Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine ¹³C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [¹³C]glucose, [¹³C]propionate, or [¹³C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that ¹³C labeling can be much lower, to the extent that δ¹³C ratios can be studied even at their natural abundances.Linking microbial phylogeny to community function provides us with insights into the roles that microorganisms play in global elemental cycling. In recent years, stable isotope-tracing approaches combined with biomarkers have been widely applied to environmental studies (8, 27, 40). Tracking stable- or radioisotope-labeled atoms from particular substrates into components of microbial cells (biomarkers) can reveal which organisms are involved in the consumption of the substrate and also yield information on rates of specific biogeochemical transformation (8).Dissimilatory sulfate reduction is a major pathway for organic carbon mineralization in coastal marine sediments, accounting for, on average, 50% of the total carbon mineralization (18, 36). Sulfate-reducing prokaryotes are a diverse and ubiquitous component of the bacterial community. The diversity of sulfate-reducing bacteria (SRB) in marine sediments has been investigated by using clone libraries of 16S rRNA (38) and dissimilatory sulfite reductase genes (11) and by fluorescence in situ hybridization-related techniques (33, 41). Desulfobacteraceae, a group of complete-oxidizing SRB belonging to the Deltaproteobacteria, have generally been found to be a major group of SRB in marine sediments.Phospholipid-derived fatty acids (PLFA) were the first type of biomarkers to be used in combination with stable isotope probing (SIP) (8). PLFA-SIP provides high sensitivity in terms of the amount of ¹³C label needed, but the phylogenetic resolution offered is low and requires reference signatures of closely related culturable relatives (8). The main advantage of DNA- and RNA-SIP is that they offer improved phylogenetic resolution (27, 40). These two methods are based on the separation of the “heavier” ¹³C-labeled nucleic acid from unlabeled nucleic acid by density centrifugation. Subsequently, organisms incorporating the greatest proportion of label into their DNA or RNA are identified by various molecular-fingerprinting techniques or by constructing clone libraries. RNA has a higher turnover rate than DNA, resulting in faster labeling, and incubation times can therefore be substantially shortened (27, 42). RNA is also more likely to reflect the phylogenetic composition of the metabolically active community, since it is highly susceptible to chemical and enzymatic degradation, and its cellular levels are often tightly regulated (19, 32), although some prokaryotes maintain high numbers of ribosomes during starvation (13).MacGregor et al. (25, 26) developed a related approach, SIP combined with magnetic-bead capture hybridization (here called Mag-SIP), which is based on the isolation of small subunit rRNA from particular phylogenetic groups and the detection of ¹³C-labeling levels by isotope ratio mass spectrometry (IRMS). rRNA is captured by hybridization with specific biotin-labeled oligonucleotide probes, followed by retrieval of hybridized target rRNA using streptavidin-coated magnetic beads. The main advantage of Mag-SIP over other nucleic acid-based SIP methods is that in principle much lower labeling levels can be applied (about 0.001% versus >10% ¹³C, respectively), as label detection is based on IRMS methods. For instance, it has been shown that the method can be used to study the effects of oil pollution on natural δ¹³C ratios of bacterial communities in sediments (37). Moreover, Mag-SIP is not based on PCR, as the isotope ratio of the target rRNA is directly measured without amplification of nucleic acid, avoiding possible PCR artifacts. However, the large amounts (1 to 10 μg) of RNA needed for an accurate isotope ratio analysis by traditional elemental-analyzer (EA)-IRMS has limited the use of Mag-SIP to general domain-specific probes (25, 26). Recently, several methods, such as liquid chromatography (LC) combined with IRMS and spooling-wire microcombustion combined with IRMS, have been introduced that allow isotopic analysis of much smaller samples than with the traditional EA-IRMS systems (20, 43).In this study, we used the wet-oxidation interface of LC-IRMS as a micro-EA (μEA)-IRMS (20). The use of μEA-IRMS substantially lowers the detection limit of isotope ratio measurements in terms of the amount of rRNA needed for an analysis but also calls for modifications of the Mag-SIP protocol in order to decrease protocol blanks (carbon from materials and reagents used in the protocol). We tested a nested set of three biotin-labeled oligonucleotide probes to capture 16S rRNA derived from Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae. The target specificities and stringencies of these probes were tested against pure cultures of both target and nontarget organisms. Moreover, phylogenetic analysis of captured 16S rRNA from environmental samples was done to check specificity and, where necessary, to adjust probe stringency. Finally, we demonstrated Mag-SIP with a study of in situ substrate use by sulfate-reducing Deltaproteobacteria in intertidal anoxic marine sediment. A generalized scheme for Mag-SIP is shown in Fig. ​Fig.11.Open in a separate windowFIG. 1.A generalized scheme for Mag-SIP. The control was sediment incubated without substrate.     

