to quickly and effectively understand a sample’s opinions about global warming. To our knowledge, SASSY has been used only within Ipsos, even though Leiserowitz et al.  have encIn this study, we found that Ipsos and MTurk significantly differed from each other on the majority of demographic variables but had a similar distribution of SASSY segments. This finding shows that even with diverse sampling frames, SASSY is a good tool for understanding sentiments towards global warming. While Ipsos had about equal regional coverage, MTurk appeared to have more in the West and less in the South than Ipsos. In addition to the geography and high levels of poverty compared to the US, Kearney and Bell  show theNeither consumer panel nor OCM sample matched the underlying US population through the US Census 2020 estimates. Overall, MTurk was younger, more educated, and had higher educational attainment but lower income compared to the US Census, whereas Ipsos was older and less educated but had higher income compared to the US Census. Howe et al.  has showWhile we focused on the US OCM market in our case study, researchers interested in using non-US OCM samples should be aware that they may differ demographically, and provide statistically significant scale parameter estimates, from consumer panels and US OCM samples . RegardlThe differences observed between sampling frames may be due to biases present within each panel. Nonresponse bias occurs when respondents choose to not participate in the survey, which makes it more difficult to draw inferences on the population estimates within the sample. Due to the politicized nature of climate change, and the low compensation rates, respondents may choose not to participate, and thus, contribute to coverage bias by not being represented within the sampling frame. However, we did try to provide a compensation rate that is higher than normal within MTurk ($2 vs. $0.05–$1.00 recommended). On the other side, participants interested in the topic area may be more likely to participate and this would contribute to selection bias in the sample. To address this, we changed the listing for the survey within MTurk, and found no significant differences between these recruitment waves.Researchers should consider the potential selection bias of internet-based methods which might be influenced by a variety of socioeconomic attributes of the respondents. While this is less of a concern in this study (since both are internet-based), researchers should still limit their inferences when generalizing to the underlying population unless they intend to use sampling weights and qualifications to achieve a nationally representative sample. Additionally, the use of convenience samples, such as OCMs and consumer panels, increases the risk of these biases within a sample. We limited the effect of this bias in our study design by providing the surveys in waves in MTurk and assessing their impact and by including attention check questions to reduce the probability of bots or automated responses.The effect of timing of the surveys could have been impacted by the COVID-19 pandemic. Ipsos was completed in April 2020, whereas MTurk was completed in July and August 2020. The collective trauma of the COVID-19 pandemic, especially during April–July 2020, may have contributed to the higher proportion of Alarmed and Concerned in the MTurk sample. However, we did compare MTurk’s SASSY segments to the 2019 Ipsos national estimates, and MTurk still had a larger portion of Alarmed and Concerned than Ipsos : 34% (31%), Concerned: 32% (26%), Cautious: 19% (16%), Disengaged: 1% (7%), Doubtful: 8% (10%), and Dismissive 6% (10%)) .Through using SASSY, researchers will be able to more comprehensively understand public opinion on climate change, which can be used for a range of climate change initiatives such as communications, messaging, and other research efforts. While non-probability sampling may have risks, the risks can be managed and accounted for in a public health researcher’s methods and sampling. We encourage public health researchers to utilize qualifications and sampling weights when using consumer panels or OCMs in their research if they intend to achieve a more nationally representative population. However, even without using premium qualifications, we achieved a similar distribution of SASSY segments. Through using SASSY and appropriate sampling techniques, researchers can understand public opinion about climate change in their sample.


Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling. Chemokine CXCL12 and growth factor EGF are important signaling molecules from the microenvironment of a primary breast tumor and distant organs. These molecules contribute to the organ-specific chemotaxis of circulating tumor cells and subsequent metastasis in breast cancer and multiple other malignancies . CrucialChemotaxis plays an important role in the migration of cancer cells and subsequent metastasis toward chemotactic molecules such as CXCL12 and EGF. Chemotaxis and other cellular functions, such as proliferation, differentiation, and cell death, are regulated by signaling oscillations. Prior research into ERK oscillatory activity has revealed the dynamic nature and integrated feedback loops of signaling networks regulating migration ,20,21,22CXCL12–CXCR4 interaction elicits G protein signaling to activate Ras and PI3K and downstream ERK and Akt, which can also be activated by EGF-EGFR. Ras and PI3K drive protrusions of cell membranes, creating positive feedback for Ras activation ,20. Ras To answer these questions, we employed an integrated approach combining live, single-cell imaging and quantitative analysis to investigate key signaling pathways and chemotactic responses of MDA-MB-231 breast cancer cells. We implemented multiplexed fluorescence reporters with automated live-cell imaging and image processing to quantify oscillatory information in signaling, morphology, and chemotaxis. Then, we used a time-dependent data processing approach to quantify relationships of signaling, morphology, and chemotaxis within a heterogeneous cell population and across populations treated with targeted inhibitors. We also employed an independent approach termed Variational System Identification (VSI), which combines computation with mathematics to infer the two components of cell motion—random migration and directed motion. Concluding from our independent approaches, we found that amplitudes of short Akt and ERK waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, amplitudes of long Akt and ERK waves, the extent of cell elongation, and nuclear polarization determine the heterogeneous directional movement of cancer cells in a chemotactic gradient, which is regulated by MEK-ERK signaling. These data provide new insights into how single cells connect dynamics of signaling response to morphologic changes associated with chemotaxis.2. Prior to loading cells for chemotaxis experiments, we precultured MDA-MB-231 cells in FluoroBrite imaging media (Thermo Fisher Scientific), 10% FBS, 1% Pen/Strep, and 1% GlutaMAX for 2 days. We used previously engineered cells that have a stable expression of histone 2B fused to mCherry (H2B-mCherry), Akt-KTR (Aquamarine), ERK-KTR (mCitrine), and CXCR4 receptor for our experiments . EGBecause our results showed that long signaling waves and aspect ratios regulate the heterogeneity of directed motion, we next asked to what extent long signaling waves are related to the aspect ratio of cells during migration. When polarized cells reorientate and modulate their morphology during mesenchymal cell migration, nuclear reorientation is important in promoting directed motion . Since Ep < 0.0001, 99% confidence intervals shown in We isolated each aspect ratio peak of individual cells and compared them with the corresponding ERK signaling. All aspect ratio peaks were aligned to the same time point to facilitate the identification of time-dependent relationships between aspect ratio and long ERK signaling waves A. Comparp < 0.0001, 99% confidence intervals shown in To quantify the extent of nuclear