In the last few decades, epigenetics has emerged as an exciting new field in development and disease, with a more recent focus towards cancer. Epigenetics has classically referred to heritable patterns of gene expression, primarily mediated through DNA methylation patterns. More recently, it has come to include the reversible chemical modification of histones and DNA that dictate gene expression patterns. Both the epigenetic up-regulation of oncogenes and downregulation of tumor suppressors have been shown to drive tumor development. Current clinical trials for cancer therapy include pharmacological inhibition of DNA methylation and histone deacetylation, with the aim of reversing these cancer-promoting epigenetic changes. However, the DNA methyltransferase and histone deacetylase inhibitors have met with less than promising results in the treatment of solid tumors. Regions of hypoxia are a common occurrence in solid tumors. Tumor hypoxia is associated with increased aggressiveness and therapy resistance, and importantly, hypoxic tumor cells have a distinct epigenetic profile. In this review, we provide a summary of the recent clinical trials using epigenetic drugs in solid tumors, discuss the hypoxia-induced epigenetic changes and highlight the importance of testing the epigenetic drugs for efficacy against the most aggressive hypoxic fraction of the tumor in future preclinical testing. The building blocks of chromatin are nucleosomes. Each nucleosome constitutes 146 base-pairs of DNA wound around a histone octamer consisting of histones H2A, H2B, H3 and H4. Nucleosomes are connected by linker DNA and the linker histone H1, extending the length of nucleosomal DNA to approximately 16–180 base-pairs. This “beads on a string” arrangement of nucleosomes is further condensed into secondary and tertiary levels of compaction , with progressively higher levels of compaction culminating in the familiar mitotic chromosome. This enables the compression of meters of DNA into a single cell while also permitting dynamic changes in chromatin structure, which is necessary to balance the need for cellular packaging with the mobilization-essential functions including transcription, replication and repair ,6,7. On Breast cancer 1 (BRCA1); PYRIN-PAAD-DAPIN domain (PYD) and caspase-recruitment domain (CARD) domain containing (PYCARD) which encodes Apoptosis-associated speck-like protein containing a CARD (ASC); and Suppressor of Cytokine Signaling (SOCS), can greatly contribute to cancer progression .,22.21,22patterns . 5-Azacy4a locus . Decitab4a locus . Decitab designs . However designs . DevelopDNA methylation is directly linked with histone deacetylation, as DNMT1 has been shown to interact with the histone deacetylase (HDAC) HDAC1 ,27. HDACDNMT and HDAC inhibitors have shown promising results against hematological malignancies. Decitabine has been FDA-approved for acute myeloid leukemia (AML) , VorinosThe microenvironment of solid tumors is characterized by regions of low oxygen (hypoxia), which plays a pivotal role in tumor progression. Tumor hypoxia arises from the high rate of tumor growth that cannot be sustained by a limited oxygen supply . HypoxiaHypoxia leads to the induction of the oxygen-sensitive master regulator Hypoxia Inducible Factor (HIF) family of transcription factors, which take control of the cellular response to hypoxia . HIF traglucose transporter protein type 1 (GLUT1), vascular endothelial growth factor (VEGF), BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) and lysyl oxidase (LOX) [GLUT1 mediates cellular glucose uptake and is important for glycolytic metabolism [VEGF plays a key role in angiogenesis and tumor vascularization [et al., Luo et al. and Rankin et al. [The HIF family of transcription factors regulate many functions, including angiogenesis, metastasis, metabolism, survival and apoptosis . HIF medse (LOX) ,66, whicrization . HIF-indrization ,69,70,71n et al. ,65,66. An et al. .p53 and VHL upon exposure to 1% oxygen, and this correlated with increased expression of HIF1α, VEGF and angiogenesis [BRCA1, RAD51, runt-related transcription factor 3 (RUNX3), p53 and VHL in hypoxia [Hypoxic tumor cells display a distinct epigenetic profile. Indeed, changes in histone acetylation have been associated with the hypoxic cellular response . Hypoxicogenesis . A decre hypoxia ,73,74,75BNIP3, which regulates hypoxia-induced cell death, was found to be repressed by DNA methylation in pancreatic, colorectal and gastric cancer [Along with histone hypoacetylation, DNA hypermethylation may also contribute to gene-silencing under hypoxic conditions . Hypermec cancer ,78. CAIXc cancer . Of the c cancer ,84.S-adenosylmethionine, the methyl donor required for DNA methylation, leading to reduced methylation of CpG islands in hepatocellular carcinoma [N-type neuroblastoma cells exposed to 1% oxygen [Contrary to the hypoxia-induced DNA hypermethylation observed at certain loci, hypoxia has been linked to a global reduction in DNA methylation. HIF-dependent methionine adenosyltransferase II, alpha (MAT2A) induction caused reduced levels of arcinoma . Hypoxiaarcinoma , and a garcinoma . Hypoxia% oxygen . TET1 ac% oxygen . Hypoxia% oxygen . However% oxygen ,87, it imutL homolog 1 (MLH1), BRCA1, RAD51, RUNX3, Ataxia Telangiectasia Mutated (ATM) and p53-associated KZNF (ZNF 420) protein (APAK) and protein phosphatase 2, catalytic subunit, alpha isozyme (PP2A-C) [MLH1, BRCA1 and RAD51 in hypoxia [Recent studies have demonstrated that oxygen levels significantly influence a change in another epigenetic mark, histone methylation, which is distinct from DNA methylation. Hypoxia has been linked to a decrease in the levels of the active histone mark H3K4me3, and an increase in the levels of the repressive marks H3K27me3, H3K9me2 and H3K9me3, at specific genes ,75,90 T. Increas(PP2A-C) ,75,76,80 hypoxia ,91,92.APAK in hypoxia [Hypoxia-induced changes in histone methylation may result from changes in the expression and activity of histone methyltransferases. Histone methyltransferases catalyze the methylation of lysine or arginine residues on histones. Lysine residues can be mono-, di- or tri-methylated, and the methyltransferases exhibit high specificity with regards to the substrate, the lysine residue and the extent of methylation . Lysine  hypoxia .In vitro, human Jumonji protein JMJD2E demonstrated an almost linear dependence on oxygen, at oxygen concentrations ranging from 0.5%–20.6% [in vitro suggesting that changes in α-ketoglutarate-levels in cancer cells can also regulate JMJD2C activity [adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) genes, which promote tumor growth in renal and colon cancer cell lines [On the other hand, hypoxia-induced histone methylation changes may also be affected by histone demethylation. Histone demethylation is carried out by histone demethylases, of which there are two types: Amine-oxidase type Lysine specific demethylases (LSD) and Jumonji C (JmjC)-domain containing oxygenases . Importa5%–20.6% . Further5%–20.6% . Interes5%–20.6% . Gliomasactivity . It is aactivity . The Jmjactivity . JMJD2C activity , and at activity . HIF-indll lines . Anotherll lines . Althougll lines , suggestll lines . Increasll lines . The H3Kll lines , explainet al. has described a role for hypoxia-induced H3K9me3 in the repression of APAK, a negative regulator of p53 [APAK can lead to the induction of p53-dependent apoptosis, which may have both positive and negative effects on the tumor. p53-dependent apoptosis may play a role in blocking the growth of tumors with functional p53 [APAK-mediated p53 activation and apoptosis may contribute to the selection of p53-mutant tumor cells [Hypoxia-induced epigenetic changes can lead to the down-regulation of tumor suppressors, providing the hypoxic tumor cells with a selective growth advantage. This is consistent with the finding that hypoxia correlates with increased aggressiveness, metastasis and therapy-resistance of tumors. However, hypoxia-induced histone modifications can also lead to the down-regulation of tumor promoting-genes. Recent work by Olcina r of p53 . H3K9me3onal p53 . Howeveror cells ,106. Thiet al. demonstrated that DNMT3a mediates silencing of endothelial PAS domain protein 1 (EPAS1), which encodes HIF2α, in renal epithelial cells, and that the loss of DNMT3a can lead to the induction of HIF2α, which provides these tumor cells with a selective growth advantage in hypoxia [Given the distinct profile of the chromatin landscape in hypoxic tumor cells, and given that the hypoxic cells tend be the most aggressive and therapy-resistant, it is essential to understand the effects of epigenetic drugs in hypoxia. Although DNMT inhibitors have undergone Phase I clinical trials in solid tumors, the efficacy of these drugs against the hypoxic fraction has not been validated. While DNA hypermethylation may play a role in silencing some genes in hypoxia, a global down-regulation of DNMTs and DNA methylation has also been observed ,87, rais hypoxia . A switc hypoxia . Given tThe effects of HDAC inhibitors under hypoxic stress have been investigated. While HDAC inhibitors cause histone hyperacetylation, increasing evidence has demonstrated a direct effect on HIF transcription factors in hypoxia, affecting both the expression and function . HDAC4 iAPAK, as described above. It would be informative to test the effects of targeting histone methylation on gene expression in hypoxia. One method of investigating the effects of histone methylation on gene expression is to inhibit the methyltransferases, and a number of histone methyltransferase inhibitors have been developed [secreted frizzled-related protein (SFRPs) and GATA family transcription factors, which were silenced in colon cancers, and this was correlated with an increase in H3K4me2 and H3K9-acetylation, and a decrease in H3K9me1 and H3K9me2 [Histone methylation is significantly impacted by oxygen levels ,80, primeveloped . The Suveveloped , and in eveloped . The LSDeveloped ,117. The H3K9me2 ,117. InvN-oxalyl glycine and its derivatives; and the hydroxamic acid-based HDAC inhibitor Suberoyl Anilide Hydroxamic Acid (SAHA) and its derivatives; and pyridine carboxylates [N-oxalyl glycine and some pyridine carboxylates may also inhibit PHD2 and FIH, which may activate the HIF pathway and confound anti-tumor activity [It is known that numerous genes are repressed in hypoxia, either though epigenetically regulated transcriptional repression  or due toxylates . It is iactivity . HoweverThere has been an increased focus on epigenetic drugs in cancer therapy. While DNMT and HDAC inhibitors have shown promising results against hematological malignancies, they have proven to be less effective against solid tumors. Most, if not all, solid tumors have regions of hypoxia, and, as discussed above, hypoxic tumor cells display a distinct epigenetic profile. In particular, hypoxic tumor cells have increased levels of the repressive histone methylation marks. We propose that when developing epigenetic drugs against solid tumors, given that the most aggressive hypoxic regions of the tumor display a distinct epigenetic profile, it is important to test the effects of these drugs under hypoxic conditions prior to clinical trials. Furthermore, given the global trend towards transcriptional silencing and the reduced function of oxygen-dependent enzymes, such as JmjC demethylases and TET hydroxylases, it is important to understand the epigenetic changes in hypoxia, and the associated biological consequences in order to design effective epigenetic drugs against solid tumors.


Geobacillus sp. isolate T6 was collected from a thermal spring in Salta, Argentina. The draft genome sequence  of this isolate is represented by one major scaffold of 3,46 Mbp, a second one of 207 kbp, and 20 scaffolds of <13 kbp. The assembled sequences revealed 3,919 protein-coding genes. Geobacillus are thermophilic Gram-positive endospore-forming bacteria, usually isolated from high-temperature environments. Thermophilic strains of this genus have attracted interest as sources of thermostable enzymes with potential biotechnological applications, including proteases, lipases, and glycosyde-hydrolases , a second scaffold of 207,976 bp, and 20 minor scaffolds of >1 kbp. The order and orientation of the contigs into scaffolds were assessed using Mega BLAST , producing unpaired and paired-end reads, respectively. A total of 218,128 single reads with an average length of 350 bp were produced by Roche 454, resulting in 16-fold coverage of the genome. Furthermore, 3,682,898 paired-end plus 3,587,489 singleton reads with an average of 126 bp were obtained using a long jumping distance library with an insert size of 8 kbp from an Illumina MiSeq 2 × 150-bp run, reaching 239-fold coverage. Geobacillus sp. isolate T6 consists of 3,767,773 bp with an average G+C content of 53%. It was subjected to automated annotation using the RAST server version 2.0 (The draft genome sequence of Geobacillus sp. isolate T6 to the genus Geobacillus. Geobacillus kaustophilus HTA426 (score 531), Geobacillus thermodenitrificans NG80-2 (score 495), and Geobacillus sp. Y412MC61 (score 495) are the closest neighbor genomes based on RAST analysis.In total, 4,011 genes were identified, including only one copy of 16s rRNA, 13 copies of 23S/5S rRNA, and 78 tRNA genes. RAST predicted 3,919 protein-coding genes, 47% of which were assigned to 452 subsystems. Phylogenetic analysis of the 16S rRNA gene confirmed the affiliation of A group of 878 enzyme-coding genes was appointed as potentially useful in several industries according to their predicted activity, including agriculture, environment, biosensor, biotechnology, medicine, biofuel, food, and other industries. Cloning and expression strategies are under way to confirm the functionality of some of these enzymes. The main goals of our work in gaining genome-based knowledge of thermophilic organisms are to characterize their thermostable enzymes and unravel the metabolic pathways adapted to extreme living conditions.LDNZ00000000. The version described in this paper is the first version, LDNZ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


The disruption of iASPP-p53 interaction by a p53-derived peptide A34 restores p53 function in cancer cells. The inhibition of iASPP phosphorylation with small molecules induces p53-dependent apoptosis and growth suppression. The mechanisms underlying aberrant expression of iASPP in human tumors should be further investigated. Reactivating WT p53 functions by targeting its novel inhibitor iASPP holds promise for potential therapeutic interventions in the treatment of WT p53-containing tumors.Although mutational inactivation of p53 is found in 50% of all human tumors, a subset of tumors display defective p53 function, but retain wild-type (WT) p53. Here, direct and indirect mechanisms leading to the loss of WT p53 activities are discussed. We summarize the oncogenic roles of iASPP, an inhibitor of WT p53, in promoting proliferation, invasion, drug or radiation-resistance and metastasis. From the therapeutic view, we highlight promising perspectives of microRNA-124, peptide and small molecules that reduce or block iASPP for the treatment of cancer. High iASPP expression enhances proliferation, aggressive behavior, the resistance to radiation/chemotherapy and correlates with poor prognosis in a range of human tumors. Overexpression of iASPP accelerates tumorigenesis and invasion through p53-dependent and p53-independent mechanisms. MicroRNA-124 directly targets  The initiation and progression of cancer is a multistep process including self-renewal, aberrant cell cycle, defective apoptosis, the induction of the epithelial to mesenchymal transition (EMT), enhanced mobility, invasion and angiogenesis, and the remodeling of the tumor microenvironment . In addiMicroRNAs (miRNAs) are characterized as endogenous, small size, non-coding RNA molecules that post-transcriptionally control the translation and stability of mRNAs . MiRNAs Slug and Snail, p53 negatively regulates EMT and suppresses cancer cell invasiveness [miR-130b, p53 transactivates this miRNA to reduce the levels of ZEB1 (a direct target gene of miR-130b), and thereby attenuates EMT and invasiveness in endometrial cancer cells [The p53 family members  play a pivotal role in the regulation of many critical biological processes including cell death, proliferation, cell cycle control and tumorigenesis , 11, 12.siveness , 20. Emesiveness , 26, 27.er cells . These ser cells .In this review, we provide a brief overview of the mechanisms by which the functions of WT p53 are inactivated and will focus on the oncogenic roles of iASPP, a negative regulator of WT p53, in human tumors. We will discuss current approaches that aim to restore p53 functions by targeting iASPP, including miR-124, a peptide derived from p53 and other small molecules.TP73 gene produces an NH2 terminally truncated isoform, ΔNp73 that lacks the transactivation domain and function as a dominant-negative inhibitor of WT p53 [The loss of p53 function in human cancers occurs either by direct or indirect mechanisms Figure . At leasf WT p53 . Other mf WT p53  and the f WT p53 . Thus, tf WT p53 .To date, intensive efforts have been made to restore WT p53 activity as an anticancer therapeutic approach . ReducinThe ASPP family consisting of three proteins  interacts with and modulates the functions of WT p53 . ASPP1/2PPP1R13L located on 19q13.2-3 [Human iASPP, which is encoded by 9q13.2-3 , has two9q13.2-3 , 50. The9q13.2-3 , 49, 50.9q13.2-3 . Of note9q13.2-3 .in vitro proliferation in human cancer cells [iASPP is overexpressed in diverse human tumors, including colorectal cancer , acute ler cells , 68, 69.er cells . These din vivo tumorigenicity and forming metastasis [in vivo tumorigenesis of prostate cancer cells [Human cancers are composed of heterogeneous cell populations. Genetic and phenotypic variation exists not only between different tumor types or between patients with the same tumor type (inter-tumor heterogeneity), but also within a single tumor (intra-tumor heterogeneity) . This intastasis , 74. Thitastasis . Importaer cells . These dOn the other hand, the concept of CSC plasticity (the model of dynamic stemness) has been also proposed , in whicp53 gain-of-function mutations [KLF17 [EZH2, MCL-1 and FOS [Despite some results showing that the activation of EMT program suppresses the tumor-initiating properties of CSCs , 77, numutations , KLF17 [s [KLF17 , EZH2, M and FOS  promote  and FOS , 22, 80  and FOS . These dThe increased iASPP expression is correlated with the resistance to radiation or chemotherapy in cervical cancer . The oveiASPP and represses the growth and invasiveness in a variety of cancer cells, including colorectal cancer, glioblastoma and prostate cancer [Bax and PUMA and tumor cell apoptosis in vitro and in vivo [The therapeutic targeting of iASPP might include targeting iASPP itself as well as modulating its upstream regulators or downstream effectors Figure . The Wnte cancer , 86, 87.e cancer , causing in vivo . Further in vivo . The ideIn addition to affecting tumor cell proliferation and survival, WT p53 can repress cancer initiation, progression and metastasis by regulating downstream genes and miRNAs-target gene networks. A more complete understanding of genetic and epigenetic mechanisms underlying aberrant expression of WT p53 inhibitor iASPP in human tumors, and detailed studies on iASPP function in various aspects of tumor progression, including EMT, stemness and metastasis, can provide a basis for new potential therapeutic applications in WT p53-containing tumors.


