The yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.Two-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions.The findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability. The Ty3 retrotransposon in budding yeast forms virus-like particles (VLPs) comprised of precursor Gag3 and Gag3-Pol3 polyproteins ,2. PreviTRP1-marked pGBK vector . Fusions of Gag3 and derivatives to the C-terminus of the Gal4-AD tagged with HA were expressed from the LEU2-marked high-copy plasmid pGAD T7 (pGAD). These fusions were constructed by amplifying the appropriate regions from Ty3 Gag3 subclones in pGEM  using polymerase chain reaction (PCR) primers containing NdeI and BamHI sites at the 5' and 3' outside ends, respectively and ligating fragments to the pGBK and pGAD vectors linearized with NdeI and BamHI. Constructs , which has ADE2, HIS3, LacZ, and MEL1 reporters regulated by the GAL4 responsive upstream activating sequences (UASs) (Clontech). Preliminary tests showed that expression of ADE2, which results in cream-colored colonies and growth in medium lacking adenine, provided the most reliable detection of two-hybrid interaction (data not shown). The yAH109 transformants containing pairwise combinations of pGAD and pGBK plasmids were selected on medium lacking tryptophan and leucine. Four isolates from each transformation were restreaked and then replica plated onto complete synthetic medium lacking adenine, tryptophan and leucine and grown at 30°C for evaluation of color development and growth. We first tested Gag3, CA, p27, and NC fusions in both vectors in all combinations. A mutant deleted for SP residues 208 to 232 (ΔSP) was also assayed . AD-CA transformants were not recovered as stable colony isolates in repeated attempts. Other constructs were shown to be positive for expression of the appropriate fusion by immunoblot analysis using rabbit polyclonal antibodies against Gal4 AD and Gal4 BD  or CA [Two-hybrid analysis  was usedA) or CA . With thHomotypical interactions (except for CA) and heterotypical interactions were assayed Figure . Gag3, Δ2EX2FX2FX3L) [Among the previously described alanine-scanning mutations in Ty3 Gag3 [FX2FX3L) -9. AlaniStudies of retroviral CA assemblies show a network of CA NTD hexamers connected via CA CTD interactions (reviewed in ). These In order to test whether mutations in Gag3 that affected assembly and homotypical and heterotypical interactions of intact Gag3 changed the exposure of NTD and CTD subdomains and therefore their ability to interact, interaction of these subdomains with mutant Gag3 was examined Table  and 9. NTwo-hybrid analysis readily detected interactions between Gag3 polyproteins. Nevertheless there were asymmetries in the interactions introduced by the expression context. These included apparent toxicity of AD-CA and AD-CTD, detection of stronger interactions of BD-P27 compared to AD-P27, and of AD-NTD compared to BD-NTD Figure . This coin vitro assembly intermediates of RSV [Recent 3D structures of full-length CA molecules in assembled HIV1 particles viewed as 2D crystals by cryoelectron microscopy (cryoEM) underscore three types of interactions with retroviral CA assembled into hexameric lattice structures: NTD-NTD and CTD-NTD intermolecular interactions and CTD-CTD interhexameric interactions ,19. NTD-s of RSV . Based oFinally, this investigation provides further support for our model that Gag3 undergoes conformational switching between unassembled and assembled states . First, The authors declare that they have no competing interests.MZ and SBS designed the study and drafted the manuscript. MZ and LZ designed and constructed Ty3 Gag3 mutant collection. MZ and VB subcloned into two-hybrid vectors and together with BI performed the two-hybrid analysis. All authors read and approved the manuscript.Sup. Fig. 1. Interactions of BD M4 mutant Gag3 D60A/R63A with Gag3 wild type, D60A/R63A, G87A, F93A, E148A/K149A, and E190A/R191A. The D60A/R63A mutation disrupts interactions with wild type and other mutant Gag3 proteins. Images of yeast strain yAH109 containing two-hybrid pGAD or pGBK vectors or expression plasmids as described in text. Cells are shown as representative streaks of independent transformants growing on synthetic dextrose medium lacking tryptophan, leucine, and adenine. Mutants are M4 (D60A/R63A), MHR2 (G87A), MHR4 (F93A), M13 (E148A/K149A), and M18 (E190A/R191A). Binding domain (BD) fusions are labeled in the top row and shown in columns; activation domain (AD) fusions are labeled in the left column and shown in rows. Scoring is based on four individual transformants (two shown). There was variability among individual BD-Gag3/AD-N-terminal domain (NTD) transformants  NTD with wild type Gag3, CA NTD, and CA CTD. BD CA NTD interacts with CA NTD and CA CTD.Click here for fileSup. Fig. 8. BD capsid (CA) NTD interaction with Gag3 wild type and D60A/R63A, G87A, F93A, E148A/K149A, and E190A/R191A. Mutations in the CA NTD and CA CTD that disrupt interactions in the Gag3 context fail to allow observation of interactions between CA NTD and Gag3.Click here for fileSup. Fig. 9. Interactions between BD capsid (CA) CTD and wild type Gag3, D60A/R63A, G87A, F93A E148A/K149A, and E190A/R191A. Mutations in Gag3 that disrupt Gag3 interactions in the Gag3 context fail to allow observation of interactions between CA CTD and Gag3.Click here for fileSup. Fig. 10. Interactions between BD Gag3 and wild type Gag3, wild type CA NTD, and capsid (CA) NTD D60A/R63A, CA NTD G87A, and CA NTD F93A. Mutations in the CA NTD that disrupt interactions in the Gag3 context fail to allow observation of interactions between CA NTD and Gag3.Click here for fileSup. Fig. 11. Interactions between BD capsid (CA) NTD and BD CA CTD with wild type CA NTD, CA NTD D60A/R63A, CA NTD G87A, and CA NTD F93A. With the exception of D60A/R63A mutations in the CA NTD that disrupt Gag3 interactions do not decrease CA NTD interactions with the CA NTD. D60A/R63A does not decrease interactions with BD CA CTD.Click here for fileSup. Fig. 12. Interactions between BD capsid (CA) CTD M13 mutant E148A/K149A and BD CA CTD M18 mutant E190A/R191A with wild type Gag3, CA NTD, CA NTD D60A/R63A, CA NTD G87A, and CA NTD F93A. Mutations in CA CTD and CA NTD that disrupt Gag3 interactions do not interfere with observation of interactions between the CA CTD and CA NTD.Click here for file


Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8  are very similar to those of phage Felix O1  and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.Based upon whole genome and proteome analysis,  Escherichia coli O157:H7 along the food chain. Our research has shown that a cocktail of virulent phages can eliminate E. coli O157:H7 from experimentally infected calves [E. coli O157:H7. Here we report on the genome and proteome of phage wV8, noting its very close similarity to the Salmonella phage Felix O1 [Bacteriophages (phages) are promising potential alternatives to antibiotics as therapeutics to reduce carriage of pathogens by food animals, thus preventing the spread of organisms such as d calves . Phage Vd calves  was renaFelix O1 -7.Myoviridae and is morphologically identical to Felix O1 and related phages. Viral particles were morphologically intact and generally have extended tails  uranyl acetate for 20 s and the particles were observed using a LEO912AB and a Philips EM 300 transmission electron microscope operating at 100 kV and 60 kV, respectively. Phage wV8 is a member of the E. coli O157:H7 PTs, the entire ECOR collection [Salmonella enterica serovars. Lytic activity on the reference E. coli O157:H7 PT strains and the Salmonella serovars was determined at multiplicities of infection (MOI) of between 0.001 and 10 in broth cultures in microplates incubated for 5 h at 37°C before inspection for complete lysis (no visible turbidity). Bacteria showing no visible lysis at any MOI were considered resistant to phage wV8, while those showing complete lysis at MOIs of 10 or less were considered sensitive to phage wV8. Strains of the ECOR collection were tested as freshly seeded bacterial lawns on agar plates spotted with 20 μl of diluted phage wV8 containing 104-106 pfu. After incubation for 18 h at 37°C, strains showing >50% lysis were considered sensitive. Phage wV8 is highly specific for E. coli O157:H7 strains, completely lyses the 12 most common E. coli O157:H7 (PTs) isolated in Canada [Salmonella strains , precipitated from clarified lysates using polyethylene glycol 8000 and purified through two rounds of CsCl equilibrium gradient centrifugation [rIIA gene so that it could be directly compared with the sequence of Felix O1. The genome was annotated using Kodon  and a variety of online tools  including tRNAScan-SE [EU877232.Phage wV8 was propagated on fugation . The DNAAScan-SE  and ARAGAScan-SE  at theirSalmonella phage Felix O1  and, indeed many of the proteins have their closest homologs with those of Felix O1 (NC_005282). This is also substantiated by SDS-PAGE analysis of the structural proteins of wV8 and Felix O1 which show considerable similarity  closely resemble those of  against NCBI databases retrieved a putative tail fibre protein from phage Felix O1 , where five sequences out of the observed 28 peptides were matched . Using a custom wV8-specific protein database, a thorough examination of all peptide sequences confirmed the protein assignment, with these five peptides providing 44.3% sequence coverage. Sequence alignment of the Felix O1 and wV8 tail fibre orthologs using ALIGN  revealed 65.7% identity.Tandem mass spectrometric (MS/MS) measurements were performed to sequence all the trypsin-digested peptides in order to obtain the high confidence protein identification in the databases. Initial MS/MS search using Mascot Tail fibre proteins from related phages typically show strong sequence similarity at the N-termini, where the protein associates with the phage tail plate. The carboxy termini, associated with receptor interaction, vary considerably. With Felix O1 and wV8, we see a completely different type of relationship: four regions of similarity separated by regions of dissimilarity, with both the C- and N-termini conserved  Reference Laboratory at the Laboratory for Foodborne Zoonoses. Approximately one-half of these still serotyped as O157:H7, while half were untypable (rough) indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.Since Felix O1 is LPS-specific , we anal. The latter program revealed that Felix O1 and wV8 share 92% of their proteins in common. Mauve analysis  this level of similarity indicates that wV8 should be classified into the newly proposed genus,"Felix O1 viruses", along with Erwinia amylovora phage φEa21-4.Based upon an extensive analysis of relationships between prokaryotic viruses  if identical, a colon (:) if conserved; and a period (.) if related.Click here for file


Voltage-clamped Xenopus oocytes injected with either 0.04 or 1.5 μg μL−1 R878C-hNav1.5 cRNA similarly showed no iNa, yet WT-hNav1.5 cRNA diluted to 0.0004–0.0008 ng μL−1resulted in expression of detectable iNa. iNa was simply determined by the amount of injected WT-hNav1.5: doubling the dose of WT-hNav1.5 cRNA doubled iNa. iNa amplitudes and activation and inactivation characteristics were similar irrespective of whether WT-hNav1.5 cRNA was given alone or combined with equal doses of R878C-hNav1.5 cRNA therefore excluding dominant negative phenotypic effects. Na+ channel function in HEK293 cells transfected with R878C-hNav1.5 was not restored by exposure to mexiletine (200 μm) and lidocaine (100 μm). Fluorescence confocal microscopy using E3-Nav1.5 antibody demonstrated persistent membrane expression of both WT and R878C-hNav1.5. Modelling studies confirmed that such iNa reductions reproduced the SSS phenotype.The four mutation carriers showed bradycardia, and slowed sino-atrial, atrioventricular and intraventricular conduction. Two also showed sick sinus syndrome; two had ST elevation in leads V1 and V2. Unlike WT-hNaClinical consequences of the novel R878C mutation correlate with results of physiological studies. SCN5A encoding the pore-forming α-subunit of the human cardiac Na+ channel have been associated with an increasingly wide range of cardiac rhythm disorders that include the long QT syndrome type 3 (LQT3), Brugada syndrome (BrS) and cardiac conduction diseases, idiopathic ventricular fibrillation, sinus node dysfunction including sick sinus syndrome (SSS), atrial standstill and sudden infant death syndrome (SIDS)  studies. For example, the gain of function, C-terminal SCN5A gene mutation (1795insD) in a large Dutch family resulted in bradycardia, conduction disease, LQT3 and BrS  and lidocaine (100 μm), manoeuvres that have previously been used to restore channel trafficking  node function then reproduced the SSS phenotype. This analysis of the R878C mutation thus extends from its clinical manifestations to physiological characterization and modelling.The present study identifies and characterizes a novel missense mutation of the highly conserved pore residue arginine to cysteine R878C in domain II S5-S6 of the cardiac NaAll investigations conformed to principles defined in the Helsinki Declaration. Seventeen members of a three-generation Chinese family were investigated see , informeGenomic DNA was extracted from 10 mL of whole blood using the Puregene Isolation kit . The coding regions of SCN5A and exon/intron boundaries were amplified from genomic DNA by polymerase chain reaction (PCR) using a comprehensive set of primers and PCR conditions as described previously  was used as template for generating R878C mutant Nav1.5 cDNA by recombinant PCR using the primer pair 5′-AAGGCATGAAAGAAGTCCATCATGTGCCAGCAAGGCAGCAGGCCTGAGTC-3′ (for R878C). The PCR product was then inserted as BseJI/Asp718I (R878C) fragment into the corresponding site of the WT-hNav1.5 clone. The resulting DNA construct was checked by restriction analysis, and nucleotide exchange was confirmed by DNA sequencing. Following Xba I digestion of the plasmid, capped cRNA was prepared by in vitro transcription reaction with SP6 RNA polymerase . The WT-hNav1.5 was subcloned into a pEGFP expression vector. pEGFP-R878C-hNav1.5 was generated by site-directed mutagenesis  using pEGFP-WT-hNav1.5 as the template for transfection of HEK293 cells used in the biophysical studies. Finally, WT-hNav1.5 was subcloned into pEGFP, pEYFP and pECFP expression vector. pEGFP-R878C-hNav1.5 and pEYFP-R878C-hNav1.5 were generated as described above, using pEGFP-WT-hNav1.5 and pEYFP-WT-hNav1.5 as templates, respectively, and used for transfection of HEK293 cells for immunochemical staining visualized by confocal microscopy.Plasmid pSP64T-hH1 coding for WT human Na1 Na+ channel subunit  using LipofectAMINE  as described previously . The pipettes were pulled with a gravity puller  and filled with a pipette solution of the following composition (in mm) KCl 10, CsF 105, NaCl 10, HEPES 10, titrated to pH 7.2 with CsOH. Pipette resistance ranged from 1.0 to 2.0 MΩ when pipettes were filled with the internal solution. The perfusion solution contained (in mm): NaCl 130, KCl 5, CaCl2 1.8, MgCl2 1.0, sodium acetate 2.8, HEPES 10, and glucose 10, titrated to pH 7.4 with NaOH. Current signals were recorded with an Axon 200B amplifier , and series resistance errors were reduced by approximately 70∼80% with electronic compensation. Signals were acquired at 20 kHz  and analysed with a PC running pclamp 9 software (Axon Instruments). All recordings were made at room temperature (20–22 °C). Whole-cell Na+ currents in Xenopus oocytes were recorded with the two-microelectrode voltage clamp technique using a commercial amplifier , as previously described (m KCl. The microelectrode resistance was between 0.2 and 0.5 MΩ. The bath solution contained (in mm): 96 NaCl, 2 KCl, 1.8 CaCl2, 10 HEPES titrated to pH 7.2 with KOH. The currents were elicited by test potentials from −80 to 40 mV using a holding potential of −120 mV. The pulsing frequency was 1 Hz. Recording and analysis of the data were performed on a PC with iso2 software . The sampling rate was generally 20 kHz.For electrophysiological recordings, the HEK293 cells were trypsinized 24 h after transfection and seeded onto a glass coverslip to a density that enabled single cells to be identified. Whole-cell Nav1.5 were fixed with 4% paraformaldehyde/PBS, mounted with fluorescent mounting medium  and viewed under confocal microscopy  at excitation wavelengths of 488 nm (for EGFP), 458 nm (for ECFP) and 514 nm (for EYFP and Cy3). The immunocytochemistry staining using E3-targeted anti-Nav1.5 antibody (Ab) and Cy3-conjugated secondary antibody  was performed as described previously . Differences were evaluated by Student’s −1 consistent with SSS, atrial ectopics and widened QRS complexes with a normal QTc   ; this wa protein , which fisoforms . Furtherisoforms . We alsoisoforms  but not + channel function. Cells were clamped at a holding potential of −120 mV and subjected to 30 ms duration depolarization pulses between −110 and +50 mV in 5 or 10 mV increments at pulse frequency of 1.0 Hz. First, HEK293 cells transiently transfected with (0.5 μg) plasmid DNA for either WT-hNav1.5 or R878C-hNav1.5 were investigated in the whole-cell configuration. Na+ currents, iNa, recorded from the HEK293 cells transfected with plasmid for WT-hNav1.5 showed average peak current densities of 432.2 ± 49.16 pA pF−1 (n = 10 cells). Similar currents were observed at a mean peak level of 334.3 ± 47.56 pA pF−1 (n = 7 cells) with transfection of half the dose of WT-hNav1.5 plasmid DNA in combination with the same dose of β1-subunit plasmid, known to increase hNav1.5 expression (+ channels. (v1.5 plasmid DNA (n = 20) did not show any detectable iNa whether in the absence , a critical region that forms part of the channel pore. There is therefore the possibility that arginine has a more specific role than simply providing a positive charge influencing the local electrostatic field essential for channel function. Such a notion would be consistent with a comparison of aligned amino acid sequences that demonstrate that R878 is highly conserved among Nav channel isoforms (v1.5 construct in which the arginine R878 was replaced by the similarly positively charged lysine (K) to give the substitution R878K, through transient transfection into HEK293 cells. iNa (n = 9).These findings thus suggest that substitution of the hydrophilic positively charged arginine with the hydrophobic uncharged amino acid cysteine results in a loss of detectable channel function. However, residue 878 in Naisoforms . This hyXenopus expression system was then used to quantitatively explore the possible effects of gene dosage and for the presence or absence of any interactions between WT and mutant gene expression. This was achieved by introduction of different combinations of a standard dose of cRNAs encoding each protein variant. The initial experiments explored the effects of identical dosages of cRNA encoding WT-hNav1.5 and mutant R878C-hNav1.5 in different combinations to determine the time course, peak amplitude and voltage dependence of the resulting whole-cell Na+ currents.The iNa is simply related to the dose of WT-hNav1.5 cRNA: the mutant R878C-hNav1.5 is non-functional even in the presence of the WT-hNav1.5 channels, and the presence or absence of mutant cRNA neither increases nor decreases the net size of iNa. Accordingly, the effects of R878C-hNav1.5 were not dependent upon the dominant negative effects of the R878C-hNav1.5 mutation on the function of WT-hNav1.5 on an otherwise normal WT-hNav1.5 expression. First, injections of cRNAs encoding (WT-hNav1.5 + H2O) (v1.5 + R878C-hNav1.5) (iNa. These were in turn doubled by the doubled dose resulting from injection of cRNA encoding (WT-hNav1.5 + WT-hNav1.5)   and (WT-hNav1.5)  resultedhNav1.5) . SecondliNa in an otherwise functional mutant for I1660V Na+ channels that have similarly been related to a Brugada phenotype  that would be expected to result in substitution of an arginine in residue 878. This amino acid residue R878 is likely to be critical for Na+ channel function. Thus, alignment analysis indicated that R878 is a residue in domain II of S5-S6 that shows a highly conserved arginine residue amongst Nav channel isoforms. Furthermore, R878 is located in the pore-forming region of domain II close to the selectivity filter DEKA and a highly conserved EEDD ring of charge  of the hNa+ channel mutations on previous occasions. This is in contrast to our recent report of a simpler clinical picture presenting a full and similar penetrance in a family pedigree showing LQT2  and adult life (uncle of the proband). All of these have nevertheless been associated with cardiac Na+ channel variant to available recent reports that have associated SCN5A mutations with a range of phenotypes extending even to SSS  in the carboxy terminus of SCN5A can result in either BrS or LQTS  or lidocaine (100 μm), manoeuvres that have previously been used to restore channel trafficking (SCN5A mutations T187I, D356N, K1578fs/52, and R1623X that were similarly linked with BrS, SSS and atrioventricular block mentioned above (iNa was also not restored by mexiletine (SCN5A. Furthermore, extracellular application of E3-targeted anti-Nav1.5 antibody yielded a significant plasma membrane expression of both WT and mutant channels. This suggests a persistent expression of mutant but non-functional Na+ channels despite the complete abolition of Na+ currents in the homozygote, consistent with a continued expression of non-functional channels rather than a total failure of expression of otherwise normal channels.Additional biophysical and immunochemical experiments provided further evidence that the presence of the mutation probably resulted in a persistent expression of non-functional Na+ currents in the patients’ cardiomyocytes should be reduced by 50%, i.e. corresponding to the level of WT-hNav1.5 channels produced from the unaffected allele. Numerical modelling demonstrated the effects upon SAN function as reflected in heart rate of a progressive reduction in Nav1.5 current resulting from increasing proportions of non-functional mutant channels in an established model of SAN-atrium tissue (iNa as seen in clinical cases, whose physiological correlates are examined here.As the affected members in the family concerned were heterozygotes, one would expect that Na+ channel in a three-generation Chinese family that complements previous studies reporting a range of, rather than a single clinical, phenotype.In conclusion, we have identified and characterized a novel mutation at a highly conserved pore residue in domain II S5-S6 of the cardiac type Na