polarization relative to ERK signaling and cell morphological change, we measured nuclear polarization as the distance of the nucleus with respect to the cell centroid. The amplitudes of long ERK waves and aspect ratio correlate with the degree of nucleus polarization. Quantile regressions between the 20th and 80th percentile illustrate that heterogeneous ERK and aspect ratio differences show a linear relationship with nuclear polarization  inhibitor, trametinib is a MEK inhibitor, and both are used clinically for cancer therapy. The inhibition of PI3K blocks downstream activation of Akt, while inhibition of MEK blocks the activation of downstream ERK. Kinase p38 has a context-dependent effect on mTORC1 ,40,41.We compared the single-cell data of MDA-MB-231 cancer cells preconditioned with either 100 nM alpelisib, 10 nM trametinib, and 10 µM SB203580 two hours prior to the addition of a CXCL12 gradient A. These We compared the effects of these compounds on various aspects of signaling and morphology amplitudes in relation to random migration and directed motion of cancer cells. We focused on amplitudes of short ERK and Akt waves relative to the heterogeneity of random migration. Despite similar reductions in mean Akt levels and no reduction in mean ERK levels B,C, the The present study investigated to what extent oscillatory signaling networks in cells are associated with heterogeneous chemotactic responses and the effects of targeted inhibitors. This single-cell chemotactic study in MDA-MB-231 cells led us to two main conclusions. First, the heterogeneity of cancer cell directed motion and random migration originates from the rate of change of kinase activities and cell morphology with oscillation period as the regulator. Second, the responsiveness of oscillatory signals and chemotaxis is regulated by the activities of MEK and p38 kinases. The fact that the heterogeneity of chemotactic response in cancer cells is determined by the oscillatory network and can be controlled by inhibition of signaling pathways suggests that potential treatment approaches for targeting fast-migrating cells should consider effects on oscillatory signaling and morphology.Three analyses provide evidence that the heterogeneity of cancer cell directed motion and random migration originate from the rate of change of signaling activities and cell morphology with oscillation period as the regulator. First, the migration speeds of individual cells are determined by the amplitude of Akt and ERK signaling waves and the morphological aspect ratio of each cell. Despite not referring to cell-to-cell heterogeneity, other previous investigations reinforce that ERK pulses are related to oncogenic transformation, collective migration, and polarization ,21,22. OThe second piece of evidence supports that the oscillation period is a regulator of the signaling and morphological amplitudes for directed motion and random migration heterogeneity. Depending on the oscillation periods of Akt and ERK signaling waves and morphological aspect ratio, cells have distinct levels of dependence on the CXCL12 gradient. Our current study shows that short Akt and ERK signaling waves with an oscillation period of 0.5–1 h contribute to random migration, whereas long signaling waves and morphological change with an oscillation period of 1.5–2 h tend to contribute to directed motion. This suggests that cell signaling networks regulate distinct modes of migration via different time scales, which was suggested previously . Random The third piece of evidence further supports that the rate of change of long ERK signaling waves and morphological aspect ratio with similar 1.5–2 h oscillation periods negatively correlate to determine the directed motion of cancer cells. Our current study shows a relationship between the extent of reduced long ERK signals and the extent of cell elongation. Cells reduce the long ERK signal and elongate at the same time and have a burst of migration speed with a delay of around 20 min. This evidence and previous investigations on short ERK pulses ,20 demonThree analyses provide evidence that activities of MEK and p38 kinases regulate the responsiveness of oscillatory signals and chemotaxis. First, the state of MEK kinase activity determines the responsiveness of both short and long signaling and morphological amplitudes with resultant impairment of cancer cell random migration and directed movement. Our study shows that the amplitudes of short and long ERK and Akt waves and aspect ratios reduce when cells are treated with a MEK inhibitor. A previous study showed that inhibitory phosphorylation of MEK is crucial for negative feedback and oscillatory ERK signaling and impacts signaling amplitudes . FurtherSecond, MEK inhibition also impacted nuclear polarization, which relates to the extent of ERK signal change, cell elongation, and directed motion acceleration. This study shows that nuclear polarization relates to single-cell changes in ERK signaling, cell elongation, and directed motion. This evidence supplements previous investigations that nuclear polarization promotes polarity and directed motion in cells ,50. HoweThird, the state of p38 kinase activity determines the responsiveness of only short Akt and ERK signaling amplitudes and blocks the random migration of cancer cells. Our study shows that the amplitudes of short ERK and Akt waves reduce when cells are treated with a p38 inhibitor. Recognizing that p38 may have context-dependent effects on mTorc1, our past work with these cells shows that p38 inhibits mTORC1 ,57. SincThis study assumes that live cell imaging reporters in cells capture all essential temporal information. However, the characterization of network dynamics is limited by imaging frequency and types of live-cell reporters. Our assumption is reasonable because the imaging frequency is five times faster than the signal dynamics that we studied. With recent advances in automated imaging techniques and live-cell reporters, the imaging frequency and the number of live-cell reporters can be increased in the future without causing phototoxicity or fluorescence crosstalk. This improvement will help reveal other mechanisms regulating heterogeneity in cancer cells, such as receptor dynamics, cytoskeletal structure, adhesion, and contractility. Cells also migrate differently in 2D versus 3D environments. We will need to confirm to what extent discoveries in this 2D chemotaxis study apply to a 3D environment. Our quantitative analysis approach focused on relationships and correlations among signaling, morphology, and migration and variations between migrating and non-migrating cells. Further studies are required to firmly establish causal relationships between these parameters. Future studies also are needed to explore pre-existing cell states and unique feedback delays in Ras/PI3K/ERK signaling and morphological networks regulating migration heterogeneity. Our study offers answers to processes in oscillatory signaling that associate with the heterogeneous responses of seemingly identical cancer cells with the same genetic mutation. In addition to the heterogeneity described here, heterogeneity also arises within a tumor and across different patients. While our study provides insights about the effective targeting of migrating cancer cells with constitutive activation of one major pathway, our findings need to be combined with other studies focusing on intratumor heterogeneity for effective prevention of metastasis.In summary, we have shown that the rate of change of ERK and Akt signaling activities and cell morphology determine the heterogeneity of cancer cell chemotaxis. We also demonstrated the effects of MEK and p38 kinases on random and directional cell movement. Our study suggests that future studies in signaling, morphology, and chemotaxis should consider the period and amplitude of oscillatory signaling outputs. Our finding paves the path towards better understanding of mechanisms regulating heterogeneous responsiveness in cancer cells and future identification of new interventions controlling cancer metastasis.