Small cell lung cancer (SCLC) is a highly malignant disease with dismal prognosis. Although great progress has been made in investigating genetic aberrations and putative drivers of this tumor entity, the mechanisms of rapid dissemination and acquisition of drug resistance are not clear. The majority of SCLC cases are characterized by inactivation of the tumor suppressors p53 and retinoblastoma (Rb) and, therefore, interchangeable drivers will be difficult to target successfully. Access to pure cultures of SCLC circulating tumor cells (CTCs) and study of their tumor biology has revealed a number of new potential targets. Most important, expression of chitinase-3-like-1/YKL-40 (CHI3L1) which controls expression of vascular epithelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) was newly described in these cells. The process switching CHI3L1-negative SCLC cells to CHI3L1-positive CTCs seems to be associated with cytokines released by inflammatory immune cells. Furthermore, these CTCs were found to promote monocyte-macrophage differentiation, most likely of the M2 tumor-promoting type, recently described to express PD-1 immune checkpoint antigen in SCLC. In conclusion, dissemination of SCLC seems to be linked to conversion of regular tumor cells to highly invasive CHI3L1-positive CTCs, which are protected by immune system suppression. Besides the classical targets VEGF, MMP-9 and PD-1, CHI3L1 constitutes a new possibly drugable molecule to retard down dissemination of SCLC cells, which may be similarly relevant for glioblastoma and other tumor entities. TM system which counts EpCAM-positive tumor cells ranged from 14 to 70% [Lung cancer is the leading cause of cancer-related deaths worldwide and, in particular, small cell lung cancer (SCLC) constitutes a highly aggressive variant accounting for approximately 15% of cases . This tu4 to 70% . A minor4 to 70% .Molecular characterization of CTCs would require expansion of CTCs in cell culture systems, so far only achieved for a few breast cancer CTCs and one colon-derived line , 13. We Screening of the permanent CTC cultures of patients for secreted cytokines revealed presence of chitinase 3-like1 (CHI3L1)/YKL-40, known to be upregulated in a range of tumor entities and to be associated with increased metastasis and decreased survival , 16. TheLevels of circulating CHI3L1 are increased in many malignancies, including cancers involving the lung, prostate, colon, rectum, ovary, kidney, breast, glioblastomas, and malignant melanoma . NumerouChitinase enzymes hydrolyse the polysaccharide chitin, an abundant architectural component in invertebrates and fungi. Most mammals encode at least two endochitinases (CHIT1 and CHIA/AMCase), as well as several homologues encoding catalytically inactive chitinase-like proteins in numerous pathological conditions . ExpansiCHI3L1 interacts with glycosaminoglycans, such as heparin and hyaluronan, and binds to collagen type I, II, and III . CHI3L1 ex vivo expanded SCLC CTCs point to these disseminated tumor cells as partial source of this protein directly produced in blood [CHI3L1 is detectable in a variety of normal cells, including macrophages, neutrophils, epithelial cells, smooth muscle cells, and chondrocytes, and its expression is stimulated by a number of cytokines, including IL13, IL6, IL1β, and IFNγ . Elevatein blood .COPD is a complex inflammatory disease involving several types of inflammatory cells and multiple inflammatory mediators. Abnormal numbers of inflammatory cells such as macrophages, dendritic cells, neutrophils, and T lymphocytes have been documented in COPD . Acidic COPD and lung cancer are closely related . The annElevated serum CHI3L1 levels in patients with metastatic cancers, including SCLC, are associated with poor prognosis, although SCLC cell lines display no or very limited CHI3L1 expression . In mostTumor cell dissemination to distant organ sites is a complex process involving multiple cell types, soluble growth factors, adhesion receptors, and tissue remodeling . In tumoin vitro [in vivo and by IL-1 in astrocytes in vitro. Thus, IL-1 and the IL-6 family of cytokines regulate CHI3L1 expression during inflammation via both STAT3 and RelB/p50 complexes.The proinflammatory cytokines, such as IL-1, TNF-α, and IL-6, modulate CHI3L1 expression in chondrocytes, macrophages, and also glioblastoma cells , 66. In in vitro . Nuclearin vitro . In contin vitro . The IL-in vitro . ExpresspSTAT3 overexpression is an important factor related to prognosis of NSCLC patients . SimilarThe immune checkpoint proteins, including the B7/CD28 receptor superfamily, have become increasingly important targets for pharmacologic blockade. Several classes of new agents have impressive clinical activity in melanoma and lung cancer , 74. NonCHI3L1 suppressed E-cadherin but induced MMP9 and cell motility in glioma cells, all of them essential features of tumor cell invasion . TargetiCHI3L1 has retained its chitosan binding activity. Correspondingly, reports indicate that N-acetyl-d-glucosamine oligomers  and d-glucosamine oligomers  have activities against cancer and inflammation , 84. COSEmploying structural chitinase data, the inhibitor bisdionin C which shows activity in the submicromolar range against GH18 enzymes was designed . A crystIn contrast to non-small lung cancer (NSCLC), targeted therapy directed to specific oncogenes is not available for SCLC and platinum-based combination chemotherapy with etoposide and second-line topotecan are standard care , 6. SCLC


In this study, the substance perillaldehyde 8,9-epoxide, a p-menthane class derivative obtained from (S)-(−)-perillyl alcohol, was selected in order to assess antitumor activity against experimental sarcoma 180 tumors. Toxicological effects related to the liver, spleen, kidneys and hematology were evaluated in mice submitted to treatment. The tumor growth inhibition rate was 38.4%, 58.7%, 35.3%, 45.4% and 68.1% at doses of 100 and 200 mg/kg/day for perillaldehyde 8,9-epoxide, perillyl alcohol and 25 mg/kg/day for 5-FU intraperitoneal treatments, respectively. No toxicologically significant effect was found in liver and kidney parameters analyzed in Sarcoma 180-inoculated mice treated with perillaldehyde 8,9-epoxide. Histopathological analyses of the liver, spleen, and kidneys were free from any morphological changes in the organs of the animals treated with perillaldehyde 8,9-epoxide. In conclusion, the data suggest that perillaldehyde 8,9-epoxide possesses significant antitumor activity without systemic toxicity for the tested parameters. By comparison, there was no statistical difference for the antitumor activity between perillaldehyde 8,9-epoxide and perillyl alcohol.Recent studies have revealed the high cytotoxicity of  Cancer is a complex genetic disease comprising specific characteristics that causes mortality in both children and adults, triggering a global health concern. More than 100 distinct types and subtypes of cancer can be found within specific organs ,3. It isThe research on derivatives of natural products has revealed new possibilities for therapeutic anticancer agents. More than two thirds of the drugs currently used in cancer treatments come directly from natural products, or have been developed using knowledge gained from the activities of their ingredients ,7,8. Attp-menthane structures are abundantly found in nature.  × 100, where NC is the average tumor weight of the negative control group and TG is the average tumor weight of the treated group [The effect on tumor growth protocol ,39. Ten-n = 10 mice/group) were determined at the first and last days of treatment. Organ weight alterations (n = 10 mice/group), and hematological and biochemical analysis (n = 5 mice/group) were performed on the last day of experimentation. All systemic toxicological analyses were also performed with 4 healthy animal groups, without the inoculation of S-180, (n = 5 mice/group) treated with 5% DMSO (i.p.), 5-FU 25 mg/Kg/day (i.p.), perillaldehyde 8,9-epoxide 100 mg/kg/day (i.p.) and perillaldehyde 8,9-epoxide 200 mg/kg/day for consecutive 7 days.Body weights  with anesthetized mice . Immediately after blood collection the animals were euthanized, and their organs  were removed and weighed. The wet mass of each organ was expressed as grams per 100 grams of body mass and compared to the negative control group.For evaluation of the effect of perillaldehyde 8,9-epoxide on the biochemical parameters aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymatic to investigate liver function alterations, creatinine and urea were measured as renal function parameters. For this test, blood samples of the animals were collected without anticoagulant, centrifuged for 10 min at 3500 rpm and 25 °C to obtain plasma.® kits  were used for biochemical evaluation of serum samples.The Clinical ChemistryFor hematological analyses, an aliquot of blood from each animal was placed in ethylene diamine tetracetic acid (EDTA). Hematological parameters, including the total as well as differential counts of leukocytes, including eosinophils, lymphocytes, neutrophils and monocytes determined by standard manual procedures using optical light microscopy.After being weighed and fixed in 10% formaldehyde; tumors, livers, spleens, and kidneys were submitted for assessment macroscopic , were cut into small pieces and, after that dehydrated in alcohol, diaphanized in xylene and paraffin-embedded. Subsequently, 5-µm-thick histological sections were obtained and stained with hematoxylin and eosin. Histological analyses were performed by light microscopy in order to assess possible damage caused by treatment.p < 0.05. All statistical analyses were carried using the GraphPad program .Data obtained were expressed as the mean ± SEM and the differences among experimental groups were evaluated using one-way analysis of variance ANOVA followed by the Student Newman-Keuls test. Were considered significant values of in vivo antitumor activity of perillaldehyde 8,9-epoxide without alterations in biochemical or histopathological parameters. Additionally, perillaldehyde 8,9-epoxide showed similar antitumor potency to perillyl alcohol. Further studies using perillaldehyde 8,9-epoxide and other structurally similar compounds may well be carried out against other tumor types.In conclusion, the data presented demonstrate the


ATM, RUNX1T1, WDR17, NTRK3, TP53, TRMT12, CACNA2D1, INTS8, and KCNH8. We observed somatic mutations in ATM (ataxia telangiectasia-mutated) in 5 out of the 12 samples and mutations in the common gamma chain (γc) signaling pathway  in 3 samples, all of which also harbored mutations in ATM. Our findings contribute insights into the genetics of PTCL and suggest a relationship between γc signaling and ATM in T cell malignancy.Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with poor response to current therapeutic strategies and incompletely characterized genetics. We conducted whole exome sequencing of matched PTCL and non-malignant samples from 12 patients, spanning 8 subtypes, to identify potential oncogenic mutations in PTCL. Analysis of the mutations identified using computational algorithms, CHASM, PolyPhen2, PROVEAN, and MutationAssessor to predict the impact of these mutations on protein function and PTCL tumorigenesis, revealed 104 somatic mutations that were selected as high impact by all four algorithms. Our analysis identified recurrent somatic missense or nonsense mutations in 70 genes, 9 of which contained mutations predicted significant by all 4 algorithms:  Peripheral T cell Lymphoma (PTCL) accounts for 10–15% of Non-Hodgkin’s Lymphoma with about 7,000 cases diagnosed per year in the United States . TraditiSTAT3, STAT5B, JAK1, JAK3, FYN, RHOA, NOTCH1, CD58, B2M, PLCG1, PTPN2, EZH2, FBXW10, TET2, DNMT3A, IDH2, ATM, CHEK2, and p53 related genes  in different subtypes of PTCL. Due to the relatively low incidence of PTCL, however, the discovery cohorts within these studies are limited, with relatively small numbers of primary PTCL samples subjected to high throughput sequencing. Given the limited number of PTCL samples sequenced relative to many other cancers, further sequencing studies serve both to validate identified driver mutations and to discover novel mutations. Therefore, it is critical to compare and analyze mutations identified across independent studies to help understand the complete role of oncogenic mutations in PTCL. We conducted whole exome sequencing of 12 PTCL cases from untreated patients, compared to patient-derived non-tumor control cells, to identify somatic mutations: potential oncogenic drivers of PTCL.To identify oncogenic mutations, and by extension, therapeutic targets in PTCL, recent studies have employed single gene, whole exome, or genome wide sequencing techniques in cases of PTCL –16. ThesSpecimens were collected for this study from patients diagnosed with PTCL at the University of Maryland Greenbaum Cancer Center with the approval of the University of Maryland, Baltimore Institutional Review Board (UMB IRB). Written consent was obtained from all patients involved in the study using a consent procedure approved by the UMB IRB. Documentation of the consent process includes patient, patient study number (samples are de-identified prior to use), principal investigator/designee signature, and date. Pathological samples used for analysis include patient blood, bone marrow, or lymph node tissue . MononucCells were stained with fluorophore-labeled antibodies to cell surface molecules for separation of malignant PTCL and non-malignant cell populations  by flow cytometry and cell sorting. Surface antigens used to distinguish PTCL cells and non-malignant cells included CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD19, CD30, CD45, and CD52 . All fluCells were washed and resuspended in PBS. After addition of Proteinase K and RNAse A (Qiagen), genomic DNA was isolated using a DNeasy kit (Qiagen) per manufacturers’ instructions.