Saussurea lappa root) exhibits potent therapeutic effect on SMMC-7721 xenografts without obvious side effects. In the in vitro experiments, VOSL inhibited HCC cell proliferation by arresting cell cycle at S and G2/M phases, and induced HCC cell apoptosis by activating the Caspase3 pathway. VOSL also decreased the capability of HCC cell migration and invasion through MMP-9 depression. Moreover, mechanistic study indicated that VOSL can act as an epithelial growth factor receptor (EGFR) inhibitor to suppress EGFR activation and then to suppress its downstream MEK/P38 and PI3-K/Akt pathways. These results suggested that VOSL may be a novel anti-HCC drug candidate.Hepatocellular carcinoma (HCC) treatment remains lack of effective chemotherapeutic drugs, therefore, discovering novel anti-HCC drugs is a very attractive and urgent task. In this study, we reported VOSL (volatile oil from  Hepatocellular carcinoma (HCC) is a most malignant cancer which generally arises from various kinds of chronic liver diseases –3. The fDuring a long time, natural products have been served as important sources of novel lead structures for discovery of anticancer agents as their diverse ‘drug-like’ structure and ‘biologically friendly’ molecular qualities –9. AccorSaussurea lappa, has been used in the treatment of cancer for thousands of years in China. It includes a variety of bioactive ingredients like volatile oil, sterols, alkaloids, organic acids and so on. Our previous studies demonstrated that the volatile oil from Saussurea lappa root (VOSL), sesquiterpene lactones-rich fraction, contains the most important anti-cancer constituents [The root of tituents . Thereintituents . Cos andtituents . Therefoin vivo. VOSL showed stronger inhibition effects on human breast cancer MCF-7 xenografts than Cos or Dehy alone. Moreover, VOSL not only inhibited the growth of tumors, but also maintained weight and vitality of tumor-burdened nude mice, which is a great significance to “tumor-burdened survival” [Our previous study also revealed that Cos and Dehy exhibited synergistic anti-cancer effect urvival” . HoweverSaussurea lappa through a bioactivity-oriented screening platform in previous study , the Jun N-terminal kinases (JNK) and the p38 MAPKs plays an important part in regulating of cancer cell biological characteristics , 15. PI3Since MEK/P38 and PI3-K/Akt is the primary downstream cascades of receptor tyrosine kinases (RTKs), meanwhile, EGFR is the most important RTK in cancer cells , 18. TheThe expression of relevant factors and the number of apoptotic cells in xenograft tumors were detected by immunohistochemistry and TUNEL assay, respectively. VOSL, C+2D, Cos, and Dehy treatments could induce SMMC-7721 cell apoptosis in xenograft tumors, among them, VOSL exerted the greatest effect on inducing cell apoptosis Figure  and 6C. HCC is one of the common malignances and has high mortality due to frequent recurrence, metastasis and multi-drug resistance , 20. OurUnlimited proliferation is a hallmark of cancer cells, thus proliferation inhibition and apoptosis induction are the two main mechanisms of chemotherapeutic agents . In thisNumerous studies indicated that Cos and Dehy can modulate cell cycle progression by activating the P21/P27 pathway, which lead to cell cycle arrest in S or/and G2/M phases , 23. In Accumulating evidence showed that MAPK activation play a vital role in HCC development . We founRTKs are glycoproteins spanning cell membrane, which interact with growth factors and in turn cause the activation of intracellular catalytic domains, whereby transmit cellular signals . EGFR isin vivo. Therefore, VOSL exhibited the therapy characteristics of multiple-components and multiple-targets, which were Cos and Dehy acted on HCC xenografts directly, and other active ingredients in VOSL may act on nude mice to exert indirectly anti-HCC activity. Figuring out the indirect anti-HCC ingredients in VOSL and exploring their associated mechanism will be our topic of further research. In conclusion, our experiments showed that VOSL suppressed HCC progression by regulating EGFR pathway  showed a synergistic anti-HCC effect in the SMMC-7721 xenografts. In addition, we speculated that there should be indirect anti-HCC compounds besides direct anti-HCC compounds, Cos and Dehy, in VOSL because VOSL exhibits better anti-HCC effect than C+2D Saussurea lappa roots were crushed into powder, and extracted with 100 mL of hexane for 1h, The filtrates were evaporated to get VOSL, and then were analyzed by ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometer . Commercial Cos and Dehy were purchased from Shanghai Yuanye Biotech CO., Ltd and its purity is more than 98%. VOSL, Cos and Dehy were dissolved in dimethyl sulfoxide to 10 mg/mL, and then diluted to appropriate concentrations with cell culture medium.VOSL was prepared as previously described . Briefly2.Human HCC cell lines, SMMC-7721, Hep3B, HepG2, Huh-7, HCC-LM3, and two normal hepatic cell lines, WRL-68 and L02, were cultured in DMEM with 10% fetal bovine serum , and incubated at 37°C, 5% COThe ratio of Cos to Dehy is about 1 to 2 by weight in VOSL, therefore, in the following experiments, we generally designed four tested groups, the Cos group, Dehy group, C+2D  group, and VOSL group, to treat HCC cells. Cell viability was detected by MTT assay. 0, 5, 10, 15, 20 μg/ml of the tested samples  were added in cultured cells. After 48 h incubation, MTT reagent was added to each well, then the absorbanc at 492 nm was detected and the IC50 value of every tested sample was calculated using the Trimmed Spearman-Karber Method .To determine long-term effects of Cos, Dehy, C+2D or VOSL on the proliferation of SMMC-7721 and Hep3B cells, cells were exposed to the tested samples at IC10, IC30 and IC50 concentrations for 48 h, respectively, and rinsed with fresh medium. The resulting adherent cells were cultured continuously for 14 days in normal culture condition. Finally, the cells were fixed and stained with crystal violet for counting the clone formation.The distribution of cell cycle was detected by propidium iodid (PI) staining. SMMC-7721 and Hep3B cells were treated with Cos, Dehy, C+2D or VOSL at various concentrations  for 48 h. The harvested cells were fixed in alcohol (70%) for 24 h at 4°C, stained with PI for 30 min, then analyzed with flow cytometry .Hoechst was used to determine the morphological change of apoptotic cells. SMMC-7721 and Hep3B cells were treated with Cos, Dehy, C+2D or VOSL at IC50 concentration for 48 h, and then stained with Hochest 33342  for 15 min. A fluorescence microscope was used to observe the morphological change of apoptotic cells.SMMC-7721 and Hep3B cells were treated with Cos, Dehy, C+2D or VOSL at IC10, IC30 and IC50 concentrations, respectively, for 48 h, and stained with Annexin V-FITC/PI  for 10 min, and then analyzed by FACS Calibur  to calculated the apoptotic ratio of cells.Scarification test was used to evaluate the migration of HCC cells. SMMC-7721 and Hep3B cells were planted in 6-well plates and wounds were created with a micro-pipette tip in the cell monolayer. The cells were exposed to Cos, Dehy, C+2D or VOSL at IC5, IC10, and IC20 concentrations, respectively, for 48 h, and their migration distances were measured by Photoshop.Transwell assay with extracellular matrix (ECM) was performed to evaluate the influence of Cos, Dehy, C+2D and VOSL on tumor invasion. SMMC-7721 and Hep3B were exposed to Cos, Dehy, C+2D or VOSL at IC5, IC10, and IC20 concentrations for 48 h, and the infiltrated cells were stained with crystal violet and statistically analyzed by microscope.SMMC-7721 and Hep3B cells were cultured and incubated with Cos, Dehy, C+2D or VOSL at IC30 and IC50 concentrations for 48 h, respectively. The harvested cells were prepared to extract total protein. The extracted protein was separated on 8% - 12% SDS-PAGE, then transferred onto PVDF membrane (Beyotime Institute of Biotechnology). The blot was blocked with non-fat milk, incubated with the primary antibodies  and then with the secondary antibodies. Signals were developed using ECL reagents . GAPDH was used as an inner control.6 cells in 150 μl PBS). Tumors were developed at 10 days after cell injection. The treatment was started when tumors reached a size of 2 - 3 mm in diameter. Thirty-six mice were randomly divided into six groups. The Cos, Dehy, C+2D and VOSL-treated groups were injected intraperitoneally at a dose of 15 mg/kg/day, respectively. The positive and negative control group were administrated with 5-Fluorouracil (5-Fu) at a dose of 15 mg/kg/day, and an equal volume of vehicle, respectively. Tumor size was monitored every three days, and tumor volume was calculated using the formula: tumor volume=0.5×a×b2, where a and b represent the largest and smaller diameters, respectively.All procedures involving animals were approved by the Experimental Animal Ethics and Management Committee. Male nude mice  were purchased from SLAC Laboratory Animal Center of Chinese Academy of Sciences . SMMC-7721 cells were injected subcutaneously into the axillary fossa of nude mice . The positive cells were observed under microscope within 5 medium-power fields at magnification 200×.in vitro experimental data were derived from at least three independent experiments, and the in vivo data were collected from 6 mice. All data were expressed as mean±SD, and the significant difference was determined by using the Student's t-test. The statistical significance was set at p < 0.05.The


