Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation

Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uraniumcontaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments

Dynamique de la communauté microbienne sulfato-réductrice d'un drainage minier acide riche an arsenic ((Carnoulès, France))

Le phénomène de Drainage Minier Acide (DMA) se produit lorsque des minéraux sulfurés sont exposés à l air et à l eau. Ils vont être oxydés entraînant l acidification des eaux, le relargage de sulfates et la lixiviation de métaux/métalloïdes associés. La production biologique d H2S par les bactéries sulfato-réductrices (BSR) et la coprécipitation de métaux/métalloïdes sous forme de sulfures métalliques étant des solutions envisagées dans les systèmes de bioréhabilitation, il s agit de mieux comprendre le rôle des BSR dans le fonctionnement des DMA. Ce travail de recherche visait à étudier la diversité des BSR au sein du DMA de la mine de Carnoulès, contaminée en arsenic, de caractériser leur dynamique spatio-temporelle et de comprendre leur rôle dans les biotransformations de l arsenic. La mise au point d une méthode de PCR nichée des gènes codant pour la sulfite-réductase dissimilatrice (dsrAB) a permis la détection des BSR au sein de cet environnement. L outil développé a servi à étudier la dynamique de la communauté le long du DMA en combinant des approches moléculaires et de corréler leur distribution en fonction des variations physico-chimiques de l écosystème. L ensemble de ces travaux a notamment permis de mettre en évidence l influence du pH, du fer et des formes de l arsenic sur la dynamique des BSR. Une approche culturale a permis d établir leur rôle potentiel dans la mobilisation des contaminants. Ainsi, une souche appartenant à une espèce non décrite auparavant a été isolée. La capacité de la souche isolée à croître à des conditions acides et à des concentrations élevées en arsenic ont permis de mieux comprendre le rôle des BSR dans les environnements miniers.Identified as one of the major problems of the world mining industry, Acid Mine Drainage (AMD) occurs when sulfur containing minerals are exposed to air and water by the process of mining extraction. Their oxidization leads to the acidification of waters, releasing of high quantities of sulfates and leaching of associated metals/metalloids such as arsenic. Because sulfate-reducing bacteria (SRB) are able to produce H2S, it is of growing interest in bioremediation systems since metals/metalloids coprecipitate in the form of metallic sulfides. It seems essential to understand the role of the SRB in the AMD functioning. This research work aimed at study the diversity of SRB in the AMD of the former Carnoulès AMD contaminated by arsenic, to characterize their spatio-temporal dynamics and to better understand their role in the bioprocessings of arsenic. The development of a nested PCR method on the dissimilatory sulfite-reductase encoding genes (dsrAB) allowed detecting SRB within this environment. This developed tool, served for studying the dynamics of the community along the AMD by combining molecular approaches and to correlate the SRB distribution according to the physico-chemical variations prevailing in the ecosystem. All these works highlighted the influence of pH, iron and arsenic forms on the SRB dynamics. Afterward, a complementary cultivation approach has been applied to study the SRB mobilization of contaminants. A new SRB strain has been isolated from Carnoulès sediments, representing a new species. The capacity of the strain to grow in acid conditions and at high arsenic concentrations allowed to better understanding the role of the SRB in mining environments.PAU-BU Sciences (644452103) / SudocSudocFranceF

Nested PCR and New Primers for Analysis of Sulfate-Reducing Bacteria in Low-Cell-Biomass Environments▿ †

New primers were designed for the amplification of dsrAB genes by nested PCR to investigate the diversity of sulfate-reducing prokaryotes (SRP) in environments with low bacterial cell density. The success of the nested PCR for the determination of SRP diversity was estimated by terminal-restriction fragment length polymorphism analysis in the Reigous, a small creek at an inactive mine (Carnoulès, France), which constitutes an extreme acidic arsenic-rich environment. Nested PCR limits were evaluated in dsrAB-rich sediments, and this technique was compared to direct PCR using either known primers (DSR1F/DSR4R) or new primers (dsr619AF/dsr1905BR). The comparison of clone libraries revealed that, even if the levels of diversity observed were not identical, nested PCR did not reduce the diversity compared to that of direct DSR1F/DSR4R PCR. Clone sequences were affiliated mainly with the Desulfobacteraceae and Desulfohalobiaceae families. Many sequences (∼30%) were related to a deeply branching lineage unaffiliated with any cultured SRP. Although this dsrAB cluster was found in all libraries, the new primers better amplified this lineage, providing more information on this unknown bacterial group. Thanks to these new primers in nested PCR, the SRP community from Carnoulès could be characterized. Specific SRP populations were obtained according to environmental characteristics. Desulfomicrobiaceae-related sequences were recovered in samples with low pH, low levels of dissolved oxygen, and high As content, while sequences belonging to the deeply branching group were found in a less extreme sample. Furthermore, for the first time, dsrAB sequences related to the latter group were recovered from freshwater

Inability of myalgic encephalomyelitis/chronic fatigue syndrome patients to reproduce VO\u3csub\u3e2\u3c/sub\u3epeak indicates functional impairment