® DNA Sample Prep Master Mix Set 1 . DNA was fragmented with the Covaris E210 focused ultrasonicator , targeting a size of 200bp, and libraries were prepared using a modified version of manufacturer’s protocol.Sequencing library construction, exome capture, sequencing, and analyses were carried out by the Genomics Resource Center (GRC) within the Institute for Genome Sciences (IGS) at the University of Maryland School of Medicine. Genomic DNA libraries with 7bp molecular barcode indexes were constructed for sequencing on the Illumina platform using the NEBNextFollowing library construction, targeted capture was performed with the Agilent SureSelect Human All Exon V4 kit following the manufacturer’s protocol. Libraries were pooled so that each received ¼ or ½ a lane of sequencing, and were sequenced with an Illumina HiSeq2000 sequencer 100PE run, generating an average of 86.8 million passed-filter reads per sample.Raw data from the sequencer was processed using Illumina’s RTA and CASAVA pipeline software and reads were truncated where the median quality score fell below Q20. Initial alignment to the hg19 human reference genome (GRch37) using BWA (v0.5.9) was followed by GATK (v1.4.5) for indel realignment and base quality score recalibration and Picard MarkDuplicates to remove artificially duplicated library fragments caused by PCR. The average on-target coverage for all samples was 94.4x and >87% of targets were covered at ≥20x with fewer than 4% of targeted bases lacking coverage. Somatic variants were predicted using both VarScan (v.2.3.7) and MuTect (v1.1.7). The resulting variant sets were annotated using ANNOVAR (v 2014-11-12). On average, VarScan found 208 somatic variants per sample, while MuTect found 264.MuTect uses a coverage cutoff of at least 14 reads in the tumor sample and at least 18 reads in the non-malignant cell sample and pre-applies filters to eliminate false positives. Only the high confidence set of calls that did not fail any of the MuTect filters were included in analysis.VarScan uses a minimum coverage of 6 reads in the tumor sample and 8 reads in the non-malignant sample. We then applied the processSomatic tool to extract a high confidence set of variants. A somatic variant was considered high confidence if the variant allele frequency of at least 10% in tumor (default) and Fisher’s Exact Test P-value was < 0.07 (default) . In addiAll variants called by Mutect and/or VarScan were then filtered by population frequency using the 1000 Genomes Project database to exclude variants with allele frequency > 0.01 in the population . Applicahttp://genetics.bwh.harvard.edu/pph2/, http://provean.jcvi.org/index.php, http://mutationassessor.org/, and http://www.cravat.us/, respectively. Indels were only analyzed by PROVEAN, as the other algorithms are equipped only for missense mutation analysis.Non-synonymous mutations identified were formatted for analysis according to the websites’ instructions and then queried by each of the following computational algorithms: Polyphen2 (Polymorphism Phenotyping v2), PROVEAN , MutationAssessor, and CHASM (Cancer-Specific High-throughput Annotation of Somatic Mutations), accessed at To identify potential oncogenic mutations in PTCL, we performed whole exome sequencing of matched tumor and non-malignant DNA samples from 12 untreated patients with PTCL. Eight different PTCL subtypes were represented in our patient cohort, including one patient each with hepatosplenic T cell lymphoma (HSTL), T-cell large granular lymphocytic leukemia (T-LGL), lymphoepithelioid T cell lymphoma (LETL), Alk(+) anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), and Sezary Syndrome (SS), and three patients each with T-cell prolymphocytic leukemia (T-PLL) and peripheral T-cell lymphoma not otherwise specified (PTCLnos).Using two different calling algorithms, Mutect and VarScan, we detected a total of 1,245 unique, high-confidence, non-synonymous SNVs and 59 indels that passed our filters, across the 12 PTCL cases with an average of 93 non-synonymous somatic mutations per PTCL sample (range 15–340). More non-synonymous mutations were independently identified by Mutect  than VarScan (499); 333 mutations were identified by both calling algorithms. The most common transition/transversion resulting in non-synonymous exonic mutation was G>T+C>A . The sigTo identify the non-synonymous SNVs from our samples most likely to influence protein function, we used three independent structure/homology-based algorithms: Polyphen2 PROVEAN, MutationAssessor, as well as a fourth algorithm, CHASM, designed to prioritize somatic missense mutations based on their representation in large-scale cancer sequencing studies –26. CHASATM, found to contain non-synonymous somatic mutations in 5 out of the 12 samples (42%). ATM has previously been shown to harbor somatic mutations in over 50% of sequenced tumor samples from patients with the T-PLL subtype of PTCL [ATM mutations in our data, three were from cases of T-PLL and the two others were from HSTL and T-LGL cases. ATM mutations in conserved residues were verified by Sanger sequencing  and WDR17 (WD repeat-containing protein 17) were identified in 3 patient samples in the cohort and predicted to be likely cancer driver mutations impacting protein function by all 4 algorithms  were also identified in 3 samples and predicted to have a significant probability of impacting protein function by Polyphen2, PROVEAN, and MutationAssessor. Mutations in MUC16 (mucin) were also observed in 3 samples and predicted to be significant by Polyphen2 and PROVEAN. Mutations in CACNA2D , INTS8 , KCNH8 (a potassium voltage-gated channel), NTRK3 (tyrosine-protein kinase receptor), TP53 (p53), and TRMT12 (a guanosine modifying transferase) were identified in 2 samples and predicted to be cancer driver mutations impacting protein function by all 4 algorithms , CCR8 (chemokine receptor), DNAH7 (dynein heavy chain), FEZF1 (fez family zinc finger protein), INTS8, LRP1B (receptor-mediated endocytosis), MTMR8 (phosphatase), MUC16, NOP2 , RASAL2 (Ras GTPase-activating protein), RUNX1T1, SDK2 (cell adhesion), SLC13A5 (sodium/citrate cotransporter), SLC16A14 (monocarboxylate transporter), SRRM2 (pre-mRNA splicing), TNFRSF19 (tumor necrosis factor receptor superfamily member), TTN, UNC79 (sodium channel complex component), and WDR17 , but we did not identify any other recurrent mutations in this subtype.Our data contains three samples from patients with T-PLL, all with a mutation in nd WDR17 . Two of Among the 70 genes found to contain somatic mutations in multiple samples, two genes contained identical somatic mutations in two different PTCL samples: TCF12 R300L  and TMEM51 E169del (transmembrane protein)  in gold.ATM has been found to be mutated, or deleted, in the majority of cases of T-PLL, with most of the identified mutations clustering near the ATM PI3Kinase domain [ATM point mutations in our study, three are from patients with T-PLL, while the final two are from patients with HSTL and T-LGL. Three of the somatic mutations identified, two from T-PLL samples and one from the HSTL sample, are in or near the highly conserved kinase domain  have previously been shown to be recurrently mutated in various subtypes of PTCL –9, 12. Wquencing .In one of our samples from a patient with T-PLL we observed a somatic mutation in a conserved region of JAK3 adjacent to the pseudokinase domain, M511I . JAK3 M5in vitro screen, then demonstrated to increase the transcriptional activity of STAT5A, which shares extensive sequence homology with STAT5B, and shown to allow cytokine independent growth of Ba/F3 cells [In the HSTL sample in our analysis, we identified a somatic mutation in a highly conserved residue in the SH2 domain of STAT5B encoding a N642H mutation, a prolific oncogenic mutation found in many hematologic malignancies . STAT5 NF3 cells . STAT5B F3 cells , 12, 47.F3 cells . AlternaF3 cells , 50. ThiF3 cells . STAT5B F3 cells . PolyPhec K315E mutation we observed in a T-PLL case is in a highly conserved residue located in the intracellular region of the γc protein C-terminal to the box1 motif, which is required for JAK binding and activation , angioimmunoblastic T-cell lymphoma (AITL), T-PLL, HSTL, SS, and T-LGL subtypes of PTCL [c signaling in T-PLL and HSTL. Other sequencing studies have revealed somatic activating mutations in JAK3 (A572V and A573V) in 38% of NKTCL, STAT3 and STAT5B in T-LGL, and recurrent gain-of-function mutations in JAK2 and STAT3 in AITL [c and to increase with γc overexpression [c K315E mutation in T-PLL, as well as an indel in the γc that was demonstrated to increase STAT5 transcriptional activity, establishes a pattern of recurrent mutations in the γc in T-PLL that may drive the development of this malignancy [c cytokine signaling, particularly in the context of IL-2, IL-7, and IL-15 signaling, is critical for T-cell differentiation, apoptosis, survival and proliferation, mutations altering this signal transduction pathway are well placed to potentially drive T cells to a cytokine-independent, malignant phenotype [c associated JAK-STAT cytokine signaling pathways are present in at least 5 subtypes of PTCL and may represent a unifying trait in a disease marked by heterogeneity between subtypes.Somatic mutations in genes encoding molecules involved in cytokine signal transduction, and specifically in γ of PTCL , 47, 53. in AITL , 53. Thepression . Our stulignancy . As γc chenotype , 56. StuIdentification of gain-of-function mutations in the common gamma chain signaling pathway suggests utility of JAK/STAT pathway inhibitors in PTCL therapy. JAK inhibitors are currently in development and several are approved for patient use, such as ruxolitinib in the treatment of myelofibrosis and tofacitinib in rheumatoid arthritis , 58. In ATM, mutated in 5 out of 12 PTCL cases and predicted by algorithms as likely to impact protein function and drive oncogenesis. Another recent whole exome sequencing study of 12 PTCL samples also identified recurrent mutations in ATM [c signal transduction components in our study were among the samples containing mutations in ATM. Our study suggests that, due to overlap of mutations in ATM and the γc signaling pathway, there is a possible correlation between these two pathways in HSTL as well as T-PLL. In a study of HPV viral replication in hepatocytes, phosphorylated STAT5B was found to activate the ATM pathway through the action of peroxisome proliferator-activated receptor gamma (PPARγ) [The most frequently recurring mutated gene identified in our study is s in ATM . One of s in ATM . PARP in (PPARγ) . The mecc signaling pathway, we also identified mutations in FYN kinase, NOTCH molecules, RHOA related molecules, and PLCG1, corroborating data published by several other studies [Our analysis has identified mutations in several of the highly recurrent mutated genes found in previous PTCL sequencing studies, supporting the importance of these pathways in PTCL oncogenesis. Aside from mutations in ATM, p53, and the γ studies , 14, 16.MUC16 and TTN. However, these genes have been found to be recurrently mutated in many different whole exome sequencing studies as they are quite long  and therefore have an increased tendency to accumulate more variants [We also identified recurrent mutations in variants . Thus, dWe used four algorithms, PROVEAN, MutationAssessor, PolyPhen2, and CHASM, as a tool to predict mutations that have a higher chance of driving PTCL oncogenesis –26. Our Publication of our data set will enable inclusion of the whole exome sequencing data from these primary PTCL patient samples in future research and analysis. Further assessments using this data, such as to determine copy number variation (CNV), may yield more insights into potential cancer drivers and therapeutic strategies. CNVs are important sources of genetic variation that involve significantly larger areas of the genome than SNVs and may lead to oncogenic phenotypes. Generally, CNV calling in exome data is more challenging and error prone than in whole genome sequencing and is still further complicated by complexity of tumor genomes. Recently developed tools to improve the accuracy of assessing CNVs in exome sequencing data, such as PatternCNV, may have utility in further identifying PTCL driver variations in the genomes of malignant T cells in this data set .c K315E, may be exposed as recurrent, potentially critical mutations in the process of PTCL tumorigenesis and its relationship to normal T-cell biology to pave the way for the discovery and development of novel therapeutic targets.Our study, together with prior findings, expand the total number of PTCL samples analyzed for somatic mutations so that less common mutations identified in one study, such as γS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.


Salmonella enterica is a pathogen of significant public health importance that is frequently associated with foodborne illness. We report the whole-genome sequences of four multidrug-resistant Salmonella enterica serovar Paratyphi B and Heidelberg strains, isolated from the Colombian poultry chain. The isolates contain a variety of antimicrobial resistance genes for aminoglycosides, β-lactams, fluoroquinolones, sulfonamides, tetracycline, and trimethoprim. Salmonella spp. continue to be one of the most important causes of foodborne gastroenteritis globally, affecting mainly infants under 1 year and children between 1 and 4 years of age  strains have also been reported as the two most prevalent serovars in Colombian poultry    and wereSalmonella enterica serovar Heidelberg  and Paratyphi B  strains. Genomic DNA from each strain was isolated from overnight cultures using the PureLink Genomic DNA minikit (Invitrogen) and DNA libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina). The libraries were prepared according to the manufacturer’s instructions and sequenced on an Illumina MiSeq instrument with 2 × 250-bp paired-end reads, according to standard Illumina protocols. The four genomes were assembled using a reference-guided assembler ARGO, developed at NCBI, and a de novo assembler SPAdes (de novo assembly in the reference-guided assembly. The reference genome GenBank accessions for the assembly were CP000886 (Paratyphi B) and CP001120 (Heidelberg). The genome sequences of strains FSAN332CC, UG1286CA, FCUN156CA, and FSAN236CA consisted of 192, 58, 56, and 58 contigs, respectively, yielding total sequences for each isolate of 5,160,508, 5,135,585, 4,900,493, and 4,996,395 bp, respectively. The overall G+C content for the isolates was determined to be 52.5%. Sequences were annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and have been deposited at GenBank. The results of the genome annotation presenting the number of genes, coding sequences (CDSs), rRNA, tRNA, CRISPR arrays, pseudogenes, and noncoding RNA (ncRNA) are summarized in Here, we report the whole-genome sequences of four r SPAdes  to creatstrA, aph(3′)-Ia, aadA1, and strB], β-lactams , fluoroquinolones (QnrB19), sulfonamides (sul1 and sul2), tetracycline [tet(A)], and trimethoprim . Future comparative analyses will advance national surveillance programs and our understanding of genome evolution and multidrug resistance in Salmonella.A search for resistance-associated genes present in all isolates was performed using ResFinder (v2.1)  with defThis whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession numbers listed in


FPA cve NFPAs , 6. The ve NFPAs , 8; for ve NFPAs , and estve NFPAs –12. The ve NFPAs , 14, andve NFPAs ; for exave NFPAs , 17. Oneve NFPAs –11. Therve NFPAs  and precve NFPAs  practice−, LH+, FSH+, and LH/FSH+; NF− means NFPA that had negative immunohistochemical stains for ACTH, FSH, GH, LH, prolactin, and TSH) versus pituitary controls (Con) -based quantitative proteomics, PTM-proteomics, bioinformatics, and pathway network-based systems biology were used to investigate the variations in proteome and protein molecular networks of an NFPA , 16, 20.ls (Con) . With coin vitro and in vivo experiments. (ii) 2DGE quantitative proteomics has obvious limitations in maximizing the coverage of an NFPA proteome. The 2DGE with an 18-cm pH3-10 NL IPG strip and 12% SDS-PAGE isolation in mentioned studies detected proteins within a range of Mr 5–150 kDa and pH 4–8  subtypes  those molecular networks derived from 2DGE quantitative proteomics data were constructed with bioinformatics and systems biology. The biological relevance of each molecular network in the pathophysiological processes of an NFPA should be further studied with d pH 4–8 . Multi-dd pH 4–8 . (iii) Pd pH 4–8 , 23 and d pH 4–8 , 24, andd pH 4–8 , 25 wered pH 4–8  and invad pH 4–8  have beed pH 4–8 , 13. An d pH 4–8 , 20, invd pH 4–8 , and fouIn summary, NFPA has high-degree heterogeneity and difficulty in its early-stage diagnosis and therapy. From the angle of systems biology strategy, exploration of variations in molecular networks that operate in an NFPA will be an important approach to the in-depth understanding of molecular mechanisms and discover effective and reliable biomarkers and therapeutic targets for personalized and precision medicine practices in an NFPA patient.XZ conceived of the concept and idea of this article, collected references, wrote, and revised the entire manuscript, was responsible for the corresponding work of the manuscript, and trained YL regarding the concepts of molecular networks and systems biology in cancer. YL participated in the collection of references, revision, and formatting manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Besides these, data clearly indicate the major role of several other salivary proteins, such as salivary carbonic anhydrase (gustin), proline-rich proteins, cystatins, alpha-amylases, histatins, salivary albumin and mucins. Other proteins like glucagon-like peptide-1, salivary immunoglobulin-A, zinc-α-2-glycoprotein, salivary lactoperoxidase, salivary prolactin-inducible protein and salivary molecular chaperone HSP70/HSPAs were also expected to play an important role. Furthermore, factors including salivary flow rate, buffer capacity and ionic composition of saliva should also be considered. In this paper, the current state of research related to the above and the overall emerging field of taste-related salivary research alongside basic principles of taste perception is reviewed.The gustatory system plays a critical role in determining food preferences and food intake, in addition to nutritive, energy and electrolyte balance. Fine tuning of the gustatory system is also crucial in this respect. The exact mechanisms that fine tune taste sensitivity are as of yet poorly defined, but it is clear that various effects of saliva on taste recognition are also involved. Specifically those metabolic polypeptides present in the saliva that were classically considered to be gut and appetite hormones ( The gustatory system plays a critical role in determining food preferences and food intake  and, con2 taste” [2 taste is understood to be a mechanism for recognizing CO2-producing sources so as to, for instance, avoid fermenting foods [There are also various complementary taste modalities like “fat-taste” or “fatty acid taste” ,7, “CO2 2 taste”  and the 2 taste” ,10. Rela2 taste” ,13,14,152 taste” ; whereasng foods .Taste buds are clusters of 50–80 (up to 100) polarized (bipolar) neuroepithelial cells that form compact, columnar pseudostratified “islands” embedded in the surrounding oral epithelium ,18. TherThe taste-sensing signals from taste buds (from taste cell receptors) are transferred through peripheral processes of unipolar nerve cells located in the genicular ganglion of the facial nerve , the inferior ganglion of the glossopharyngeal nerve and the inferior ganglion of the vagus nerve . Throughreceptor cells” earlier, it is now abundantly clear that roughly half or fewer of all these cells are indeed receptors only [Although taste buds in all regions respond to sweet, salty, sour, bitter and very likely also to umami (though this has not been studied as thoroughly), there are differences in their sensitivities to these tastes ,24. Presors only . Besidesors only . Their sors only .Despite type I cells being the most abundant cells of taste buds, their functions are not well characterized ,17,18. Ii.e., ASICs) taste receptors  have also been reported in several extraoral sites such as the gastrointestinal system [Interestingly, cells expressing sweet/umami (T1R) and/or bitter (T2R) and/or sour ; the rel system ; the urol system ; the repl system ,40; and l system . Recentll system . These dl system ; and alsl system ,45.In general, sweet, salty, sour, bitter, and umami are considered to be the basic taste qualities. Recent studies have proposed candidate receptors for these five basic tastes ,47,48 whi.e., cells expressing PKD2L1) possess synaptic structures [Interestingly, only a few taste bud cells have synaptic contact with nerve fibers in the taste buds . It is lructures  and may ructures ,48,52,53ructures ,18. Regaructures ,18, becaructures ,55. The Although the majority (60%–70%) of taste cells respond exclusively to one of the basic taste qualities ,57, theri.e., sugars, artificial sweeteners, sweet amino acids, and sweet proteins), whereas the T1R1/T1R3 heterodimer is activated primarily by monosodium l-glutamate in humans [i.e., mouse) [l-glutamate (MSG) can be considered as the prototypic umami stimulus in humans [i.e., aspartate), many short peptides, some organic acids , and expectably also by other compounds [Sweet and umami taste are recognized by the T1R receptor family  that belongs to family C of GPCRs . T1Rs asn humans  and by a, mouse) ,59,61. An humans ,62,63, uN-terminals to which glutamate still binds, albeit with reduced affinity compared to those receptors expressed in the brain [i.e., in the food) compared to the circumstances in the brain [T1R receptor-independent sweet and/or umami taste receptors may also exist in taste cells . A potenhe brain ,63. Conche brain .Recent studies have shown that sensitivity of sweet taste cells can be modulated by hormones and other endogenous factors  also diri.e., T2R41, T2R42, T2R45, T2R48, T2R60) are still unknown [Bitter taste is recognized by the T2R receptor family that belongs to family A of GPCRs . In huma unknown . T2Rs ar unknown ,69, poss unknown .2+ signaling pathway [2+ stimulates transient receptor potential channel M5 (TRPM5) which depolarizes the taste cell leading to the generation of action potentials by means of the voltage-gated sodium channels (VGSC) of cells [Sweet, umami, and bitter tastes are recognized by different GPCRs but use a common signaling pathway after activation of these receptors ,71,72: t pathway ,48,71,72of cells ,25,48. Tof cells ,18,25.− channels [Sour and salty taste may be mediated by ion channel-type receptors. Sour taste is clearly initiated by protons acting at the sour taste receptor ,73,74. Hchannels , and polchannels ,47,52. cchannels ,80, but channels .