Bacillus aryabhattai strain SQU-R12 was isolated from date palm seedlings, where it showed a growth-promoting capacity by being able to synthesize indole-3-acetic acid phytohormone and reduce ethylene biosynthesis by producing 1-aminocyclopropane-1-carboxylic acid deaminase. The draft genome sequence of this strain is reported here. Bacillus aryabhattai strain SQU-R12 was generated in order to uncover the underlying growth-promoting mechanism of this strain, which was previously identified based on functional and biochemical characterizations . Genes within scaffolds were identified using Glimmer , and theThe DNA sequencing produced 6,310,670 total reads, including 6,231,215 mapped reads in pairs. The assembly results showed that the genome consisted of 5,584,636 bp, assembled into 118 scaffolds ranging in length from 319 to 1,177,917 bp, with an average length of 47,327 bp and a GC content of 37.74%. The genome included 5,635 putative coding sequences and 91 pseudogenes. There were 4,931 genes with a known function, representing 1,552 enzymes localized on the 639 Kyoto Encyclopedia of Genes and Genomes pathways. The list also contained 88 RNA genes, including 5 complete rRNA genes and 75 tRNAs.Bacillus oleronius based on a partial 16S rRNA gene sequence ; therefore, this strain was designated B. aryabhattai SQU-R12.The reported strain was previously identified as belonging to the species sequence ; howeverescribed . The resd-cysteine desulfhydrase  and nitroreductase proteins ; tryptopfhydrase ; severalfhydrase ; and fivfhydrase . In addifhydrase  revealedNHZZ00000000. The version described in this paper is the first version, NHZZ01000000.This whole genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number


Salvia miltiorrhiza. In this study, 28 ESTs that contained the GRAS domain were identified from a S. miltiorrhiza cDNA library. Of these, full-length sequences of five genes were cloned and sequence analysis indicated that all five proteins contain only one GRAS domain and therefore, belong to the GRAS family. The five genes were designated S. miltiorrhiza GRAS1-5 (SmGRAS1-5), which belong to groups I (SmGRAS2 and SmGRAS4), II (SmGRAS3), III (SmGRAS1), and VIII (SmGRAS5) respectively. Additionally, SmGRAS1-5 have different expression patterns in the reed head, stems, leaves, flowers, and roots of S. miltiorrhiza. In this study, the expression of SmGRAS1-5 was sensitive to Gibberellin (GA) stress and that of SmGRAS1, SmGRAS4 and SmGRAS5 was sensitive to Ethephon (Eth) stress respectively. Moreover, S. miltiorrhiza copalyl diphosphate synthases 1 (SmCPS1) and S. miltiorrhiza kaurene synthase like 1 (SmKSL1), which are two key enzymes gene in the diterpenoid biosynthesis pathway, were also response to GA and Eth stress. In addition, Dihydrotanshinone (DT-I) and Tanshinone I (T-I) content were enhanced by GA and Eth stress, Tanshinone IIA (T-IIA) content was increased by GA stress, and the accumulation of Cryptotanshinone (CT) was insensitive to both GA and Eth stress. Together, these results provide insights into functional conservation and diversification of SmGRASs and are useful information for further elucidating SmGRAS functions.The gibberellin-responsive element binding factor (GRAS) family of proteins plays an important role in the transcriptional regulation of plant development and hormone signaling. To date, there are no reports on GRAS family proteins expressed in  DELLA, HAIRY MERISTEM (HAM), SCARECROW-like (SCL) PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1), LATERAL SUPPRESSOR (LS), SCARECROW (SCR), SHORTROOT (SHR), SCARECROW LIKE3 (SCL3) [SCR and SHR genes are involved in radial organization of the root through the formation of a SCR/SHR complex [SCL3 acts as an integrator downstream of the GA/DELLA and SCR/SHR pathways, mediating GA-promoted cell elongation during root development [PAT1, SCL5, SCL13 and SCL21 genes are highly homologous in Arabidopsis thaliana and are involved in light signaling pathways. Meanwhile, PAT1, SCL5, SCL21 are positive regulators of phytochrome-A signal transduction, while SCL13 mainly participates in phytochrome-B signal transduction [AtRGL2 is involved in seed germination and is up-regulated during A. thaliana seed imbibition [Plant growth and yield is strongly influenced by hormones such as GA, Jasmonate (JA), Salicylic acid (SA) and Eth . Plants 3 (SCL3) . The SCR complex . SCL3 acelopment , 11. Thesduction –14. AtRGbibition .GRAS in response to abiotic and biotic stress. For example, SlGRAS (Solanum lycopersicum) is expressed in response to the stress induced by ethephon (Eth), gibberellic acid (GA), indole acetic acid (IAA), salicylic acid (SA), and abiotic factors in tomato [GRAS gene VaPAT1 (Vitis amurensis) enhances cold, drought, and salt tolerance in transgenic A. thaliana by modulating the expression of a series of stress-related genes [LiGRAS (Lotus japonicas) can display a phylogenetic pattern characteristic of symbiotic genes [There are many reports on n tomato . The stred genes . In respic genes .S. miltiorrhiza, is a major ingredient in traditional Chinese medicines.[GRAS genes have an affect on MEP and MVA pathways. For example, AtSCL3 influences GA signaling and homeostasis [AtPAT1, AtSCL13, and AtSCL6 are involved in phytochrome A, phytochrome B, and GA signaling, respectively [SmGRAS proteins in tanshinone metabolism, we searched and amplified SmGRAS genes from the S. miltiorrhiza transcriptome database. This research focused on the prediction of the functions of these SmGRAS genes and their response to GA and Eth treatments. Additionally, downstream of the MEP and MVA pathways that are involved in the tanshinone synthesis pathway in S. miltiorrhiza, SmCPS1 catalyzes -geranyl- -geranyl diphosphate (GGPP) to produce copalyl diphosphate (CPP), and then SmKSL1 catalyzes CPP to form miltiradiene, which is a precursor for tanshinone biosynthesis [SmCPS1 and SmKSL1 under GA and Eth stress. The changes in tanshinone content were detected under the GA and Eth stress. Finally, SmGRAS response to GA and Eth signaling and its involvement in tanshinone biosynthesis were analyzed.Tanshinone, obtained from the roots of edicines.. Tanshinedicines.. Have maedicines.–25. Meaneostasis , 11. Furectively , 14. To ynthesis . SmCPS1 ynthesis , 26, 27;S. miltiorrhiza sterile plantlets seedlings, which preserved in our lab, were cultured on an MS medium (pH 5.8) that contained 7% agarose. The hairy roots of S. miltiorrhiza were derived from these plantlets, infected with Agrobacterium rhizogenes (ATCC15834 strain), and sub-cultured every 30 days.The S. miltiorrhiza hairy roots were incubated in 6,7-V liquid medium in a shaker  with 50 mg/L GA and 200 μg/L Eth (Sigma-C0143) for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 72 h, and 8 d. Treated samples were harvested separately for each incubation period. S. miltiorrhiza hairy roots grown in liquid 6,7-V medium without GA or Eth were used as controls. Here we set three biological replicates, and each replicate contained five hairy roots.For GA and Eth treatments, 18-day-old SmGRAS gene expression, leaves, roots, stems, flowers, and reed heads were collected when the S. miltiorrhiza seedlings were at the flowering stage.To analyze tissue-specific S. miltiorrhiza GRAS (SmGRAS) were extracted from the Danshen transcriptional Resource Database (DsTRD: http://bi.sky.zstu.edu.cn/DsTRD/home.php). The full-length SmGRAS coding sequences (CDSs) were amplified by PCR using the primers listed in supplementary http://genes.mit.edu/GENSCAN.html). The theoretical isoelectric point (pI) and molecular weight (Mw) were predicted using the pI/Mw tool on the ExPASy server (http://web.expasy.org/computepi/). Phylogenetic trees were constructed using the maximum likelihood method with MEGA6.0. The resulting tree topology was reassembled by blasting the A. thaliana database [http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLSMapper–form.cgi.The full-length ORF sequences of five database . ConservS. miltiorrhiza Bunge roots were extracted using the TIANGEN reagent according to the manufacturer’s instructions . The DNase-treated RNA was reverse transcribed using SuperScript reverse transcriptase  according to the manufacturer’s instructions. Real-time quantitative PCR was performed on an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems) using SYBR Premix ExTaq . The PCR method was programmed as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. The SmActin gene was used as the endogenous control. The quantitative RT-PCR primer is shown in supplementary Total RNAs from The high performance liquid chromatography (HPLC) method involved a reference and was performed according to the method established in our laboratory . We addep<0.05 or p<0.01. Error bars indicate the standard deviation obtained from three different experiments. The figures were drawn using Origin 9.0 software.Experiments were performed with three biological and technical replicates, respectively. The relative gene quantification method (delta-delta Ct) was used to evaluate quantitative variation in gene expression. The ANOVA statistical analysis of HPLC data, and 2-ΔΔCq was used to assess the qRT-PCR results. ANOVA  followed by the least significant difference (LSD) test were calculated in SPSS  and considered statistically different at S. miltiorrhiza cDNA library. Of these, full-length coding sequences (CDS) of five genes were cloned and our analysis indicated that all five proteins contained only one conserved GRAS domain, suggesting that they belong to the GRAS protein family. We designated the genes as gibberellin-responsive element binding factor 1–5 (SmGRAS1–5). The SmGRAS1–5 gene IDs are KY435886, KY435887, KY435887, KY435888, KY435889, and KY435890, respectively. The ORFs of the five genes did not contain introns and encoded 489, 459, 748, 526, and 335 amino acids, respectively. Furthermore, nucleus location signals (NLS) were identified in the five SmGRAS genes. These five SmGRAS proteins belong to groups I (SmGRAS2 and SmGRAS4), II (SmGRAS3), III (SmGRAS1), and VIII.