Over the last few decades, benefitting from the sufficient sensitivity, high specificity, nondestructive, and rapid detection capability of the surface-enhanced Raman scattering (SERS) technique, numerous nanostructures have been elaborately designed and successfully synthesized as high-performance SERS substrates, which have been extensively exploited for the identification of chemical and biological analytes. Among these, Ag nanorods coated with thin metal oxide layers (AgNRs-oxide hybrid array substrates) featuring many outstanding advantages have been proposed as fascinating SERS substrates, and are of particular research interest. The present review provides a systematic overview towards the representative achievements of AgNRs-oxide hybrid array substrates for SERS applications from diverse perspectives, so as to promote the realization of real-world SERS sensors. First, various fabrication approaches of AgNRs-oxide nanostructures are introduced, which are followed by a discussion on the novel merits of AgNRs-oxide arrays, such as superior SERS sensitivity and reproducibility, high thermal stability, long-term activity in air, corrosion resistivity, and intense chemisorption of target molecules. Next, we present recent advances of AgNRs-oxide substrates in terms of practical applications. Intriguingly, the recyclability, qualitative and quantitative analyses, as well as vapor-phase molecule sensing have been achieved on these nanocomposites. We further discuss the major challenges and prospects of AgNRs-oxide substrates for future SERS developments, aiming to expand the versatility of SERS technique. W4–107 [2 ,18,19. B4–107 [2 ,21, chem4–107 [2 ,22, clin4–107 [2 ,24, envi4–107 [2 ,26, and 4–107 [2 ,28.9 [Recently, nanofabrication approaches based on oblique angle vapor deposition (OAD) ,30,31 an9 , and hav9 ,33,37, b9 ,44, viru9 ,46, amin9 , uranyl 9 , polychl9 ,34, and 9 ,50, whic9 ,52,53, a9 ,55,56, l2O3, TiO2, SiO2, HfO2, and so on. Taking advantages of the excellent stability and multi-functions of oxide layers, AgNRs-oxide hybrid array substrates provide superior SERS sensitivity and reproducibility, high thermal stability, long-term activity in air, corrosion resistivity, and intense chemisorption of target molecules. These advantages contribute to recyclable and cost-effective SERS substrates for both qualitative and quantitative analyses. This review introduces the synthesis of AgNRs-oxide arrays as versatile SERS substrates, summarizes their structural, physical, and chemical properties, and highlights their practical applications. We further discuss the major challenges and prospects of AgNRs-oxide substrates for future SERS developments.To overcome these inevitable limitations, covering AgNR arrays with thin metal oxide layers has been proposed as a strategy to solve the above problems ,59,60,61θ (>70°) with respect to the substrate normal [AgNR arrays are synthesized based on OAD technique. OAD is a physical vapor deposition technique in which the vapor atoms are deposited at a large incident angle e normal ,37. The e normal ,57. As se normal ,31,37,582O3 over AgNRs using trimethylaluminum (TMA) and water as ALD precursors [2 was adopted as the carrier and purge gas. Typically, one complete reaction cycle consisted of four steps: (1) TMA reactant exposure; (2) N2 gas purging; (3) water vapor exposure; and (4) N2 gas purging. This reaction cycle was repeated for different times so as to control the Al2O3 thickness. TiO2 and HfO2 have also been successfully deposited over AgNRs by ALD [2 layers have been coated onto AgNRs to form AgNRs-SiO2 core-shell nanostructures [2 more compact, and its thickness was tailored by varying the coating time. Moreover, Huang et al. proposed to cover AgNRs with a thin TiO2 layer directly through OAD fabrication [2, and did the deposition again at the same substrate orientation. Because this coating approach only requires the OAD system instead of many complicated apparatuses, it is very efficient and cost-effective and thus promising for real-world fabrication.Several approaches have been exploited to produce oxide layers over AgNR arrays. Because SERS is a highly localized effect that depends significantly on the distance between metal surfaces and target molecules ,63,64, tecursors . High pus by ALD ,60, withructures . The sherication . To be s2O3, TiO2, and HfO2 shells fabricated by ALD is about 0.6–0.8 nm per ALD cycle [2 grown by hydrolysis reaction is approximately 0.27 nm/min [2 capping prepared by the OAD method in LD cycle ,57,60, a7 nm/min . For TiO7 [It has been long recognized that the SERS enhancement of metal nanostructures depends strongly on the distance between metal surfaces and adsorbed molecules. We therefore investigate the coating effect on the sensitivity of SERS substrates. The results in 7 ,57,60, c7   are robust in morphology at 200 °C, but melt partly at 300 and 400 °C. For AgNRs coated by 2-cycle Al2O3 of ~1.5 nm thick, no obvious structural change is observed after being heated at 300 and 400 °C [2 or HfO2 shell also sustain their morphology and SERS efficiency at 300–400 °C [2 of ~5 nm, the Ag mass transport from tips to sides is slowed down. They preserve their shapes well at 100 °C, while they coarsen to some extent at 200 °C [High-temperature SERS detection is a vital part for routine applications, which can be utilized for monitoring many in situ reactions, such as thermal crystallization , structud 400 °C . The mor0–400 °C ,60. Moret 200 °C . These r2O3 presents a slight signal decrease after 50 days, which can be explained by the pinhole-containing feature of the ultrathin oxide shell. While the substrates coated by ~1.5 nm or thicker Al2O3 remain almost constant in SERS response during the whole test period. As a result, a thin but compact coating layer could sufficiently passivate the internal Ag NRs so as to stabilize their SERS activity under atmospheric conditions for a long period. Similar conclusion has been verified on AgNRs-TiO2 substrates [The effect of oxide coating on the temporal stability of AgNRs in air is investigated as a function of shelf time, see details in bstrates .2O3 deposition, the chemical stability of coated substrates is substantially improved, even if the oxide shell is sub-nanometer and has a few pinholes [2O3 shell prevents the internal AgNRs from direct contact with external etchants such as NaCl and H2O2, retaining sufficiently their morphology and SERS efficiency. Additionally, for strong acidic media where Al2O3 will dissolve quickly, ultrathin HfO2 film has been implemented to protect AgNRs from failure [To function as reliable SERS sensors, the chemical stability is of essential importance especially at erosive environments. Because chloride ions ,72,73, spinholes . As show failure . This ki2 [2. Strong target signals are observed during SERS sensing, and they almost vanish after UV irradiation. More importantly, the results from the subsequent three circulations show that the Raman intensities are fully recovered at each detection step, which suggests that the AgNRs-TiO2 structure is capable of enduring multiple UV irradiations with considerable robustness. Another point to note is that the molecule degradation capability not only comes from the photocatalysis of TiO2, it also benefits from the intensive interaction between Ag and TiO2 that optimizes the separation of photo-excited charge carriers and as a result facilitates the degradation efficiency  functional groups, ascribed to the strong polar interaction [2O3 substrates to enhance the absorbability and correspondingly SERS detection efficiency of carboxylic acids. Dipicolinic acid (DPA) is a commonly used biomarker for the recognition of bacterial spores [2O3 as the SERS platform, with the limit of detection (LOD) down to 10−8 M. Meanwhile, the partial least squares regression (PLSR) [−8 to 1 × 10−5 M, which provides a calibration for the quantification of trace DPA. In addition to analytes that can directly react with Al2O3 surfaces, for those who have no or weak interaction with oxides  ,86 model cyanide ,88,89), tability . These r2 substrate with acid resistance is applicable for their identification [From a more realistic perspective, AgNRs-oxide substrates have been adopted to sense food antiseptics . It is kfication . The LOD2 substrate is placed in a homemade gas detection system presented in 2 is utilized as the carrier gas. During gas detection, N2 is injected into the analyte solution, and target molecules in the vapor phase are carried out together with the N2 flow and are captured by the AgNRs-HfO2 platform. SERS spectra are recorded simultaneously during gas passing. It is shown in 2 substrate for gas detection. Furthermore, we explore the renewability of AgNRs-HfO2 arrays during gas recognition, i.e., after SERS measurement, the substrate is heated on a hot plate at 250 °C for 30 s to desorb molecules. The results in −1 peak escalate with vapor passing and after each annealing treatment, the substrate is free of 2-NAT while it is totally