+-specific salt taste receptor [+, K+, NH4+ and Ca2+, and is amiloride insensitive [+-specific salt taste receptor (ENaC) and are likely to be expressed by different taste cell types [not Na+-specific components of salt taste responses was suggested to be mediated by a taste variant (TRPV1t) of the transient receptor potential (TRP)-type nonselective cation channel-coupled vanilloid receptor-1 (TRPV1) [In the case of salt taste, the epithelial sodium ion channel (ENaC) is believed to be the primary receptor ,48,81 bereceptor . In addiensitive ,87. Thisll types ,87,88. A (TRPV1) ,81,89; h (TRPV1) .In the case of sour and salty taste sensitive cells, in which channel type receptors are activated by the taste compounds, the signal transduction occurs by means of the ion channel activation induced depolarizations of these cells, which elicit action potentials that depolarize the taste cell leading to the generation of action potentials by means of the voltage-gated sodium channels (VGSC) of cells . The gen2 taste” [2-taste [There are also various receptors of the complementary taste modalities like “fat taste” or “fatty acid taste”  “CO2 tas2 taste”  and tast2 taste” ,10. Howe2 taste”  and astr[2-taste  will be Saliva is a body fluid, primarily produced by three pairs of major salivary glands  and by many minor salivary glands ,92,93. PEntering the mouth, ductal saliva of the salivary glands are blended, and supplemented with various constituents originating from intact or destroyed mucosal cells, immune cells, and oral microorganism ,92,93. IWhole saliva is a major determinant of the environment on all the oral surfaces. On tooth surfaces, saliva plays an important role in acquired pellicle formation, which is a thin layer of several salivary proteins with calciumhydroxide-binding properties ,92,93. TSaliva also takes part in the maintenance of the structures of taste buds and taste-sensing cells. In this respect, the effect of salivary epidermal growth factor EGF , in addiAs mentioned above, saliva is primarily produced by three major salivary glands—the parotid, the submandibular and the sublingual glands—and by numerous minor salivary glands ,92. In rtastant-binding proteins” were hypothesized to function as necessary cofactors in taste perceptions by concentrating and delivering hydrophobic sapid molecules (hydrophobic tastants) to the receptors of taste sensing cells [The secretory fluid of these glands besides others contains certain hydrophobic molecule transporter (lipophilic−ligand carrier) proteins expected as “tastant-binding proteins” ,100 and ng cells  may be iSimilar “tastant-binding proteins” and other proteins with various protein-binding abilities are conceivably also present in the whole saliva. It is also likely that whole saliva fulfills all those functions in relation with other taste buds, which are mentioned above in conjunction with the saliva of von Ebner’s gland in the case of taste buds of circumvallate and foliate papillae.It is likely that the flow rate of saliva can modify the concentration of tastants and various soluble taste perception-related mediators because of a dilution effect ,103. Fur+, but also of others like K+, NH4+ and Ca2+) is detected only when above salivary concentrations, saliva influences salt taste threshold levels [+, K+, Cl−, HCO3− and the charged salivary proteins [Besides flow rate and buffer capacity, ionic composition of saliva may also play a significant role in taste sensation. Since salt taste of various ions  on basal oral epithelial cells [There is increasing evidence that taste perception can be modulated by premised salivary polypepides interacting with their respective receptors expressed in the taste cells; in this way, they influence food intake through fine tuning of taste perception ,119,123.Leptin is a 16 kDa hormone first reported as being produced by adipocytes, but it is also expressed by other cell types, including several cells of mammary gland and placenta and also chief cells of stomach ,126. LepLeptin acts mainly on the hypothalamus, where it exerts a strong neuroendocrine effect on food intake and energy metabolism, but may also act on several other peripheral organs ,126 becaBesides its endocrine action, leptin may also act in an exocrine manner . Leptin i.e., mucosal transudate) may also be possible. Salivary leptin seems to be also rather stable in the oral cavity [Leptin was also detected in saliva ,126, andl cavity , therebyl cavity , it may Ghrelin is a 28-amino-acid peptide hormone, and similar to many other peptide hormones, it is processed from a larger precursor (94-amino-acid) by prohormone convertase PC1/3 . GhrelinGhrelin is conventionally considered to be an appetite-regulating hormone. Ghrelin also has many other actions linked to feeding behavior, energy homeostasis, reproduction, sleep regulation, corticotrope secretion and regulation of gastro-entero-pancreatic functions ,134,135.As indicated above, ghrelin is processed from a larger precursor (94-amino-acid) by prohormone convertase PC1/3 , and botO-acyltransferase (GOAT) [i.e., types I, II and III) of taste cells [i.e., type I, II and III) of taste cells [As indicated above, to activate GHSR (the cognate receptor), ghrelin must be acylated with an eight-carbon fatty acid at serine 3 by ghrelin e (GOAT) ,131,132;Ghrelin and the two receptor isoforms, GHS-R 1a and GHS-R 1b, are also produced by the human salivary glands, with subsequent excretion of the hormone into saliva . Concenti.e., acrylated) and “nonactive”  forms [Importantly, there are both acylated and des-acylated ghrelin in saliva ; latter d) forms , in relad) forms , and as Insulin is the main hormone controlling carbohydrate and lipid metabolism, produced by pancreatic B-cells of the islets of Langerhans . Insulinl-endocrine cells in the distal gut epithelia [Neuropeptide Y (NPY) and peptide YY (PYY) belong to a family of peptides sharing similar hairpin-like PP-fold structural homology and evolutionary history ,141. NPYpithelia ,123,141.pithelia ,123,141.pithelia ,123.i.e., transudate from blood) and taste bud cells [3–36 and PYY3–36 and thereby changing their binding specificity to YRs [Significantly, both neuropeptide Y (NPY)  and peptud cells ,142. In ud cells ,144) truy to YRs ,123. Howy to YRs ,123 becay to YRs  and may 3–36 and PYY3–36 peptides, thereby suggesting the possible role of salivary NPY3–36 and PPY3–36 in modulating taste perception. YRs were colocalized primarily with those neuronal markers which are expressed in type II cells [3–36 and PYY3–36 peptides may play a role in the modulation of one or another type III cell-dependent taste modalities , and perhaps also in the modulation of sour taste.It is significant that all tested YR subtypes  are prominently expressed in mice taste cells and showed preferential apical distribution within most of the cells ,123. ThiII cells , and to II cells . It is t3–36 likely not being involved in the modulation of bitter taste [3–36 is likely to be involved in the modulation of sweet taste [3–36 is likely to play a role in the modulation of a complementary taste modality referred to as fat/fatty acid taste [salivary PYY3–36 resulted in a significant reduction in food intake providing further evidence for a complementary oral pathway of action of PYY3–36 [3–36).Available data point to salivary PYYer taste ,124, wheet taste . Interesid taste ,124. In  PYY3–36 ,119,124 Glucagon-like peptide-1 (GLP-1) is typically considered as a hormone produced by the endocrine cells of the gut . Its priImportantly, GLP-1 is produced in sweet and umami taste-sensing cells ,121, andIn contrast to the other metabolic polypeptides discussed above, GLP-1 was not yet found in the human whole saliva . It is rThe hypothesis that a certain amount of GLP-1 may be present in the saliva, or at least around certain taste buds, is supported by the finding that local oral  administration of GLP-1  induced reduction of food intake in murine very likely via direct action on GLP-1 receptors of taste cells ,119. Thu2 + H2O ↔ HCO + H [i.e., a high amount of salivary CA-VI is supplied to the gastrointestinal tract together with swallowed saliva). CA-VI was also linked to taste function because a previous study reported that hypogeusic subjects had salivary CA-VI as low as 20% of that of normal subjects [The carbonic anhydrases (CAs) are an expanding family of zinc-containing enzymes, which participate in the maintenance of pH homeostasis in the human body, catalyzing the reversible reaction: CO HCO + H . Carboni HCO + H ,147, whi HCO + H ,149,150. HCO + H ,152, as  HCO + H , and it  HCO + H  and in n HCO + H ,147  recognition [2 taste [Importantly, CA-VI was identified as gustin , a previognition ,159,160.[2 taste . Higher [2 taste , indicatProline-rich proteins (PRPs) form a major fraction of salivary proteins. The PRPs are highly phosphorylated proteins . The varN-terminal region, and a somewhat different repeated sequence compared to basic PRPs [Acidic PRPs contain a longer and highly acidic sic PRPs . Acidic sic PRPs ,93,165. sic PRPs ,167. PRPsic PRPs .i.e., were upregulated in saliva) in rats exposed to a tannic acid diet [Salivary PRPs were expected to play a role in the perception of astringency (see below) based on their premised high polyphenol-binding affinity ,168,169 n-propylthiouracil)-induced bitter taste sensation indicate that the salivary level of certain members (namely Ps-1 and II-2) of the basic proline-rich protein family were higher in the unstimulated saliva of PROP super-tasters and PROP medium tasters compared to PROP non-tasters [Importantly, salivary PRPs were also recently linked with bitter taste recognition ,170. Stu-tasters ,170. Dat-tasters . FurtherCystatins are cysteine protease inhibitors that block the action of endogenous, bacterial and parasitic protozoan proteases ,172. Cysi.e., were upregulated in saliva) in rats exposed to atannic acid diet [Although data are still contradictory as to whether increased  or decrecid diet ; its indcid diet . These dHistorically, the perception of dietary fat was thought to be based on olfactory and textural  properties ; howeverThe mechanism how fat/fatty acid-sensing occurs has not yet been established. There are various candidate fatty acid receptors expressed in taste cells, like fatty acid translocase CD36 and G protein-coupled receptors GPR126 or GPR40 . One canIt is very likely that CD36 is involved in the recognition of fatty acids in the oral cavity , and ava3–36  plays an important role in the modulation of a fat/fatty acid taste [3–36 on Y2R type PP-fold peptide-binding receptors (Y2Rs) localized at the apical parts of taste cells [i.e., 6-n-propylthiouracil/PROP/) recognition [Available data clearly indicate the importance of saliva in fat/fatty acid taste sensation . Althougid taste ,124. It ognition ,159,160.ognition ,159,160.tactile sensation [Astringency is a ensation ,10 descrensation . Therefoensation ,168.It is generally accepted that key steps of astringency elicitation are phenol/salivary protein interactions ,186, leai.e., unstimulated) conditions [Thus, it is generally well accepted that the gradual lowering of (soluble and/or surface-attached) polyphenol-precipitating proteins under polyphenol exposure implies an increasing involvement of the deeper mucosal surface-attached protein layer, leading to the rupture of the lubricating film and the consequent appearance of an astringency sensation ,191. Accnditions ,168.In good accordance with this, data indicate that the ability to maintain a nearly constant protein concentration and an unchanged capacity to bind and precipitate polyphenols after masticatory and/or taste stimulation(s), characterize subjects with lower sensitivity to astringency, whereas a strong reduction in the values of both these salivary characteristics characterize more sensitive subjects ,168. Furi.e., salivary mucins and salivary albumin), is likely able to increase the availability of lipophilic polyphenol tastants for taste cells, and in this way can significantly improve taste sensation.Whole saliva and also salivary albumin and mucin are able to solubilize lipophilic plant polyphenols ,193 whicIncreased concentration of certain salivary proteins such as salivary alpha-amylase, salivary albumin and salivary immunoglobulin-A (sIgA) also seems to be coupled with improved taste perception namely in relation with the bitter taste sensation .i.e., for sIgA) and zinc-α-2-glycoprotein [Lower bitter taste acceptance (expectably increased bitter taste perception) of infants was also associated with higher salivary concentration of the secretory component  were co, leptin ,119,123.Moreover, the data clearly indicate the major role of several other salivary proteins: salivary carbonic anhydrase  ,8,114, sA role of certain other proteins like glucagon-like peptide-1 (GLP-1) , salivarAlthough it was suggested nearly a hundred years ago that salivary composition might be responsible for taste differences among people , the exa