These results demonstrate that a polymorphism within the TS gene, probably through its effect on TS expression levels, can influence the survival benefit obtained by CRC patients from 5-FU-based chemotherapy. © 2001 Cancer Research Campaignhttp://www.bjcancer.comHigh levels of thymidylate synthase (TS) expression have been associated with poor survival of colorectal cancer (CRC) patients to 5-fluorouracil (5-FU)-based chemotherapy. Recent evidence suggests that a polymorphism within the enhancer region of the TS gene promoter can influence TS expression, with the triple repeat homozygote (3R/3R) being associated with significantly higher tumour TS levels than either the double repeat homozygote (2R/2R) or heterozygotes (2R/3R). In the present study we investigated whether TS genotype was associated with the degree of survival benefit from chemotherapy in 221 Dukes' C stage CRC patients. Patients with the 3R/3R polymorphism (


Ixeris repenes (L.) A. Gray, a common dune plant, for the isolation of gibberellin secreting endophytic fungi.Endophytic fungi are known plant symbionts. They produce a variety of beneficial metabolites for plant growth and survival, as well as defend their hosts from attack of certain pathogens. Coastal dunes are nutrient deficient and offer harsh, saline environment for the existing flora and fauna. Endophytic fungi may play an important role in plant survival by enhancing nutrient uptake and producing growth-promoting metabolites such as gibberellins and auxins. We screened roots of Ixeris repenes and screened them for growth promoting secondary metabolites. The fungal isolate IR-3-3 gave maximum plant growth when applied to waito-c rice and Atriplex gemelinii seedlings. Analysis of the culture filtrate of IR-3-3 showed the presence of physiologically active gibberellins, GA1, GA3, GA4 and GA7  along with other physiologically inactive GA5, GA9, GA12, GA15, GA19, GA20 and, GA24. The plant growth promotion and gibberellin producing capacity of IR-3-3 was much higher than the wild type Gibberella fujikuroi, which was taken as control during present study. GA5, a precursor of bioactive GA3 was reported for the first time in fungi. The fungal isolate IR-3-3 was identified as a new strain of Penicillium citrinum (named as P. citrinum KACC43900) through phylogenetic analysis of 18S rDNA sequence.We isolated 15 endophytic fungi from the roots of Penicillium citrinum from the sand dune flora is interesting as information on the presence of Pencillium species in coastal sand dunes is limited. The plant growth promoting ability of this fungal strain may help in conservation and revegetation of the rapidly eroding sand dune flora. Penicillium citrinum is already known for producing mycotoxin citrinin and cellulose digesting enzymes like cellulase and endoglucanase, as well as xylulase. Gibberellins producing ability of this fungus and the discovery about the presence of GA5 will open new aspects of research and investigations.Isolation of new strain of  Gibberella fujikuroi, a known pathogen of rice plants  GAs internal standards  and the fungal gibberellins were monitored simultaneously. The retention time was determined using hydrocarbon standards to calculate the KRI (Kovats Retention Index) value, while the GAs quantification was based on peak area ratios of non-deuterated (extracted) GAs to deuterated GAs.Fungal gibberellins (GAs) were extracted from culture filtrates after 7 days of incubation in Czapek broth according to an established protocol . ExtractAn efficient method was developed for the isolation of genomic DNA from endophytic fungi, because usual CTAB extraction method and mycelial grinding was causing DNA shearing. Rich mycelial culture was obtained by growing fungus in Czapek culture broth (supplemented with 1% glucose and peptone) for 7 days on rotary shaking incubator (120 rpm and 28°C), and lyophilized for 24 hrs. A 0.5 g of lyophilized sample was broken carefully in 2 ml eppendorf, with blunt end spatula or glass rod. Double volume of lysis buffer  containing 1% of 2- mercaptoethanol was added. The mixture was vortexed briefly (30 sec) to obtain homogeneity and left to incubate for 2 hr in water bath set at 55°C. 250 μl/ml of pre-heated 4% CTAB extraction buffer was added to lysed cells mixture and incubated further at 65°C for 1 hr. Chloroform extraction followed by iso-propanol precipitation yielded condensed strand of nucleic acid, which was cleaned from RNA using 10 ul of RNase A for 2 hr of incubation at 37°C. The isolated DNA was suspended in 50 ul of autoclaved deionized distilled water and tested for purity and quantity by agarose gel electrophoresis.E. coli mutant cells (RBC) by overnight incubation (37°C). Plasmids with inserts were extracted using SolGent Plasmid mini-prep kit, and sequenced.Fungal isolate was identified by sequencing internal transcribed region (ITS) of 18S rDNA, using universal primers ITS-1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS-4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). A 25 μl of PCR mixture contained 2.5 μl of dNTPs and Ex-Taq buffer, 2 μl of each primer, 0.5 μl of DNA sample, and 0.2 μl of Ex-Taq polymerase. Rest volume was adjusted with 15.3 μl of autoclaved deionized distilled water. The reaction cycle consisted of 2 min of initial denaturation at 95°C, followed by 35 cycles of 30 sec denaturation time (95°C), 60 sec of annealing (55°C) and 30 sec of extension (72°C), and a final extension time of 5 min at 72°C. The resultant product was gene cleaned using Nucleogen gene clean kit, ligated in T-vector using Takara Perfect T-cloning kit, and then inserted into DHα  was used to look for nucleotide sequence homology for 18S ITS (1/4) region for fungi. The obtained sequences were aligned by ClustalW using MEGA version 4.0 software [The BLAST search program software , and theSAK and MH participated equally in overall experimentation and manuscript preparation. JGK, WSK and BML participated in design of the study. YSC and UHY participated in sampling area selection, plant collection and identification. SJS, SKH and JMK helped in root screening for fungal isolation. MH, HYK and IJL participated in gibberellins extraction and quantification. HY and JGK helped in gene isolation and identification. All authors read and approved the final manuscript.1 in culture filtrate of fungal isolate IR-3-3GC-MS SIM spectra for GA. Arrow indicates the peak of fungal GA1 that coincides with that of internal standard GA1.Click here for fileGC-MS SIM spectra for GA3in culture filtrate of fungal isolate IR-3-3. Arrow indicates the peak of fungal GA3 that coincides with that of internal standard GA3.Click here for fileGC-MS SIM spectra for GA4in culture filtrate of fungal isolate IR-3-3. Arrow indicates the peak of fungal GA4 that coincides with that of internal standard GA4.Click here for fileGC-MS SIM spectra for GA7in culture filtrate of fungal isolate IR-3-3. Arrow indicates the peak of fungal GA7 that coincides with that of internal standard GA7.Click here for file5 in culture filtrate of fungal isolate IR-3-3GC-MS SIM spectra for GA. Arrow indicates the peak of fungal GA5 that coincides with that of internal standard GA5.Click here for file5 in culture filtrate of wild type G. fujikuroiGC-MS SIM spectra for GA. Arrow indicates the peak of fungal GA5 that coincides with that of internal standard GA5.Click here for file


Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs. A key event in regulating gene expression involves changes in the acetylation status of core histones. Regulation is accomplished by a balance between the addition of acetyl groups by histone acetyltransferase enzymes and removal of the moieties by deacetylases. These changes are essential in regulating cellular differentiation and proliferation and, likewise, disruption results in a variety of pathologies, including cancer. In addition, these key regulators are targeted by herpesviruses to ensure persistent infection during the life of the host. In the case of the herpesvirus human cytomegalovirus (HCMV), changes in histone acetylation have been implicated in the choice between latent and acute phases of infection. We have used a focused proteomics approach to identify proteins that are interacting with and regulating the histone deacetylase 1 (HDAC1) protein during acute cytomegalovirus infection. Our studies identified numerous cellular and viral proteins including HCMV pUL29/28. This protein bound to components of the nucleosome remodeling and deacetylase complex, NuRD, and functional NuRD components were necessary for HCMV gene expression and infection. Our study demonstrates a new tool for studying host-pathogen interactions as well as provides new insights into the complex regulation of HDAC1 during HCMV replication. Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that causes life threatening disease in immunocompromised adults, specifically individuals undergoing solid organ or hematopoietic cell transplant and individuals with Acquired Immunodeficiency Syndrome (AIDS) The lytic HCMV replication cycle proceeds through a highly coordinated series of events. At the very start of infection, cellular defenses are inhibited and viral immediate-early gene expression is facilitated by proteins and RNAs that are delivered to cells as constituents of virions Upon entry, the HCMV genome rapidly becomes associated with cellular histones nucleosome remodeling and deacetylase (NuRD) metastasis-associated (MTA) protein family. The histone demethylase, LSD1, has also been found within the NuRD complex HDACs have been indentified within multi-protein complexes. HDAC1 is found in the Regulation of HDACs plays important roles in viral replication and pathogenesis. In HCMV, the virus-coded IE2 protein interacts with HDAC1 We recently reported that constituents of the NuRD complex can be captured from infected cell extracts with a tagged HCMV UL38-coded protein at 24 hpi 2 at 37°C. Primary human foreskin fibroblast  All cells were propagated in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen) in 5% COwt, was obtained from an infectious bacterial artificial chromosome clone of the AD169 strain, termed pAD/Cre inUL29F and BADinUL38Tap viruses as well as the pUL28-, pUL29- and pUL38-deficient mutants, BADsubUL28, BADsubUL29 and BADsubUL38, have been previously described subUL38 which was propagated on pUL38-expressing fibroblasts 2). Viral titers were determined by using an immunofluorescence assay to quantify pUL123 (IE1)-expressing cells et al.Wild-type HCMV, BAD5′-ATAGAATTCAAGCTTGCCACCATGGCGCAGACGCAGGGC-3′ and 5′-CCTTGCTCACGGCCAACTTGACCTCCTC-3′) from an expression plasmid kindly provided by E. Seto (University of South Florida). PCR amplification of gfp used an enhanced GFP expression plasmid (Clontech) (primers: 5′-CTCCTCCAGTTCAACCGGGTGAGCAAGGGCGAGGAGCTG-3′ and 5′-TTAAGATCTTACTTGTACAGCTCGTCCA-3′) (Integrated DNA Technologies). The PCR products were combined, amplified using the terminal primers to each gene, and introduced into pLXSN using the restriction enzymes, EcoRI and BamHI. The pUL38 expression vector, pLXSN-UL38, was produced by PCR amplification of the UL38 gene using HCMV genomic DNA (primers: 5′-ATAGAATTCGCCACCATGACTACGACCACGCATAG-3′ and 5′-ATACTCGAGCTAGACCACGACCACCATCTG-3′) and introduced into pLXSN. The myc-tagged pUL29/28 expression vector, pLXSN-UL29/28myc, was constructed by PCR amplification UL29/28 from cDNA produced from RNA isolated at 6 hpi 5′-ATAGTTAACGCCACCATGGAACAAAAGCTAATATCAGAAGAAGACCTATCCGGCCGTCGCAAG-3′ and 5′-ATAGGATCCTCACGACGCCCCCGTGC-3′). Additional plasmids used in these studies have been described elsewhere, including pCGN-UL29/28 and pCGN-UL29 The identification of HDAC1-interacting proteins used fibroblasts (HF-HD1gfp cells) stably expressing an HDAC1-enhanced green fluorescent protein (gfp) fusion protein, in which the gfp was fused at the C-terminal end of the cell protein. These cells were generated by transduction using a retrovirus produced from pLXSN-HDAC1gfp and a pure population of GFP-positive cells was isolated by using a FACSVantage flow cytometer (BD Biosciences). pLXSN-HDAC1gfp was constructed by PCR amplification using Expand High Fidelity PCR System (Roche) of the HDAC1 gene  Tween-20, 1 µM ZnCl2, 1 µM CaCl2, 0.5% Triton-X100, 250 mM NaCl, 1/100 protease inhibitor cocktail (Sigma)), homogenized, and insoluble material was removed by centrifugation. The supernatant was used for affinity purification by incubation with 6 mg magnetic beads pre-conjugated with anti-GFP antibodies for 1 h at 4°C, as described 4OH, 0.5 mM EDTA solution, resolved by 1-D gel electrophoresis on a 4–12% NuPAGE Novex Bis-Tris gel (Invitrogen), and stained with Coomassie Blue (Pierce).HF-HD1gfp cells were grown to ∼70% confluency, infected with BADwt.As a control , an equiPLUS, Thermo Finnigan) Homo sapiens (152010 sequences) and Viruses (346953 sequences) species using the XProteo (http://www.xproteo.com) algorithm, as described Gel lanes were cut into 25 sections, diced, protein was digested with trypsin (Promega), and the resulting peptides were concentrated on reverse phase resin  and eluted onto a MALDI (matrix assisted laser desorption ionization) target as described Immunoprecipitation experiments were performed as previously described N-lauroylsarcosine, and protease inhibitors [Roche]; and then cells were disrupted and DNA was fragmented by sonication. Aliquots of lysate containing 3 µg of viral DNA were diluted 1∶10 in 0.01% sodium dodecyl sulfate [SDS], 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.0], 167 mM NaCl, and protease inhibitors and incubated with antibody overnight at 4°C. The immunocomplexes were captured with protein G-agarose, eluted from the beads at room temperature with buffer containing 1% SDS and 100 mM NaHCO3, crosslinks were reversed by incubation at 65°C for 5 h, proteins were digested with proteinase K (40 µg/ml) for 2 h at 55°C, and DNA recovered by using QiaQuick purification columns (Qiagen) and assayed by quantitative PCR (qPCR) with a Power SYBR green PCR kit (Applied Biosystems) and a primer pair specific for the HCMV MIEP (5′-AACAGCGTGGATGGCGTCTCC-3′ and 5′-GGCACCAAAATCAACGGGACTTT-3′). The efficiency of amplification for the primers was determined by generating a standard curve with 10-fold serial dilutions of a known concentration of viral DNA. The slope values of the standard curve for the primer pair amplicons ranged from −3.5 to −3.2, indicating 90 to 100% efficiency. Dissociation curves were performed to ensure that the primers generated a single amplification product, a template control was included, and each sample was run in duplicate.Chromatin immunoprecipitation (ChIP) analysis was performed as previously described For immunofluorescent analysis, fibroblasts were grown to confluence on glass coverslips, infected with HCMV at a multiplicity of 0.01 PFU/cell and fixed for 15 min in 2% paraformaldehyde at 24 hpi. Slides were treated for 15 min in 0.1% Triton X-100, washed with PBST (PBS containing 0.2% Tween 20), and incubated for 30 min in PBS-blocking buffer . Proteins were identified by incubating slides with the indicated primary antibody in PBS blocking buffer for 1 h at room temperature. After further washing with PBST, slides were incubated for 30 min using a secondary conjugated to Alexa 488 or 546 (Invitrogen). Cells were washed with PBST, mounted and viewed using a Zeiss LSM510 confocal microscope (Carl Zeiss). Quantitation of protein colocalization was performed using the Velocity software package (Improvision).Antibodies used in these studies were: mouse anti-HDAC1 IgG1 (Millipore), rabbit anti-HDAC1 polyclonal (Millipore), rabbit anti-MTA2 polyclonal (Santa Cruz), mouse anti-FLAG M2 IgG1 (Sigma), mouse anti-Myc IgG1(Millipore), rabbit anti-mSin3A polyclonal (Santa Cruz), rabbit anti-TSC2 polyclonal (Santa Cruz), mouse anti-HA IgG1 (Sigma), mouse anti-tubulin IgG1 (Sigma), rabbit anti-protein A (Sigma), mouse anti-IE1 (clone 1B12) 5′-ATAGCCAGTGTGCAGCTCCCTAAT-3′ and 5′-AACGGGCCCTGTTTACTTCTCCTT-3′; set 2: 5′-AGGAAGGCTATGGGCTTTCTTGGA-3′ and 5′-TGCCGTATGCCCTGTAAAGATGGT-3′) and CHD4 , and firefly luciferase gene (5′-ATTTATCGGAGTTGCAGTTGCGCC-3′ and 5′-GCTGCGAAATGCCCATACTGTTGA-3′). PCR quantitation was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems). Quantities for unknown samples were defined relative to a standard curve consisting of 10-fold serial dilutions of a single sample and completed for each primer pair. The results were normalized to either β-actin DNA or GAPDH cDNA levels within each sample.RNA levels in infected cells were determined by quantitative real-time reverse transcription-PCR (qRT-PCR) as previously described 5′-CCGGGCTGACACAGTTATTATCTATCTCGAGATAGATAATAACTGTGTCAGCTTTTT-3′) or RBBP4 (5′-CCGGCCCTTGTATCATCGCAACAAACTCGAGTTTGTTGCGATGATACAAGGGTTTTTG-3′). The control shRNA consisted of a scrambled sequence (5′-TCAGTCGCGTTTGCGACTGGTTCAAGAGACCAGTCGCAAACGCGACTGTTTTTGGAAAC-3′) and was a kind gift from P. Traktman . Recombinant lentiviruses were produced by co-transfecting 293T cells with lentivirus expression plasmid and packaging plasmids Disruption of cellular CHD4 and RBBP4 expression utilized short hairpin RNAs (shRNA) stably expressed in primary human fibroblasts or U-2 OS cells. The shRNA clones were expressed from the lentivirus vector, pLKO.1-Puro (Sigma Mission Clones), containing an shRNA sequence against either CHD4  and a Victor3 luminometer (Perkin-Elmer). Activity was measured by using equal protein amounts within each lysate and normalized to the luciferase activity from empty vector. Expression of pUL29/28 and pUL38 was assayed by Western blot analysis using the same lysates.As discussed above, regulation of the viral chromatin state, including deacetylation of histones, plays a pivotal role in efficient HCMV infection. We were interested in determining what viral proteins might influence these events through interactions with HDACs. We focused on HDAC1, a 55 kDa nuclear protein. HDAC1 previously has been shown to influence herpesvirus replication as well as latency We constructed a retrovirus vector expressing the neomycin resistance gene and the HDAC1 open reading frame (ORF) with the enhanced green fluorescent protein (GFP) ORF fused at its C-terminus (pHDAC1gfp). For these studies, we used life-extended fibroblasts, which can be maintained for extended periods in culture and were demonstrated to support efficient HCMV replication Overexpression of HDAC proteins occurs in many cancers, and can affect cell proliferation wt). At 24 hpi, pHDAC1gfp and associated proteins were isolated from cell extracts by using one-step immunoaffinity purification on magnetic beads coated with antibodies to GFP potential HDAC1-interacting proteins.To identify cellular and viral proteins that interact with HDAC1, we infected HF-HD1GFP cells with wild-type HCMV  or UL29 (BADsubUL29) ORFs, both of which fail to express normal levels of immediate-early genes Because pUL29/28 interacts with both pUL38 and HDAC1 , we nextinUL29F and collected whole cell lysates during the course of HCMV infection beginning at 6 hpi. Using an antibody against HDAC1, both pUL29/28 and pUL38 were co-precipitated beginning at 6 hpi and at all times tested through 72 hpi  or pUL29/28-deficient (BADsubUL29) virus in the presence or absence of the HDAC antagonist, trichostatin A (TSA). Because we previously observed an abnormality in particle to PFU ratio for BADsubUL38 2-fold decrease in virus yield for BADsubUL29 and an ∼104-fold decrease for BADsubUL38, consistent with our previous observations It is well established that inhibition of HDACs during cytomegalovirus infection stimulates immediate-early gene expression wt or BADsubUL29 and quantified IE1 RNA accumulation  targeting two specific components of the complex that were detected in the pHDAC1gfp capture experiment: CHD4 and RBBP4 . This apTo test for a role of the NuRD subunits in HCMV replication, we infected fibroblasts expressing the scrambled control, CHD4, RBBP4 or both shRNAs with wild-type virus expressing a GFP marker gene at a multiplicity of 0.25 PFU/cell, and visualized GFP-positive cells 96 h later . The shRTo confirm that pUL29/28 and/or pUL38 influence the accumulation of IE1 by modulating activity of the MIEP, which controls expression of UL123 mRNA, and to test whether the promoter was controlled by the viral proteins outside the context of the viral genome, we performed assays using a reporter in which luciferase expression is controlled by the MIEP. We have previously demonstrated that pUL29/28 alone can activate the MIEP in HEK-293T cells cis-acting elements contribute to full MIEP induction during infection ISRE and NF-κB inUL29F virus, we infected primary fibroblasts and immunoprecipitated crosslinked DNA using an antibody to the MTA2 component of NuRD as well as to the FLAG epitope in pUL29/28. Both antibodies immunoprecipitated more MIEP DNA than a control antibody in three independent experiments  experiments. Using the eriments .We conclude that pUL29/28 works with components of the NuRD complex to activate the MIEP independently of pUL38, and both proteins interact with the MIEP during infection.Histone acetylation/deacetylation plays a pivotal role in the regulation of HCMV gene expression In addition to these HCMV proteins, we detected peptides corresponding to the viral kinase pUL97 in the HDAC1 capture experiment . AlthougWe have previously shown that pUL29/28 and pUL38 are needed for efficient HCMV replication Our data demonstrate that components of the NuRD complex, with which pUL29/28 interacts, are needed for efficient replication. This is in contrast to the consequences of drug-mediated, global HDAC inhibition during infection, which enhances immediate-early gene expression pUL38 interacts with NuRD components only in the presence of pUL29/28 ; and pULsnailwnt4brca1Snail and wnt4 are rapidly down regulated during HCMV infection et al. How does pUL29/28 modulate the NuRD complex to favor expression of the MIEP? The HCMV genome undergoes chromatin remodeling and changes in acetylation during the course of infection through unknown mechanisms Previously, we have demonstrated that HCMV pUL38 also interacts with and antagonizes the TSC complex during infection Table S1List of proteins isolated with HDAC1 at 24 h following infection with HCMV. Protein name, gi number, number of peptides detected in the MS spectra, sequence coverage (%), XProteo score (d'), and amino acid sequences of peptides confirmed by MALDI IT CID are shown for the isolated proteins. Only proteins confirmed by the CID fragmentation of at least two peptides were included in the table. M* indicates an oxidation at methionine; # indicates unique peptides in protein isoforms.(0.06 MB XLS)Click here for additional data file.Figure S1Probing for non-specific isolations due to interactions with the GFP tag or resin. Affinity purification of GFP were performed from fibroblasts expressing free GFP, infected with HCMV, and harvested at 24 hpi. Isolated proteins were resolved by 1-D gel electrophoresis on a 4–12% gradient gel, stained with Coomassie Blue, and identified by mass spectrometry.(0.42 MB TIF)Click here for additional data file.