(SmGRAS5) These groups may play a role in phytochrome A and B, light, and GA signal transduction pathways during plant growth and development [In this study, 28 ESTs whose amino acid sequences contained a GRAS domain were identified from a SmGRAS phylogenetic tree construction, was identified from A. thaliana protein databases. The phylogenetic tree showed that the five SmGRAS proteins developed from changes in gene structure over the course of S. miltiorrhiza evolution. The SmGRAS1 clustered into group III, SmGRAS2 and SmGRAS4 clustered into group I, SmGRAS3 clustered into group II and SmGRAS5 clustered into groups VII , and then decreased significantly after 8 days . The gene expression SmGRAS2 at 1 h and 24 h and SmGRAS4 at 1 h significantly were upregulated by the GA treatment (p<0.01). SmGRAS3 expression decreased in the initial stage of treatment and then increased until 24 h after treatment (p<0.01). The gene expression level changes showed that SmGRAS5 was sensitive to exogenous GA stress, especially after 2 h and 8 h (p<0.01) of GA treatment. In addition, the gene expression levels of SmCPS1 and SmKSL1, which encode the key enzymes in tanshinone biosynthesis, were measured. The expression of SmCPS1 was significantly up-regulated after 1 h, but not at 8 h and 12 h of GA treatment (p<0.01) . Thus, the expression of the SmGRAS1-5 and SmCPS1 and SmKSL1 genes were all responsive to GA stress and exhibited spatiotemporal relationship to each other.ynthesis , 11, 13;r 8 days . The gen(p<0.01) ; howeverSmGRAS genes respond to Eth signaling, we measured gene expression levels of SmGRAS1-5 during Eth treatment. The gene expression of SmGRAS1 decreased after 1 h, 8 h, and 72 h (p<0.01), and significantly increased at 2 h and 4 h (p<0.01) of treatment. However, SmGRAS2 decreased at 1 h, 4 h, 8 h, and 12 h, but then increased after 24 h of Eth treatment. The lowest expression level of SmGRAS2 was measured at 1 h of treatment and was the highest after 8 days of Eth exposure (p<0.01). SmGRAS3 increased at 2 h and 8 days (p<0.01), but the expression decreased at the other treatment times. SmGRAS4 was significantly upregulated by Eth treatment after 2 h (p<0.01) and SmGRAS5 showed a wave-like response to Eth stress at 2 h, 8 h, and 72 h (p<0.01). In addition, the gene expression level of SmCPS1 significantly increased at the 1 h, 2 h, 4 h, and 8 day time points under Eth stress (p<0.01). Like the expression pattern of SmCPS1, SmKSL1 was significantly induced by Eth stress at 2 h, 24 h, and 8 days, respectively , cryptotanshinone (CT), tanshinone I (T-I), and tanshinone IIA (T-IIA) compounds in S. miltiorrhiza. Our study used exogenous GA and Eth to treat S. miltiorrhiza hairy roots. The results showed an accumulation of DT-I and T-I induced by Eth (p< 0.01) on the eighth day of treatment (p< 0.05 or p< 0.01). Conversely, the accumulation of CT was insensitive to exogenous GA signaling. Our results showed that the accumulation of tanshinone compounds was not significantly different from the control at the preliminary stages of exogenous GA and Eth stress, but did differ significantly in their accumulation after eight days of treatment with either exogenous compound. These results suggest that the accumulation of tanshinone compounds is sustainable under exogenous GA and Eth stress.The secondary metabolism of reatment . Howeverreatment , especiaMYB, bHLH, AP2/ERF, WRKY, SPL and NAC have been verified [GRAS family genes, which specifically respond to GA signaling, play important roles in the regulation of diterpenoid biosynthesis [GRAS family is found in several plant species, including Populus trichocarpa, A. thaliana, Oryza sativa, Brassica pekinensis, Prunus mume, and Pinus radiata [GRAS protein family is functionally characterized in Zea mays, Petunia hybrids, Medicago truncatula, and Lilium longiflorum [AtSCL14 interacts with a TGA product, and this complex seems to be involved in the activation of a general broad-spectrum detoxification network upon challenge with xenobiotics like auxin and SA [Many TFs take part in plant secondary metabolite regulation , and theverified –32. In aynthesis , 11, 14. radiata –36; howegiflorum –40. For n and SA .GRAS family is one type of plant specific transcription regulatory factors. The originally discovered members of the family are GAI, RGA and SCR, which named by the characteristics of the alphabet [SHR, SCR, LS, HAM, PAT1, and DELLA, which are involved in biological metabolism processes of root radial growth, the growth of axillary buds, meristem maintenance, phytochrome signaling pathways, and GA signaling pathways [A. thaliana, the group AtSHR, AtPAT1, AtSCL1, AtSCL11 and AtSCL6 are related to phytochrome A, phytochrome B, shoot-stem development, abiotic stress response and GA signaling, respectively, however, AtSCL13 function is unknown [S. miltiorrhiza. In this study, the SmGRAS1-5 have different expression patterns in the reed head, stem, leaf, flower, and root. Indeed, SmGRAS2, SmGRAS3, SmGRAS4, and SmGRAS5 had the highest expression levels in leaves than in any other tissues. This was consistent with the phylogenetic results that arranged these genes closed to the SCL subgroup, suggested they might have a relationship with phytochrome A, phytochrome B and GA signaling [SmGRAS1 was expressed in all tested tissues and was closely aligned with AtSHR, indicating that it might be involved in shoot-stem development [SmGRAS4 was highly expressed in the flower, which further implies its role in phytochrome A and B function.The alphabet . The GRApathways . By anal unknown , 42. Thuignaling . Howeverelopment . In addiGRAS family members are important in responding to biotic and abiotic stresses. Earlier reports show that GRAS family members were expressed in response to low temperature, drought, infiltration, and hormones in A. thaliana, rice, bergamot, P. euphratica, and tobacco [SCARECROW-LIKE3 (SCL3), which enables the regulation of GA feedback [GRAS gene (NtGRAS1) that is significantly up-regulated under H2O2 and salicylic acid stress [SmGRAS1-5 gene expression was sensitive to exogenous GA and Eth stress  and S. miltiorrhiza 1-deoxy-d-xylulose- 5-phosphate synthase (SmDXSII), and the co-expression of S. miltiorrhiza hydroxy-3methylglutary CoA reductase (SmHMGR) and SmGGPPS, which produces higher levels of tanshinone in S. miltiorrhiza hairy roots [Ophiorrhiza pumila [Anisodus acutangulus hairy roots [SmCPS1 and SmKSL1 are genes that encode key enzymes for diterpene biosynthesis [SmGRAS1-5, SmCPS1, and SmKSL1 genes respond to GA and Eth stress. Combined with previous results, the stress-inducible expression patterns of the SmGRAS genes suggest that they might have a relationship with the gene expression regulation of SmCPS1 and SmKSL1. As both tanshinone and GA are diterpenoids, their biosynthesis pathways are similar. These results implicate that SmGRASs are very likely to be transcriptional regulation activators of SmCPS1 and SmKSL1. Moreover, GRAS protein function may be involved in diterpenoid biosynthesis; for example, Phyllostachys edulis SCL3 (PeSCL3) may influence root development by regulating GA biosynthesis [SmGRAS1-5 expression correlates with tanshinone biosynthesis increase under GA and Eth stress.Tanshinone is a secondary metabolite product of ry roots –49. Co-oa pumila . In addiry roots . The resry roots , 30. Theynthesis , 26, 27.ynthesis . DELLA pynthesis . Thus, o+, and yeast elicitors (YE) increased accumulation of tanshinone in S. miltiorrhiza hairy roots and transgenic S. miltiorrhiza hairy root lines [SmGRAS1-5 participation in the regulation of tanshinone biosynthesis, which is mediated by GA and Eth signaling.Reports on exogenous treatment with plant hormones or yeast components can elicit enhanced tanshinone accumulation in plant cell or tissue culture. For example, treatment with exogenous methyl jasmonate (MJ), salicylic acid (SA), Agot lines –55. SimiSmGRAS genes from of S. miltiorrhiza and named them SmGRAS1-5, respectively. SmGRAS1 was expressed in all tissues and has a close phylogenetic relationship with AtSHR. SmGRAS2-5 were expressed at the highest levels in leaves, and share a close relationship with AtPAT and AtSCL. Our research shows that SmGRAS1-5 expression is sensitive to exogenous GA, and SmGRAS1, SmGRAS4, and SmGRAS5 is sensitive to exogenous Eth stress, while the SmGRAS2 and SmGRAS3 are either up-regulated or down-regulated at different time-points of Eth treatment. Interestingly, the gene expression of SmCPS1 and SmKSL1 increased with exogenous GA and Eth stress, as did the accumulation of DT-I and T-I content, while T-IIA content was induced only by GA stress after eight days. Overall, we show that SmGRAS1-5 might participate in the regulation of tanshinone biosynthesis and are mediated by GA or Eth signaling. Thus, this study provides a resource for the selection of candidate genes for the further characterization of S. miltiorrhiza and enhancement of tanshinone production through the engineering of secondary metabolic pathways that could positively manipulate the production of target molecules in this “heal-all” medicinal plant.We amplified five 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.


Pseudomonas sp. strain Ep R1, isolated from the roots of the medicinal plant Echinacea purpurea. The elucidation of this genome sequence may allow the identification of genes associated with the production of antimicrobial compounds.In this announcement, we detail the draft genome sequence of the  Pseudomonas sp. strain Ep R1, a strain isolated from the roots of Echinacea purpurea, an MP with immunomodulant, antiviral, and antimicrobial activity  may contribute to the therapeutic properties of these plants . Here weactivity . The E. onducted . In partdophytes  and of c complex . Moreove complex .Pseudomonas sp. Ep R1 was determined by a 2- × 300-bp paired-end approach using the MiSeq sequencing system . A total of 1,148,852 paired-end reads were obtained, representing approximately 100× coverage of the whole genome. De novo assembly was performed using SPAdes 2.3  through MeDuSa scaffolder . The G+C content is 65.5%, similar to that of other Pseudomonas genomes sequenced so far. Automated annotation of the Pseudomonas sp. Ep R1 draft genome sequence using the NCBI Prokaryotic Genome Annotation Pipeline detected 6,001 protein-coding genes, 67 RNA-coding genes , and 173 pseudogenes.The genome sequence of Ades 2.3 , which gPseudomonas Ep R1 genome harbors 6 clusters involved in the biosynthesis of streptomycin, stenothricin, pimaricin, type 3 polyketide synthase (T3PKS), siderophore (desferrioxamine B), and nonribosomal peptide synthetase (NRPS) (amychelin). Moreover, the genome sequence was analyzed through CARD  putatively involved in antibiotic resistance, some conferring specific resistance to fluoroquinolone, mupirocin, beta-lactam, aminocoumarin molecules, and others involved in regulatory or inactivating systems and efflux pumps.Genes involved in the biosynthesis of secondary metabolites with antimicrobial activity were searched. The analysis was performed within an antiSMASH shell , which rugh CARD , which lMWTQ00000000. The version described in this paper is the version MWTQ00000000.1.This whole-genome shotgun project has been deposited in GenBank under the accession no.


