Current production by non-methanotrophic bacteria enriched from an anaerobic methane-oxidizing microbial community.

In recent years, the externalization of electrons as part of respiratory metabolic processes has been discovered in many different bacteria and some archaea. Microbial extracellular electron transfer (EET) plays an important role in many anoxic natural or engineered ecosystems. In this study, an anaerobic methane-converting microbial community was investigated with regard to its potential to perform EET. At this point, it is not well-known if or how EET confers a competitive advantage to certain species in methane-converting communities. EET was investigated in a two-chamber electrochemical system, sparged with methane and with an applied potential of +400 mV versus standard hydrogen electrode. A biofilm developed on the working electrode and stable low-density current was produced, confirming that EET indeed did occur. The appearance and presence of redox centers at -140 to -160 mV and at -230 mV in the biofilm was confirmed by cyclic voltammetry scans. Metagenomic analysis and fluorescence in situ hybridization of the biofilm showed that the anaerobic methanotroph 'Candidatus Methanoperedens BLZ2' was a significant member of the biofilm community, but its relative abundance did not increase compared to the inoculum. On the contrary, the relative abundance of other members of the microbial community significantly increased (up to 720-fold, 7.2% of mapped reads), placing these microorganisms among the dominant species in the bioanode community. This group included Zoogloea sp., Dechloromonas sp., two members of the Bacteroidetes phylum, and the spirochete Leptonema sp. Genes encoding proteins putatively involved in EET were identified in Zoogloea sp., Dechloromonas sp. and one member of the Bacteroidetes phylum. We suggest that instead of methane, alternative carbon sources such as acetate were the substrate for EET. Hence, EET in a methane-driven chemolithoautotrophic microbial community seems a complex process in which interactions within the microbial community are driving extracellular electron transfer to the electrode.

Recommendations for the treatment of established fungal infections.

Evidence-based guidelines for the treatment of established fungal infections in the adult haematology/oncology setting were developed by a national consensus working group representing clinicians, pharmacists and microbiologists. These updated guidelines replace the previous guidelines published in the Internal Medicine Journal by Slavin et al. in 2004. The guidelines are pathogen-specific and cover the treatment of the most common fungal infections including candidiasis, aspergillosis, cryptococcosis, zygomycosis, fusariosis, scedosporiosis, and dermatophytosis. Recommendations are provided for management of refractory disease or salvage therapies, and special sites of infections such as the cerebral nervous system and the eye. Because of the widespread use newer broad-spectrum triazoles in prophylaxis and empiric therapy, these guidelines should be implemented in concert with the updated prophylaxis and empiric therapy guidelines published by this group.

Cryoplasty for arterial restenosis.

OBJECTIVE: A prospective follow-up study of patients with arterial restenosis undergoing cryoplasty. MATERIALS & METHODS: Between May 2004 and June 2005, 10 patients with restenosis following ilio-femoral endovascular treatment underwent twelve cryoplasty procedures. All patients had had at least one previous episode of stenosis treated by conventional endovascular methods and had suffered further restenosis. The indications for treatment were grafts at risk (n=5) and symptomatic in-stent restenosis (n=5). Two patients underwent re-cryoplasty. Cryoplasty was performed in accordance with manufacturer's instructions using 6-8mm balloons. All patients had Doppler ultrasound evaluation at 1, 3, 6 and 12 months. RESULTS: All procedures had angiographically successful immediate outcome with <30% residual stenosis. Non flow limiting dissection was evident in two cases. In six procedures (50%), restenosis was evident within 6 months post-procedure, whilst in the other six, there was progressive restenosis appearing between 6-12 months. Five cryoplasty procedures have needed endovascular re-intervention due to symptomatic high-grade restenosis and a sixth is awaiting surgery. CONCLUSION: Cryoplasty is of no value in patients with restenosis in the iliofemoral segment with half the procedures failing within six months and all of them within the first year. Evidence to support the use of cryoplasty in the peripheral arterial restenotic lesions is lacking.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands.

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.

The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.

The Mouse Genome Database (MGD): integrating biology with the genome.

The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.

The application of 1H HR-MAS NMR spectroscopy for the study of structures and associations of organic components at the solid-aqueous interface of a whole soil.

High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) allows the application of solution-state NMR experiments to samples that are not fully soluble and contain solids. Only the species in contact with the solvent system employed become NMR observable. In this study utilizing D2O as the solvent system we show it is possible to examine the structures at the solid-aqueous interface of a whole soil. Combining one- and two-dimensional HR-MAS NMR allowed, for the first time, the identification of fatty acids, aliphatic esters, and ethers/ alcohols as prominent species at the solid-aqueous interface of the soil with signals from sugars and amino acids also apparent. Few, if any signals from aromatic protons were observed when the soil was swollen in aqueous media, although these signals are observed in extracts from the same soil and when the soil is swollen with a more penetrating solvent(DMSO-d6)which is known to disassociate hydrogen bonds. These findings indicate that the soil aromatic moieties are protected in hydrophobic regions which are not water accessible. Furthermore, when the soil was amended with a herbicide (trifluralin), direct observations of interactions between the protons on a xenobiotic and the surrounding soil matrix were possible for the first time. HR-MAS promises to be a method that can be widely applicable to a range of complex environmental samples without the need for extraction, pretreatment, or purification.

Quality of life in HCV-infection: lack of association with ALT levels.

OBJECTIVES: To examine the impact of HCV infection in an Australian clinic population and identify the relationships between morbidity, psychosocial variables and one clinical indicator of HCV activity, alanine aminotransferase (ALT). METHOD: Ninety-five untreated HCV-infected patients (21-69 years) in infectious and liver diseases clinics who were all positive for HCV-RNA and had no significant comorbidities or coinfections completed a survey containing the Short Form 36 (SF36), as well as the six-item Social Support Questionnaire (SSQ6), demographic items and questions concerning respondents' perceptions of their mode and duration of infection. Nine volunteers from this group participated in semi-structured qualitative interviews aimed at exploring the social impact of HCV status. These data were compared with serum ALT levels. SF36 scores were compared to population norms and according to participant variables. RESULTS: Mean SF36 scores were significantly lower, across all modalities, than population norms. SF36 scores differed significantly according to age, sex, mode of infection, alcohol and methadone use, and satisfaction with social support. They did not differ significantly according to perceived or actual ALT level or pattern of ALT activity. Worry about ALT was prevalent (>50%) and this was independent of perceived ALT level. CONCLUSIONS AND IMPLICATIONS: HCV-infection is associated with significantly reduced quality of life and includes the perception of substantial social discrimination. ALT levels are of limited usefulness in ascertainment of a person's sense of wellbeing and quality of life in HCV-infection. Increased support and information for affected individuals and measures aimed at countering social discrimination are important recommendations of the current study.

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy.

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.

Expression of human endogenous retrovirus k envelope transcripts in human breast cancer.

PURPOSE: We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer. EXPERIMENTAL DESIGN: Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis. RESULTS: No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed >97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed. CONCLUSIONS: These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine.

Carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human adenocarcinomas has been a target for cancer immunotherapy. Limited information is available regarding the ability of patients to mount an antibody response to this self-antigen following vaccination. Recombinant vaccinia viruses encoding full-length or internally deleted cDNAs for human CEA were used to vaccinate 32 patients with CEA-expressing adenocarcinomas, predominantly of colorectal origin. CEA-specific autoantibodies were induced by vaccination in 7 of 32 patients. None of the patients had CEA antibodies detected before vaccination. CEA specificity of the antibodies initially identified by ELISA was confirmed by competitive inhibition analysis as well as recognition of recombinant CEA produced in baculovirus-infected insect cell cultures and human cell cultures by Western blot. The CEA autoantibodies were predominantly IgG1, with a minority of patients also demonstrating IgM autoantibodies. CEA antibodies were of low titer and low avidity, based on competitive inhibition assays. These autoantibodies did not affect clinical serum CEA protein quantitation. Furthermore, elevated serum CEA levels commonly encountered in patients with advanced adenocarcinoma did not hinder detection of low avidity polyclonal CEA antibodies. CEA antibodies such as those induced in these pilot trials are projected to have modest antitumor activity. Thus, additional Phase I/II trials of recombinant vaccinia-CEA with alternative prime-boost approaches and/or augmentation strategies are warranted in an effort to enhance the frequency and avidity of CEA-specific autoantibodies and cytolytic T cells before Phase III trials.

Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples.

Enzyme-linked immunosorbent assay (ELISA) data from surface water reconnaissance were compared to data from samples analyzed by gas chromatography for the pesticide residues cyanazine (2-[[4-chloro-6-(ethylamino)-1,3,5-triazin-2-yl]amino]-2-methylpropanenitrile) and metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide). When ELISA analyses were duplicated, cyanazine and metolachlor detection was found to have highly reproducible results; adjusted R2s were 0.97 and 0.94, respectively. When ELISA results for cyanazine were regressed against gas chromatography results, the models effectively predicted cyanazine concentrations from ELISA analyses (adjusted R2s ranging from 0.76 to 0.81). The intercepts and slopes for these models were not different from 0 and 1, respectively. This indicates that cyanazine analysis by ELISA is expected to give the same results as analysis by gas chromatography. However, regressing ELISA analyses for metolachlor against gas chromatography data provided more variable results (adjusted R2s ranged from 0.67 to 0.94). Regression models for metolachlor analyses had two of three intercepts that were not different from 0. Slopes for all metolachlor regression models were significantly different from 1. This indicates that as metolachlor concentrations increase, ELISA will over- or under-estimate metolachlor concentration, depending on the method of comparison. ELISA can be effectively used to detect cyanazine and metolachlor in surface water samples. However, when detections of metolachlor have significant consequences or implications it may be necessary to use other analytical methods.

