Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA+ and OMPLA- H. pylori variants.The association between H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment.Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA+/-did not affect the acute inflammatory response to H. pylori.In whole genome analysis of the epithelial response to  H. pylori is well established as the primary cause of peptic ulcer disease and the initiator of the multistep cascade leading to gastric adenocarcinoma. Although gastric cancer is the fourth most common cancer worldwide and second only to lung cancer in causing cancer related deaths [H. pylori prevalence in the western world, but this phenomenon does not completely explain the great geographical differences in gastric cancer distribution. The reason why only 1-2% of H. pylori-infected individuals develop gastric malignancies remains unexplained, and includes both differences in bacterial strains, most importantly cagA status, host genetics and environmental aspects.d deaths , there aH. pylori carcinogenesis involves indirect action of the bacteria through chronic inflammation of the gastric corpus mucosa, and also direct action of H. pylori on epithelial cells. Persistent inflammation is associated with enhanced production of several pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-7 and IL-8 [H. pylori on epithelial cells may also promote carcinogenesis. + H. pylori cagAstrains inject bacterial products into epithelial cells through a sophisticated type IV injection process, which activates intracellular signaling pathways, in particular the mitogen-activated protein kinase family (MAPK) pathway [H. pylori on gastric epithelial apoptosis has showed conflicting evidence. Several in vitro studies have showed that H. pylori stimulate apoptosis [+ H. pylori cagAinfection.and IL-8  which in pathway  and nucl pathway , all of  pathway ,6, wherepoptosis ,10. CagApoptosis  and intepoptosis . ASPP2 iH. pylori pldA gene, coding for bacterial outer membrane phospholipase A (OMPLA), displays phase variation resulting in 'ON' (OMPLA+) and 'OFF' (OMPLA-) switching of OMPLA activity due to a spontaneous slippage in a homopolymer (C) tract of the gene [+ variant was associated with increased bacterial survival in an acidic environment, adherence, hemolysis and release of urease and VacA compared to the OMPLA- variant [Tannæs et al. have previously reported that the the gene . The OMP variant . OMPLA h variant , and a l variant .H. pylori exposure has been subjected to many experiments since the discovery of the bacterium in 1984 [H. pylori strains, and no authors have compared the epithelial cell response to OMPLA+ against OMPLA- bacteria. The aim of the current study was to investigate the temporal gene expression response of gastric epithelial cells exposed to a clinically obtained H. pylori strain, and to examine the contribution of OMPLA on the inflammatory response. Emphasis has been put on the most important biological responses using Gene Ontology (GO) terms and associated cellular signaling pathways.Although the gastric epithelial cell response to  in 1984 , only a  in 1984 -29. AlmoH. pylori infection at 3 and 6 h, non-exposed and H. pylori exposed cells were stained and examined with immunofluorescence microscopy . We therefore concluded that analysis of the results could be performed without further consideration of differences in phase variation.To study the cellular morphology following y Figure . At bothH. pylori exposed AGS cells were compared against non-infected control cells at six separate time points within 24 h. 7498 chip probes corresponding to 6237 human genes showed differential expression in the infected cells compared to control cells at no less than 1 time point (p < 0. 05)  signaling pathways as well as many cancer-related pathways and immunological pathways. At 1 h, IL-8 was involved in most of the affected signal pathways. At 3 and 6 h, most of the highest ranked pathways had several genes in common, such as NFKB1, NFKB2, NFKBIA, NFKBIE, BIRC2, BIRC3, JUND, CCND1 and AKT3. The phosphatidylinositol signaling system is assigned a high IF at 6 h due to the significance of one single gene, PIK3C2B, which is down-regulated by a log2FC of -0.58 and plays a key role in this pathway. At 12 h, the most affected cellular pathways were the leukocyte transendothelial migration, cell adhesion molecules, DNA replication pathway, p53 signaling pathway as well as several cancer-related pathways. Relatively similar results are seen at 24 h, however some of the cancer-related pathways are represented further down the list  signal pathways by Pathway Express at each time point. Significantly impacted pathways and corresponding Impact Factor (IF) are presented in Table 2FC > 1.5 in the GO analysis, excluding lesser significantly expressed genes that were likely to result in erroneous GO ranking. Only terms categorized under Biological Processes are included , at a distance threshold of 0.54, . Table IL-8 and CXCL2 seemed to dictate many of the acute inflammatory processes like chemotaxis, immune response and neutrophil activation.Hierarchical clustering of the 245 genes with a log, Figure . Each cl, Figure . GO analJUN, GADD45A, DDIT3, MKNK2, DUSP1, RPS6KA5, FLNC, and RASGRP were also involved in MAPK signaling. Furthermore, CSF2RA, IL24, IL20R and the oncogene PIM1 were involved in Jak-STAT signaling and cytokine-cytokine signaling.Cluster C comprised the largest cluster, and contained 150 genes that did not show any change until after 6-12 h. The GO terms apoptosis, cell cycle arrest and stress response genes were markedly enriched, and many of these genes such as EDN1, one of the isoforms of the potent vasoconstrictor endothelin, which enriched virtually all of the listed GOs. NFKB2, one of two NF-κB subunits, HBEGF and ETS1 were also included in this cluster.Cluster D showed a moderate increase peaking at 12 h, followed by a decrease towards 24 h. 13 genes were assigned to this cluster, including FGFR3 and several heat shock protein genes that were involved in the MAPK signaling pathway and apoptosis inhibition. Also, several GO biosynthetic processes were enriched.Cluster E demonstrated 71 genes that showed down-regulation after 6-12 h and included IL-8, as this was the single most differentially regulated gene in the study. mRNA and protein were sampled at the same time points and studied by rt-PCR and ELISA  demonstrated increased cell growth, whereas at MOI of 200:1, apoptosis was stimulated [H. pylori on apoptosis: caspase inhibitors HSPA5 and DHCR24 showed similar late down-regulation as heat shock genes HSPA1B, HSPB1, which are also associated with apoptosis stimulation . MCL1, an anti-apoptotic gene expressed in response to CagA injection [BCL-2 and very little increase in BAX expression in our study, two important genes that determine the sensitivity of cells to other apoptotic stimuli [TP53BP2, an important tumor suppressor gene (TSG) in human cancer, primarily stimulating p53 promotion of apoptosis genes. On the other hand, TP53BP2 is coding ASPP2 protein, which has also been shown to stimulate apoptosis independently of p53 [TP53BP2 expression seen in our study, might therefore potentiate this effect by increasing the CagA-ASPP2 interaction to cause increased inhibition of p53-mediated apoptosis. In fact, the current study showed that p53 target genes involved in apoptosis [FAS, DR4, TNFRSF10B , DCR1, DCR2, P53AIP1, CASP6, APAF1 and BNIP3L did not show any significant increase, and BNIP3L, CASP6 and APAF1, BID and BAX showed only little increase. p53 target genes regulating non-apoptotic cellular processes including MDM2, GADD45A, CDKN1A , EGFR, CCND1, CCNG2 and TGFA demonstrated moderate to marked up-regulation. This differential gene expression identified among the p53 target genes in this study, may indicate selective inhibition of p53-mediated apoptosis due to increased CagA-ASPP2 interaction, consistent with Buti's findings.poptosis ,8, wherepoptosis ,10. VacApoptosis -33, wherpoptosis ,12,34. Bimulated . CagA maimulated . Apoptosnjection , demonst stimuli -39. Notey of p53 -42. Howepoptosis  such as H. pylori exposure, and the cluster analysis suggests that there is some biological coordination of gene expression regulating apoptosis. This may explain some of the complex carcinogenic mechanism of H. pylori in gastric adenocarcinoma. There is strong association between H. pylori infecton, in particular the cagA+ genotype [H. pylori [cagA+ H. pylori infection, in particular pathways in cancer, bladder cancer, prostate cancer, small cell lung cancer and the MAPK pathway. Several individual oncogenes and cancer related genes were also increased during, and at the end of the study, including ANGPT2, CEBPB, ECGF1, MMP7, MMP10, JUN, FOSB, EGFR, CTNNB1, ANXA1, CD55, CLDN1, KLK6, KRT7, LCN2, MYC, PIM1, PIM2, PIM3 and ATF3.Nevertheless, this study was not designed to assess whether the overall sum of inhibitory and stimulatory signals facilitated apoptosis or proliferation of epithelial cells. The current results illustrate the complexity of apoptosis regulation in epithelial cells in response to genotype , and gasgenotype ,46, and . pylori ,48. FurtIL-8 appears paramount in the acute inflammatory response to H. pylori infection, as this gene is involved in all significant response pathways in the initial cellular response to infection. Several authors have demonstrated increase in IL-8 in response to H. pylori in both in vivo [IL-8 mRNA in vitro peaks between 2 and 4 h before decreasing over the next hours under similar conditions [H. pylori-induced IL-8 secretion may be explained by both stimulation of the MAPK signaling system [H. pylori exposure. Regulators of NF-κB; TNFAIP3, RELB and BIRC3, which could also have explained the IL-8 expression, show increasing expression after 3 h  and a truncated, non-functional phospholipase A (OMPLA-) respectively. The isogenicity of the variants was previously confirmed by amplified fragment length polymorphism [vacA genotype and were cagA positive displaying an ABC EPIYA genotype [cagα, cagβ, cagE, cagL, cagM, cagX and cagY genes indicated that the variants harboured an intact cag pathogenicity island (cagPAI) and were capable of CagA translocation (unpublished data). Both variants displayed a truncated LPS.The bacterial strain, at -70°C . The twomorphism . The vargenotype ,80. The 600. Aliquots of the OMPLA+ and OMPLA- bacterial suspensions were transferred to separate cell culture flasks at appropriate concentrations. Dilutions of the suspensions were also plated onto blood agar plates. After 5 days of microaerobic incubation, the colonies were counted and inspected for any OMPLA phase shifts.The bacteria were cultured on blood agar plates under microaerobic conditions at 37°C for 48 h. After cultivation, the bacteria were harvested and suspended in phosphate buffered saline (PBS). Bacterial concentrations were estimated by measuring OD2 incubator at a gas composition of 5% CO2 and 20% O2. When cells grew to a confluent monolayer of approximately 5,1 × 106 cells/flask (60%) the medium was changed to RPMI supplemented with 2 mM L-glutamine only. After an equilibration period of about 30 min, bacteria in PBS were added. To study AGS cell gene expression during the first 24 h, the cells were co-cultured with the H. pylori at a multiplicity of infection (MOI) of 300:1. The two phase variants (OMPLA+ and OMPLA-) were assigned to separate co-cultures, to allow the investigation of the whole genome response to H. pylori infection per se, and also to study possible differences in the response to the OMPLA+ and OMPLA- variants. Co-cultured cells were incubated for 30 min, 1, 3, 6, 12 and 24 h, before RNA was stabilized by RNAlater , and the cells were harvested.The gastric epithelial cell line AGS (American Type Culture Collection no: CRL 1739) was grown on RPMI supplemented with 2 mM L-glutamine and 10% foetal calf serum at 37°C in a COlater followed by harvesting of the cells. Non-infected AGS cells served as a negative control. Both the time-course and the dose-response experiments were carried out in three cell culture replicates and independently performed twice on separate days.To ensure that the obtained gene response was adequate, a dose-response experiment was performed, adding bacteria to AGS cells at a MOI of 15:1, 150:1, 300:1, 600:1, 900:1 and 1200:1. Cells were co-incubated for 3 h, before being immersed in RNAHelicobacter antibody  for 1 h at room temperature. Subsequently, the AGS cells were morphologically examined using a fluorescent microscope  under a 40x objective.Briefly, the bacteria were added to AGS cells grown on glass coverslips at a MOI of 300:1. The cells were co-incubated for 3 and 6 h and then fixed by 4% formalin. Following blocking with 5% bovine serum albumin in PBS for 30 min, the bacteria were stained with rabbit anti-Total RNA was isolated using RNeasy Mini  according to the manufacturer's protocol. RNA concentration and quality were determined using a NanoDrop ND-1000 spectrophotometer  and Agilent 2100 Bioanalyzer . For real-time PCR, cDNA was prepared using a First-Strand cDNA Synthesis Kit , according to standard protocol.The Illumina TotalPrep RNA amplification Kit  was used to amplify RNA for hybridization on Illumina BeadChips. To synthesize first strand cDNA by reverse transcription, we used total RNA from each sample collected above. Following the second strand cDNA synthesis and cDNA purification steps, the in vitro transcription to synthesize cRNA was prepared overnight for 12 h.IL-8 was analyzed using custom IL-8 primer and probe . Mean cycle time (Ct) was calculated, and the comparative Ct-method [Each sample was tested in triplicate by real-time quantitative PCR (rt-PCR) on the 7900HT Fast Real-Time PCR system (Applied Biosystems). Expression of t-method  was utilIL-8 protein was measured in the cell culture supernatant by the Quantikine Human CXCL8/IL-8 enzyme linked immunosorbent assay (ELISA) kit, according to manufacturer's instructions . The test samples were not diluted. Serial dilutions of recombinant human IL-8 were used for standard curves. The optical density of the wells was determined using a microtitre plate reader  set to a wavelength of 450 nm, with wavelength correction set to 540 nmThe gene expression profiles were measured using Illumina Human HT-12 v3 Expression BeadChip , which enables genome-wide expression analysis  of 12 samples in parallel on a single microarray. 35967 of the probes were designed using the RefSeq  library and 12.837 probes were derived from the UniGene (build 199) database ,86.2-transformed and quantile normalized. Moderated t-tests [p-values were calculated by controlling the false discovery rate (FDR), using the Benjamini-Hochberg procedure [p-value  < 0.05. The analysis was performed using the R statistical package [R/BioConductor ,88, with t-tests  were therocedure . The dif package  and the 2 fold change (log2FC) > 1.5 was applied to the total genelist of 6237 significant genes  software  for hierhttp://www.ebi.ac.uk/arrayexpress.The microarray data are available under the accession number E-MTAB-846 in the ArrayExpress database LLE, YE and TMT performed inoculation and co-incubation of cells and bacteria, as well as performed ELISA and rt-PCR analysis. YE and TMT carried out immunofluorescence and microscopy. IRKB participated in the design of the study, and GB coordinated the study and helped to draft the manuscript. LLE carried out the microarray data analysis and wrote the main manuscript. All authors read and approved the final manuscript.Table S1. The list of genes that showed significant differential expression at no less than 1 time point in H. pylori exposed AGS cells (p < 0.05).Click here for fileTable S2. The list of genes that showed significant log2 fold change > 1.5 in H. pylori exposed AGS cells at no less than 1 time point (p < 0.05).Click here for file


Altogether, these advances in podocyte research indicate that calcineurin-NFAT signal or calcineurin-synaptopodin axis has a direct proteinuric effect on podocytes which raises the possibility of developing specific antiproteinuric drugs that lack the unwanted effects of calcineurin or NFAT inhibition.The antiproteinuric effect of cyclosporine A(CsA) has been believed to result from its immunosuppressive effect on the transcription factor NFAT in T cells. However, current evidences supporting this hypothesis are missing. A recent study showed that CsA has a direct antiproteinuric effect on podocytes, suggesting a novel non-immunosuppressive mechanism for CsA's antiproteinuric effect. Conditional NFATc1 activation in podoyctes per se is sufficient to induce proteinuria in mice, indicating that NFAT activation in podocytes is a critical pathogenic molecular event leading to podocyte injury and proteinuria. Meanwhile, evidence showed that TRPC6-mediated Ca Clinically, calcineurin inhibitors  have been used to reduce proteinuria in focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and other proteinuric kidney diseases . T-cell α-actinin-4 [Proteinuria, a cardinal sign and a prognostic marker of kidney disease, affects several hundred million people worldwide . Proteinctinin-4 , CD2AP [ctinin-4 , 17, nepctinin-4 , PLCE1 [ctinin-4 , podocinctinin-4 , TRPC 6 ctinin-4 , 22, forctinin-4 , and MYOctinin-4  lead to ctinin-4 , 25. Othctinin-4 , 27, podctinin-4 , FAT1 [2ctinin-4 , Nck1/2 ctinin-4 , and synctinin-4 .TRPC6 is a member of the large transient receptor potential superfamily of nonselective cation channels , 33. MutThe actin-binding protein synaptopodin, which is highly expressed in podocytes , is a keβ3 integrin colocalize in podocytes and thus form a complex with β3 integrin, thereby causing the activation of β3 integrin. In vivo gene delivery of constitutively active β3 integrin is sufficient to induce proteinuria in mice; conversely, inhibition of uPAR expression and β3 integrin activation has an antiproteinuric effect [Recently, urokinase receptor (uPAR) and its soluble form (suPAR) have been shown to be involved in the pathogenesis of proteinuria and FSGS , 40. uPAc effect .β3 integrin on podocyte foot processes may be the mechanism of injury caused by high suPAR blood concentrations [Interestingly, suPAR, a soluble form of uPAR has been identified as a circulating FSGS factor that is elevated in the serum of approximately two-thirds of primary FSGS patients. suPAR-mediated activation of trations .2+-dependent serine/threonine phosphatase composed of a catalytic subunit, CnA, and a regulatory subunit, CnB [The calcineurin is ubiquitously expressed in all mammalian tissues and is a Canit, CnB , 44. Calnit, CnB , 46, incnit, CnB , 44.Clinically, CsA is used to reduce proteinuria in kidney diseases, such as idiopathic nephrotic syndrome . The immunosuppressive effect of CsA results from inhibition of signaling by the transcription factor NFAT in T cells , and thi2+/calcineurin signals [Although Faul et al. argued against an antiproteinuric role of CsA through the suppression of T cells, it is possible for the involvement of NFAT proteins downstream of calcineurin in podocyte injury, since the NFAT transcription factors are the most extensively studied calcineurin substrates and the major regulators of transcription in response to Ca signals , 52.2+ signaling through ion channels has recently emerged as a potential modulator of podocyte function, and several Ca2+-permeable channels have been identified inpodocytes [2+, calcineurin dephosphorylates the NFAT proteins that reside in the cytoplasm in resting cells. This dephosphorylation exposes the concealed nuclear localization signals of the NFAT proteins, leading to the cytoplasm to nucleus translocation of these proteins. In the nucleus, the NFATc proteins form NFAT transcription complex with their nuclear partners to control the transcription of target genes.Caodocytes , 54. Gen2+ influx pathways for the nonselective, cationic current in podocytes [2+ signaling, podocyte dysfunction [2+ channels, including TRPC6 [2+/calcineurin signals mediated podocyte injury. Inhibition of calcium channels and chelation of extracellular calcium reduced protamine sulfate-induced damage, suggesting that calcium signaling plays a critical role in the initial stages of glomerular injury. Calcineurin inhibitors (FK506 and CsA) inhibited protamine sulfate-mediated barrier changes, indicating that calcium signaling acts, in part, through calcineurin-dependent cleavage of synaptopodin. Meanwhile, mutations in TRPC6 enhance the amplitude and duration of the Ca2+ channel current which cause NFAT activation, indicating the activation of the calcineurin-NFAT pathway as a potential mediator of FSGS [TRPC6 mutations were found in families with hereditary FSGS, and TRPC5 and TRPC6 channels are now known as the Caodocytes . Mutatiofunction , 22, andng TRPC6 , 56. Vasng TRPC6  showed t of FSGS .TetO sequence of the NFATc1NUCTetO- transgene. The mice carrying three alleles  were referred as mutants. Thus, results from Wang et al. [Importantly, the study of Wang et al.  demonstrg et al.  provided2+ influx stimulates NFAT-dependent TRPC6 expression, is involved in angiotensin II (Ang II)-associated podocyte injury. In vitro and in vivo models, Ang II, a key contributor to the pathogenesis of glomerular disease, increases TRPC6 expression in podocytes. The regulation of TRPC6 expression by AngII is dependent on TRPC6-mediated Ca2+ influx and the activation of the Ca2+-dependent calcineurin/NFAT signaling. Tian et al. [2+ influx through TRPC5 and TRPC6 channels and synaptopodin signaling in podocytes. Schlöndorff et al. [Using a similar model for conditional NFAT activation in podocytes, Nijenhuis et al.  demonstrn et al.  showed tf et al.  showed tIn addition, another study showed that calcineurin induces podocyte apoptosis in a genetic model of type 1 diabetes mellitus (Akita mice). In cultured podocytes, activated NFAT promotes podocyte apoptosis in a calcineurin-dependent fashion, and induction of apoptosis by either angiotensin II or endothelin-1 was blocked by a calcineurin inhibitor (FK506). This induction of apoptosis appears to require NFAT-induced gene transcription .nucNFATc1 transgenic mice, making them potentially direct targets of NFATc1[nucNFATc1 transgenic mice, indicating that the upregulation of Wnt signaling seems to be the pathogenesis of NFAT activation-induced podocyte injury and FSGS.In other cell populations, NFATc1 activation leads to distinctive changes in transcription and cellular behavior –65. Inteof NFATc1. The uprof NFATc1, 67, mayof NFATc1, and WntnucNFATc1 transgenic mice and Akita mice showed NFAT activation-induced podocyte apoptosis and injury, and NFAT-dependent TRPC6 expression, the mutations of which have been shown to be associated with proteinuria and glomerulosclerosis in human patients. In summary, recently, there is the possibility that CsA might have a coupled nonimmunological antiproteinuric effect: one as demonstrated by Faul et al. on synaptopodin and the podocyte cytoskeleton, another separate one through the inhibition of NFAT as NFAT induces proteinuria and podocyte apoptosis (The antiproteinuric effect of CsA is attributed to its immunosuppressive effect. However, recently published researches showed that the effect of CsA on proteinuria is not dependent on NFAT inhibition in T cells, but rather results from its effects on kidney podocytes, including the stabilization of synaptopodin. Moreover, in vivo evidence from poptosis .


TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44+/HER2+, a metastatic variant), and HR5  cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ERhigh/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44 Triple-negative breast cancer (TNBC) is considered the most aggressive breast cancer subtype because it is associated with the greatest probability of early relapse and death -3. It isAlthough it is highly sensitive to chemotherapy, the progression-free time of TNBC, however, is generally short, and has greater recurrence rates than those of non-TNBC tumors during the first and third years after their initial diagnosis, as well as a higher 5-year mortality rate ,4. The hBRCA1 and BRCA2. The combination of the mutation of BRCA and PARP inhibition attributed to so-called synthetic lethality [BRCA1 and BRCA2 mutations account for slightly more than 10% of breast cancers that are triple-negative [EGFR), mammalian target of rapamycin (mTOR), the RAS-mitogen-activated protein kinase signaling pathway (Raf/Mek/MAP), and Src tyrosine kinase [high/CD24-/low have cancer stem cell properties [in vitro and in vivo [No currently accepted therapeutic target is known for TNBC, unlike some other subtypes of breast cancer . ER-exprethality ,7. The iethality . Althougnegative . Other te kinase ,9. Howevoperties . Such caoperties . It has  in vivo -17. Thusin vitro and in vivo conditions [MAP kinase, Src tyrosine kinase , RSK kinases [et al. [Kinases play an essential role in the processes of protein phosphorylation and are deregulated in many diseases, such as cancer. Numerous studies have proved that many kinases are critical in cancer cell survival under both nditions -20. Kinanditions ,22. For  kinases ,9,23,24. kinases ,26. Proc [et al. ,27 foundhigh population in SUM149 after siRNA treatments were identified and tested directly against sorted CD44high/CD24-/low cells of SUM149. The most impressive kinase lead was polo-like kinase 1 (PLK1). Therefore, we focused on PLK1 inhibition as the best potential therapeutic lead for TNBC by showing that it is highly expressed in breast cancer cell lines, and its inhibition by PLK1 siRNA as well as BI 2536, an ATP-competitive inhibitor designed to inhibit PLK1 [high/CD24-/low population and induced apoptosis. Combined treatment with drugs and BI 2536 greatly inhibited the growth of TNBC. Therefore, it offers potential as a better therapeutic target for TNBC.In this study, we first performed a genome-wide human kinase siRNA library screen against a TNBC cell line SUM149 for growth inhibition. A panel of selected active kinases was then further tested on four different breast cancer cell lines to confirm the spectrum of growth inhibition. Several kinases that also inhibited the tumor-initiating CD44bit PLK1 , killed M HEPES (Sigma-Aldrich), and 5% fetal bovine serum . MDA-MB-231 and MCF7 were purchased from ATCC and cultured in Dulbecco Modified Eagle medium  with 10% FBS. BT474-M1, a metastatic variant of BT474, was a gift of Dr. Mien-Chie Hung . HR5, which is derived from BT474 and is resistant to trastuzumab, was from Dr. Carlos Arteaga . They were both cultured in DMEM-F12 (1:1) with 10% FBS. AU565, HCC1937, and T47D (ATCC) were cultured in RPMI-1640 media supplemented with 5% FBS (Gibco/Invitrogen), 10 mM HEPES, 4.5 g/L glucose, 1 mM sodium pyruvate, and 100 units/ml penicillin/streptomycin (Sigma-Aldrich). All the cells were incubated at 37°C with 5% CO2, and subcultured twice weekly during the experimental period.SUM149 cells were purchased from Astrand  and cultured in F-12 (Ham) media  supplemented with 5 μg/ml insulin , 1 μg/ml hydrocortisone (Sigma-Aldrich), 10 mM on arrival by following the manufacturer's instructions and stored at -20°C before use.The siRNA library (Vision 2) of 691 human kinases was purchased from Qiagen . Two different sequences of siRNA target each of the genes in the library. The siRNA stock samples were diluted to working stocks at 2 μM for 72 hours. Cells were then fixed in 2% paraformaldehyde (Sigma-Aldrich) with nuclear dye, Hoechst 33342 (1 μg/ml) (Sigma-Aldrich). After a gentle wash with phosphate-buffered solution (PBS), the cells were kept in fresh PBS, and the plates were kept at 4°C in the dark before analysis on the ArrayScan high-content screening system . Twenty view fields per well were scanned and analyzed. The screen was repeated once to confirm the activity of siRNAs. Cells treated with Lipofectamine RNAiMAX alone without siRNA served as controls. Additionally, scrambled siRNAs and green fluorescent protein siRNAs, which were included in the library, served as internal references in each assay plate. Apoptosis was identified by nuclear morphology and Hoechst dye intensity by the HCS system [The screening methods were previously described . In brieS system , which aA panel of 28 active kinases was selected from the hit list, based on their activity and classes, and silenced by their corresponding siRNAs in four breast cancer cell lines, MDA-MB-231, SUM149, BT474-M1, and HR5. Cell lines MDA-MB-231 and SUM149 are TNBC, whereas the latter two are HER2 positive. Unless otherwise stated, all growth assays in the study were done in replicates of three or five and repeated at least once to confirm the activity.M, as described in a previous section. After 72 hours of treatment, the cells were fixed in 2% paraformaldehyde with nuclear dye, Hoechst 33342, at room temperature for 30 minutes. The cells were then washed gently 3 times with PBS and stained with 40 μl/well of mouse anti-human CD44-PE conjugated antibody  at room temperature for 1 hour in the dark. The samples were then washed with PBS and kept at 4°C in the dark before analysis with the HCS system for the CD44high cells surviving the siRNA treatments.SUM149 cells were treated with the selected siRNAs at 5 nhigh/CD24/-low subpopulation as described [SUM149 cells were cultured and sorted for the CD44PLK1 protein expression in eight breast cancer cell lines, SUM149, MDA-MB-231, BT474, HR5, HR6, MCF7, HCC1937, and AU565, was investigated with Western blot, as previously described . In briePLK1 siRNA on PLK1 expression, SUM149 and MBA-MB-231 were seeded into six-well culture plates (BD) at 350,000 cells/well in 2 ml corresponding media. PLK1 and control siRNAs were added to achieve 5 nM final concentration, and Lipofectamine RNAiMAX alone without siRNA served as the control. The sample plate was then incubated for 72 hours. After harvesting the cells and extracting the proteins, PLK1 expression was detected with immunoblotting , as described earlier.To confirm the silencing efficacy of high cells that were also PLK1high was calculated.To explore the possible connection between PLK1 and CD44, SUM149 cells were seeded onto eight-chamber slides (BD), washed with PBS, fixed with 2% formaldehyde for 20 minutes, rinsed twice with PBS, and then incubated with PBS containing 0.1% Triton X-100 (Sigma-Aldrich) for 30 minutes. Next, the slides were washed with PBS and incubated with mouse anti-CD44  and rabbit anti-PLK1  antibodies diluted in buffer containing 10% bovine serum albumin and 2% goat serum overnight at 4°C in a humidified container. After washing 3 times with PBS, glass slides were incubated with Alexa Fluor 546 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies  for 1 hour, washed 3 times, and then mounted by using Prolong Gold (Invitrogen) with 4ʹ,6-diamidino-2-phenylindole . Cells were observed with a Zeiss AX10 microscope and photographed by using an Olympus DP72 digital camera. All cells in three randomly selected view fields (×10 magnification) were surveyed for CD44 and PLK1 expression, and the percentage of CD44M or 100 nM for 72 hours. Fixed cells were then stained with rabbit anti-phospho-cyclin B1 (S133) , which is a known downstream substrate of PLK1 [The effect of PLK1 inhibitor on PLK1 activity was studied with an immunofluorescence method. SUM149 cells were seeded on glass coverslips in six-well dishes and treated with dimethyl sulfoxide (DMSO) or BI 2536 at 25 n of PLK1 . This waM in 96-well plates for 72 hours. Fixed cells were then stained with the cyclin B1 antibody, as described earlier, except that Hoechst was used, and the cells were kept in PBS before analyzing with the HCS system.For quantitative analysis of PLK1 activity, SUM149 cells were seeded at 3,000 cells/well overnight and treated with DMSO or BI 2536 at 10 to 100 nM in the medium for 72 hours. Propidium iodide  and Hoechst dye solution were added 40 minutes before the end of treatments to each well at a final concentration of 1 μg/ml for each dye. The sample plates were then scanned live with the HCS system. Growth inhibition was calculated as a percentage of the control without the DMSO and the drug, and the samples treated with DMSO alone served as a reference. To address whether a longer period of treatment would increase the efficacy of the drug compound, SUM149 cells were treated with BI 2536 for 10 days. The methods were the same as stated earlier, except that the seeding density was only 1,000 cells/well, and the media with BI 2536 were later replaced with fresh media containing BI 2536 at days 4 and 7 of the treatments.Prior studies reported that BI 2536 is highly selective for PLK1 when tested against 1,000 related kinases . BI 2536PLK1 siRNAs, sorted CD44high/CD24-/low cells of SUM149 were seeded at a density of 3,000 cells/well in 96-well plates. They were then treated with BI 2536 at concentrations ranging from 1 to 100 nM for 72 hours.To determine whether BI 2536 has a similar inhibitory effect on TICs as do the M or 25 nM) was added at time of seeding . Serial passaging was performed as per Subculture of Mammospheres protocol (StemCell Technologies). In brief, after 7 days in culture, mammospheres were counted, collected in a conical tube, and centrifuged at 350 g for 5 minutes. Pellets were triturated with trypsin-EDTA (Invitrogen) to break up mammospheres to single cells. Cold PBS with 2% FBS was added, and cells were centrifuged at 350 g for 5 minutes. Pellets were resuspended in Mammocult media, and cell counts were performed. The mammosphere assay was reseeded by using the same cell densities and treatments as described earlier.Mammosphere assays were performed with SUM149, as well as with MDA-MB-231 cells, which highly expresses CD44 in about 90% of its population, in ultra-low attachment six-well culture plates  in complete Mammocult media , as previously described . DMSO cohigh/CD24-/low, SUM149 cells were seeded at 1,000 cells/well in 96-well plates overnight. Taxol, Dox, or 5FU (Sigma-Aldrich) at different concentrations were then added the following day, and the plates were incubated for 72 hours. One of the plates was then fixed and stained for Hoechst, CD44 APC  and CD24 FITC  antibodies, as described earlier, and analyzed with an HCS system for growth and CD44high/CD24-/low cells. The medium in the second plate was removed and washed once with fresh medium. Then the medium with BI 2536 at different concentrations was added to the plate and incubated for another 4 days. The plate was fixed and analyzed with HCS, as described.Chemotherapeutic drugs like paclitaxel (Taxol), doxorubicin (Dox), and 5-fluorouracil (5FU) had been reported to induce resistance of cancer cells, and to this is probably attributed their induction of TICs in the surviving population ,15,31,32To investigate apoptosis caused by BI 2536 on breast cancer cells of SUM149, MDA-MB-231, BT474-M1, and HR5, the cells after drug treatment were stained with PI or phospho-H2AX for quantification of apoptosis . In brieM siRNAs for 72 hours under the experimental conditions  on SUM149 cells once they were silenced by 5 nPLK1, GCK, SKP2, PLAU, RPS6KA2, PI4K2B, and LOC392265. In particular, these kinases are significantly active on HR5, a trastuzumab-resistant model. The results indicated that these kinases offer potential applications not only in TNBC but also in other subtypes of breast cancer.Although the initial kinase siRNA library screen was done on SUM149 cells, most of the 28 selected active kinases, once silenced by their corresponding siRNAs, showed a strong and broad spectrum of inhibitory effect on the growth of all four cell lines tested, SUM149, MDA-MB-231, BT474-M1, and HR5 , conversely, significantly induced CD44high cells after siRNA silencing.SUM149 cells consist of about 5% CD44l Figure . In parthigh/CD24-/low cells of SUM149 by silencing them with corresponding siRNAs at 5 nM for 72 hours, all of them, as expected, significantly inhibited the growth of the TICs compared with control  of CD44high cells were also PLK1high, whereas the CD44low cells failed to express high levels of PLK1 .feration ,31. We t1 Figure . The higM or higher concentrations led to aberrant accumulation of phospho-cyclin B1 in the nuclear area of SUM149 cells .Both qualitative and quantitative studies showed that PLK1 inhibition by BI 2536 at 25 ns Figure  and 3E. M with 80% to 90% growth reduction at 10 to 25 nM for most cancer cell lines after a 72-hour treatment. In particular, HR5, a trastuzumab-resistant cell line, is similarly sensitive to BI 2536 as is BT474-M1. A longer period of treatment of SUM149 cells with BI 2536 killed almost all cells at concentrations of 25 nM or higher , which has a wild-type p53, is equally sensitive to PLK1 inhibition as MDA-MB-231 (TNBC), which is p53 mutant. Similarly, of the three slightly less-sensitive cell lines, SUM149 (TNBC) is p53 mutant, whereas MCF7 and T47D (ER+ and HER2-) are both p53 wt. The results indicate that sensitivity to PLK1 inhibition may not be linked directly to p53 status. Although a normal cell line was not included in the study for comparison, numerous studies, both in vivo and even clinical trials, have established that PLK1 inhibition by siRNA or BI 2536 is well tolerated, with neutropenia being the main side effect [PLK1 siRNA with chemotherapeutic drugs also enhanced the sensitivity toward Taxol and trastuzumab (Herceptin) in a synergistic manner [PLK1 with TICs in breast cancer. Of the 28 selected kinases in our focused studies, PLK1 is the leading candidate, based on its activity in inhibiting cancer cell growth, and in particular, its activity against the TICs once silenced by siRNA or by the small-molecule inhibitor, BI 2536. Fillmore and Kuperwasser [high/CD24-/low cells under the experimental conditions. Interestingly, Gleixner et al. [In this study, the expression of ts exist ,40. In te effect ,39,47-49e effect ,51. Combc manner . Most imerwasser  reportedr et al.  recentlyPLK1 is the focus of our study for its significant growth inhibition on breast cancer, availability of small-molecular inhibitors, and the safety data in clinical trials of different cancer treatment [SKP2 and PLAU (uPA), which inhibited the growth of sorted CD44high/CD24-/low cells of SUM149. Indeed, uPA/PAI-1 is the only biomarker to have been conferred with LOE-1 as a definitive prognostic marker of poor disease outcome in early breast cancer [+ cells [- and HER2-) and might play a role in the development of resistance to anti-estrogens [Although reatment ,40,49, st cancer . Furthert cancer . The firt cancer ,54. Evid[+ cells . Concept[+ cells , and as [+ cells . High lestrogens . Overexpstrogens . FurtherThe inhibition of PLK1 led to significant growth inhibition, either alone or in combination with other drugs, on different breast cancer cells and TICs, making them promising therapeutic targets in the treatment of TNBC and other breast cancers.5FU: 5-fluorouracil; DAPI: 4ʹ,6-diamidino-2-phenylindole; DMEM: Dulbecco Modified Eagle medium; DMSO: dimethyl sulfoxide; Dox: doxorubicin; ER: estrogen receptor; FBS: fetal bovine serum; HCS: high content screening; HER2: human epidermal growth factor 2; PBS: phosphate-buffered solution; PI: propidium iodide; siRNA: small interfering RNA; Taxol: paclitaxel; TICs: tumor-initiating cells; TNBC: triple-negative breast cancer.The authors declare that they have no competing interests.KH performed siRNA screens, growth assays, cyclin B1 assays, apoptosis measurements, and prepared the manuscript. JL performed mammosphere formation and immunoblotting assays. AF performed immunofluorescence of PLK1 and cyclin B1. SED conceived the study, arranged research funding, and prepared the manuscript. All authors read and approved the final manuscript.Complete list of active kinases identified in siRNA library screen. Table M for 72 hours under the test conditions.Click here for file


The special class IIb histone deacetylase, HDAC6, plays a prominent role in many cellular processes related to cancer, including oncogenesis, the cell stress response, motility, and myriad signaling pathways. Many of the lessons learned from other cancers can be applied to ovarian cancer as well. HDAC6 interacts with diverse proteins such as HSP90, cortactin, tubulin, dynein, p300, Bax, and GRK2 in both the nucleus and cytoplasm to carry out these cancerous functions. Not all pro-cancer interactions of HDAC6 involve deacetylation. The idea of using HDAC6 as a target for cancer treatment continues to expand in recent years, and more potent and specific HDAC6 inhibitors are required to effectively down-regulate the tumor-prone cell signaling pathways responsible for ovarian cancer. Clinical trials show HDAC inhibitors to be effective anti-tumor drugs [Rpd3: specifically HDAC1, HDAC2, HDAC3, and HDAC8. Class 2 is related to the yeast gene Hda1p and includes HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10. Class 3 includes the Sirtuin family of enzymes, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7. Class 4 is only made up of HDAC11. Class 2 HDACs are further divided into Class 2a, which includes HDAC4, HDAC5, HDAC7 and HDAC9, and class 2b, which includes HDAC6 and HDAC10. One unique characteristic of the HDACs is their ability to shuttle between the cytoplasm and the nucleus. Because of this shuttling capacity, it was soon discovered that they act on more than just histones [Histone acetyltransferases (HATs) and deacetylases (HDACs) have opposing effects on the acetylation status of their substrates, which include core histones and non-histone proteins. Thus, HDACs may be a misnomer since they deacetylate more substrates than just the histones in the nucleus. Either way, DNA wraps around histones in the nucleus. When HATs act on histones, gene expression levels tend to be up-regulated due to the increase in docking sites where transcription factors can bind. HDACs have a reverse effect . Thus, Hor drugs . There aor drugs . Class 1N-terminal tails of histones and other substrates [HDACs catalyze the deacetylation of lysine residues in the bstrates . Nucleosbstrates . Lysine bstrates . A groupbstrates .N-terminal and C-terminal deacetylase domain [C-terminal deacetylase domain is highly active in vitro [HDAC6 is unique in having two deacetylase domains. One report showed that both of the deacetylase domains are required for its deacetylase activity . This ree domain . A histie domain . Thus, tin vitro . HDAC6 ain vitro ,13, a nuin vitro . The unbin vitro ,13,15. Tin vitro . The tetin vitro ,17. TherHDAC6 is a cancer drug target because of its role in transforming normal cells to cancer cells. Excess HDAC6 is associated with tumorigenesis and cell survival; therefore, HDAC6 can be used as a marker for prognosis. In multiple myeloma cells, blocking expression of HDAC6 can cause apoptosis. In breast cancer MCF-7 cells, HDAC6 helps lead to metastasis by up-regulating cell motility . HDAC6 aThe average age of diagnosis for ovarian cancer is 63, and there were 22,280 new cases in 2012. As of 1 January, 2012, there were 192,750 women alive who had been diagnosed with ovarian cancer. The American Cancer Society estimates 14,030 deaths from ovarian cancer in 2013 [Treating ovarian cancer can cause many side-effects including a decrease in bone density, cardiovascular diseases, cognitive defects, fear of cancer recurrence, distress, pain and infertility . Common p53, BRAF, β-catenin, Rb, BRCA1, BRCA2, MSH2 and MLH1 and overexpression of epithelial growth factor receptor (EGFR), Akt and HER2. HDAC inhibitors are promising drugs for ovarian cancer treatment, and there are several currently in clinical trials. Epithelial ovarian cancers, including serous, mucinous, clear cell, and endometrioid, constitute 80%–90% of ovarian cancers [Ovarian neoplasms can arise from the surface epithelium, the gonadal or sex-cord stroma, germ cells, fallopian tubes or from metastasis from other tumors . Endomet cancers . In addi cancers . RecentlHDAC6 gene expression [HDAC6 is located on the Xp11 chromosome, and HDAC6 seems to be found in the brain, breast, colon, ovary, pancreas, prostate and heart, and may be up-regulated in cancers of the brain, breast, ovary, and pancreas [HDAC6 because they can be more susceptible to HDAC inhibitor treatment [HDAC6 were found in oral squamous cell carcinomas (OSCC) than in the normal oral keratinocytes (NOKs). The cell lines investigated included Ca9-22, OK92, Ho1-N-1, HSC2, HSC3, HSC4, SAS, and Sa3 [In many murine cell lines  there is much more HDAC6 in the cytoplasm than in the nucleus. During cell differentiation and proliferation in B16 cells, many HDAC6 proteins are found to translocate to the nucleus as well . MCF-7 bpression . HDAC6 ipancreas . Additioreatment . Greater and Sa3 . Acute m and Sa3 .Measuring mouse embryonic fibroblasts (MEFs) in an anchorage-independent fashion, researchers from Duke found that HDAC6 is required for oncogenesis . They alA link was discovered between protein acetylation and ubiquitination when HDAC6 was found to interact with proteins of the ubiquitin signaling pathway, p97/VCP/Cdc48p and phospholipase A2-activating protein. HDAC6 has a zinc-finger ubiquitin-binding domain, which can bind a mono- or polyubiquitin and ubiquitinated proteins ,37. AbnoWhen the proteasome is inhibited, the E3-ubiquitin ligase TRIM50 can send polyubiquitinated proteins to an aggresome for storage. This action is mediated by HDAC6 and p62 . An aggrAfter the aggresomes are formed, HDAC6 also plays a role in their clearance by autophagy, which leads to degradation by lysosomes. When the cell was stressed with MPP(+) (1-methyl-4-phenylpyridinium) and HDAC6 was silenced, aggresome formation and autophagy were decreased . SequestRAS or p53 is mutated, HSF1 can help protect mice from developing tumors. In cells, however, HSF1 may play a supportive role in tumorigenesis through proliferation, survival, and protein synthesis mechanisms. Indeed, some cancer cell lines seem to be at least partially dependent on HSF1 function [As aforementioned, molecular chaperones can counterbalance the proteasome as the cell finds the middle ground for its protein levels . One majfunction .HDAC6 also plays an important role in cell migration and mobility through its action on the cytoskeleton . It has The cytoskeleton is partially made up of microtubules and actin filaments. In addition to microtubules, as it turns out, HDAC6 also deacetylates cortactin, a protein that binds to actin ,85, F-acOther proteins involved with cell motility include dysferlin, GRK2, and calpain. Dysferlin has an inhibitory interaction with HDAC6, leading to increased tubulin acetylation . With caCell motility is used as a model for studying cancer metastasis . It seemSo far we have explored several signaling pathways that lead to cancer. There are many more, some including Ku70, Tat, and CYLD. Ku70 may be the most important in neuroblastomas . It is iMany more HDAC6-related proteins remain. One such protein is Runx2, the first transcription factor found to bind to HDAC6. HDAC6 regulates the activity of Runx2 by repressing its activation of p21 . Also, pHDAC6 knockout MEFs are resistant to transformation [HDAC6 knockout mice might be less prone to cancer. Third, normal microtubule dynamics are essential for cellular functions; microtubule acetylation will disturb such functions. Several chemotherapeutic agents such as paclitaxel, which targets