In Situ Determination of the Effects of Lead and Copper on Cyanobacterial Populations in Microcosms

Background

Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth.

Methodology/Principal Findings

Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations.

Conclusions/Significance

The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants.     

<Emphasis Type="Italic">Endohyalina</Emphasis>, the genus in the Physciaceae to accommodate the species of the <Emphasis Type="Italic">Rinodina ericina-</Emphasis>group

The genus Endohyalina is characterized by crustose, autonomous, or obligately lichenicolous thalli, lecideine apothecia with a hymenium often more or less inspersed with oil droplets and a brown hypothecium, Bacidia-type asci, small Dirinaria-type ascospores developing with type B ontogeny, bacilliform conidia and containing diploicin as the major secondary metabolite. The genus is based on four species previously included in Rinodina—R. ericina s. lat., R. insularis, R. interjecta and R. kalbii—and on two lichenicolous species from the Canary Islands described here as new, Endohyalina brandii and E. diederichii. The generic type, Endohyalina rappii, is reduced to synonymy with E. ericina whereas E. circumpallida is excluded from the genus and returned to Buellia s. lat. Except for the thalline growth form and the common lichenicolous habit, the diagnostic characters of Endohyalina are akin to those of Diploicia. New chemical data on Endohyalina insularis and E. kalbii are reported, and a simple method for determining the secondary chemistry of lichenicolous fungi is provided.     

Hypothesis testing approaches to the exon prediction problem

MOTIVATION: Many gene identification methods assign scores to gene elements prior to their assembly into predicted genes. The scoring system is often based on log-likelihood ratios. These methods usually perform well but it is difficult to interpret how significant a score is. RESULTS: We have developed several tests of significance for the scores: (1) a sum-of-scores test (SST), (2) an intersection-union test (IUT), based on a multiple hypothesis testing interpretation of an exon's score and (3) a meta-analytical approach (MA), which combines several P-values, corresponding to the exon's parts, to yield a global P-value. We performed simulation studies, which show that the MA has better sensitivity and specificity than other methods and is easier to interpret by non-expert users. This is an improvement over other methods and is especially relevant for users who would like to predict incomplete gene sequences.     

Activation of AMP-dependent protein kinase by hypoxia and hypothermia in the liver of frog Rana perezi

We have investigated different signaling molecules that could be activated by temperature acclimation and hypoxia, using an experimental approach consisting in submerging frogs in a water-filled box maintained at 2-4 degrees C at ambient oxygen levels or supplied with 98% N2:2% CO2 for normoxia or hypoxia conditions, respectively. The results obtained showed no significant changes in the expression of heat shock protein 70. The phosphorylation state of AMP-dependent activated protein kinase, the down-stream component of a protein kinase cascade that acts as an intracellular energy sensor, was significantly increased in both experimental conditions, showing higher values in the absence of oxygen. Similarly, the phosphorylation state of one of its known substrates, elongation factor 2, was also increased, consistent with the arrest of protein synthesis. These results point out an important role of this kinase, adjusting the rates of ATP-consuming and ATP-generating pathways, in the survival strategies to hypoxia and hypothermia.     

Effects of putrescine accumulation in tobacco transgenic plants with different expression levels of oat arginine decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two possible ways to synthesize putrescine (Put) in plants. In previous work ( Masgrau et al. 1997 ), we observed an altered phenotype (growth inhibition, leaf chlorosis and necrosis) in tobacco transgenic plants ( Nicotiana tabacum L. var. Wisconsin-38) containing the oat ADC cDNA under the control of a tetracycline inducible promoter, the severity of which was correlated with Put content. Now we have analysed the T₂ generation of a selected transgenic line (line 52), which in previous generations was characterized by presenting a moderate increase in ADC activity and polyamine levels, but no phenotype alterations. Studying two selected individuals, one with a high expression level of the transgene and the other with a moderate expression level, we demonstrate that only the one with increased polyamine content displays the altered (toxic) phenotype. The possible causes of toxicity have been analysed. The results suggest that either Put or its oxidation products, via diamine oxidase (DAO; EC 1.4.3.6), are the responsible factors for the deleterious effects observed in the transgenic plants.     

Recognition of conserved amino acid motifs of common viruses and its role in autoimmunity

The triggers of autoimmune diseases such as multiple sclerosis (MS) remain elusive. Epidemiological studies suggest that common pathogens can exacerbate and also induce MS, but it has been difficult to pinpoint individual organisms. Here we demonstrate that in vivo clonally expanded CD4+ T cells isolated from the cerebrospinal fluid of a MS patient during disease exacerbation respond to a poly-arginine motif of the nonpathogenic and ubiquitous Torque Teno virus. These T cell clones also can be stimulated by arginine-enriched protein domains from other common viruses and recognize multiple autoantigens. Our data suggest that repeated infections with common pathogenic and even nonpathogenic viruses could expand T cells specific for conserved protein domains that are able to cross-react with tissue-derived and ubiquitous autoantigens.     

Influence of reagent formulation on mRNA quantification by RT-PCR using imported external standard curves

Use of an imported external standard curve is common in real-time quantitative RT-PCR. Two practical strategies for long-term experiments include importing a grand mean standard curve to all accumulated runs or using daily imported standard curves, fixing the slope at the beginning of the experiment and calibrating successive runs with curves generated from this imported slope, adding a single standard that registers the variation in the y-intercept. This study determines the influence that a change in reagent lots has on these two calibration approaches when determining mRNA copy numbers of the ornithine decarboxylase and porphobilinogen deaminase genes. Two sets of determinations were run with the use of lot A and lot B. A marked decrease in the crossing points (Cp) in the standards for both genes at all concentration levels was observed with the change in lots. A grand mean standard curve was generated for each gene and each set and comparisons between the sets were performed. Statistically significant differences were found with respect to the y-intercept but not the slope, suggesting that the change of reagent lot affected the detection sensitivity but not the efficiency of the reaction. The excellent correlation coefficients obtained for these curves for each gene were not achieved when overall data from both sets were combined to generate an overall grand mean standard curve. We conclude that when faced with a change of RT-PCR reagent lot that will affect the detection sensitivity of the method, samples should be calculated with either the daily imported standard curves or with the respective grand mean standard curve for each lot.     

Chemokines and Toll-like receptors in Lyme disease pathogenesis

Lyme disease is a tick-transmitted inflammatory disorder, caused by the spirochete Borrelia burgdorferi (Bb). Recent discoveries cast new light on Bb dissemination and the ensuing pathogenesis of inflammation. Although the strong proinflammatory Bb lipoproteins have been implicated in the induction of inflammation, they do not seem to act exclusively through Toll-like receptor (TLR) engagement. In fact, mice that are deficient for MyD88, a component of the TLR signaling pathway, manifest similar or increased recruitment of cells into Bb-infected tissues. By contrast, the absence of the chemokine receptor CXCR2 results in reduced inflammation. Overall, these findings highlight the complexity of Lyme disease pathogenesis and identify chemokine pathways as novel therapeutic targets for the control of Bb-induced inflammation.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCas^E murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies.