Persistence of lung inflammation and lung cytokines with high-resolution CT abnormalities during recovery from SARS

Background

During the acute phase of severe acute respiratory syndrome (SARS), mononuclear cells infiltration, alveolar cell desquamation and hyaline membrane formation have been described, together with dysregulation of plasma cytokine levels. Persistent high-resolution computed tomography (HRCT) abnormalities occur in SARS patients up to 40 days after recovery.

Methods

To determine further the time course of recovery of lung inflammation, we investigated the HRCT and inflammatory profiles, and coronavirus persistence in bronchoalveolar lavage fluid (BALF) of 12 patients at recovery at 60 and 90 days.

Results

At 60 days, compared to normal controls, SARS patients had increased cellularity of BALF with increased alveolar macrophages (AM) and CD8 cells. HRCT scores were increased and correlated with T-cell numbers and their subpopulations, and inversely with CD4/CD8 ratio. TNF-α, IL-6, IL-8, RANTES and MCP-1 levels were increased. Viral particles in AM were detected by electron microscopy in 7 of 12 SARS patients with high HRCT score. On day 90, HRCT scores improved significantly in 10 of 12 patients, with normalization of BALF cell counts in 6 of 12 patients with repeat bronchoscopy. Pulse steroid therapy and prolonged fever were two independent factors associated with delayed resolution of pneumonitis, in this non-randomized, retrospective analysis.

Conclusion

Resolution of pneumonitis is delayed in some patients during SARS recovery and may be associated with delayed clearance of coronavirus, Complete resolution may occur by 90 days or later.     

Remediation of Metal-Contaminated Soil by an Integrated Soil Washing-Electrolysis Process

Chelating agents such as EDTA and DTPA are often used to remove metals from soil. However, their toxicity, bio-recalcitrance, and problems with recovery of heavy metal and chelating agents severely limit their applications. A biodegradable chelating agent, LED3A, and two surfactants, SDS and Triton X 100, were evaluated as potential alternatives for remediation of metal-contaminated soil.

LED3A alone only removed 40% of cadmium the addition of surfactant significantly enhanced its cadmium removal capacity up to 80% for a wide range of pH (5 to 11). The enhancement increased with both surfactant concentrations and LED3A concentrations. Because LED3A had a much higher removal capacity for copper, the synergistic effect of surfactant-LED3A mixture was less obvious. Sequential extraction analysis indicated that the LED3A not only removed copper from carbonate and Fe-Mn oxide fraction, but also from organic fractions. A three-dimension electrolysis reactor could effectively recover both metals and LED3A-SDS within thirty minutes. The combined soil washing by LED3A-surfactants and electrolysis provides a potential approach for remediation of copper- and cadmium-contaminated soils.     

Predicting enzyme subclass by functional domain composition and pseudo amino acid composition

As a continuous effort to use the sequence approach to identify enzymatic function at a deeper level, investigations are extended from the main enzyme classes (Protein Sci. 2004, 13, 2857-2863) to their subclasses. This is indispensable if we wish to understand the molecular mechanism of an enzyme at a deeper level. For each of the 6 main enzyme classes (i.e., oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase), a subclass training dataset is constructed. To reduce homologous bias, a stringent cutoff was imposed that all the entries included in the datasets have less than 40% sequence identity to each other. To catch the core feature that is intimately related to the biological function, the sample of a protein is represented by hybridizing the functional domain composition and pseudo amino acid composition. On the basis of such a hybridization representation, the FunD-PseAA predictor is established. It is demonstrated by the jackknife cross-validation tests that the overall success rate in identifying the 21 subclasses of oxidoreductases is above 86%, and the corresponding rates in identifying the subclasses of the other 5 main enzyme classes are 94-97%. The high success rates imply that the FunD-PseAA predictor may become a useful tool in bioinformatics and proteomics of the post-genomic era.     