recovered in the following “vapor exposure-thermal cleaning” cycles. We believe this highly robust and versatile SERS platform could act as a recyclable sensor for in situ monitoring of complex gases from realistic environments, such as air pollutants [Aside from revealing the feasibility of SERS determination in liquids, AgNRs-oxide arrays also provide real-time monitoring of vapor-phase molecules at ultralow concentrations . To captllutants ,95, explllutants ,97, volallutants ,99, and llutants ,101.This review provides insights for the synthesis, characterization, and applications of AgNRs-oxide hybrid array substrates as SERS sensing platforms, all in order to emphasize our understanding and utilization of the SERS technique. AgNR arrays are prepared based on the OAD method, and oxide shells are readily coated onto AgNRs by diverse approaches with controllable thickness and excellent uniformity. The characterization of these nanocomposites is detailed in aspects of shell growth, SERS sensitivity, and reproducibility, as well as thermal, temporal, and chemical stability. By virtue of the ultrathin thickness, uniformity, and stability of oxide layers, AgNRs-oxide substrates possess large SERS EFs, outstanding reproducibility, and excellent stability. In addition, the reusability, quantification, and gas sensing have been achieved on the substrates, which hold great potential for the identification of trace analytes in real systems. 2O3 substrate. As for the SERS-based immunoassay and nucleic acid detection, AgNRs-oxide arrays offer potent capture substrates to bind SERS nanotags. The close proximity of the substrate–nanotag interface provides additional plasmonic coupling that further increases the SERS enhancement. It is thus urgent to demonstrate the SERS determination of bacteria, viruses, nucleic acids, and proteins on AgNRs-oxide arrays before the substrates can be routinely applied. Second, surface functionalization [2 [Although AgNRs-oxide array substrates have proved their extraordinary advantages, they are still in their infancy for practical SERS applications, and several challenges are yet to be addressed. First, AgNRs-oxide hybrid arrays possess satisfactory reproducibility and reliability exceeding the performance of commonly used nanoparticles, which are crucial for biosensing. The qualitative and quantitative detection of DPA, an anthrax biomarker, has been achieved on the AgNRs-Allization ,103,104 lization ,106,107 ation [2 ,109, is


ADCYAP1, ARNTL, ARNTL2, BHLHE40, CLOCK, CRY1, CRY2, CSNK1D, CSNK1E, GSK3B, HCRTR2, KLF10, NFIL3, NPAS2, NR1D1, NR1D2, PER1, PER2, PER3, REV1, RORA, RORB, RORC, SENP3, SERPINE1, TIMELESS, TIPIN, VIP, and VIPR2) are associated with MetS and its individual components independently and/or through complex interactions in a Taiwanese population. We also analyzed the interactions between environmental factors and these genes in influencing MetS and its individual components. A total of 3,000 Taiwanese subjects from the Taiwan Biobank were assessed in this study. Metabolic traits such as waist circumference, triglyceride, high-density lipoprotein cholesterol, systolic and diastolic blood pressure, and fasting glucose were measured. Our data showed a nominal association of MetS with several single nucleotide polymorphisms (SNPs) in five key circadian clock genes including ARNTL, GSK3B, PER3, RORA, and RORB; but none of these SNPs persisted significantly after performing Bonferroni correction. Moreover, we identified the effect of GSK3B rs2199503 on high fasting glucose (P = 0.0002). Additionally, we found interactions among the ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, and RORB rs972902 SNPs influenced MetS (P < 0.001 ~ P = 0.002). Finally, we investigated the influence of interactions between ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, and RORB rs972902 with environmental factors such as alcohol consumption, smoking status, and physical activity on MetS and its individual components (P < 0.001 ~ P = 0.002). Our study indicates that circadian clock genes such as ARNTL, GSK3B, PER3, RORA, and RORB genes may contribute to the risk of MetS independently as well as through gene-gene and gene-environment interactions.Increased risk of developing metabolic syndrome (MetS) has been associated with the circadian clock genes. In this study, we assessed whether 29 circadian clock-related genes (including  ARNTL), clock circadian regulator (CLOCK), cryptochrome circadian clock 1 (CRY1), cryptochrome circadian clock 2 (CRY2), neuronal PAS domain protein 2 (NPAS2), period circadian clock 1 (PER1), period circadian clock 2 (PER2), and period circadian clock 3 (PER3) genes [Circadian rhythms are naturally recurring cycles that regulate the timing of biological events such as the sleep-wake cycle and energy metabolism . The int3) genes .Clock and Arntl, may result in metabolic abnormalities of lipid and glucose homeostasis, hallmarks of MetS [CLOCK [CRY2 [CLOCK gene was associated with MetS, although there were no significant associations between any of these three SNPs and MetS individually [CRY2 rs75065406 had a nominally significant association with MetS, but this association did not remain significant after correcting for multiple testing [ARNTL gene was associated with hypertension, an individual component of MetS [NPAS2 rs11541353 was associated with hypertension and PER2 #10870 was linked to blood glucose levels [CRY1 haplotype (rs4964513 and rs12821586); however, the significance did not persist after adjusting for multiple testing [ARNTL, CRY1, and PER2 are expressed in human adipose tissue, and their gene expressions were related to the individual components of MetS such as waist circumference [Arntl, Clock, or Per3 genes, suggesting miscommunication between the circadian clock and metabolic pathways may lead to metabolic disorders [Accumulating evidence indicates that the circadian clock genes may contribute to the development of metabolic syndrome (MetS). Animal studies showed that a loss-of-function mutation of the circadian clock genes, such as  of MetS , 6. In hS [CLOCK  and CRY2CK [CRY2  genes. Pvidually . Additio testing . Moreove of MetS . Furtherisorders .ADCYAP1), ARNTL, ARNTL2, basic helix-loop-helix family member e40 (BHLHE40), CLOCK, CRY1, CRY2, casein kinase 1 delta (CSNK1D), casein kinase 1 epsilon (CSNK1E), glycogen synthase kinase 3 beta (GSK3B), hypocretin receptor 2 (HCRTR2), Kruppel like factor 10 (KLF10), nuclear factor interleukin 3 regulated (NFIL3), NPAS2, nuclear receptor subfamily 1 group D member 1 (NR1D1), nuclear receptor subfamily 1 group D member 2 (NR1D2), PER1, PER2, PER3, REV1 DNA directed polymerase (REV1), RAR related orphan receptor A (RORA), RAR related orphan receptor B (RORB), RAR related orphan receptor C (RORC), SMT3 specific peptidase 3 (SENP3), serpin family E member 1 (SERPINE1), timeless circadian clock (TIMELESS), TIMELESS interacting protein (TIPIN), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide receptor 2 (VIPR2) genes.While there have been several studies examining the relationship between single nucleotide polymorphisms (SNPs) in the circadian clock genes and MetS, to our knowledge, none have been evaluated in terms of gene-gene interactions. Furthermore, the interplay between the circadian clock genes and health-related behaviors, such as alcohol consumption, smoking status, and physical activity, has not been fully assessed in previous association studies. Given that gene-gene and gene-environment interactions may play a pivotal role in the development of MetS, we hypothesized that the circadian clock genes may contribute to the etiology of MetS and its individual components independently and/or through complex interactions. The gene panel consisted of 29 circadian clock-relevant genes  selectedThis study incorporated Taiwanese subjects from the Taiwan Biobank, which gathered information and specimens from participants in recruitment centers across Taiwan in 2013–2015 –27. The Current alcohol drinkers were defined as persons who reported drinking more than 150 ml of alcohol per week during the last six months . CurrentMeasurements of metabolic traits  were obtained when participants underwent general health examinations –24. To aADCYAP1, ARNTL, ARNTL2, BHLHE40, CLOCK, CRY1, CRY2, CSNK1D, CSNK1E, GSK3B, HCRTR2, KLF10, NFIL3, NPAS2, NR1D1, NR1D2, PER1, PER2, PER3, REV1, RORA, RORB, RORC, SENP3, SERPINE1, TIMELESS, TIPIN, VIP, and VIPR2.DNA was isolated from blood samples using a QIAamp DNA blood kit following the manufacturer’s instructions . The quality of the isolated genomic DNA was evaluated using agarose gel electrophoresis, and the quantity was determined by spectrophotometry . SNP gen2 goodness-of-fit test with 1 degree of freedom . Multiple testing was adjusted by the Bonferroni correction. To address the issue of multiple testing, we also calculated Q values and false discovery rates (FDRs) using the qvalue function in an R package [Categorical data were evaluated using the chi-square test. We conducted