The proposed C235 system offers a unified representation for the efficient encoding and effective manipulation of color. It can be applied to designing a high-rate LCD system and colorizing objects with multiple attributes and DNA codons, opening the door to manipulating colors and lights for even broader applications.RGB and CYMK are two major coloring schemes currently available for light colors and pigment colors, respectively. Both systems use letter-based color codes that require a large range of values to represent different colors. The problem is that these two systems are hard to use for manipulating any operations involving combinations of colors, and they lack the capacity for inter-changeability or unification. Based on prime number theory and Goldbach’s conjecture, this study presents a universal color system (C 235 color system unifies RGB, CMYK and HSV wheels with high compression rates and low compression errors.The C Originating from Newton’s theory1, RGB , a light-color structure that contains 3 × 256 values of letter symbols, and CMYK , a pigment-color structure that contains 4 × 100 values of letter symbols3, have become the most popular color frames used today. Most of the other color frames, such as HSV  are derived from RGB and CMYK3.Numbers and colors are powerful tools for expressing objects such as people, goods, and DNA. The former can quantify objects and the latter can represent them visually. Isaac Newton’s theory of light claims that all colors can be generated from three basic colors: red, green, and bluer, g, b). In the CMYK frame, each of C, M, Y, and K has 100 values, expressed as , and is coded as .In the RGB frame, each of R, G, and B colors has 256 values expressed as , and is coded as ((i)Expression problems: R, G, B and C, M, Y, K are letter symbols; it is hard to use them to explicitly express the relationship between colors. Take the RGB framework as an example. Based on key colors R, G, and B, another nine colors {RY, Y, YG, GC, C, CB, BM, M, MR} can be deduced, where Y stands for yellow, C for cyan, and M for magenta. However, it is not easy for a user to directly realize the components of color from these letter symbols. Difficulties arise in various application contexts without a specific mechanism for mathematical operations. For instance, what is the complement color of R? What are the triad-complementary pairs within these 12 colors?(ii)Computing problems: Letter symbols in the current color frames are hard to use for color computation. For instance, what is the resulting color after blending four colors of RY, GC, CB, and MR? Moreover, what is the reflecting color of an apple if we use a blue light to irradiate a green apple?(iii)4.Unification problems: Letter symbols are hard to use for unifying pigment colors and light colors, same for unifying RGB, CMYK, and HSV frames together. Such issues may cause ineffective conversions among different colors(iv)c, m, y, and k may assume 100 values, while in an RGB frame, each of r, g, and b may assume 256 values. Take RGB as an example. Each of the R, G, and B colors has 256 values, thus resulting in 3 × 2562 hues, which makes it challenging to distribute and allocate these many colors and hues on a color wheel4,5. In addition, the large number of color values may cause a huge computational burden for combining some of them to generate preferred colors6.Size problems: In a CMYK frame, each of The weakness of the current CMYK and RGB frames are given below.5 screen requires 3 × 8 = 24 pulses to generate R, G, and B lights7, which is both time- and energy-consuming. Moreover, the transformation between lights and colors is so complicated that no usable smart system is currently available9.The weakness of RGB and CMYK is further reflected in emerging applications, such as cell phones, PCs, and TVs, in which RGB and CMYK are widely applied. For current technologies, each pixel on an LCD11 and Goldbach’s conjecture12, referred to as C235, to encode colors and colorize objects. The aim is to solve bottlenecks inherent in existing methods. Prime number theory claims that any natural number larger than 1 can be uniquely expressed as the product of other prime numbers. This result is widely used for classifying information in the field of cryptography13 and optimization16. Goldbach’s conjecture further claims that any even number larger than 2 can be expressed as the sum of two prime numbers.In this study, we present a new color framework based on the prime number theory2 represents gray. The exponent of 30 is used to indicate the gray level. Therefore, 302 is darker than 301.In our study, we choose the first three prime numbers 2, 3, and 5, to represent the three basic colors of red, green and blue, respectively. Since the white light is a combination of red, green, and blue lights, we may denote the white light by 30 = 2 × 3 × 5. Cyan is composed of green (3) and blue (5), so 3 × 5 = 15 is used to denote the cyan color. Similarly, 2 × 5 = 10 represents magenta, 2 × 3 = 6 represents yellow, and 15 × 10 × 6 = 30Furthermore, we can use seven numerical symbols to replace seven basic letter symbols, i.e.,  =  and another six numerical symbols for the corresponding letter symbols:2 × 3 = 12, 32 × 2 = 18, 32 × 5 = 45, 52 × 3 = 75, 52 × 2 = 50, 22 × 5 = 20) =  to express 12 key hues, and use number 30 to express grayness. In addition, we can use Consequently, we can use 12 numbers , code <15> = <3 × 5> is for color cyan (C), code <18> = <3 × 6> is for color yellow-green (YG), and code <45> = <3 × 15> is for color cyan-green (CG). Color in the C235 system is also associated with a gray level for its lightness/thickness. Since <30> = <2 × 3 × 5> represents a white light, we use the powers of 30  to indicate the grayness levels. The general rule is that higher power means a darker/thicker color.A C235 color system of 36 hues/colors with three gray levels organized in three rings and 12 sectors—the inner circle has three gray codes  surrounded by 36 hues/color codes  spreading in three rings that belong to the 12 sectors  outside the big circle. This C235 system makes plotting a specific color more convenient. For instance, <2 × 30> = <2 × 2 × 3 × 5> represents a color composed of hue <2> located in the first ring with a gray level <30> that belongs to the R sector. Hence it is “light red”. For another instance, <2 × 52 × 303> is a color composed of hue <50 = 2 × 52> and a gray level <303> . Noting that hue <50> is located at ring 2 of the BM sector, we know that the color is a dark blue magenta with more blue than red.Figure 235 system are described as follows.The steps for building the C2 to represent gray color. Table Step 1: Choose the smallest three prime numbers 2, 3, and 5 to denote three basic colors. Since the red light, green light, and blue light are irreducible, we denote 2, 3, and 5 as red, green, and blue, respectively. Because the white light is a combination of red, green, and blue lights, we denote 30 = 2×3×5 as the white and gray light. Then, we use 3 × 5 = 15 to denote the cyan color, since cyan is composed of green (3) and blue (5). Similarly, we use 2 × 5 = 10 to represent the magenta color, and use 2 × 3 = 6 to represent the yellow color, and use 15 × 10 × 6 = 30S be the set composed of 0 and the first 18 prime numbers, i.e.,Step 2: Express pigment-color values based on Goldbach’s conjecture. Let S; and any odd number between 0 and 99 can be expressed as the sum of the two numbers in S plus 1. This means that by