microtubules, are currently used to treat cancer patients. Based on the above information, one would expect that inhibition of HDAC6 would exert minimal side-effects and can effectively augment the current anti-tumor drugs. Indeed, researchers have been working very hard at inhibiting HDAC6 activity selectively and effectively. Early pan-HDAC inhibitors include trichostatin A (TSA) and suberolyanilide hydroxamic acid (SAHA). These inhibitors can inhibit cell growth and prevent the formation of tumors in mice models [Several lines of evidence suggest that HDAC6 is an ideal target for cancer therapy. First, mice lacking HDAC6 display hyperacetylated tubulin in many tissues tested but are viable and develop normally . Second,ormation ; HDAC6 ke models . Anothere models . This sae models . Hydroxae models . Acting e models . When tue models . HDAC6 se models . Tubuline models .H)-one and piperazine-2,5-dione aryl hydroxamates were synthesized [The search for potent HDAC6 inhibitors to block cancer cell proliferation continued strong through 2007. Many pan-HDAC inhibitors had been found by this time, and researchers were becoming more interested in less-toxic, more-selective, single isoform inhibitors to more deftly regulate the development of oncocells . A few tthesized .Still in 2009, clinical trials found many side-effects associated with these inhibitors, and so the search continued for the magic bullet . A good Recently, HDAC inhibitors have shown promise as agents against ovarian cancer. In 2007, a group found that the class 1 HDAC inhibitor, R306465, worked well in preventing the growth of A2780 ovarian, H460 lung, and HCT116 colon carcinomas . In 2008As we have seen, HDAC6 plays a role in many cellular processes; it is one of the most important histone deacetylases in the cytoplasm and also controls interesting processes in the nucleus (HDAC6 signaling is summarized in


Fossils of a marsupial mole  are described from early Miocene deposits in the Riversleigh World Heritage Area, northwestern Queensland, Australia. These represent the first unequivocal fossil record of the order Notoryctemorphia, the two living species of which are among the world's most specialized and bizarre mammals, but which are also convergent on certain fossorial placental mammals (most notably chrysochlorid golden moles). The fossil remains are genuinely ‘transitional', documenting an intermediate stage in the acquisition of a number of specializations and showing that one of these—the dental morphology known as zalambdodonty—was acquired via a different evolutionary pathway than in placentals. They, thus, document a clear case of evolutionary convergence  between only distantly related and geographically isolated mammalian lineages—marsupial moles on the island continent of Australia and placental moles on most other, at least intermittently connected continents. In contrast to earlier presumptions about a relationship between the highly specialized body form of the blind, earless, burrowing marsupial moles and desert habitats, it is now clear that archaic burrowing marsupial moles were adapted to and probably originated in wet forest palaeoenvironments, preadapting them to movement through drier soils in the xeric environments of Australia that developed during the Neogene. Until the discovery of the extinct taxon described here, no fossil notoryctemorphians were known [Notoryctemorphia, the marsupial moles, is the least diverse but most extraordinarily distinct of the four orders of living Australian marsupials. It currently comprises one family (Notoryctidae), one genus (re known . In partre known –4. Most re known ), Peramere known ,7) or bore known –13.Notoryctes is unique in exhibiting a distinctive molar morphology termed zalambdodonty [Notoryctes, several living and fossil therian groups have zalambdodont molars  become increasingly ‘channelled’ by an underlying morphological/genetic developmental constraint as they move down this pathway, or can these highly specialized patterns be achieved in completely different ways? That is, do they arise by parallelism (independent acquisition via the same evolutionary pathway) or convergence (independent acquisition via a different pathway)? While most placental mammals have achieved zalambdodonty via hypertrophy of the paracone and suppression of the metacone [Among living marsupials, bdodonty ,15. Zalabdodonty . Two cret molars ; these iarsupial ,18) and atherian ,20). Witmetacone , until nNotoryctes and chrysochlorids share similar fossorial specializations of the skeleton [Notoryctes, some  chrysochlorids occupy sandy desert environments [Modern notoryctids are confined to deserts in Australia and exhibit numerous anatomical specializations that appear exceptionally well suited to burrowing through desert sands. These include a conical skull, extreme modifications of the axial and appendicular skeleton  and soft tissue features such as a ‘nasal shield’, a lack of eyes and external ears, and a tubular body shape . As a reskeleton ,26 and cronments . This faronments ) that thHigher level systematic nomenclature used in this paper follows . Dental 2.Mammalia Linnaeus, 1758Marsupialia Illiger, 1811Notoryctemorphia Aplin & Archer, 1987Notoryctidae Ogilby, 1892Naraboryctes philcreaseri new genus and species.(a)naraba ‘to drink’ , in reference to its fossorial specializations and close relationship to Notoryctes. The species name honours Phil Creaser for many contributions to Riversleigh and other palaeontological research at the University of New South Wales including establishment of the Coalition for Research into Australian Terrestrial Ecosystems (CREATE) Fund.From ensland; ), in ref(b)a–d).QM F23717 (Queensland Museum) from Upper Site, left dentary with i1-3 (i1 root only) c1 p1-3 m1-4, missing mandibular condyle and medially inflected portion of angular process a–d.Figu(c)k); QM F23716, partial right maxilla with P1-3 M1 (l). Referred specimens: from Upper Site, QM F54502 ; QM F51323 , QM F51324 , QM F51325 , QM F51327 , QM F23718 , QM F51329 , QM F51330 , QM F54559 , QM F54560 ; from Wayne's Wok Site, QM F23719 , QM F51328 (left dentary).Paratypes that are also topotypes: QM F51322, partial left premaxilla with I1-4 k; QM F23 P1-3 M1 l. Referr(d)The Type Locality is Upper Site, Godthelp Hill, Riversleigh World Heritage Area, Boodjamulla National Park, northwestern Queensland . Wayne's Wok Site is on Hal's Hill and 250 m from Upper Site. Both have been interpreted as Faunal Zone B sites –35 and a(e)Naraboryctes differs from Notoryctes spp. in the following combination of features: retention of small paracone; postmetacrista much longer than premetacrista; medial cusp on postmetacrista variably present; well-developed three-cusped talonid which is slightly smaller than trigonid on m2-4; anterior cingulid present; complete adult dental formula I1-5/1-3, C1/1, P1-3/1-3, M1-4/1-4; proportionately larger coronoid process of dentary; medial epicondyle of humerus less enlarged; capitulum and trochlea continuous; supracondyloid foramen present; elongated olecranon process of ulna less hooked; flat (rather than concave) medial anconeal process of ulna; anconeal sides of capitular notch and trochlear notch of ulna continuous (rather than separated by a ridge).The only known species of Rediagnosis of family Notoryctidae: small-bodied (<100 g) marsupials; complete adult dental formula I1-5/1-3, C1/1, P1-3/1-3, M1-4/1-4; upper molars sub- or fully zalambdodont with paracone much smaller than metacone or missing altogether but with large protocone; lower molars with reduced or absent talonids; postcranium with fossorial adaptations. A detailed description of the dentition, cranial and postcranial elements of this new taxon is provided in the electronic supplementary material, appendix S1.3.Menura, Orthonyx, Hypsiprymnodon, dactylopsiline petaurids and phalangerin phalangerids; e.g. [Previous authors ,36–40 hads; e.g. –43). Amods; e.g. ,45. Rive4.Naraboryctes philcreaseri to Notoryctemorphia because of the extreme reduction of the paracone, anteroposterior compression of its upper molars and reduction of the talonid on its lower molars. Collectively, these apomorphies represent an incipiently zalambdodont morphology that anticipates the fully zalambdodont condition in species of Notoryctes. This familial attribution is further supported by cranial and isolated postcranial specimens referable to N. philcreaseri in addition to the premaxilla, humerus and ulna noted above [Notoryctes [Notoryctes, the delto-pectoral ridge is also markedly displaced distally, resulting in increased mechanical advantage of the musculature, and the enlarged lateral supracondylar ridge provides for increased musculature in the lower arm. Massively expanded articular surfaces help strengthen the joints during digging by spreading high mechanical forces over a greater surface area [N. philcreaseri /1-3; C1/1; P1-3/1-3; M1-4/1-4. This is the same as the plesiomorphic dental formula for Australian marsupials and, except for the loss of one lower incisor, the same as the most plesiomorphic dental formula known for any marsupial. All known Australian marsupials have lost i4 and all except some peramelemorphians and probably N. philcreaseri have lost I5. The dental formula for species of Notoryctes is controversial because of considerable polymorphism in tooth number, both between specimens and within the same specimen. While Archer [maximum dental formula of N. typhlops is I1-4/1-3 C1/1 P1-3/1-3 M1-4/1-4, loss of an upper incisor and at least one upper and one lower premolar is not uncommon. Thomas [N. caurinus as I1-3/1-2; C1/1; P1-2/1-2; M1-4/1-4. The tiny size of P1 in N. philcreaseri compared with P2-3 suggests that it is P1 rather than P3 that is lost in N. caurinus and some specimens of N. typhlops. An apparent autapomorphy of N. philcreaseri is the presence of a cuspule near the middle of the postmetacrista, although it is possible that this cuspule has been secondarily lost in species of Notoryctes.e Archer  stated t. Thomas  gave theN. philcreaseri unequivocally demonstrates that zalambdodonty evolved in notoryctemorphians from a tribosphenic precursor by loss of the paracone . This demonstrates that highly specialized morphological patterns, such as zalambdodonty, can be achieved in very different ways in different mammalian clades and can result from convergent rather than parallel evolution  have suppressed the metacone. However, perhaps the simplest explanation is that the metacone is typically larger than the paracone in marsupials and other metatherians, whereas the reverse is usually true for placentals and other eutherians , and it metacone .N. philcreaseri remain unclear. However, based on the mechanical properties of food items  than those of species of Notoryctes. Insect larvae and worms are common below ground; if zalambdodonty is indeed an adaptation for feeding on soft-bodied invertebrates, this may explain why many zalambdodont mammals are fossorial  or semi-fossorial , Beck [ems . The cotebrates . In batstebrates ,60). TheN. philcreaseri appear to be less-specialized for feeding on soft-bodied invertebrates and hence they may have spent relatively less time feeding underground. However, because powerful digging in this species, as indicated by the hypertrophied, notoryctid-like olecranon process, is combined with incipient zalambdodonty, burrowing in notoryctids probably preceded evolution of fully developed zalambdodonty as well as a concomitant shift in preference for softer subterranean foods.Collectively, the dentition and mandible of N. philcreaseri described here demonstrate that notoryctids were incipiently zalambdodont and at least semi-fossorial by the early Miocene. The early Miocene Riversleigh Faunal Zone B faunal assemblages containing N. philcreaseri appear to represent rainforest biotas for reasons discussed above. This led some of us  b,13 b11,1us e.g. ,61) to s to sN. pus (e.g. ), that nus (e.g. , may actus (e.g. ,24,62.