Coupling interaction between thromboxane A2 receptor and alpha-13 subunit of guanine nucleotide-binding protein

G protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins that play an essential role in modulating many vital physiological events, such as cell communication, neurotransmission, sensory perception, and chemotaxis. Understanding of the 3D (dimensional) structures of these receptors and their binding interactions with G proteins will help in the design of drugs for the treatment of GPCR-related diseases. By means of the approach of structural bioinformatics, the 3D structures of human alpha-13 subunit of guanine nucleotide-binding protein (G alpha 13) and human thromboxane A2 (TXA2) receptor were developed. The former plays an important role in the control of cell growth that may serve as a prototypical G protein; the latter is a target for nitric oxide-mediated desensitization that may serve as a prototypical GPCR. On the basis of the 3D models, their coupling interactions were investigated via docking studies. It has been found that the two proteins are coupled with each other mainly through the interaction between the minigene of G alpha 13 and the 3rd intracellular loop of the TXA2 receptor, consistent with the existing deduction in the literatures. However, it has also been observed via a close view that some residues of the TXA2 receptor that are sequentially far away but spatially quite close to the loop region are also involved in forming hydrogen bonds with the minigene of G alpha 13. These findings may provide useful information for conducting mutagenesis and reveal the molecular mechanism how the human TXA2 receptor interact with G alpha 13 to activate intracellular signaling. The findings may also provide useful insights for stimulating new therapeutic approaches by manipulating the interaction of the receptor with the relevant G proteins.     

Functional analysis of all nonribosomal peptide synthetases in Cochliobolus heterostrophus reveals a factor, NPS6, involved in virulence and resistance to oxidative stress

Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.     

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.     

Reconstruction of burn scar of the upper extremities with artificial skin

The management of upper-extremity burn contractures is a major challenge for plastic surgeons. After approval by the Food and Drug Administration, artificial skin (Integra) has been available in Taiwan since 1997. From January of 1997 to July of 1999, the authors applied artificial skin to 13 severely burned patients for the reconstruction of their upper extremities, resulting in an increased range of motion in the upper-extremity joints and improved skin quality. An additional benefit was the rapid reepithelialization of the donor sites. There were no complications of infection throughout the therapeutic course, and the overall results were satisfactory. During the 2-year study, scar condition was monitored between 8 and 24 months, and a good appearance and pliable skin were obtained according to the Vancouver Scar Scale. According to this evaluation of Oriental skin turgor, normal pigmentation was restored about 6 months after the resurfacing procedure. For patients with severe burns in whom there is insufficient available skin for a full-thickness skin graft or another appropriate flap for scar revision, Integra is an alternative. The two major concerns in dealing with artificial skin are (1) a 10- to 14-day waiting period for maturation of the neo-dermis, necessitating a two-stage operation, and (2) prevention of infection with antibiotics and meticulous wound care.     

Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation

A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by two general promoters (cytomegalovirus and medaka -actin) and one muscle-specific promoter (zebrafish -actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, 34.6% (81/234), 10% (10/60), and 18% (38/212) showed homogenous GFP-expression for the derivative constructs of the cytomegalovirus, -actin, and -actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was (a) extended for an entire lifetime without degradation, and (b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye.     

Efficient combination of multiple word models for improved sequence comparison

MOTIVATION: Studies of efficient and sensitive sequence comparison methods are driven by a need to find homologous regions of weak similarity between large genomes. RESULTS: We describe an improved method for finding similar regions between two sets of DNA sequences. The new method generalizes existing methods by locating word matches between sequences under two or more word models and extending word matches into high-scoring segment pairs (HSPs). The method is implemented as a computer program named DDS2. Experimental results show that DDS2 can find more HSPs by using several word models than by using one word model. AVAILABILITY: The DDS2 program is freely available for academic use in binary code form at http://bioinformatics.iastate.edu/aat/align/align.html and in source code form from the corresponding author.     

Predicting subcellular localization of proteins in a hybridization space

MOTIVATION: The localization of a protein in a cell is closely correlated with its biological function. With the number of sequences entering into databanks rapidly increasing, the importance of developing a powerful high-throughput tool to determine protein subcellular location has become self-evident. In view of this, the Nearest Neighbour Algorithm was developed for predicting the protein subcellular location using the strategy of hybridizing the information derived from the recent development in gene ontology with that from the functional domain composition as well as the pseudo amino acid composition. RESULTS: As a showcase, the same plant and non-plant protein datasets as investigated by the previous investigators were used for demonstration. The overall success rate of the jackknife test for the plant protein dataset was 86%, and that for the non-plant protein dataset 91.2%. These are the highest success rates achieved so far for the two datasets by following a rigorous cross-validation test procedure, suggesting that such a hybrid approach (particularly by incorporating the knowledge of gene ontology) may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology. AVAILABILITY: The software would be made available on sending a request to the authors.