using 4 × 18 = 72 values , we can express all CMYK colors.Then, we know from Goldbach’s results that any even number between 4 and 99 can be expressed as the sum of the two numbers in i> be the code of color i. We use codes i is denoted as Code(i):li = min{Code(i) asID(i) is an integer.Step 3: Let <235 frame. This renders potential usages of free color conversion between RGB and CMYK systems. In the meantime, Expression (1) and Expression (2) can also unify the RGB and CMYK frames, thus inducing their potential use in color conversions between RGB and CMYK frames.The key merit of Expressions (1) and (2) is that both pigment colors and light colors can be unified in the Ck = 2, 3, 5) are expressed asStep 4: The idea of the merger of light color with pigment color is expressed using the following example. Suppose there is an incident light k = 2, 3, 5.k = 2, 3, 5.Suppose Suppose Suppose the cellophane is red  and (2) are based on an 8-bit color frame, where Once a Example 1: Consider two pigment colors, i and j with i, we have j, we first convertl be the merger of color i and color j, then we have Code(l) = Code(l) = For color Example 2: Where to find the three colors of Example 1 in the ring form of a C235 system?i being located at ring 71, color j at ring 100, and color l at ring 170. Also, we know color l is darker than color j than color i.235 color frame are summarized below:(i)235 color system can code and allocate up to 2563 colors on a two-dimensional disk.A basic C(ii)235 color coding frame can unify the RGB and CMYK frames and provide easy conversion between different color frames.The C(iii)235 color frame allows easy manipulation of various color operations.The number-based CCompared to the current RGB and CYMK color codes, the merits of C3. However, the RGB wheel and CMYK wheel are not effective enough for the following reasons: Firstly, an RGB wheel contains 3 × 2562 color hues and a CMYK wheel contains 3 × 1002 hues. The number of hues is too large for manipulating or displaying colors6. Secondly, CMYK and RGB need 3 × 256 and 4 × 100 values, respectively, for representing colors. Thirdly, the CMYK wheel and RGB wheel are not interchangeable. This study, therefore, designs a compressed color wheel, called C235 wheel, which may integrate CMYK and RGB wheels with a compression error rate of less than 1.2%.Color wheel is a powerful tool for displaying and manipulating colors235 wheel is designed to unify CMYK, RGB, and HSV color systems using a much smaller number of raw colors  to represent all 2563 colors, as illustrated in Fig. 3 contains 3 × 2562 color hues using 768 (= 3 × 256) key values, while a CMYK wheel contains 3 × 1002 hues using 400 (= 4 × 100) key values  using only 99 key values. The compression rate of C235 color wheel versus the RGB wheel is 235 wheel is shown in Fig. By performing the next five tasks, we can construct a compressed C235 color wheel can be used in the following tasks:The design of a CTask 1: (33 Primes) Express pigment-color values based on Goldbach’s conjecture. Let T be the set composed of 0 and the first 33 prime numbers, i.e.,T; and any odd number between 0 and 255 can be expressed as the sum of the two numbers in T plus one. This means that we can represent all RGB colors using 3 × 33 = 99 key values.Then, we know that any even number between 4 and 255 can be expressed as the sum of the two numbers  in Task 2: (Reduction of primes) Form a subset T was obtained in Task 1U consists of 18 prime numbers as followsSubset Task 3: (Attractors) Generate attractors for the compressed C235 color wheel.2 = 5292 attractors, each of which is expressed as There are 3 × 42w-Code(i) as the color code of an attractor i on a color wheel, expressed asDenote Task 4: (Attractor assignment) Assign each of 2 = 5292 blocks on a C235 wheel.Task 5: (Compression rate and error) The compression rate of the C235 color wheel versus the RGB wheel is Because the distance from the center of a block to the 4 attractors of the block is bounded by 235 wheel uses a much smaller number of hue blocks to express codes than RGB and CMYK3 do. The comparison is listed in Table 235 wheel is more effective in unifying colors than RGB, CMYK, and HSV are, as illustrated in Fig. 235 wheel, as shown in Fig. a, b, c, d, e, and f be six reference points on the wheel. The dash-line enclosed area located at the top of the wheel is zoomed in as shown in the square under the wheel Each color is expressed in a universal format that can be converted into an RGB, CMYK, or HSV frame. For instance, given b and c.For (ii)a, b, and c. The code is (iii)Notice that Expression 3) indicates that the C indicateeel Fig. . Several235 wheel, and assigned to attractor point a. Moreover, the value 1.17 reflects that 7. Interestingly, the C235 color system allows users to conveniently merge lights and colors and facilitates the design of smart lighting systems by adjusting users’ preferences. In fact, such a system can be widely used in fashion shows, painting exhibitions, and commodity displays19. Suppose there is a natural light LCD  technology is widely adopted for making cellular phones, tablets, TVs, and many other electronic products. Most such involved displays use LED as the light source. A typical LED is fed with pulsed high currents for a short period of time using the pulse width modulation (PWM) technique to create modulated electronic pulses of the desired widthA user will face decisions under different scenarios. Suppose a natural light a2 = a3 = a5 = 100 and b2 = 100, b3 = 100, b5 = 0. Suppose that we want the apple to show the orange color , then we know 120 = (c2 + 100)100/256 to get c2 ≐ 207. Similarly, 60 = (c3 + 100)100/256 implies that c3 ≐ 53. Therefore, the control light should be <2207353>, as shown in Fig. Assume that a yellow apple is irradiated by a white light, as shown in Fig. The present study is related to the design of PWM, especially for LCD. Consider the following application scenario.Example 3: When a user wants to let an LCD pixel emit color 9 demands more logical bits for representing colors and a sophisticated hardware design, as well as higher power consumption for emitting color lights. Please refer to Supplementary B for the technical details of the traditional PWM. The design proposed in this study is accomplished by employing an extended Goldbach conjecture in a C235 color system such that the nine pulse widths used for presenting each of R, G, or B at a pixel in the current 9-bit LCD display can be reduced to only two widths. Hence the new design may significantly reduce the technological complexity in associated devices.A traditional PWM modulator14. In the current LCD system, for any integer X between 0 and 256, X can be expressed asOne promising application of our color coding is for the LCD industry. Currently, to emit the R, G, and B lights of 256 colors, each pixel on the LCD screen has three LEDs under the control of a pixel circuit. All pixel circuits follow the order of an LCD central computing centerX can be explored by C235 asy and z are primes, 235 can generate all even numbers by adding the two closest prime numbers. Each prime represents a pulse width. Therefore, the LCD control center first computes the required prime value to generate a specific light for a pixel, and then orders the pixel circuits to control each of its R, G, and B LEDs to emit two pulse widths. The total pulse widths generated is 235 can use two pulses only to generate any pulse between 0 and 256. Figure 235 LCD PWM is much less than that in the current LCD PWM, the required time and energy can be reduced significantly. This scheme could be valuable for designing high-rate future LCDs. Notably, 24 pulse widths are needed to generate a traditional PWM. With the C235 PWM, we can generate In contrast, in our case, based on Goldbach’s conjecture, for an integer between 6 and 254, An extended Goldbach conjecture can help us overcome the drawbacks of the current LCDs that demand many pulse widths for generating R, G, and B brightness. Goldbach’s conjecture states that any even number can be given as the sum of two prime numbers. From Goldbach’s conjecture, we have the following proposition.