the Student’s t test to compare the difference in the means from two continuous variables. To estimate the association of the investigated SNP with MetS, we conducted a logistic regression analysis to evaluate the odds ratios (ORs) and their 95% confidence intervals (CIs), adjusting for covariates including age and gender . Further package . The criTo investigate gene-gene and gene-environment interactions, we employed the generalized multifactor dimensionality reduction (GMDR) method . We testhttp://biostats.usc.edu/Quanto.html).Based on the effect sizes in this study, the power to detect significant associations was evaluated by QUANTO software (−5). The related Q values and FDRs were listed in ARNTL rs10832020, GSK3B rs2199503, PER3 , RORA , and RORB rs972902 (ARNTL rs10832020 (P = 0.0065), GSK3B rs2199503 (P = 0.007), PER3 rs10746473 (P = 0.001), RORA rs8034880 (P = 0.0002), and RORB rs972902 (P = 0.0087). In addition, the genotype frequency distributions for the ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, and RORB rs972902 SNPs were in accordance with the Hardy–Weinberg equilibrium among the subjects .Among the 881 SNPs assessed in this study , there wrs972902 . For furARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, and RORB rs972902 after adjusting for covariates, indicating an increased MetS risk among the subjects . Similarly, there was an indication of an increased risk of MetS among the subjects after adjustment of covariates for genetic models in the ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, and RORB rs972902 SNPs  high waist circumference vs. normal waist circumference subjects; (b) high triglyceride vs. normal triglyceride subjects; (c) low HDL vs. normal HDL subjects; (d) high blood pressure vs. normal blood pressure subjects; and (e) high fasting glucose vs. normal fasting glucose subjects. As shown in GSK3B rs2199503 (P = 0.0002), RORA rs8034880 (P = 0.02), and RORB rs972902 (P = 0.015) SNPs, there was an indication of an increased risk of high fasting glucose among the subjects after adjustment of covariates for genetic models. The effect of GSK3B rs2199503 on high fasting glucose remained significant after Bonferroni correction (P < 0.05/75 = 0.0007). Additionally, there was a significantly increased risk of high waist circumference (P = 0.0013) among the subjects for PER3 rs10746473; however, this association did not persist after Bonferroni correction (P < 0.05/75 = 0.0007).Next, ARNTL rs10832020 and RORA rs8034880 (P < 0.001), GSK3B rs2199503 and RORA rs8034880 (P < 0.001), GSK3B rs2199503 and RORB rs972902 (P = 0.002), PER3 rs10746473 and RORA rs8034880 (P < 0.001), and PER3 rs10746473 and RORB rs972902 (P < 0.001). The effects of these two-way models remained significant after Bonferroni correction (P < 0.05/10 = 0.005), indicating a potential gene-gene interaction between RORA and ARNTL, between RORA and GSK3B, between RORA and PER3, between RORB and GSK3B, and between RORB and PER3 in influencing MetS. Similarly, there were two-way gene-gene interaction models in influencing individual components such as high waist circumference (P = 0.003) or high fasting glucose (P = 0.007) as shown in In addition, the GMDR analysis was used to assess the impacts of combinations between the five SNPs in MetS and its individual components including age and gender as covariates. RORB rs972902 and smoking (P < 0.001). The effect of the two-way model remained significant after Bonferroni correction (P < 0.05/18 = 0.0028), indicating a potential gene-environment interaction between RORB and smoking in influencing MetS. Additionally, there was a significant three-way model (P = 0.001) after Bonferroni correction (P < 0.0028), indicating a potential gene-environment interaction among PER3, RORB, and smoking in influencing MetS. Similarly, there were significant two-way up to four-way gene-environment interaction models  in influencing individual components such as low HDL after Bonferroni correction (P < 0.0028). In addition, there were significant three-way and four-way models  in influencing high fasting glucose after Bonferroni correction (P < 0.0028).Furthermore, ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, or RORB rs972902 with MetS among the MetS and non-MetS subjects.Finally, statistical power analysis revealed that the present study had a 99.9% power to detect associations of ARNTL, GSK3B, PER3, RORA, and RORB to assess gene-gene and gene-environment interactions although the association with MetS did not remain significant after performing Bonferroni correction (P < 6 x 10−5). Here, we report for the first time that gene-gene interactions of the ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, and RORB rs972902 SNPs may contribute to the etiology of MetS. Moreover, there were gene-environment interactions between ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, and RORB rs972902 with health-related behaviors, such as alcohol consumption, smoking status, and physical activity. Finally, our data revealed that the GSK3B rs2199503 SNP was associated with the individual component of MetS such as high fasting glucose.Our association study is the first to date to examine whether the main effects of 881 SNPs in 29 circadian clock-related genes are significantly associated with the risk of MetS and its individual components independently and/or through gene-gene interactions among Taiwanese individuals. We also investigated the relationship between these genes and health-related behaviors to examine whether these genes confer a risk of MetS according to its effect on gene-environment interactions. A growing body of evidence indicates that genetic association studies using individual genes may overlook the associations that can only be identified if gene-gene and gene-environment interactions are explored . TherefoARNTL gene, we found a positive association of MetS with 4 SNPs in the ARNTL gene, especially the rs10832020 SNP. The ARNTL gene is located on chromosome 11p15 and encodes a basic helix-loop-helix protein to build the ARNTL/CLOCK heterodimeric protein with CLOCK, another basic helix-loop-helix protein [ARNTL gene has been associated with metabolic regulation [ARNTL gene as an indicator of hypertension [Arntl gene induced MetS and dyslipidemia in an animal model [ARNTL rs3789327 (P = 0.0346), but not with ARNTL rs6486121 and rs969485 (data not shown). In addition, we did not detect an association of MetS with ARNTL rs6486121, rs3789327, and rs969485 in this study.Regarding the  protein . The ARNgulation , 13. Woortension . MoreoveGSK3B gene, particularly the rs2199503 SNP. We also suggested an association of GSK3B rs2199503 with high fasting glucose. The GSK3B gene is located on chromosome 3q13.3 and encodes a subfamily of glycogen synthase kinase (GSK), which comprises two isoforms GSK3A and GSK3B. It has been implicated that GSK3 regulates numerous cellular functions such as metabolism, circadian rhythms, neurological degeneration, and immune responses [Further, five potential candidate SNPs for MetS were identified at the esponses . Chen etesponses .PER3 gene, notably the rs10746473 SNP. Our analysis also indicated that there was an association of PER3 rs10746473 with high waist circumference. The PER3 gene, located on chromosome 1p36.23, encodes components of the circadian rhythms of locomotor activity, metabolism, and feeding behavior [Per3 mutant mice led to higher body mass on a high fat diet, suggesting Per3 may play a role in body mass regulation [Per3 knock-out mice displayed glucose intolerance as well as altered body composition (such as increased adipose and decreased muscle), indicating Per3 as potent mediator of cell fate [Per3 was tissue specific such that the loss of functional PER3 had an effect on circadian period in about half of the analyzed tissues, demonstrating Per3 as an important player in the mammalian circadian system [On another note, there was an association of MetS with 6 SNPs within the behavior . Dallmangulation . In addiell fate . In an an system .RORA gene and 6 SNPs in the RORB gene may contribute to the susceptibility for MetS, especially RORA  and RORB rs972902. Our results also suggested an association of high fasting glucose with the RORA rs8029848, RORA rs8034880, and RORB rs972902 SNPs; however, the significance did not persist after adjusting for Bonferroni correction. The proteins encoded by RORA (located on chromosome 15q22.2) and RORB (located on chromosome 9q2) constitute a subfamily of nuclear hormone receptors [RORA and RORB genes have been implicated in the regulation of a wide variety of physiological processes, including metabolism, circadian rhythm, and inflammatory responses [Rora-deficient mice showed a markedly reduced susceptibility to age- and diet-induced MetS, implicating Rora plays a critical role in the regulation of several aspects of MetS [Moreover, our results are the first to raise the possibility that 43 SNPs in the eceptors . The ROResponses , 41. KanCLOCK gene [CRY2 rs75065406 [NPAS2 rs11541353 with hypertension [PER2 #10870 with high fasting glucose [CRY1 haplotype (rs4964513 and rs12821586) with elevated blood pressure [CLOCK , CRY1 (rs4964513 and rs12821586), CRY2 rs75065406, NPAS2 rs11541353, and PER2 #10870 due to lack of these SNPs in the custom chip. It is worth mentioning that the minor allele frequencies of CRY2 rs75065406 and NPAS2 rs11541353 are all 0% in Asian populations (such as Japanese and Han Chinese) as shown in the public data from the 1000 Genomes Project (http://www.1000genomes.org). Moreover, it should be noted that CRY1 (rs4964513 and rs12821586) are annotated within the mitochondrial transcription termination factor 2 (MTERF2) gene, which is in the vicinity of the CRY1 gene, as shown in the dbSNP database (https://www.ncbi.nlm.nih.gov/). On the other hand, we identified other potential candidate SNPs for MetS, including 3 SNPs in the CRY1 gene and 6 SNPs in the NPAS2 gene.Previously, circadian clock genes have been reported to associate with MetS, such as a three-marker haplotype  of the rtension , an assoARNTL, GSK3B, PER3, RORA, and RORB in influencing MetS by using the GMDR approach. To our knowledge, no other study has been conducted to evaluate gene-gene interactions between these genes. Besides the statistical significance, the potential biological mechanism under the interaction models was our concern. The functional relevance of the interactive effects of ARNTL, GSK3B, PER3, RORA, and RORB on MetS remains to be elucidated. In mammals, the molecular clock mechanism is currently viewed as a complex interplay of transcriptional feedback regulatory loops involving various circadian clock genes to generate and maintain circadian rhythms [ARNTL encodes the ARNTL protein, which is a basic helix-loop-helix protein that forms heterodimers with either CLOCK or NPAS2, two other basic helix-loop-helix proteins [PER1, PER2, PER3, CRY1, and CRY2 via E-box elements in the promoters of target genes [ARNTL gene and provide a good timing of the core clock machinery [Intriguingly, another finding was that we further inferred the epistatic effects between  rhythms . The corachinery .PER3, RORB, and smoking in influencing MetS. Similarly, we tracked down several significant gene-environment interaction models in influencing individual components such as low HDL and high fasting glucose. It has been highlighted that common diseases are known to have a considerable genetic contribution, although only a proportion of complex diseases overall is explained by the established candidate genes, pointing out that environmental effects and gene-environment interactions will be our key concern in the outlook for genomic studies [Remarkably, the GMDR analysis of gene-environment interactions reflected the interplay among  studies .In terms of the average ages in the MetS and non-MetS groups, subjects with MetS were significantly older than those without MetS in this study . ConsistBHLHE41) gene due to lack of those genes in the custom chip. In future work, prospective clinical trials with other ethnic populations are needed to examine a thorough evaluation of the association and interactions of the investigated genes with MetS and its individual components [This study has both strengths and limitations. The main weakness was that our observations require much further research to validate whether the findings are replicated in diverse ethnic groups –47. Thismponents , 49. On ARNTL, GSK3B, PER3, RORA, and RORB genes may affect the prevalence of MetS independently and/or through complex gene-gene and gene-environment interactions. Furthermore, the circadian clock genes may be a determinant of MetS component factors. Independent replication studies with larger sample sizes will likely demonstrate further insights into the role of the circadian clock genes found in this study.In conclusion, we conducted an extensive analysis of the association as well as gene-gene and gene-environment interactions of the circadian clock genes with MetS and its individual components in Taiwanese subjects. Our findings indicate that the S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.


The role of interleukins (ILs) and oxidative stress (OS) in precipitating neurodegenerative diseases including sporadic Alzheimer’s disease (AD), requires further clarification. In addition to neuropathological hallmarks—extracellular neuritic amyloid-β (Aβ) plaques, neurofibrillary tangles (NFT) containing hyperphosphorylated tau and neuronal loss—chronic inflammation, as well as oxidative and excitotoxic damage, are present in the AD brain. The pathological sequelae and the interaction of these events during the course of AD need further investigation. The brain is particularly sensitive to OS, due to the richness of its peroxidation-sensitive fatty acids, coupled with its high oxygen demand. At the same time, the brain lack robust antioxidant systems. Among the multiple mechanisms and triggers by which OS can accumulate, inflammatory cytokines can sustain oxidative and nitrosative stress, leading eventually to cellular damage. Understanding the consequences of inflammation and OS may clarify the initial events underlying AD, including in interaction with genetic factors. Inflammatory cytokines are potential inducers of aberrant gene expression through transcription factors. Susceptibility disorders for AD, including obesity, type-2 diabetes, cardiovascular diseases and metabolic syndrome have been linked to increases in the proinflammatory cytokine, IL-18, which also regulates multiple AD related proteins. The association of IL-18 with AD and AD-linked medical conditions are reviewed in the article. Such data indicates that an active lifestyle, coupled to a healthy diet can ameliorate inflammation and reduce the risk of sporadic AD.  Alzheimer’s disease (AD), and particularly sporadic late-onset AD (LOAD), is already a major health issue globally, with AD incidence continually increasing. Partially it is explainable by aging and lengthened lifespan. A number of genetic factors and female gender are important contributors to AD, but even those cannot fully explain the worldwide rise of AD cases. The increasing incidence of LOAD indicates a significant role for environmental and lifestyle factors. Some medical conditions, partially related to lifestyle, increase AD risk, including obesity and type-2 diabetes (T2DM), where both chronic low-grade inflammation and oxidative stress (OS) are present. Chronic inflammation and OS increases lipid peroxidation and free-radical mediated cell death, with relevance to the etiology and course of many neurodegenerative diseases, including AD ,4,5,6. HReactive oxygen species (ROS) are normally produced as by-products of oxidative metabolism, including during the course of normal mitochondrial respiration. However, they are also generated during pathological events, such as inflammation. Brain metabolism requires relatively large amounts of oxygen, with the oxidative burden further increasing during the course of normal aging ,9. Even 2−), hydroxyl radicals (OH˙), hydrogen peroxide (H2O2), and nitric oxide (NO) [2O2 reacting with Fe2+ to give rise to the highly reactive OH˙. Among free radical species, H2O2, released for instance in oxidative burst by microglia, seems to be a relatively stable compound that can diffuse freely across membranes. O2− in turn is highly produced by mitochondria and is rapidly converted to H2O2, which can readily diffuse also within cells. These properties make H2O2 particularly toxic to the cells, which is further potentiated if converted to the highly reactive OH˙ [2O2 can also be detected in the rat brain at micromolar concentrations following ischemia/reperfusion [ROS can act as free radicals or they can react with other molecules to form free radicals. ROS include superoxide O−, hydroxide (NO) . Neuronstive OH˙ . H2O2 caerfusion . Nevertherfusion , which cIt is also important to note that the inflammatory and defense profile in humans differs from that of rodents ,16. RodeActivated microglia, the principal defense cells in the central nervous system (CNS), and reactive astrocytes are the primary cellular sources of brain inflammation. Activated microglia and reactive astrocytes surround extracellular Aβ-plaques as well as dead or damaged neurons, including in AD ,22,23. A2+ or Fe3+, producing H2O2 and OH˙ [ROS damages and increases the rigidity of all cell membranes, leading to modifications in proteins and lipids, as well as cell membrane receptors, which alters their function, intracellular signaling and clustering with other membrane receptors. For instance, many pro-inflammatory processes, including IL-18, activate the transcription factor, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), leading to the induction of β-site amyloid precursor protein cleaving enzyme 1 (BACE-1) ,26. BACE and OH˙ . Aβ can  and OH˙ ,29,30. I and OH˙ , resultiROS can activate the nucleotide-binding domain, leucine-rich repeat -family and pyrin domain-containing protein 3 (NLRP3) inflammasome protein