Many tumors sustain elevated levels of reactive oxygen species (ROS), which drive oncogenic signaling. However, ROS can also trigger anti-tumor responses, such as cell death or senescence, through induction of oxidative stress and concomitant DNA damage. To circumvent the adverse consequences of elevated ROS levels, many tumors develop adaptive responses, such as enhanced redox-protective or oxidatively-generated damage repair pathways. Targeting these enhanced oxidative stress-protective mechanisms is likely to be both therapeutically effective and highly specific to cancer, as normal cells are less reliant on such mechanisms. In this review, we discuss one such stress-protective protein human MutT Homolog1 (MTH1), an enzyme that eliminates 8-oxo-7,8-dihydro-2’-deoxyguanosine triphosphate (8-oxodGTP) through its pyrophosphatase activity, and is found to be elevated in many cancers. Our studies, and subsequently those of others, identified MTH1 inhibition as an effective tumor-suppressive strategy. However, recent studies with the first wave of MTH1 inhibitors have produced conflicting results regarding their cytotoxicity in cancer cells and have led to questions regarding the validity of MTH1 as a chemotherapeutic target. To address the proverbial "elephant in the room" as to whether MTH1 is a bona fide chemotherapeutic target, we provide an overview of MTH1 function in the context of tumor biology, summarize the current literature on MTH1 inhibitors, and discuss the molecular contexts likely required for its efficacy as a therapeutic target. MTH1 germline knockout mouse is developmentally normal and displays no pathological phenotypes, barring low incidences of spontaneous late age-onset lung, liver, and stomach tumors [MTH1-null mouse, the MTH1-null/OGG1-null mouse, or mouse embryonic fibroblasts (MEFs) derived from these animals with oxidant-generating chemicals such as the mitochondrial poison 3-nitropropionic acid or hydrogen peroxide has been reported to increase mitochondrial DNA oxidation and induce neurotoxicity or general cytotoxicity [Human MutT Homolog 1  is an 18 kD Nudix pyrophosphatase that hydrolyzes the oxidized purine deoxyribonucleotides, 8-oxo-dGTP, as well as the less frequently-occurring 2-OH-dATP and 8-oxo-dATP, and their ribonucleotide analogs into their monophosphate equivalents ,4. Theseh tumors . Treatintoxicity ,17,18. TUnlike normal cells, tumors produce high levels of oxidants due to chronically-hyperactivated mitogenic and pro-survival signaling, as well as metabolic alterations . MTH1 prMTH1-null animals is a result of increased mutagenic burden in the MTH1-null mouse [MTH1-null animals do not possess elevated levels of the hallmark G -> T tranversions expected due to increased genomic 8-oxoGua incorporation [OGG1-null background [MTH1-null mice likely arise through alternate mechanisms, potentially through MTH1 inhibition-induced genomic instability in the setting of aging-associated oxidative stress. Consistent with this idea, we were the first to show the acute effects of MTH1 inhibition in human cells are irreparable genomic strand breaks that are capable of inducing p53-dependent cell senescence under high oxidant, but not low oxidant, environments [MTH1 mRNA levels positively correlate with KRAS levels, even in early-stage human NSCLC tumors, and that introduction of oncogenic RAS is sufficient to upregulate MTH1 mRNA and protein expression [Due to its canonical anti-mutator function, the prevailing notion was that MTH1 plays a tumor-suppressive role in cells, and that the elevated late-onset spontaneous tumor formation observed in ll mouse ,15. Howeporation  and, in ckground . Thus, tronments . Researcronments . We thenronments ,32. Convronments . Importaronments . Based oronments , an inheronments . Congruepression .Consistent with our results, meta-analyses of public datasets indicate that RAS-driven tumors, such as pancreatic cancer or lung adenocarcinoma, show poor disease prognosis and higher relapse rates in tumors where MTH1 is elevated . NotablyBuilding on our observations regarding the role of MTH1 in preventing genomic DNA breaks and OIS ,31, the The first-in-class MTH1 inhibitors, TH287 and TH588, reported in 2014 , consistThe (S)-enantiomer of crizotinib, a well-known Met/ALK inhibitor, was also reported in 2014 as a nanomolar suppressor of MTH1 activity . By cont15 and 19) showed better potency and engagement vis-à-vis TH588. (S)-crizotinib, by contrast, showed much weaker target engagement in their comparison. This study also evaluated the effects of MTH1 depletion via siRNA and CRISPR knockout on U2OS and SW480 cells, respectively. Unlike the studies of Gad et al. and Huber et al. [19 and assessed a panel of DNA damage markers via Western blotting (see 1 in their study) induced the elevation of pSer15-p53, total p53, and cleaved PARP1, re-capitulating the original results with this compound [Following these first-in-class inhibitors, several other groups published studies describing their own MTH1 inhibitors. Kettle et al.  at Astrar et al. ,36, theyr et al. ,36. Kettr et al.  also treting see . They focompound , their o1) treatment, which generated γH2AX-containing DSB foci in the original study [19 elevated total p53 levels (indicating induction of a genotoxic response) and, although cleaved PARP levels were observed only for TH588 (compound 1), they did not assay for proliferation defect markers such as p21cip1 or p27kip1, which can be induced downstream of p53 and may have occurred with their compound 19. The use of population-based, rather than single cell, assays renders their results inconclusive rather than negative. They also observed no effects on viability with a commercial siRNA against MTH1, but were able to do so with the siRNA sequence used by Gad et al. [While these results  may seemal study . Interesd et al. . In and d et al. . Thus, td et al.  is not cd et al. ,27,30,31d et al.  and foun19, and found all three compounds killed cells transfected with the siMTH1 sequence used by Gad et al. [Kettle et al.  treated d et al. . They cod et al. ). Thus, d et al. . WithoutAccording to proteomics profiling carried out by Kawamura et al. , the TH5Petrocchi et al. at MD Anderson also reported their in-house MTH1 inhibitors, IACS-4619 and IACS-4759 . Both ofRecently, Helleday and colleagues published their best-in-class inhibitor, TH1579, which was able to reduce viability in a number of cell lines, concomitantly elevating p53 and DNA DSB markers . In thisWarpman et al.  also carTaken collectively, the reported discrepancies among recent studies characterizing MTH1 inhibitors in cancer models reinforce the idea (supported in part by our findings) ,27 that MTH1-null vs. wild-type animals [Cancer cells are characterized by metabolic plasticity, which affects the cellular redox state, as well as ROS production. In this regard, it was recently reported that the loss of the Von-Hippel Lindau (VHL) factor, a tumor suppressor and E3 ubiquitin ligase that degrades the hypoxia-inducible factor HIF under normoxic conditions, sensitizes cells to MTH1 inhibition . The una animals  potentia animals , could m animals . Thus, a animals  and may Finally, we note that a key difference between the studies that show a tumor-suppressive effect of MTH1 inhibition (including ours) ,35,36,42In conclusion, the role of MTH1 biology in tumors is still in its nascent stages and the utility of its therapeutic inhibition, the proverbial current “elephant in the room”, cannot be evaluated without a comprehensive understanding of the complex molecular factors that governs its function in tumor cells . While t


Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.The shell of the pearl oyster ( Patinopecten yessoensis) [Hyriopsis schlegelii) [Pinctada fucata) [Calmodulin (CaM) seems to play important roles in regulating the uptake, transport, and secretion of calcium in molluscan shell formation, such as yesso scallop (soensis) , freshwalegelii) , and pea fucata) . The pea fucata)  and may  fucata) . The pea fucata) , 7, wher fucata) . The highttp://www.oysterdb.com/), as described in Six organ types  were collected from slaughtered Pacific oysters, snap-frozen in liquid nitrogen, and stored in the Ultra-low Temperature Freezer (−80°C) until extracting RNA. The total RNA was extracted using Trizol reagent . All of the primers were designed referring to one of the predicted CaM sequences (CGI_10006482) deposited in OysterBase  was got using SuperScript II (Invitrogen) and the primer GSP1; after synthesizing the cDNA, the deoxycytidine triphosphate and terminal deoxynucleotide transferase were used to add a poly-C tail to the 3′ end of the cDNA; the primer pair of GSP2 and AAP was used to carry out the first PCR, the product of which was reamplified by GSP3 and AUAP according to the same thermal profile, which includes 2 minutes of an initial denaturation at 94°C, then 35 cycles comprising 30 seconds of denaturation at 94°C, 30 seconds of annealing at 55°C, and 2 minutes of extension at 72°C, 7 min of final extension at 72°C, and holding at 4°C at last. For the 3′-RACE, Oligo dT-3 sites Adaptor Primer (Takara) and AMV Reverse Transcriptase XL (Takara) were used to get the RACE-ready cDNA. The temperature profile employed was as follows: 10 minutes of at 30°C, 30 minutes at 50°C, 5 minutes at 95°C, and 5 minutes at 5°C. All of the RACE-ready cDNA products were amplified using sense primers (GSP-3′ RACE) and antisense primers , where the temperature profile was as follows: 5 minutes at 95°C; 30 cycles including 30 seconds at 94°C, 30 seconds at 55°C, and 5 minutes at 72°C; and then 7 minutes at 72°C. The PCR products of 5′-RACE and 3′-RACE were purified, ligated into the pMD18-T vector, and sequenced, respectively.μL volume, which contained 10 μL of SYBR Green Master Mix (Tiangen), 5 pmol of each primer, 7 μL of water, and 1 μL of cDNA obtained by reverse transcription from each tissue  RNA using Moloney murine leukemia virus reverse transcriptase . Each cDNA sample was amplified with the primers CaM-F and CaM-R in three duplicates, each cDNA sample with the primers eLF1-F and eLF1-R in three duplicates, and the negative control in three duplicates on the same 96-well microplates. The PCR program was used according to the recommendation in the manufacturer. The specificity of the PCR products was evaluated by melting-curve analysis. The data were analyzed using the 2−ΔΔCt method.The mRNA levels of the cgCaM gene were measured using the method of Quantitative Real-time PCR. The quantities of cgCaM mRNA in different organs were standardized against that of elongation factor 1 (eLF1) mRNA to compensate for variations in volume of input mRNA. All of the reactions were carried out in a 20 http://ca.expasy.org/tools/pi_tool.html), and another online tool, the PSORT II program, was used to predict the N-terminal signal and cell location (http://psort.ims.u-tokyo.ac.jp/). Functional domain (IPR) prediction was performed at http://prosite.expasy.org/.The online tool, Compute pI/Mw, was used to predict the isoelectric point and molecular weight of cgCaM protein . A multiple sequence alignment was obtained using CLUSTALW. Phylogenetic tree was constructed using the neighbor-joining algorithm of MEGA 6 [The CaM sequences of other mollusks were downloaded from NCBI and the CaM sequence fromf MEGA 6 . The robhttp://pfam.xfam.org/clan/CL0220, converted them into HMM file using hmmbuild tool in HMMER software and searched these motifs in the genomes of Caenorhabditis elegans, Schistosoma ansoni, Schistosoma haematobium, Capitella teleta, Helobdella robusta, Culex quinquefasciatus, Drosophila melanogaster, Octopus bimaculoides, Daphnia magna, Daphnia pulex, Ciona intestinalis, Branchiostoma floridae, Callorhinchus milii, Saccoglossus kowalevskii, Aplysia californica, Biomphalaria glabrata, Pinctada fucata, Lingula anatina, Acropora digitifera, Strongylocentrotus purpuratus, Lottia gigantea, Homo sapiens, Gallus gallus, Crassostrea gigas, Chrysemys picta, Pelodiscus sinensis, Lepisosteus oculatus, and Salmo salar using hmmsearch tool and calculated the motif numbers in these genomes. According to the degree of calcification, Caenorhabditis elegans, Schistosoma ansoni, Schistosoma haematobium, Capitella teleta, Helobdella robusta, Culex quinquefasciatus, Drosophila melanogaster, Octopus bimaculoides, Daphnia magna and Daphnia