<Use Goldbach Conjecture for expressing RGB>P that contains all prime numbers between 3 and 149:Any even integer between 8 and 256 can be expressed as a sum of two elements in the following prime set For instance, 256 = 149 + 107, 200 = 103 + 97, and 90 = 67 + 23.It uses 34 primes to express any even number between 4 and 256. We are interested in forming a smaller prime set to express all numbers between 4 and 256 within a tolerable margin of error.Suppose that there is a set of integers, expressed in the form ofn belongs to the integer set n in N can be expressed as the sum of two primes in 16:In other words, A program for finding P, subject to the restriction that all integers in N can be expressed as the sum of two primes. The above is a linear 0-1 program that can be efficiently solved by most commercial optimization software. The solution to the above program isThis program intends to minimize the number of primes in set P.We then deduce the following proposition modified from Goldbach’s conjecture:<Extended Goldbach conjecture for 8-bit lighting>n in an 8-bit RGB system. For (i)n can be expressed Any value (ii)n.Any value (iii)p and q represent key color values in R, G, and B. Table p and q satisfying n = p + q. For instance, 10 = 5 + 5, 40 = 23 + 17, 220 = 113 + 107,…, 256 = 149 + 107. It is noted that 220 can also be expressed as 149 + 71. Since 113 and 107 are closer to each other than 149 and 171, we adopt the expression 220 = 113 + 107. We can also approximately express 218, 219, 221, and 223 as the sum of 113 and 107 with an error within Prime Consider any value Following the International Commission on Illustration, the tolerable rate of R, G, B brightness is 10%. Therefore, a brightness level larger than 30 is an admissible estimate within the tolerable error.8-bit LCD PWMA light color The error of the generated light is smaller than 15. If we colorize objects corresponding to attributes and allocate these colorized objects on a ring, we can visualize the corresponding relationships. By modifying a C235 wheel, we can design a C235 ring. Our method is also suitable for visualizing the relationship among them. By modifying a C235 color wheel, we can design a C235 ring for this purpose.The world is full of multi-attribute objectsx with a color code Code(x) and a number Number(x) as given in the following:(i)a is represented by number 1, since c h is numbered as 32 with color code Person (iii)c and person d are complementary with each other, where i, n, and o are triad-complementary becausePerson Consider a set of 16 people, identified as 235 method can be harnessed to colorize DNA codons18 for revealing the relationship between 22 amino acids and 64 genetic codons. There are 22 amino acids in a DNA codon, where each codon acid is composed of A, G, C, T17. For instance, acid 1 is composed of GCT, GCC, GCG, and GCA. By utilizing C235, we can assign each acid a color code and a number, and thus allocate all 22 amino acids on a C235 ring. If we denote the al, gl, cl, and tl, the color codes of A, G, C, T at position l for l = 1, 2, 3, then we haveMore interestingly, our C8  and the outer circle the color of 3020, i.e., gray at level 20  to classify millions of items of merchandise worldwide20. The current HS classification is a six-digit code displayed on a large text table. By utilizing the C235 color system, we can assign colors to HS merchandise codes to help people easily recognize each of these goods.Other than DNA codons, the CSupplementary Information for Unifying Colors by Primes


Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and its incidence is steadily increasing. The development of HCC is a complex, multi-step process that is accompanied by alterations in multiple signaling cascades. Recent years have seen advancement in understanding molecular signaling pathways that play central roles in hepatocarcinogenesis. Aberrant activation of YAP/TAZ, Hedgehog, or Wnt/β-catenin signaling is frequently found in a subset of HCC patients. Targeting the signaling pathway via small molecule inhibitors could be a promising therapeutic option for the subset of patients. In this review, we will introduce the signaling pathways, discuss their roles in the development of HCC, and propose a therapeutic approach targeting the signaling pathways in the context of HCC.Hepatocellular carcinoma (HCC) is a major health concern worldwide, and its incidence is increasing steadily. To date, receptor tyrosine kinases (RTKs) are the most favored molecular targets for the treatment of HCC, followed by immune checkpoint regulators such as PD-1, PD-L1, and CTLA-4. With less than desirable clinical outcomes from RTK inhibitors as well as immune checkpoint inhibitors (ICI) so far, novel molecular target therapies have been proposed for HCC. In this review, we will introduce diverse molecular signaling pathways that are aberrantly activated in HCC, focusing on YAP/TAZ, Hedgehog, and Wnt/β-catenin signaling pathways, and discuss potential therapeutic strategies targeting the signaling pathways in HCC. The incidence of liver cancer has been steadily increasing, posing a major global health problem. The World Health Organization reported that about 800,000 deaths were due to liver cancer, making it the fourth leading cause of cancer-related death . HepatocMolecular target therapy has been intensively studied in recent years as a promising treatment option for patients with advanced HCC ,6,7. TheGiven the extreme heterogeneities in the molecular signature of HCC and difficulties in the development of globally-effective therapeutics for the disease, identification of molecular signaling pathways dysregulated in HCC would be fundamental to developing therapeutic targets as a personalized medical approach. In this review, we will introduce molecular signaling pathways beyond RTK that are aberrantly activated in HCC, that is, YAP/TAZ, Hedgehog, and Wnt/β-catenin signaling pathways, and discuss potential therapeutic strategies targeting the signaling pathways in the context of HCC.YAP/TAZ signaling is majorly activated when a tumor-suppressive Hippo signaling pathway is inactivated. A variety of mechanical signals such as cell shape and extracellular matrix (ECM) stiffness, as well as cell–cell interactions, elicit signals regulating the Hippo signaling cascade ,15,16. TThe Hippo–YAP/TAZ signaling pathway consists of mammalian sterile 20-like kinase 1  and MST2 , large tumor suppressor kinase 1 (LATS1) and LATS2, the adaptor proteins Salvador 1 (SAV1), MOB1A and MOB1B, and yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1  ,20 Figu. The sigYAP/TAZ signaling is involved in multiple facets of carcinogenesis, including the promotion of cellular proliferation, maintenance of cancer stem cells (CSCs), and induction of tissue invasion of tumor cells, as