Burkholderia phenoliruptrix strain AC1100 (ATCC 53867) degrades a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetate. The molecular mechanism of 2,4,5-trichlorophenoxyacetate degradation has been extensively studied. Here we present a 7.8-Mb assembly of the genome sequence of this 2,4,5-trichlorophenoxyacetate-degrading strain, which may provide useful information related to the degradation of chlorinated aromatic compounds. Burkholderia occupy a wide range of ecological niches and have versatile properties of bioremediation, biocontrol, and plant growth promotion (Burkholderia phenoliruptrix AC1100 (ATCC 53867) (formerly Pseudomonas cepacia AC1100 and Burkholderia cepacia AC1100) was the first pure culture that used 2,4,5-trichlorophenoxyacetate as a sole source of carbon and energy . The reads were de novo assembled with Velvet software to 286 contigs (>500 bp), providing 44-fold coverage  . The genome of strain AC1100 has 1 rRNA operon and 52 tRNA loci. There are 493 subsystems represented in the genome sequence , and the metabolic network of AC1100 (determined by the RAST server) was reconstructed . We haveASXI00000000. The version described in this paper is the first version, ASXI01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Winogradskyella psychrotolerans strain RS-3T, isolated from a sediment sample of a marine transect of Kongsfjorden, Ny-Ålesund, Svalbard, Arctic Ocean, is reported.The 4.3-Mb genome of  Winogradskyella psychrotolerans strain RS-3T is a Gram-negative, nonmotile, rod-coccus shaped bacterium. It was isolated from a marine transect sample from Kongsfjorden, Ny-Ålesund, Svalbard, Arctic Ocean. Winogradskyella pacifica and Winogradskyella thalassocola were reported to be the most closely related species by the 16S rRNA gene sequence analysis, with sequence similarities to the type strains of these species of 98.5 and 97.7%, respectively  was used for the assembly of the raw sequencing data, which yielded a genome of 4,342,100 bp in 219 contigs, with a G+C% mol of 37.70%. The genome annotation was performed using the Rapid Annotations using Subsystems Technology (RAST) server (The Roche 454 (FLX Titanium) pyrosequencing platform was used to sequence the genome of The annotation has a total of 4,068 genes, including 44 RNA genes. The annotation has 54 genes providing resistance against different antibiotics and toxic compounds. There are 107 genes for membrane transport, including 5 ABC transporters and 5 cation transporters. The genome also has 67 genes for different stress responses, including 33 genes for oxidative stress, 14 heat shock response genes, and 5 cold shock response genes.W. psychrotolerans RS-3T to be Flavobacteriales bacterium ALC-1, Croceibacter atlanticus HTCC2559, Cellulophaga sp. MED134, Dokdonia donghaensis MED134, and Zunongwangia profunda SM-A87, with similarity score values of 513, 391, 358, 344, and 343, respectively.A functional comparison of the genome was performed using the SEED framework  which reWhole-genome sequencing of this bacterium will help in understanding the metabolism and evolution of bacteria under extreme weather conditions, like the Arctic Ocean.W. psychrotolerans RS-3T has been deposited in DDBJ/EMBL/GenBank under the accession no. ATMR00000000. The version described in this paper is the first version, ATMR01000000.The draft genome sequence of


Recent investigations highlighted strong similarities between neural crest migration during embryogenesis and metastatic processes. Indeed, some families of axon guidance molecules were also reported to participate in cancer invasion: plexins/semaphorins/neuropilins, ephrins/Eph receptors, netrin/DCC/UNC5. Neuropilins (NRPs) are transmembrane non tyrosine-kinase glycoproteins first identified as receptors for class-3 semaphorins. They are particularly involved in neural crest migration and axonal growth during development of the nervous system. Since many types of tumor and endothelial cells express NRP receptors, various soluble molecules were also found to interact with these receptors to modulate cancer progression. Among them, angiogenic factors belonging to the Vascular Endothelial Growth Factor (VEGF) family seem to be responsible for NRP-related angiogenesis. Because NRPs expression is often upregulated in cancer tissues and correlated with poor prognosis, NRPs expression might be considered as a prognostic factor. While NRP1 was intensively studied for many years and identified as an attractive angiogenesis target for cancer therapy, the NRP2 signaling pathway has just recently been studied. Although NRP genes share 44% homology, differences in their expression patterns, ligands specificities and signaling pathways were observed. Indeed, NRP2 may regulate tumor progression by several concurrent mechanisms, not only angiogenesis but lymphangiogenesis, epithelial-mesenchymal transition and metastasis. In view of their multiples functions in cancer promotion, NRPs fulfill all the criteria of a therapeutic target for innovative anti-tumor therapies. This review focuses on NRP-specific roles in tumor progression. While NA major distinction between these two members of the NRP family is based on their ligand specificities. NRPs were originally described as high-affinity cell-surface receptors for axon guidance molecules such as class-3 semaphorins (Sema) . Indeed,Several analyses using mutant mice lacking NRPs function subsequently conferred to semaphorin/neuropilin an essential role in axon guidance during nervous system development -11.In vivo models using NRPs transgenes also suggested other essential functions of NRPs. Indeed, overexpression of NRP1 in chimeric mice generated an excess of capillaries and blood vessels, suggesting an important role of NRP1 in angiogenesis and vasculogenesis [−/− NRP2−/−) constitutes the most severe phenotype observed, impairing any blood vessel development and causing earliest death in utero at E8.5 [ogenesis . In contogenesis  and in togenesis ,14. NRP2ogenesis ,11. Moreogenesis . However165, PIGF-2 (Placenta Growth Factor), VEGF-B, VEGF-C, VEGF-D and VEGF-E [145, VEGF165, PIGF-2 [Because Vascular Endothelial Growth Factor (VEGF) plays a central role in the development of vascular network, interactions between NRPs and VEGF were rapidly considered. NRPs were indeed found to be receptors for several members of the VEGF family. NRP1 can effectively bind VEGFd VEGF-E , whereas, PIGF-2 ,22, VEGF, PIGF-2 ,22, and , PIGF-2 . NRPs ar, PIGF-2 ,24 , NRP2a(22)) whereas NRP2b is characterized by insertions of 0 or 5 amino acids after amino acid 808 (NRP2b(0), NRP2b(5)) ) . NRP2a s2.2.11NRP1 and s12NRP1) and one of NRP2 (s9NRP2) were cloned by Rossignol and collaborators [sIIINRP1 and sIVNRPI were characterized [et al., reported that s11NRP1 is capable of tumor cell apoptosis by antagonizing VEGF binding, suggesting that sNRPs and NRPs have opposite functions [Two soluble forms of NRP1   in vivo . In this in vivo . NRP1 is in vivo . When ex in vivo . Howeverin human . On the in human . Becausein human ,36.3.3.et al. isolated NRP1 from endothelial cells and tumor tissues [The contribution of NRPs in angiogenesis prompted the investigation of NRP's role in oncogenesis. Besides the presence of NRPs on tumor-associated vessels, authors have reported the wide expression of NRPs among different human tumors, suggesting a potential role of this molecular network in cancer progression. In 1998, Soker  tissues . Indeed, tissues ,38, conf tissues , melanom tissues , lung ca tissues ,42, brai tissues ,44 colon tissues , pancrea tissues -49, brea tissues , myeloid tissues , salivar tissues , infanti tissues , ovarian tissues  and blad tissues   are subdivided into eight classes, on the basis of structural similarities. Class 1 and 2 constitutes invertebrate semaphorins, whereas classes 3 to 7 comprise vertebrate semaphorins . All semXenopus tadpoles nervous tissue [Plexin family is the first class of co-receptor identified. Plexins have been identified like NRPs, from immunostaining of s tissue . While ps tissue  by formis tissue ,92, plexs tissue ,94. Nines tissue -97. Mores tissue -100. Thes tissue . Membrans tissue .Like type-B plexins, NRPs contain a binding site for PDZ domains in the C-terminal domain. Indeed, the PDZ domain of NIP, also called GIPC (GAIP interacting protein at the C terminus), is thought to be implicated in interaction with NRPs and plexins, activating small GTPase-activating proteins . In partSemaphorins are reported to be very often down-regulated or mutated in human cancers, allowing massive VEGF/NRPs interactions. Because semaphorins are frequently inactivated by allele loss or promoter methylation, they have been rapidly considered to function as a TSG (tumor suppressor gene). Indeed, deletions occur in the region 3p21.3 of the short arm of chromosome 3, a region encoding for Sema3B and Sema3F in various cancers, including lung cancer and even ovarian cancer -104. Mor4.1.1.et al. reported that Semaphorin 3A (Sema3A) mediates cell repulsion and can even induce cell death in a neuroectodermal progenitor cell line, both effects depending on interactions with NRP1 [165, cell survival decreases. NRP1 is implicated in this Sema3A-mediated apoptosis [First, Bagnard ith NRP1 .When Sempoptosis . Moreovepoptosis . After apoptosis   expression decreases colony formation, proliferation, and even tumorigenicity in murine xenograft experiments ,115. Sim4.1.3.First observations that Semaphorin 3F (Sema3F) might have a role in cell motility and cell invasion was suggested by Brambilla and colleagues, in lung cancer cells . Then, sin vivo and decreased the number of intra-tumor vessels, suggesting that Sema3F has huge potential in anti-angiogenic and anti-metastasis therapies [A role of Sema3F in tumor angiogenesis was then suggested. Implantation of BHK-21 (Baby Hamster Kidney-21) cells transfected with Sema3F concomitantly with cells producing VEGF-165 inhibited tumor-related angiogenesis in mice whereas no effect on angiogenesis was observed when BHK-21 cells transfected with empty vector were implanted with the same VEGF-165 producing cells . Moreoveherapies   is described as an enhancer of tumor growth and metastasis er cells   leads to a reduction of VEGFC-mediated migration of Lymphatic Endothelial cells (LEC) in vitro and to an inhibition of lymphangiogenesis in vivo [B treatment [nrp2+/−vegfr2+/− mice have normal lymphatic development unlike double-heterozygous nrp2+/−vegfr3+/− mice, indicating that Nrp2 partners with VEGFR3 to modulate lymphatic vessel sprouting and lymphangiogenesis [First, Soker esponses . Later, esponses . Moreoveesponses . NRP2 enesponses ,46. /scatter factor (SF)/c-Met signaling [Because heparin growth factors FGF and HGF have been recently identified as NRPs ligands, they are believed to contribute to NRP-mediated angiogenesis too. Indeed, NRP1 potentiates HGF and FGF2 induced proliferation, survival, invasion in human umbilical vein endothelial cells (HUVEC), glioma cells, pancreatic cancer cells ,137,138.ignaling ,137. NRPignaling ,137,138 4.2.4.et al. show that NRP1 associates with TGFRI and TGFRII to enhance TGFβ1 signaling in cancer cells [More recently, a study suggested that NRP1 is a receptor for both active TGFβ1 and TGFβ1-LAP. In addition, NRP1-TGFβ1 interactions on T cells resulted in enhanced T regulator activity . Then oter cells   with 8-fold increased affinity for VEGF compared to wild-type B domain of NRP2 [Since naturally occurring soluble forms of neuropilins are described to inhibit tumor progression, researchers tend nowadays to develop soluble peptides preventing VEGF-binding on neuropilins. For this purpose, Geretti  of NRP2 . This Mu of NRP2 .in vitro [Furthermore, screening of phage libraries expressing random peptides binding to various cancer cells has allowed the identification of amino acid sequences especially binding NRPs. Indeed, Sugahara and collaborators reported two tissue-penetrating peptides binding human integrins and NRP1 capable of penetrating into tumor tissue and cells ,151. Conin vitro -155.6.3.A) or coagulation factors V/VIII domains (anti-NRP1B) of NRP1 have been first generated. [Genentech has very recently developed monoclonal antibodies targeting NRP1. In particularly, high-affinity monoclonal antibodies targeting either CUB domains (anti-NRP1nerated.  