pulex were placed into the lowly calcified group; Ciona intestinalis, Branchiostoma floridae, Callorhinchus milii, Saccoglossus kowalevskii, Aplysia californica, Biomphalaria glabrata, Pinctada fucata, Lingula anatina, Acropora digitifera, Strongylocentrotus purpuratus, and Lottia gigantea were placed into moderately calcified group; Homo sapiens, Gallus gallus, Crassostrea gigas, Chrysemys picta, Pelodiscus sinensis, Lepisosteus oculatus, and Salmo salar were placed into highly calcified group.We downloaded the Alignment file (Stockholm format) of the EF hand clan members from The full-length cgCaM cDNA, which comprised 648 nucleotides, was obtained using the RACE technique. As shown in  Pinctada fucata (pfCaM and pfCaLP).The cgCaM peptide was predicted to have an isoelectric point of 4.24 and a molecular weight of 12.86 kDa using the Compute pI/Mw tool. According to the PSORT II program, the cgCaM peptide was predicted to have no N-terminal signal peptide and it was expected to be located in the cytoplasm (probability = 60.9%), which is similar to the CaM and CaLP proteins inAfter functional domain prediction, it was found that the cgCaM protein contained three EF-hand calcium-binding domain (CA_BIND) domains at amino acids 8–43, 45–80, and 81–113, and pfCaM also had three CA_BINDs . We did p < 0.05).The mRNA expression levels of the cgCaM gene were examined by real-time PCR and compared indifferent organs . As shown in As shown in It was found that the numbers of some EF hand family members, including EFhand_Ca_insen, EF-hand_1, EF-hand_2, EF-hand_3, EF-hand_4, EF-hand_5, EF-hand_6, EF-hand_7, EF-hand_8, EF-hand_9, EF-hand_10, EF-hand_11, EF-hand_like, and IQ, in highly calcified species were higher than those in lowly and moderately calcified species . InteresThe shell is one of the most important features of mollusks because it is related to their health and growth. Thus, elucidating the mechanism of shell formation will be beneficial for mollusk culture. PfCaM and pfCaLP are believed to play important roles in shell formation of the pearl , 11 oyst Hydroides elegans [The mantle is an important organ for shell formation , 13, alt elegans  (Figure It was found that highly calcified species had more EF hand clan members in their genomes , hinting


Lonicera macranthoides is an important medicinal plant widely used in traditional Chinese medicine. Luteoloside is a critical bioactive compound in L. macranthoides. To date, the molecular mechanisms underlying luteoloside biosynthesis are still largely unknown. In this work, high performance liquid chromatography (HPLC) was employed to determine the luteoloside contents in leaves, stems, and flowers at different developmental stages. Results showed that senescing leaves can accumulate large amounts of luteoloside, extremely higher than that in young and semi-lignified leaves and other tissues. RNA-Seq analysis identified that twenty-four differentially expressed unigenes (DEGs) associated with luteoloside biosynthesis were significantly up-regulated in senescing leaves, which are positively correlated with luteoloside accumulation. These DEGs include phenylalanine ammonia lyase 2, cinnamate 4-hydroxylase 2, thirteen 4-coumarate-CoA ligases, chalcone synthase 2, six flavonoid 3′-monooxygenase (F3′H) and two flavone 7-O-β-glucosyltransferase (UFGT) genes. Further analysis demonstrated that two F3′Hs (CL11828.Contig1 and CL11828.Contig2) and two UFGTs (Unigene2918 and Unigene97915) might play vital roles in luteoloside generation. Furthermore, several transcription factors (TFs) related to flavonoid biosynthesis including MYB, bHLH and WD40, were differentially expressed during leaf senescence. Among these TFs, MYB12, MYB75, bHLH113 and TTG1 were considered to be key factors involved in the regulation of luteoloside biosynthesis. These findings provide insights for elucidating the molecular signatures of luteoloside accumulation in L. macranthoides. Lonicera macranthoides, a member of the Caprifoliaceae family, is a medicinal plant primarily distributed in Southern China. It has been widely used as a critical raw material in traditional Chinese medicine for thousands of years, because it can effectively treat H1N1, respiratory syndrome, and hand-foot-and-mouth disease . Furthermore, L. macranthoides extracts are utilized as important and indispensable ingredients in functional foods, beverages, wine and cosmetics [L. macranthoides [L. macranthoides remain controversial because of the distinctive accumulation patterns of bioactive compounds, especially flavones in flower buds. Luteolin and luteoloside are the main flavones in Lonicera. Previous studies demonstrated that the flavones are accumulated at high levels in L. japonica, which are approximately two fold higher than those in flower buds of L. macranthoides [L. macranthoides was distinguished from L. japonica indicated by the report that luteoloside is less abundant in flowers of L. macranthoides than in those of L. japonica . However, Yuan et al. [L. japonica and L. macranthoides. To date, there has been a longstanding controversy regarding the luteoloside accumulation in L. macranthoides. Hence, the knowledge of quantitative profiling for luteoloside is of major importance for characterizing molecular basis of luteoloside biosynthesis and has significant implications for health benefits of L. macranthoides.nthoides . These cnthoides ,5,6. Hownthoides . Accordin et al.  found noLonicera by using engineering strategies. Luteoloside is derived from the phenylpropanoid pathway, which can be divided into two stages [trans-cinnamic acid via phenylalanine ammonia-lyase  and then to p-coumaric acid via trans-cinnamate 4-hydroxylase . Subsequently, p-coumaroyl-CoA is generated via 4-coumarate-CoA ligase , which is the precursor for a variety of important secondary metabolites [PAL1, C4H2 and 4CL2 positively correlates with the content of luteolin, the substrate of luteoloside biosynthesis, in the leaves and flower buds of L. japonica. These findings are consistent with the reports showing a positive correlation was found between the enzyme activities of PAL, C4H and 4CL and the accumulation of luteoloside [L. japonica. Chalcone synthase  and chalcone isomerase  are the key regulatory enzymes required for the conversion of p-coumaroyl-CoA to naringenin [O-β-glucosyltransferase  [L. japonica [L. japonica [LmFNSII-1.1, might be the key gene involved in flavone accumulation in the buds of L. macranthoides. Despite recent advances, the publications concerning the molecular evidence for a relationship between luteoloside abundance and organ development of L. macranthoides are limited. It remains a mystery that which enzymes or genes are responsible for luteoloside accumulation in L. macranthoides.Luteoloside is an important plant flavone , which eo stages . First, abolites  observedjaponica ,14. VeryMYB11/12/111 in Arabidopsis [VvMYBF1 in Vitis vinifera [EsMYBF1 in Epimedium sagittatum [MdMYBA [MdMYB1 [MdMYB10 [VvMYBA1 and VvMYBA2 in grape [PyMYB10 in pears [AtMYBL2 [AtMYB4, AtMYB60 [AmMYB308 [FaMYB1 [Lonicera are available [Flavonoid biosynthesis involves a multitude of regulatory proteins. Some transcription factors (TFs), such as R2R3-MYB, bHLH and WD40, can regulate the genes encoding biosynthetic enzymes by binding their promoters. To date, MYBs controlling flavonoid biosynthesis have been identified in many plant species. bidopsis ,16, VvMYvinifera  and EsMYgittatum  were cha [MdMYBA , MdMYB1  [MdMYB1  and MdMY[MdMYB10  in applein grape ,23, and in pears  control [AtMYBL2 , AtMYB4, AtMYB60 ,27, AmMYAmMYB308  and FaMY [FaMYB1 . Furthervailable . Thus, iL. macranthoides was performed using the Illumina HiSeq™ 2000 platform to explore the key genes encoding biosynthetic enzymes and regulator proteins associated with the luteoloside biosynthesis in L. macranthoides. These results provide genomic resources for future enhancement of luteoloside production by genetic strategies in L. macranthoides.In the present study, luteoloside in different tissues and developmental stages was determined using high performance liquid chromatography (HPLC) and transcriptome-wide sequencing in Luteoloside contents in the leaves, stems and flowers were quantified by HPLC . The conL. macranthoides, two libraries including SL and YL were established for RNA-Seq analysis. The raw data generated by sequencing for each library ranged from 82.4 to 87.5 million reads. After filtering, a total of 76.8 to 82 million clean reads were obtained, accounting for 93% of the raw reads, with an average GC % of 44.19% for all clean reads being  showed significant similarity to known proteins. Among them, 101,618, 68,308, 68,713, 67,523, 55,312 and 56,734 unigenes can be annotated in the NCBI nonredundant (Nr), nucleotide sequence (Nt), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of proteins (COG), and GO databases, respectively , 16 for cellular component . Several metabolic pathways, including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, lipid and fatty acid metabolism, starch and sucrose metabolism, tryptophan metabolism, cutin and wax biosynthesis, and flavone and flavonol biosynthesis displayed outstanding enrichments . This fiectively , by whicPAL, one C4H, fourteen 4CL, eight CHS, two CHI, one FNS, eight F3′H and seven UFGT genes (PAL2 (Unigene109136), C4H2 (CL11118.Contig2) and the majority of 4CL genes (13 out of 14) were significantly upregulated in senescing leaves compared with young leaves. We also found that CHS2 (CL19869.Contig1), six F3′H , two UFGT (Unigene2918 and Unigene97915) genes displayed dramatic increases in transcript levels during leaf senescence. Meanwhile, CHI and FNS paralogous genes appeared remarkable down-regulation in senescing leaves, compared with those in young leaves  in several species and also displayed amino acid sequence similarities with UF7GTs in Arabidopsis and Scutellaria (To further characterize the molecular properties of the  species . Two UFGtellaria .MYBs (16 out of 30) underwent significant down-regulation in senescing leaves, such as those orthologous to TT2, AtMYB12, PsMYB1, SlMYB46, VvMYB44, and VvMYB306. Meanwhile, the remaining differentially expressed MYBs, including those homologous to AtMYB48, VvMYB1R1, VvMYB75, AmMYB305, EsMYB13, and GhMYB9, showed dramatically increased transcripts in senescing leaves , 38% identity with TTG in Medicago (XP_003624554) and 37% identity with TTG1 in Malus (AHM88209). QRT-PCR analysis also showed that the transcripts of several unigenes, including those homologous to MYB, bHLH and TTG1, were in dramatic regulation during leaf senescence . Our study demonstrated that luteoloside was accumulated at extremely higher level in senescing leaves of L. macranthoides  coincided precisely with the high level of luteoloside in senescing leaves  and apigenin-7-O-glucoside in FNSII-overexpression transgenic lines in Lonicera [L. japonica (LjFNSII-1.1) exhibits an apparently higher catalytic activity (approximate 4-fold) than that in L. macranthoides (LmFNSII-1.1). They concluded that the less abundance of flavones in flowers of L. macranthoides compared with that in L. japonica might be attributed to the weak catalytic efficiency of LmFNSII. However, in our dataset, a FNSII gene (Unigene2335) exhibiting 99% identity with LmFNSII-1.1 (deletion of a leucine at position 22 in LmFNSII-1.1) displayed lower transcript abundance in senescing leaves than in young leaves (Unigene2918 and Unigene97915), homologs of AtUF7GT and SbUF7GT  to produce eriodictyol and then FNSII converts eriodictyol to luteolin, which can be metabolized to luteoloside via UFGTs. Therefore, although the transcript levels of FNSII in young leaves were approximately 7-fold higher than that in senescing leaves , which are homologs of AtMYB12, were more abundantly expressed in young leaves  through formation of highly dynamic MYB/bHLH/WD40 (MBW) complexes [AtbHLH113 homologs (CL3655.Contig4 and Unigene11884) and four AtbHLH78 homologs, displayed higher mRNA levels in senescing leaves than that in young leaves, which is positively correlated with the accumulation of luteoloside, suggesting their involvements in the regulation of luteoloside generation. This result is in accordance with the findings in Arabidopsis that AtbHLH113 is predicted to interact with PAP1/MYB75 modulating anthocyanin biosynthesis [bHLH, the key component