well as drug resistance and recurrence of cancer ,27,28,29Human HCC cells with high YAP or TAZ levels exhibit poor differentiation ,34. MoreIn addition to the roles played by YAP/TAZ in cell proliferation, apoptosis and migration, YAP/TAZ are also involved in hepatic fibrosis. Considering significant correlations between hepatic fibrosis and liver cancer, it is noteworthy that YAP/TAZ could initiate and promote hepatocarcinogenesis via upregulation of stromal activation and hepatic fibrosis ,26. FinaThe precise roles of the Hippo–YAP/TAZ signaling pathway in hepatocarcinogenesis have been studied using genetically engineered mouse models in which various components of the signaling cascade were deleted or activated . The stuflox/flox mice and AAV-CRE  led to the elevation of YAP/TAZ levels in the liver . In. In87]. The synthesis of hedgehog ligands is stimulated by diverse factors that trigger liver regeneration, which include various liver mitogens and cell stressors. The Hedgehog ligands are released from ligand-producing cells into the local environment where they engage receptors on Hedgehog responsive cells. In particular, Sonic Hedgehog (Shh), the representative Hedgehog ligand, enhances the viabilities of Hedgehog target cells, serving as viability and proliferative factors for various types of liver cells ,94. Shh Shh is strongly expressed in the ventral foregut endoderm where liver, pancreas and lung buds develop . Shh is The elevated expression of Shh and its target genes such as Ptch and Gli was detected in HCC tissues ,108,109.Various studies employing animal models have demonstrated that the Hedgehog signaling pathway plays an important role in hepatocarcinogenesis. A transgenic mouse model was developed in which Shh is secreted from hepatocytes, which mimics histopathological features of patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) . Secreteflox/flox; αSMA-CreERT2 mice in which treatment with tamoxifen induces nuclear import of Cre recombinases in myofibroblasts and leaves out an exon of Smo gene [flox/flox; αSMA-CreERT2 mice with tamoxifen showed a blockade of Hedgehog signaling selectively in myofibroblasts and inhibited liver fibrosis [Genetic ablation of Smo in myofibroblasts can be achieved using SMOSmo gene . The trefibrosis . This suPreclinical studies targeting the Hedgehog signaling pathway mainly focus on the inhibition of the activity of Smo. For example, cyclopamine can block the Hedgehog signaling pathway by inhibiting Smo. It is demonstrated that cyclopamine increased apoptosis and decreased cells growth and proliferation in various HCC cell lines including SMMC-7721, SK-Hep1, Hep3B, Huh7, and PLC ,108,116.Taccalonolide A is a Hedgehog antagonist. Treatment with Taccalonolide A decreased mRNA levels of Hedgehog signaling molecules and led to reduced viability of HCC cells . GANT61 The Wnt/β-catenin signaling is a signal transduction pathway that initiates with the binding of Wnt protein to its receptor Frizzled and then transmits the signal through β-catenin. In the absence of Wnt, β-catenin is downregulated by a destruction complex which consists of Glycogenesis kinase3β (GSK3β) and casein kinase1α (CK1α), tumor suppressor adenomatous polyposis coli (APC), and scaffold protein AXIN  122,123123. The The binding of Wnt to receptors Frizzled on the cell surface recruits phosphoprotein Dishevelled (Dvl) to the receptor, providing a binding site for Axin and GSK3β. This event leads to phosphorylation of transmembrane low-density lipoprotein receptor-related protein5/6 (LRP5/6), and more importantly inhibits phosphorylation of β-catenin by GSK3β . UnphospCTNNB1 gene (coding for β-catenin), rendering the protein resistant to degradation [CTNNB1 mutations are caused by missense substitution which occurs at exon 3 at amino acid position 29–49 [Studies have shown that there is a strong correlation between the level of nuclear β-catenin and the tumor grade of HCC ,142. Eleradation ,145. Thron 29–49 ,146,147.on 29–49 ,149. ThuCTNNB1 strongly suggests that loss-of-function mutation in negative regulators of β-catenin such as APC or AXIN should be present in HCC. In line with the speculation, a loss-of-function mutation in AXIN occurs at a frequency of 3–16% in HCC, which is the second most common mutation in HCC that leads to activation of the Wnt/β-catenin signaling pathway [CTNNB1 among 100 primary HCC tissues. Of note, they found 6 mutations in AXIN1 in the 87 tumor samples that had no CTNNB1 mutations, underlining the significance of AXIN1 mutation in human HCC [CTNNB1, 37.5% of mutations found in AXIN are nonsense mutations, while only 30% of mutations are attributed to missense mutations [The fact that the accumulation of β-catenin in HCC is more frequently found than mutations in  pathway ,146,150. pathway ,151,152.uman HCC ,151,152.utations ,136. G12V) alone did not induce HCC, while the co-expression of NRASG12V with an activated mutant form of β-catenin led to the formation of HCC [Although aberrant activation of β-catenin is frequently found in human HCC, the expression of a constitutively active form of β-catenin alone was not enough to develop HCC in murine models ,154. Thin of HCC . The simn of HCC . Considen of HCC ,159.The co-activation of both the Wnt/β-catenin and Akt/mTOR pathways is found in about 10% of patients with HCC who frequently show poor survival. Toh et al. reported that the co-expression of constitutively active forms of human AKT and β-catenin led to the formation of a high HCC tumor burden in both male and female FVB/N mice with 100% penetrance . Of noteBased on the frequent activation of Wnt/β-catenin signaling in HCC, various molecules targeting the signaling pathway have been tested for HCC treatment. In an attempt to block the initiation step of the signaling cascade, monoclonal antibodies such as OMP-18R5 have been developed which bind to Frizzled receptors and inhibit its interaction with Wnt ligands. OMP-18R5 showed suppression of tumors in xenograft models of various types of cancers . OMP-54FConsidering that Frizzled receptor 7 (FZD7) is most frequently overexpressed in HCC among the 10 human Frizzled receptors , small iCGP049090, PKF118-310, and PKF115-584 are small molecules that inhibit the interaction between Tcf4 and β-catenin, and thus suppress the transcriptional activity of the Tcf4/β-catenin complex. In in vitro studies, treatment with the inhibitors induced apoptosis and cell cycle arrest in human hepatoma cell lines, HepG2, Hep40, and Huh-7 ,169. FurTankyrase acts as an activator of Wnt/β-catenin signaling by mediating the degradation of AXIN1 and AXIN2 which are negative regulators of β-catenin . NVP-TNKThe development of HCC is a complex, multi-step process accompanied by alterations in multiple signaling cascades. A fundamental understanding of molecular signaling pathways to tumorigenesis can help predict patient response to targeted therapies, which can have a substantial impact on clinical decision-making. Of all the tumor driver genes, those responsible for oncogenic addiction are important ,176. AccMost studies targeting the YAP/TAZ, Hedgehog, or Wnt/β-catenin signaling pathways in HCC are currently under preclinical stages in which the target therapies have reduced cell viability, proliferation and migratory potentials of HCC. Phase I and/or II clinical trials targeting the signaling pathways have been completed or are undergoing . With thAll authors have read and agreed to the published version of the manuscript.