These annerated. . Later, nerated. .One of them, a full human antibody targeting NRP1, MNRP1685A is actually in phase-1 of development alone or in combination with bevacizumab with or without paclitaxel for treatment of advanced solid tumors .B monoclonal antibodies decrease the number of tumor-associated lymphatic vessels and metastasis in sentinel lymph node and in distant organs in mice xenograft experiments [Monoclonal antibodies targeting the b1/b2 domains of NRP2 have been recently developed. By blocking binding of VEGF and VEGFC to NRP2, these anti-NRP2eriments .6.4.NRPs role in tumorigenesis is more complex than initially thought and appears to depend on the nature of the ligand. In the context of cancer, it appears that semaphorins and VEGF are competing for NRPs binding, although they bind different NRPs sub-units. While semaphorins are responsible for inhibition of tumor growth, proliferation and even induction of apoptosis in cancer cells, VEGF tends to oppositely enhance angiogenesis and tumor growth. As described above, some semaphorins such as Sema3B and Sema3F are considered as TSG and are very often downregulated in cancer cells ,104,120.7.NRPs are multifunctional non-tyrosine kinase receptors for class-3 semaphorins and VEGF family members implicated in both physiological development and pathological situations. NRPs are expressed in endothelial cells and in many types of cancer cells. Through their direct interactions with plexins or growth factor receptors, NRPs have rapidly emerged as key regulators of angiogenesis, lymphangiogenesis, EMT and tumor progression. In many cancers, expression of one or both has been correlated with tumor progression and/or poor prognosis. As a consequence, several strategies have been used in pre-clinical studies to inhibit NRPs function, such as knockdown strategies with siRNAs, small peptide inhibitors, and blocking antibodies. However, the molecular mechanisms by which NRPs modulate cancer progression are still poorly understood. Understanding the interactions between VEGF, VEGFRs, semaphorins and NRPs should provide additional data for the rational development of novel anti-tumor strategies.


The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading,  As parman diet ,5. AlthoDe novo synthesis of PUFA in microalgae is largely through an aerobic pathway involving sequential addition of double bonds to saturated fatty acids, mainly 18:0 (and 16:0), via Δ9 and Δ12 (or ω6) desaturases to produce 18:2n-6 , which can then be further desaturated by Δ15 (or ω3) desaturase to give 18:3n-3  [n-3 followed by Δ8 desaturation, and Δ17 desaturation of 20:4n-6 to produce EPA has also been shown to occur [n-3 and 18:5n-3, which are abundant in some classes of marine microalgae including prymnesiophytes and dinoflagellates, do not fit on the conventional pathway and, together with DHA, can be the main PUFA in these species [Shewanella sp. that, along with Vibrio sp., comprise the majority of the PUFA-producing bacterial species isolated from the guts of fish and invertebrates. A review of PUFA in marine bacteria had concluded that conventional aerobic pathways must be used to synthesize PUFA [Schizochytrium, a thraustochytrid-like marine protist that accumulates large amounts of C22 LC-PUFA, DHA and 22:5n-6 [Schizochytrium (named PUFA synthase) has sequence homology to 8 of the 11 domains of Shewanella PKS and comprises three genes that account for the production of DHA and 22:5n-6 [Schizochytrium showing that enzymes of the aerobic pathway are also present in these organisms [n-3 → 18:5n-3 → 20:6n-3 → DHA, while LNA, 18:4n-3, 20:4n-3 and EPA do not lie on the PKS pathway [n-3 and 18:5n-3 in certain microalgae and the fact that they are associated with DHA but not EPA. It may also explain the presence of PUFA > C22 such as 28:7n-6 and 28:8n-3 that have been reported in some species of marine dinoflagellates [Sphaeroforma arctica was demonstrated to possess seven enzymes of PUFA biosynthesis including Δ12, ω3, Δ8, Δ5, and Δ4 desaturases, and two elongases, one with efficient elongating activity towards C18 and C20 substrates, and another with specific affinity for elongation of C20 PUFA [Primary production of PUFA in the marine environment can occur in photosynthetic microalgae, heterotrophic protists, and bacteria. 8:0 and 1:0, via Δ8:0 and 1:0, via Δ 22:5n-6 . The enz pathway . The PKSgellates  as both 18:0 and :0, via Δde novo from saturated and monounsaturated fatty acids and so PUFA are essential dietary nutrients [n-3 to produce 18:4n-3 that is elongated to 20:4n-3 followed by Δ5 desaturation, with synthesis of ARA from 18:2n-6 using the same enzymes [Oncorhynchus mykiss) [Siganus canaliculatus) and Senegalese sole  have Δ4 desaturases that would enable a more direct route for the synthesis of DHA [Saccharomyces cerevisiae, which has no endogenous expression of LC-PUFA Fad or Elovl genes, and determining the enzyme activities through incubation of the transformed yeast with appropriate PUFA substrates. The cDNAs for Δ6 Fad have been cloned and characterized from all fish so far investigated including marine species [S. senegalensis, nutritional and biochemical data suggest one should be present [Salmo salar) [Fish, like all vertebrates, cannot synthesize PUFA utrients . However enzymes   , but recs of DHA ,24. Thers of DHA . The mol species . Althougo salar) . Zebrafio salar)  and a sio salar) . Howevero salar) . In mammo salar) . The cDNo salar) . Functioo salar) , may be o salar) . Therefode novo synthesis or trophic upgrading of PUFA in the levels between the primary producers and fish, which are largely filled by invertebrates [n-3 PUFA with highest prevalences (>50%) from two species of bivalve Chlamys islandica and Astarte sp., and the amphipod Gammarus wilkitzkii. This emphasizes the importance of studying biosynthetic pathways and mechanisms at a molecular level. In this context, the present paper focuses on recent research into the molecular and biochemical mechanisms producing LC-PUFA in marine invertebrates, particularly cephalopod molluscs, that are of particular interest due to their potential as aquaculture species.The interest in LC-PUFA biosynthesis pathways in fish has been stimulated by the drive towards increasing sustainability of aquaculture through replacing the traditional major feed ingredients, fish meal and fish oil, derived from pelagic (reduction) fisheries with more sustainable alternatives such as plant meals and vegetable oils. As vegetable oils do not contain LC-PUFA, but are rich in C18 PUFA, there has been considerable research into the pathways of endogenous production of EPA and DHA in fish species ,18,25,32tebrates . Indeed,tebrates . The stutebrates . In totaMolluscs are arguably the group among marine invertebrates in which biosynthesis of PUFA has been most extensively investigated, in part due to commercial interest and perceived nutritional value of molluscs as sources of “omega-3” for humans . Comprehn-7 or ∆916:1) and oleic acid (18:1n-9 or ∆918:1), from palmitic acid (16:0) and stearic acid (18:0), respectively [Octopus vulgaris, was a ∆5-like desaturase  involved in the biosynthetic pathways of PUFA including NMI fatty acids. Thus, ty acids . Moreovety acids , and othectively ,46. Furt sources . Elongasonoensis . Interesonoensis .in silico searches by the authors revealed that two transcripts encoding putative elongases with potential roles in the biosynthetic pathways of LC-PUFA in the owl limpet, Lottia gigantea, can be identified, one with high homology to the octopus Elovl (jgi|Lotgi|224291|), and another to Elovl4 proteins (jgi|Lotgi|178149|). Similarly, assembly of EST hits enabled us to deduce the partial amino acid sequences of Elovl-like enzymes in the molluscs Mytilus galloprovinciallis (gb|FL495089.1| and gb|FL499406.1|), C. gigas , Euprymna scolopes (gb|DW256301.1|), Lymnaea stagnalis , and Aplysia californica . A similar strategy enabled us to identify sequences of putative Fads from H.discus hannai (gb|ADK38580.1| and gb|ADK12703.1|), A. californica (gb|XP_005090573.1| and gb|XP_005090577.1|), C. gigas (gb|EKC33620.1|), and L. gigantea (jgi|Lotgi|113523| and jgi|Lotgi|143229|). It is noteworthy that more than one Fad-encoding gene appears to exist in the genomes of some molluscs, such as H.discus hannai and L. gigantea, as this contrasts with previous assumptions on the presence of a single Fad gene in molluscs [The increasing availability of DNA sequence data has revealed the presence of elongase-like genes in the genomes of several species of molluscs. Thus, O. vulgaris), which experiences massive mortalities at the paralarval developmental stage [O. vulgaris [Sepia officinalis [Long-chain PUFA have been regarded as essential nutrients for cephalopods, particularly in early life-cycle stages . Howeveral stage . In ordevulgaris ,62, thisicinalis .O. vulgaris [5-like domain, and the haem-binding motif, HPGG, similar to vertebrate desaturases of the “Fads” family [S. cerevisiae, the octopus Fad exhibited Δ5-desaturase activity on both saturated and polyunsaturated fatty acyl substrates. The yeast’s endogenous 16:0 and 18:0 were efficiently converted to the ∆5-desaturated monoenes 16:1n-11 (∆516:1) and 18:1n-13 (∆518:1), respectively. This ability of octopus Fad was consistent with the fatty acid compositions reported for other molluscs including Littorina littorea and Lunatia triseriata that showed the presence of ∆5-desaturated monoenes including 18:1n-13 and 20:1n-15 (∆520:1) [n-3 and 20:3n-6 were converted to EPA and ARA, respectively  . The ∆5-ectively . In contpathways . Similarectively . Howeverhalopods . Second,g to DHA , as the O. vulgaris [n-3 to 24:5n-3 required for DHA biosynthesis through the Sprecher pathway, and further supports the abovementioned hypothesis that endogenous biosynthesis of DHA is not possible, suggesting DHA is an essential nutrient in cephalopods. In order to investigate if other elongases could possibly complement the functions that Elovl5/2 plays in the LC-PUFA biosynthesis in cephalopods, we have recently cloned a second elongase with high homology to vertebrate Elovl4 proteins, elongases with demonstrated roles in the biosynthesis of very long-chain (C > 24) PUFA in vertebrates [We have recently confirmed the existence of a cDNA encoding