complex, composed of an intracellular sensor, such as a Nod-like receptor (nucleotide-binding oligomerization domain-like receptor), the precursor pro-caspase-1 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Inflammasome is primarily driven by caspase-1 , whereasIL-18 is a member of a rather large IL-1 gene family. In comparison to IL-1β, IL-18 expression seems to be higher in the AD brain, partly due to its longer half-life . AlthougIL-18, directly or via OS, also modulates a number of key central processes, including antioxidative enzymes, such as peroxiredoxins 2, 3 and 6 , protein deglycase DJ-1  and regulatory biliverdin reductase A (BVRA). Protein levels of dimethylarginine dimethylaminohydrolase 2 (DDAH2) can also be regulated by IL-18 . In addi2O2 as a second messenger molecule, as well as cell proliferation. PRX2 is expressed solely in neurons [2O2, which commonly leads to cell death. PRX2 can also interact with presenilin-1 (PS-1), as can the anti-apoptotic Bcl-2 and Bcl-xL proteins. These interactions may regulate processing of APP to Aβ and protect against neuronal apoptosis [The multifunctional antioxidant thioredoxin-dependent PRXs form a family PRX1-6), with members that are differentially localized intracellularly, with additional variation across different cell types . In gene-6, with poptosis ,49. IL-1poptosis , which m2O2, PRX6 can also reduce short chain organic fatty acids and phospholipid hydroperoxides, indicating the importance of PRX6 in phospholipid turnover [Levels of mitochondrial PRX3 are decreased in the AD brain and leakage may explain this reduction since mitochondria are commonly damaged in AD, due to multiple causes . Howeverturnover . In AD, turnover . Like Aβturnover ,42, and turnover . Both uPturnover  and reguturnover . 2O2 and functions as a redox-sensitive chaperone [IL-18 down-regulates protein levels of an atypical PRX-like peroxidase DJ-1 , which shaperone ,57. DJ-1haperone ,58. Howehaperone . For inshaperone . Intereshaperone . DJ-1-dehaperone . IL-18 rEvolutionarily conserved, multifunctional BVR and heme oxygenase (HO) are crucial in the defense against OS. BVR is expressed in all tissues; BVRA is dominant in adults and BVRB in the fetus . HO is rl-arginine (ADMA) and N(G)-monomethyl-l-arginine (MMA). Both ADMA and MMA act as inhibitors for nitric oxide synthase (NOS) [Protein levels of DDAH are increased in the frontal lobes of AD patients, which can partially be driven by IL-18 . DDAH isse (NOS) ,71. Whense (NOS) . In addise (NOS) , whilst se (NOS) . Howeverse (NOS) ,75. FiguActivated MMPs regulate BBB integrity, having a role in the digestion of tight junctions and BBB basement membrane proteins and are therefore critical contributors to different brain diseases ,77. AlthROS are key mediators of increased BBB permeability . Free raSince MMPs contribute to ROS and inflammation-induced BBB dysfunction and leakage, MMPs can also promote the progression of several CNS disorders, including AD, stroke, PD, multiple sclerosis and cerebral aneurysms ,93. IschLate-onset AD (LOAD) represents 90–95% of all AD cases, with increasing data indicating that both LOAD and AD are related to metabolic diseases and thereby closely linked to vascular and related factors. Such factors include total cholesterol and other lipid parameters, as well as hypertension and metabolic alterations, such as glucose utilization impairments in the brain, insulin responsiveness and energy metabolism. Metabolic syndrome (MetS) covers all these conditions, namely abdominal obesity, increased blood pressure, elevated fasting plasma glucose, and low high-density lipoprotein (HDL) levels or high serum triglyceride levels. When these parameters occur together, there is an increased risk of cardiovascular disease, stroke and T2DM, as well as cognitive impairment and dementia, with the most important risk factor being hyperglycemia ,98.Factors involved in MetS generate chronic systemic low-grade inflammation, “metaflamation”, which interferes with adipose tissue homeostasis and insulin signaling. In the brain, insulin and insulin-like growth factor (IGF) signaling are involved in synaptic maintenance, neuroprotection and neuronal growth ,100. DurPartly as a consequence of mitochondrial loss or damage due to Aβ and OS, AD also shows evidence of glucose hypometabolism. MetS and diabetes affect mitochondrial dynamics, suggesting mitochondria as critical sites in the progression of AD. Mice fed a high fat diet develop obesity, lower glucose and insulin tolerance, and elicit insulin resistance in the brain, versus mice fed with a normal diet. The brains of these mice also exhibit biochemical changes associated with increased Aβ deposition and NFT formation, as well as decreased synaptic plasticity . SucroseAmong the multiple activators, the saturated FFA, palmitate, induces the activation of the NLRP3 inflammasome protein complex. In this activation process, FFA palmitate signals through an adenosine monophosphate activated protein kinase (AMPK)-autophagy-mitochondrial ROS pathway , with thENOA decline can increase ROS, mainly generated through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together can affect many cellular processes, including cell growth and senescence . In mildENOG levels are increased significantly in an array of medical conditions, including Creutzfeldt-Jakob disease, cerebral trauma, brain tumors and cardiovascular disease. Glycolytic ENOG also has additional roles, including functioning as a neurotrophic-like factor, leading to neuronal growth, regeneration, differentiation and survival. This occurs after its translocation and binding to the plasma membrane ,118,119.14-3-3 proteins belong to a highly conserved protein family of ubiquitous cytoplasmic chaperones that play important role in metabolism, signal transduction, intracellular trafficking, cell cycle control and apoptosis ,123. IL-Dementia is also associated with low education and low levels of book reading, with other lifestyle activities, such as learning new things and having an active social network, able to delay dementia onset, most likely by maintaining or improving the neural plasticity and synaptic network. However, these lifestyle factors do not seem to significantly influence the expression of AD pathophysiology ,135. AnoPhysical inactivity is a risk factor for AD, as well as the development of MetS and cardiovascular diseases. Regular physical exercise enhances metabolism and circulation, including in the brain, with resultant improvements in neurogenesis. Increased cardiorespiratory fitness is also associated with improved memory performance and reduced hippocampal atrophy. In AD patients, exercise improves cognitive function, decrease neuropsychiatric symptoms and slow the decline in activities of daily living (ADL). Therefore, regular physical exercise can be recommended as a prevention strategy for AD as well as one type of treatment for pre-clinical and late stage AD. Aerobic exercise in early AD is particularly associated with benefits in cognitive functioning ,137,138.2+, cAMP response element-binding protein (CREB), peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and NF-κB. These signaling pathways can stimulate mitochondrial biogenesis and cellular stress resistance. Neurons counteract the damaging challenges also by up-regulating antioxidant defenses, autophagy/mitophagy and DNA repair [Among people with MetS, IL-18, in comparison to other pro-inflammatory cytokines, seems to be the best marker for inflammation . High-inA repair . HoweverA repair . Aging mIn addition to physical activity, good nutrition throughout life can also prevent or delay chronic diseases and disabilities in old age, including AD ,143,144.Important targets for OS and nitrosative stress mediated damage and function alterations are cell membranes with their variety of protein receptors, commonly harbored in lipid rafts. Nutrition influences the composition and function of cell membranes, including lipid rafts. Lipid rafts are microdomains, composed of cholesterol and glycosphingolipids, which are crucial components and regulators of cellular signaling and neurotransmission . ActivitElevated total cholesterol in midlife has been associated with increased risk of dementia in later life, whereas the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) has been shown to cause AD-like pathology in cell culture models . FurtherA significant decline in the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) seems to be associated to both natural aging and AD . AlthougIncreased IL-18 levels , linked In people with MetS, high-intensity aerobic interval training can significantly reduce serum IL-18 levels . RegularIn conclusion, a lifetime of moderate exercise, in combination with a healthy diet containing enough antioxidative components and omega-3, as well as an intellectually challenging lifestyle may be effective in the prevention of LOAD, with efficacy that is at least partially mediated via the central and systemic regulation of IL-18 and ROS.


NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R promoters were methylated in 80.5%, 79.2%, 67.1%, 73.2%, 35.1%, 36.4%, 38.5%, and 35.9% of the samples, respectively. In a multivariate Cox proportional hazards analysis, the odds ratio for recurrence was 2.044  when the NPY2R promoter was methylated. In patients without lymph node metastasis (n = 100), methylation of NPY2R (compared with methylation of the other seven genes) best correlated with poor disease-free survival (DFS) . In patients with oral cancer (n = 69), methylated NPY1R and NPY2R were independent prognostic factors for poor DFS, both individually and, even more so, in combination . Similar findings were observed for NPY2R and NPY4R in patients with oropharyngeal cancer (n = 162) .Staging and pathological grading systems are useful but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). To identify potential prognostic markers, we examined the methylation status of eight neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinomas. The  Head and neck cancer is the eighth most common cancer worldwide with approximately 650,000 new cases reported annually . More thTechnological advances revealed aberrant expression of G protein-coupled receptors (GPCRs) in human tumors owing to events such as gene point mutations, gene silencing via promoter methylation, and changes in gene copy number . The GPCAberrant promoter methylation, a hallmark of cancer cells, accounts for the inactivation of many tumor suppressor genes. In HNSCC, methylation of gene promoters is a common mechanism of transcriptional silencing –9. NotabNPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R) encode neuropeptide receptors and are in the Class A β subgroup. This study is the first to implicate neuropeptide receptors in the genesis of HNSCC.The aim of this study was to determine the methylation status of eight GPCR-encoding genes in HNSCCs and its relationship to recurrence, survival, and clinical characteristics . All eight genes  was used to assess the promoter methylation status of eight genes encoding neuropeptide receptors in 231 primary HNSCC samples. At least one of these genes was methylated in almost all samples . The mean number of methylated genes per sample was 4.46  Figure . The met% Figure . Relativ% Figure .The methylation index (MI) was defined as the ratio of the number of methylated genes and the number of tested genes in each sample. Continuous marker methylation analyses showed no association between the MI for any of the eight target genes and age at disease onset, sex, alcohol consumption, smoking status, tumor size, lymph node status, clinical stage, or recurrence Figure .HCRTR2 promoter significantly correlated with lymph node metastasis (P = 0.040), methylation of the NPY1R promoter significantly correlated with smoking status (P = 0.041), and methylation of the NPY2R promoter significantly correlated with age (P = 0.040) and recurrence (P = 0.004).Associations between the methylation status of the target genes and the clinicopathological features of the patients are summarized in Table HCRTR2 was unmethylated (P = 0.016) and when NPY2R was methylated (P = 0.001). Additional analysis of only the patients without lymph node metastasis (n = 100) revealed shorter DFS times for methylated versus unmethylated NPY2R (P = 0.026), but no differences for the other seven genes , the adjusted odds ratio for recurrence was 2.044    and 2.93 , respectively, and the hazard rate was 3.9 times higher   and together .Odds ratios for recurrence according to tumor origin were also determined. When the NPFFR2, HCRTR2, NPY1R, NPY2R, and NPY5R promoter methylation correlated inversely with their respective mRNA levels in both the HNSCC and normal tissue samples (HCRTR1 promoter than in those with a methylated HCRTR1 promoter (P = 0.038)  . mRNA ex samples . To vali= 0.038) .NPFFR, HCRTR, and NPYR genes is important for understanding how tumors arise and whether they will recur. Using real-time PCR, we examined the methylation status of these genes, all of which encode G protein-coupled neuropeptide receptors, in 231 HNSCCs originating in the hypopharynx, larynx, oropharynx, or oral cavity. We also compared the methylation status of matched HNSCC and normal samples using data from TCGA database. We found that aberrant methylation of the NPY1R, NPY2R, and NPY4R promoters correlated positively with recurrence in patients with HNSCC.Identifying epigenetic modifications of the HCRTR1 and HCRTR2 genes, respectively. Orexins transiently increase intracellular calcium levels through Gq-dependent and -independent pathways [HCRTR1 mRNA regardless of their location or Dukes stage, whereas adjacent normal colonocytes did not express HCTRTR1 mRNA [HCRTR2 correlates with hypermethylation of HCRTR2 in endometrial cancer compared with normal endometrium [Members of the GPCR family include the neuropeptide FF (NPFF) receptors, of which there are two subtypes, NPFFR1 and NPFFR2, which bind the RF-amide related peptides and FF neuropeptides, respectively . NPFFR1 pathways  and markpathways . In a prTR1 mRNA . Loss ofometrium .NPY6R gene is functional in rabbits and mice, it is absent in rats and considered a pseudogene in primates and pigs [NPY2R gene have been observed in smoking-unrelated urothelial carcinomas [in vitro [Neuropeptide Y (NPY) activates five GPCRs, namely, NPY1R, NPY2R, NPY4R, NPY5R, and NPY6R . It is oand pigs . NPY regand pigs . Via NPYand pigs . Signifircinomas . NPY2R ircinomas , and NPYrcinomas . NPY4R ircinomas . The spein vitro , and genin vitro .GALR1, GALR2, TACR1, and SSTR1 genes, respectively, and the methylation frequencies of these genes are 51.0%, 37.6%, 34.0%, and 64.0%, respectively [GALR1 promoter hypermethylation significantly and inversely correlates with DFS time [GALR2 promoter is methylated versus unmethylated [TACR1 or SSTR1 methylation and prognosis in HNSCC patients regardless of tumor origin [Type 1 and 2 galanin receptors, type 1 tachykinin receptors, and type 1 somatostatin receptors, which are also neuropeptide GPCRs, are encoded by the ectively –29. In or origin , 28.NPY1R, NPY2R, and NPY4R methylation with tumor recurrence in oral and oropharyngeal cancers. This finding may facilitate HNSCC screening and the development of surveillance algorithms. Simultaneous analysis of the methylation status of multiple neuropeptide GPCR-encoding genes will allow us to better predict tumor-related events, assess biological behavior, and design targeted therapies for HNSCCs.Our study associates GPCRs and their signal transduction networks affect physiological, immunological, and endocrinological processes and stem cell biology . This isNPY1R, NPY2R, and NPY4R genes is an independent indicator of DFS in patients with oral and/or oropharyngeal cancers. Our findings support the use of methylation markers in patient selection for adjuvant therapy after initial surgical treatment and may aid oropharyngeal cancer screening and surveillance programs. However, they are preliminary and hence need to be validated in larger and more homogeneous HNSCC patient cohorts.Studies involving human specimens and high-throughput profiling platforms may be susceptible to measurement bias from a variety of sources. The present study suggests that the methylation status of the Two hundred and thirty-one primary HNSCC samples were obtained from patients during surgery at the Department of Otolaryngology, Hamamatsu University School of Medicine. All patients provided written informed consent, and the study protocol was approved by the Institutional Review Board of the Hamamatsu University School of Medicine. Pertinent information including age, sex, smoking status, alcohol consumption, lymph node status, tumor site, tumor size, and clinical stage was obtained from the patients’ medical records . The malTM Methylated HeLa gDNA (TaKaRa). The normalized methylation value (NMV) was determined as follows: NMV = (Target gene-S/Target gene-FM)/(ACTB-S/ACTB-FM), where Target gene-S and Target gene-FM represent target gene methylation levels in the tumor sample and universal methylated DNA control, respectively, and ACTB-S and ACTB-FM represent ACTB (which encodes β-actin) methylation levels in the sample and control, respectively. Analysis was performed using the software (version 1.03A) for the Thermal Cycler Dice Real Time System TP800 according to the manufacturer's directions [Extraction and bisulfite conversion of genomic DNA from 231 primary HNSCC and 36 noncancerous mucosal samples were performed using a MethylEasy Xceed Rapid DNA Bisulfite Modification Kit  . The metrections .glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression.Total RNA was isolated with RNeasy Plus Mini kit , and cDNA was synthesized with ReverTra Ace qPCR RT kit . Primer sequences are shown in http://methhc.mbc.nctu.edu.tw/php/index.php) [http://www.cbioportal.org/) databases [Aberrant DNA methylation data contained in TCGA (available in May 2017) were collected from the MethHC (dex.php)  and cBioatabases  using thReceiver-operator characteristic (ROC) curve analysis was performed using the NMVs for 36 HNSCC and 36 adjacent normal mucosal samples and the Stata/SE 13.0 system . The area under the ROC curve indicated the optimal sensitivity and specificity cutoff levels for distinguishing between the methylation levels in normal and HNSCC tissue, and the NMV thresholds were calculated for each target gene. The cutoff values were used to determine the methylation frequencies of the target genes . The oveAssociations between the variables were assessed by using Student's t-test. Disease-free survival (DFS) was measured from the date of the initial treatment to the date of diagnosis of locoregional recurrence or distant metastasis. The Kaplan-Meier test was used to calculate survival probabilities, and the log-rank test was used to compare survival rates. The prognostic value of the methylation status was assessed by performing a multivariate Cox proportional hazards analysis adjusted for age (≥70 versus <70 years), sex, alcohol intake, smoking status, and tumor stage (I–III versus IV) . Differe


Among its regulators, protein phosphatase-1 (PP1), a Ser/Thr phosphatase, appears to be critical in controlling CaMKII-dependent neuronal signaling. In postsynaptic densities (PSDs), CaMKII is required for hippocampal long-term potentiation (LTP), a cellular process correlated with learning and memory. In response to Ca2+ elevation during hippocampal LTP induction, CaMKIIα, an isoform that translocates from the cytosol to PSDs, is activated through autophosphorylation at Thr286, generating autonomous kinase activity and a prolonged Ca2+/CaM-bound state. Moreover, PP1 inhibition enhances Thr286 autophosphorylation of CaMKIIα during LTP induction. By contrast, CaMKII nuclear import is regulated by Ser332 phosphorylation state. CaMKIIδ3, a nuclear isoform, is dephosphorylated at Ser332 by PP1, promoting its nuclear translocation, where it regulates transcription. In this review, we summarize physio-pathological roles of CaMKII/PP1 signaling in neurons. CaMKII and PP1 crosstalk and regulation of gene expression is important for neuronal plasticity as well as survival and/or differentiation.Ca In the brain, Ca) kinase , regulatasticity . CaMKII asticity ,5.2+ elevation by extracellular stimuli, Ca2+-CaM binding to CaMKII displaces autoinhibitory domains to allow ATP and exogenous substrates access to the active site. Immediately after activation, Thr286 in the autoinhibitory domain of the α isoform  is autophosphorylated by the neighboring kinase domain. This event increases Ca2+-CaM binding affinity and blocks interaction of autoinhibitory and catalytic domains, thereby generating autonomous kinase activity and prolonging the Ca2+/CaM-bound state. CaMKIIα autonomy is critical for induction and maintenance of hippocampal long-term potentiation (LTP), both of which underlie learning and memory [CaMKII has attracted substantial attention due to its function in synaptic plasticity, an activity that occurs at postsynaptic densities (PSDs) ,7,8. In d memory ,10.2+ stimulus, two adjacent CaMKII monomers are simultaneously bound by Ca2+/CaM. In these conditions, one subunit serves as a substrate for the other, resulting in Thr286/Thr287 phosphorylation. Once the first subunit is phosphorylated, subsequent phosphorylation within the holoenzyme is more likely to occur, as lower Ca2+ levels are required for the second phosphorylation. Thus, in this scenario, CaMKII remains active, even when Ca2+ levels return to basal levels, until it is dephosphorylated. If the number of phosphorylated subunits exceeds a threshold and the phosphorylation rate is greater than the dephosphorylation rate, then CaMKII activity is sustained [The 12 subunits of the CaMKII holoenzyme assemble into two coplanar rings, each containing six subunits . These rustained .328KKRKS332), is reportedly phosphorylated by the CaMK family members CaMKI or CaMKIV, blocking association of CaMKII with the NLS receptor m-pendulin and thereby preventing nuclear localization [In contrast to postsynaptic CaMKII function, the physiological relevance of nuclear activity of CaMKII isoforms in the central nervous system (CNS) remains unclear. Alternative splicing of CaMKII generates a multitude of isoforms for each CaMKII subunit . Among tlization .The Ser/Thr phosphatase Protein Phosphatase-1 (PP1), a key regulator of CaMKII signaling, forms a heterodimer comprised of a catalytic (PP1c) and a regulatory subunit. PP1c can form a complex with over 50 regulatory or scaffolding proteins that dictate substrate specificity and subcellular localization . In mammIn this review, we focus on the role of CaMKII/PP1 signaling in both neuronal plasticity at PSDs and gene expression in the nuclei. We also discuss how imbalanced CaMKII/PP1 activity may underlie neuronal pathologies, such as mental disorders and neurodegeneration.PSDs are localized in the tips of dendritic spine heads and contain multiple classes of proteins that function in neuronal signaling in response to presynaptic neurotransmitter release, such as glutamate . CaMKII One factor governing this persistent “on-state” is PP1 localization to dendritic spines and PSDs ,33. PP1 Once activated, CaMKII remains in an active conformation throughout the LTP maintenance phase, an observation that forms the basis of the hypothesis that CaMKII is critical for memory formation ,10. HoweIn animal models of Parkinson’s disease, striatal dopamine depletion increases CaMKIIα autophosphorylation at Thr286 in parallel with decreased PP1γ1 activity and increased PP1γ1 binding to spinophilin ,43,44. M2+ signaling, and nuclear Ca2+ transients are some of the most potent regulators of neuronal gene expression [Transduction of signals from synapses to the nucleus is primarily mediated by Capression . Nuclearpression ,49 throupression ,51, methpression , activatpression ,54, CCAApression ,56, and pression .2+-induced CaMKII activation in primary cultured neurons stimulates CREB phosphorylation at Sers 133, 142, and 143 [2+-induced CREB-dependent transcription [2+ influx trigger CaMKII-dependent MeCP2 phosphorylation at Ser421 [Specifically, CaMKII phosphorylates CREB at Ser133 and Ser142 in vitro . Moreove and 143 . CREB phcription . Howevercription . The tracription . Neuronat Ser421 . Knock-it Ser421 ,62, unde328KKRKS332), is phosphorylated by CaMKI or CaMKIV, prohibiting nuclear localization [Until recently, mechanisms underlying substrate phosphorylation by nuclear CaMKII remained unclear. Thus, we investigated nuclear-cytoplasmic shuttling of the nuclear isoform CaMKIIδ3. Previously, others had reported that CaMKIIδ3 Ser332, which is C-terminal to the NLS (lization . To inve2+/CaM to the nucleus following depolarization of cultured superior cervical ganglion neurons and that the Ca2+/CaM-CaMKIIγ complex is dephosphorylated at Ser334 by calcineurin, allowing it to shuttle to the nucleus. Nuclear delivery of Ca2+/CaM activates nuclear CaM kinases, including CaMKIV and CaMKK, driving CREB phosphorylation and transcription of its target genes [Others have reported nuclear activity of CaMKIIαB and CaMKIIγA in neurons ,64. For CaMKII-PP1 signaling transcriptionally regulates BDNF, a factor vital for neuronal survival, growth, and maintenance, in brain circuits functioning in emotion and cognition . MeCP2 mWe also previously revealed that nuclear CaMKII/PP1 signaling is important for neuronal survival and differentiation . We repo2+ signaling to PSDs or nuclei. However, the composition of the dodecameric CaMKII holoenzyme affects CaMKII localization [CaMKII/PP1 signaling plays a crucial role in many different aspects of synaptic plasticity in PSDs and in activity-regulated transcription in nuclei. CaMKII alternative splicing generates numerous subtypes of each CaMKII isoform. lization ,79. The lization . Nuclearlization  and/or Flization  to facil


Acidic reagents extracted more P from TSP than PRs, while the strip removed equal amounts from the two sources. The P removed by the three tests was related significantly to dry matter yield (DMY), but only in the first harvest, except for B1. Established critical P levels (CPLs) differed for TSP and PRs. In PR-fertilized soils, CPLs were 27, 17, and 12 mg P kg-1soil for Meh3, B1, and strip, respectively, and 42, 31, and 12 mg P kg-1 soil, respectively, in TSP-fertilized soils. Thus, the strip resulted in a common CPL for TSP and PRs (12 mg P kg-1 soil). This method can be used effectively in soils where integrated nutrient sources have been used, but there is need to establish CPLs for different crops. For cost-effective fertilizer P recommendations based on conventional soil tests, there is a need to conduct separate calibrations for TSP- and PR-fertilized soils.The effectiveness of different soil tests in assessing soil phosphorus (P) in soils amended with phosphate rocks (PRs) is uncertain. We evaluated the effects of triple superphosphate (TSP) and PRs on extractable P by conventional soil tests (Mehlich 3 [Meh3] and Bray-1 [B1]) and a nonconventional test . Extracted amounts of P were in the order: Meh3 >B1 > strip. All the tests were significantly correlated (