Colaphellus bowringi, an economically important pest of Cruciferous vegetables. As an initial step, 14 genes from the extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (P38) cascades were knocked down using RNA interference (RNAi). The results revealed that RNAi knockdown of MAPK-ERK kinase (MEK), ERK, Kinase suppressor of RAS 2 (KSR2), and P38 induced ovarian development stagnation, low fecundity, and decreased longevity, which indicate that ERK and P38 signaling pathways are important for female C. bowringi survival and reproduction. The potential regulatory role of ERK and P38 pathways in the female reproductive process was investigated using quantitative real-time PCR. We found that ERK pathway possibly regulated ecdysone biosynthesis and P38 pathway possibly involved in the germline stem cell (GSC) development and differentiation. Our findings demonstrated the importance of the MAPK signaling pathway in the female reproduction of insects, and further enhanced the molecular mechanism of female reproductive regulation in insects.The mitogen-activated protein kinase (MAPK) signaling pathway is a well-conserved intracellular signal transduction pathway, and has important roles in mammalian reproduction. However, it is unknown whether MAPK also regulates insect reproductive mechanisms. Therefore, we investigated the role of the MAPK signaling pathway in ovarian growth and oviposition in the cabbage beetle  In eukaryotic organisms, the mitogen-activated protein kinase (MAPK) signaling pathway is a well-known and well-conserved intracellular signal transduction pathway. This pathway is essential for yeast to mammalian cell differentiation, proliferation, apoptosis, and metabolism ,2,3. ExtDrosophila melanogaster [Bactrocera dorsalis [According to the above explanation, ERK, JNK, and P38 pathways are involved in various physiological processes via different mechanisms. In amphibians, fish, and mammals, the importance of the MAPK signaling pathway in reproduction has been thoroughly studied, for instance, ERK cascade regulates the meiosis of oocytes ,13, the nogaster ,19. In adorsalis . HoweverD. melanogaster, germline stem cells (GSC) differentiated into various cells such as oocytes and nurse cells, demonstrating the importance of GSC development and differentiation [protein nanos (Nanos) and DEAD-Box Helicase Vasa (Vasa) being the most commonly used marker genes [Several studies have linked female insect reproduction to population growth and maintenance. The ovary is a major reproductive organ in female insects, and its development is usually divided into two stages: previtellogenesis and vitellogenesis. It has been demonstrated that during the previtellogenesis process in ntiation ,22, wither genes ,24. Viteer genes ,26. Prever genes ,28,29. Mer genes ,31. Seveer genes ,33,34. HC. bowringi, an economically significant pest of Cruciferous vegetables. The larvae and adults of C. bowringi mainly fed on leaves of host vegetables, reducing the yield of vegetables and causing economic losses [C. bowringi ovarian development by regulating Vg synthesis [C. bowringi, expanding our understanding of female reproductive regulation in insects.In this study, we first identified 14 candidate genes from ERK, JNK, and P38 signaling pathways before performing RNAi knockdown on the cabbage beetle c losses , which ic losses . Most prynthesis ,38,39. TC. bowringi adults were captured from the natural population in Xiushui County , Jiangxi Province, China. The beetles were raised at 25 °C and 70% relative humidity and were fed radish leaves (Raphanus sativus var. longipinnatus), according to previous study [Approximately 500 us study . The newC. bowringi transcriptome database (PRJNA575933) was used to retrieve the sequences of 14 candidate genes from the activator, MAPKKK, MAPKK, and MAPK members of ERK, JNK, and P38 cascades  was used to deduce all corresponding protein sequences. MEGA 6 software created 14 rooted phylogenetic trees using the neighbor-joining method based on these sequences. The Simple Modular Architecture Research Tool (SMART)  predicted the functional protein domains.Based on a reference pathway (map04013) from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and previous research ,42, C. bcascades . Open reribosomal protein L19 (RPL19) and Actin1 were used as reference genes [−ΔΔCT method was used to assess relative expression levels [Total RNA was extracted using TRIzol  per manufacturer specifications. The PrimeScript RT reagent Kit with gDNA Eraser  was used to perform cDNA reverse transcription according to manufacturer’s instructions. qRT-PCR was then conducted on an ABI QuantStudio 6 Flex  using the corresponding primers  and TB Gce genes . The 2−Δn levels  with thrAll double-stranded RNAs (dsRNAs) were synthesized using RNAi primers  and a T7n = 18–37) and ovarian sizes (n = 10–13) were recorded and measured, as instructed in previous studies [Tissues from 15 females were obtained at 4 days PE for each biological replicate, including heads, midguts, fat bodies, and ovaries. For total RNA isolation and qRT-PCR, each treatment had three biological replicates. At 4-day PE, ovarian development grades  were used to represent values.All experimental results were analyzed statistically using SPSS 14  and GraphPad Prism 8 . One-way ANOVA was used to determine the significance of tissue expression patterns, followed by Tukey’s LSD tests (α = 0.05). To determine the significance of survival rates, a Kaplan–Meier (KM) survival estimate with a log-rank test was used. For other variables, significance differences were achieved using an Independent-samples C. bowringi clustered with homologous genes from other Coleoptera species such as Tribolium castaneum , resulting in the death of all beetles at 10 days PE. Knocking down ERK significantly increased mortality (p < 0.0001), whereas silencing of KSR2 (p = 0.0404) and P38 (p = 0.0052) resulted in a modest but significant decline in adult longevity  and Shade are critical enzymes that catalyze the production of JH and 20E, whereas Krüppel homolog 1 (Kr-h1) and E74 are key factors that respond to JH and 20E signaling ,37,38,39JHAMT1 and Kr-h1 transcript levels between the dsGFP control and the dsMEK, dsKSR2, dsERK and dsP38 treatments  synthesis in the swimming crab Portunus trituberculatus [RAC1, TAK1, MKK7, and JNK did not result in any defects in yolk deposition or ovarian development in C. bowringi, implying that the JNK signaling pathway is not required for female insect reproduction. To the best of our knowledge, this is the first time that the function of the MAPK signaling pathway in insect ovarian development is systematically demonstrated by gene function loss.In vertebrates, the role of the MAPK signaling pathway in reproductive regulation has been explained. The ERK signaling pathway, for example, regulates critical physiological events in reproduction such as oocyte meiosis, follicle growth, and ovulation ,50,51. Hrculatus , the knoERK causes low ovariole number and fecundity in the green lacewing Chrysopa pallens [Drosophila loss-of-function P38 MAPKK homolog mutant [P38 deficiency results in immature ovaries and low egg production, implying that P38 signaling pathway is critical for female reproduction in insects. Furthermore, targeting ERK and P38 cascades reduced longevity, most likely due to inhibition of cell proliferation and reactive oxygen species signaling [Previous research on insects suggested that ERK signaling pathway was involved in meiotic metaphase II arrest , vitello pallens , consistg mutant . Howeverignaling ,60. ThesMEK, ERK, KSR2, and P38 tissue expression patterns revealed that their expression levels were higher in comparison to other tissues, indicating that ERK and P38 pathways are important for ovarian development in cabbage beetles. We detected the expression levels of critically biosynthetic and inducible JH and 20E signaling genes to further investigate the possible mechanism of these four key genes regulating female reproduction. The JH and 20E are important regulators in female insects during previtellogenesis and vitellogenesis [C. bowringi [D. melanogaster [Bombyx mori [Leptinotarsa decemlineata larvae [MEKK4 and MAP2K6 participated in 20E synthesis in the female adults of B. dorsalis, suggesting that the MAPK signaling pathway is also involved in 20E synthesis in the ovaries [ERK and P38. Previous research concluded that JH activated ERK and P38 [Nanos and Vasa, which are important in GSC development and differentiation. Nanos and Vasa were highly conserved in vertebrates and invertebrates. They were discovered in D. melanogaster and both responsible for primordial germ cell (PGC) determination, migration, GSC differentiation, and development [ERK resulted in a slight decrease in Vasa expression, whereas silencing P38 resulted in a decrease in Nanos and Vasa expression. In the year 1999, scientists proposed that P38 was downstream of decapentaplegic signaling and appears to be involved in the localization and translation of Nanos and Vasa [MEK and KSR2 had no effect on JH and 20E biosynthesis or GSC development and differentiation. This finding implied that there might be functional compensation in the ERK signaling pathway, and MEK and KSR2 probably play important roles in female reproduction independent of the ERK signaling pathway. Therefore, a further study that investigating how these two genes regulate female reproduction in C. bowringi would be interesting.The ogenesis , which hbowringi ,38,39,45nogaster ,62, Bombbyx mori , and Lepa larvae . A recen and P38 ,65, whilelopment ,66,67. Tand Vasa ,69. Our In conclusion, the current study systematically identified the genes of MAPK signaling pathways and examined the functional differentiation of ERK, JNK, and P38 pathways in cabbage beetle female reproduction at the transcriptional level. According to our findings, the ERK signaling pathway may influence reproduction by regulating 20E biosynthesis, whereas the P38 signaling pathway most likely regulates GSC development and differentiation. This study unveiled the importance of the MAPK signaling pathway in female insect reproduction and advanced our understanding of the molecular mechanisms of insect reproductive regulation.


Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development and plays a key role in tumor proliferation, invasion and metastasis. Numerous long non-coding RNAs (lncRNAs) could regulate the occurrence and development of EMT through various complex molecular mechanisms involving multiple signaling pathways in PCa. Given the importance of EMT and lncRNAs in the progression of tumor metastasis, we recapitulate the research progress of EMT-related signaling pathways regulated by lncRNAs in PCa, including AR signaling, STAT3 signaling, Wnt/β-catenin signaling, PTEN/PI3K/AKT signaling, TGF-β/Smad and NF-κB signaling pathways. Furthermore, we summarize four modes of how lncRNAs participate in the EMT process of PCa  Prostate cancer (PCa) is one of the most common malignancies in males worldwide and has become a global “killer” threatening the health of elderly men. The incidence and mortality of PCa rank second and fifth in men worldwide, respectively . Accountβ-catenin and Snail protein, are up-regulated, thereby allowing the cells to migrate or metastasize to different organs  sensitivity via inhibiting AR signaling . The stuet al.  found thTaken together, the interaction network between AR and lncRNAs is multi-directional. AR can induce the transcription of certain lncRNAs, and lncRNAs could also regulate the expression abundance of AR at mRNA or protein level. Such reciprocal mode inside the AR-lncRNAs network may indicate us more potential methods to improve current anti-androgen therapies.et al. family. STAT family has dual functions of signal transduction and transcriptional activation. In JAK-STAT signal pathway, JAK (Janus kinase) and STATs are intracellular and receptor binding proteins, which complete signal transduction from cytoplasm to nucleus . Generalet al., 95% of et al.  and T-cell factor/lymphoid enhancing factor (TCF/LEF), respectively. It plays an important role in maintaining cell adhesion and the morphological structure of adjacent tissues.  is a recently identified lncRNA that could promote PCa metastasis via regulating Wnt/β-catenin . The ceRet al.  reportedMoreover, besides the canonical Wnt/β-Catenin pathway, the non-canonical Wnt pathway also participates in the EMT progression of PCa. As a non-canonical Wnt signaling pathway ligand, Wnt5a works with its receptor FZD2 and induces the expression of EMT-related genes in PCa. LncRNA MCM3AP-AS1 has been demonstrated abnormally up-regulated in PCa tissues and could up-regulate the expression of Wnt5a at mRNA and protein level via sponging miR-876-5p in PCa cells .The deficiency of PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor and the oncogenic activation of PI3K  signaling axis are among the most common altered signaling pathways in primary PCa that facilitate tumor occurrence, disease progression and therapeutic resistance . Recent via activation of ERK and PI3K/AKT signaling pathways , lymph-node metastasis (p = 0.035), Gleason score (p = 0.001), tumor stage (p = 0.036) and recurrence-free survival of PCa patients. In vitro biological experiments have further proved that knockdown of LINC01296 can inhibit the proliferation, migration and invasion of PCa cells by inhibiting the activity of the PI3K/AKT/mTOR pathway. Another study , phosphorylation of IκBα (activated by IKK complex), degradation of IκBα protein and release of NF-κB and the nuclear shuttle of NF-κB. The aberrant activation of NF-κB signaling pathway is also reported to participate in the alteration of diverse tumor phenotypes . In humaet al.  reportedThe above is the main lncRNAs-regulated signaling pathways that were mostly reported to participate in the EMT process of human PCa cells, while researches about other signaling pathways, especially the pathways targeting EMT process, are not so abundant. Here, we selected the three most typical ones to make a brief discussion. The first one is NORAD. In addition to regulating Wnt/β-catenin signaling pathway, lncRNA NORAD also could regulate the release of extracellular vesicles (EVs) to promote the bone metastasis of PCa cells . Via regIn our review, we discuss the role of lncRNAs in participating in the EMT process in PCa via regulating multiple signaling pathways, including AR signaling pathway, STAT3 signaling pathway, Wnt/β-catenin signaling pathway, PTEN/PI3K/AKT signaling pathway, TGF-β/Smad pathway and NF-κB signaling pathway. Based on our discussion, there are four modes of how lncRNAs participate in the EMT process of PCa via regulating diverse signaling pathways: 1) Forming a ceRNA network via sponging miRNAs to alleviate the repression of downstream target genes and therefore activate or inactivate related signaling pathways, such as MAGI2-AS3 in regulating STAT3 signaling pathway and NORAD in regulating Wnt/β-catenin signaling pathway shown in ; 2) FormMoreover, considering that metastasis is the main lethal cause of PCa patients, we hope clinical doctors and researchers could benefit from our current literature review. From our standpoint, among the signaling pathways we discussed above, lncRNAs targeting AR signaling pathway are of much more values and here we raised several unsolved points that deserve further exploration. 1) Bioinformatic analysis or clinical researches about lncRNAs that could predict the metastasis-free survival (MFS) is absent. As distant metastasis is universally regarded as the terminal status of PCa patients, researches that are lack of quantitative analysis of MFS generally failed to evaluate the progress of PCa patients, since only MFS has been shown to be a surrogate endpoint for overall survival (OS) . 2) Rese