Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Growth-induced mass flows in fungal networks

Cord-forming fungi form extensive networks that continuously adapt to maintain an efficient transport system. As osmotically driven water uptake is often distal from the tips, and aqueous fluids are incompressible, we propose that growth induces mass flows across the mycelium, whether or not there are intrahyphal concentration gradients. We imaged the temporal evolution of networks formed by Phanerochaete velutina, and at each stage calculated the unique set of currents that account for the observed changes in cord volume, while minimizing the work required to overcome viscous drag. Predicted speeds were in reasonable agreement with experimental data, and the pressure gradients needed to produce these flows are small. Furthermore, cords that were predicted to carry fast-moving or large currents were significantly more likely to increase in size than cords with slow-moving or small currents. The incompressibility of the fluids within fungi means there is a rapid global response to local fluid movements. Hence velocity of fluid flow is a local signal that conveys quasi-global information about the role of a cord within the mycelium. We suggest that fluid incompressibility and the coupling of growth and mass flow are critical physical features that enable the development of efficient, adaptive biological transport networks.     

Phagocytic function in cyclists: correlation with catecholamines and cortisol.

Flow cytometer measurements were made of the basal variations in peripheral blood functional monocytes and granulocytes over the course of a training season (January to November) of a cycling team. Parallel determinations were made of plasma concentration of catecholamines (chromatography) and cortisol (RIA) in a search for neuroendocrine markers. The results showed the greatest phagocytic capacity to occur in the central months (March, May, and July), coinciding with the greatest number and highest level of competitive events with good correlation with a peak in epinephrine during these months (r(2) = 0.998 for monocytes and r(2) = 0.674 for granulocytes). No good correlations were found between phagocytosis and norepinephrine or cortisol. The highest values for phagocytosis and epinephrine concentration were found in May. These results suggest that blood epinephrine concentration could be a good neuroendocrine marker of sportspeople's phagocytic response.     

A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens     

Interaction of the organic anion 1-aniline 8-naphthalene sulfonate (ANS) with isolated rat hepatocytes

The interaction of ANS with rat hepatocytes in time was studied by fluorescence spectroscopy. The intercept of the first linear portion of the time curve of interaction showed a positive value over all the ANS concentration range employed. This value was maintained after cellular disruption by homogenization. It was affected by ionic strength, pH, and divalent cation in the incubation medium, all conditions affecting the cellular surface. These data suggest that this phenomenon might be a binding of the compound to the hepatocytes surface. Due to the time constant and its disappearance after cellular disruption the other slower component of the curve seems to correspond to a process of translocation across the membrane.     

Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.     

The spread of somatosensory-evoked potentials within the nervous system

Four thalamic and cortical recordings were carried out in 5 patients. The thalamic-evoked potentials were typical and revealed a triphasic complex, but their latencies showed a relatively high standard deviation. They could be divided into two groups according to their latencies, both of which had low SD. These data suggested that there could be two types of latency of thalamic SEP, because the 4 patients' body sizes were very similar. More detailed surface, cortical and depth recordings are needed to resolve these questions.