Modifications of the histone amino-terminal tails affect access of regulatory factors and complexes to chromatin and thereby influence biological processes. Cancer cells are characterized by prominent epigenetic dysregulation, including histone modifications. However, the functional roles of the histone methyltransferases (HMT) in cancer remain unclear.p16 and RASSF1A were not activated in KD cells.We studied RNAi-based inhibition  of 2 different H3K9 HMTs, SUV39H1 and G9a. Knockdown of the 2 HMTs in PC3 cancer cell line markedly inhibited cell growth and caused profound morphological changes with loss of telomerase activity and shortened telomeres. SUV39H1 KD cells showed substantial increase in G2/M fraction. G9a KD cells showed increased DNA content (1.7-fold in 2 independent clones) compared with FACS analyses to control. Karyotype analyses showed that this was due to an increased number of chromosomes (from 61 to 102) in G9a KD cells compared to parental PC3. Intriguingly, we found abnormal centrosome morphology and number in about 25% of the G9a KD cells, while centrosomes were morphologically normal in control cells. Microarray analyses after KD of SUV39H1 or G9a showed very few genes up-regulated among the 39,000 genes. The silenced tumor-suppressor genes These data suggest that the 2 HMTs, SUV39H1 and G9a are required to perpetuate the malignant phenotype. Furthermore, G9a plays a critical role in regulating centrosome duplication presumably through chromatin structure rather than through affecting gene expression in cancer cells. Targeting these histone methyltransferases may be of therapeutic benefit in cancers. Chromatin assembly is a critical process related to DNA replication, gene expression and progression through the cell cycle. Specific modifications are associated with certain DNA template-mediated processes Regulation of higher-order chromosome structure affects mitotic fidelity and ensures balanced chromosome segregation. Heterochromatin assembly at centromeres facilitates both kinetochore formation and sister chromatid cohesion Cancer cells are characterized by prominent epigenetic dysregulation, including altered chromatin modification. Previously we found increased level of G9a in human cancers 2 at 37°C. Cells were split 12–24 h before 5-Aza-2′-deoxycytidine  treatment. Cells were then treated with either 1 µM of DAC, or PBS (control) daily for 3 days.The prostate cancer cell line PC3, the lung cancer cell line H1299 and the breast cancer cell line MCF7 were obtained from the American Type Culture Collection . They were grown in RPMI medium , plus 10% FBS in plastic tissue culture plates in a humidified atmosphere containing 5% CO5′-AAGATTGAGCCTCCGCTGATT-3′ and 5′-AATCTCAAGTGTGTGCGTATC-3′ for G9a and SUV39H1, respectively). As a control, we used shRNAs for Luciferase (Luc) synthesized by BD Biosciences or shRNA vector without hairpin oligonucleotides.We designed a retrovirus vector  encoding a small hairpin RNA (shRNA) directed against G9a and SUV39H1 in PC3 (target sequences of ChIP assays were performed based on a modification of previously published methods Immunoblotting was performed with primary antibodies for anti-G9a (Abcam), anti-SUV39H1, anti-K9 dimethylated histone H3, anti-K9 trimethylated histone H3 (Upstate Biotechnology), histone H3 (Abcam) and ß-actin (Sigma).Total RNA was isolated using TRIzol (Invitrogen), and 2 µg was reverse transcribed with MLV-reverse transcriptase (Invitrogen). The primer sequences used are described in We performed bisulfite treatment as reported previously PC3 cells infected with retrovirus encoding G9a-shRNA, SUV39H1-shRNA or control-shRNA were examined for SA-ß-gal activity as previously described Total RNA was extracted from cells infected with either G9a-shRNA, SUV39H1-shRNA or control-shRNA as described above. Targets for microarray hybridization were generated from the RNA according to the manufacturer's instructions . The human U133A gene chip (Affymetrix) which contains ∼33,000 transcripts was used for gene expression profiling. Hybridization, washing, scanning, and analysis of gene chips were performed according to the manufacturer's instructions. Expression levels were analyzed by the statistical algorithm in the Microarray Analysis Suite (MAS) 5.0 software (Affymetrix) using the default parameters. The data from the control-shRNA treatment and parental PC3 were used as a baseline expression for comparison with either G9a-shRNA or SUV39H1-shRNA treated sample.Telomerase activity was analyzed by a modified method of the standard telomerase repeat amplification protocol (TRAP), the TRAPeze Telomerase Detection Kit  according to the manufacturer's instructions. Reaction products were run on a 15% native polyacrylamide gel and stained with ethidium bromide.G9a-shRNA, SUV39H1-shRNA or control-shRNA treated PC3 cells were grown on Falcon culture slides . Anti-CENP-A monoclonal antibody (Abcam), anti-K9 dimethylated histone H3 and anti-K9 trimethylated histone H3 polyclonal antibodies (Upstate Biotechnology) were used as primary antibodies. Centrosomes were detected with a γ-tubulin polyclonal antibody (Sigma) using a standard protocol G9a-shRNA, SUV39H1-shRNA or control-shRNA-treated PC3 cells were evaluated for telomere shortening using the Telo-FISH method to visualize telomeres in interphase cell populations, as described previously To evaluate the role of SUV39H1 and G9a in cancers, first we established clones of PC3, in which either of these 2 HMTs were stably downregulated by shRNA. We used PC3, since endogenous SUV39H1 and G9a were highly expressed in this cell. In these knockdown (KD) clones, expression of the 2 HMTs was reduced by 80–90% . KD cellBoth SUV-KD and G9a-KD showed markedly inhibited cell growth and profound morphological changes, enlarged and flattened shapes. . SenesceRASSF1A, which is a target of DNA methylation in PC3, and found that there was no effect on gene reactivation in either the G9a-KD or the SUV-KD cells. Given that DNA methylation, a crucial epigenetic mechanism for silencing tumor suppressor genes in human neoplasia, has been mechanistically linked to histone 2MeH3K9 methylation Induction of cellular senescence in response to stimuli depends primarily on the p53 or p16-pRB pathways Klotho and growth differentiation factor 8 (GDF8), were truly silenced in the control cells and up-regulated in both G9a-KD and SUV-KD cells (data not shown). Thus, very few genes are up-regulated after inhibition of the 2 HMTs in PC3. Although, G9a and SUV39H1 are generally involved in gene silencing, we found that 12 and 2 genes (2 genes were common) were down-regulated in the G9a-KD and the SUV-KD cells, respectively . Then we searched for genes with signal intensities increased more than 2 times after KD of either G9a or SUV39H1. After removal of self-inconsistent probes among the same gene, we could identify only 4 and 6 genes (3 genes are common) in the G9a-KD and the SUV-KD cells, respectively. We examined those 7 genes by RT-PCR and found that only 2 genes, ectively . Down-reP<0.01) than the SUV-KD cells (63 chromosomes) and control cells (65 chromosomes). This was true regardless of the cell line types used. Both stable G9a-KD clones of the breast cancer cell line MCF7 and the lung cancer cell line H1299 also showed an increased number of chromosomes compared to control cells  and the SUV-KD cells evidenced substantial increases in G2/M fraction (from 29% to 40%) compared to the control . ConsistCentrosomes are essential for proper cellular polarity and balanced distribution of chromosomes The chromatin structure at telomeres is also important to maintain the length of telomere repeats The current study demonstrates that expression of SUV39H1 and G9a is important to support the growth of malignant cells, although they play distinct roles in cancer cells. Unexpectedly, very few genes are up-regulated after inhibition of the 2 HMTs, suggesting that H3K9 methylation associated silencing, which has been mechanistically linked to DNA methylation Centromere mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint Recent reports demonstrated that centromeric chromatin specific combinations of histone modifications and the three-dimensional organization of chromosomes could also be important for recruitment of cohesion complexes to heterochromatin near sister kinetochores Centrosomes start to duplicate at the late G1/early S phase of the cell cycle, and two functional centrosomes are formed during G2 G9a appears to be required for hTERT expression and telomere maintenance. SUV39H1 is also required for control telomere regulation. In mouse model, embryonic fibroblast from mice null with both Suv39h1 and Suv39h2 showed abnormal telomere elongation Figure S1Downregulation of G9a methyltransferase in MCF7 and H1299 cell lines. A, Real-time PCR shows that shRNA interference effectively downregulates G9a in MCF7 and H1299. Error bars in real-time PCR indicate standard deviation of the expression in 3 independently isolated clones. B, FACS analyses showing DNA content is increased 1.5–2 times in the G9a-KD cells compared to the mock control in an isolated clone. Increased DNA content was observed in two independently-isolated clones (data not shown). C, karyotype analyses showed small and increased chromosomes in both MCF7-G9a-KD and H11299-G9a-KD cell lines. D, Centrosomes were detected using an antibody against γ-tubulin. In the control cells, 1–2 centrosomes appearing as point-like structures are seen. Abnormal centrosome morphology and number are observed in around 20% of the G9a-KD cells.(2.67 MB EPS)Click here for additional data file.Table S1Summary of the primers(0.04 MB DOC)Click here for additional data file.Table S2List of up-regulated and down-regulated genes in G9a-KD or SUV39H1-KD(0.05 MB DOC)Click here for additional data file.