an Elovl involved in the production of LC-PUFA in the cephalopod vulgaris . Phylogevulgaris . This invulgaris . It was vulgaris . More spvulgaris ,32,53. Ctebrates ,67,68,69As alluded to above, marine invertebrates are a possibly unique source of unusual PUFA, the NMI fatty acids, as opposed to the common methylene-interrupted PUFA, in that their double (ethylenic) bonds are separated by more than one methylene group. First discovered (reported) in the 1970s, these ubiquitous, although generally minor, components of the lipids of invertebrates have been extensively studied ever since . AltΔ5,1120:2, Δ7,1320:2, Δ5,11,1420:3, and Δ7,1322:2, using GC-MS methodologies [Δ5,1120:2, Δ7,1320:2, and Δ7,1322:2, can be speculated to proceed as follows. De novo biosynthesis of 16:0 and 18:0 is followed by desaturation via Scd (Δ9-desaturase) to produce 16:1n-7 (Δ916:1) and 18:1n-9 (Δ918:1), respectively. Preliminary data have confirmed that O. vulgaris expresses an Scd with the necessary enzymatic abilities [n-7 (Δ1118:1) and 20:1n-9 (Δ1120:1) and further Δ5-desaturation results in the formation of Δ5,1118:2 and Δ5,1120:2, and a further elongation would result in the production of Δ7,1320:2 and Δ7,1322:2. Direct evidence for the production of Δ5,11,1420:3 was obtained through functional assays on the octopus Δ5 Fad [n-6  was efficiently converted into the NMI fatty acid Δ5,11,1420:3  to Δ5,11,14,1720:4  revealed the presence of four NMI fatty acids that were identified as dologies . The biobilities . Subsequs Δ5 Fad . Yeast e1,1420:3 . The oct4,1720:4 , anotherEuryspongia rosea [In silico searches for putative Fad and Elovl genes in the genome of the demosponge Amphimedon queenslandica [Fatty acids from sponges, particularly of the class Demospongiae, have unique structural characteristics that include long fatty acyl chains (up to 34 carbons), presence of branched chains and functional groups, relatively high degrees of unsaturation (up to six double bonds), and a particular pattern of unsaturation within the fatty acyl chain ,78. Amonia rosea  revealedslandica  revealedEucyclops serrulatus had the ability to endogenously produce DHA when fed on microalgae devoid of this fatty acid [Dropanopus forcipatus and Euphausia superba [Data available in the literature demonstrate that the ability of crustaceans for LC-PUFA biosynthesis varies among species, with no obvious patterns associated with phylogenetics (classes/subclasses) or habitat (freshwater/marine). For example, harpacticoid copepods, but not calanoid species, have the ability to biosynthesize LC-PUFA, possibly related to poor quality of food available in detritus-rich habitats. Thus, the presence of desaturases and elongases involved in the biosynthesis of PUFA have been demonstrated in harpacticoid copepods ,83,84,85tty acid . On the  superba ,87,88.Eriocheir sinens [E. sinens.While the ability for endogenous LC-PUFA biosynthesis in some crustaceans has often been indicated through indirect analytical  approaches, more direct  evidence demonstrating the above mentioned enzymatic activities are scarce and thus the specific biosynthetic pathways remain elusive. Recent investigations have shown the existence of a putative desaturase from the Chinese mitten crab, r sinens ,90. Whilr sinens . This reDaphnia pulex, which showed increased LC-PUFA content in response to low temperature exposure [Artemia, with an apparent ability to interconvert fatty acids from the n-6 to n-3 series. [n-6) was converted to LNA (18:3n-3), were particularly interesting as they implied the existence of an n-3 (or Δ15) desaturase in crustaceans, enzymes that were believed to be absent in animals but now confirmed to exist in some invertebrates like the nematode C. elegans [n-3 desaturases in the copepod Caligus rogercresseyi (gb|ACO10720.1|), an ectoparasite that causes major issues in the Atlantic salmon farming industry. In addition, within this species, other sequences for potential candidate enzymes were identified including desaturases (gb|ACO10922.1), annotated as “Delta-5 fatty acid desaturase”, and elongases homologous to vertebrate Elovl2 (gb|ACO10776.1|) and Elovl4 (gb|ACO11542.1|).Some ability for endogenous production of LC-PUFA has also been reported in other crustaceans including the branchiopoda exposure , and Art series. ,93. The  elegans ,95. InteSinularia sp. and hard coral Acropora sp. provided clues to what biochemical pathways of PUFA synthesis were present in the different fractions [n-6, 18:4n-3, EPA, 22:5n-3 and DHA were mainly synthesized by the zooxanthellae, and that 20:3n-6, ARA, and 22:4n-6 were synthesized in the polyp tissue. Soft coral polyps were also able to synthesize 24:5n-6 and 24:6n-3. These tetracosapolyenoic fatty acids are chemotaxonomic markers of soft corals [n-7, 16:3n-4, and 16:4n-1. The authors speculated that the biosynthesis of 16:2n-7 in Sinularia sp. and 18:3n-6 in Acropora sp. was catalyzed by a Δ6 desaturase, and that the relatively even distribution of 18:2n-6, 18:3n-6, and 16:2n-7 among the different fractions indicated translocation between zooxanthellae and coral polyps [n-7, perhaps suggesting the action of a C16 elongase in the coral. In contrast, a recent study with the model anemone, Aiptasia pulchella, using enriched stable isotopic (13C) incorporation from dissolved inorganic carbon, showed fatty acid synthesis rates were attributed to only a complex integration of lipogenesis pathways within the dinoflagellate symbionts, and that there was no evidence of symbiont-derived enriched isotope fatty acids being directly utilized in host PUFA synthesis [The presence of other organisms, including primary producers, within some invertebrates, which complicates the unequivocal identification of PUFA and/or LC-PUFA biosynthetic pathways, is particularly pertinent when considering cnidarians that have symbiotic interactions with dinoflagellate microalgae (zooxanthellae) that are able to synthesize PUFA . Furtherractions . The rest corals , regardlt corals , showingl polyps . Consistynthesis .Ciona intestinalis, was one of the first published a decade ago. A search of the C. intestinalis genome using the Δ6 elongase sequence from the moss Physcomitrella patens as a query, indicated the presence of an elongase, and functional characterization of the ORF in the yeast S. cerevisiae revealed it was capable of elongating both 18:4n-3 and EPA [Little is known about PUFA biosynthesis and metabolism in tunicates, formerly known as urochordates. However, embryonic development in ascidian tunicates (sea squirts) is simple, rapid, and easily manipulated which, along with transparency, has made them suitable models for studying the fundamental developmental processes of chordates. As a result, good genomic resources are available and, indeed, the genome of the sea squirt,  and EPA . HoweverC. intestinalis (gb|NP_001029014.1|), the polychaete worm (Annelida) Capitella teleta (gb|ELT94279.1|), and the Acorn worm (Hemichordata) Saccoglossus kowalevskii (gb|XP_002739666.1), among others. Putative Elovl2- and Elovl5-like elongases include those from the polychaete C. teleta (ELU02248.1) and the purple sea urchin (Echinodermata) Strongylocentrotus purpuratus (gb|XP_789039.2|).As mentioned previously, increasing availability of genomic data from invertebrate organisms is a useful tool for the identification of possible desaturase and elongase genes with potential roles in the biosynthesis of PUFA and/or LC-PUFA. Thus, some desaturases with high homology to vertebrate Fads-like proteins, and to the ∆5-like desaturases from molluscs, can be found in the GenBank public database. These include desaturases in Almost all PUFA originate in microalgae, bacteria and heterotrophic protists inhabiting aquatic ecosystems. Fish, occupying relatively high trophic levels within aquatic systems, can significantly contribute to trophic upgrading as they have the ability to metabolize PUFA to produce LC-PUFA. However, the pathways of PUFA biosynthesis in invertebrates, organisms occupying trophic levels between the primary producers and fish in aquatic ecosystems, have remained largely unexplored. Particularly challenging in the study of fatty acid metabolism in invertebrates is the potential presence of microflora and symbiotic organisms with active metabolic pathways that can hinder the unequivocal assignment of enzymatic activities to the host invertebrate. Molecular studies clearly emerge as a valuable strategy to address this challenge and, thus, significant progress is now being made to understand the biosynthetic pathways and mechanisms producing LC-PUFA in marine invertebrates.Euryspongia rosea [n-3 (or Δ15) desaturase, an enzyme catalyzing the interconversion of n-6 to n-3 fatty acids [Data available in the literature provide evidence that marine invertebrates possess and express genes encoding desaturase and elongase enzymes with a role in the endogenous production of LC-PUFA. Ever-increasing genomic data derived from sequencing projects conducted for a wide range of marine invertebrates is providing an excellent source of molecular evidence supporting the existence of these biosynthetic pathways. While potential candidates for both desaturase and elongase genes have been identified in sponges, cnidarians, annelids, echinoderms, hemichordates, and tunicates, a more robust set of data combining analytical, biochemical and molecular evidence is available for crustaceans and, particularly, molluscs. Using the common octopus as model species, we have begun to elucidate pathways for the biosynthesis of PUFA in cephalopods and possibly other molluscs. To this end, several genes encoding desaturases and elongases that are directly involved in the production of PUFA have been identified by molecular cloning, and functionally characterized by heterologous expression in yeast. A Δ5 desaturase has been confirmed in the cephalopods, octopus and cuttlefish ,65, and ia rosea  was founty acids ,93. FurtIn silico searches for Elovl-like sequences in the genome of marine invertebrates indicate that homologs to vertebrate Elovl2, Elovl4, and Elovl5 exist. Interestingly, while the proteins are distinguished as three distinct families in vertebrates [Amphimedon queenslandica, the copepod Caligus rogercresseyi, the polychaete Capitella teleta, and the purple sea urchin Strongylocentrotus purpuratus. Functionally, the cephalopod Elovl5/2 is similar to that of vertebrate Elovl5, with ability to efficiently elongate C18 and C20 PUFA substrates, but no activity on C22 PUFA. These results reveal an interesting evolutionary scenario predicting the divergence of the Elovl5/2 basal protein into distinct protein families in vertebrates. Ongoing investigations in O. vulgaris suggest that the cephalopod Elovl4-like protein participates in the biosynthesis of very LC-PUFA (34 carbons).Fatty acid elongation systems have also been described in marine invertebrates. tebrates , only twStudies of the molecular mechanisms underlying PUFA and LC-PUFA biosynthesis in marine invertebrates are not only illuminating alternative and unusual biosynthetic pathways and metabolism, but are also providing insights to gene and pathway evolution, as well as being a resource that can supply potentially valuable molecular tools in the form of genes involved in PUFA biosynthesis and metabolism.