Similarly, patients with non-truncating NF1 mutations displayed two- and six-fold higher risk of developing CHD : 1.1162–3.4814, p = 0.0193) and pulmonary valve stenosis , respectively. Noteworthy, all but one patient  with pulmonary valve stenosis, and 18/35 (51.4%) patients with other CHDs displayed Noonan syndrome (NS)-like features. Present data confirm the significant frequency of CHD in patients with NF1, and provide further evidence for a higher than expected prevalence of NF1 in-frame variants and NS-like characteristics in NF1 patients with CHD, particularly with pulmonary valve stenosis.The aim of this study was to assess the prevalence and type of congenital heart disease (CHD) and the associated mutation spectrum in a large series of patients with neurofibromatosis type 1 (NF1), and correlate the mutation type with the presence and subgroups of cardiac defects. The study cohort included 493 individuals with molecularly confirmed diagnosis of NF1 for whom cardiac evaluation data were available. CHD was reported in 62/493 (12.6%) patients. Among these patients, 23/62 (37.1%) had pulmonary valve stenosis/dysplasia, 20/62 (32.3%) had mitral valve anomalies, and 10/62 (16.1%) had septal defects. Other defects occurred as rare events. In this NF1 subcohort, three subjects carried a whole-gene deletion, while 59 were heterozygous for an intragenic mutation. A significantly increased prevalence of non-truncating intragenic mutations was either observed in individuals with CHD  or with pulmonary valve stenosis , when compared to individuals without CHD  ( Neurofibromatosis type 1 , also known as von Recklinghausen disease, is one of the most common autosomal dominant disorders with multisystem involvement. NF1 affects approximately 1/2000 live births .NF1 gene, which is located on chromosome 17q11.2, and contains 57 constitutive and three alternatively spliced exons [NF1 encodes neurofibromin, a protein with tumor suppressor function belonging to the family of GTPase activating proteins (GAPs). Neurofibromin is expressed in several cells, including neurons, and Schwann and glial cells, and has an important modulatory role in the control of cellular proliferation, differentiation, apoptosis, and migration. Indeed, promoting the conversion from active GTP-bound RAS to inactive GDP-bound RAS, neurofibromin negatively regulates RAS signaling [NF1 is caused by heterozygous mutations of the ed exons ,5. NF1 eignaling .NF1 gene mutations include intragenic mutations, and less than 10% are represented by large deletions encompassing the entire NF1 gene  and its flanking genomic regions at 17q11.2 [NF1 deletions, which are quite heterogeneous in terms of size and gene content, and may occur as either germline or post-zygotic mutations [The large majority 90–95%) of disease-causing  17q11.2 ,9. So fa0–95% of utations .NF1 gene. These patients usually have a milder phenotype characterized by CaLS, SF, and complete absence of any cutaneous, subcutaneous, or superficial plexiform NF [NF1 mutations, heterozygous variants in the SPRED1 gene, encoding another negative regulator of RAS–MAPK signaling, were reported in Legius syndrome, an autosomal dominant disorder that shows some similarities to NF1 but is less severe [Only a few clinically relevant genotype–phenotype correlations in NF1 are characterized thus far, since they are very difficult to identify because the mutation spectrum is broad, with the majority of individuals having rare/private mutations, the expression of the phenotype is extremely variable, and the disorder is progressive in nature. The first association involves patients heterozygous for germline type-1 large deletions. These patients have worse manifestations. Indeed, they frequently exhibit dysmorphic features, overgrowth/tall-for-age stature, significant delay in cognitive development, congenital heart disease (CHD), and a greater number of cutaneous, subcutaneous, and plexiform NFs as compared to the general NF1 population ,10. Moreiform NF ,12. The iform NF ,14, a geiform NF ,16. A foiform NF . Recentliform NF . Clinicas severe ,20.NF1 gene mutations, generally non-truncating variants—missense changes and in-frame deletions [NF1 and PTPN11, the major gene contributing to NS [NF1 is associated with a clinical phenotype that may overlap NS. Indeed, some NF1 mutations, e.g., c.2970–2972delAAT deletion [Previously, we observed an increased prevalence of non-truncating mutations in patients with neurofibromatosis Noonan syndrome , a condition characterized by a clinical phenotype with features overlapping NF1 and NS . The majeletions . In a ming to NS ,16, or ing to NS ,26,27,28deletion ,29 or mideletion , were redeletion , most ofdeletion ,32, was NF1 gene [The prevalence of CHD in individuals with NF1 is not well defined, ranging from 2% to 27%, based on medical histories reported to an international database ,34 or onNF1 gene ,10. AdulTo further evaluate the type and prevalence of CHD in NF1 and better define the clinical and molecular characteristics of NF1 patients with CHD, we retrospectively revised the prevalence and type of cardiac and non-cardiac features, as well the mutation spectrum, in a large series of patients with molecularly confirmed NF1, and correlated the mutation type with the presence and subgroups of CHD.n = 440) or to the Laboratory of Medical Genetics of Bambino Gesù Pediatric Hospital  (n = 53), and for whom cardiac evaluation data were available. Of these, 67 patients were clinically evaluated at the Medical Genetics Section of Bambino Gesù Pediatric Hospital , 120 patients were evaluated at the Department of Translational Medicine, Federico II University, Pediatric Section , and the remaining 306 subjects were evaluated at other medical centers in Italy. Clinical data were collected through a shared clinical questionnaire that included family history, sex, age at last follow-up, presence or absence of CaLS, SF, cutaneous and subcutaneous NFs, plexiform NFs, LN, OPG, skeletal malformations, cardiovascular malformations, learning and intellectual disability, cerebrovascular malformations, NS facial features, growth, thoracic anomalies, macrocephaly, urogenital anomalies, hypertension, and occurrence of neoplasms other than NFs or OPG. Informed consent was obtained from all patients or their legal guardians. Clinical and genetic analyses were conducted with the approval of the institutional review boards of the participating institutions (n° 315/18).The study cohort included a total of 493 individuals with molecularly confirmed diagnosis of NF1, who were referred for genetic testing because of a clinical suspect of the disease, either to the Molecular Diagnostic Laboratory of CSS-Mendel Institute  , combined with multiplex ligation-dependent probe amplification (MLPA) to screen for the presence of NF1 intragenic deletions/duplications, and WGDs. Parallel sequencing libraries were prepared using either the HaloPlexHS target enrichment kit  or the SureSelect target enrichment system , whereas sequencing was carried out on a MiSeq platform . Variant calling and data analyses were performed using an in-house implemented pipeline, as previously described [Mutation analysis was performed by either direct sequencing or parallel sequencing scan of all escribed . All varescribed .p-values. A p-value <0.05 was considered significant. The odds ratio, its standard error, and its 95% confidence interval (CI) were calculated according to Reference [Categorical variables were analyzed and compared using either the chi-square or Fisher’s exact test. Results are expressed as eference .The study cohort included 153 familial cases belonging to 130 apparently unrelated families, 168 sporadic cases, and 172 cases with unknown family history. In total, 217 patients were males and 276 were females. The average age at the time of the observation was 21 years and four months (range: four months to 64 years).NF1 variant. Of these, 158/493 (32.0%) had nonsense mutations, 130/493 (26.4%) carried frameshift indels, 74/493 (15.0%) subjects were heterozygous for splice site changes, and 6/493 (1.2%) exhibited intragenic CNVs, whereas 14/493 (2.8%) carried large deletions encompassing the entire gene. Finally, 111/493 (22.6%) subjects had missense variants or in-frame indels. Mutation analysis was performed on germline DNA, and only mutations in the classical splice sites or known to alter splicing were classified as splicing mutations; therefore, the prevalence of this type of mutation could be an underestimation since it is well established that many NF1 mutations alter splicing through non-canonical splicing sites [All 493 patients enlisted in the study were heterozygous for an ng sites . Among pp > 0.05). The most common CHD was PVS, which was diagnosed in 21/62 patients (33.9%). Among individuals with PVS, 15/21 (71.4%) were females and 6/21 (28.6%) were males. Pulmonic valve dysplasia (PVD) without pressure gradient accounted for 2/62 (3.2%) individuals. In one case, PVD was combined with aortic valve dysplasia.CHD was present in 62/493 patients (12.6%). Among these, 42/62 (67.8%) were females and 20/62 (32.2%) were males. The prevalence of males and females in patients with CHD was not statistically different from that observed in the total cohort  individuals, mostly represented by cases with mitral valve prolapse (MVP), mitral valve insufficiency (MVI), or both. Aortic valve insufficiency and polyvalvular dysplasia were observed in one case each . Taken together, valvular heart diseases accounted for 45/62 (72.6%) individuals in the present series. Septal defects were observed in 10/62 (16.1%) patients. Two patients had ASD, two had VSD, and six showed patent foramen ovale (PFO) with minimal left-to-right shunt. One of the patients with VSD had also PFO. Two patients had patent ductus arteriosus (PDA). Dextrocardia, transposition of the great arteries, tetralogy of Fallot, HCM, and lipomatous hypertrophy of the interatrial septum were seen in one patient each. Type and prevalence of CHDs in the study are summarized in NF1 mutation was significantly less represented among the patients with intragenic mutations without CHD   vs. truncating mutations: 37/368 (10.1%), odds ratio = 1.9713, 95% CI: 1.1162–3.4814, p = 0.0193)  compared to patients with intragenic mutations , but the difference was non-significant , likely because of the small number of patients with these NF1 lesions. Moreover, patients with CHD showed a significantly higher prevalence of NS-like facial features compared to patients without CHD (p < 0.00001), and a relatively higher, but non-significant prevalence of learning disabilities, macrocephaly, thoracic anomalies, and short stature, as well as a non-significant lower prevalence of plexiform neurofibromas  carried a WGD and 59/62 (95.2%) had an intragenic mutation. Among patients with intragenic mutations, 22/59 (37.3%) had non-truncating (in-frame) mutations. Remarkably, this type of .038502) a. Consis 0.0193) b. Indivip = 0.033785). Conversely, anomalies of the mitral valve were more frequent in individuals with truncating mutations  than in those with in-frame mutations , even though this difference was not significant (p = 0.064013) (see p > 0.05). However, when we grouped together patients with CHD other than PVS , e.g., patients with septal defects (ASD (n = 2), VSD (n = 2), PFO (n = 6)), conotruncal (TOF (n = 1)), or laterality (TGA (n = 1), dextrocardia (n = 1)) defects, and with cardiac hypertrophy (n = 1), and other cardiac defects (patent ductus arteriosus (PDA) (n = 2), lipomatous hypertrophy of the interatrial septum (n = 1)), we noticed a slightly higher, but non-significant, prevalence of in-frame changes  than in patients without CHD  (p > 0.05). Non-cardiac clinical characteristics of NF1 patients with and without CHD are reported in Of note, among patients with CHD, anomalies of the pulmonary valve were significantly more prevalent in patients with non-truncating mutations  compared with patients with truncating mutations  (NF1 mutations were found in 22/23 (95.7%) patients with pulmonary valve anomalies, whereas one patient harbored a WGD. Similar to NF1 patients with CHD and intragenic mutations, a significantly higher prevalence of non-truncating mutations was observed in patients with PVS  compared to patients without PVS  (p = 0.00185) (p = 0.0001)  individuals of the total cohort, and in 23/62 (37.1%) individuals with CHD. In two cases, the pulmonary valve anomaly was combined with an ASD. Among patients with pulmonary valve anomalies, 21 had PVS with or without dysplastic leaflets, whereas two showed pulmonary valve dysplasia without pressure gradient, associated with dysplasia of the aortic valve in one case. Intragenic 0.00185) c. Of not 0.0001) d.p = 0.002175). Non-cardiac clinical characteristics of NF1 patients with and without PVS are reported in Remarkably, all but one (19/20) patient with PVS had NS facial features. In addition to facial NS characteristics, NF1 patients with PVS showed also a higher, but non-significant prevalence of other NS-related features like learning disabilities, short stature, macrocephaly, and thoracic anomalies. Interestingly, even if lower than in patients with PVS, patients with CHD other than PVS  also had a high frequency of NS facial features compared with patients without CHD  , a repository for collecting comprehensive information on the clinical manifestations and natural history of neurofibromatosis , left heNF1 WGD by echocardiography identified three patients who developed HCM [NF1 mutations in HCM, conditional knock-out mice lacking the murine ortholog, Nf1, in myocardium, were shown to have normal embryonic cardiovascular development but marked cardiac hypertrophy, progressive cardiomyopathy, and fibrosis in the adult, together with hyperactivation of Ras and downstream pathways [One patient of the present series was reported to have isolated HCM, while two other individuals showed a mild septal hypertrophy in conjunction with either mitral valve insufficiency or polyvalvular dysplasia. Out of these three cases, one had a WGD and two showed a truncating mutation. Although HCM was not observed in patients from the NFFID database , this caoped HCM . Consistpathways . It is pSimilarly to that observed in the NNFID ,34 and oNF1 in-frame changes, the existence of other genotype–phenotype correlations cannot be excluded. Grouping together all patients with cardiac structural anomalies other than those affecting valves , we noticed that the frequency of NF1 in-frame changes was slightly higher compared with that of patients without CHD. The present cohort has limited statistical power to detect small associations; therefore, we cannot exclude that the same NF1 mutations that predispose to PVS could also predispose, to a lesser extent, to other CHDs .Although the most striking finding of this study was the association between PVS and NF1 mutations was previously described by our group [NF1 in-frame mutations, together with the increased incidence of NS-like related features in NF1 patients with PVS, favors the hypothesis that some NF1 specific mutations may predispose more toward an NS-like phenotype rather than to a classic NF1 phenotype. Indeed, patients with NFNS generally tend to show less penetrance for neurofibromas, the typical hallmark of NF1 [NF1 missense mutations affecting p.Arg1809 [NF1 mutations may contribute in a similar manner to some aspects of the disease, e.g., neurofibroma development, but may have different roles in other aspects of the disease, e.g., predisposition to PVS or other NS-related features [PVS is the characteristic CHD of patients with NFNS . In the ur group  and otheur group ,47,48,49ur group . Considek of NF1 ,22. This.Arg1809 ,14, toge.Arg1809 ,22,29, a.Arg1809 , another.Arg1809 . In partfeatures .NF1 variants, whereas NF1 patients with mitral valve anomalies have a mutation spectrum overlapping that of the NF1 general population. This evidence confirms that all children with NF1 should have a careful cardiac examination, in particular those showing signs of NFNS and/or harboring mutations that are consistently associated with either NFNS and/or PVS. In parallel, current findings confirm the importance of persisting in searching for genotype–phenotype correlations in NF1, favoring the use of large and clinically well-characterized cohorts, as well as studies aimed at dissecting the effects of specific mutations on neurofibromin functioning.Present data confirm the significant frequency of CHD in patients with NF1, mostly represented by pulmonary valve stenosis and mitral valve anomalies, and more rarely by septal defects or other CHDs. Moreover, they clearly show that the majority of NF1 patients with PVS display NS-like characteristics and have a higher than expected frequency of in-frame


Sus scrofa) during the weaning stage is important to their disease susceptibility and later growth. Quantitative real-time PCR (RT-qPCR) is commonly used to screen for differentially expressed genes and, for accurate results, proper reference housekeeping genes are essential. Here we assessed the mRNA expression of 18 well-known candidate reference genes at different parts of the gastrointestinal tract (GIT) of piglets during the weaning process by RT-qPCR assay. GeNorm analysis revealed that B2M/HMBS/HPRT1 were the three most stable reference genes and GAPDH was the least stable gene in the duodenum, jejunum, ileum, colon, and whole GIT. BestKeeper analysis found that B2M/HMBS/PGK11, HMBS/B2M/HPRT1, B2M/HMBS/HSPCB, B2M/HPRT1/HMBS, and B2M/HMBS/HPRT1 were the most stable genes in the duodenum, jejunum, ileum, colon, and whole GIT, respectively, whereas GAPDH, B-actin, and 18S rRNA were the least stable genes at different parts of the GIT. To confirm the crucial role of appropriate housekeeping genes in obtaining reliable results, we analyzed the expression of ALP using each of the 18 reference genes to normalize the RT-qPCR data. We found that the expression levels of ALP normalized using the most stable reference genes (B2M/HMBS/HPRT1) differed greatly from the expression levels obtained when the data were normalized using the least stable genes . We concluded that B2M/HMBS/HPRT1 were the optimal reference genes for gene expression analysis by RT-qPCR in the intestinal mucosal development stages of piglets at weaning. Our findings provide a set of porcine housekeeping reference genes for studies of mRNA expression in different parts of the pig intestine.The intestinal mucosal development of piglets ( The weaning process is a widespread health concern in the swine industry ,2 becausB-actin (beta-actin), GAPDH , and 18S , are context-dependent because their expression levels may vary significantly with the experimental conditions being investigated  [To further assess the efficacy of the selected reference genes for normalization, the relative mRNA expression of a selected target gene , and differences were considered significant at P < 0.05.Normalization was performed with the 18 references genes and the relative mRNA expression of 2) ranged between 0.998 and 1.000  and B2M (P >0.05) were not significantly altered at the different parts of the GIT or at the different growth stages, whereas the other 16 reference genes (P <0.05) were significantly changed at the different parts of GIT and at different growth stages  would be sufficient to normalize the data across all the samples.Additionally, GeNorm determined the optimal number of reference genes needed to normalize RT-qPCR data on the basis of the average value of pairwise variations (Vn/n+1). In this study, when the cut-off M value was < 0.24, the inclusion of an additional reference gene did not further improve the normalization. On the basis of this threshold, the values of V3/4 values fell below 0.24 for different parts of the GIT Figs –7B and dB2M, HMBS, and PGK11 were the most stable reference genes with R values of 0.97, 0.92, and 0.89 respectively. In the jejunum, HMBS, B2M, and HPRT1 were the most stable with R values of 0.87, 0.81, and 0.62 respectively. In the ileum, B2M, HMBS, and HSPCB were the most stable with R values of 0.82, 0.82, and 0.77 respectively. In the colon, B2M, HPRT1, and HMBS were the most stable with R values of 0.79, 0.67, and 0.61 respectively. In the whole GIT, B2M, HMBS, and HPRT1 were the most stable with R values of 0.78, 0.70, and 0.59 respectively.The correlation of repeated pairwise variations was calculated to assess the optimal reference genes using BestKeeper. BestKeeper requires that the SD values of selected reference genes should not exceed 1.00, and the most stable reference gene also should have a Pearson coefficient of correlation (R) close to 1.00. PGK11/HPRT1/B2M , HPRT1/HMBS/TBP , HPRT1/PGK11/B2M , and B2M/HMBS/HPRT1  were the most stable reference genes at 0, 7, 14, and 21 dpw, respectively.At different growth stages , PGK11/HB2M, HMBS and HPRT1), and we then used ALP as a target gene to assess the reference genes by normalizing its expression against all the reference genes as well as the geometric mean of B2M/HMBS/HPRT1  instead of one individual reference gene  would be more sufficient to normalize the data across all the samples.According to the results of GeNorm and BestKeeper, the optimal number of reference genes in this study are three  and the geometric mean of B2M/HMBS/HPRT1  for ALP expression at the peak of relative expression at 0 dpw. In the ileum, compared with normalization against B-actin and 18S, the normalization against the geometric mean of B2M/HMBS/HPRT1, gave a much lower value for ALP expression at 14 and 21 dpw. In the colon, the ALP expression obtained by normalized against the geometric mean of B2M/HMBS/HPRT1 and B-actin was significantly lower than its expression normalized against 18S at 0 dpw. In the whole GIT, the peak of the ALP expression normalized against 18S and B-actin did not significantly change when compared with normalization against the geometric mean of B2M/HMBS/HPRT1. Thus, at different parts of the GIT, normalization against some commonly used reference genes produced dramatic differences in ALP expression that were not seen when normalized against the geometric mean of B2M/HMBS/HPRT1.Furthermore, we also assessed the relative mRNA expression of BS/HPRT1 . In the Sus scrofa  that were related to mRNA gene expression analysis using miscellaneous technologies were available in NCBI’s PubMed database , GAPDH , and 18S . Less than 25% of the publications used more than one endogenous control gene to assess gene expression, although normalization with a single housekeeping gene can produce false results [B2M, HMBS, and HPRT1 as the optimal housekeeping genes for normalization of RT-qPCR expression data of genes involved in the intestinal development of piglets because of their high expression abundance and high stability in different parts of the GIT .Because gene expression analysis by RT-qPCR assay has high specificity, accuracy, and is cost-effective, it is commonly used to identify genes associated with novel metabolic or signaling pathways involved in multiple biological processes in pig, which is a good model for human health. It is therefore critical to select appropriate housekeeping reference genes for RT-qPCR data normalization in gene expression analysis . A totaldatabase . In thes results . In thisB2M, is ubiquitously expressed in nucleated cells and is present in most biological fluids, such as urine, synovial fluid, and serum [B2M also plays multiple roles in the immune system of pigs [B2M was a commonly used reference gene in longissimus thoracis et lumborum muscle [HPRT1 is conserved across prokaryotes and eukaryotes and plays a vital role in the generation of purine nucleotides via the purine salvage pathway [HPRT1 is considered to be a somatic cell genetic marker with clinical significance in human disease, and is therefore one of the best characterized genes in human [HPRT1 is a frequently used housekeeping gene in different pig tissues, such as diaphragm, heart, lung [HPRT1 was the least stable housekeeping gene due to its inconsistent expression patterns [HMBS gene sequence is about 10-kb long, has 15 exons, and encodes a major protein involved in the heme biosynthetic pathway that regulates autosomal dominant disorders, such as acute intermittent porphyria [HMBS also stimulates transferase activity that catalyzes the conversion of guanine to guanosine monophosphate and hypoxanthine to inosine monophosphate via the transfer of the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate [HMBS was identified as the most stable housekeeping gene in the duodenum of piglets [nd serum ,24. Besim muscle  and duodm muscle , but it m muscle , left vem muscle , ovary [m muscle , and skem muscle . HPRT1 i pathway . HPRT1 iin human . HPRT1 irt, lung , skeletart, lung , right art, lung , and duort, lung . Howeverpatterns . The HMBorphyria . HMBS alhosphate . HMBS wa piglets , but was piglets  and uter piglets .GAPDH, B-actin, and 18S [GAPDH, B-actin, and 18S, have been shown to be significantly affected by biological processes, different experimental treatments, and different cell types and tissues [The most frequently used housekeeping genes,  and 18S , were no and 18S , Cinar e and 18S , Zhang e and 18S , Gessner and 18S , Li et a and 18S , and Wan and 18S , but con and 18S , Kuijk e and 18S , and Li  and 18S . Because tissues . The opt tissues , tissues tissues ,32,34, d tissues ,29, and  tissues ,15. RefeB2M/HMBS/HPRT1 were the optimal housekeeping genes in the duodenum, jejunum, ileum, and colon as well as the whole GIT. According to BestKeeper, B2M/HMBS/PGK11, HMBS/B2M/HPRT1, B2M/HMBS/HSPCB, B2M/HPRT1/HMBS, and B2M/HMBS/HPRT1 had the highest stability in the duodenum, jejunum, ileum, colon and the whole GIT, respectively. In addition to the GeNorm and BestKeeper analyses, we assessed the impact of normalization on the expression pattern of a target gene (ALP) using all the reference genes and the geometric mean. ALP is an important regulator involved in the primary digestive and absorptive functions of the small intestine and is expressed mainly in the intestinal mucosa of pig [ALP is involved in the hydrolysis of monophosphate esters and transcellular solute transport, and participates in the absorption of fat, which is crucial to gut mucosal structures and functions in weaned-piglets [ALP as the target gene to validate the reference genes. Intriguingly, the mRNA expression of ALP at different parts of the GIT, normalized against B2M, HMBS, HPRT1, and the geometric mean of all the reference genes, was similar at all post-weaning growth stages tested. However, compared with the results obtained by normalized against B2M, HMBS, HPRT1, and the geometric mean of all the reference genes, underestimation or overestimation of ALP expression occurred when the ALP RT-qPCR expression data were normalized to other commonly used reference genes, including GAPDH, B-actin, or 18S. Thus, substantial differences in the relative expression levels of ALP were obtained when different reference genes were chosen for normalization, indicating the need for accurate validation of reference genes.To validate the stability of the housekeeping genes we used GeNorm and BestKeeper. According to GeNorm, a of pig . ALP is -piglets . Because-piglets , we seleB2M/HMBS/HPRT1 were the optimal reference genes in duodenum, jejunum, ileum, and colon, as well as the whole GIT of pig, whereas other commonly used reference genes  were the least stable. Our results provide a set of optimal endogenous control genes for normalization of RT-qPCR expression data for intestinal studies of pig during the weaning stage.The results of the GeNorm and BestKeeper analyses indicated that S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 FigHMBS, 2: ALP, 3: 18S, 4: TBP, 5: RPL19, 6: PPARGC1A, 7: 5S, 8: CANX, 9: HSPCB, 10: B-actin, 11: UBC, 12: B2M, 13: HPRT1, 14: PGK1, 15: YWHAZ, 16: GAPDH, 17: PPIA, 18: RPL32, and 19: ALDOA.Agarose gel (2%) electrophoresis shows a band at the expected size for each reference gene. M: DNA Marker I (MD101) (100–600 bp), 1: (DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigA) Rank order of gene expression stability based on average expression stability values (M) for the reference genes from least stable (left) to most stable (right). B) Pairwise variation analysis (V) to determine the optimal number of reference genes for RT-qPCR data normalization.(DOCX)Click here for additional data file.S4 FigA) Rank order of gene expression stability based on average expression stability values (M) for the reference genes from least stable (left) to most stable (right). B) Pairwise variation analysis (V) to determine the optimal number of reference genes for RT-qPCR data normalization.(DOCX)Click here for additional data file.S5 FigA) Rank order of gene expression stability based on average expression stability values (M) for the reference genes from least stable (left) to most stable (right). B) Pairwise variation analysis (V) to determine the optimal number of reference genes for RT-qPCR data normalization.(DOCX)Click here for additional data file.S6 FigA) Rank order of gene expression stability based on average expression stability values (M) for the reference genes from least stable (left) to most stable (right). B) Pairwise variation analysis (V) to determine the optimal number of reference genes for RT-qPCR data normalization.(DOCX)Click here for additional data file.


Taiwania cryptomerioides Hayata. In this research endeavor, we studied the anti-cancer effect of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral squamous cancer cells (OSCC), and elucidated the underlying intricacies. OSCC were treated with Taiwanin E and analyzed through MTT assay, Flow cytometry, TUNEL assay, and Western blotting for their efficacy against OSCC. Interestingly, it was found that Taiwanin E significantly attenuated the cell viability of oral cancer cells (T28); however, no significant cytotoxic effects were found for normal oral cells (N28). Further, Flow cytometry analysis showed that Taiwanin E induced G1cell cycle arrest in T28 oral cancer cells and Western blot analysis suggested that Taiwanin E considerably downregulated cell cycle regulatory proteins and activated p53, p21, and p27 proteins. Further, TUNEL and Western blot studies instigated that it induced cellular apoptosis and attenuated the p-PI3K/p-Akt survival mechanism in T28 oral cancer cells seemingly through modulation of the ERK signaling cascade. Collectively, the present study highlights the prospective therapeutic efficacy of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral cancer.Taiwanin E is a bioactive compound extracted from  Over the years, complementary and alternative medicine therapies have garnered great attention all across the globe , 2. AccuTaiwania cryptomerioides Hayata) represents one of the most economically relevant plant species endemic to Taiwan. Numerous bioactive compounds have been derived from this plant species. Many of them have been demonstrated to exhibit potent activity against bacteria, fungi, termites, mites, and cancers  is the most common malignant epithelial neoplasm that can afflict the oral cavity. It is thought that more than 90% malignancies arising from the head and neck tissue section are OSCC . DespiteInterestingly, it was found that Taiwanin E significantly attenuated the cell viability of oral cancer cells (T28) in a dose- and time-dependent fashion; nevertheless, no cytotoxic effects were found for normal oral cells (N28). Moreover, it was observed that Taiwanin E induces G1 cell cycle arrest in T28 cells, as was evident through Flow cytometry studies, and, further, Western blot analysis suggested that Taiwanin E considerably downregulated cell cycle regulatory proteins and activated p53, p21, and p27 proteins. In addition, TUNEL staining showed that Taiwanin E induced apoptosis in T28 oral cancer cells. Furthermore, it was found that the cell survival proteins, such as p-PI3K, p-Akt, and the antiapoptotic protein Bcl-xL, were considerably reduced following treatment with Taiwanin E; nevertheless, the pro-apoptotic proteins, such as Bax, Cyt C, and c Cas 3, were, however, considerably enhanced. Further, understanding the underlying intricacies; mechanistically, it was found that Taiwanin E modulated the expression of ERK and resulted in cellular apoptosis in T28 oral cancer cells. Taken together, the data convincingly ascertained the promising candidature of Taiwanin E against oral cancer.All chemicals and reagents were procured from Sigma Aldrich Co.  unless otherwise mentioned.Taiwania cryptomerioides Hayata. The procedures for isolation, purification, and characterization of Taiwanin E was performed following our previously published reports with slight modifications .3.N28 and T28 cells were cultured in Dulbecco's minimum essential medium (D7777)  supplemented with 10% charcoal-treated FBS , 1% penicillin/ streptomycin , L-Glutamine, and NaHCOFor treatment, cells were seeded in triplicate in cell culture plates and treated with varying concentrations of Taiwanin E  for different time intervals , and thereafter, the cells were harvested and analyzed for respective parameters. For activator experiments, cells were treated with Taiwanin E (10 μM) and co-treated with increasing concentrations of ERK activator  for 24 h. Thereafter, they were analyzed by MTT assay and Western blotting, following the standard procedures as described below.Cell viability was assessed as reported in our previous studies . Basical5 cells/well) were seeded in tissue culture plates; thereafter, they were treated with varying doses of Taiwanin E  for 24 h and treated with Taiwanin E (10 μM) for different time intervals . Following treatment, cells were harvested and subsequently fixed in chilled ethanol overnight at 4°C. Consequently, cells were washed with PBS, centrifuged at 600 ×g for 5 min and thereafter resuspended in PBS containing 10 mg/ml RNase A and incubated with 30 mg/ml of PI for 30 min at RT. Finally, the samples were acquired with flow cytometry .Cell cycle study was performed with flow cytometry. Briefly, T28 oral cancer cells  assay following manufacturer-recommended procedures  and processed through Adobe Photoshop.Results are represented as mean ± SD. Statistical analysis was done with Graph Pad Prism5 statistical software . Multiple comparisons were assessed through ANOVA. Normal oral cells N28 and cancer cells T28 were treated with increasing concentrations of Taiwanin E for a 24 h time interval; thereafter, cell viability was ascertained by MTT assay. MTT data showed that Taiwanin E exhibited cytotoxic effects on T28 oral cancer cells, especially at 10 μM concentration, whereas no cytotoxic effects were observed for N28 normal oral cells . In addiFurther, N28 and T28 cells were treated with Taiwanin E (10 μM) for different time periods from 0 to 48 h, and cell viability was determined through MTT assay; interestingly, it was observed that Taiwanin E exhibited cytotoxic effects on T28 cells in a time-dependent manner. However, no such effects were observed for normal N28 oral cells . FurtherCollectively, these results suggested that Taiwanin E exhibited cytotoxic manifestations toward tumor oral cells T28 in a dose- and time-dependent fashion; however, no cytotoxic effects were observed for normal oral cells N28. Moreover, Taiwanin E treatment in T28 cells modulated their normal regulation of cell cycle progression. Nevertheless, no significant effects were observed for normal oral cells N28 (data not shown).We further investigated whether Taiwanin E affects cell cycle-related proteins in T28 oral cancer cells. To this end, cells were treated with Taiwanin E (10 μM) for different time periods from 0 to 48 h and thereafter analyzed through Western blotting. Interestingly, it was found that the cell cycle regulatory proteins, such as cyclin B1, cyclin D1, and cyclin E, were considerably decreased , whereasWe further investigated whether Taiwanin E could induce cellular apoptosis in T28 oral cancer cells. As evident through TUNEL assay, considerable apoptosis was observed following treatment with Taiwanin E . To gainFurther, the effect of Taiwanin E on the MAPK signaling cascade was investigated. As could be seen from the figure, following treatment with Taiwanin E, a considerable reduction in the p-ERK1/2 was observed; no significant effects, however, were observed for p-JNK and p-p38 proteins . CollectTo further investigate the underlying intricacies of modulation of ERK in the efficacy of Taiwanin E, the effect of the ERK activator on T28 cell proliferation was ascertained. Intriguingly, it was observed that ERK activator co-treatment with Taiwanin E in T28 cells considerably reversed the effect of Taiwanin E in a dose-dependent fashion . In addiCollectively, it could be envisaged that Taiwanin E considerably modulated ERK, which eventually led to the inhibition of cancer cell survival, induced cell cycle arrest at G1, and apoptosis in T28 oral cancer cells. Collectively, the data convincingly demonstrated that Taiwanin E may potentially become an effective small molecular drug against oral cancer in the near future.Various bioactive compounds  derived from Taiwania has been shown to be effective against various debilitating diseases. Accumulating evidences have shown their efficacy against various forms of cancers, including A-549 lung carcinoma, MCF-7 breast adenocarcinoma, and HT-29 colon adenocarcinoma . In thisTaiwania cryptomerioides Hayata upregulated p53, phosphorylated p53, p21, and p27, and, contrastingly, downregulated the G(2)/M checkpoint mediators cyclins, leading to the induction of G(2)/M cell-cycle arrest in MCF-7 cells  and camptothecin derivatives (from Camptotheca acuminate), are widely explored for cancer treatment, and, as Taiwanin E displayed lower cytotoxicity against normal oral cells, it could be envisaged as a promising drug to treat oral cancer in the near future, thus warranting future investigation.Collectively, keeping in mind the fact that various phytochemicals, such as taxol (from The datasets generated for this study are available on request to the corresponding author.S-HW and C-YH contributed toward the conception and design of the study. S-HW, H-CW, and KB performed the experiments and data analysis. KB, C-JC, and C-YH contributed to the manuscript writing, editing, and proofreading. Y-HK, Y-PC, H-HH, D-TB, VV, Y-HC, and P-JL supported the experiment design, data analysis, and the manuscript writing and proofreading.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. The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors C-YH and Y-HC.


Background: In mice, postnatal immune development has previously been investigated, and evidence of a delayed maturation of the adaptive immune response has been detected. Methods: In this study, the effects of red grape polyphenol oral administration on the murine immune response were explored using pregnant mice (TAG/F3 transgenic and wild type (wt) mice) as the animal model. The study was performed during pregnancy as well as during lactation until postnatal day 8. Suckling pups from polyphenol-administered dams as well as day 30 post-weaning pups (dietary-administered with polyphenols) were used. Polyphenol effects were evaluated, measuring splenic cytokine secretion. Results: Phorbol myristate acetate-activated splenocytes underwent the highest cytokine production at day 30 in both wt and TAG/F3 mice. In the latter, release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α was found to be higher than in the wt counterpart. In this context, polyphenols exerted modulating activities on day 30 TAG/F3 mice, inducing release of interleukin (IL)-10 in hetero mice while abrogating release of IL-2, IFN-γ, TNF-α, IL-6, and IL-4 in homo and hetero mice. Conclusion: Polyphenols are able to prevent the development of an inflammatory/allergic profile in postnatal TAG/F3 mice.  In mice, the post-natal immune system is not completely developed, and maternal milk seems to play an important yet protective function . TherefoWithin the lymphoid tissue, the thymus represents the major site for T lymphocyte maturation , which oAs for the spleen, by the end of the gestation, this organ is still immature from an immunological point of view, but by 3–7 days postnatally, the splenic periarteriolar lymphocyte sheet (mainly composed by T cells) starts developing, with the marginal sinus becoming more evident by day 7 . PrimaryThe development of the murine adaptive immunity, which occurs in the immediate postnatal period, has been addressed in a few studies. In neonatal mice, reduced T cell number has been demonstrated, when compared to adult mice ,11. ThisTaken together, these data point out a certain degree of vulnerability of neonatal mice to infections, which may result from impaired cell-mediated adaptive immune response imparted by Th1 cells. To overcome delayed murine Th1 cell maturation, dietary manipulations with prebiotics, galacto-oligosaccharides, fructo-oligosaccharides, fish oil, and synbiotics have been demonstrated to be very effective ,20,21,22Polyphenols (flavonoid and nonflavonoid compounds) are largely present in fruits, vegetables, cereals, wine, and extra virgin olive oil . These nTaking advantage of the abovementioned polyphenol activities, in this study, splenic immune responsiveness was evaluated in suckling pups from mothers orally administered with polyphenols between 8 and 30 days after weaning. TAG/F3 mice, a transgenic line undergoing delayed neural development as a consequence of upregulation of the Contactin 1 adhesive glycoprotein expression, were employed ,32,33. OIL-12 is a Th1-induced cytokine, released by splenic dendritic cells upon antigenic activation . As showIL-2 is a major growth factor released by activated Th1 cells . At postIFN-γ is a predominantly Th1-derived cytokine, which plays a fundamental role in host protection against intracellular pathogens .At postnatal day 8, this cytokine could not be detected in control and active supernatants from both wt homo and hetero pups, respectively (data not shown). At postnatal day 30 , minute IL-4 was identified as the major cytokine produced by Th2 cells . The corIL-10 is an anti-inflammatory cytokine released by Treg cells . As showAt day 30, no evidence of IL-10 release was recorded in all experimental setups (data not shown).At days 8 and 30, IL-17, an inflammatory cytokine produced by Th17 cells , was notIL-6 is an acute phase protein considered as an expression of innate immune response .At day 8, IL-6 was not detected in all pups (data not shown). As indicated in TNF-α is another cytokine mostly produced by macrophages and implicated in chronic inflammatory disease . Data reThe present data confirm and extend previous attainments on murine neonatal immune system maturation ,5. IndeeTo the best of our knowledge, this is the first observation on immune system development in TAG/F3 transgenic mice. In the present discussion, the main arising evidence is that at days 8 and 30 in both wt and TAG/F3 transgenic mice, unstimulated cells are unable to release any type of cytokine. Therefore, only data related to PMA-stimulated cells with or without polyphenol administration were analyzed. At postnatal day 8, IL-12, a cytokine produced by antigen presenting cells   is detecAs far as IFN-γ is concerned, at day 8, this cytokine is undetectable in all mice, while at day 30, it is produced in higher amounts in both homo (35 ± 0.95 pg/mL) and hetero mice (31 ± 0.82 pg/mL) when compared to the wt counterpart (7 ± 0.82 pg/mL). IFN-γ is a protective cytokine which promotes the microbicidal function of macrophages as well as cytotoxic functions of T lymphocytes (CD8+ cells) and natural killer (NK) cells which, in turn, exert anti-neoplastic (CD8+ and NK cells) and antiviral functions (CD8+ cells), respectively . All togWith regard to IL-4 a Th2-related cytokine , at day Of note, IL-4 is involved in allergic response (IgE production) and in the conversion of the primary (IgM) to the secondary (IgG) immune response .IL-10 is a cytokine predominantly produced by Treg cells, provided with anti-inflammatory and immune modulating activities . In wt mIL-17, an inflammatory cytokine predominantly released by Th17 cells , is undeIL-6 is an acute phase protein also involved in Th17 cell activation . Its relTNF-α is an inflammatory cytokine, which actively participates to the maintenance of chronic and auto-immune disease . At day In synthesis, all mice seem to be protected against external and internal stimuli, even if the absence of IL-10 production may result in exaggerated inflammatory responses. These data also suggest a powerful response of the innate immunity at early stages.Previous studies on TAG/F3 transgenic mice ,32,43 deAs previously stated in this paper, polyphenols are endowed with anti-inflammatory and immune modulating activities in various experimental and clinical settings ,26,27,28In The generation of the TAG/F3 transgenic mice strain, which undergoes delayed neural development as a consequence of Contactin 1 and subsequent Notch pathway upregulation, has already been described ,33. MiceCanosina red grape from Nero di Troia is an autochthonous Vitis vinifera grape cultivar, growing in Apulia , characterized by thick-skinned and small-sized berries. Frozen seeds from berries were extracted by percolation with ethanol/water (70:30). The extract was first analyzed by liquid chromatography with diode array detection to define the polyphenol composition. Thereafter, it was purified on a synthetic adsorbent brominated resin, and the polyphenol content percentage was determined. Extracts were evaluated for their potential antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl assay, which measures the ability of test agents to scavenge radicals -penicillin (100 UI)) , and 30% fetal bovine serum . Cell suspensions were counted using trypan blue dye exclusion test, and cell density was adjusted at 1 × 10g for 10 min. Then supernatants were carefully discarded and, finally, pellets were resuspended in 500 µL of PBS. Samples were acquired using the FACS DIVA software (Becton Dickinson), which allows distinguishing different types of cytokines according to some properties, such as forward scatter, side scatter, and mean fluorescence of the following fluorochromes: PE, APC, and APC-Cy7. A standard curve for all cytokines was initially prepared, and the FCAP software (Becton Dickinson) was then used to quantify all cytokines in standards and samples.Cell supernatants obtained from single mice were incubated with the cytometric bead array (CBA) mouse soluble protein Flex Set kit . This permitted to quantify the following cytokines: IL-12; IL-2; IFN-γ; IL-4; IL-10; IL-17; IL-6; and TNF-α. The capture beads provided in each CBA mouse soluble protein Flex Set were dispensed in each cell supernatant and, after 1 h incubation in the dark, a mouse soluble protein Flex Set PE detection was added for 2 h in the dark. Afterwards, each sample was suspended in 1 mL PBS and centrifuged at 800× p < 0.05.Statistical analysis was performed using Bonferroni’s test for comparison between groups. GraphPad Prism statistical software release 5.0 for Windows Vista was used. The statistical significance was set at


Aging and various age-related diseases are associated with reductions in melatonin secretion, proinflammatory changes in the immune system, a deteriorating circadian system, and reductions in sirtuin-1 (SIRT1) activity. In non-tumor cells, several effects of melatonin are abolished by inhibiting SIRT1, indicating mediation by SIRT1. Melatonin is, in addition to its circadian and antioxidant roles, an immune stimulatory agent. However, it can act as either a pro- or anti-inflammatory regulator in a context-dependent way. Melatonin can stimulate the release of proinflammatory cytokines and other mediators, but also, under different conditions, it can suppress inflammation-promoting processes such as NO release, activation of cyclooxygenase-2, inflammasome NLRP3, gasdermin D, toll-like receptor-4 and mTOR signaling, and cytokine release by SASP (senescence-associated secretory phenotype), and amyloid-β toxicity. It also activates processes in an anti-inflammatory network, in which SIRT1 activation, upregulation of Nrf2 and downregulation of NF-κB, and release of the anti-inflammatory cytokines IL-4 and IL-10 are involved. A perhaps crucial action may be the promotion of macrophage or microglia polarization in favor of the anti-inflammatory phenotype M2. In addition, many factors of the pro- and anti-inflammatory networks are subject to regulation by microRNAs that either target mRNAs of the respective factors or upregulate them by targeting mRNAs of their inhibitor proteins. Aging is associated with manifold changes. These comprise declined secretion of hormones such as melatonin ,2, reduc+ concentration, which depends on the activity of nicotinamide phosphoribosyltransferase (NAMPT) [+ concentration and, therefore, SIRT1 activity. Moreover, BRCA1 overexpression upregulated SIRT1 expression and decreased NAD+ levels and SIRT1 activity. This divergence also seems to be relevant to aging. SIRT1 expression was not generally shown to be decreased in the course of aging, but rather, it was shown to often increase. Nevertheless, SIRT1 activity was found to be reduced because of lowered NAD+ levels [+ concentration (recommended) or to at least test the effects of sirtuin inhibitors [For example, the effects of melatonin in the immune system can be either pro- or anti-inflammatory ,16,17. G (NAMPT) ,20,21,22 (NAMPT) . Suppres+ levels ,25. Howe+ levels . Moreove+ levels ,37,38,39hibitors .The changes in melatonin secretion and SIRT1 activity also have a circadian dimension. Both of them are under circadian control and exhibit cycles of high amplitudes. Moreover, either of them can influence circadian oscillators. Apart from its known chronobiotic actions via the suprachiasmatic nucleus (SCN), melatonin also influences peripheral oscillators . SIRT1 hThese reservations also have to be kept in mind when considering the effects of melatonin and SIRT1 in the regulation of pro- and anti-inflammatory processes. These are of particular relevance to aging and age-related diseases, especially as proinflammatory mechanisms gain increasing importance in the course of senescence. The overlapping effects of melatonin and SIRT1 in the field of inflammation regulation will be discussed in this article.In the course of studies that revealed numerous effects of melatonin beyond the control of circadian oscillators, immunological actions of melatonin were also discovered ,44,45,462 in human lymphocytes [Many of the earlier investigations on melatonin in the immune system revealed proinflammatory effects, as repeatedly summarized ,15,17,44phocytes .However, this short overview of the inflammation-promoting effects does not yet sufficiently describe the actions of melatonin within the proinflammatory network. In fact, these actions also comprise numerous suppressive changes in this network and, thereby, can turn out to result in an anti-inflammatory balance. To appropriately judge the complex influences of melatonin, it is necessary to view it under these different, additional perspectives: (1) changes in tissues and their main cellular constituents; (2) cell specificity; (3) effects on clonal expansion, differentiation, and polarization in the immune system; (4) contributing proinflammatory effects of non-immune cells; and (5) conditionality, with regard to high-grade or low-grade inflammatory challenges that may occur in the progression of aging or by experimental procedures.As recently summarized, melatonin-induced elevation of proinflammatory cytokines, such as IL-1β, IL-2, IL-6, IL-12, TNFα, and IFNγ, has been repeatedly observed in monocytes, monocyte-derived cell lines, and type 1 T-helper cells . One can•NO-mediated interplay with microglia and astrocytes; (3) amyloid-β (Aβ) toxicity; (4) brain insulin resistance; (5) diabetes and metabolic syndrome; (6) senescence-associated secretory phenotype (SASP) of non-immune cells with DNA damage response (DDR); (7) garb-aging; (8) reduced antioxidant protection by decreased melatonin; and, presumably, also (9) metabolic malfunction because of poorly coordinated and weakened circadian rhythms. Notably, all these pathophysiological alterations are associated with oxidative and nitrosative/nitrative stress and mitochondrial malfunction. They have multiple interconnections and, thereby, display the potential of forming vicious cycles [The proinflammatory network comprises numerous components, which are regulated differently by melatonin. Moreover, the responses are often highly contextual. Findings obtained by applying pro-oxidant or proinflammatory challenges, such as administration of oxidotoxins, mitotoxins, bacterial lipopolysaccharides (LPS) or experimental sepsis, may not lead to results comparable to those found in aging. Therefore, it seems important to focus on changes that really occur in aging or age-related diseases and can be mostly classified as low-grade inflammation ,17,70. Ss cycles ,17,70. •NO, which can activate microglia, astrocytes, and other neurons that also release •NO at elevated rates. These responses contribute to a spreading of excitation and to the release of oxidants and proinflammatory mediators, especially by microglia. High levels of •NO and superoxide anions (O2•−) generate the formation of their adduct, peroxynitrite (ONOO−), a highly reactive intermediate that causes damage to mitochondria and additionally generates free radicals  [2•− formation. Notably, the release of proinflammatory mediators in response to Aβ is not restricted to microglia. Even neurons have been shown to respond to Aβ peptides by upregulating TNFα, IL-1β, COX-2, and the T-cell and monocyte attractant chemokine CX3CL1 [Immunosenescence, which is primarily associated with thymic involution and, additionally, exhaustion of leukocyte subpopulations upon lifelong exposure to foreign antigens, typically leads to a shift toward a proinflammatory phenotype, which is evident by increased levels of proinflammatory cytokines that may result in a so-called immune risk profile ,71,72,73+ CO3•−) ,75. Thes+ CO3•−) ,76,77. A+ CO3•−) ,81,82,83+ CO3•−) ,85 and, + CO3•−)  and neur+ CO3•−) ,87, whice CX3CL1 ,89,90,91e CX3CL1 . Circadie CX3CL1 ,94. Numee CX3CL1 ,96,97,98e CX3CL1  has parte CX3CL1 , inhibite CX3CL1 , and thee CX3CL1 . The shie CX3CL1 ,103. In e CX3CL1 ,105. Howe CX3CL1 , findinge CX3CL1 ,108. Fore CX3CL1 ,109. Thie CX3CL1 . Also, be CX3CL1 ,111,112,e CX3CL1 , which hN-formylated peptides and proteins) to the cytosol [Sting gene polymorphism that is associated with low-risk of aging-related diseases, presumably by reducing inflammaging [Another aspect of proinflammatory effects concerns the role of non-immune cells in inflammaging. This comprises mainly three processes, SASP, garb-aging, and release of macromolecules from dying cells. In SASP, DNA-damaged cells are arrested in terms of proliferation, but continue to participate in the metabolism of the tissue. However, they release several signal molecules, including proinflammatory cytokines and chemokines, thereby inducing local, low-grade inflammation ,117,118. cytosol ,131,132, cytosol . Melaton cytosol . Unfortu cytosol . cGAS apammaging .Activation of the NLRP3 inflammasome in various systems, under different conditions and counteractions by melatonin, have been recently reviewed . These fAnother proinflammatory route is based on TLR4 (toll-like receptor 4) activation, e.g., via the IFNγ adaptor protein, TRIF (toll-receptor-associated activator of interferon). In the macrophage-like cell line RAW264.7, melatonin has been shown to suppress the release of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, and IL-8, by TRIF and TLR4 inhibition . As TLR4A further possible proinflammatory pathway that is inhibited by melatonin concerns mTOR (mechanistic target of rapamycin) activation. However, most respective information is not directly related to inflammation, but rather to mitophagy or apoptosis. Interestingly, an mTOR inhibiting action by melatonin was also shown to be suppressed by inhibition of PIN1 . MoreoveWhile melatonin is partially acting by either stimulating or inhibiting components of the proinflammatory network, it also upregulates molecules of an anti-inflammatory network. Some of them are negatively correlated with proinflammatory agents. For instance, NF-κB, a transcription factor involved in prooxidant and, thereby, proinflammatory responses, is inversely coupled to antioxidant and anti-inflammatory regulators, in particular, Nrf2 ,150,151.Sirt1 siRNA [An especially important anti-inflammatory regulator under control by melatonin is SIRT1. It has been classified as a secondary signaling molecule that mediates several effects of melatonin ,42. In nt1 siRNA , notablyt1 siRNA . The relt1 siRNA  and may,t1 siRNA . With thSirt1 siRNA, are summarized elsewhere [This overlap becomes obvious from two lines of evidence, (1) the interference of sirtuin-related agents with melatonin effects, and (2) similar actions of melatonin and SIRT1. In the former context, reductions of NLRP3 inflammasome activation and IL-1β levels by melatonin were blocked by the sirtuin inhibitor EX527 in a rat COPD (chronic obstructive pulmonary disease) model . The samlsewhere .Numerous studies have demonstrated antioxidant and anti-inflammatory actions by SIRT1 that are also known from melatonin . This coSirt1 siRNA [SIRT1 also displays several additional anti-inflammatory effects, which cannot yet be matched with corresponding data from melatonin treatment. As mentioned in the previous section, some reports on melatonin effects on mTOR signaling have not been directly related to inflammation. Other studies on actions of melatonin on mTORC1 (mTOR complex 1) activity in cancer cells led to contradictory results ,183,184 t1 siRNA ,30,31.Another recently reported anti-inflammatory action of SIRT1 has been observed in macrophages after treatment with LPS. The lncRNA-CCL2, which is related to the gene locus of the chemokine CCL2 and stimulates the release of proinflammatory cytokines, was downregulated by SIRT1 . No corr1 receptor-mediated activation of JAK2 (Janus kinase 2), which phosphorylates STAT3  [Despite the fact that many proinflammatory effects of melatonin are mediated by lymphocytes ,49,54,56ption 3) ,196,197.ption 3) ,199,200.ption 3) ,201. Thiption 3) . Howeverption 3) ,204, actption 3) ,194. A pption 3) . With reption 3) ,154,155.ption 3) . 1/2-dependent cascade of pIRS-2, GRB2, and pSTAT6. pSTAT6 dimers are responsible for M2-specific gene expressions, partially in conjunction with KLF4 (krüppel-like factor 4), which interacts with pSTAT6. Additional effects of melatonin seem to contribute, such as MT1/2-dependent inhibition of PI3K (phosphoinositide 3-kinase), thereby blocking the proinflammatory Akt/mTORC1 cascade [M2 polarization of macrophages, and presumably in similar ways of microglial cells, is largely promoted by STAT6 phosphorylation, in pathways regulated by melatonin and anti-inflammatory cytokines, especially IL-4 and IL-13 . A key s cascade . Moreove cascade . Serine  cascade .The discovery of countless noncoding RNAs (ncRNAs) with regulatory properties has substantially changed our understanding of regulation. Many of these RNAs interfere with posttranscriptional processes. This is especially the case in most microRNAs (miRNAs), which target mRNAs, and snoRNAs , which are involved in RNA processing ,208. OthA complete consideration of all ncRNAs with positive or negative actions on inflammation would go beyond the scope of this article. A comprehensive list of miRNAs that affect pro- or anti-inflammatory cytokines has been recently published . With reThe pleiotropic targeting of various mRNAs by a single miRNA does not only open the possibility of jointly downregulating several mRNAs that are functionally connected, but may also reflect multiple functions in different contexts. This problem of overlapping actions of miRNAs with roles in both inflammation and cancer is also evident in the findings summarized in The examples in + levels or inhibition by sirtuin inhibitors have not been determined. In reports in which upregulation by melatonin was associated with downregulation of SIRT1 (miR-30a) or Nrf2 and upregulation of NF-κB (miR-146a), this combination may be interpreted as a route of melatonin’s proinflammatory arm. The example of miR-24, which is suppressed by melatonin, but enhances SIRT1 expression, may be taken as a tumor-specific relationship, because this would conform to melatonin’s known suppression of SIRT1 in cancer cells [A few special types of connections between melatonin and the three possible downstream factors shall be briefly discussed. The levels of miR-7 have been shown to increase during aging, and it was assumed that it may downregulate SIRT1 . On the er cells ,365. Theer cells ,18. Unfoer cells . H19, a relationship to miR-675  mRNA, which was concluded to cause downregulation of p53 and p21, thereby inhibiting premature senescence by oxidative stress [H19 and miR-675 by melatonin was also observed in protection experiments against brain injury after subarachnoid hemorrhage [H19 expression and, therefore, also that of miR-675-3p, whereas H19 was shown to suppress miR-200a [H19 in inflammatory processes (not cited), however, they did not consider melatonin. Another type of interaction between lncRNA and miRNA was based on sponging. The lncRNA MEG3 was reported to enhance pyroptosis in atherosclerotic models, such as human aortic endothelial cells exposed to oxidized LDL (low density lipoprotein). MEG3 was found to sponge miR-223 and to activate NLRP3, whereas melatonin was shown to reduce pyroptosis by suppressing NLRP3 activation and downstream factors, such as NF-κB, gasdermin D, IL-1β, and IL-18 [MTC, which was associated with proliferation and also NF-κB activation [CPS1-IT1, with a downstream effect of reduced HIF-1α activity [RAD51-AS1, which interacts with RAD51 mRNA, and the findings were interpreted as a sensitization to chemotherapeutic drugs [TERRA (telomeric repeat-containing RNA), has been investigated in the context of melatonin. This RNA interacts with PARP-1 (polyADP-ribose polymerase-1), a sensor of DNA damage. This interaction was shown to stimulate SASP, but melatonin suppressed SASP-related gene expression and, therefore, an important aging-promoting process [Available information concerning melatonin effects on other noncoding RNAs is, unfortunately, limited. A few publications have addressed such actions on lncRNAs. In the case of the lncRNA -675 cf.  exists,  miR-675 . The mole stress . Upregulmorrhage . In a stmiR-200a . Meanwhind IL-18 . In hairtivation . This fiactivity . In anotic drugs . A funct process . CDR1as , a few recent publications indicate relationships to NF-κB signaling [CDR1as deficiency in the brain [Work on the mediation of melatonin effects on lncRNAs can be expected to become highly important in the future ,370, altignaling ,385,386 ignaling , and thehe brain .The decrease of melatonin in aging and in various aging-related diseases ,2 has diSirt1 siRNA. Moreover, some mitochondrial actions of melatonin were shown to be absent in Sirt1−/− mice [+ levels, hormones such as triiodothyronine and glucocorticoids, oncogenes, lncRNAs such as HOTAIR, and various miRNAs not regulated by melatonin. The incoherence of melatonin effects on miRNAs and their targeting of typically otherwise melatonin-controlled transcription factors, such as Nrf2 and NF-κB, is evident from Under other conditions, melatonin has been shown to preferentially exert anti-inflammatory, antioxidant, and other beneficial actions in aging ,393,394.−/− mice . The inc


MAMLD1 is one of the recognized DSD genes. However, its role is controversial as some MAMLD1 variants are present in normal individuals, several MAMLD1 mutations have wild-type activity in functional studies, and the Mamld1-knockout male mouse presents with normal genitalia and reproduction. We previously tested nine MAMLD1 variants detected in nine 46,XY DSD patients with broad phenotypes for their functional activity, but none of the mutants, except truncated L210X, had diminished transcriptional activity on known target promoters CYP17A1 and HES3. In addition, protein expression of MAMLD1 variants was similar to wild-type, except for the truncated L210X. We hypothesized that MAMLD1 variants may not be sufficient to explain the phenotype in 46,XY DSD individuals, and that further genetic studies should be performed to search for additional hits explaining the broad phenotypes. We therefore performed whole exome sequencing (WES) in seven of these 46,XY patients with DSD and in one 46,XX patient with ovarian insufficiency, who all carried MAMLD1 variants. WES data were filtered by an algorithm including disease-tailored lists of MAMLD1-related and DSD-related genes. Fifty-five potentially deleterious variants in 41 genes were identified; 16/55 variants were reported in genes in association with hypospadias, 8/55 with cryptorchidism, 5/55 with micropenis, and 13/55 were described in relation with female sex development. Patients carried 1-16 variants in 1-16 genes together with their MAMLD1 variation. Network analysis of the identified genes revealed that 23 genes presented gene/protein interactions with MAMLD1. Thus, our study shows that the broad phenotypes of individual DSD might involve multiple genetic variations contributing towards the complex network of sexual development.Disorders/differences of sex development (DSD) are the result of a discordance between chromosomal, gonadal, and genital sex. DSD may be due to mutations in any of the genes involved in sex determination and development in general, as well as gonadal and/or genital development specifically.  Disorders/differences of sex development (DSD) occur when there is a discordance between chromosomal, gonadal, and genital sex . DSD mayMAMLD1  is one of the recognized DSD-related genes  some pulation ; b) the enotypes ; c) MAMLe family ; d) seve studies ; and e) oduction .MAMLD1 is expressed in human fetal and adult testis and in human ovaries  and reproduce similarly to wild-type mice  (NR5A1 variants) and 46,XX POF females (NR5A1 and TBX2) (SEMA3A and AKR1C4)  has changed the genetic approach in research and diagnostics. Whole-exome sequencing (WES) has led to the discovery of many new genes and has given insight into complex traits. Oligogenic inheritance is currently discovered for several disorders by HTS. In the field of sex development, digenic inheritance has recently been suggested in a 46,XY DSD patient with gonadal dysgenesis (ariants) ; in a fand TBX2) ; as well AKR1C4) . Similarming WES . In addiming WES .MAMLD1 variants. WES data were filtered by common tools and a disease-tailored algorithm including MAMLD1-related and DSD-related known and candidate genes. Additional hits in likely disease-causing genes were detected in all eight MAMLD1 carriers. Our results suggest that oligogenic origin of disease may contribute towards the broad phenotypes of human MAMLD1.Therefore, in this study, we performed WES in seven 46,XY patients with DSD  and one MAMLD1 variant were analyzed using WES. Clinical and genetic characteristics of 46,XY patients were previously reported in detail in The study was approved by the Ethics Committee of Hospital Universitari Vall d'Hebron  (CEIC: PR(IR)23/2016). Written informed consent was obtained from the patients for the publication of their cases. Eight DSD patients  each carrying one DNA was extracted from blood leukocytes using QiaCube  or manually using a DNA isolation kit (Qiagen). WES was performed by CNAG . Libraries were prepared with a SureSelect Human All Exon V5 capture kit  and sequenced with a HiSeq™ 2000 sequencing system . Putative candidate variants were confirmed by Sanger sequencing.http://annovar.openbioinformatics.org/) (https://www.broadinstitute.org/igv/ (http://gnomad.broadinstitute.org/about)   and filtered with the functional annotation of genetic variants from HTS data  , visualiorg/igv/ . Frequeng/about)  and the st 2018) . gnomAD MAMLD1-related and DSD-related known and candidate genes (n = 606) similar to the algorithm previously set up for MAMLD1-related genes by searching in published literature and databases. DSD-related genes are part of our DSD-gene database and tools (http://string-db.org/)   and the db.org/)  were usein-silico predictors were accessed through ANNOVAR . Functional exonic predictors were CADD  (http://sift.jcvi.org/), PolyPhen-2 , Provean (http://provean.jcvi.org), MutationAssessor (http://mutationassessor.org/r3/), Mutation Taster (http://www.mutationtaster.org/), LRT, FATHEMM, Fathmm-MKL, PROVEAN, VEST3 (Variant Effect Scoring Tool), MetaSVM, MetaLR, MCAP, DANN and fitCons. Exonic predictors on evolutionary conservation were: GERP++, phyloP , phastCons  and SiPhy. Splicing predictors were: splicing predictors from dbscSNV ADA and RF and SPIDEX splicing predictor (DPSI), and those splicing predictors from Alamut Visual software were: SSF, MaxEnt, NNSPLICE, GeneSplicer and Ex-Skip.We used 30 pathogenic predictors to predict possible impact of amino acid substitutions on the structure, function and evolutionary conservation of corresponding human proteins and to predict impact on splicing. These  ANNOVAR  annotation.edu/) , SIFT , VarSome , ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and Alamut Visual 2.11 . We searched for reported  disease-causing variants with the Human Gene Mutation Database  and dbSNP (http://www.ncbi.nlm.nih.gov/snp/). We used STRING for the search for interactions within genes carriers of interesting variants (DSD-related and/or MAMLD1-related). Data from STRING are extracted from known interactions , predicted interactions  and other inferred evidences such as text mining, co-expression and protein homology. We used Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/) and OMIM (https://www.omim.org) to build our DSD gene list and for further data analysis. The datasets generated for this study are publicy available in dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewBatch.cgi?sbid=1063030.The following bioinformatics software tools were used for the interpretation and classification of variants: InterVar (in dbSNP : https:/MAMLD1- and DSD-related known and candidate genes. B) We kept variants with MAF  ≤0.015 or not detected in gnomAD, and variants with the following predicted type, consequences and locations: splicing (intronic or exonic), exonic, intergenic, regulatory. C) We confirmed the correct annotation and location of variants by checking their alignment data in IGV  (data not shown). D) We excluded variants that were considered non-relevant for our study: E.g. 1) variants found in more than two patients, 2) variants in repeat regions, 3) variants in genes or gene regions with high variability, 4) variants with low coverage and/or low quality, 5) variants with non-similar allelic depths. E) We revised variants with the annotated pathogenic predictors: functional exonic, evolutionary-conservation and splicing predictors , as previously described. F) We run InterVar and VarSome to classify the variants, searched for reported  human disease-causing variants with the HGMD, and revised evidences of relationship with DSD, sex development and clinical phenotype of each patient with literature and database search. G) We used STRING to find out interactions among genes carriers of interesting variants (DSD-related and/or MAMLD1-related)  Each patient’s WES data were first filtered by our related) 2. H) WeMAMDL1 variants revealed several candidate gene variants that potentially contribute to each patient’s phenotype. A detailed summary of patients’ characteristics and number of variants and genes is shown in WES performed in eight unrelated subjects  harboring hemizygous/heterozygous MAMLD1 patients  and RECQL4 (associated to syndromic hypospadias)  and 7; IRX5 in patients 5 and 6; MAML1 in patients 4 and 6; MAML2 in patients 5 and 8; NOTCH2 in patients 5 and 7; RECQL4 in patients 2 and 3 and WDR11 in patients 6 and 7  and NOTCH1 . Furthermore, RIPK4 presented 2 variants in patient 1. Finally, BNC2 variant c.1868C>A:p.(Pro623His) (MAF = 0.002) was detected in 2 patients (patient 1 and 7) and MAML3 variant c.881A>G:p.(Asn294Ser) (MAF = 0.0028) in patients 7 and 8 , and 5 genes have been found in patients with micropenis . Also, 15 genes have been described in other types of DSD   . Thirteen of these genes have been found involved in female gonadal development and 46,XX DSD , 8 of which in patient 8 , while other genes  may have both AD and autosomal recessive (AR) inheritance. FLNA and CUL4B are X-linked (XLR), while CYP1A1, FREM2, EVC, HSD3B2, IRX5, PROP1, RAS1, RECQL4, RIPK4 and ZBTB16 are known for AR inheritance. No information on inheritance is currently available for the remaining genes including ATF3, BNC2, GRID1, DAPK1, IRX6, IGFBP2, MAML1, MAML2, MAML3, NRP1, PIK3R3, WNT9A and WNT9B.According to OMIM, almost all of our patients presented at least one variant in a gene with autosomal dominant inheritance (AD)  to variable degrees of hypospadias, cryptorchidism and small penis . Patient 6, who presented with a right aortic arch, was found to carry variants in five genes  that have been previously described in patients with heart and/or vascular anomalies  have so far only been related to female gonadal development  for sex development. In our DSD individuals carrying MAMLD1 variants, three genes seemed prominent in the network analysis, NOTCH1, NOTCH2 and GLI3. NOTCH signaling is a highly conserved signaling pathway and comprises 4 transmembrane receptors. It is essential for the regulation of embryonic development of multiple organ systems including gonadal development (NOTCH1 cause the Adams–Oliver syndrome (OMIM 616028), while autosomal dominant mutations in NOTCH2 are reported in the Alagille syndrome 2 (OMIM 610205) and in the Hajdu–Cheney syndrome (OMIM 102500). By contrast, GLI3 is a zinc-finger transcription factor belonging to the desert hedgehog (DHH) signal transduction pathway. DHH signaling is essential for driving Leydig cell differentiation  or in the Greig cephalopolysyndactyly syndrome (OMIM 175700).A search for an underlying network comprising variants in the identified genes related to ractions 2 suggeselopment . Accordielopment . NOTCH selopment . Autosomntiation . Thus, Nntiation . AutosomMAMLD1 individuals share variants in 19 genes comprised in such DSD panels, including ATF3, BNC2, CUL4B, EVC, FLNA, FRAS1, FREM2, GLI3, HOXA13, HSD3B2, IRX5, NOTCH2, PROP1, PTPN11, RECQL4, RET, RIPK4, WDR11 and ZBTB16. By contrast, through our work 22 new genes are now added for considering with differences in sex development: CDH23, COL9A3, CYP1A1, DAPK1, EMX2, EYA1, FGF10, GLI2, GRID1, IGFBP2, IRX6, MAML1, MAML2, MAML3, MYO7A, NOTCH1, NRP1, PIK3R3, PPARGC1B, TGFBI, WNT9A and WNT9B.Taken together, our results expand the landscape of genes possibly involved in DSD by revealing both new and old players. Genetic platforms for DSD diagnostics currently consider about 270 genes that have been identified with monogenetic forms of DSD in (mostly) several independent individuals . Our eigIdeally, genetic variants are tested functionally for proof of their disease-causing effect in model systems. However, when finding multiple variants, which may all contribute only partially, such testing is no longer feasible. Therefore, the likelihood of disease-causing effect of identified variants was assessed in our study by established bioinformatic tools for genetics and by assessing the genotype-phenotype correlation in each patient with current knowledge from literature and databases in the field. In future studies with bigger sample size, next-generation statistical genetic analyses may be employed to identify associations between a group of variants and the complex trait of sex development .MAMLD1 patients may be due to additional variants in other DSD-related genes. We found up to 55 additional genetic hits that may contribute to the DSD phenotype making an oligogenic causation plausible. Bioinformatic network analysis can help in interpreting complex genetic data and put identified single candidate genes into a greater perspective to understand their possible role in DSD biology.In summary, HTS analysis indicates that the broad DSD phenotypes of https://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewBatch.cgi?sbid=1063030.The datasets generated for this study are publicly available in dbSNP : https:/The study was approved by the Ethics Committee of Hospital Universitari Vall d'Hebron  (CEIC: PR(IR)23/2016). Written informed consent was obtained from the patients for the publication of their cases.CF: Conceptualization, funding acquisition, investigation, methodology, interpretation, project administration, resources, supervision, writing – original draft preparation, writing – review and editing. LA: Interpretation, supervision, resources, visualization, writing – review and editing. MF-C: Investigation, writing – review and editing. K-SS: Validation, interpretation, writing – review and editing. IM: Resources, interpretation, writing – review and editing. LC: Resources, writing – review and editing. IE: Resources, writing – review and editing. NC: Conceptualization, data-curation, formal analysis, investigation, methodology, interpretation, project administration, supervision, visualization, writing – original draft preparation, writing – review and editing.http://www.snf.ch) (320030-146127) to CF, the Instituto de Salud Carlos III  Centro de Investigación Biomédica en Red de Enfermedades Raras  U-712 to MF-C, the Agency for Management of University and Research Grants , Barcelona, Spain (2009SGR31) to LA, and by the Beatriu de Pinós Fellowship 2014 BP-B 00145 , the Instituto de Salud Carlos III  Centro de Investigación Biomédica en Red de Enfermedades Raras  U-712 to NC.This work was supported by grants of the Swiss National Science Foundation (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.


Lepisosteus oculatus), as a non-teleost ray-finned fish, represents a fish lineage that diverged before the teleost genome duplication (TGD), providing an outgroup for exploring the evolutionary mechanisms of circadian clocks after whole-genome duplication. In this study, we interrogated the spotted gar draft genome sequences and found that spotted gar contains 26 circadian clock genes from 11 families. Phylogenetic analysis showed that 9 of these 11 spotted gar circadian clock gene families have the same number of genes as humans, while the members of the nfil3 and cry families are different between spotted gar and humans. Using phylogenetic and syntenic analyses, we found that nfil3-1 is conserved in vertebrates, while nfil3-2 and nfil3-3 are maintained in spotted gar, teleost fish, amphibians, and reptiles, but not in mammals. Following the two-round vertebrate genome duplication (VGD), spotted gar retained cry1a, cry1b, and cry2, and cry3 is retained in spotted gar, teleost fish, turtles, and birds, but not in mammals. We hypothesize that duplication of core clock genes, such as (nfil3 and cry), likely facilitated diversification of circadian regulatory mechanisms in teleost fish. We also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the per1 or per2 duplicated paralogs derived from the TGD in the teleost fish, implicating possible subfuctionalization cases. Together, these findings help decipher the repertoires of the spotted gar’s circadian system and shed light on how the vertebrate circadian clock systems have evolved.Circadian rhythms are biological rhythms with a period of approximately 24 h. While canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. The spotted gar ( Clock, bmal1, Period, Cryptochrome) [Period and Cryptochrome genes by binding to E-box motifs (CACGTG) in their promoter regions. PERIOD and CRYPTOCHROME then form another heterodimer to repress the transcriptional activities of CLOCK: BMAL1 heterodimers [Circadian clocks regulate various cellular and physiological activities and processes in organisms ranging from cyanobacteria to mammals, allowing for them to adapt to the day–night cycle on Earth . A circaochrome) ,5. CLOCKrodimers ,7. The crodimers ,9. CLOCK1 and NPAS2/CLOCK2 are two known paralogs that were generated by the 2R genome duplication event [clock family . Tetraodon has clock1a and clock1b genes, while medaka and stickleback have clock1 and clock2 genes. clock1a/ clock1b is are a duplicated pair that resulted from the third round of whole genome duplication that occurred within the ray-finned fish lineage prior to the radiation of the teleost fish (approximately 300 Mya) [Circadian systems have undergone a genetic revolution. Like many other multigene families, the circadian genes that are found in single copies in invertebrates are duplicated in vertebrates . Gene duon event . Zebrafi300 Mya) .L. o.), a large, air-breathing ray-finned North American fish, suggested that its lineage has diverged from the teleost lineage before the TGD [Teleost fish are attractive for the study of many evolutionary questions related to diverse aspects of biology. Fish show a remarkable level of diversity in their morphology, ecology, behavior, and genomes, as well as multiple other facets of their biology . Whole g the TGD ,26. Thus the TGD ,24.Clock, Bmal, Per, Cry, Csnk1e, Nr1d, Ror, and Tim. A number of other transcription factors also thought to function in the circadian regulation of gene expression, including Dbp, Nfil3, and Dec [bmal, clock, period, cry, dec, csnk1e, nr1d, ror, par, nfil3 and tim) involved in vertebrates’ circadian rhythm pathways. We found that spotted gar contains 26 circadian clock genes from the 11 families. Nine of these 11 families of spotted gar are the same as those of humans. However, the orthologs of nfil3 and cry are different between spotted gar and humans. These analyses strongly support the notion that spotted gar resemble humans to a great extent, without the TGD, but still contain extra copies of genes that mammals lack.A major question in molecular evolution is how duplicate genes are retained in a genome. Several studies have attempted to account for the evolutionary fates of duplicate genes ,22,27,28 and Dec ,30,31. W and Dec . To furtperiod, clock, bmal, cry, dec, csnk1e, nr1d, ror, par, nfil3, and tim. These circadian clock genes from representative species were uncovered from Ensembl (Release 95) (http://www.ensembl.org/index.html). The final data set included 365 gene sequences , to better reflect their evolutionary history and increase genome connectivity. All of the old names and corresponding new names of these genes are listed in Ac, Anolis carolinensis; Dm, Drosophila melanogaster; Dr, Danio rerio; Ga, Gasterosteus aculeatus; Gg, Gallus gallus; Hs, Homo sapiens; Lc, Latimeria chalumnae; Lo, L. o.; Mu, Mus musculus; Oa, Ornithorhynchus anatinus; Ol, Oryzias latipes; On, Oreochromis niloticus; Pm, Petromyzon marinus; Ps, Pelodiscus sinensis; Tg, Taeniopygia guttata; Tn, Tetraodon nigroviridis; Tr, Takifugu rubripes; and Xt, Xenopus tropicalis.We selected 11 circadian clock gene families including equences . Based uhttp://bio.ieo.eu/fancygene/) [http://www.ebi.ac.uk/interpro/).Exonic boundaries of the coding regions of these genes were determined according to Ensembl. The exon–intron structures were presented using the FancyGene visualization software (cygene/) . The funhttp://www.genomicus.biologie.ens.fr/genomicus) [Gene synteny for the circadian clock genes was compared to the syntenic region using the Genomicus database (nomicus) . Spottedper genes in vertebrates were obtained from Ensembl. The transcription factor binding elements were predicted using the JASPAR (http://jaspar.genereg.net/) dataset (profile score threshold 99%).The upstream regions at approximately 3000 bp for period, clock, bmal, tim, dec, csnk1e, nr1d, and ror, have the same number of genes as humans without extra duplicate copies . The potential ortholog of dbp is present as an unannotated sequences on an unassembled scaffold  in the current version (LepOcu1) of the spotted gar genome .In the nfil3 family genes showed that there is only one nfil3 ortholog in flies . Almost all fish, including coelacanth and spotted gar, have the other two nfil3 members, nfil3-2 and nfil3-3. Intriguingly, lizards have only nfil3-2, while frogs have only nfil3-3. However, both nfil3-2 and nfil3-3 are completely lost in mammals and birds. Further, nfil3-2 and nfil3-3 were duplicated to nfil3-2a/nfil3-2b and nfil3-3a/nfil3-3b in teleost fish via TGD, respectively. As expected, some of these nfil3 duplicates were lost in some fish lineages—for instance, nfil3-2a and nfil3-3a in fugu, nfil3-3a in tetraodon, nfil3-3b in medaka, and nfil3-3b in cave fish were all gone. Moreover, zebrafish and cave fish possess additional nfil3-1b.1 and nfil3-1b.2 genes, while tilapia and platyfish only have nfil3-1b.1, and tetraodon only has nfil3-1b.2, which appears to derive from local tandem duplication and cannot be found in other species  and two hlf genes , which form monophyletic groups with spotted gar orthologs, respectively  in the spotted gar genome . Dbp orthologs are also conserved in most vertebrates. There is only one Dbp gene in mammals, which was duplicated to dbpa and dbpb in teleost fish.The ectively A. The pohlf genes are conserved from spotted gar to humans (four coding exons), but the structure of tef in gar (six coding exons) is different between zebrafish and humans (four coding exons) (The exon structures of g exons) B.per genes between spotted gar, teleost fish, and mammals. There are some motifs, such as NFE2L1::MafG, Prrx2 and SOX10, in upstream regions of the orthologus genes from spotted gar to mammals. Interestingly, we found that the Ahr::Arnt motif exists in unduplicated per1 and per2 ortholog genes in spotted gar, some teleosts, and mammals, but this motif only exists in one of the per1 or per2 duplicated pairs in teleost fish  lineage [Teleost fish provide an attractive model for studying a multitude of questions related to evolution. This may be linked to the apparent, considerable, plasticity of their genome, manifested, for example, by a high variability in genome size and chromosome number . Particu lineage ,41,42,43 lineage ,24,25. C lineage . Our ananfil3 genes in vertebrates. The phylogenetic and the syntenic analyses support the notion that the teleost fish and tetrapod nfil3 have a common ancestor. There is only one nfil3 ortholog in lampreys, and nfil3 genes have increased to three in coelacanth and spotted gar. During vertebrate evolution, the second round of vertebrate genome duplication (VGD) arose after the divergence between agnatha and teleost fish. The most parsimonious explanation is that the ancestral nfil3 gave rise to nfil3-1/2 and nfil3-3/4 by tandem duplication. Then, the clusters were duplicated to nfil3-1, nfil3-2, nfil3-3, and nfil3-4 genes in the VGD2, with the quick loss of some orthologs . The TGD generated extra copies of cry1a in teleost fish. For instance, zebrafish has retained cry1aa, cry1ab, cry1ba, cry1bb, cry2, and cry3 genes, which are consistent with our previous findings [The findings .per1a/per1b or per2a/per2b have preserved some functions compared to their ancestors . Interesting, the duplicated copies of the per1 gene found in zebrafish showed distinct patterns of temporal and spatial expression [per1a gene is expressed in the retina and in both the telencephalon and the diencephalon of the forebrain in a dark environment, when there is no detectable expression of per1b. However, the expression of per1a is significantly up-regulated, and the per1b gene is constitutively expressed throughout the head region when the fish are in a light environment [per genes may provide evidence that the mutations of these binding motifs caused gene subfunctionalization.It is important to understand the evolutionary fates of circadian clock genes. According to the duplication-degeneration-complementation (DDC) model, one of the potential fates of duplicate gene pairs with multiple regulatory regions is that each duplicate may experience a loss or reduction of its expression for different subfunctions by degenerative mutations . The compression . The perironment . This paironment . Our ananfil3 and cry families. It appears that these genes duplicated before the TGD. In mammals, NFIL3 is a homologue of vrille (vri), which functions as a key negative component of the circadian clocks in Drosophila [nfil3 and cry) in teleost fish diversified the circadian regulatory mechanisms in fish.Compared to mammals, the extra copies of core clock genes in spotted gar were found primarily in the osophila ,49,50,51osophila ,53. Althosophila ,55,56. Tosophila . The duposophila . Hence, nfil3-1, nfil3-2.1, and nfil3-2.2. While nfil3-1 is the ortholog of mammalian Nfil3-1, the nfil3-2 and nfil3-3 orthologs have been lost in mammals. For the cry gene family, spotted gar has two cry1 genes, cry1a, and cry1b, which likely derived from local (tandem) duplication in their ancestor and are co-orthologs of mammalian Cry1. Similar to teleosts, spotted gar also contain cry3, which mammals do not have. In addition, we also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the per1 or per2 paralogs in the teleost fish, suggesting that subfunctionalization occurred in the teleost fish per family. These results strongly support the notion that spotted gar genomically resembles humans to a great extent (without the TGD), but still contain extra copies of genes that mammals lack.In conclusion, we uncovered 26 circadian clock genes from 11 families among spotted gar, and nine of these 11 families of spotted gar circadian clock genes possessed the same number as humans, without an extra duplicate copy. The spotted gar contains