Sema7A and plexinC1 expression in the developing rat nervous system.Although originally identified as embryonic axon guidance cues, semaphorins are now known to regulate multiple, distinct, processes crucial for neuronal network formation including axon growth and branching, dendritic morphology, and neuronal migration. Semaphorin7A (Sema7A), the only glycosylphosphatidylinositol-anchored semaphorin, promotes axon growth in vitro and is required for the proper growth of the mouse lateral olfactory tract in vivo. Sema7A has been postulated to signal through two unrelated receptors, an RGD-dependent α1β1-integrin and a member of the plexin family, plexinC1. β1-integrins underlie Sema7A-mediated axon growth and Sema7A function in the immune system. Sema7A-plexinC1 interactions have also been implicated in immune system function, but the neuronal role of this ligand-receptor pair remains to be explored. To gain further insight into the function(s) of Sema7A and plexinC1 during neural development, we present here a detailed analysis of Sema7A and plexinC1 in multiple neuronal systems including: the olfactory system, the hypothalamo-hypophysial system, the hippocampus, the meso-diencephalic dopamine system, and the spinal cord. Within these systems, Sema7A and plexinC1 are often expressed in specific neuronal subsets. In general, Sema7A transcript levels increase significantly towards adulthood, whereas plexinC1 expression decreases as development proceeds.In situ hybridization revealed select expression of PlexinC1, but not Sema7A, is strongly expressed by distinct populations of migrating neurons. In addition to neuronal expression, Sema7A and plexinC1 transcripts were detected in oligodendrocytes and ependymal cells, respectively.Sema7A and plexinC1 expression patterns are consistent with these proteins serving both cooperative and separate functions during neural development. The prominent expression of plexinC1 in several distinct populations of migrating neurons suggests a novel role for this plexin family member in neuronal migration. SEMA7A gene were recently found to be associated with bone mineral density and fracture risk in postmenopausal women -dCTP (Amersham Pharmacia Biotech) using the Prime-It II random labelling kit (Stratagene) and membranes were hybridized following standard procedures. A 362 base pair rat Sema7A cDNA probe was used to detect Sema7A [plexinC1 coding region), and then for GAPDH [Sema7A and plexinC1 probes do not cross-hybridize with the mRNA of other semaphorins and plexins, respectively.Northern blot analysis was performed as described before . In brieed with 3P-dCTP , mouse plexinC1; 5'-tggtggtgacgaggtacaaa-3' and 5'-tactgcactgctccatcagg-3' (468 bp), and rat GAPDH; 5'-ctcatgaccacagtccatgc-3' and 5'-atgtaggccatgaggtccac-3' (473 bp). The PCR products were run in an agarose gel and visualized with ethidium bromide. PCR amplification specificity was confirmed by sequencing.Total RNA was isolated from E15, E19, P0, P14 or adult brain using TRIzol reagent (GibcoBRL). RT-PCR was performed as described before using primers specific for  control . PCR conSema7A and plexinC1 in tissue sections. Non-radioactive in situ hybridization was performed using alkali-hydrolyzed digoxigenin-labelled cRNA probes transcribed from rat Sema7A (a 362 base pair fragment corresponding to nucleotides 364–726 of coding region) [plexinC1 (a ~2.25 kb fragment encoded by EST AA940270) [tyrosine hydroxylase  [Sema7A, plexinC1 and TH. In addition, we have performed experiments using a mouse Sema7A probe [Although antibodies have been raised against mouse Sema7A and plexinC1 ,17, thes region)  cDNAs. T region) . SectionSema7A or plexinC1 and immunohistochemistry for TH was performed as described previously [Immunohistochemistry was performed on cryosections as described previously . In brieeviously  using poRJP carried out the experiments for figures Sema7A and plexinC1 expression in the embryonic meso-diencephalic system. Description: In situ hybridization for TH, Sema7A, plexinC1 and sense control on consecutive coronal sections of the E15 meso-diencephalic dopamine (mdDA) system. (A) TH labels mdDA neurons. (A-C) Whereas no significant Sema7A expression is detected within the area of TH labelling, plexinC1 and TH expression clearly overlaps. (D) The sense control shows no specific labelling. Thus, at E15 plexinC1 but not Sema7A is expressed by mdDA neurons. Scale bar 60 μm (A-D).Click here for fileSema7A and plexinC1 expression in the embryonic and adult cerebellum. Description: In situ hybridization for Sema7A and plexinC1 on consecutive sagittal sections of the E19  and adult cerebellum .  Sema7A is widely expressed throughout the developing cerebellum, whereas plexinC1 signals are enriched in the rhombic lip (arrow) and external granule cell layer (EGL).  In the adult cerebellum, Sema7A is expressed in Purkinje cells (P) and granule cells, whereas plexinC1 only labels few cells in the granule cell layer (GCL). Scale bar 150 μm , and 245 μm .Click here for file


During untreated, chronic HIV-1 infection, plasma viral load (VL) is a relatively stable quantitative trait that has clinical and epidemiological implications. Immunogenetic research has established various human genetic factors, especially human leukocyte antigen (HLA) variants, as independent determinants of VL set-point.p<0.01) but not in SCs. In contrast, favorable HLA variants, mainly A*74, B*13, B*57 (or Cw*18), and one HLA-A and HLA-C combination (A*30+Cw*03), dominated in SCs; their independent associations with low VL were reflected in regression beta estimates that ranged from −0.47±0.23 to −0.92±0.32 log10 in SCs (p<0.05). Except for Cw*18, all favorable variants had diminishing or vanishing association with VL in SPs (p≤0.86).To identify and clarify HLA alleles that are associated with either transient or durable immune control of HIV-1 infection, we evaluated the relationships of HLA class I and class II alleles with VL among 563 seroprevalent Zambians (SPs) who were seropositive at enrollment and 221 seroconverters (SCs) who became seropositive during quarterly follow-up visits. After statistical adjustments for non-genetic factors (sex and age), two unfavorable alleles (A*3601 and DRB1*0102) were independently associated with high VL in SPs (Overall, each of the three HLA class I genes had at least one allele that might contribute to effective immune control, especially during the early course of HIV-1 infection. These observations can provide a useful framework for ongoing analyses of viral mutations induced by protective immune responses. Polymorphic human leukocyte antigen (HLA) molecules facilitate immune surveillance by presenting a wide spectrum of self and foreign antigens to T-cells and by serving as ligands for killer immunoglobulin-like receptors (KIRs) on natural killer (NK) cells. The extensive HLA allelic diversity + T-cell depletion) Differential impact of HLA alleles on HIV-1 viral load (VL) was first observed among seroconverted men in the Multicenter AIDS Cohort Study In our own work, several HLA alleles and haplotypes have been associated with low (favorable) or high (unfavorable) VL in adult Zambians predominantly infected with HIV-1 clade C (HIV-1C) viruses 10 VL (p≥0.12). Between the two patient groups, statistically significant (p≤0.05) differences were seen for sex ratio, age, and the distribution of three VL categories previously shown to be highly predictive of viral transmission potential 10 VL was well within the boundary (0.30 log10) of biological and epidemiological relevance For this study, 784 HIV-1 seropositive Zambian adults consisted of 563 SPs who were seropositive at enrollment and 221 SCs who became seropositive during quarterly follow-up visits Table 1.HLA-A, HLA-B, and HLA-C) resolved most alleles to their 4-digit (high resolution) designations while leaving some at their 2-digit  specificities. HLA class I allele groups with multiple common alleles were limited to A*02, A*30, A*68, B*14, B*15, B*42, B*44, B*57, B*58, Cw*02, Cw*03, and Cw*07. The overall frequencies of HLA class I alleles and haplotypes  in the study population were highly comparable to those seen in 429 discordant couples  analyzed previously for heterosexual HIV-1 transmission For the 784 HIV-1 seropositive Zambians, molecular genotyping for the three HLA class I genes (Table S1). Correlation coefficients (r) in pairwise LD tests ranged from 0.17 to 0.97 . Only two pairs of alleles, B*39 with Cw*12 plus B*42 with Cw*17, were considered as tagging for each other . Accordingly, the vast majority of individual HLA class I alleles required independent testing in association analyses. Three common HLA class I combinations, A*23+B*14, A*23+Cw*07, and A*30+Cw*03, were also tested selectively because they were considered as probable haplotypes associated with HIV-1 VL in Zambians Tests of linkage disequilibrium (LD) among HLA class I alleles focused on common allele groups seen in at least 16 individuals (∼2% of the study population). A total of 32 common, 2-locus haplotypes were identified  among alleles at these two neighboring class II loci allowed unambiguous assignment of 2-locus haplotypes in these subjects , leading to the identification of 19 common haplotyes  (Table S1). Only the neighboring alleles DRB1*0302 and DQB1*0402 were in sufficient LD to tag each other . One common haplotype, DRB1*1301-DQB1*0501, appeared to be unique to the Zambian population. The frequency of the haplotype DRB1*1101-DQB1*0602 exceeded that of DRB1*1101-DQB1*0301 in Zambians; that excess has not been reported in other populations of African ancestry Alleles for two HLA class II genes, 10 VL and two contrasting VL categories (high versus low) as alternative outcome measures in generalized linear models (GLMs) and logistic regression models to screen the effects of common HLA alleles and haplotypes, including fully resolved HLA class I alleles  implicated in