A developmental bias in reading frame usage by human fetal thymic TCRBDJ transcripts is not present in genomic TCRBDJ rearrangements.

We have previously reported that reading-frame usage and functional diversification is developmentally regulated, with virtually all TCRB DJ mRNA transcripts using a single reading frame at 8 weeks of gestational age, tapering to 50% by adult life. We used the polymerase chain reaction to create genomic libraries of DJ rearrangements in the TCRB locus from thymuses at 7.7, 10, and 16 weeks of gestational age, and from adult thymuses. Clones were randomly picked and sequenced to determine junctional sequences and reading-frame utilization. The resulting data address the hypothesis that cells bearing genomic joints in reading frame one are preferentially selected during fetal life. This hypothesis predicts that reading-frame bias would also be observed among genomic DJ joints. Instead, we observed random utilization of the three possible D-region reading frames among genomic D1s1 ==> J1s1 joints during fetal life. Similar results were obtained at 7.7 weeks of gestational age in a second thymus in which both RNA and DNA were simultaneously isolated and used to create libraries of TCRBDJ transcripts or rearrangements. We conclude that reading-frame utilization is random among genomic D1s1-JB1s1 rearrangements and that the preferential usage of a single reading frame among mRNA transcripts of TCRB DJ transcripts is the result of preferential transcription of genomic TCRB DJ joints in a single reading frame, or that TCRB DJ transcripts have a longer half-life than transcripts in reading frames two or three.

Hepatitis C genotyping by direct sequencing of the product from the Roche AMPLICOR test: methodology and application to a South Australian population.

The Roche AMPLICOR RT-PCR amplifies a 244 nucleotide sequence within the 5' non coding region (5'NCR) of the viral genome and is a widely used commercial test for the qualitative determination of hepatitis C RNA from sera. This paper describes a routine procedure for the purification of the PCR product, and its use in automated DNA sequencing, for determining the genotype of hepatitis C virus (HCV) isolates. Direct sequencing of the purified product was possible for 86% of samples, whilst 14% required additional amplification using a nested PCR method in order to read the resulting electropherogram. This method of genotyping is considerably less expensive than currently available commercial kits, and is convenient for the increasing number of laboratories that have access to automated DNA sequencers. The highly conserved nature of the 5'NCR limited differentiation of types and subtypes to an extent comparable to commercial HCV typing methods. Using this method on available laboratory samples and on patients about to commence interferon therapy, we found a predominance of genotype 1 (59%) and 3a (31%). Analysis of data on the interferon patients showed the median length of time from first exposure to diagnosis to be significantly longer for patients with genotype 1 than genotype 3a.

Effects of phosphorothioate oligodeoxyribonucleotide and oligoribonucleotides on human complement and coagulation.

We have synthesized and studied the effects of phosphorothioate (PS) oligodeoxyribonucleotide (DNA) and oligoribonucleotides (RNA, 2'-O-methyl-RNA and 2'-5'-RNA) on complement activation and prolongation of activated partial thromboplastin time (aPTT) in vitro. These results suggest that a PS-DNA prolongs aPTT, and inhibits complement lysis more than do the PS-RNA analogs.

Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.

In the present study, using the C24 bile acid chenodeoxycholic acid as substrate, rat liver bile acid CoA ligase activity (rBAL) was purified 200-fold from detergent-solubilized microsomes using a combination of Q-Sepharose anion exchange, hydroxyapatite, and CM-Sepharose chromatography. Purified rBAL had a molecular weight of 65 kDa by SDS-PAGE analysis. Gel filtration of purified rBAL indicated that rBAL activity forms a complex with other proteins with an apparent aggregate molecular weight of 243 kDa. A monoclonal antibody raised against the 65-kDa protein and covalently coupled to 6B-Sepharose completely absorbed rBAL activity from a semipurified preparation of rat liver microsomes. Western blot analysis confirmed the elution of the 65-kDa protein from the affinity phase at low pH. Optimum rBAL activity was found at pH 8.5, and activity was dependent on the divalent cation Mg2+. In the presence of 50 microM CoA and 2.5 mM MgCl2, kinetic analysis revealed that the apparent K(m)s of ATP and chenodeoxycholic acid of the purified enzyme were 548 +/- 247 and 18.0 +/- 6.2 microM, respectively, and the apparent Vmax was 9.53 +/- 2.0 nmol min-1 mg protein-1. The formation of chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic bile acids (the C24 monohydroxy bile acid lithocholic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, the C27 homolog of cholic acid), but only weakly by cholic acid. Chenodeoxycholyl-CoA and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrospray ionization mass spectrometry.