involved in the regulation of flavonoid biosynthesis, was observed to activate late biosynthetic genes    in L. maynthesis . In our Lonicera macranthoides Hand-Mazz (cv. Yu Lei 1#) used in the experiments was planted in a greenhouse of Chongqing University of Arts and Sciences . Plant tissues including leaves, stems and flowers at different developmental stages were collected from 5-year-old seedlings. Leaves and stems at three developmental stages including young leaves and stems, semi-lignified leaves and stems, senescing leaves and stems were obtained and illustrated in v/v) by ultrasonication for 30 min. The extracts were cooled to room temperature and centrifuged at 4000 rpm for 10 min. Afterward, the supernatant was filtered through a 0.22 µM microfiltration membrane for luteoloside analysis by HPLC.Tissue samples were subjected to lyophilization and homogenization with a grinding miller. The powder was then passed through a 40-mesh (420 μm) standard sieve before extraction. A total of 0.25 g homogenized samples was extracted with 20 mL of ethanol . A 15% B linear gradient was conducted from 0.01 min to 8.00 min, followed by a 15~50% B linear gradient from 8.01 min to 25.00 min, and finally a 50% B linear gradient from 25.01 min to 40.00 min, at a flow rate of 0.7 mL/min. The column and the detector were operated at 35 °C. A volume of 20 μL supernatant was injected and the detection wavelength was monitored at 360 nm. Luteoloside standard (>98%) was purchased from SIGMA . The luteoloside contents were analyzed in triplicate and calculated based on peak area measurements. Statistical significance was performed with SPSS using Duncan’s new multiple range test.HPLC was performed on a Shimadzu LC-20A HPLC analyzer , equipped with a LC-20AT pump, a SIL-20A auto sampler, a CBM-20A system controller, a SPD-M20A diode array detector (DAD)and a CTO-20A column oven. Separation was carried out on a Shimadzu Shim-Pack VP-ODS C18 column  using gradient elution. Mobile phase A was 2% formic acid in deionized water, while phase B was 2% formic acid in methanol  according to manufacturer’s instruction. RNA-Seq was performed at Hangzhou 1GENE Technology Co., Ltd. . Illumina TruSeq™ RNA Sample Prep Kit (Cat# RS-122-2001)  was employed to construct cDNA library according to the manufacturer’s protocol. Briefly, Poly(A) mRNA was enriched from 5 µg total RNA using oligo (dT) magnetic beads and fragmented in a thermomixer. The short fragments were used as templates to synthesize first- and second-strand cDNAs. Afterward, cDNA was end-repaired, A-tailed and ligated with Illumina-specific adaptors. Finally, the fragments were size selected and PCR amplified to generate the cDNA library. RNA-sequencing was performed using the Illumina HiSeq™ 2000 platform. Each sample was analyzed in two biological replicates in this RNA-Seq experiment.http://trinityrnaseq.github.io/) and TGICL software (version r2013-04-11) (https://sourceforge.net/projects/tgicl/) were employed to assemble our clean data. The clean reads from the SL and YL libraries were first processed independently. To obtain complete reference sequences, the clean reads from all the samples were mixed and assembled again. To assess the quality of de novo assembly, the length distribution of assembled contigs and unigenes were collected. To obtain functional annotation of a given unigene, the sequence was aligned against protein sequence database entries including those in the Nr, Swiss-Prot, KEGGand COG databases using BLASTx . The unigenes were also aligned against the Nt database using BLASTn and we declared sequences similar if the corresponding E-value for the alignment was less than 10−5.The raw reads were filtered by removing adaptors and low-quality reads. After that, the clean reads were generated. Trinity (version 2.0.6)  were used to determine the threshold of p value. We set “FDR < 0.001 and the absolute value of log2Ratio > 1” as the threshold to judge the significance of gene expression difference according to described method in the literature [Gene expression was calculated using the number of reads aligned to a single gene and the total number of reads aligned to reference sequences in the reads per kb per million reads (RPKM) method . R packaterature .https://www.genscript.com/ssl-bin/app/primer)   and a meThe raw transcriptome reads reported here have been deposited in the NCBI Short Read Archive under the Accession Nos. SAMN08289417 (SL1), SAMN08289418 (SL2), SAMN08289419 (YL1), and SAMN08289420 (YL2).L. macranthoides can accumulate very large amounts of luteoloside, which was extremely higher than those in other leaf samples and organs including stems and flowers. Transcriptome analysis of senescing leaves and young leaves screened a subset of candidate genes associated with luteoloside biosynthesis. The elevated mRNA levels of twenty-four unigenes including PAL2, C4H2, thirteen 4CLs, CHS2, six F3′Hs and two UFGTs were considered to contribute to luteoloside accumulation in senescing leaves with respect to young leaves. Thus, we hypothesized that luteoloside may be preferentially biosynthesized using the following route: naringenin-eriodictyol-luteolin. Furthermore, several unigenes encoding MYB, bHLH and WD40 TFs, such as MYB12, MYB75, bHLH113 and TTG1, involved in flavonoid biosynthesis, were coexpressed with luteoloside biosynthetic unigenes in our dataset. Their roles in regulating luteoloside biosynthesis in L. macranthoides will be characterized via genetic and biochemical approaches in vitro and in vivo in our future work.In this study, we demonstrated senescing leaves in


HLA class I  and class II  genes. A total of 56 Arab populations comprising 10,283 individuals were selected from several databases, and were compared with 44 Mediterranean, Asian, and sub-Saharan populations. The most frequent alleles in Arabs are A*01, A*02, B*35, B*51, DRB1*03:01, DRB1*07:01, DQB1*02:01, and DQB1*03:01, while DRB1*03:01-DQB1*02:01 and DRB1*07:01-DQB1*02:02 are the most frequent class II haplotypes. Dendrograms, correspondence analyses, genetic distances, and haplotype analysis indicate that Arabs could be stratified into four groups. The first consists of North Africans , and the first Arabian Peninsula cluster , who appear to be related to Western Mediterraneans, including Iberians; this might be explained for a massive migration into these areas when Sahara underwent a relatively rapid desiccation, starting about 10,000 years BC. The second includes Levantine Arabs , along with Iraqi and Egyptians, who are related to Eastern Mediterraneans. The third comprises Sudanese and Comorians, who tend to cluster with Sub-Saharans. The fourth comprises the second Arabian Peninsula cluster, made up of Omanis, Emiratis, and Bahrainis. It is noteworthy that the two large minorities (Berbers and Kurds) are indigenous (autochthonous), and are not genetically different from “host” and neighboring populations. In conclusion, this study confirmed high genetic heterogeneity among present-day Arabs, and especially those of the Arabian Peninsula.This is the first genetic anthropology study on Arabs in MENA (Middle East and North Africa) region. The present meta-analysis included 100 populations from 36 Arab and non-Arab communities, comprising 16,006 individuals, and evaluates the genetic profile of Arabs using  HLA) system plays a key role in self-nonself recognition, and is divided into class I  and class II  loci, and comprises 220 genes in a 3.6 Mb region found on the short arm of chromosome 6. HLA system is highly polymorphic, and in excess of 17,000 alleles were detected. For example, there are 4,828 B, 3,968 A, and 3,579 C class I alleles, compared with 2,103 DRB1, and 1,142 DQB1 class II alleles. Several HLA alleles were associated with various auto-immune and infectious diseases  17]. P. PHLA alHLA allele frequencies must be obtained by molecular typing, and that subjects should be typed for at least one of the following: HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1. Publications were excluded in case of serological data; sample size less than 35 individuals, typed individuals (or controls) were either related and not randomly selected, presentation of duplicate data sets. Studies were also excluded if they presented incomplete/partial allele frequencies, or there were significant ambiguities in the typing.All included studies met the following criteria. HLA-A, -B, -C, -DRB1, and -DQB1 allele frequencies, haplotype frequencies, region, country, and typed loci.Studies were independently selected by two authors (H.A and H.L). An external referee was invited in case of disagreements not resolved by both reviewers. Data extracted from selected papers included publication year, study type , sample size, A three-dimensional correspondence analysis and bi-dimensional representation were performed using VISTA V5.02 software . PhylogeHLA datasets, of which 315 were deemed relevant to the study. Of these, 42 articles and 11 HLA datasets containing information on 56 Arab populations, and meeting the study criteria, were included. The study flow is illustrated in HLA datasets which meet the criteria of this study, containing complete information on 44 other populations were selected, but without going through systematic review. The populations used in the comparison were chosen mainly from neighboring Arab countries. This study relied on a database consisting of 100 populations (of which data of 11 populations were extracted from association studies) from 36 countries Arab and worldwide countries, and belonging to Asia, Europe, and Africa. The distribution of populations by region is illustrated in The use of more than fifty key words allowed identification of 5,456 papers and HLA datasets  selected provided information on 56 populations   . The stuForty-four worldwide populations , 68–85 cHLA-A, -B, -DRB1, or DQB1.Data of only three populations , 75, 84 HLA-A and -B alleles in Arab populations. A*02 was the most prevalent allele, and its frequency exceeded 25% in some populations, such as Saudis (30.4%) , and Saudis (0.0231).HLA A-B haplotypic data are extremely rare in Arabs. The most frequent A-B haplotypes in Arabs are shown in A*02:01-B*50:01 (9.0%) and A*02:01-B*44:02/03 (7.5%) were the haplotypes with the highest frequencies in Berbers of Zrawa. Diversity in A-B haplotype frequencies are found among Arabs, hence demonstrating comparable frequencies of A-B haplotype in Arab populations, which did not exceed 5.3% in Gabesians (Tunisia). For example, while A*34:02-B*08:01 and A*29:01-B*45:01 characterize Tunisians, A*01-B*57(02.9%), A*30-B*18 (01.50%), and A*33:01-B*14:01 (02.50%) characterize Algerians. Several haplotypes identified in Arabs were also seen in other Mediterraneans. For example, A*32:01-B*40:02 was seen in Greeks (2%) [A*02:01-B*50:01 was seen in Italians (2%) [A*24:02-B*08:01 (4.75%) and A*30:02-B*53:01 (3.48%) were only identified in Saudis.eks (2%)  and Spaneks (2%) , while Aans (2%) , Portuguans (2%) , and Morans (2%) . A*24:02DRB1-DQB1 haplotypes with significant LD in Arabs are listed in DRB1*03:01-DQB1*02:01 haplotype was the most frequent DRB1-DQB1 haplotype in Arabs  [DRB1*07:01-DQB1*02:02 is also frequent in Arabs, such as Moroccans (16.70%), and is reportedly common in Spaniards (17.3%) [DRB1*07:01-DQB1*02:01 is also a common DRB1-DQB1 haplotype, and its frequency exceeds 4% in several Arab populations.The most frequent in Arabs , and its (17.5%) , Morocca (17.5%) , Algeria (17.5%) , and Cre (17.5%) . In addi (17.3%) , and Mor (17.3%) , but rarDRB1*16:01-DQB1*05:01 and DRB1*04:01-DQB1*03:02, rare in neighboring populations and Mediterraneans, were identified only in Lebanese and Bahraini Arabs. The high frequency of DRB1*11:01-DQB1*03:01 haplotype (31.42%) among Lebanese is noteworthy, since it is the highest in all populations studied, but rare in Saudi (2.2%). Furthermore, DRB1*11:01/04-DQB1*03:01, identified in Arabs, is also frequent in Cretans (18.5%) [DRB1*01:02-DQB1*05:01 was seen in Spaniards (6.30%) [DRB1*13:01-DQB1*06:03 was also reported for Spaniards (13.23%) [DRB1*15:01-DQB1*06:02 was observed in Cretans (2.6%) [In addition,  (18.5%)  and Basq (18.5%) , while D (6.30%) . Varied 13.23%) , Cretans(13.23%) , and Ger(13.23%) . Likewiss (2.6%) , German s (2.6%) , and Sou% , CretaHLA alleles and haplotypes between North Africans and Arabian Peninsula