C16orf59, DAP3, ETV4, GABARAPL1, PPAR, RADIL, RSPO1, SERTM1, SRPK1, ST6GALNAC6, and TNXB. Our findings imply a multilayered lung adenocarcinoma regulome in which transcript-/exon-level regulation may be dissociated from gene-level regulation. Our described method may be used to identify potentially important genes/transcripts/exonic regions for the tumorigenesis of lung adenocarcinoma and to construct accurate tumor vs. normal classification systems for this disease.Lung adenocarcinoma is one of the most deadly human diseases. However, the molecular mechanisms underlying this disease, particularly RNA splicing, have remained underexplored. Here, we report a triple-level  analysis of lung adenocarcinoma transcriptomes from 77 paired tumor and normal tissues, as well as an analysis pipeline to overcome genetic variability for accurate differentiation between tumor and normal tissues. We report three major results. First, more than 5,000 differentially expressed transcripts/exonic regions occur repeatedly in lung adenocarcinoma patients. These transcripts/exonic regions are enriched in nicotine metabolism and ribosomal functions in addition to the pathways enriched for differentially expressed genes . Second, classification models based on rationally selected transcripts or exonic regions can reach accuracies of 0.93 to 1.00 in differentiating tumor from normal tissues. Of the 28 selected exonic regions, 26 regions correspond to alternative exons located in such regulators as tumor suppressor (GDF10), signal receptor (LYVE1), vascular-specific regulator (RASIP1), ubiquitination mediator (RNF5), and transcriptional repressor (TRIM27). Third, classification systems based on 13 to 14 differentially expressed genes yield accuracies near 100%. Genes selected by both detection methods include  As the most deadly human cancer in the world, lung cancer was responsible for 1.59 million deaths in the year 2012 alone , which aA major goal of lung cancer studies is early clinical detection of the disease. Numerous molecular methods have been developed for this purpose. For instance, the serum concentrations of CEA and CYFRA 21-1 have been proposed as markers of lung cancer , 11, altRecently, transcriptomic approaches have been widely applied to the identification of biomarkers in lung cancer . Most prAberrant splicing has been implicated in apoptosis, evasion from immune surveillance, cell proliferation, the Warburg effect, angiogenesis, metastasis, and response to anticancer drugs , 31. Theet al. were differentially expressed between lung cancer and normal tissues  CRL-5870), the embryonic kidney cell line HEK-293T (ATCC CRL-3216), and the lung adenocarcinoma cell line CL1-5 (provided by Dr. Cheng-Wen Wu ). Cells ETV4 (NM_001986) was amplified from the cDNA of CL1-5 cells by polymerase chain reaction (PCR), using forward primer (F) gggaattcGG ATGGAGCGGA GGATGAA and reverse primer (R) gggcggccGC TGGGGGCTAG TAAGAGT, where gggaattc and gggcggcc were sequences including the restriction enzyme cutting site. The gene was cloned into the EcoRI and NotI sites of pcDNA6 (Invitrogen). ETV4 shRNA clones (TRCN0000013933 and TRCN0000013936) and the luciferase shRNA control clone (TRCN0000072244) were purchased from the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. All plasmids were confirmed by DNA sequencing.Full-length human ETV4, H1435 lung cancer cells were transfected with ETV4-overexpressing plasmid or control vector (pcDNA6) for 48 h by using the Fugene Reagent (Roche). For lentiviral production, the envelope plasmid (pMD2.G), packaging plasmid (pCMV-deltaR8.91), and target gene (ETV4 shRNA clones and luciferase shRNA control clone) were transfected into HEK-293 cells by using Polyethylenimine Reagent (Sigma) for 24 h. The media was changed to Complete Medium, which included 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. After 48 h, the supernatant was collected and filtered by a 0.2-μm syringe filter . For lentiviral infection, CL1-5 cells were infected with 2 ml of lentiviral supernatant and 2 μl of polybrene  for 48 h. These transient transfectants were further assayed according to the purpose of each experiment.For overexpression of ETV4 , ALDH , CD133 , SOX2 (F: atgggttcggtggtcaagt and reverse primer: actctggggctccttcttg), and GAPDH . Relative gene expression was calculated by 2−ΔCT .Total RNA was isolated by using Trizol (Invitrogen) and used as a template for reverse transcription into cDNA by using the M-MLV Reverse Transcription Kit (Invitrogen). To quantify mRNA expression, qRT-PCR was performed with a LightCycler 480 (Roche) for the following genes (primer pairs): 3 cells) were seeded in 96-well tissue culture plates and incubated for 6 h. MTT (5 mg/ml) was added for further 4 hours after the indicated time points. Supernatant was removed, and 100 μl DMSO were added to dissolve the crystal. The absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay reader, with subtraction of background at 630 nm.Cell proliferation was assessed by the MTT  -2, 5-diphenyltetrazolium bromide) assay. Cells , followed by flow cytometric analysis in the green fluorescence channel, FL1. The aldefluor-specific inhibitor, diethylaminobenzaldehyde, was added to the sample as a control to define the gates of ALDH+ region.The


Gossypium barbadense chromosomes with data from its recurrent parent TM-1 during fiber elongation (from 5 DPA to 20 DPA).Cotton fiber, a highly elongated, thickened single cell of the seed epidermis, is a powerful cell wall research model. Fiber length, largely determined during the elongation stage, is a key property of fiber quality. Several studies using expressed sequence tags and microarray analysis have identified transcripts that accumulate preferentially during fiber elongation. To further show the mechanism of fiber elongation, we used Digital Gene Expression Tag Profiling to compare transcriptome data from longer fiber chromosome introgressed lines (CSILs) containing segments of various A large number of differentially expressed genes (DEGs) involved in carbohydrate, fatty acid and secondary metabolism, particularly cell wall biosynthesis, were highly upregulated during the fiber elongation stage, as determined by functional enrichment and pathway analysis. Furthermore, DEGs related to hormone responses and transcription factors showed upregulated expression levels in the CSILs. Moreover, metabolic and regulatory network analysis indicated that the same pathways were differentially altered, and distinct pathways exhibited altered gene expression, in the CSILs. Interestingly, mining of upregulated DEGs in the introgressed segments of these CSILs based on D-genome sequence data showed that these lines were enriched in glucuronosyltransferase, inositol-1, 4, 5-trisphosphate 3-kinase and desulfoglucosinolate sulfotransferase activity. These results were similar to the results of transcriptome analysis.This report provides an integrative network about the molecular mechanisms controlling fiber length, which are mainly tied to carbohydrate metabolism, cell wall biosynthesis, fatty acid metabolism, secondary metabolism, hormone responses and Transcription factors. The results of this study provide new insights into the critical factors associated with cell elongation and will facilitate further research aimed at understanding the mechanisms underlying cotton fiber elongation.The online version of this article (doi:10.1186/1471-2164-15-838) contains supplementary material, which is available to authorized users. Cotton is a commercial fiber crop, producing the most prevalent natural fibers used by the textile industry. Cotton fibers are single-celled seed trichomes that develop from epidermal cells of the ovule, 30% of which differentiate into spinnable fibers , 2. CottGossypium hirsutum during the elongation period [G. hirsutum, G. barbadense and G. arboretum cotton species [Cotton fiber is an excellent model for studying the mechanisms of plant cell elongation, with peak rates of expansion of >2 mm/day in n period , 8, 9. I species , 10–12.  species , 13–15.  species , 17, eth species , 18 and  species  are invo species , 21. Som species  and down species . Several species –26. Tran species –29.G. hirsutum, which represents over 95% of cultivated cotton worldwide, is characterized by high yield and moderate fiber quality. G. barbadense, acultivated extra-long staple tetraploid cotton, is characterized by low yield and increased fiber quality (fineness and strength). Chromosome segment introgression line (CSIL) production is an effective method for combining the high yield of G. hirsutum with the superior fiber properties of G. barbadense. With the exception of a single, homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSIL is the same as that of the recipient parent [Lycopersicon esculentum (tomato), Oryza sativa (rice), Triticum aestivum (wheat) and Gossypium (cotton) [t parent . CSILs c(cotton) –34.G. barbadense chromosome segments in the background of the standard genetic line G. hirsutum cv. TM-1, developed in our laboratory [In this study, we analyzed the transcriptome profiles of longer fiber CSILs containing inserts of various boratory , using tG. hirsutum cv. TM-1, the genetic standard line for upland cotton [G. barbadense cv. Hai7124, extra-long staple cotton, is widely grown in China [G. barbadense chromosomal segments were different in all four lines [d cotton , was obtin China , 36. Thein China . The intur lines .The sampTotal RNA was extracted from frozen tissue using an improved CTAB extraction protocol . RNAs weDigital gene expression (DGE) libraries were constructed using an Illumina Gene Expression Sample Preparation Kit according to the manufacturer’s instructions. A total of 24 libraries derived from immature fibers at 5, 10, 15 and 20 DPA were constructed and sequenced using the Solexa Genome Sequencing Analyzer system provided by BGI , which was described in detail previously .G. raimondii (http://www.phytozome.net). The remaining high quality sequences were then mapped to this database; only a single mismatch was allowed, and more than one match was excluded. Gene expression levels were the summation of tags aligned to different positions of the same gene. Expression levels were expressed as TPM, transcripts per million. To identify DEGs during fiber elongation, pairs of DEG profiles from different libraries were compared. Four fiber developmental periods for the five CSILs were compared with the same period for TM-1, and 20 comparisons were obtained. P- and Q-values were also calculated for every comparison [2Ratio| ≥ 1, to judge the significance of differences in transcript abundance.Raw data reads were filtered by the Illumina pipeline to produce clean data. All low-quality data, such as short tags (<21 nt) and singletons, were removed. A database of 21-base-long sequences was produced beginning with CATG using 37,505 reference genes from the diploid species mparison . DEGs wehttp://www.tm4.org/mev.html) [http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm).Genes expressed in more than half of the libraries were used for PCC analysis, and clustering of log2-transformed TPM values of these genes was performed with the “Self-organizing tree algorithm”  . Clusterhttp://www.blast2go.com/b2ghome).Mapman was also used to analyze gene enrichment  and metahistone3 gene (ACC NO.AF024716) was used as an internal control for normalization of expression levels . The relative gene expression levels were presented as 2-ΔCT. The Pfaffl method was used to analyze expression data [Quantitative RT-PCR assays were performed in a 7500 Real-Time PCR system . The reactions were performed in a final volume of 20 μL containing 2 μL of diluted cDNA, 10 μL of 2× SYBR mix  and 200 nM of forward and reverse primers. Primer lengths were designed to range from 18 to 24 nt using Beacon Designer 7, and PCR amplicon lengths were designed to range from 100 bp to 150 bp. The thermal cycling conditions were 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. All reactions were run in triplicate, and the cotton ion data .G. barbadense chromosome segment(s), were identified. The average fiber length of CSIL-35431, CSIL-31134, CSIL-31068 and CSIL-31044 was 31.33, 30.63, 31.00 and 29.97 mm, respectively, which was significantly longer than that of TM-1, while CSIL-35368 had a shorter fiber length than TM-1, at 27.67 mm  were expressed in more than half of the 24 libraries. To examine the relationship between the experimental samples, Pearson correlation coefficient (PCC) analysis was performed on these genes obtained from all 24 libraries. As shown in Figure To examine gene expression patterns during fiber development, the 22,153 genes were classified into four groups, along with an unclassified group  in the CSILs, we examined 20 comparison groups between CSILs and TM-1 from 5 DPA to 20 DPA. The number of DEGs between the same developmental stage ranged from 4,500 to 8,000  showed similar results, according to genes in Additional file 81 genes Phytohormones play an important regulatory role in various plant growth and developmental processes. In the present study, DEGs involved in hormone biosynthesis and signal transduction pathways were identified in the CSILs. Numerous genes involved in hormone signal transduction and biosynthesis of auxin, BR, ethylene, JA and ABA were upregulated in the CSILs , cell wall, lipid, starch and sucrose metabolism exhibited altered expression at 10 DPA in all three CSILs  at 10 DPA and 15 DPA in the four longer fiber CSILs ; therefore, quantitative real-time PCR confirmed the accuracy and reliability of the expression levels determined by DGE analysis.To validate the transcriptome results, we selected 13 genes in two plant materials for quantitative real-time PCR analysis. The results of pair-by-pair comparisons between the transcriptome data and qRT-PCR analysis and qRT-PCR primers are shown in Additional file Cotton fiber cell elongation is a complex and highly regulated process involving metabolic pathways, signal transduction and transcriptional regulation. To date, the roles of carbohydrate metabolism, phytohormones, lipid metabolism and transcription factors in promoting cotton fiber elongation have been underexplored. CSILs with longer fibers provide an excellent system for studying cotton fiber elongation. First, our results support the notion that these metabolic and signaling pathways are significantly upregulated and cooperate during cotton fiber elongation. In addition, we obtained detailed information about DEGs involved in cell wall precursor biosynthesis , cell wall-related protein , very-long-chain fatty acid biosynthesis , phenylpropanoid pathway  and so on. We also identified new candidate genes in introgressed segments by combing the transcriptome data. Our integrated analysis provided additional details and new insights into the mechanisms of fiber elongation.Function enrichment, KEGG analysis and metabolic pathway analysis showed that the categories carbohydrate metabolism and cell wall biosynthesis were highly enriched in the CSILs, and the categories cell wall processor synthesis, cellulose synthesis, hemicellulose synthesis, cell wall proteins  and pectin were enriched as well  genes are required for cellulose synthesis [GhCESA5–GHCESA10) were identified; CSEA1, CESA3, CESA6, CESA9 and CESA10 are involved in primary cell wall biosynthesis [COBRA is critical for the biosynthesis of cell wall components [Cellulose synthase genes were significantly upregulated in CSIL-35431, such as genes encoding CESA3, CESA6 and CESA7. In ynthesis . Recentlynthesis . In addimponents , 49.Pectin is a polysaccharide component of the primary cell wall, and pectin modification enzymes play an important role in fiber elongation , 22, 50.GhFLA1 improves fiber initiation and elongation by affecting the integrity of the primary cell wall matrix [Arabinogalactan proteins (AGPs), which are involved in many aspects of plant development, are abundant in developing fibers and are involved in fiber elongation. l matrix . We founExpansins and tubulins are important for cell wall-loosening and fiber elongation , 52, 53.G. barbadense segments also showed that genes involved in glucuronosyltransferase, inositol-1, 4, 5-trisphosphate 3-kinase and desulfoglucosinolate sulfotransferase (SOT) activity were upregulated in the CSILs, indicating the important roles of these enzymes in carbohydrate metabolism. Several SOT proteins have been characterized in Flaveria sp. and Brassica napus L. that show substrate specificity for several flavonols, steroids and brassinosteroids, and these genes were involved in various physiological processes, such as growth, development and adaptation to stress [Mining of DEGs in introgressed o stress –58. Alsoo stress .Fatty acids are another important factor involved in fiber elongation , 61. SevSecondary metabolism-related genes were among the most statistically significant differentially expressed categories between CSILs and TM-1 during fiber elongation. The phenylpropanoid pathway participates in the biosynthesis of many plant cell wall phenolics, which are responsible for the biosynthesis of a variety of products including lignins, lignans, hydroxycinnamic acid conjugates, flavonoids and other related constituents, and cinnamyl alcohol dehydrogenase (CAD) is considered to be a key enzyme in the biosynthesis of these products . Our resGhTUB1, GhTUB3 and GhTUB9 in cultured cotton ovules [In this study, a large group of genes response to hormones including auxin, BR, ethylene, JA and ABA were upregulated in the CSILs, which were shown to play roles in fiber cell elongation  under the accession number SRP047139.Additional file 1: Table S1: Categorization and abundance of tags. (XLS 36 KB)Additional file 2: Figure S1: Statistics of DEGs between CSILs and TM-1 from 5 DPA to 20 DPA. Orange bar, upregulated genes compared to TM-1; blue bar, downregulated genes compared to TM-1, green square, total DEGs. CSILs included CSIL-35431, CSIL-31134, CSIL-31068, CSIL-31044 and CSIL-35368, compared with TM-1. 5, 5 DPA; 10, 10 DPA; 15, 15 DPA; 20, 20 DPA. (TIFF 248 KB)Additional file 3: Figure S2: Functional enrichment analysis of common DEGs between four CSILs with superior fiber quality. (A) Common DEGs between four CSILs from 5 DPA to 20 DPA. A, B, C, D indicated CSIL-35431, CSIL-31134, CSIL-31068, CSIL-31044, respectively. (B) Gene enrichment analysis of common DEGs. Color from blue to red means function enriched with smaller p-value. (TIFF 491 KB)Additional file 4: Table S2: GO enrichment analysis of common upregulated DEGs in four CSILs. (XLS 34 KB)Additional file 5: Figure S3: GO enrichment analysis of genes downregulated only in CSIL-35368. (A) Common DEGs of four CSILs. A, B, C, D indicated CSIL-35431, CSIL-31134, CSIL-31068, CSIL-35368, respectively. 5, 5DPA; 10, 10 DPA; 15, 15 DPA; 20, 20 DPA. (B) GO enrichment of DEGs only down-regulated in CSIL-35368 from 5 to 20 DPA. (TIFF 464 KB)Additional file 6: Table S3: Pathway analysis of all DEGs in four CSILs with superior fiber quality. (XLS 215 KB)Additional file 7: Figure S4: Pathway analysis of common DEGs between four CSILs from 5 to 20 DPA. All the DEGs used to analysis pathway were according to Figure Additional file 8: Table S4: Pathway analysis of common DEGs between four CSILs. (XLS 72 KB)Additional file 9: Table S5: Expression levels of DEGs involved in hormones, cell wall biosynthesis and TFs. (XLS 144 KB)Additional file 10: Figure S5: Overview of metabolism-related DEGs in different CSILs at 10 DPA. (A) Overview of metabolism-related DEGs in CSIL-35431. (B) Overview of metabolism-related DEGs in CSIL-31134. (C) Overview of metabolism-related DEGs in CSIL-35368.1, cell wall proteins, HRGP; 2, cell wall proteins, AGPs; 3, cell wall proteins, LRR; 4, minor CHO metabolism, callose; 5, cell wall precursor synthesis; 6, cell wall pectin esterases and PME; 7, cellulose synthesis; 9, FA synthesis and FA elongation. 10, starch synthesis; 11, starch degradation; 12, sucrose degradation. Blue square, downregulated gene; Red square, upregulated gene. (TIFF 970 KB)Additional file 11: Table S6: List of DEGs that participate in different metabolic pathways. (XLS 48 KB)G. barbadense chromosome segments. (XLS 42 KB)Additional file 12: Table S7: Upregulated DEGs located in introgressed G. barbadense chromosome segments. Every square indicated one molecular function with GO term number. From up to down, the rank level of GO term became lower. With the color deepened, from white to red, the molecular functions were enriched more significantly with smaller P-value. DEGs involved the GO term were listed. (TIFF 305 KB)Additional file 13: Figure S6: GO term graph of upregulated DEGs located in introgressed Additional file 14: Table S8: Correlation between quantitative real-time PCR and DGE analysis of 13 DEGs. (XLS 30 KB)


Bacillus anthracis strain Stendal, isolated from one of the diseased cows.In July 2012, an anthrax outbreak occurred among cattle in northern Germany resulting in ten losses. Here, we report the draft genome sequence of  Bacillus anthracis is a rare disease in Germany, with only three reported outbreaks between 2009 and the end of 2015 . For the WGS library, 1,705,145 reads with a total of 460 Mb were generated. Bowtie-2 (Whole-genome shotgun (WGS) sequencing of Bowtie-2 , where nB. anthracis Stendal was 5,227,419 bp with a 63-fold coverage for the chromosome (137-fold for pXO1 and 103-fold for pXO2), and the mean G+C content was 35.5%. For initial annotation, the assembled contigs were submitted to the RAST annotation pipeline , CP014178 (pXO2), and CP014179 (chromosome). The versions described in this paper are the first versions.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers