Evolution and classification of the CRISPR-Cas systems

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.     

Long-term intracellular chromium partitioning with subsurface bacteria

Subsurface bacteria were used to study the kinetics of chromate uptake and the internal distribution of the chromium that is taken up by these cells using two equilibration periods (1 and 50 days). Cells that were exposed to chromate for 50 days (to simulate in-situ conditions) were able to sequester up to 200% more chromium per unit mass of cells than were cells that were exposed for only 1 day. Chromium distributions showed an increase in chromium sorption by the cell wall, by the membrane and ribosome, and by the soluble fraction after a 50-day equilibration period compared to after 1 day of equilibration. Killed cell controls suggest that active transport of chromate is the method of intracellular accumulation during the 50-day equilibration period.     

A preliminary study of the seagrass Thalassodendron ciliatum (Forssk.) den hartog from Eastern Indonesia. Biological results of the snellius II expedition

Two dense meadows of the seagrass Thalassodendron ciliatum (Forssk.) den Hartog were sampled during the Indonesian—Dutch Snellius II expedition to Eastern Indonesia. Production data were obtained from one of these meadows. The production of leaf biomass was measured by the leaf marking technique of Zieman and by the plastochrone interval method. The two methods reached comparable results. The production of leaf tissue was 4.2 mg ADW shoot^?1 day^?1. The production of rhizome biomass was calculated in a similar way, based on the plastochrone interval of rhizome nodes. The production of the meadow, exclusive of the production of roots and fruits, amounted to 4.5 g ADW m^?2 day^?1. A significant correlation between the growth rates of rhizomes and leaves was observed. Biomass data from the second site are given.     

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.     

Dimethylarginine Levels in Cerebrospinal Fluid of Hyperacute Ischemic Stroke Patients are Associated with Stroke Severity

We hypothesise that asymmetric and symmetric dimethylarginine (ADMA, SDMA) are released in cerebrospinal fluid (CSF) due to ischemia-induced proteolysis and that CSF dimethylarginines are related to stroke severity. ADMA and SDMA were measured in CSF of 88 patients with ischemic stroke or TIA within 24 h after stroke onset (mean 8.6 h) and in 24 controls. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS) score at admission. Outcome was evaluated by institutionalization due to stroke and the modified Rankin scale. Dimethylarginine levels were higher in patients with stroke than in TIA patients, who had higher levels than controls and correlated with the NIHSS. Logistic regression analysis confirmed that dimethylarginines were independently associated with stroke severity. The SDMA/ADMA ratio did not differ significantly between controls and stroke patients. CSF dimethylarginine levels are increased in hyperacute ischemic stroke and are associated with stroke severity. R. Brouns is a research assistant of the Fund for Scientific research Flanders (FWO-Vlaanderen).     

A comparison of the annual production and biomass in three monospecific stands of the seagrass Thalassia hemprichii (Ehrenb.) aschers.

Annual production and biomass data were collected in three seagrass communities of Thalassia hemprichii (Ehrenb.) Aschers. from Papua New Guinea. Leaf growth rates, determined by the marking technique, resulted in a growth rate of 8.3 mm day^?1 for the youngest leaves. Production of above-ground plant parts was assessed by the plastochrone interval. The annual mean values were 9.3, 10.0 and 9.9 days for Sites 1, 2 and 3, respectively. Annual mean total above-ground production amounted to 2.1 mg ADW shoot^?1 day^?1 at Site 1, and 5.5 and 4.5 mg ADW shoot^?1 day^?1 for Sites 2 and 3, respectively; 73–89% of the total net production was contributed by the leaves. Rhizome production was correlated to the plastochrone interval of the leaves. Annual mean biomass of leaves amounted to 16–27% of the total biomass. The mean biomass of the other plant parts remained constant during the year. The annual mean turnover time of the different plant parts (above- and below-ground) varied considerably between the sites.