populations. On the contrary, key differences were noted between Levant Arabs , and other Arab populations, highlighted by high frequencies of A*24, B*35, DRB1*11:01, DQB1*03:01, and DRB1*11:01-DQB1*03:01 haplotype in Levantine Arabs compared to other Arab populations. Class I haplotype frequencies are lower than Class II haplotypes, because of weak LD between A and B loci, due to long physical distance between them, compared to DRB1 and DQB1 loci. The identification of shared haplotypes between Arabs and other Mediterranean and Asian populations is attributed to the higher admixture of Mediterraneans and Asians in Arab populations.This meta-analysis is the first genetic anthropology study in MENA region, and included 100 populations from 36 Arab and neighbouring countries, and comprising in excess of 16,000 individuals. A main outcome of the study is the lack of striking differences in the distribution of The relatedness between North Africans and Iberians was previously discussed , 86, 88.HLA markers prompted the conclusion that all Berbers of North Africa constitute a homogeneous genetic unit, except for small isolates, such as the Berbers of Djerba, who display a Berber genetic profile.The analyses performed showed that current North Africans are closely related to Tunisian (Zrawa and Matmata) and Moroccan (Sousse-Agadir and Eljadida) Berbers, suggesting that North Africans have a genetic Berber profile. On the contrary, North Africans displayed a greater distance from the Arabs of Levant , indicating low genetic contribution of Phoenician and Levant Arab invasion of North Africa. These observations based on HLA data on Eastern Saudi Arabia that shed light on pre-Islamic history; some ancient people may have originated from old Persians, but quantification is difficult and undetermined [HLA-A,-B, -DRB1, and DQB1 markers support the notion that Saudis along with the Kuwaitis and Yemenis are closely related to North Africans.Saudi populations used in this study originated from Eastern Saudi Arabia, especially from Riyadh province. There is no reliable termined . The genThe most plausible explanation for West Arabia and Yemen clustering with Iberian/North Africans is a possible important massive migration that occurred when Sahara underwent desiccation in all directions , 93. CulHLA Kuwait genetic similarity to this group seems more difficult to achieve but interaction between Arabian Peninsula and Mesopotamia through this strategic Kuwait area is documented since 6,500 years BC (Ubard Period) [Explanation to  Period) .HLA class I  and class II markers (DRB1 and DQB1) analysis. However, Levant Arabs are distant from North African Arabs  and Iberians . The strong relatedness between Levant Arab populations is explained by their common ancestry, the ancient Canaanites, who came either from Africa or Arabian Peninsula via Egypt in 3300 BC [Using genetic distances, correspondence analysis and NJ trees, we showed earlier , 62 and  3300 BC , and set 3300 BC . The relHLA markers, may be due to three reasons. First, Egypt is a neighbor to Levant Arab countries, and historically part of the Levant. Second, the Egyptians invaded the Levant several times throughout history; the most significant was 1468 BC invasion, where they settled for 12 centuries [The close relatedness of Levant Arabs to Egyptians, as confirmed genetic distances using enturies . Third, Historically, Levant is a wider region that included countries along the Eastern Mediterranean with its islands, and extended from Greece to Cyrenaica . BroadlySudanese are close to sub-Saharan Africans , and North Africans, in particular Egyptians, suggesting that the genetic profile of Sudanese is the admixture between North Africans  and sub-Saharan Africans throughout history. The close relatedness of Sudanese to sub-Saharan Africans suggests a reduced genetic effect of Arabs on Sudanese. Also, the Comorians  are close to sub-Saharan Africans  , EgyptiaA*30-B*18-Cw*5 basic HLA haplotype is very high in Sardinia, Iberia, and North Africa) [Bahrainis, Emiratis, and Omanis are geographically similar populations, which explains their genetic relationship as demonstrated in this study. These three populations tend to form a heterogeneous group with Pakistanis, Indians, Iranian Arabs (Famoori), Sardinians , Egyptians, and some sub-Saharan Africans, such as Congolese. These populations appear close to certain Eastern Mediterranean populations including Greeks, Macedonians, and those further, in particular North Africans, hence explaining their intermediate grouping, and distinction from two main clusters. Collectively, this suggests high admixture in these populations brought about by their commercially important position. Sardinia is a relative genetic isolate “founded” by Iberian Norax/Nora  and Iberians/North Africans may be genetically related to Sardinians ( Africa) .The Kurds and Berbers are the two major ethnic minorities in Arab world. Berbers are indigenous North African ethnic group found over a vast area stretching from Atlantic Ocean to Siwa Oasis in Egypt, and from Mediterranean Sea to Niger River. Berbers number about 20 million people, and constitute 40–45% of Moroccans, 20–25% of Algerians, and 2–7% in both Libya and Tunisia. The Kurds live in the northern regions of Iraq (15–20%) and Syria (10%). They constitute an Indo-European ethnic group, and speak Kurdish. Less important minorities include Armenians, Nubians, Assyrians, and Turkmen .Berbers populations used in this work are closely linked to each other, as well as to present-day North Africans, and to Western Mediterranean populations, especially Iberians. Indeed, the Moroccan Berbers are not genetically different from the current Moroccans, nor those of neighboring populations, like Algerians and Tunisians. This also applies to Tunisian Berbers, except those of the island of Djerba, who appear to be related to Eastern Mediterranean populations, including Levant Arabs. This suggests that North African Berbers are in perfect harmony with their environments, and that differences between them are cultural rather than genetic due to 7th century Arabization of the region.Clustering and genetic distances analyses demonstrated that Iraqi and Iranian Kurds are not genetically different from Iranians or neighboring populations, including Levant Arab, and are close to Turks and other Eastern Mediterranean populations. This suggests that Kurds originate from the region, and are in genetic harmony with neighboring populations, despite the clear cultural differences. This suggests that Kurds, Syrians, Jordanians, Palestinians, Iraqis, Lebanese, and Iranians probably share the same genetic profile, with few differences. Accordingly, our findings confirm the results of an earlier study of Arnaiz-Villena on Iraqi Kurds .Sunni Muslims constitute the majority (80%) of Arab populations, followed by Shi'a Muslims (10%) who are present in parts of Iraq, Lebanon, Saudi Arabia, Kuwait, Yemen, and Bahrain. Non-Muslims make up about 10% of all Arabs, and Christianity (6%) is the second largest religion among Arabs, with about 20 million Christians living in Lebanon, Egypt, Iraq, Syria, and Jordan. Other minor religions (4%) such as Judaism, Druze and others are practiced on a much smaller scale .HLA data on Sunni and Shiite Arabs are not available, same as comparison of Muslims to Christians. The only available data are those concerning Arab Jews. In this study, data are available for three Jewish populations, including two from North Africa (Moroccan and Libyan Jews) and one from the Arabian Peninsula (Yemenite Jews). While genetic distances separating these three groups of Jews are small , Saudis, Kuwaitis, and Yemenis, with relatedness to Western Mediterraneans, including Iberians. The second includes Levantine Arabs , Iraqi, and Egyptians, who appear to be related to the Eastern Mediterranean and Iranians, who in turn belonged to 'Great Levant' historically described. The third consists of Sudanese and Comorians who associate with Sub-Saharan Africans. Finally, the fourth group of Arabs comprises Omanis, Emiratis, and Bahrainis. This group associates with heterogeneous populations . Lastly, the two main indigenous minorities, Berbers and Kurds, are not genetically different from the ‘host’ and neighboring populations.S1 Checklist(DOC)Click here for additional data file.S1 FigPopulations’ data were taken from references detailed in Tables (TIF)Click here for additional data file.S2 FigPopulations’ data were taken from references detailed in Tables (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Phosphate binding with sevelamer can ameliorate detrimental histomorphometric changes of bone in chronic renal insufficiency (CRI). Here we explored the effects of sevelamer-HCl treatment on bone strength and structure in experimental CRI.Forty-eight 8-week-old rats were assigned to surgical 5/6 nephrectomy (CRI) or renal decapsulation (Sham). After 14 weeks of disease progression, the rats were allocated to untreated and sevelamer-treated (3% in chow) groups for 9 weeks. Then the animals were sacrificed, plasma samples collected, and femora excised for structural analysis .Sevelamer-HCl significantly reduced blood pH, and final creatinine clearance in the CRI groups ranged 30%-50% of that in the Sham group. Final plasma phosphate increased 2.4- to 2.9-fold, and parathyroid hormone 13- to 21-fold in CRI rats, with no difference between sevelamer-treated and untreated animals. In the femoral midshaft, CRI reduced cortical bone mineral density (-3%) and breaking load (-15%) , while sevelamer increased bone mineral density (+2%) and prevented the deleterious changes in bone. In the femoral neck, CRI reduced bone mineral density (-11%) and breaking load (-10%), while sevelamer prevented the decrease in bone mineral density (+6%) so that breaking load did not differ from controls.In this model of stage 3–4 CRI, sevelamer-HCl treatment ameliorated the decreases in femoral midshaft and neck mineral density, and restored bone strength despite prevailing acidosis. Therefore, treatment with sevelamer can efficiently preserve mechanical competence of bone in CRI. Efficient control of hyperphosphatemia is a cornerstone in the treatment of chronic kidney disease-mineral bone disorder (CKD-MBD) –5. If diIn a 2-year study with haemodialysis patients, sevelamer treatment prevented the decrease in trabecular bone density in thoracic vertebrae , while iWhile sevelamer treatment has provided benefits to bone density and histology in CRI, information about the influence of sevelamer on the mechanical competence of bone is lacking. Since the principal task of bones is to bear skeletal loads without breaking , 17, we The study design was approved by Tampere University Animal Experimentation Committee, and Provincial Government of Western Finland, Department of Social Affairs and Health, Finland. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. As high mortality rates (>80%) had been previously observed in experiments where 5/6 nephrectomized rats were treated with sevelamer-HCl for 6 months , the AniForty-eight 8-week old male Sprague-Dawley rats were subjected to surgical 5/6 nephrectomy (n = 30) or sham-operation  –26. PostDuring the last study week 24-hour urine was collected. At close, the rats were anaesthetized by intraperitoneal urethane (1.3 g/kg), carotid artery was cannulated, and blood samples were drawn –26. BothThe general condition  of the study animals was assessed at least 3 times daily. Premature sampling was applied in 5 CRI and 6 CRI+Sev rats  using th2 and 1,25(OH)2D3 using radioimmunological assays . Plasma fibroblast growth factor-23 (FGF-23) was determined using ELISA  .