The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs  were randomly selected for SDS–PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.Calcium ion (Ca A steep Ca2+ gradient across the plasma membrane as well as the inner membrane system enclosing the cellular Ca2+ storages is established under the controls of stimulus responsive Ca2+ channels, pumps and transporters . The loop is enriched in negatively charged residues, and a Gly at position six allows the loop to wrap around the Ca2+ ion, a feature critical for high-affinity Ca2+ binding  normalized data] in different tissues of soybean were downloaded from soybean transcriptome atlas . MicroarGlycine max var. Williams 82) were soaked in sterilized water for 4 h, and then germinated at room temperature in the dark between two layers of filter paper moistened with sterilized water (-2 s-1). After cultivating for 8 days, soybean seedlings were used for various stress treatments. For salt stress, the roots of seedlings were immersed in nutrient solution containing a relative high concentration of NaCl (200 mM) (Soybean seeds (ed water . After 4(200 mM) . Plant r9. Primer specificity was then confirmed by blasting each primer sequence against the soybean genome. Total RNA was extracted from soybean tissues using RNApure Plant Kit (with DNase I)  and digested with DNaseI to eliminate genomic DNA contamination according to the manufacturer’s instruction. cDNA was synthesized from 1.0 μg total RNA by SuperRT Reverse Transcriptase  using oligo(dT) primers in a 20 μL reaction system. Quantitative RT-PCR (qRT-PCR) was performed on a real-time PCR system  as described previously (GmACTIN11 (Gm18g290800). The gene-specific primers are listed in Supplemental Table Gene-specific primers were designed using NCBI Primer-BLASTeviously . AmplifiGmCML1, GmCML13, GmCML39, and GmCML95 by PCR from cDNA of soybean seedlings. AtCaM7 were cloned from Arabidopsis cDNA. The gene-specific primers are listed in Supplemental Table Escherichia coli BL21(DE3) pLysS (Stratagene). Bacterial cultures were grown under antibiotic selection in Luria-Bertani medium with agitation at 37°C to an OD600 of 0.4–0.5 and protein expression was induced over the course of 3 h by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). His-tagged recombinant proteins were purified using Ni-NTA agarose affinity beads (Qiagen) as described by the manufacturer. The electrophoresis mobility-shift assay was performed as described .A total of at least 262 putative EF-hand-containing proteins were found in soybean genome by using the methods described in the “Materials and Methods” section. These proteins were checked for the presence of EF-hands by using InterPro database  Supplem. The numFigure 2). For example, chromosome 2, 5, and 17 contained the highest number of 17 EF-hand proteins, whereas only 6 EF-hand proteins were found on chromosome 9 and 15. Tandem duplication is a factor contributing to the evolution of many gene families .The 262 EF-hand proteins identified in this study were distributed on all of the 20 soybean chromosomes. However, the number of EF-hand proteins on each chromosome appeared to be uneven, ranging from 6 to 17 . The schematic diagrams of representative EF-hand proteins of various groups containing different number of EF-hands were shown in Figure 4. As can be seen, the CaMs, CMLs, and CBLs have no functional domain other than EF-hands, while the other proteins have additional domains, such as protein kinase domain, TIR domain, mitochondrial carrier domain, actin-binding domain, and FAD-binding domain .Sequences of all the identified EF-hand proteins were aligned using ClustalW and a phylogenetic tree was constructed using the neighbor-joining method. The phylogenetic tree of the overall 262 EF-hand proteins was shown in Arabidopsis thaliana CaM2 (AtCaM2) (98.0–98.7%), six genes were found to encode two isoforms of CaMs in soybean , both are 149 amino acid (AA) in length. In addition, there were at least 144 genes encoding for CML proteins; these CMLs share at least 12.8% overall amino acid identity with AtCaM2, and have no other known functional domains except the EF-hands .Based on the extremely high amino acid identity to GmCBL1-GmCBL15, based on their amino acid identity to AtCBL1 .A total of 15 genes encoding putative CBLs were found in soybean genome, and they showed high sequence identity with AtCBL1 (48.8–77.5%). These genes were sequentially named Figure 7 shows the unrooted phylogenetic tree for CDPKs/CRKs from soybean and Arabidopsis, and also CCaMKs from soybean, Medicago truncatula and Lotus japonicus, based on the comparisons of their amino acid sequences. These GmCDPK/CRK/CCaMK proteins are closely related to their orthologs in Arabidopsis and other plants.The soybean genome contains 50 CDPKs, 2 CCaMKs, and 13 CRKs. The detailed characteristics are shown in Supplemental Tables Figure 8).A total of 17 Rboh genes were found in the soybean genome. They were successively named GmRbohA-Q based on their homology to AtRbohD , UEP9 (Gm13g272100), and UEP10 (Gm13g272300) are tandemly duplicated genes encoding EF-hand proteins with putative sodium/calcium exchanger function .In addition, there are genes in soybean genome encoding 15 EF-hand proteins which are not belonging to CaM/CMLs, CBLs, CDPK/CRK/CCaMKs, and Rbohs, and they are named UEPs, . Here we surveyed the presence of eight classes of cis-elements , and UEP12 (Gm15g030100)  . However, 17 genes were found to have no expression in any of these tissues; these genes are CML11, CML15, CML16, CML23, CML42, CML47, CML54, CML60, CML70, CML72, CML81, CML86, CML130, CML139, CML142, CBL2, and CBL3. These genes could be pseudogenes or could be expressed only under specific developmental stages or environmental conditions which were not met in these studies. As shown in the heat maps, some genes were ubiquitous in various tissues, while some genes are tissue-specific . For example, CML6, CML18, CML26, CML48, CML61, CBL6, CBL11, CDPK18, CDPK19, and CDPK20 were constitutive expressed in various tissues; CML20, CML30, CML71, CML78, CML87, CML113, CML118, and CDPK28 were specifically expressed in flower tissue; CaM2, CML2, CML9, CML19, CML24, CML55, CML77, CML137, CDPK6, CDPK14, CCaMK1, CCaMK2, and RbohN were preferentially expressed in roots/root tips/root hairs .Transcriptome data derived from Illumina sequencing were used to assess the expression patterns of EF-hand genes in different tissues of soybean. The transcriptome atlas provided expression data for 55,616 putative soybean genes in eight types of tissues and organs, including root tips, roots, root hairs, nodules, leaves, shoot apical meristems, flowers and green pods . The expBradyrhizobium japonicum inoculation . For example, CML59, CML102, CML117, and RbohB were induced by cold, drought, and flooding; CML109 and UEP6 (Gm12g227800) were repressed by cold and flooding; CML82 and UEP11 (Gm14g017400) were induced by cold but repressed by flooding; CML28, CDPK45, and UEP10 (Gm13g272300) were only responsive to cold stress.In order to investigate whether the EF-hand genes are responsive to environmental stresses, we retrieved the published soybean microarray and/or deep sequencing data reflecting soybean responses to various stresses including cold, drought, flooding, phosphate deficiency, and culation . Of all Figure 12). Nearly all of these genes were responsive to diverse stresses. Most of them were induced by salt stress, but were repressed by various nutrient deficiencies, and eight genes of them were responsive to PEG treatment (dehydration), four were induced  and four  were repressed .In addition, we also selected 14 EF-hand genes to confirm their transcriptional responses to environmental stresses including salt, dehydration, phosphate deficiency, iron deficiency, and zinc deficiency by qRT-PCR  to confirm their Caenomenon . As show protein  for negaunctions .2+ mediated signaling is involved in various developmental processes and responses to a variety of abiotic and biotic stresses. The Ca2+ binding EF-hand motif is highly conserved in most of the Ca2+ sensors. CaMs/CMLs, CBLs, and CDPKs are the major subfamilies of EF-hand Ca2+ sensors  have been analyzed in model plants and some major crops, no detailed information about these Ca2+ sensors in soybean is systemically reported so far . The EF-hand genes in soybean include 6 CaM genes, 144 CML genes, 15 CBL genes, 50 CDPK genes, 13 CRK genes, 2 CCaMK genes, 17 Rboh genes, and 15 UEP genes  (Ca sensors . Althougd so far . Using dables 2+ . Consistigure 1) .2+ sensor may be a little different between legume and non-legume plants.The number of total EF-hand proteins in Arabidopsis and rice plants were previously found to be 250 and 243, respectively . The numFigures 9, 10). Ca2+ sensor subfamilies, such as CaM/CMLs and CDPKs also show tissue and developmentally specific expression patterns  , Ca2+-dependent ATPase MCA8, the cyclic nucleotide-gated channels (CNGCs) and CCaMK are required for the establishment of root symbiosis in legumes . Notably, CML45 and CML110 were responsive to the inoculation of rhizobia (B. japonicum) . Whether these genes are possibly associated with symbiosis establishment or nodule development should deserve further researches.The expression patterns of EF-hand genes in different tissues revealed that these genes were diversely expressed in various tissues of soybean  ; CBL-intprotein) ; CDPK28 protein) ; Arabidochannels . Ca2+ si legumes . Interescam3 mutant was more sensitive to heat stress  . It is notable that most of these genes (85%) contain at least one type of stress-related cis-elements in their promoters  ; overexpnd NO3-) ; Arabidond NO3-) ; rice CPnd NO3-) ; overexpnd NO3-) . In the CaM genes, 144 CML genes, 15 CBL genes, 50 CDPK genes, 13 CRK genes, 2 CCaMK genes, 17 Rboh genes, and 15 UEP genes. Most of these genes (87.8%) contain one or multiple hormonal signal and/or stress-responsive cis-elements in the -1500 bp promoter regions. Expression profiling revealed that these EF-hand genes were broadly expressed in different organs of soybean. Expression pattern analyses also revealed that nearly half of these genes (43.1%) could be induced or repressed under various environmental or nutritional stresses, indicating their potential role in stress responses. Further in-depth functional characterization will enhance our understanding of Ca2+-mediated signaling underling plant responses to environmental and nutritional stresses and facilitate the development of stress resistant crops.In this study, a total of at least 262 genes encoding proteins with varying number of EF-hand motifs were identified in soybean genome, including 6 HZ conceived and designed the study, and wrote the manuscript. HZ, YxZ, and XZ performed the bioinformatic analysis and the experiments. EP and YyZ participated in the data analysis. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.