The grant is what I eat: the politics of social security and disability in the post-apartheid South African state

In South Africa, disability grant allocation has been under review and tensions are evident in government rhetoric stressing welfare provision on the one hand, and encouraging 'rationalization' on the other. This ambiguity is traced down to the level of grant negotiations between doctors and 'clients' in a psychiatry clinic in Khayelitsha. Here 'having nerves' embodies the distress associated with harsh circumstances and is deemed by supplicants as sufficient to secure a grant. The paper illustrates how national discourses influence the presentation and experience of suffering and the way in which doctors mediate diagnoses. The implications of local understandings of 'health citizenship' for expectations of the post-apartheid state are explored.     

Moderate potassium chloride supplementation in essential hypertension: is it additive to moderate sodium restriction?

Twenty patients with mild or moderate essential hypertension and not receiving any drug treatment, who had been moderately restricting their sodium intake to around 70 mmol(mEq) a day for at least one month and whose mean blood pressure was then 163/103 mm Hg, were entered into a double blind, randomised crossover study to compare one month''s treatment with slow release potassium chloride tablets (64 mmol potassium chloride a day) with one month''s treatment with a matching placebo. Mean (SEM) urinary sodium excretion on entry to the study was 68 (6.8) mmol/24 h. Mean urinary potassium excretion increased from 67 (6.9) mmol(mEq)/24 h with placebo to 117 (4.6) mmol/24 h with potassium chloride. Supine and standing systolic and diastolic blood pressures did not change significantly with potassium chloride supplementation when compared with pressures while receiving placebo or before randomisation. In patients who are able moderately to restrict their sodium intake doubling potassium as a chloride salt has little or no effect on blood pressure.     

Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents.

Structural features of the mouse and rat manchette and the role of the manchette in shaping the spermatid nucleus were investigated. Rod-like elements about 10 nm in diameter and 40-70 nm in length were seen linking the innermost microtubules of the manchette and the outer leaflet of the nuclear envelope in step 8 through step 11 rat and mouse spermatids that either had been routinely fixed for electron microscopy or had been isolated and detergent extracted. Rod-like linkers were also seen joining the nuclear ring to the plasma membrane and nuclear envelope. These linkers may ensure that under normal conditions the manchette remains in a defined position relative to these membranous components. A variety of compounds (taxol, cytoxan, and 5-fluorouracil) were found to perturb the manchette and to affect nuclear shaping. In addition, sys and azh mutant mice were used to determine the consequences of defective manchette formation. These genetic conditions and chemical treatments either produced manchettes that were not in their normal position (azh, sys, and taxol) and/or caused the manchette to appear abnormal (azh, sys, cytoxan, 5-fluorouracil, and taxol), and all resulted in a deformation of the step 9-11 spermatid nucleus. In all instances where the manchette was present, either in normal or ectopic locations, the sectioned nuclear envelope was parallel to the long axis of the microtubules of the manchette. In general, areas of the nuclear envelope where the manchette was not present, or where it was expected to be present but was not, were rounded (normal animals, sys, cytoxan). In addition, there are indications using certain compounds (cytoxan and 5-fluorouracil) as well as in the azh and sys mouse that the manchette may exert pressure to deform the nucleus. It is suggested that the rod-like linkages of the manchette ensure that the nuclear envelope remains at a constant distance from the manchette microtubules and that this is a major factor acting to impart nuclear shape changes on a region of the head caudal to the acrosome during the early elongation phase of spermiogenesis. The manchette microtubules, which are also known to be linked together, may act as a scaffold to deform this part of the nucleus from its spherical shape, perhaps in concert with forces initiated by other structural elements. Evidence from sys animals indicates that structural elements, such as the acrosomal complex over the anterior head (acrosome-actin-nuclear envelope), may affect nuclear shaping over the acrosome-covered portion of the spermatid head.(ABSTRACT TRUNCATED AT 400 WORDS)     

A general-purpose electronic model for arbitrary configurations of neurons

This paper describes a general-purpose electronic model for simulating the electrical activity in small groups of nerve cells arranged in arbitrary configurations. The model consists of 44 realizations of two basic modules (the “cell”, and the “axon with synapses”) which can be connected together in various arrangements by way of snapper-capped wires. The activity of the individual units is displayed via flashing lights atop the constituent cells; also there are taps on each cell from which one can obtain and display more detailed information concerning the electrical activity of each and all cells, including the graded “generator” potential of the triggering section, on for example an oscilloscope. The modules include realistic approximations to basic mechanisms of neuronal activity, but the main advance over previous models is the emphasis on and ability to deal with the network context of neuroelectric signals.Illustrative applications to mutually-inhibiting centers and to our ladder net theory for reticular-like networks are presented. The primary advantages of the electronic model are: actual physical representation of various configurations, asynchronous timing, flexibility with respect to overall configuration and with respect to network parameters, immediate turnaround, and cost.     

Reversal by verapamil of defect in sodium transport in leucocytes in essential hypertension.

The effect of treatment with verapamil on cell sodium transport was studied in the leucocytes of patients with essential hypertension. Previously described abnormalities of sodium efflux rate constant and intracellular sodium content were confirmed, the component of the sodium efflux rate constant sensitive to ouabain being lower and the intracellular sodium content higher in the patients compared with controls. Verapamil reversed these abnormalities and reduced blood pressure.