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a multi-system illness characterized, in part, by increased fatigue following minimal exertion, cognitive impairment, poor recovery to physical and other stressors, in addition to other symptoms. Unlike healthy subjects and other diseased populations who reproduce objective physiological measures during repeat cardiopulmonary exercise tests (CPETs), ME/CFS patients have been reported to fail to reproduce results in a second CPET performed one day after an initial CPET. If confirmed, a disparity between a first and second CPET could serve to identify individuals with ME/CFS, would be able to document their extent of disability, and could also provide a physiological basis for prescribing physical activity as well as a metric of functional impairment. Methods: 22 subjects diagnosed with ME/CFS completed two repeat CPETs separated by 24 h. Measures of oxygen consumption (VO ), heart rate (HR), minute ventilation (Ve), workload (Work), and respiratory exchange ratio (RER) were made at maximal (peak) and ventilatory threshold (VT) intensities. Data were analyzed using ANOVA and Wilcoxon\u27s Signed-Rank Test (for RER). Results: ME/CFS patients showed significant decreases from CPET1 to CPET2 in VO peak (13.8%), HRpeak (9 bpm), Ve peak (14.7%), and Work at peak (12.5%). Decreases in VT measures included VO at VT (15.8%), Ve at VT (7.4%), and Work at VT (21.3%). Peak RER was high (≥1.1) and did not differ between tests, indicating maximum effort by participants during both CPETs. If data from only a single CPET test is used, a standard classification of functional impairment based on VO peak or VO at VT results in over-estimation of functional ability for 50% of ME/CFS participants in this study.Conclusion: ME/CFS participants were unable to reproduce most physiological measures at both maximal and ventilatory threshold intensities during a CPET performed 24 hours after a prior maximal exercise test. Our work confirms that repeated CPETs warrant consideration as a clinical indicator for diagnosing ME/CFS. Furthermore, if based on only one CPET, functional impairment classification will be mis-identified in many ME/CFS participants. © 2014 Keller et al.; licensee BioMed Central Ltd. 2 2 2 2

A pair of identical twins discordant for myalgic encephalomyelitis/chronic fatigue syndrome differ in physiological parameters and gut microbiome composition

Objective: Unknown ethiology Background: Patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) present with profound fatigue, flu-like symptoms, pain, cognitive impairment, orthostatic intolerance, post-exertional malaise (PEM), and exacerbation of some or all of the baseline symptoms. Case Report: We report on a pair of 34-year-old monozygotic twins discordant for ME/CFS, with WELL, the non-affected twin, and ILL, the affected twin. Both twins performed a two-day cardiopulmonary exercise test (CPET), preand post-exercise blood samples were drawn, and both provided stool samples for biochemical and molecular analyses. At peak exertion for both CPETs, ILL presented lower VO peak and peak workload compared to WELL. WELL demonstrated normal reproducibility of VO @ventilatory/anaerobic threshold (VAT) during CPET2, whereas ILL experienced an abnormal reduction of 13% in VAT during CPET2. A normal rise in lactate dehydrogenase (LDH), creatine kinase (CK), adrenocorticotropic hormone (ACTH), cortisol, creatinine, and ferritin content was observed following exercise for both WELL and ILL at each CPET. ILL showed higher increases of resistin, soluble CD40 ligand (sCD40L), and soluble Fas ligand (sFasL) after exercise compared to WELL. The gut bacterial microbiome and virome were examined and revealed a lower microbial diversity in ILL compared to WELL, with fewer beneficial bacteria such as Faecalibacterium and Bifidobacterium, and an expansion of bacteriophages belonging to the tailed dsDNA Caudovirales order. Conclusions: Results suggest dysfunctional immune activation in ILL following exercise and that prokaryotic viruses may contribute to mucosal inflammation and bacterial dysbiosis. Therefore, a two-day CPET and molecular analyses of blood and microbiomes could provide valuable information about ME/CFS, particularly if applied to a larger cohort of monozygotic twins. 2

Eukaryotes in the gut microbiota in myalgic encephalomyelitis/chronic fatigue syndrome

Patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often suffer from gastrointestinal symptoms and many are diagnosed with irritable bowel syndrome (IBS). Previous studies, including from our laboratory, have demonstrated that the ME/CFS gut bacterial composition is altered and less diverse when compared to healthy individuals. Patients have increased biomarkers of inflammation and leaky gut syndrome. To further investigate dysbiosis in the ME/CFS gut microbiome, we sought to characterize the eukaryotes present in the gut of 49 individuals with ME/CFS and 39 healthy controls. Using 18S rRNA sequencing, we have identified eukaryotes in stool samples of 17 healthy individuals and 17 ME/CFS patients. Our analysis demonstrates a small, nonsignificant decrease in eukaryotic diversity in ME/CFS patients compared to healthy individuals. In addition, ME/CFS patients show a nonsignificant increase in the ratio of fungal phyla Basidiomycota to Ascomycota, which is consistent with ongoing inflammation in ME/CFS. We did not identify specific eukaryotic taxa that are associated with ME/CFS disease status

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome : a pilot study

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls. We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed. ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association. Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CF

Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation.

Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments

Clerodendron paniculatum L.

原著和名: シマヒギリ科名: クマツヅラ科 = Verbenaceae採集地: 台湾 日月潭〜嘉義 (台湾省 日月潭〜嘉義)採集日: 1968/8/3採集者: 萩庭丈壽整理番号: JH048968国立科学博物館整理番号: TNS-VS-99896

Additional file 2: Figure S2. of Reduced diversity and altered composition of the gut microbiome in individuals with myalgic encephalomyelitis/chronic fatigue syndrome

Principal Coordinate Analysis (PCoA) plot of healthy controls versus subjects with ME/CFS. Distances were calculated with weighted UniFrac (a) and unweighted UniFrac (b). Data were evenly sampled at 32223 sequences per sample. (PDF 2631 kb