Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.     

Study of the inhibition of cyclin-dependent kinases with roscovitine and indirubin-3′-oxime from molecular dynamics simulations

Molecular dynamics simulations were performed to elucidate the interactions of CDK2 and CDK5 complexes with three inhibitors: R-roscovitine, S-roscovitine, and indirubin-3′-oxime. The preference of the two complexes for R-roscovitine over the S enantiomer, as reported by the experiment, was also found by the simulations. More importantly, the simulations showed that the cause of the stronger affinity for the R enantiomer is the presence of an important hydrogen bond between R-roscovitine and the kinases not found with S-roscovitine. The simulations also showed two amino acid mutations in the active site of CDK5/R-roscovitine that favor binding-enhanced electrostatic contributions, making the inhibitor more effective for CDK5 than for CDK2. This suggests that the effectiveness of roscovitine-like inhibitors can be improved by enhancing their electrostatic interaction with the kinases. Finally, molecular mechanics–Possion–Boltzmann/surface area calculations of the CDK5/indirubin-3′-oxime system in both water-excluded and water-included environments gave significantly different electrostatic contributions to the binding. The simulations detected the displacement of a water molecule in the active site of the water-included CDK/indirubin-3′-oxime system. This resulted in a more conserved binding pattern than the water-excluded structure. Hence, in the design of new indirubin-like inhibitors, it is important to include the water molecule in the analysis. Figure Hydrogen bonding networks at the active sites of both CDK5/R-roscotivine (light grey) and CDK2/R-roscovitine (black).     

Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: D_eff, the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. D_eff for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.     

The mycetisms and their relevancy in medicine.

Poisonous mushrooms play an important role in public health, since the poisonings caused by their toxins have very high morbidity-mortality rates. No real data are available on this problem because diagnosis is often difficult. This work points out the important role of poisonous mushrooms. An analysis is included of the clinical features of the most important mycetisms, the mushrooms that produce them and the toxins that cause this pathology. A guide to aid clinical doctors in the diagnosis and treatment of mycetisms and recomendations to prevent these poisonings are provided.     

Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.     

Changes in structure,activity and metabolism of aerobic granules as a microbial response to high phenol loading

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m^–3 day^–1. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m^–3 day^–1, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m^–3 day^–1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m^–3 day^–1, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

An electron microscopic study of neuronal degeneration and glial cell reaction in the retina of glaucomatous rats

The present investigation was focused on the ultrastructural changes in the neurons and glial cells in the retina of rats with experimentally-induced glaucoma. An experimental glaucoma model was created by limbal-derived vein cauterization. Animals were sacrificed at 1, 3 weeks and 3 months post-operation. Retinae were dissected and processed for electron microscopy. Neuronal degeneration was observed in all the different layers of the retina at both 1 and 3 weeks post-operation. Some degenerating neurons were found in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). And the dying neurons presented apoptotic-like more than necrotic neurons. Many degenerating axons and axon terminals were observed between neurons in the GCL, inner plexiform layer (IPL), INL, and outer plexiform layer (OPL). Activated astrocytes and microglial cells were present in close association with degenerating neurons and axons. The Müller cells in the INL also presented longer and darker processes with more microfilaments than in normal cells. Degenerating neuronal debris, degenerating axonal profiles and electron-dense bodies were often found in the cytoplasm of macrophages. The results suggest that both microglial cells and astrocytes are activated in the process of neuronal degeneration in the retina of experimentally-induced glaucomatous rats. It is hypothesized that they may play a protective role in removing degenerating neuronal elements in the retina after the onset of glaucoma.     

Intestinal candidiasis. A clinical report and comments about this opportunistic pathology

An eight-years-old girl, who presented with recurrent upper respiratory tract infections, was treated with broad-spectrum antibiotics. Afterward she presented with intestinal candidiasis. The isolated species was identified as Candida albicans by differential tests. Treatment given was with 500,000 IU of oral nystatin every 8 hours for 10 days and intestinal normal microbiota restoratives. Evolution has been satisfactory, although concomitantly type A hepatitis developed. Rest and a soft diet were recommended. The child is now perfectly healthy with normal liver function tests. CONCLUSION: Prolonged treatments with broad-spectrum antibiotics destroyed the indigenous intestinal microbiota, which provoked intestinal C. Albicans proliferation and adversely affected the immunological system of the patient, thus facilitating the establishment of a viral infection.