Increased dietary fiber consumption has been associated with many beneficial effects, including amelioration of obesity and insulin resistance. These effects may be due to the increased production of short chain fatty acids, including propionate, acetate and butyrate, during fermentation of the dietary fiber in the colon. Indeed, oral and dietary supplementation of butyrate alone has been shown to prevent high fat-diet induced obesity and insulin resistance. This review focuses on sources of short chain fatty acids, with emphasis on sources of butyrate, mechanisms of fiber and butyrate metabolism in the gut and its protective effects on colon cancer and the peripheral effects of butyrate supplementation in peripheral tissues in the prevention and reversal of obesity and insulin resistance. One of the widely recognized benefits of fiber consumption is the production of the short chain fatty acids (SCFAs) butyrate, propionate and acetate via fermentation in the colon ,2,3. SpeThis review attempts to integrate the available literature relating butyrate to measurable health outcomes. Herein, we describe dietary sources of butyrate and the observed effects of butyrate in the gastrointestinal tract, including its trophic, chemopreventive and anti-inflammatory roles. We also review the action of butyrate in peripheral tissues with respect to its ability to prevent obesity and insulin resistance which may have translatable effects for the clinical treatment and prevention of obesity and type 2 diabetes (T2D).sn-3 position and susceptible to cleavage by pancreatic lipase in the small intestine [sn-2 position, as very few dibutyryl diacylglycerols have been verified via gas chromatography [sn-1 and sn-3 positions [Bovine milk fat is a particularly rich source of butyrate, with butyrate contributing approximately 4% wt/wt . The fatntestine ,28,29. Btography ,29,30. Tositions . As a coositions ,34,35,36Human breastmilk has also been examined as a potential source of butyrate for neonates and a modulator of the colonic microbiota ,38,39. RAlthough dietary fiber does not contain butyrate, its fermentation by microbes in the cecum and distal colon generates SCFAs, including butyrate, which can be utilized by the host organism ,44. ThisThe digestibility of food starches depends not only on chain length and extent of branching but also upon method of cooking, chemical pretreatments  and cooling . For exaThe interactions between dietary fiber and the colonic microbiome are likely bidirectional. The efficiency of resistant starch fermentation is dependent on the bacterial communities present in the colon  and commFaecalibacterium prausnitzii (a member of Clostridium cluster IV) and Eubacterium rectale/Roseburia (Clostridium cluster XIVa) have currently received the most attention as they constitute 5–10% of total bacteria in fecal samples collected from healthy adults [F. prausnitzii may ultimately enhance butyrate production [F. prausnitzii was co-cultured with Bifidobacterium breve Yakult or Bifidobacterium adolescentis and oligofructose (a particular inulin-type fructan) provided as the energy source butyrate was produced in appreciable quantities (between ~12 mM and ~30 mM after 48 h of fermentation) [F. prausnitzii with Bifidobacterium angulatum or Bifidobacterium longum was dependent upon the energy substrate provided; oligofructose encouraged greater butyrate production than other inulin molecules in both co-culture models, which the investigators attributed to energy competition among the bacterial strains as well as less bioavailable acetate as a co-substrate of butyrate synthesis [+ ratios that were 16-fold lower than their conventionally raised counterparts, as well as 56% lower ATP levels [Butyrivibrio fibrisolvens, their energy status improved [Although numerous bacterial strains have been analyzed for their butyrate-producing capacities, y adults . In addioduction . When F.ntation) . In contynthesis . SCFA prP levels . When thimproved . These d+ concentration gradient to efficiently sequester SCFAs within colonocytes have been identified as a mechanism of SCFA uptake [Due to the overall hydrophobicity and low molecular weights of the SCFAs in their protonated forms, acetate, propionate and butyrate can be readily absorbed via nonionic diffusion across the apical membrane of colonocytes ,63. Yet,A uptake ,43,64. WA uptake ,64. In aA uptake ,65,66,67A uptake ,62,68.i/o and the Gq pathways, whereas GPR109A and FFAR3 only utilize the Gi/o pathway [q family, DAG and IP3 may also activate protein kinase C (PKC) to ultimately stimulate the downstream activities of the extracellular signal-regulated kinase 1/2 (ERK-1/2) and c-Jun N-terminal kinase (JNK) pathways [At the cell surface butyrate acts as a ligand for metabolite-sensing G-protein coupled receptors (GCPRs), including GPR43, GPR41 and GPR109A, on intestinal epithelial cells ,69,70,71 pathway ,72. In tpathways . Butyratpathways ,75. Actipathways ,77. GLP-pathways . PYY affpathways ,79,80. Tpathways ,82. In apathways ,84. Thuspathways . Because2 is plentiful [Mpc1) gene, an outcome that is observed among several cancers [Butyrate is known to promote growth of the colonic epithelium, yet it exerts a predominately inhibitory effect on colorectal cancers ). Emergilentiful ,86. Upta cancers . Neverth cancers . Under c cancers ,90. Buty cancers . In nonc cancers . In addi cancers , thereby cancers , therebySlc5a8) gene, which codes for a sodium-dependent butyrate transporter, has been observed in both cancerous colonocytes as well as aberrant crypt foci [l-carnitine supplementation also mitigated cancer cachexia symptoms in BALB/c mice injected with adenocarcinoma cells [The ability of butyrate to accumulate in the cytoplasm of cancerous colonocytes appears to be related to the coordinated downregulation of fatty acid uptake into their mitochondria. Although it was historically established that SCFAs did not require the carnitine palmitoyltransferase proteins  for entry into the mitochondria of hepatic and cardiac tissues , it is iypt foci . These dypt foci ,96,97,98ypt foci ,100. Orama cells ,102.−/− cells was not markedly inhibited by incubation with TGF-β and 3H-thymidine incorporation only decreased modestly (25% decrease vs. 61% for control) [Smad3 mRNA content, with the longest incubation period (48 h) producing the greatest expression relative to control; moreover, sodium butyrate exhibited a dose-dependent effect on SMAD3 protein expression over a 24-h time course, with intermediate doses (2.5 and 5.0 mM concentrations) having the most potent effects [With the HDACi and acetylation effects of butyrate widely recognized, attention has now turned to the identification of signaling pathways through which butyrate may exert anti-proliferative and pro-apoptotic effects in cancerous tissues. For example, the transforming growth factor β (TGF-β) signaling pathway has been implicated in cell sensitization to pro-apoptotic mechanisms in noncancerous colonocytes but persistent downregulation of its downstream modulators such as mothers against decapentaplegic homolog 3 (SMAD3) has been implicated in cancer progression ,104. In control) . MicroRNcontrol) . Sodium  effects . Interes effects . Pretrea effects . The com effects . These d effects . In a si effects ,110. Alt effects ,112. Unf effects ,113. Thi effects .Although the tumor suppressor effects of butyrate have been attributed to its HDACi activity, it is important to note that recent studies have found that butyrate may act through the GPR109A receptor, independently of HDAC inhibition, in colon cells to prevent cancer . For exaButyrate is not only responsible for the energy requirements of the colonic epithelium but it also preserves such tissues by mitigating chronic inflammatory responses through activation of its target GPCRs and its HDACi activity ,116,117.2+ [The effects of butyrate on IL-8 are dependent upon dose and time effects, as well as the cell type under investigation. For example, Gibson and Rosella isolated colonic crypt cells from patients diagnosed with colorectal cancer, Crohn’s disease, or ulcerative colitis and assessed IL-8 secretion in response to butyrate treatment (1 mmol/L) over a 24 h time course; in all disease groups butyrate administration significantly lowered IL-8 concentrations in comparison to control . Importa2+ . A simil2+ . When th2+ .2) and greater than one-third of the adult population is obese (BMI ≥ 30 kg/m2) [Obesity and its associated pathologies represent one of the greatest emerging healthcare challenges in the developed world. In the United States alone, at least two-thirds of the adult population is classified as overweight (defined as a body mass index (BMI) ≥ 25 kg/m0 kg/m2) ,133,134.0 kg/m2) . Obesity0 kg/m2) . Obesity0 kg/m2) ,137,138.0 kg/m2) ,141,142,0 kg/m2) ,145,146,0 kg/m2) ,148, rep0 kg/m2) ,150 and 0 kg/m2) ,152. In 0 kg/m2) . Once T20 kg/m2) . Interes0 kg/m2) ,160,161.C. butyricum not only reduced body weight gain and mitigated fat pad size over a 12 week HFD intervention but it also reduced free fatty acid content in the liver, suggesting diminished ectopic hepatic lipid deposition relative to the HFD controls [Dietary butyrate supplementation has been shown to mitigate weight gain through attenuating increases in adiposity in animals fed high fat diets (HFD) ,155,156.Importantly, the anti-obesogenic effects observed following butyrate supplementation can also be achieved through dietary interventions involving resistant starches and other fermentable fibers ,12,13. FA similar study utilized a 2 by 2 factorial design to assess high-amylose maize resistant starch type II (HAM-RS2) and sodium butyrate dietary interventions in male Sprague-Dawley rats over the course of 12 weeks . Signifi®)) on body weight and metabolic parameters in C57BL/6J mice and two polygenic murine models of obesity: NONcNZO10/LtJ and Non/ShiLtJ mice, respectively [Gpr41 mRNA content in the adipose tissue to a greater extent than any of the other SCFAs and to a level higher than the HFD or low-fat diet  controls [Finally, a study was conducted comparing the effects of two dietary fat levels (7% vs. 11% wt/wt) and starch compositions  [C. butyricum probiotics in conjunction with the HFD exhibited significantly lower fasting serum insulin levels and lower blood glucose levels at both 30 and 120 min following an intraperitoneal glucose tolerance test (GTT) [C. butyricum supplementation [In addition to directly supplementing butyrate into the diet, multiple studies have illustrated that supplementing the diet with butyrogenic bacterial strains in the form of a well-tolerated probiotic can exert similar metabolic effects ,156,161.entation . In compentation . Additioentation . In a diThese studies clearly indicate that butyrate supplementation through various means may have beneficial effects on preventing obesity and whole-body insulin resistance, which are characterized by aberrant metabolism in peripheral tissues. For example, perturbations of mitochondrial β-oxidation of fatty acids, leading to incomplete β-oxidation and ectopic lipid deposition in liver and skeletal muscle and excessive accumulation in adipose tissue is observed in the obese, insulin resistant state ,169,170.The mechanisms through which butyrate acts to attenuate and ameliorate obesity and insulin resistance are not fully understood but clearly involve contributions from various peripheral tissues, including the liver, skeletal muscle and adipose tissue . Yet, thw/w sucrose supplementation) for 6 weeks [C. butyricum (5 × 108 CFU) intragastrically in male ICR mice for 5 days prior to acute liver injury induced via carbon tetrachloride (CCl4) intraperitoneal injection [C. butyricum exerted a prophylactic effect against CCl4 injection: ICR mice that received the bacterial strain showed improvements in markers of oxidative stress, including significantly greater nuclear factor (erythroid derived-2)-like 2 (NRF-2) protein content and elevated superoxide dismutase (SOD) and catalase enzyme activity and markers of pro-inflammation, including reduced TNF-α and increased anti-inflammatory cytokine IL-10 [Obese, diabetic models in a simplistic context exhibit fatty, insulin resistant livers due to increased triglyceride content and decreased β oxidation within the tissue, which is also associated a pro-inflammatory state. Supplementation of butyrate or butyrogenic bacterial strains is associated with the reduction of ectopic lipids in hepatic tissue ,174,175. 6 weeks . Despite 6 weeks . A simil 6 weeks . Protein 6 weeks . Additio 6 weeks . In a muAdipor1/Adipor2) and uncoupling proteins 2 and 3 (Ucp2/Ucp3) in butyrate-supplemented mice [Pgc-1α gene expression that was associated with prevention of obesity and insulin resistance as well as improved mitochondrial function and a higher percentage of type 1 oxidative fibers in skeletal muscle [Skeletal muscle represents one of the largest insulin responsive tissues and utilizes a significant proportion of blood glucose during metabolism . In caseted mice . Finallyted mice . With reted mice . Westernted mice . Sodium ted mice . For exal muscle . Thus buThe anti-obesogenic and diabetic effects of butyrate supplementation may be partially attributed to its metabolic effects on adipose tissue . ButyratAlthough in vitro and in vivo animal studies support the ability of butyrate supplementation to prevent colon cancer, obesity and insulin resistance, few studies in human participants have been conducted to date to corroborate the beneficial results of butyrate supplementation. Human studies have primarily emphasized changes in gut microbial diversity  following dietary intake of resistant starches or other fermentable carbohydrates ,195,196,Butyrate, a SCFA essential for the proper growth and function of the gastrointestinal epithelium, is primarily derived from the fermentation of dietary fibers and resistant starches by the colonic microbiota. Beyond its HDACi action butyrate serves as a fuel source for enterocytes and colonocytes, facilitates apoptosis of colonic cancer cells and reduces gut inflammation. Although additional work is needed to determine the levels of butyrate reaching and affecting peripheral tissues and a mechanism of action within these tissues, the literature has shown that butyrate supplementation has the ability to prevent obesity and insulin resistance through its actions in peripheral tissues. Mainly butyrate supplementation acts to decrease ectopic lipid deposition and inflammation and produces mitochondrial adaptations that increase β-oxidation of fatty acids. Given the beneficial effects of butyrate found in animal and clinical studies, dietary strategies that can increases butyrate levels may serve as likely treats to mitigate cancer, obesity and T2D. Thus, additional translational and clinical studies in these areas are warranted.


Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing.After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis.Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression.This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for investigation of the genetic structure, function, and regulatory mechanisms of the PLE gene family. Pig liver carboxylesterase, also known as pig liver esterase (PLE), consists of a family of enzymes composed of various isozymes. PLE is indispensable in the field of organic synthesis owing to its unique asymmetric hydrolytic activity, and nearly a century of research on PLE has centred on its applications in this field ,2. PLE cPLE is the most complex family of carboxylesterases in mammals, consisting of many members with similar physical and chemical properties. Hence, it is very difficult to isolate and purify isozymes containing a single component using physical chemistry methods. Extraction of natural PLE from pig liver had already been attempted in the 1960s, but the PLE extracted from pig liver was found to comprise a mixture of multiple isozymes, each with different hydrolytic characteristics ,6,7. ForIn our previous study, more than 54 isoenzymes of PLE cDNA were obtained from Tongcheng pig and Large white pig, and from each individual of pig, about 8–15 isoenzymes were found, and the main isoenzymes were also expressed and their hydrolysis activity also were examined, and it turn out that each enzyme has different activity (data wait for publication). Taking trimmer is required for these enzyme to be active , the numIn this study, we used a previously constructed Rongchang pig BAC genomic library , and priPCR amplification to validate primer sets was performed using pig genomic DNA as a template and amplification products were characterised by 0.8% agarose gel electrophoresis . PrimersA three-step PCR screening method was employed, and primer pairs 1, 2, 4, 5, and 6 were used to identify five positive BAC clones from the library, designated BAC-10, BAC-70, BAC-75, BAC-119, and BAC-206. NANODROP 2000C spectrophotometer (Thermo Scientific). The concentrations of the five clones were 3000-5000ng/μL, the A260/A280 ratios were 1.91–1.96, and the A260/A230 ratios were 2.1–2.2. Each isolated clone (5 μl) was subject to electrophoresis on a 0.8% agarose gel . Each sample migrated as a complete band without any indication of contamination by impurities or of degradation  were transferred to LB liquid culture containing 12.5μg/mL chloramphenicol and BAC DNA isolated according to the instructions provided in the BAC/PAC DNA Isolation Kit. DNA concentration was measured using a radation . Thus thTM Library was approximately 10kb. The accuracy of the sequencing data was 99.999%, demonstrating its reliability. Bioinformatics was then applied to analyse and assemble the sequences of BAC-10, BAC-70, BAC-75, BAC-119, and BAC-206, and a total of five BAC sequences were obtained, with lengths of 109,591 bps, 127,733 bps, 106,999 bps, 127,294 bps, and 116,854 bps, respectively.After purification and concentration of the five BAC samples, a library composed of 20kb fragments derived from each sample was constructed. After SMRT sequencing, the original output data was filtered, and high-quality sequences of BAC-10, BAC-70, BAC-75, BAC-119, and BAC-206 were generated, with totals of 41,228,895bps, 1,013,780,514bps, 1,487,229,841bps, 307,626,930bps, and 191,394,912bps, respectively. The average read length of the SMRTcellOur previous study obtained 54 PLE subtypes from Tongchong pig and Large White pig (data wait for publication), which were divided into A, B, C, D, E, F, G groups according to the similarity of their amino acid sequence, and among the same group, different isotypes were distinguished by Araba numbers. PLE genes found in this study are named in the same way.The sequences of the assembled BAC DNA samples were aligned with that of γ-PLE cDNA (GenBank: X63323) using BLAST. The five BAC sequences were found to contain a total of four complete PLE genes; BAC-10 contained the complete sequence of the PLE-G2 gene, BAC-70 contained two complete PLE genes (PLE-1 and PLE-B9), BAC-75 contained PLE-B9 and an unknown 63kb gene sequence, BAC-119 contained PLE-C4 and an unknown 82kb gene sequence, and BAC-206 contained PLE-G2 and PLE-1. The lengths of the PLE-1, PLE-B9, PLE-C4, and PLE-G2 gene sequences were 33,759bps, 28,384bps (BAC-75) /28,272bps (BAC-70), 27,835bps, and 30,819bps, respectively; these sequences were submitted to GenBank, and their accession numbers are KX577727, KX577728, KX577729, and KX577730, respectively. The average length of the spacer sequences between the PLE genes was approximately 22kb. In addition, sequence alignment revealed that the PLE-B9 genes obtained from BAC-70 and BAC-75 showed 100% amino acid sequence homology (with only one nucleotide difference). Sequence alignment of the regions 6082bps upstream of these genes revealed 99% homology , revealing the sequence obtained is reliable. Sequence information for each BAC clone is presented in Using the γ-PLE cDNA sequence (GenBank: X63323) as a reference, and based on the GT-AG rule of bases at the start and end of introns, the exons, introns, noncoding sequences, and spacer sequences were identified for each PLE gene. The four PLE genes were found to have 14 exons and 13 introns, of which exons 1, 2, 5, 7, 10, 11, 13, and 14 were relatively highly conserved.The regulatory regions of the four PLE genes contained two regions of reverse complement sequence of 720bps in length, taking an example of PLE1, one at nucleotides -5132bp /-4413bp and the other at -802bp /-82bp, separated by 3611bps. These two sequences can be complementary to form Loop ring structure, which is likely to be very significant for the regulation of gene expression. The regulatory sequences of the different PLE genes exhibited a high degree of homology. Using the regulatory region 6000bps upstream of the PLE-1 gene start codon as a reference, homology with the PLE-G2 upstream sequence was up to 99%, and that with PLE-B9 and PLE-C4 was approximately 96%. In addition, the size and sequence of introns and the sequence of exons of the different PLE genes differed to some extent; however, the sequences of all exon-intron junctions were highly conserved and followed the AG-GT rule. Alignment of the amino acid sequences of the four PLE genes revealed that the homology between the four isoforms and γ-PLE was ≥ 96%, and all had identical signal peptide sequences of 18 amino acids and C-terminal ER retention signals (HAEL). Intron alignment showed that the introns of PLE-1 and PLE-G2 were the most similar; the homologies between all introns were ≥ 97% in these two genes, except for intron 2 because of different sizes (PLE-1 is 5403bps but PLE-G2 is 2195bps). The introns of PLE-B9 and PLE-C4 were relatively similar in size, with homologies of≥ 96%. The introns of the two PLE-B9 genes obtained from BAC-70 and BAC-75 were ≥ 99% homologous. Details of the exon and intron sizes of the four PLE genes are presented in Tables Meanwhile, a BLASTP comparison between the known γ-PLE isoenzyme with PLE1、PLE-B9、PLE-C4、PLE-G2 isoenzymes and PICE was also performed, which confirmed that the difference amino acids are not randomly distributed in the sequences, but are in several distinct regions with total of 25 AA different , PICE waAs the peptide sequence of PLE isoenzymes reported was different in 25 AA positions, compariSus scrofa carboxylesterase APLE isoform gene is located on chromosome 6 at position 27,276,898–27,308,831 bp, has a length of 31,934 bps (Gene ID: 397478), and encodes 565 amino acids. Comparative analysis of the sequences of carboxylesterase in different species using BLAST revealed that the amino acid sequences of porcine PLE-1, PLE-G2, PLE-B9, and PLE-C4 have 77–78% homology with hCES1, 72% with mCES1, and 97% with Sus scrofa APLE. The N-terminal signal peptide sequence and C-terminal ER retention signal sequences of the PLE genes are involved in mediation of the proper transport and localization of carboxylesterases. Alignment of the human, mouse, and porcine carboxylesterase amino acid sequences revealed that the signal peptide amino acid sequences differed greatly among different species. C-terminal ER retention signals were also different; the third amino acid from bottomin the ER retention signals in human, mouse, Sus scrofa carboxylesterase APLE, and PLE are I, V, A, and A, respectively.Comparative genomic analysis helps to explain genetic evolution, assists in mining for potentially functional gene regions, and has become an important bioinformatics technology in the study of complex gene structures and potential regulatory regions. Using these methods, it was determined that the gene for human carboxylesterase 1 (hCES1) is located on chromosome 16 at position 55,802,851–55,833,186 bp, has a length of 30,336 bps (Gene ID: 1066), and encodes 568 amino acids; the house mouse carboxylesterase 1 (mCES1) gene is located on chromosome 8 at position 93,302,369–93,337,209 bp, has a length of 34,841 bps (Gene ID: 12623), and encodes 566 amino acids; the http://www-bimas.cit.nih.gov/molbio/proscan/) to analyse transcription factor binding sites in these regions, relatively few were found in the sequence at -5000 to -2000 bp, whereas several different transcription factor binding sites were found in the region from -2003bp to +1077 bp (see The sequences upstream (-5000 to -1 bp) of the four PLE genes exhibited 99% homology. Using Proscan software. No simple tandem repeats of the same gene were identified. Furthermore, our results confirm that, in the pig liver carboxylesterase gene family, one gene encodes one polypeptide, which is obviously different compared to antibody gene family.In summary, our analyses reveal the complete sequences of four PLE genes, thereby filling the gaps in the database regarding PLE gene sequences and providing the necessary foundation for analysis of the genomic structure, and regulation mechanisms of gene expression of the PLE gene family.The study was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Hubei Province, China, and the protocol was approved by the Committee on the Ethics of Animal Experiments at the College of Veterinary Medicine, Huazhong Agricultural University.E.coli strain and the pCC1BAC vector (8128 bps) were used for library construction ; 5× loading buffer, DL2000, DL1500 ; agarose ; DNA gel extraction kit ; yeast extract, tryptone, NaCl, agar ; chloramphenicol ; BAC/PAC DNA Isolation Kit .Chloramphenicol stock solution: 0.25 g chloramphenicol was dissolved in methanol (99.9% purity) to obtain a 25 mg/ml solution, which was sterilized by filtering through a 0.22 μm filter, aliquoted, and stored at -20°C.2O, and brought to a total volume of 1000 mL. The broth was autoclaved at 121°C for 15 min before use.Lysogeny broth (LB) liquid medium: 5 g yeast extract, 10 g tryptone, and 10 g NaCl were dissolved in 800 mL ddH2O and brought to a total volume of 1000 mL. The medium was autoclaved at 121°C for 15 min before use. After cooling the medium to approximately 55°C, chloramphenicol was added on a clean bench . After mixing, the medium was poured into sterile disposable culture plates. The plates were stored at 4°C after medium solidification.LB solid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, and 15 g agar were dissolved in 800 mL ddH2O were fully dissolved in 700 mL deionized water. Glacial acetic acid (57.1 mL) was added and stirred thoroughly to ensure mixing, and then the total volume was brought to 1 L. The buffer was stored at room temperature before use.50× TAE nucleic acid electrophoresis buffer: 242 g Tris base and 37.2 g Na-EDTA·2HThe known γ-PLE cDNA sequence (GenBank: X63323) was analysed along with incomplete PLE gene sequences available in NCBI databases. Based on the AG-GT rule , the PLE coding region was found to be composed of 14 exons. Alignment of partial PLE gene exon and intron sequences using BLAST, demonstrated that some exons of the PLE gene were highly conserved. In addition, the introns of different PLE isoforms showed significant differences in size. Based on these results, eight primers spanning the upstream and downstream junctions of the conserved exons were designed .PCR amplification was performed using the primers described above with Tongcheng pig genomic DNA  as a template. Appropriate primers were identified based on the presence or absence, and size of the resulting amplicons. The PCR system is presented in A commonly used three-step PCR screening method, with less stringent conditions, was selected. Primers verified as suitable were used to screen for PLE-positive BAC clones from the Rongchang pig BAC genomic DNA library. The detailed procedure was as follows:The library (consisting of 500 384-well plates) was removed from storage at -80°C and placed at 4°C to thaw. A 384-pin replicator was used to inoculate LB agar plates containing 12.5μg/mL chloramphenicol with all BAC clones from the 384-well plates. LB plates were incubated overnight at 37°C and colony growth observed. Next, 5 mL of sterile LB liquid medium containing 12.5μg/mL chloramphenicol was added to each LB agar plate. After evenly spreading and mixing the clones with the medium, using a sterile triangular cell spreader, the broth mixture was tipped to one side of the culture plate, aspirated with a sterile pipette tip, and transferred to an EP tube for later use.Each 384 culture plate colony mixture from the previous step was transferred to a LB liquid medium (containing 12.5μg/mL chloramphenicol). The culture was expanded by overnight culture at 37°C with shaking, and the BAC DNA harvested using the BAC/PAC DNA Isolation Kit .PCR amplification was performed using the BAC DNA clone mixture from the 384-well plates as templates  was added to 24 1.5 mL centrifuge tubes. A toothpick was used to transfer each set colonies (16 colonies in a set) to the centrifuge tubes, and these samples were then boiled for 5 min. PCR amplification was performed directly using this mixture as the template (for reaction conditions see section e above) were used as templates for colony PCR (for reaction conditions see section Sixteen colonies containing positive clones were identified. The remaining colonies that had been stored on LB agar medium (see Colonies identified as containing positive clones were transferred to LB liquid medium containing 12.5μg/mL chloramphenicol and incubated at 37°C with shaking (200 rpm) overnight (16 h) to expand the culture, and obtain cultures of BAC clones containing PLE genes.BAC DNA clones were isolated using the BAC/PAC DNA Isolation Kit . To measure DNA concentration and purity, the plasmids were sent to Tianjin Biochip Corporation  and Biomarker Technologies  after characterisation by 0.8% agarose gel electrophoresis, where they were further purified and concentrated to 69ng/μL with a total DNA of 34.5 μg. Using PacBio third-generation sequencing, each BAC was allocated to a single sequencing unit; a total of five sequencing units were used, in order to ensure accurate separation of sequencing data.Oropetium thomaeumwith 72-fold coverage. A draft genome was constructed which contained telomeric and centromeric sequences, long terminal repeats, retrotransposons, tandem repeat genes, and other genomic elements that are difficult to sequence, with over 99.999% accuracy [PacBio third-generation sequencing produces an average read length of 10–15 kb, and a single sequencing unit can process up to 1 Gb . In 2015accuracy . These d


Geobacillus thermoglucosidasius has the potential to produce chemicals and fuels from biomass-derived sugar feedstocks. Here, we present the genome sequence of strain NCIMB 11955, the progenitor of an ethanologenic industrial strain, revealing 11 single-nucleotide polymorphisms and 2 indels compared to strain DSM 2542 and two novel plasmids.The industrially important thermophile  Geobacillus thermoglucosidasius is a Gram-positive, thermophilic, spore-forming bacterium that performs a typical mixed-acid fermentation , producing 1.66 Gb of data consisting of a total of 801,181,830 nucleotides . Using CLC Genomics Workbench version 7.0.4 (CLCbio), 3,203,678 reads were produced through trimming with a quality score limit of 0.05 and maximum ambiguous nucleotide of 2. The genome sequence was then derived by mapping to DSM2542 (CP012712)  as a reference and de novo assembled contigs, the 321-bp gap was closed through PCR amplification and Sanger sequencing of the missing region. A total of 11 single-nucleotide polymorphisms (8 nonsynonymous) and 2 indels were identified compared to DSM 2542. Affected genes included those encoding an MFS transporter , rpoE , a transposase , YggS , CodY , CheA , RimP , and adenine phosphoribosyltransferase .The entire reference was covered by 92% of the reads (average coverage of 189.38 ± 41.51) with four gaps . While the first three were closed through identification of Geobacillus plasmids. Plasmid pNCI001 shared homology with pGS18  and pLW1071 , whereas plasmid pNCI002 was homologous to pGG01 , pGEOTH01 , and pGY4MC101 .BLAST analysis of two unmapped contigs designated pNCI001  and pNCI002 , revealed homology to a number of G. thermoglucosidasius C56-YS93 (Genome annotation was carried out by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) service, resulting in a total of 3,844 coding sequences . The chromosome shared identical features to that of DSM 2542 , 12. TheC56-YS93 .CP016622 (chromosome), CP016623 (pNCI001), and CP016624 (pNCI002).The complete genome sequence has been deposited in GenBank under the accession numbers


Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes  were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress. Stipa grandis P. Smirn. is a C3 perennial bunchgrass [S. grandis steppe acts as a natural green barrier and plays an important role in sandstorm prevention [S. grandis have mostly focused on ecological aspects, such as the relationship between the photosynthetic capacity of S. grandis and the soil moisture gradient [S. grandis [2 on plant growth [S. grandis to high temperature and drought have also been reported [S. grandis related to grazing and location [S. grandis in a typical grassland; the results showed that many stress-related genes had differential expression under long-term grazing conditions [S. grandis’s responses to grazing and related stresses remain unclear. To further understand the stress response mechanism of S. grandis, details of the expression patterns of more stress-related genes are necessary.nchgrass  that is nchgrass . This spnchgrass . The S. gradient , the eff grandis , and thet growth . The phylocation , 3, 7. Tnditions . HoweverrRNA, GAPDH , ACT (β or γ actin), TUB (α or β tubulin), EF1α (elongation factor 1α), and UBQ (poly-ubiquitin) have been used most frequently as internal control genes [CUL (Cullin), UBCP (ubiquitin carrier protein) [CAP (adenylyl cyclase-associated protein), SKIP1 (ASK-interacting protein 1) [F-BOX (F-box/kelch-repeat protein) [PP2A (protein phosphatase 2A) [SAMDC (s-adenosyl methionine decarboxylase) [SNF (sucrose non fermenting-1 protein kinase) [Quantitative real-time reverse transcription PCR (qRT-PCR) is an efficient tool for expression analysis because of its high-throughput, accuracy, sensitivity, and ease of use compared with traditional methods, such as northern blotting and semi-quantitative reverse transcription-PCR –11. The ol genes , 17. Recotein 1) , F-BOX (protein) , PP2A (ptase 2A) , SAMDC (oxylase) , 25, and kinase) . However kinase) , 27. ThuS. grandis under various experimental conditions.In this study, 15 candidate reference genes that could be used potentially as internal control genes to normalize gene expression were selected. The expression stabilities of these reference genes under various abiotic stresses and three hormone treatments were assessed using four algorithms . According to our analysis, we were able to recommend the most suitable reference genes to quantify gene expression in S. grandis seeds were collected from the Baiyinxile Livestock Farm of Xilinhot, Inner Mongolia . The seeds were sown in pots filled with a soil mixture , and incubated in a growth chamber at 23°C with a 16-h light/8-h dark cycle.No specific permits were required for the described field studies.S. grandis plants were removed from the soil gently, washed clean with tap water, and then immediately subjected to the different treatments. For plant hormone, high pH and salinity treatments, the plants were dipped separately in solutions of 100 μM salicylic acid (SA), 100 μM jasmonic acid (JA), 100 μM abscisic acid (ABA), high pH (pH 10), and 300 mM NaCl. For the drought treatment, the plants were placed on filter paper and incubated in a growth chamber. For the wounding treatment, plants growing in pots were wounded using tweezers and placed in a growth chamber. For cold and heat treatments, plants growing in pots were maintained at 4°C or 42°C, respectively. Untreated plants were used as the control. Shoot samples (from four to five plants) were taken at 1, 3, 6, 12, 24, and 48 h time points for all treatments, and an additional 0.5-h time point for drought and wounding treatments, and were frozen immediately in liquid nitrogen and stored at −80°C until RNA extraction. Three independent biological experiments were performed.Four-week-old plants were used in the following experiments, and the different treatment conditions were mainly achieved according to methods described previously , 28, 29.Total RNA was extracted from the samples using the Trizol regent. The purity and integrity of the RNA samples were estimated by calculating their A260/280 absorbance ratios and by agarose gel electrophoresis analysis. Total RNA (0.5 μg), pretreated with RNase-free DNase I , was reverse transcribed into cDNA using an M-MLV reverse transcriptase kit (Takara) and oligo (dT) 18 primers (Takara) following the manufacturer’s instructions. The generated cDNAs were diluted 20-fold for qRT-PCR analysis.EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2. According to these sequences  with a final volume of 20 μL per reaction, as previously described . Each 20First, three analytical tools, geNorm (version 3.5) , NormFingeNorm determines the most stable reference genes from a set of tested genes in a given sample set under a certain experimental condition and calculates the gene expression normalization factor via geometric averaging of all the reference genes . geNorm http://fulxie.0fees.us/?type=reference), which is a comprehensive web-based tool that integrates geNorm, NormFinder, BestKeeper, and the comparative delta Ct method, was applied to determine the most suitable reference genes for the overall final ranking [Finally, RefFinder , phenylalanine ammonia-lyase (PAL), late embryogenesis abundant protein (LEA), lipoxygenase (LOX), heat shock protein 90 (HSP90), and pathogenesis-related protein 1 (PR1) encoding genes were obtained from the  library  and usedSgPOD and SgPAL under all treatments, SgLEA under drought, cold, salt, high pH and ABA treatments, SgLOX under wounding and JA treatments, SgHSP90 under heat treatment, and SgPR1 under SA treatment were quantified using the most and least stable reference genes, respectively. The primers were as follows:The transcript levels of SgPOD: TCGACTTCGCTGTTCCCACT/CCTTTTGCGGTGAAGTTTCCSgPR1: GTCGTCTGCAACAACAACGG/GGGACTACTGCGAAGCAATCASgPAL: AGCAGCACAACCAGGACGTA/GTCACGCAGTTCTTGACGGASgLEA: CTTCCACTTCCATGATTTACGC/CATCCATACAGACCCCAGACACSgLOX: CTGGAGGGCATCGAGCAC/GGGTAGAGCAGCATGTAGGGSgHSP90: CGGATCTTGTGAACAACCTCG/GTAGAAACCAACACCAAACTGACC−ΔΔCT method [The 2T method  was usedS. grandis transcriptome data [Fifteen fragments of candidate reference genes , obtaineome data , that exSamDC exhibited the highest expression abundance, with a mean Ct of 16.91, while SKIP5 had the lowest expression level, with a mean Ct of 26.08 across all samples.The expression levels of the candidate reference genes were determined by qRT-PCR across all samples  , and a wSamDC (3.40 cycles) and UCH (4.33 cycles) showed the smallest expression variations across all the samples. In contrast, SKIP5 (6.34 cycles) and SKIP11 (6.21 cycles) exhibited the largest relative expression variations among all the samples.UBC15 and UBC17 were the most stably expressed genes with the lowest M value of 0.0844. For ABA, salt, and JA treatments, the most stably expressed genes were EF1beta and UBC15, with M values of 0.096, 0.106, and 0.117, respectively. For heat treatment, HERC2 and ACT had the lowest M value of 0.137. Under cold treatment, UBC17 and SKIP5 had the lowest M value, and for high pH treatment, UBC17 and CUL4 were the most stable genes. After SA and wounding treatments, the M values of the combinations EF1beta and HERC2, and UBC2 and UBC15 were the lowest, respectively. Under heat and cold treatments, the least stable genes were SKIP5 and SKIP1, respectively. In the other seven treatments, SKIP11 was the most unstable gene. Across all samples, UBC17 and CUL4 ranked as the most stable genes, while SKIP11 was the least stable gene.The geNorm results are shown in The optimal number of reference genes for normalization was calculated by pairwise variation (V). Under various conditions, the Vn/n+1 values were lower than 0.15 , which sCUL4, UBC15, and UBC17; under cold treatment were SKIP5, HERC2, and UBC17; under heat treatment were ACT, SKIP1, and GAPDH; under JA treatment were UBC17, EF1beta, and UBC15; under salt treatment were UBC15, EF1beta, and HERC2; under high pH treatment were UBC15, EF1beta, and SKIP5; under SA treatment were HERC2, UBC17, and EF1beta; under wounding treatment were UBC2, UBC15, and UBC17; and under drought treatment were EF1beta, UBC15, and GAPDH. According to all the samples in the study, UBC15, HERC2, and EF1beta were the most stable genes. In addition, the least stable genes ranked by NormFinder and geNorm were consistent: for cold and heat stresses they were SKIP1 and SKIP5, and for the other treatments it was SKIP11.Based on the results calculated by NormFinder , the thrUBC17 as the most stably expressed gene following cold, wounding, and ABA treatments, while UCH had the lowest SD value under drought and salt stresses. GAPDH, SamDC, UBC2, and SKIP1 were ranked as the most stably expressed genes under heat, high pH, JA, and SA treatments, respectively. When all the samples were analyzed together, UCH was rated as the most stable gene with the lowest SD value.As shown in EF1beta (drought and JA), ACT (heat), SKIP5 (cold), UBC15 , UBC2 (wounding), and HERC2 (SA) were ranked as the top stable genes under the different treatments. Across all samples, EF1beta, HERC2, and UCH were suggested as the three best reference genes for normalizing the expression of target genes.RefFinder, which combines the geNorm, NormFinder, and BestKeeper outputs, was applied to generate a comprehensive final ranking of the candidate references. As shown in SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90, and SgPR1, in response to different treatments were examined. When normalization was performed using the two most stable genes under each treatment, EF1beta and UBC15 (drought), SKIP5 and UBC17 (cold), UBC15 and EF1beta , ACT and GAPDH (heat), UBC15 and HERC2 (high pH), UBC2 and UBC15 (wounding), EF1beta and UBC17 (JA), UBC15 and CUL4 (ABA), and HERC2 and UBC17 (SA), the expression of SgPOD was affected by different treatments  with the quantitative real-time polymerase chain reaction (qPCR) . The accS. grandis. Four statistical algorithms, geNorm, NormFinder, BestKeeper, and RefFinder, were used to evaluate the stability of these genes. All the genes displayed different expression patterns following the various treatments. Moreover, high stability levels were observed using geNorm analysis; all the M values were below the accepted threshold of 1.5  [ACT was identified as the best reference gene by the geNorm and RefFinder programs under heat stress  . In our t stress .UBC genes  were analyzed in this study. Notably, these UBC genes performed well under different treatments (excluding heat stress), especially when analyzed by geNorm and RefFinder  encoding gene [UBC genes could be used more often as an internal control for normalizing gene expression under various stress treatments. SKIP, a spliceosome component and transcriptional activator, is conserved in eukaryotes and has diverse functions in plant development associated with responses to environmental changes [SKIP5 was ranked as the most stable gene under cold stress using geNorm , E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases) . AccumulefFinder . UBC15 aing gene , also sh changes . Among tg geNorm , NormFing geNorm , and Refg geNorm , and SKIanalysis . Conversprograms , except 2O2 [S. grandis defense against stressful conditions. The late embryogenesis abundant protein encoding gene LEA has been widely reported to participate in responses to drought, cold, and salt stresses, and ABA treatment, and plays an important role in plant tolerance to abiotic stress [SgLEA under drought, cold, salt, high pH and ABA treatments Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Text(TXT)Click here for additional data file.


The HIPK2 (serine/threonine homeodomain-interacting protein kinase 2) is a “caretaker” gene, its inactivation increases tumorigenicity while its activation inhibits tumor growth. This report reviews the anti-tumorigenic mechanisms of HIPK2, which include promotion of apoptosis, inhibition of angiogenesis in hypoxia, prevention of tumor invasion/metastasis and attenuation of multidrug resistance in cancer. Additionally, we summarize conditions or factors that may increase HIPK2 activity. Since being discovered in 1998 by Kim et al. using a yeast two-hybrid screen designed to characterize novel molecules that bind to homeoproteins, the homeodomain-interacting protein kinases (HIPKs), have been proven to be a tumor suppressor and one of the highly conserved factors regulating signaling and gene expressions. They control a wide spectrum of biological functions such as DNA damage response, apoptosis, hypoxia, cell proliferation and invasion , 3, 4.The HIPK family members 1 to 3 are structurally similar, with HIPK4 being remotely related to them. HIPK1 to 3 were originally characterized as molecules that interact with, homeobox transcription factors NKx-1.2. Approximately 90% of the amino acid sequences that make up their kinase domains are conserved across HIPK1-3. Additionally, the architecture of their noncatalytic regions is also conserved , 6. HIPKHIPK2, a nuclear body localized 1189-amino-acid protein, belongs to the DYRK serine/threonine homeodomain-interacting kinase family . HIPK2 cIt has been reported that HIPK2 is a tumor-inhibiting factor and DNA damage monitoring kinase by promoting apoptosis through targeting p53 and its family members, p73 and p63 , anti-apvia modulating expression of certain genes that impact growth arrest, senescence, apoptosis and DNA repair ..90].Upon sensing UV damage, HIPK2 is activated and causes Ser46 phosphorylates of p53, which next facilitates its acetylation at lysine 382, resulting in p53-dependent apoptosis . After eHIPK2 is activated by various anti-cancer drugs, including cisplatin (CDDP), adriamycin (ADR) and roscovitin, to form HIPK2/p53Ser46 apoptotic signaling pathway. Therefore, HIPK2 is the key factor of p53 activity in response to chemotherapeutic drugs . In chemIn addition to DNA damaging agents, HIPK2 can be activated by zinc in hypoxia and in chemoresistance. Zinc restored protein levels of HIPK2 and the chemo-sensitivity in cancer cells . Zinc upThe reason Traditional Chinese medicine comes to our attention in this review is because of a study revealing inhibition of HIF-1 and S100A4 by saponin extracts of ginsenoside (Ginsen) and Gynostemma, as well as coix polysaccharides, resulting in the suppression of cancer cell migration and invasion . VerbascHIPK2 is a multi-functional signaling molecule and a tumor suppressor that mediates growth, regulation and apoptotic cellular responses. HIPK2 induces cell death by activating p53-dependent , 31, 32


Our data revealed that DGLHD normalized glucose and insulin level, increased the expression of adiponectin, diminished fat accumulation and lipogenesis, and promoted glucose uptake. Metabolomic analysis also demonstrated that DGLHD decreased isoleucine, adenosine, and cholesterol, increased glutamine levels in liver and visceral adipose tissue (VAT) of ob/ob mice. Importantly, DGLHD promoted the shift of pro-inflammatory to anti-inflammatory cytokines, suppressed T lymphocytes proliferation, and enhanced regulatory T cells (Tregs) differentiation. DGLHD also inhibited dendritic cells (DCs) maturation, attenuated DCs-stimulated T cells proliferation and secretion of IL-12p70 cytokine from DCs, and promoted the interaction of DCs with Tregs. Further studies indicated that the changed PI3K/Akt signaling pathway and elevated PPAR-γ expression were not only observed with the ameliorated glucose and lipid metabolism in adipocytes and hepatocytes, but also exhibited in DCs and T cells by DGLHD. Collectively, our results suggest that DGLHD exerts anti-insulin resistant and antisteatotic effects by improving abnormal immune and metabolic homeostasis. And DGLHD may be a novel approach to the treatment of obesity-related insulin resistance and hepatic steatosis.Dangguiliuhuang decoction (DGLHD) is a traditional Chinese medicine (TCM) formula, which mainly consists of angelica, radix rehmanniae, radix rehmanniae praeparata, scutellaria baicalensis, coptis chinensis, astragalus membranaceus, and golden cypress, and used for the treatment of diabetes and some autoimmune diseases. In this study, we explored the potential mechanism of DGLHD against insulin resistance and fatty liver  Obesity is defined as a pathological condition associated with numerous health problems and a reduced life expectancy, reaching epidemic proportions worldwide -1, peroxisome proliferator activated receptor (PPAR)-γ, and phosphatidyl inositol 3-kinase (PI3K)/protein kinase B (Akt) signaling Ye, . Impairmin vivo pathophysiologic setting.However, inflammation is not the only mechanism of insulin resistance and fatty liver in obesity, various abnormalities of lipid metabolism can also impair insulin signaling. These lipotoxic effects include the production of intracellular lipid mediators, such as diacylglycerols, as well as more direct effects of circulating saturated fatty acids (SFAs)   and eosinophils) and pro-inflammatory cells , neutrophils, CD8While, accumulating evidence suggests that the functional status of DCs is involved in the promotion of Tregs differentiation  formulae, often composed of mixtures of biologically active compounds, have a medical practical history for more than 2000 years, and are widely used for a variety of diseases  analysis and biological studies, the 0.13 g/ml nominal concentration decoction was filtered (0.2 μm), sterilized, and diluted or concentrated in the proper buffer or medium to the required final concentration.Dangguiliuhuang decoction (DGLHD) was provided by the Department of Traditional Chinese Medicine, the Central Hospital of Wuhan. According to the 2015 Edition of the Chinese pharmacopeia, the formula contains angelica sinensis, radix rehmanniae, radix Quality control of DGLHD was analyzed by HPLC on a reverse-phase analytical column . The mobile phase was made up of (A) 100% water with 0.1% phosphoric acid and (B) 100% acetonitrile. Gradient elution from 5 to 45% B for 40 min was used at 10 min intervals. The flow rate was 1 mL/min and the detection wavelength was 280 nm. Chemical standards included calycosin-7-glucoside, verbascoside, ferulic acid, coptisine, berberine, baicalin, wogonoside, baicalein, and wogonin purchased from the National Institutes of Food and Drug Control . Their purity was verified by HPLC. And GW9662 (PPAR-γ antagonist) was purchased from MedChem Express .2.3T3-L1 preadipocytes and HepG2 cells were obtained from Cell Center of Tongji Medical College. They were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum , 100 U/ml penicillin, and 100 ug/ml streptomycin  at 37°C and 5% COTo yield insulin-resistant 3T3-L1 adipocytes, preadipocytes were induced into mature adipocytes by incubation in DMEM containing 10 μg/mL insulin, 1 μM dexamethasone (DEX), and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h. Subsequently, 3T3-L1 adipocytes were cultured in DMEM with 10 μg/mL insulin and 1.0 μM DEX for 96 h. For establishing a steatosis-like phenotype, HepG2 cells were incubated in DMEM medium with 1 mM oleic acid and 1 mM palmitic acid for 24 h.ob/Lepob (ob/ob) mice and male C57BL/6J mice were purchased from Beijing Huafukang Bio-Technology Co., Ltd., and maintained at the experimental animal center of Tongji Medical College  under specific pathogen-free conditions. The animals were housed at 23 ± 2°C in a 12 h light/dark cycle with free access to standard diet and water.Animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Male LepObesity-related insulin resistance and hepatic steatosis were evaluated in spontaneously obese ob/ob mice. Either a solution of DGLHD  or an equivalent volume of water were administered intragastrically to ob/ob mice once a day for 8 weeks. A Tregs neutralizing mouse model was established by injecting intraperitoneally LE/AF-purified anti-mouse CD25 antibody  into ob/ob mice, at a dose of 125 μg/mouse once a week for 3 weeks. To investigate adverse effects of DGLHD, C57BL/6J mice were treated with water or DGLHD (6.0 g/kg) by intragastrical gavage for 8 weeks.Body weights and mean food consumption of ob/ob mice in each group were monitored once a week for 8 weeks. After the prescribed time period, mice were fasted overnight and then operated appropriately for tissues and serum.Oral glucose tolerance tests (OGTT) and insulin tolerance tests(ITT)were performed after ob/ob mice received DGLHD or water for 8 weeks. For the OGTT, mice were fasted for 16 h and given D-glucose (2 g/kg) intragastrically. Blood glucose was then measured at 0, 15, 30, 60, 90, and 120 min after glucose administration using a Contour blood glucose meter . For ITT, mice were fasted for 6 h, then injected intraperitoneally with insulin (1 U/kg). Blood glucose was then measured at 0, 15, 30, 60, 90, and 120 min after insulin injection.Liver and VAT were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Hepatic lipid content was also determined by Oil Red O staining. Steatosis areas were quantified by histomorphometry using a computerized image analysis system. Steatosis score was assigned under double-blinded conditions and the degree of steatosis was determined. Scores of 0, 1, 2, and 3 corresponded to steatosis areas of <5, 5–33, 33–66, and >66%, respectively , triglyceride (TG), and total cholesterol (TC) in serum, liver and HepG2 cells were measured by commercial detection kits  according to the manufacturer's protocols. Glucose levels in the cell suspension were determined using the glucose oxidase method.Serum from DGLHD-treated ob/ob mice and control mice, and supernatant from T cells, DCs, and 3T3-L1 and HepG2 cell lines—with or without DGLHD treatment—were reserved at −80°C until quantification. The concentrations of interleukin (IL)-10, transforming growth factor (TGF)-β1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-12p70, insulin, and adiponectin were measured by commercial ELISA kits .Dendritic cells (DCs) derived from bone marrow were prepared as reported previously  was used to assess the immunostimulatory function of DCs and was estimated using a 3--2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. In brief, DCs from ob/ob mice as stimulators were treated with 0.5 mg/mL mitomycin C to prevent proliferation. Then DCs and allogeneic T cells were co-cultured at the ratio of 1:10 for 48 h.T cells were isolated from spleen by anti-CD4 immunomagnetic beads  and cultured in RPMI 1640 supplemented with 10% FBS. T cells were stimulated with anti-CD3 antibody (5 μg/ml) and anti-CD28 antibody (5 μg/ml) in the absence or presence of DGLHD for 3 days. T lymphocyte proliferation was evaluated by MTT assay at 490 nm.Cell suspensions were prepared from spleen, lymph nodes, VAT, or liver of ob/ob mice as previously described  buffer, and protein concentration was quantified with a BCA Protein Quantitation Kit . Lysate samples were separated by SDS-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% non-fat milk, and incubated with indicated antibodies overnight at 4°C. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were applied for 1–2 h at room temperature, prior to detection with an enhanced chemiluminescence kit. Polyclonal antibodies to anti-PI3K, anti-p-PI3K (Ser 192), anti-Akt, anti-p-Akt (Thr 183/Tyr 185), and anti-PPAR-γ were purchased from Cell Signaling Technology . The amount of protein expression was normalized by β-actin.Quantitative real-time PCR (qRT-PCR) was performed as described previously -administrated mice—a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) system was applied. The samples were pre-processed before detection according to previous report  analysis identified four major compounds in the DGLHD decoction: 75.17 mg/L berberine, 160.70 mg/L baicalin, 86.53 mg/L wogonoside, and 49.45 mg/L wogonin Figure .The ob/ob mouse carries a spontaneous mutation in the leptin gene, which results in a leptin deficiency, subsequently leading to hyperphagia, inactivity, obesity, insulin resistance, and hepatic steatosis , aromatic amino acids, and purines, as well as cholesterol and glucose Figure . Other cIn order to characterize the metabolic alterations effected by DGLHD in more detail, we further conducted pathway-enhancement analysis for different metabolites. We found that treatment of ob/ob mice with DGLHD strongly influenced pathways in adipose tissue that control levels of ABC transporters, metabolism of glycine, serine, threonine, arginine, alanine, glutamate, phenylalanine, and purines, and the biosynthesis of aminoacyl-tRNA Figure . In liveGiven that obesity is considered as a kind of chronic low-grade inflammation characterized by increased cytokine levels in the circulation and at local sites , fatty acid synthase (FAS), and cluster of differentiation 36 (CD36), and sterol regulatory element-binding protein-1 (SREBP-1)—is elevated in adipose and hepatic tissues of obese mice (Hardy et al., in vivo, we examined the direct effects of DGLHD on insulin resistance and steatosis of adipocytes and hepatocytes in vitro. In insulin-resistant 3T3-L1 adipocytes, we observed that DGLHD increased glucose uptake, adiponectin levels, PI3K/Akt activation, and attenuated lipid biosynthesis (Figure Considering that adipose and liver are major determinants of lipid and glucose metabolism s Figure . DGLHD ws Figure . In HepGs Figure . All of Obesity and its metabolic abnormalities disrupt immune homeostasis, increase inflammatory cell infiltration, and reduce insulin sensitivity, leading to insulin resistance and liver steatosis; at the same time, persistent inflammation with excessive insulin secretion often results in β cell destruction (Lee and Lee, The main chemical components in DGLHD detected by HPLC are baicalin, berberine, wogonoside, and wogonin. In TCM, baicalin is employed to treat inflammation, hypertension, cardiovascular diseases, and bacterial and viral infections. Recent studies also indicate that baicalin improves glucose tolerance and insulin resistance, enhances insulin sensitivity, and attenuates lipid deposition, the mechanisms of which are related to the activation of Akt/GSK-3β, Akt/Glut4, and PGC1α/Glut4 pathway as well as enhanced PPAR-γ and p-Akt expression (Xi et al., in vivo and in vitro, the latter of which benefits for improved insulin resistance and steatosis (Tilg and Moschen, Here, treatment with DGLHD decreased the extent of fat accumulation and improved systemic and local metabolism, as evidenced by lowered fat content in liver and reduced levels of TC, TG, FFA, and increased adiponectin Consistent with the genes related with lipid biosynthesis regulated by DGLHD, the metabolites in liver and VAT were also impacted. Specific alterations in metabolite levels may be markers of the progression of diseases involving altered insulin sensitivity. For example, changes in glycine and glutamine levels in plasma correlated negatively with insulin resistance and an increased glutamine/glutamate ratio in plasm predicted a reduced risk of diabetes (Cheng et al., Inflammation and immune response are closely linked to altered metabolic homeostasis in obesity-induced insulin resistance and liver steatosis Ye, . Our resImmature DCs typically express low levels of MHC-II and costimulatory molecules to promote T cell anergy and Tregs generation, both of which are important for the maintenance of immune homeostasis (Hubert et al., Tregs are characterized by their ability to suppress proliferation of effector T cells and induce T cell anergy, which may be beneficial for the improvement of insulin resistance and hepatic steatosis (Ilan et al., Immune cell activation including DCs and Tregs, is coupled to profound changes in metabolism of them, and these metabolic alterations modulate their fate and function (Everts and Pearce, As mentioned above, the enhancement of immature DCs and Tregs initiated by DGLHD is closely associated with the activation of PPAR-γ. PPAR-γ is a key transcription factor controlling metabolism, which negatively affects the maturation of DCs (Kim et al., It has been reported that oral DGLHD 15.6 g/day/per subject significant attenuated dental ulcers and gastrointestinal discomfort in patients (Zheng and Quan, In summary, DGLHD exerts beneficial effects on immune and metabolic balance through regulating PI3K/Akt pathway or PPAR-γ, playing potent anti-inflammatory, anti-insulin resistant, and anti-steatotic effects in ob/ob mice. DGLHD directly suppressed the activation and function of DCs, and promoted Treg generation and DCs-Treg interactions in ob/ob mice with insulin resistance and steatosis by altering their metabolism. DGLHD also favorably modulated metabolites level associated with the development of insulin resistance in VAT and liver. These results provide experimental evidence for the therapeutic utility of DGLHD as an agent against insulin resistance and hepatic steatosis in humans. Clearly identifying the active components in the DGLHD concoction is a subject of future investigations.Parts of this study has been submitted in abstract form at IMMUNOLOGY 2017™, Washington, D.C., May 12-16, 2017.HC drafted the manuscript and prepared the figures. HC and LT performed experiments, analyzed and interpreted the data. YT, ZX, and RF performed experiments. YL, YQ, JL, and ZY provided suggestions and material support. MX designed the study, obtained the funding, supervised the whole project and reviewed 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.


U2AF1 mutation was significantly associated with non-response to azacitidine, which was consistent in multivariate analysis . Regarding overall survival, mutations in DNMT1 (P=0.031), DNMT3A (P=0.006), RAS (P=0.043), and TP53 (P=0.008), and two clinical variables  were independent predicting factors of poor prognosis. For AML-free survival, mutations in DNMT3A (P<0.001), RAS (P=0.001), and TP53 (P=0.047), and two clinical variables  were independent predicting factors of poor prognosis. By combining these mutations and clinical predictors, we developed a quantitative scoring model for response to azacitidine, overall- and AML-free survival. Response to azacitidine and survival rates became worse significantly with increasing risk-scores. This scoring model can make prognosis prediction more reliable and clinically applicable.Although hypomethylating therapy (HMT) is the first line therapy in higher-risk myelodysplastic syndromes (MDS), predicting response to HMT remains an unresolved issue. We aimed to identify mutations associated with response to HMT and survival in MDS. A total of 107 Korean patients with MDS who underwent HMT (57 responders and 50 non-responders) were enrolled. Targeted deep sequencing  was performed for 26 candidate MDS genes. In multivariate analysis, no mutation was significantly associated with response to HMT, but a lower hemoglobin level  and low platelet count  were independent markers of poor response to HMT. In the subgroup analysis by type of HMT agents,  Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms that are defined by clonal stem cell disorders and characterized by ineffective hematopoiesis and an increased risk of progression to acute myeloid leukemia (AML) . This syVarious prognostic scoring systems have been developed to estimate prognosis which can support decision making for selecting therapeutic options . Among tTET2, DNMT3A and ASXL1 mutations have been reported to be associated with treatment response in MDS patients  and secondary AML) and CMML, which suggests their roles in disease progression. Similarly, in this study, SETBP1 mutations were identified especially in high-risk MDS (two of RAEB-2 and one of CMML).ding MDS , 24, 29,TP53, EZH2, RUNX1, ETV6, ASXL1, SRSF2, U2AF1, and SF3B1 have shown prognostic relevance [ASXL1, TP53, and PTPN11 were suggested as markers of inferior survival, whereas SF3B1 mutation was a marker of favorable survival [TP53, TET2, and DNMT3A were predictors of poorer survival [TP53, RAS (KRAS and NRAS), DNMT1, and DNMT3A were independent predictors of poor survival after HMT. Taken our results and previous reports together, TP53 and DNMT3 mutations seem to be associated with poorer survival, but the prognostic roles of mutations in other genes, such as DNMT1, RAS, and TET2 remain to be validated.Regarding the effects of genetic mutations on survival, mutations in elevance , 30–33 asurvival , 13, 34.survival . Our stuBy merging significant genetic and clinical factors, we developed risk scoring systems for predicting response to azacitidine and survival. Response to azacitidine and survival rates became worse significantly with increasing risk-scores, suggesting that this scoring model can predict the treatment outcomes in more detailed fashion. However, we could not perform an independent validation of our model in this study. Further studies with larger independent samples will be required to validate its clinical validity and applicability.U2AF1 which was associated with poorer response to azacitidine. Furthermore, the mutations in TP53, RAS, DNMT3A, and DNMT1 were identified as independent predictors of poorer OS, and DNMT3A, TP53 and RAS mutations as predictors of poorer AFS following HMT. Based on our findings, we developed a quantitative scoring model for response to HMT and survival, which can make prognosis prediction more reliable and clinically applicable.In summary, this study identified the independent molecular markers for the prediction of response to HMT or survival following it. We discovered a hotspot mutation of the A total of 107 Korean patients with MDS including CMML were enrolled in this study. The clinical and molecular variables were identified prior to HMT. All patients were treated with one of two HMAs: azacitidine (n=66) or decitabine (n=41). The median age of the cohort was 59 (range 23-76 years). All patients were recruited from the Asan Medical Center  and Seoul St. Mary's Hospital . We only included the patients whose pre-HMT bone marrow (BM) samples were available to avoid potential bias from molecular aberrations acquired during and after HMT. The baseline characteristics and treatment outcomes are shown in Table DNMT3A, TET2, EZH2, RUNX1, ASXL1, STAG2, CBL, TP53, SRSF2, SF3B1, U2AF1, LAMB4, DNMT1, ETV6, KRAS, NF1, NPM1, NRAS, PRPF8, IDH1, IDH2, JAK2, FLT3, SETBP1, ATRX, and ZRSR2) were analyzed by targeted deep sequencing in the 107 MDS genomes (57 responders and 50 non-responders). In brief, sequencing libraries were generated using AmpliSeq Library Kit 2.0 with a customized target panel  according to the manufacturer's instructions. This customized panel consists of 1,088 amplicons covering 98.4% of all coding exons in 26 target genes. Sequencing was performed on a P1 chip on the Ion Torrent Proton (Life Technologies) according to the manufacturer's instructions. Sequencing reads were aligned to GRCh37/hg19 and genomic variants were called using the Torrent Suite 4.2. The information of sequencing alignments  were summarized in A total of 26 well-known genes in MDS (P less than 0.05 were considered significant in all statistical analyses.Categorical variables were compared using the Fisher's exact test or chi-square test as appropriate, and continuous variables were compared using the Student's t test. For survival analysis, time-to-event was defined as duration from the date of HMT to the date of death from any cause OS or the date of AML progression AFS. In univariate survival analysis, survivals were calculated according to the Kaplan-Meier method. Differences in survival curves were assessed with the log-rank test. Stepwise multiple logistic regression and Cox proportional hazards models were used for multivariate analysis.


CLOCK rs3749473 (p = 0.0017), NPAS2 rs17655330 (p = 0.0013), RORA rs13329238 (p = 0.0009), and RORB rs10781247 (p = 7.9 × 10−5). We also found that interactions between CLOCK rs3749473, NPAS2 rs17655330, RORA rs13329238, and RORB rs10781247 affected cognitive aging (p = 0.007). Finally, we investigated the influence of interactions between CLOCK rs3749473, RORA rs13329238, and RORB rs10781247 with environmental factors such as alcohol consumption, smoking status, physical activity, and social support on cognitive aging (p = 0.002 ∼ 0.01). Our study indicates that circadian clock genes such as the CLOCK, NPAS2, RORA, and RORB genes may contribute to the risk of cognitive aging independently as well as through gene-gene and gene-environment interactions.Previous animal studies have indicated associations between circadian clock genes and cognitive impairment . In this study, we assessed whether 11 circadian clockgenes are associated with cognitive aging independently and/or through complex interactions in an old Taiwanese population. We also analyzed the interactions between environmental factors and these genes in influencing cognitive aging. A total of 634 Taiwanese subjects aged over 60 years from the Taiwan Biobank were analyzed. Mini-Mental State Examinations (MMSE) were administered to all subjects, and MMSE scores were used to evaluate cognitive function. Our data showed associations between cognitive aging and single nucleotide polymorphisms (SNPs) in 4 key circadian clock genes,  ARNTL), clock circadian regulator (CLOCK), cryptochrome circadian clock 1 (CRY1), cryptochrome circadian clock 2 (CRY2), neuronal PAS domain protein 2 (NPAS2), nuclear receptor subfamily 1 group D member 1 (NR1D1), period circadian clock 1 (PER1), period circadian clock 2 (PER2), period circadian clock 3 (PER3), RAR related orphan receptor A (RORA), and RAR related orphan receptor B (RORB) genes [ARNTL encodes the ARNTL protein, which is an elementary helix-loop-helix protein that forms heterodimers with either CLOCK or NPAS2, two other primary helix-loop-helix proteins [PER1, PER2, PER3, CRY1, and CRY2 via E-box elements in the promoters of the target genes [ARNTL gene and thus contribute to the timing of the core clock machinery [Circadian rhythms are naturally recurring cycles that influence the timing of biological events such as sleep-wake cycles, hormone release, and energy metabolism . The intArntl [Clock [Cry1 [Cry2 [Npas2 [Per2 [ARNTL and CLOCK genes are associated with AD risk [Previous works have shown that the circadian clock is involved in regulation of brain cognitive functions such as memory, mood, and adaptation to novelty –6. FurthArntl , Clock [l [Clock , Cry1 [82 [Npas2 , and Pers2 [Per2 . In addis2 [Per2 . Therefos2 [Per2 , 12. Spe AD risk –16.ARNTL, CLOCK, CRY1, CRY2, NPAS2, NR1D1, PER1, PER2, PER3, RORA, and RORB genes.While several encouraging findings on the relationship between circadian clock genes and cognitive aging have emerged, to our knowledge, human data is scarce in terms of SNPs. Moreover, the interplay between circadian clock genes and environmental factors such as alcohol consumption, smoking status, physical activity, and social support, has not been comprehensively evaluated in previous association studies. Given that circadian rhythms and their relevant genes may play a key role in the development of cognitive aging, we hypothesized that core circadian clock genes may contribute to the etiology of cognitive aging independently and/or through complex interactions. The gene panel investigated here comprises the 11 aforementioned core circadian clock genes , namely Table P < 0.05) with MMSE scores (Table RORB rs10781247 SNP with MMSE scores nearly reached significance after applying a Bonferroni correction (P < 0.05/644 = 7.8 × 10−5). As shown in Table RORB rs10781247 SNP indicated an association with MMSE scores among subjects after adjusting for covariates such as age, gender, and education in genetic models, including the additive model (P = 0.0021) and dominant model (P = 7.9 × 10−5).First, we investigated the association between cognitive aging and the 11 circadian clock genes mentioned above. Among the 644 SNPs assessed in this study , there wes Table . HoweverCLOCK rs3749473, NPAS2 , RORA , and RORB rs2273975 (Table CLOCK rs3749473 (P = 0.0017), NPAS2 rs17655330 (P = 0.0013), RORA rs13329238 (P = 0.0009), and RORB rs10781247 (P = 7.9 × 10−5). The genotype frequency distributions of these four SNPs were in accordance with the Hardy–Weinberg equilibrium among the subjects .Next, we identified a nominal association of MMSE scores with 10 SNPs, including NPAS2 rs17655330 and RORB rs10781247 (P = 0.003), RORA rs13329238 and RORB rs10781247 (P = 0.003), NPAS2 rs17655330 and RORA rs13329238 (P= 0.004), and CLOCK rs3749473 and RORA rs13329238 (P = 0.042). These results indicate potential gene-gene interactions between NPAS2 and RORB, RORA and RORB, NPAS2 and RORA, and CLOCK and RORA, respectively, in influencing cognitive aging. Additionally, there were a three-way model (P = 0.001) and a four-way model (P = 0.007) indicating potential gene-gene interactions among CLOCK, NPAS2, RORA, and RORB in influencing cognitive aging.We then employed categorized MMSE scores as an outcome  for gene-gene and gene-environment analysis. First, generalized multifactor dimensionality reduction (GMDR) analysis was used to assess the impacts of combinations between the four key SNPs in cognitive aging, including age, gender, and education as covariates. Table CLOCK rs3749473 and environmental factors, namely smoking (P = 0.004), alcohol consumption (P = 0.01), and social support (P = 0.011), indicating potential gene-environment interactions between CLOCK and environmental factors in influencing cognitive aging. Similarly, there was a significant two-way model involving RORA rs13329238 and environmental factors such as smoking (P = 0.01), alcohol consumption (P = 0.002), physical activity (P = 0.01), and social support (P = 0.002). Finally, there was a significant two-way model involving RORB rs10781247 and the environmental factors alcohol consumption (P = 0.005) and physical activity (P = 0.035). However, there was no significant two-way model involving NPAS2 rs17655330 and environmental factors.Table CLOCK, NPAS2, RORA, and RORB genes. Table CLOCK, NPAS2, RORA, and RORB genes.Finally, Table CLOCK rs3749473, NPAS2 rs17655330, RORA rs13329238, and RORB rs10781247, may play an important role in the modulation of cognitive aging in old adults in a Taiwanese population. Notably, the association of the RORB rs10781247 SNP with MMSE scores nearly persisted after correcting for multiple testing (P < 7.8 × 10−5). Additionally, our data revealed that gene-gene interactions between CLOCK, NPAS2, RORA, and RORB may contribute to the etiology of cognitive aging. Our data also indicated that there were gene-environment interactions between CLOCK, RORA, and RORB with environmental factors such as alcohol consumption, smoking status, physical activity, and social support.To our knowledge, our study is the first to date to assess whether 644 SNPs in 11 circadian clock genes are significantly associated with the risk of cognitive aging independently and/or through gene-gene interactions among old Taiwanese individuals. We also examined the relationship between these genes and environmental factors to investigate whether these genes confer a risk of cognitive aging according to its effect on gene-environment interactions. Here, we report for the first time that several SNPs of the circadian clock genes, including RORA gene and 6 SNPs in the RORB gene may contribute to susceptibility to cognitive aging, especially the RORA SNPs rs13329238, rs8040450, rs11638929, rs17237563, rs17303341, rs146660446, and the RORB SNPs rs10781247 and rs2273975. 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 circadian rhythm, immunity, cellular metabolism, embryonic development, and inflammatory responses [RORB gene and a test of verbal intelligence [RORA gene is distinctly up-regulated in the hippocampi of AD-affected postmortem human brains, a brain region key to memory and learning, indicating a potential link between RORA and AD [RORA gene was reported to increase the risk of acquiring psychiatric and neurological disorders, including bipolar disorder [RORB gene was also previously linked to psychiatric and neurological disorders, such as bipolar disorder [To our knowledge, our results are the first to raise the possibility that 42 SNPs in the esponses . By usinlligence . ConsistA and AD . In termdisorder , and posNPAS2 gene, especially rs17655330 and rs62152925. The NPAS2 gene, located on chromosome 2q11.2, encodes a member of the basic helix-loop-helix family of transcription factors. The functions of the NPAS2 and CLOCK proteins are partially redundant [CRY1, CRY2, PER1, PER2, and PER3 gene expression [Npas2-deficient mice may have impaired brain function and that NPAS2 may activate the neuronal gene expression required for the acquisition of long-term memory, indicating its importance for the execution of complex cognitive tasks [In addition, an intriguing finding was a positive association of cognitive aging with 19 SNPs in the CLOCK gene, particularly CLOCK rs3749473. The CLOCK gene is located on chromosome 4q12 and encodes a basic helix-loop-helix protein that constitutes the ARNTL/CLOCK heterodimeric protein with ARNTL, another basic helix-loop-helix protein [CLOCK gene  with AD in Chinese populations [Clock gene showed declines in cognitive performance, including increased rearing activity and impaired intersession habituation [On another note, we also observed that there was an association of cognitive aging with 3 SNPs in the  protein . Evidenculations –15; howeCLOCK, NPAS2, RORA, and RORB in influencing cognitive aging by using the GMDR approach. To our knowledge, no other study has been conducted to evaluate gene-gene interactions between these genes. The functional relevance of the interactive effects between CLOCK, NPAS2, RORA, and RORB on cognitive aging remains to be elucidated. At the molecular level, the circadian clock consists of an interlocking network of several positive and negative feedback loops [Clock/Clock mutant mice demonstrated that CLOCK is not indispensable for circadian rhythms, suggesting that NPAS2, as a substitute for CLOCK, can contribute to circadian rhythms in the absence of CLOCK [Arntl, Cry1, Nr1d1, and Per2 genes [RORA, RORB, and NR1D1 genes associated with an increased risk of bipolar disorder by using the multifactor dimensionality reduction method [Remarkably, another intriguing finding was that we further inferred the epistatic effects between ck loops . While tck loops . Additiock loops . Previouof CLOCK , 28. By r2 genes . Furthern method . These sCLOCK gene and environmental factors , between the RORA gene and environmental factors , and between the RORB gene and environmental factors . This interplay may manifest itself functionally through epigenetic changes. In previous animal studies, circadian deregulation was shown to affect epigenetic parameters such as DNA methylation, histone modifications, and miRNAs, suggesting that epigenetic alterations might be linked with age-related cognitive decline [In the GMDR analysis of gene-environment interactions, we tracked down the interplay between the  decline –31. It hP < 0.05) between cognitive aging and four other genes, the ARNTL, CRY2, PER2, and PER3 genes, which are located on chromosomes 11p15, 11p11.2, 2q37.3, and 1p36.23, respectively. The ARNTL gene encodes a basic helix-loop-helix protein that forms the ARNTL/CLOCK heterodimeric protein with CLOCK [CRY2 gene encodes a flavin adenine dinucleotide-binding protein that stimulates the circadian clock [PER2 and PER3 genes are members of the Period family, which encodes components of the circadian rhythms of locomotor activity, metabolism, and feeding behavior [ARNTL gene in a Chinese population was previously observed [PER2 and PER3 genes in a Brazilian population [CRY2 gene. It is worth mentioning that multiple potential defects underly the discordant results found among these studies, including sample size, sample deviation, environmental control, study design, covariate adjustment, phenotype definitions, population stratification, and various ethnicities [In this study, we further found evidence of potential association , which includes questions based on the five domains of orientation, registration, attention and calculation, recall, and language. We analyzed MMSE as a continuous outcome, as well as according to categories based on previously defined MMSE thresholds : MMSE scARNTL, CLOCK, CRY1, CRY2, NPAS2, NR1D1, PER1, PER2, PER3, RORA, and RORB.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 , 51. SNPX2 goodness-of-fit test with 1 degree of freedom . In order to correct for multiple testing, we applied a conservative Bonferroni correction factor for the number of SNPs employed in the analysis. The criterion for significance was set at P < 0.05 for all tests. Data are presented as the mean ± standard deviation.In this study, we evaluated the association of the investigated SNP with MMSE scores by a general linear model using age, gender, and education as covariates , 33. TheTo investigate gene-gene and gene-environment interactions, we employed the generalized multifactor dimensionality reduction (GMDR) method . We test