earlier studies of native Africans or African-Americans We used logHLA-A and HLA-C allele combinations were significantly associated with HIV-1 VL in this study population (p≤0.04) , were the most favorable alleles (mean β estimates ≤−0.34 log10); A*36 and DRB1*0102 were most unfavorable (mean β close to 0.30 log10 for both). These VL differences approached and usually exceeded the quantitative threshold  considered biologically and epidemiologically significant p = 0.08) was explained by its LD  with an established unfavorable allele B*45 (p = 0.05).In univariate GLMs, eight individual HLA variants and two (p≤0.04) Table 2.p = 0.13, 0.08, and 0.11, respectively). Among the seven associations supported by logistic regression models, A*36 and DRB1*0102 were among the most unfavorable, while Cw*18 and B*57 were among the most favorable. These seven associations were further confirmed in alternative GLMs, where the parameter estimates improved for all (Table S2). For all 10 HLA variants associated with VL using either outcome, their rankings based on respective beta estimates and on odds ratios had a strong correlation . However, the same analytic approach failed to confirm the associations of 16 HLA variants with HIV-1 disease control, as previously reported for populations of European and African ancestry (Table S3).Despite almost 43% reduction in the total sample size, the logistic regression models provided confirmatory findings for seven of 10 HLA variants detected in GLMs Table 2.p≤0.02 in univariate Cochran-Armitage trend test in each patient group) . Modest LD between A*29 and B*13 (r = 0.27) (Table S1) appeared to account for the association of A*29 in SCs (p = 0.05): in the absence of B*13, the association of A*29 was no longer statistically significant (p = 0.09).For separate analyses of SPs and SCs, we first assessed the relationships across three categories of HIV-1 VL. Three alleles  showed highly consistent effects on VL in both SPs and SCs (t group) Table 3.Table S3) Table S3). Only B*57, represented primarily by B*5703 in Zambians, showed both strong internal consistency (between SPs and SCs) and unequivocal external corroboration Among HLA class I alleles associated with HIV-1 VL in either SPs or SCs or both groups, A*36 and B*5802 were unfavorable, while A*29, A*74, B*57, and Cw*18 were favorable; all of these were consistent with our earlier observations based on a smaller number of Zambians 10 HIV-1 VL in both SPs and SCs (p≤0.05)  and favorable for B*8101, Cw*18, and A*30+Cw*03 . In addition, A*29 and A*74 were favorable only in SCs .By GLM, including statistical adjustments for the effects of sex and age on HIV-1 VL, only B*57 (favorable) and B*5802 (unfavorable) were associated with log(p≤0.05) Table 4.10) in VL were observed for only three most favorable HLA alleles  in SCs, their regression beta estimates ranged from −0.42±0.17 to −0.62±0.20 log10 (10 (a two-fold difference). Although strong LD between B*57 and Cw*18  would have predicted similar impact of these two alleles in both patient groups, the impact of B*57 appeared stronger in SCs. B*13  was clearly favorable in SCs , but its effect vanished in SPs .Biologically and epidemiologically meaningful differences (0.30 log20 log10 Table 4.Table S3) [The effect sizes of the unfavorable A*36 and B*5802 varied little between SPs and SCs. For B*45 and DRB1*0102, the unfavorable effect observed in SPs was absent in SCs Table 4.10, respectively, p<0.01 for both)  (10), and A*74 showed more modest impact (−0.20±0.08 log10). Cw*18 could replace B*57 and B*8101 as an independent factor (p<0.0001), but the association of B*57 and B*8101 was no longer statistically significant when the three variants were forced into the same model (results not shown).In a reduced multivariable model for SPs, A*36 and DRB1*0102 continued to show unfavorable association with VL (+0.25 and +0.38 logor both) Table 5,10 (∼3.0- to 8.3-fold) (p<0.05). These effects were ranked as B*13>B*57>A*74>(A*30+Cw*03) or B*13>A*74>Cw*18>(A*30+Cw*03) when B*57 was replaced Cw*18 in the reduced multivariable model. The adjusted effect sizes attributed to all five favorable HLA variants in aggregate exceeded the effect sizes for age and sex. In contrast to the model for SPs, a model for SCs that included both B*57 and Cw*18 indicated a non-significant association with Cw*18 (data not shown).In SCs, favorable HLA class I alleles and one 2-allele combination dominated the independent effects, with mean beta estimates ranging from −0.47 to −0.92 logHLA-A and HLA-B supertypes, including those that have been added or refined recently In further assessment of favorable and unfavorable HLA variants retained in the multivariable models Table 5,10 copy number) or as a categorical outcome measure in adult Zambians confirmed most of the favorable and unfavorable HLA associations that we previously reported Table S2). In resource-limited settings that require concentration on patients with extreme outcomes Our expanded analyses of VL as a quantitative trait  had even stronger associations, none of which could be explained by effects of their accompanying HLA-B and HLA-C alleles. Both alleles have been confirmed in recent analyses of African-Americans . Thus, although the influences of multiple HLA-B variants on HIV-1 pathogenesis and evolution have been more readily detected and confirmed, several HLA-A alleles can exert comparable effects.Plasma VL during untreated, chronic HIV-1 infection reflects an equilibrium between viral replication and immunologically mediated viral clearance. Population-based studies have clearly established the predictive value of VL for heterosexual HIV-1 transmission HLA-B and HLA-C genes are located in a genomic segment (the beta block) rarely disrupted by recombination hot spots HLA-C alleles (Cw*16 as unfavorable and Cw*18 as favorable) with VL in Zambians could be partially or completely explained by their strong LD with HLA-B alleles: B*45 for Cw*16 and B*57 plus B*8101 for Cw*18. However, as we noted earlier HLA-C alleles in HIV-1 infection, because none of these alleles individually had a clear association with VL. Assuming that alleles at HLA-C and HLA-A can act in concert to confer genetic advantage during HIV-1 infection (or disadvantage in other settings), differences in peptide-binding and in HLA-C allelic expression can offer two plausible explanations + T-helper (TH) cells. Their peptide-binding grooves can be loaded with protein degradation products (13–25 amino-acid residues) generated in the lysosome. CD4+ T-cells that effectively respond to HIV-1 antigens can facilitate immune control through cytokines and T-cell-dependent antibody responses, but activated TH cells, especially those specific to HIV-1 antigens, are lost rapidly because they are preferentially targeted by the virus + T-cells in patients with chronic HIV-1C infection + T-cells. The largely inconclusive findings for HLA class II alleles and haplotypes might reflect their promiscuity in antigen presentation.HLA class II molecules specialize in presenting exogenous antigens for immune surveillance by CD4Within the context of HIV-1C infection, the Zambian cohort has produced some of the first evidence that HLA factors can mediate HIV-1 VL as well as heterosexual transmission 10 value (a continuous variable), VL differences greater than 0.30 log10 (∼2-fold) are often considered biologically and epidemiologically significant gag variants When expressed as a logThis study complied with the human experimentation guidelines of the United States Department of Health and Human Services, and all enrolled patients provided written informed consent. The work presented here was further approved by the Institutional Review Boards at University of Alabama at Birmingham, under Protocols X051108005 and X071119002.Beginning in 1995, HIV-1 seropositive Zambians were recruited and followed by the Rwanda/Zambia HIV-1 Research Group in Lusaka, Zambia. Procedures for quarterly medical examination, voluntary counseling and testing, and viral sequencing have been described elsewhere HLA-B and HLA-C alleles because of their known, strong linkage disequilibrium (LD) in the study population HLA-DRB1 and HLA-DQB1 alleles in the same way and for the same reason r). In a given individual, simultaneous assignments of two haplotypes with a statistical probability ≥70% by the EM algorithm were considered reliable. Individuals with unreliable haplotype assignments (probabilities <70%) were excluded from formal testing of haplotypic associations.Using the expectation-maximization (EM) algorithm in SAS Genetics , common HLA class I B-C haplotypes were assigned for t-test , and χ2 test .The overall characteristics of HIV-1 seroprevalent Zambians (SPs) and seroconverters (SCs) with complete HLA genotyping were summarized in http://www.graphpad.com/prism/Prism.htm). Summary statistics in association analyses included: 1) proportional odds ratio (pOR) and 95% confidence intervals (CIs); 2) regression beta estimates, expressed as means and standard errors (SE), and 3) univariate and multivariable (adjusted) p values. A p value≤0.050 was considered statistically significant because most HLA alleles and haplotypes highlighted in this work have been implicated in earlier studies. The overall emphasis was on biologically and epidemiologically significant (>0.30 log10) differences in VL The statistical approach to association analysis followed the strategies established in other related work Table S1HLA class I and class II alleles and haplotypes found in at least 16 out of 784 adult Zambians.(0.04 MB DOC)Click here for additional data file.Table S210 HIV-1 viral load (VL) based on alternative generalized linear models.HLA variants associated with log(0.05 MB DOC)Click here for additional data file.Table S3Dismissal of 16 candidate HLA variants reported in earlier studies and common in Zambians.(0.07 MB DOC)Click here for additional data file.


Thiophenes and mono-aromatics, especially the methylated species, are strongly enriched in fluids emissions related to hydrothermal systems. Addition of hydrogen sulphide to dienes and electrophilic methylation involving halogenated radicals may be invoked for the formation of these species. On the contrary, the formation of furans, with the only exception of C4H8O, seems to be favoured at oxidizing conditions and relatively high temperatures, although mechanisms similar to those hypothesized for the production of thiophenes can be suggested. Such thermodynamic features are typical of fluid reservoirs feeding high-temperature thermal discharges of volcanoes characterised by strong degassing activity, which are likely affected by conspicuous contribution from a magmatic source. The composition of heteroaromatics in fluids naturally discharged from active volcanoes and geothermal areas can then be considered largely dependent on the interplay between hydrothermal vs. magmatic contributions. This implies that they can be used as useful geochemical tools to be successfully applied in both volcanic monitoring and geothermal prospection.The composition of non-methane organic volatile compounds (VOCs) determined in 139 thermal gas discharges from 18 different geothermal and volcanic systems in Italy and Latin America, consists of C On the contrary, little attention was devoted to heavier organic compounds for similar purposes.Chemical constituents released from gas discharges in active and quiescent volcanic complexes and geothermal systems can be related to: 1) primary (magma degassing) and 2) secondary  sources. The ratio between magmatic eillance . Magma d and CH4 ,6. Magmaally SO2 –9. Seconally SO2 ,11, are ally SO2 –15. ThesIn the present work, 139 gas emissions from active volcanoes and geothermal systems set in different geodynamical environments were analysed for the determination of non-methane VOC (Volatile Organic Compound) composition, especially that of heteroaromatic and aromatic compounds. On the basis of this dataset, the main goals were to: 1) assess the origin of thiophenes and furans in naturally discharged fluids and 2) evaluate the possible use of these compounds as geochemical tracers to discriminate different fluid source regions in volcanic-hydrothermal environment.2.2.1.2 and characterised by variable amounts of SO2 (from 1.2 to 569394 μmol/mol).A representative composition of the main gas components in fluid discharges from the volcanic and geothermal systems investigated in the present study is listed in 4 (<690 μmol/mol) and high concentrations of HCl (from 351 to 74540 μmol/mol) and H2 (up to 32591 μmol/mol), indicate that the gas chemistry of these systems is strongly controlled by magma degassing [2 below the instrumental detection limit (≈0.01μmol/mol: samples #48–138), relatively low outlet temperatures <118 °C, high CH4 concentrations (up to 64103 μmol/mol), and HCl not exceeding 500 μmol/mol.Such features, coupled with relatively low concentrations of CHegassing . This hyegassing –20 that egassing –23 (sampegassing –31 (samp2.2.Cl-bearing (13 compounds), O-bearing  and heteroaromatic (7 compounds) groups, were determined (M gases) are relatively low (from 61 to 1664 ppbv), whereas they range from 180 to 1235942 ppbv (H gases). Hydrothermal reservoirs, even when associated with volcanic systems, are commonly recharged by fluids circulating within organic-bearing sedimentary rocks. This organic source is then transformed into VOCs through biogenic and thermogenic processes [H gases are expected. On the contrary, the organic-rich component constitutes a minor fraction of the M gases, since it is generally destroyed by high-temperature, oxidizing fluids released from the magmatic melts [M and H gases: in the M gases, alkane, Cl-bearing, aromatic, heteroaromatic and alkene compounds are present in almost comparable amounts , whereas cyclic and O-bearing species represent a small VOC fraction (<0.04%)  with minor cyclic, alkene, heteroaromatic and O-bearing compounds (from 0.7 to 1.4%), and traces (<0.04%) of Cl-bearing compounds , aromatic (21 compounds), cyclic (17 compounds), alkene (15 compounds), 942 ppbv  in thoserocesses . Therefo(<0.04%) . The orgompounds . These e species ,38. Pyro species ,40, wher species . The pre species  or, alte species ,45. Conv species ,47.2.3.4H4O, 3-C5H6O, C4H8O, 2-C5H10O, C4H4S, 3-C5H6S and 2,5-C6H8S, and those of the simplest aromatics (C6H6 and C7H8), are reported in 4H4S is largely the most abundant S-bearing compound (up to 1.9 ppbv), whereas C4H4O (up to 31 ppbv) dominates the furan composition. Conversely, the H gases have relatively high concentrations of C4H4S and 3-C5H6S , minor 2,4-C6H8S (up to 13 ppbv), and no furans, with the only exception of those from Deception, Nisyros, Vesuvio, Copahue and El Chichon volcanoes. As shown in 2S,  of Con phase ,49. Moreon phase ,51. AccoMt. Etna .4+ alcohols or carbonyls with CS2 over alkali-promoted alumina; 2) reaction of unsaturated aldehydes with H2S over an alkali-promoted alumina; 3) reaction of C4+ alkyl hydrocarbons or olefins with CS2, S, and H2S over alkali-promoted alumina; 4) catalytic dehydrogenation of tetrahydrothiophene; 5) synthesis from furan and H2S over alumina [2S as sulphurising agent [2S to dienes in the presence of H+ and metal catalysts  reaction of C alumina –56. The ng agent . Generalatalysts .M gases heteroaromatics and inorganic sulphur-bearing gases, the latter being constituted by SO2 and H2S at comparable concentrations  at hydrothermal conditions mono-aromatics and thiophenes are efficiently produced by similar genetic processes; 2) all these compounds have a similar behaviour in response to thermodynamic conditions caused by presence of oxidizing and high temperature (>400 °C) magmatic fluids.The C 3-C5H6S  and C6H6H6S , whose formation likely occurs in both geothermal and volcanic fluid reservoirs where halogenated species are abundant [4H4S is the only methyl-thiophene recognized in both geothermal and volcanic gases  occurring in the M gases , typical of hydrothermal environments, tend to favour the consumption of C4H4O to produce C4H8O through catalytic hydrogenation  fumarolic vents and 2) boiling pools, respectively. During sampling, CO2, SO2 and HCl dissolved into the alkaline solution, water vapour condensed, and H2S reacted with Cd2+ to form insoluble CdS, allowing the residual gases  to be concentrated in the head-space [®).Gas samples for the determination of the main gas species were collected into pre-evacuated 60 mL glass flasks filled with 20 mL of a 4N NaOH and 0.15 M Cd(OH)ad-space –64. Gas 3.2.2 and H2 were analysed with a Shimadzu 15A gas-chromatograph equipped with Thermal Conductivity Detector (TCD) and a 9 m, 5A molecular sieve column. Methane and C1–C4 alkanes and alkenes were analysed with a Shimadzu 14a gas-chromatograph equipped with Flame Ionization Detector (FID) and a 10 m long stainless steel column (φ = 2 mm) packed with Chromosorb PAW 80/100 mesh coated with 23% SP 1700. The alkaline solution, separated from the solid precipitate by centrifugation at 4,000 rpm for 30 min, was used for the determination of: 1) CO2 as CO32− by titration with 0.5 N HCl solution; 2) SO2 as SO42−, after oxidation with H2O2, by ion-chromatography (Metrohm Compact 761); 3) HCl, as Cl− by ion-chromatography. The solid precipitate was oxidized by H2O2 to determine H2S as SO42− by ion-chromatography [Nitrogen, Ar, Otography ,64. The m/z. The transfer-line temperature was set at 230 °C. The mass detector was equipped with EI set at 70 eV. The source temperature was 250 °C. The gas chromatograph was equipped with a split/splitless injection port operating in the splitless mode with a dedicated SPME liner (0.75 mm i.d.). Analytes were desorbed from the SPME fiber through direct exposure for 2 min in the GC injection port, heated at 230 °C. The chromatographic column was a 30 m × 0.25 mm i.d. 0.25 μm film thickness TV1-MS fused silica capillary column (Thermo). The carrier gas was helium set to a flow-rate of 1.3 mL/min in constant pressure mode. The column oven temperature program was the following: 35 °C (hold 10 min), rate 5.5 °C/min to 180 °C (hold 3 min), rate 20 °C/min to 230 °C (hold 6 min) [The VOCs were pre-concentrated and transferred from the sampling vials into the column headspace of a Thermo Trace GC Ultra gas chromatograph by using a manual SPME (solid-phase micro-extraction) device introduced through the silicon membrane of the glass vial to expose the gas mixtures to a divinylbenzene (DVB)-Carboxen-polydimethylsiloxane (PDMS), 50/30 μm, 2 cm long fibre assembly  for 15 min . The used 6 min) . Compouni.e., alkanes, alkenes, aromatics, cyclics, chlorofluorocarbons, ketones, aldehydes and heteroaromatics. The values of the Relative Standard Deviation (RDS), calculated from seven replicate analyses of a gaseous mixture in which the compounds of interest were present at a concentration of 2 ppmv, are <7%. Eventually, the detection limits were determined by linear extrapolation from the lowest standard in the calibration curve using the area of a peak having a signal/noise ratio of 5 [The VOCs identified by mass spectrometry were quantified using an external standard calibration procedure performed on the basis of calibration curves created by analyzing gaseous standard mixtures of the main VOC groups, tio of 5 .4.4H4O seems to be favoured at highly acidic and oxidizing conditions that are determined by the presence of fluids from magmatic degassing. Methylated and hydrogenated heteroaromatics are also preferentially associated with hydrothermal conditions. According to these considerations, the composition of O- and S-bearing heteroaromatics can be utilized in both volcanic and geothermal systems to evaluate contributions of fluids produced in different “natural dominions”, i.e., hydrothermal and magmatic. These results may imply useful applications in volcanic monitoring and geothermal prospection, although the existing dataset should be expanded to better constrain the behaviour of these new geochemical tracers. Experimental runs, able to test the mechanisms of formation and stability of heteroaromatics at temperature, redox and catalytic conditions resembling those of a volcano-hydrothermal environment, would probably useful to better constrain their behaviour.The distribution of thiophenes and furans in gases from hydrothermal and magmatic-hydrothermal systems have been revealed to be strongly dependent on the physical-chemical conditions acting on fluid reservoirs, where VOCs are produced via a complex series of catalytic processes, involving organic matter buried in sedimentary formations. Thiophene seems to be efficiently produced at hydrothermal conditions and tend to be destroyed in presence of hot, highly oxidizing fluids from a magma source. On the contrary, the formation of C