–35.33–35max). The CVrms of Fmax for femoral neck compression is 7.6% [The proximal part of each specimen was used for femoral neck compression test , 34. Pro is 7.6% .Statistical analyses were performed using one-way and two-way analyses of variance (ANOVA), and the least-significant difference test. If variable distribution was skewed, the Kruskal-Wallis test, supported by Mann-Whitney U-test in the post-hoc analyses, was used, and the p values were corrected with the Bonferroni equation. Spearman correlations were calculated, as appropriate. The results were reported as mean±SEM, and p<0.05 was considered significant.The stresses in lower extremity bones stem from weight-bearing, bending, and torsional loading produced by muscles , 38. To 3 and 1,25-(OH)2D3 concentrations. Plasma calcium level was not reduced, which can be attributed to the secondary hyperparathyroidism in CRI. Although CRI resulted in lower final body weight, no effect was observed on longitudinal bone growth, as femoral lengths were similar in all study groups  and creatinine clearance (P = 0.059) were not significantly different between the CRI vs. CRI+Sev groups . Of note, sevelamer-HCl treatment reduced plasma 25-OH-D3 and 1,25-(OH)2D3 levels and blood pH in both Sham and CRI rats, but was without effect on plasma FGF-23 levels , while sevelamer treatment prevented the decrease in cBMD , BMC (-5.1%), increased diameter in the ML direction (+3.0%), and decreased breaking load (-10.4%), while no significant changes in the craniocaudal diameter  and tCSA  were observed . In ratsP = 0.054, We explored the effects of sevelamer-HCl treatment on femoral bone strength and structure in a model of advanced CRI. To mimic the clinical situation in renal disease, the first 14 weeks comprised a progression period of renal insufficiency and hyperphosphatemia, followed by a 9-week treatment period. The study protocol was successful, since characteristic plasma chemistry of advanced CRI was observed –25, and Hyperphosphatemia is an essential factor for the development of CKD-MBD , 42, theIn rodent models of CRI, the harmful influence of hyperphosphatemia, and beneficial effect of sevelamer, on bone histology have been previously reported , 13, 45.Prompted by the above favorable influences of sevelamer on bone density and histology, and the fact that increased susceptibility to fractures is the most important clinical manifestation of metabolic bone disorders , 16, 19,crude plasma levels of phosphate and PTH between untreated and sevelamer-treated rats with CRI call for elaboration  in sevelamer-treated versus untreated rats with CRI. When the results were adjusted for the levels of plasma creatinine, the lowering effect of sevelamer-HCl on plasma phosphate and PTH in rats with CRI became apparent , an effect that is not explained by differences in renal function, or plasma concentrations of phosphate, PTH, and FGF-23 [2D3 was reduced even in the sham-operated control rats. The plasma 25OH-D3 concentrations were also reduced in the Sham+Sev (-20%) and CRI+Sev (-33%) groups when compared with respective controls. One possibility for these changes is reduced chow intake in the sevelamer-HCl groups, but unchanged weight gain does not support this notion. Sevelamer also interferes with the absorption of fat-soluble vitamins in the gut [3 could explain the far greater reductions in 1,25(OH)2D3 following sevelamer-HCl therapy . Since acidosis is known to inhibit 1,25(OH)2D3 synthesis in the rat [2D3 concentrations following sevelamer-HCl treatment.We observed reduced plasma level of 1,25(OH)d FGF-23 . It is i the gut , 50, and the rat , reducedAlthough treatment with sevelamer-HCl has been reported to reduce plasma FGF-23 concentrations in the adenine-induced model of severe CRI , sevelamLike in the clinical setting, mortality in advanced experimental CRI is high , 25. Mor3 and 1,25-(OH)2D3 concentrations were clearly reduced. These experimental results suggest that sevelamer treatment can efficiently preserve the mechanical competence of bone in CRI. Importantly, the present beneficial effects on bones were not explained by influences of sevelamer-HCl on renal function.In summary, the phosphate binder sevelamer-HCl effectively prevented the experimental CRI-induced changes in femoral bone mineral density and breaking load, in spite of advanced renal insufficiency and acidosis. In healthy control rats, sevelamer administration was without any major effects on bone, although plasma 25OH-DS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


We found that individuals carrying the polymorphic rs17071138 and rs8089104 are more susceptible to oral cancer . Among oral cancer-related risk factor exposures, the individuals carrying the polymorphic rs17071138 had 4.26- , 2.34- , and 2.34-fold  higher risks of developing oral cancer.In total, 1342 participants, including 601 healthy controls and 741 patients with oral cancer, were recruited for this study. Allelic discrimination of TC of the SERPINB5 rs17071138 polymorphism may be a factor that increases susceptibility to oral cancer. Interactions of gene-to-gene and gene-to-oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.Heterozygous  SERPINB5 (a mammary serine protease inhibitor), a 42-kDa cytoplasmic protein that belongs to the serine family of protease inhibitors (serpins) is expressed in normal human mammary epithelial cells and has a protective role in the oncogenic process by inhibiting cell proliferation, development, invasion, angiogenesis, and metastasis –7.th most prevalent malignancy  and then quantitated by measuring the absorbance at an optical density of 260 nm . SERPINB5 rs17071138 (assay ID: C_33627662_20), rs3744941 (assay ID: C_27493638_10), and rs8089104 (assay ID: C_29202434_30) polymorphisms were assessed using an ABI StepOne™ Real-Time PCR System and analyzed using SDS vers. 3.0 software (Applied Biosystems). A genotyping fluorescence-based TaqMan SNP assay was used for the analysis. The final volume for each reaction was 10 μL and contained 5 μL of the TaqMan Universal PCR Master Mix, 0.25 μL of the primer/TaqMan probe mix, and 10 ng of genomic DNA. The real-time PCR consisted of an initial denaturation step at 95°C for 10 min followed by 40 cycles consisting of 92°C for 15 s and 60°C for 1 min [Allelic discrimination of or 1 min . The resp<0.05, our sample size had at least an 80% power to detect a 1.5-fold increased risk in susceptibility to oral cancer associated with the SERPINB5 rs17071138, SERPINB5 rs3744941, and SERPINB5 rs8089104 genetic polymorphisms.Based on the results of Kim et al. , assuminp value of <0.05 was considered significant. Data were analyzed with SAS statistical software .Adjusted odds ratios (AORs) with their 95% confidence intervals (CIs) of the association of genotype frequencies with oral cancer risk and clinical characteristics were estimated by multiple logistic regression models after controlling for other covariates. A This study estimated differences in demographic characteristics, such as gender, age, area of residence, race, and alcohol, tobacco, and areca (betel) nut consumption between oral cancer patients and controls. Significantly different distributions of gender, age, and alcohol, tobacco, and areca nut consumption between oral cancer patients and controls were found (data not shown). These variables with a statistically significant difference between oral cancer patients and controls were included in the multiple logistic regression models.rs17071138 TC polymorphism and with CC homozygotes of the SERPINB5 rs8089104 polymorphism had 1.57-  and 1.58-fold  greater risks of developing oral cancer, respectively, compared to those with wild-type homozygotes of SERPINB5 rs17071138 TT and SERPINB5 rs8089104 TT after adjusting for confounding factors. A gene-to-gene interaction effect on the increased susceptibility to oral cancer was also found, the AORs and 95% CIs increased to 4.26-  and 5.91-fold  greater risks of developing oral cancer for participants with at least one of the following, either TC or CC of rs17071138, CT or TT of rs3744941, or CT or CC of rs8089104, and for participants with TC or CC of rs17071138, and CT or TT of rs3744941, and CT or CC of rs8089104 compared to participants with TT of rs17071138, CC of rs3744941, and TT of rs8089104 , 2.34- , and 2.34-fold  increased risks of developing oral cancer compared to those with WT homozygotes TT of SERPINB5 rs17071138 among areca nut, alcohol, and tobacco consumers, respectively, after adjusting for confounding factors (p = 0.01), 7.93- , and 3.32-fold  increased risks of developing oral cancer for participants with at least one of the following, TC or CC of rs17071138, or CT or TT of rs3744941, or CT or CC of rs8089104, and 10.61- , 12.67- , and 5.02-fold  increased risks of developing oral cancer for participants with TC or CC of rs17071138, and CT or TT of rs3744941, and CT or CC of rs8089104 compared to participants with TT of rs17071138, CC of rs3744941, and TT of rs8089104 when consuming areca nut, alcohol, and tobacco, respectively  or intestinal-type gastric cancer (n = 178) and 406 unaffected controls to examine relationships of the SERPINB5 rs17071138 T/C, rs3744941C/T, and rs8089104 T/C gene polymorphisms with susceptibility to gastric cancer. They found that none of the SNPs was associated with intestinal-type gastric cancer susceptibility; however, the presence of the C allele of rs3744941 C/T and the T allele of rs8089104 T/C, significantly increased the susceptibility to diffuse-type gastric cancer [SERPINB5 genetic polymorphisms on the HCC risk. They detected that the SERPINB5 rs17071138 T/C, rs3744941 C/T, and rs8089104 T/C genetic polymorphisms were not associated with HCC susceptibility [It was reported that genetic factors play important roles in oral cancer susceptibility, and discovery of potential oral cancer-related SNPs was recommended for the early detection of possible candidates for oral cancer development , 13–15. c cancer  and hepac cancer  recruiteTC heterozygotes of the SERPINB5 rs17071138 T/C polymorphism and CC homozygotes of the SERPINB5 rs8089104 T/C polymorphism were significantly associated with susceptibility to oral cancer. Moreover, gene-to-gene interactions significantly increased the risk of oral cancer susceptibility among participants with TC or CC of rs17071138, CT or TT of rs3744941, and CT or CC of rs8089104 polymorphisms of SERPINB5. It was demonstrated that oncogene-induced senescence contributed to repressing aberrant proliferation of oral squamous cells at risk of neoplastic transformation [TC heterozygotes of rs17071138 T/C and CC homozygotes of SERPINB5 rs8089104 T/C can decrease promoter activity resulting from alterations in transcription factor binding, which could diminish the production of SERPINB5 [In the present study, ormation . SERPINBormation . We suggSERPINB5 , 21, 22.TC heterozygotes of the SERPINB5 rs17071138 polymorphism and who were areca nut, alcohol, or tobacco consumers had respective 4.26-, 2.34-, and 2.34-fold significantly increased risks of developing oral cancer compared to those with WT TT homozygotes of SERPINB5 rs17071138, after adjusting for confounding factors. Moreover, gene-gene interactions increased risks of oral cancer susceptibility by 4.03-, 7.93-, and 3.32-fold among subjects exposed to oral cancer-related risk factors, for participants with at least one of the following, TC or CC of rs17071138, CT or TT of rs3744941, or CT or CC of rs8089104 as well as increased risks of 10.61-, 12.67-, and 5.02-fold for participants with TC or CC of rs17071138, CT or TT of rs3744941, and CT or CC of rs8089104 compared to participants with TT of rs17071138, CC of rs3744941, and TT of rs8089104 when exposed to areca nut, alcohol, and tobacco consumption, respectively. Increased SERPINB5 gene expression was reported to play an important role in protecting individuals from cancer development by inhibiting cell proliferation, development, invasion, angiogenesis, and metastasis [rs17071138 TC genotype compared to individuals with the rs17071138 TT WT homozygous genotype [rs17071138 TT WT homozygotes had higher SERPINB5 gene expression, which contributed to inhibition of oral cancer development; however, individuals with heterozygotes TC of the SERPINB5 rs17071138 polymorphism exhibited decreased SERPINB5 protein function or expression, which contributed to a lack of response to oncogene-induced senescence and consequently promoted the development of oral cancer [The individuals exposed to oral cancer-related environmental risk factors, including areca nut, alcohol, and tobacco consumption, were demonstrated to have increased risks of oral cancer by inducing mucosal fibroblast proliferation, oral epithelial hyperplasia, and dysplasia –29. Moregenotype –30. Indil cancer –30. In al cancer –30.TC of the SERPINB5 rs17071138 polymorphism may be a factor that increases susceptibility to oral cancer. Interactions of genes and oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.In conclusion, our results suggested that heterozygotes