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OBJECTIVE: This retrospective chart review describes the epidemiology and clinical features of 40 patients with culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. METHODS: Patients with positive M. pneumoniae cultures from respiratory specimens from January 1997 through December 1998 were identified through the Microbiology records. Charts of patients were reviewed. RESULTS: 40 patients were identified, 33 (82.5%) of whom required admission. Most infections (92.5%) were community-acquired. The infection affected all age groups but was most common in infants (32.5%) and pre-school children (22.5%). It occurred year-round but was most common in the fall (35%) and spring (30%). More than three-quarters of patients (77.5%) had comorbidities. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common symptoms, and crepitations (60%), and wheezes (40%) were the most common signs. Most patients with pneumonia had crepitations (79.2%) but only 25% had bronchial breathing. Immunocompromised patients were more likely than non-immunocompromised patients to present with pneumonia (8/9 versus 16/31, P = 0.05). Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications, 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities. CONCLUSION: our results were similar to published data except for the finding that infections were more common in infants and preschool children and that the mortality rate of pneumonia in patients with comorbidities was high. | [
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] |
Inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO•) and increased indices of NO• -dependent oxidative stress. Although NO• is known to have anti-microbial, anti-inflammatory and anti-oxidant properties, various lines of evidence support the contribution of NO• to lung injury in several disease models. On the basis of biochemical evidence, it is often presumed that such NO• -dependent oxidations are due to the formation of the oxidant peroxynitrite, although alternative mechanisms involving the phagocyte-derived heme proteins myeloperoxidase and eosinophil peroxidase might be operative during conditions of inflammation. Because of the overwhelming literature on NO• generation and activities in the respiratory tract, it would be beyond the scope of this commentary to review this area comprehensively. Instead, it focuses on recent evidence and concepts of the presumed contribution of NO• to inflammatory diseases of the lung. | [
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] |
Surfactant protein-D (SP-D) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. SP-D is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. SP-D, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. SP-D also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. In addition, SP-D binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. Consistent with these diverse in vitro activities is the observation that SP-D-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. The phenotype of macrophages isolated from the lungs of SP-D-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. The expression of SP-D is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized SP-D might contribute to the pathogenesis of a variety of human lung diseases. | [
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Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease. | [
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] |
Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner. | [
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Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis. | [
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] |
Recent evidence suggests that critically ill patients are able to tolerate lower levels of haemoglobin than was previously believed. It is our goal to show that transfusing to a level of 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. Although many questions remain, many laboratory and clinical studies, including a recent randomized controlled trial (RCT), have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. In addition, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of red blood cells (RBCs), save blood overall, and decrease health care costs. | [
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The 21st International Symposium on Intensive Care and Emergency Medicine was dominated by the results of recent clinical trials in sepsis and acute respiratory distress syndrome (ARDS). The promise of extracorporeal liver replacement therapy and noninvasive ventilation were other areas of interest. Ethical issues also received attention. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of a high standard. The meeting was marked by a sense of renewed enthusiasm that positive progress is occurring in intensive care medicine. | [
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] |
Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states. | [
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This report describes the design and implementation of the Real-time Outbreak and Disease Surveillance (RODS) system, a computer-based public health surveillance system for early detection of disease outbreaks. Hospitals send RODS data from clinical encounters over virtual private networks and leased lines using the Health Level 7 (HL7) message protocol. The data are sent in real time. RODS automatically classifies the registration chief complaint from the visit into one of seven syndrome categories using Bayesian classifiers. It stores the data in a relational database, aggregates the data for analysis using data warehousing techniques, applies univariate and multivariate statistical detection algorithms to the data, and alerts users of when the algorithms identify anomalous patterns in the syndrome counts. RODS also has a Web-based user interface that supports temporal and spatial analyses. RODS processes sales of over-the-counter health care products in a similar manner but receives such data in batch mode on a daily basis. RODS was used during the 2002 Winter Olympics and currently operates in two states—Pennsylvania and Utah. It has been and continues to be a resource for implementing, evaluating, and applying new methods of public health surveillance. | [
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Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals. | [
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Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis. | [
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The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays. | [
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] |
We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function. | [
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|
There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients. | [
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] |
INTRODUCTION: The pathogenicity of late respiratory infections with herpes simplex virus type 1 (HSV-1) in the critically ill is unclear. METHODS: In four critically ill patients with persistent pulmonary infiltrates of unknown origin and isolation of HSV-1 from tracheal aspirate or bronchoalveolar lavage fluid, at 7 (1–11) days after start of mechanical ventilatory support, a pulmonary leak index (PLI) for (67)Gallium ((67)Ga)-transferrin (upper limit of normal 14.1 × 10(-3)/min) was measured. RESULTS: The PLI ranged between 7.5 and 14.0 × 10(-3)/min in the study patients. Two patients received a course of acyclovir and all survived. CONCLUSIONS: The normal capillary permeability observed in the lungs argues against pathogenicity of HSV-1 in the critically ill, and favors that isolation of the virus reflects reactivation in the course of serious illness and immunodepresssion, rather than primary or superimposed infection in the lungs. | [
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BACKGROUND: Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. METHODS: We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. RESULTS: We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. CONCLUSIONS: Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. | [
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BACKGROUND: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH. METHODS: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H(2)O(2). Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. RESULTS: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O(2 )toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. CONCLUSION: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O(2 )and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O(2 )and its metabolites. | [
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BACKGROUND: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes. RESULTS: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system. CONCLUSIONS: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment. | [
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In the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries. | [
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Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. | [
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Horizontal DNA transfer is an important factor of evolution and participates in biological diversity. Unfortunately, the location and length of horizontal transfers (HTs) are known for very few species. The usage of short oligonucleotides in a sequence (the so-called genomic signature) has been shown to be species-specific even in DNA fragments as short as 1 kb. The genomic signature is therefore proposed as a tool to detect HTs. Since DNA transfers originate from species with a signature different from those of the recipient species, the analysis of local variations of signature along recipient genome may allow for detecting exogenous DNA. The strategy consists in (i) scanning the genome with a sliding window, and calculating the corresponding local signature (ii) evaluating its deviation from the signature of the whole genome and (iii) looking for similar signatures in a database of genomic signatures. A total of 22 prokaryote genomes are analyzed in this way. It has been observed that atypical regions make up ∼6% of each genome on the average. Most of the claimed HTs as well as new ones are detected. The origin of putative DNA transfers is looked for among ∼12 000 species. Donor species are proposed and sometimes strongly suggested, considering similarity of signatures. Among the species studied, Bacillus subtilis, Haemophilus Influenzae and Escherichia coli are investigated by many authors and give the opportunity to perform a thorough comparison of most of the bioinformatics methods used to detect HTs. | [
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Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis. | [
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] |
Using differential display RT-PCR, we identified a gene of 2750 bp from human adult testis, named H-Lse, which encoded a putative protein of 523 amino acids and molecular weight of 58 kd with structural characteristics similar to that of mouse lysosome sialic-acid-specific 9-O-acetylesterase. Northern blot analysis showed a widespread distribution of H-Lse in various human tissues with high expression in the testis, prostate, and colon. In situ hybridization results showed that while H-Lse was not detected in embryonic testis, positive signals were found in spermatocytes but not spermatogonia in adult testis of human. The subcellular localization of H-Lse was visualized by green fluorescent protein (GFP) fused to the amino terminus of H-Lse, showing compartmentalization of H-Lse in large dense-core vesicles, presumably lysosomes, in the cytoplasm. The developmentally regulated and spermatogenic stage-specific expression of H-Lse suggests its possible involvement in the development of the testis and/or differentiation of germ cells. | [
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] |
BACKGROUND: The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis. We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM). METHODS: Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted. The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis. RESULTS: Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis. WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM. CONCLUSION: Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis. | [
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Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10(−5) to 4 × 10(−5). This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus. | [
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] |
mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot. | [
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Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ∼60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ∼1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences. | [
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While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data. | [
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The ratio between proteins P27 and replicase of Cocksfoot mottle virus (CfMV) is regulated via a −1 programmed ribosomal frameshift (−1 PRF). A minimal frameshift signal with a slippery U UUA AAC heptamer and a downstream stem–loop structure was inserted into a dual reporter vector and directed −1 PRF with an efficiency of 14.4 ± 1.9% in yeast and 2.4 ± 0.7% in bacteria. P27-encoding CfMV sequence flanking the minimal frameshift signal caused ∼2-fold increase in the −1 PRF efficiencies both in yeast and in bacteria. In addition to the expected fusion proteins, termination products ending putatively at the frameshift site were found in yeast cells. We propose that the amount of premature translation termination from control mRNAs played a role in determining the calculated −1PRF efficiency. Co-expression of CfMV P27 with the dual reporter vector containing the minimal frameshift signal reduced the production of the downstream reporter, whereas replicase co-expression had no pronounced effect. This finding allows us to propose that CfMV protein P27 may influence translation at the frameshift site but the mechanism needs to be elucidated. | [
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In Australia, compared with other developed countries the many and varied programs which comprise public health have continued to be funded poorly and unsystematically, particularly given the amount of publicly voiced political support. In 2003, the major public health policy developments in communicable disease control were in the fields of SARS, and vaccine funding, whilst the TGA was focused on the Pan Pharmaceutical crisis. Programs directed to health maintenance and healthy ageing were approved. The tertiary education sector was involved in the development of programs for training the public health workforce and new professional qualifications and competencies. The Abelson Report received support from overseas experts, providing a potential platform for calls to improve national funding for future Australian preventive programs; however, inconsistencies continued across all jurisdictions in their approaches to tackling national health priorities. Despite 2004 being an election year, public health policy was not visible, with the bulk of the public health funding available in the 2004/05 federal budget allocated to managing such emerging risks as avian flu. We conclude by suggesting several implications for the future. | [
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] |
GIDEON (Global Infectious Diseases and Epidemiology Network) is a web-based computer program designed for decision support and informatics in the field of Geographic Medicine. The first of four interactive modules generates a ranked differential diagnosis based on patient signs, symptoms, exposure history and country of disease acquisition. Additional options include syndromic disease surveillance capability and simulation of bioterrorism scenarios. The second module accesses detailed and current information regarding the status of 338 individual diseases in each of 220 countries. Over 50,000 disease images, maps and user-designed graphs may be downloaded for use in teaching and preparation of written materials. The third module is a comprehensive source on the use of 328 anti-infective drugs and vaccines, including a listing of over 9,500 international trade names. The fourth module can be used to characterize or identify any bacterium or yeast, based on laboratory phenotype. GIDEON is an up-to-date and comprehensive resource for Geographic Medicine. | [
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This debut editorial of Globalization and Health introduces the journal, briefly delineating its goals and objectives and outlines its scope of subject matter. 'Open Access' publishing is expected to become an increasingly important format for peer reviewed academic journals and that Globalization and Health is 'Open Access' is appropriate. The rationale behind starting a journal dedicated to globalization and health is three fold: Firstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health – be they a SARS virus or a predilection for fatty foods – have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing. Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve. And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic. A balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first. | [
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] |
In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNA(Ser)CAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m1G37 is critical for leucylation activity. This finding was supported by the fact that the tRNA(Ser)CAGs possessing the leucylation activity always have m1G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNA(Ser)CAGs possessing m1G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNA(Ser)CAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a 'polysemous codon'. | [
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At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication. | [
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] |
BACKGROUND: Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein σ3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating. RESULTS: The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis. CONCLUSION: Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections. | [
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The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA. | [
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Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(−3)–10(−5) (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. | [
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BACKGROUND: Epitopes can be defined as the molecular structures bound by specific receptors, which are recognized during immune responses. The Immune Epitope Database and Analysis Resource (IEDB) project will catalog and organize information regarding antibody and T cell epitopes from infectious pathogens, experimental antigens and self-antigens, with a priority on NIAID Category A-C pathogens () and emerging/re-emerging infectious diseases. Both intrinsic structural and phylogenetic features, as well as information relating to the interactions of the epitopes with the host's immune system will be catalogued. DESCRIPTION: To effectively represent and communicate the information related to immune epitopes, a formal ontology was developed. The semantics of the epitope domain and related concepts were captured as a hierarchy of classes, which represent the general and specialized relationships between the various concepts. A complete listing of classes and their properties can be found at . CONCLUSION: The IEDB's ontology is the first ontology specifically designed to capture both intrinsic chemical and biochemical information relating to immune epitopes with information relating to the interaction of these structures with molecules derived from the host immune system. We anticipate that the development of this type of ontology and associated databases will facilitate rigorous description of data related to immune epitopes, and might ultimately lead to completely new methods for describing and modeling immune responses. | [
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] |
BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon. | [
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Zoonoses, diseases that jump from animals to humans, are a growing health problem around the world. Understanding their causes and their effects on humans have therefore become an important topic for global public health | [
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BACKGROUND AND METHODS: Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR. RESULTS: We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10(5 )viral copies/ml in nasal lavage and 1.88 × 10(5 )viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD. CONCLUSION: HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely. | [
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The BIG project looks at some of the ethical concerns surrounding globalization and health. | [
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BACKGROUND: The present study aimed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. METHODS: A nationwide representative sample of 2,000 Japanese aged 20 or older was asked about their perception and level of concern about various environmental and genetic risk factors in relation to cancer prevention, as a part of an Omnibus Survey. Interviews were conducted with 1,355 subjects (609 men and 746 women). RESULTS: Among 12 risk factor candidates, the attributable fraction of cancer-causing viral and bacterial infection was considered highest (51%), followed by that of tobacco smoking (43%), stress (39%), and endocrine-disrupting chemicals (37%). On the other hand, the attributable fractions of cancer by charred fish and meat (21%) and alcohol drinking (22%) were considered low compared with other risk factor candidates. For most risk factors, attributable fraction responses were higher in women than in men. As a whole, the subjects tended to respond with higher values than those estimated by epidemiologic evidence in the West. The attributable fraction of cancer speculated to be genetically determined was 32%, while 36% of cancer was considered preventable by improving lifestyle. CONCLUSION: Our results suggest that awareness of the attributable fraction of cancer causes in the Japanese general population tends to be dominated by cancer-causing infection, occupational exposure, air pollution and food additives rather than major lifestyle factors such as diet. | [
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BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device. | [
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Illicit drug abuse and HIV/AIDS have increased rapidly in the past 10 to 20 years in China. This paper reviews drug abuse in China, the HIV/AIDS epidemic and its association with injection drug use (IDU), and Chinese policies on illicit drug abuse and prevention of HIV/AIDS based on published literature and unpublished official data. As a major drug trans-shipment country with source drugs from the "Golden Triangle" and "Gold Crescent" areas in Asia, China has also become an increasingly important drug consuming market. About half of China's 1.14 million documented drug users inject, and many share needles. IDU has contributed to 42% of cumulatively reported HIV/AIDS cases thus far. Drug trafficking is illegal in China and can lead to the death penalty. The public security departments adopt "zero tolerance" approach to drug use, which conflict with harm reduction policies of the public health departments. Past experience in China suggests that cracking down on drug smuggling and prohibiting drug use alone can not prevent or solve all illicit drug related problems in the era of globalization. In recent years, the central government has outlined a series of pragmatic policies to encourage harm reduction programs; meanwhile, some local governments have not fully mobilized to deal with drug abuse and HIV/AIDS problems seriously. Strengthening government leadership at both central and local levels; scaling up methadone substitution and needle exchange programs; making HIV voluntary counseling and testing available and affordable to both urban and rural drug users; and increasing utilization of outreach and nongovernmental organizations are offered as additional strategies to help cope with China's HIV and drug abuse problem. | [
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] |
Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nm1240) contains supplementary material, which is available to authorized users. | [
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BACKGROUND: Current national preparedness plans require local health departments to play an integral role in responding to an influenza pandemic, a major public health threat that the World Health Organization has described as "inevitable and possibly imminent". To understand local public health workers' perceptions toward pandemic influenza response, we surveyed 308 employees at three health departments in Maryland from March – July 2005, on factors that may influence their ability and willingness to report to duty in such an event. RESULTS: The data suggest that nearly half of the local health department workers are likely not to report to duty during a pandemic. The stated likelihood of reporting to duty was significantly greater for clinical (Multivariate OR: 2.5; CI 1.3–4.7) than technical and support staff, and perception of the importance of one's role in the agency's overall response was the single most influential factor associated with willingness to report (Multivariate OR: 9.5; CI 4.6–19.9). CONCLUSION: The perceived risk among public health workers was shown to be associated with several factors peripheral to the actual hazard of this event. These risk perception modifiers and the knowledge gaps identified serve as barriers to pandemic influenza response and must be specifically addressed to enable effective local public health response to this significant threat. | [
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BACKGROUND: As a number of commentators have noted, SARS exposed the vulnerabilities of our health care systems and governance structures. Health care professionals (HCPs) and hospital systems that bore the brunt of the SARS outbreak continue to struggle with the aftermath of the crisis. Indeed, HCPs – both in clinical care and in public health – were severely tested by SARS. Unprecedented demands were placed on their skills and expertise, and their personal commitment to their profession was severely tried. Many were exposed to serious risk of morbidity and mortality, as evidenced by the World Health Organization figures showing that approximately 30% of reported cases were among HCPs, some of whom died from the infection. Despite this challenge, professional codes of ethics are silent on the issue of duty to care during communicable disease outbreaks, thus providing no guidance on what is expected of HCPs or how they ought to approach their duty to care in the face of risk. DISCUSSION: In the aftermath of SARS and with the spectre of a pandemic avian influenza, it is imperative that we (re)consider the obligations of HCPs for patients with severe infectious diseases, particularly diseases that pose risks to those providing care. It is of pressing importance that organizations representing HCPs give clear indication of what standard of care is expected of their members in the event of a pandemic. In this paper, we address the issue of special obligations of HCPs during an infectious disease outbreak. We argue that there is a pressing need to clarify the rights and responsibilities of HCPs in the current context of pandemic flu preparedness, and that these rights and responsibilities ought to be codified in professional codes of ethics. Finally, we present a brief historical accounting of the treatment of the duty to care in professional health care codes of ethics. SUMMARY: An honest and critical examination of the role of HCPs during communicable disease outbreaks is needed in order to provide guidelines regarding professional rights and responsibilities, as well as ethical duties and obligations. With this paper, we hope to open the social dialogue and advance the public debate on this increasingly urgent issue. | [
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] |
The anticancer potential of catechins derived from green tea is not well understood, in part because catechin-related growth suppression and/or apoptosis appears to vary with the type and stage of malignancy as well as with the type of catechin. This in vitro study examined the biological effects of epicatechin (EC), epigallocatechin (EGC), EC 3-gallate (ECG) and EGC 3-gallate (EGCG) in cell lines from human gender-specific cancers. Cell lines developed from organ-confined (HH870) and metastatic (DU145) prostate cancer, and from moderately (HH450) and poorly differentiated (HH639) epithelial ovarian cancer were grown with or without EC, EGC, ECG or EGCG. When untreated cells reached confluency, viability and doubling time were measured for treated and untreated cells. Whereas EC treatment reduced proliferation of HH639 cells by 50%, EGCG suppressed proliferation of all cell lines by 50%. ECG was even more potent: it inhibited DU145, HH870, HH450 and HH639 cells at concentrations of 24, 27, 29 and 30 µM, whereas EGCG inhibited DU145, HH870, HH450 and HH639 cells at concentrations 89, 45, 62 and 42 µM. When compared with EGCG, ECG more effectively suppresses the growth of prostate cancer and epithelial ovarian cancer cell lines derived from tumors of patients with different stages of disease. | [
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BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) can experience 'exacerbations' of their conditions. An exacerbation is an event defined in terms of subjective descriptors or symptoms, namely dyspnoea, cough and sputum that worsen sufficiently to warrant a change in medical management. There is a need for reliable markers that reflect the pathological mechanisms that underlie exacerbation severity and that can be used as a surrogate to assess treatment effects in clinical studies. Little is known as to how existing study variables and suggested markers change in both the stable and exacerbation phases of COPD. In an attempt to find the best surrogates for exacerbations, we have reviewed the literature to identify which of these markers change in a consistent manner with the severity of the exacerbation event. METHODS: We have searched standard databases between 1966 to July 2004 using major keywords and terms. Studies that provided demographics, spirometry, potential markers, and clear eligibility criteria were included in this study. Central tendencies and dispersions for all the variables and markers reported and collected by us were first tabulated according to sample size and ATS/ERS 2004 Exacerbation Severity Levels I to III criteria. Due to the possible similarity of patients in Levels II and III, the data was also redefined into categories of exacerbations, namely out-patient (Level I) and in-patient (Levels II & III combined). For both approaches, we performed a fixed effect meta-analysis on each of the reported variables. RESULTS: We included a total of 268 studies reported between 1979 to July 2004. These studies investigated 142,407 patients with COPD. Arterial carbon dioxide tension and breathing rate were statistically different between all levels of exacerbation severity and between in out- and in-patient settings. Most other measures showed weak relationships with either level or setting, or they had insufficient data to permit meta-analysis. CONCLUSION: Arterial carbon dioxide and breathing rate varied in a consistent manner with exacerbation severity and patient setting. Many other measures showed weak correlations that should be further explored in future longitudinal studies or assessed using suggested mathematical modelling techniques. | [
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|
BACKGROUND: The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. RESULTS: We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis. CONCLUSION: The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies. | [
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The development of DNA microarray technology a decade ago led to the establishment of functional genomics as one of the most active and successful scientific disciplines today. With the ongoing development of immunomic microarray technology—a spatially addressable, large-scale technology for measurement of specific immunological response—the new challenge of functional immunomics is emerging, which bears similarities to but is also significantly different from functional genomics. Immunonic data has been successfully used to identify biological markers involved in autoimmune diseases, allergies, viral infections such as human immunodeficiency virus (HIV), influenza, diabetes, and responses to cancer vaccines. This review intends to provide a coherent vision of this nascent scientific field, and speculate on future research directions. We discuss at some length issues such as epitope prediction, immunomic microarray technology and its applications, and computation and statistical challenges related to functional immunomics. Based on the recent discovery of regulation mechanisms in T cell responses, we envision the use of immunomic microarrays as a tool for advances in systems biology of cellular immune responses, by means of immunomic regulatory network models. | [
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BACKGROUND: Because smallpox (variola major) may be used as a biological weapon, we reviewed outbreaks in post-World War II Europe and North America in order to understand smallpox transmission patterns. METHODS: A systematic review was used to identify papers from the National Library of Medicine, Embase, Biosis, Cochrane Library, Defense Technical Information Center, WorldCat, and reference lists of included publications. Two authors reviewed selected papers for smallpox outbreaks. RESULTS: 51 relevant outbreaks were identified from 1,389 publications. The median for the effective first generation reproduction rate (initial R) was 2 (range 0–38). The majority outbreaks were small (less than 5 cases) and contained within one generation. Outbreaks with few hospitalized patients had low initial R values (median of 1) and were prolonged if not initially recognized (median of 3 generations); outbreaks with mostly hospitalized patients had higher initial R values (median 12) and were shorter (median of 3 generations). Index cases with an atypical presentation of smallpox were less likely to have been diagnosed with smallpox; outbreaks in which the index case was not correctly diagnosed were larger (median of 27.5 cases) and longer (median of 3 generations) compared to outbreaks in which the index case was correctly diagnosed (median of 3 cases and 1 generation). CONCLUSION: Patterns of spread during Smallpox outbreaks varied with circumstances, but early detection and implementation of control measures is a most important influence on the magnitude of outbreaks. The majority of outbreaks studied in Europe and North America were controlled within a few generations if detected early. | [
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BACKGROUND: Case definitions have been recognized to be important elements of public health surveillance systems. They are to assure comparability and consistency of surveillance data and have crucial impact on the sensitivity and the positive predictive value of a surveillance system. The reliability of case definitions has rarely been investigated systematically. METHODS: We conducted a Round-Robin test by asking all 425 local health departments (LHD) and the 16 state health departments (SHD) in Germany to classify a selection of 68 case examples using case definitions. By multivariate analysis we investigated factors linked to classification agreement with a gold standard, which was defined by an expert panel. RESULTS: A total of 7870 classifications were done by 396 LHD (93%) and all SHD. Reporting sensitivity was 90.0%, positive predictive value 76.6%. Polio case examples had the lowest reporting precision, salmonellosis case examples the highest (OR = 0.008; CI: 0.005–0.013). Case definitions with a check-list format of clinical criteria resulted in higher reporting precision than case definitions with a narrative description (OR = 3.08; CI: 2.47–3.83). Reporting precision was higher among SHD compared to LHD (OR = 1.52; CI: 1.14–2.02). CONCLUSION: Our findings led to a systematic revision of the German case definitions and build the basis for general recommendations for the creation of case definitions. These include, among others, that testable yes/no criteria in a check-list format is likely to improve reliability, and that software used for data transmission should be designed in strict accordance with the case definitions. The findings of this study are largely applicable to case definitions in many other countries or international networks as they share the same structural and editorial characteristics of the case definitions evaluated in this study before their revision. | [
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Ventilator associated pneumonia (VAP) is the leading cause of morbidity and mortality in intensive care units. The incidence of VAP varies from 7% to 70% in different studies and the mortality rates are 20–75% according to the study population. Aspiration of colonized pathogenic microorganisms on the oropharynx and gastrointestinal tract is the main route for the development of VAP. On the other hand, the major risk factor for VAP is intubation and the duration of mechanical ventilation. Diagnosis remains difficult, and studies showed the importance of early initiation of appropriate antibiotic for prognosis. VAP causes extra length of stay in hospital and intensive care units and increases hospital cost. Consequently, infection control policies are more rational and will save money. | [
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] |
BACKGROUND: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region. METHODS: We established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs. RESULTS: During this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information. CONCLUSION: Knowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease. | [
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|
Live-virus vaccines activate both humoral and cell-mediated immunity, require only a single boosting, and generally provide longer immune protection than killed or subunit vaccines. However, growth of live-virus vaccines must be attenuated to minimize their potential pathogenic effects, and mechanisms of attenuation by conventional serial-transfer viral adaptation are not well-understood. New methods of attenuation based on rational engineering of viral genomes may offer a potentially greater control if one can link defined genetic modifications to changes in virus growth. To begin to establish such links between genotype and growth phenotype, we developed a computer model for the intracellular growth of vesicular stomatitis virus (VSV), a well-studied, nonsegmented, negative-stranded RNA virus. Our model incorporated established regulatory mechanisms of VSV while integrating key wild-type infection steps: hijacking of host resources, transcription, translation, and replication, followed by assembly and release of progeny VSV particles. Generalization of the wild-type model to allow for genome rearrangements matched the experimentally observed attenuation ranking for recombinant VSV strains that altered the genome position of their nucleocapsid gene. Finally, our simulations captured previously reported experimental results showing how altering the positions of other VSV genes has the potential to attenuate the VSV growth while overexpressing the immunogenic VSV surface glycoprotein. Such models will facilitate the engineering of new live-virus vaccines by linking genomic manipulations to controlled changes in virus gene-expression and growth. | [
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] |
RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described. | [
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] |
Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electro-chemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays. | [
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] |
BACKGROUND: The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. METHODS: Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. RESULTS: From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. CONCLUSION: We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. | [
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BACKGROUND: EpiFlex is a flexible, easy to use computer model for a single computer, intended to be operated by one user who need not be an expert. Its purpose is to study in-silico the epidemic behavior of a wide variety of diseases, both known and theoretical, by simulating their spread at the level of individuals contracting and infecting others. To understand the system fully, this paper must be read together in conjunction with study of the software and its results. EpiFlex is evaluated using results from modeling influenza A epidemics and comparing them with a variety of field data sources and other types of modeling. EpiFlex is an object-oriented Monte Carlo system, allocating entities to correspond to individuals, disease vectors, diseases, and the locations that hosts may inhabit. EpiFlex defines eight different contact types available for a disease. Contacts occur inside locations within the model. Populations are composed of demographic groups, each of which has a cycle of movement between locations. Within locations, superspreading is defined by skewing of contact distributions. RESULTS: EpiFlex indicates three phenomena of interest for public health: (1) R(0 )is variable, and the smaller the population, the larger the infected fraction within that population will be; (2) significant compression/synchronization between cities by a factor of roughly 2 occurs between the early incubation phase of a multi-city epidemic and the major manifestation phase; (3) if better true morbidity data were available, more asymptomatic hosts would be seen to spread disease than we currently believe is the case for influenza. These results suggest that field research to study such phenomena, while expensive, should be worthwhile. CONCLUSION: Since EpiFlex shows all stages of disease progression, detailed insight into the progress of epidemics is possible. EpiFlex shows the characteristic multimodality and apparently random variation characteristic of real world data, but does so as an emergent property of a carefully constructed model of disease dynamics and is not simply a stochastic system. EpiFlex can provide a better understanding of infectious diseases and strategies for response. | [
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] |
BACKGROUND: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. METHODS: MultiCode(®) PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in ~3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. RESULTS: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. CONCLUSIONS: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting. | [
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Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels. | [
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] |
The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a α-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a −1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed −1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea. | [
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Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly >200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. | [
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BACKGROUND: Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. RESULTS: We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. CONCLUSION: A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at . | [
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In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2(−/−)mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2(−/−)mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2(−/−)mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2(−/−)mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination. | [
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There is an increasing recognition that detailed nucleic acid sequence information will be useful and even required in the diagnosis, treatment and surveillance of many significant pathogens. Because generating detailed information about pathogens leads to significantly larger amounts of data, it is necessary to develop automated analysis methods to reduce analysis time and to standardize identification criteria. This is especially important for multiple pathogen assays designed to reduce assay time and costs. In this paper, we present a successful algorithm for detecting pathogens and reporting the maximum level of detail possible using multi-pathogen resequencing microarrays. The algorithm filters the sequence of base calls from the microarray and finds entries in genetic databases that most closely match. Taxonomic databases are then used to relate these entries to each other so that the microorganism can be identified. Although developed using a resequencing microarray, the approach is applicable to any assay method that produces base call sequence information. The success and continued development of this approach means that a non-expert can now perform unassisted analysis of the results obtained from partial sequence data. | [
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] |
Accuracy in translation of the genetic code into proteins depends upon correct tRNA–mRNA recognition in the context of the ribosome. In human [Formula: see text] three modified bases are present in the anticodon stem–loop—2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms(2)t(6)A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm(5)s(2)U34) and pseudouridine (ψ) at position 39—two of which, ms(2)t(6)A37 and mcm(5)s(2)U34, are required to achieve wild-type binding activity of wild-type human [Formula: see text] [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390–13395]. Molecular dynamics simulations of nine tRNA anticodon stem–loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms(2)t(6) modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms(2)t(6)A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to ψ39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps. | [
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] |
Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection. | [
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BACKGROUND: Planning for the next pandemic influenza outbreak is underway in hospitals across the world. The global SARS experience has taught us that ethical frameworks to guide decision-making may help to reduce collateral damage and increase trust and solidarity within and between health care organisations. Good pandemic planning requires reflection on values because science alone cannot tell us how to prepare for a public health crisis. DISCUSSION: In this paper, we present an ethical framework for pandemic influenza planning. The ethical framework was developed with expertise from clinical, organisational and public health ethics and validated through a stakeholder engagement process. The ethical framework includes both substantive and procedural elements for ethical pandemic influenza planning. The incorporation of ethics into pandemic planning can be helped by senior hospital administrators sponsoring its use, by having stakeholders vet the framework, and by designing or identifying decision review processes. We discuss the merits and limits of an applied ethical framework for hospital decision-making, as well as the robustness of the framework. SUMMARY: The need for reflection on the ethical issues raised by the spectre of a pandemic influenza outbreak is great. Our efforts to address the normative aspects of pandemic planning in hospitals have generated interest from other hospitals and from the governmental sector. The framework will require re-evaluation and refinement and we hope that this paper will generate feedback on how to make it even more robust. | [
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INTRODUCTION: Acute respiratory distress syndrome (ARDS) has heterogeneous etiologies, rapid progressive change and a high mortality rate. To improve the outcome of ARDS, accurate diagnosis is essential to the application of effective early treatment. The present study investigated the clinical effects and safety of open lung biopsy (OLB) in patients with early-stage ARDS of suspected non-infectious origin. METHODS: We undertook a retrospective study of 41 patients with early-stage ARDS (defined as one week or less after intubation) who underwent OLB in two medical intensive care units of a tertiary care hospital from 1999 to 2005. Data analyzed included baseline characteristics, complication rate, pathological diagnoses, treatment alterations, and hospital survival. RESULTS: The age of patients was 55 ± 17 years (mean ± SD). The average ratio of arterial partial pressure of oxygen (PaO(2)) to fraction of inspired oxygen (FiO(2)) was 116 ± 43 mmHg (mean ± SD) at biopsy. Seventeen patients (41%) were immunocompromised. Postoperative complications occurred in 20% of patients (8/41). All biopsies provided a pathological diagnosis with a diagnostic yield of 100%. Specific pathological diagnoses were made for 44% of patients (18/41). Biopsy findings led to an alteration of treatment modality in 73% of patients (30/41). The treatment alteration rate was higher in patients with nonspecific diagnoses than in patients with specific diagnoses (p = 0.0024). Overall mortality was 50% (21/41) and was not influenced by age, gender, pre-OLB oxygenation, complication rate, pathological results, and alteration of treatment. There was no surgery-related mortality. The survival rate for immunocompromised patients was better than that for immunocompetent patients (71% versus 33%; p = 0.0187) in this study. CONCLUSION: Our retrospective study suggests that OLB was a useful and acceptably safe diagnostic procedure in some selected patients with early-stage ARDS. | [
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The distribution of multi-host pathogens over their host range conditions their population dynamics and structure. Also, host co-infection by different pathogens may have important consequences for the evolution of hosts and pathogens, and host-pathogen co-evolution. Hence it is of interest to know if the distribution of pathogens over their host range is random, or if there are associations between hosts and pathogens, or between pathogens sharing a host. To analyse these issues we propose indices for the observed patterns of host infection by pathogens, and for the observed patterns of co-infection, and tests to analyse if these patterns conform to randomness or reflect associations. Applying these tests to the prevalence of five plant viruses on 21 wild plant species evidenced host-virus associations: most hosts and viruses were selective for viruses and hosts, respectively. Interestingly, the more host-selective viruses were the more prevalent ones, suggesting that host specialisation is a successful strategy for multi-host pathogens. Analyses also showed that viruses tended to associate positively in co-infected hosts. The developed indices and tests provide the tools to analyse how strong and common are these associations among different groups of pathogens, which will help to understand and model the population biology of multi-host pathogens. | [
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BACKGROUND: Contact tracing plays an important role in the control of emerging infectious diseases, but little is known yet about its effectiveness. Here we deduce from a generic mathematical model how effectiveness of tracing relates to various aspects of time, such as the course of individual infectivity, the (variability in) time between infection and symptom-based detection, and delays in the tracing process. In addition, the possibility of iteratively tracing of yet asymptomatic infecteds is considered. With these insights we explain why contact tracing was and will be effective for control of smallpox and SARS, only partially effective for foot-and-mouth disease, and likely not effective for influenza. METHODS AND FINDINGS: We investigate contact tracing in a model of an emerging epidemic that is flexible enough to use for most infections. We consider isolation of symptomatic infecteds as the basic scenario, and express effectiveness as the proportion of contacts that need to be traced for a reproduction ratio smaller than 1. We obtain general results for special cases, which are interpreted with respect to the likely success of tracing for influenza, smallpox, SARS, and foot-and-mouth disease epidemics. CONCLUSIONS: We conclude that (1) there is no general predictive formula for the proportion to be traced as there is for the proportion to be vaccinated; (2) variability in time to detection is favourable for effective tracing; (3) tracing effectiveness need not be sensitive to the duration of the latent period and tracing delays; (4) iterative tracing primarily improves effectiveness when single-step tracing is on the brink of being effective. | [
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BACKGROUND: The time delay between the start of an influenza pandemic and its subsequent initiation in other countries is highly relevant to preparedness planning. We quantify the distribution of this random time in terms of the separate components of this delay, and assess how the delay may be extended by non-pharmaceutical interventions. METHODS AND FINDINGS: The model constructed for this time delay accounts for: (i) epidemic growth in the source region, (ii) the delay until an infected individual from the source region seeks to travel to an at-risk country, (iii) the chance that infected travelers are detected by screening at exit and entry borders, (iv) the possibility of in-flight transmission, (v) the chance that an infected arrival might not initiate an epidemic, and (vi) the delay until infection in the at-risk country gathers momentum. Efforts that reduce the disease reproduction number in the source region below two and severe travel restrictions are most effective for delaying a local epidemic, and under favourable circumstances, could add several months to the delay. On the other hand, the model predicts that border screening for symptomatic infection, wearing a protective mask during travel, promoting early presentation of cases arising among arriving passengers and moderate reduction in travel volumes increase the delay only by a matter of days or weeks. Elevated in-flight transmission reduces the delay only minimally. CONCLUSIONS: The delay until an epidemic of pandemic strain influenza is imported into an at-risk country is largely determined by the course of the epidemic in the source region and the number of travelers attempting to enter the at-risk country, and is little affected by non-pharmaceutical interventions targeting these travelers. Short of preventing international travel altogether, eradicating a nascent pandemic in the source region appears to be the only reliable method of preventing country-to-country spread of a pandemic strain of influenza. | [
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] |
Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody. | [
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BACKGROUND: Recent international and national events have brought critical attention to the Canadian public health system and how prepared the system is to respond to various types of contemporary public health threats. This article describes the study design and methods being used to conduct a systems-level analysis of public health preparedness in the province of Alberta, Canada. The project is being funded under the Health Research Fund, Alberta Heritage Foundation for Medical Research. METHODS/DESIGN: We use an embedded, multiple-case study design, integrating qualitative and quantitative methods to measure empirically the degree of inter-organizational coordination existing among public health agencies in Alberta, Canada. We situate our measures of inter-organizational network ties within a systems-level framework to assess the relative influence of inter-organizational ties, individual organizational attributes, and institutional environmental features on public health preparedness. The relative contribution of each component is examined for two potential public health threats: pandemic influenza and West Nile virus. DISCUSSION: The organizational dimensions of public health preparedness depend on a complex mix of individual organizational characteristics, inter-agency relationships, and institutional environmental factors. Our study is designed to discriminate among these different system components and assess the independent influence of each on the other, as well as the overall level of public health preparedness in Alberta. While all agree that competent organizations and functioning networks are important components of public health preparedness, this study is one of the first to use formal network analysis to study the role of inter-agency networks in the development of prepared public health systems. | [
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] |
Inflammation in the middle ear mucosa, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions and reflux, is the crucial event in the pathogenesis of otitis media with effusion (OME). Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic inflammatory processes and stimulate the chronic condition of OME. Cytokines are the central molecular regulators of middle ear inflammation and can switch the acute phase of inflammation in the chronic stage and induce molecular-pathological processes leading to the histopathological changes accompanying OME. In this review we present cytokines identified in otitis media, immunoregulatory [interleukin (IL)-2, IL-10, transforming growth factor-beta]) and allergy associated (IL-4, IL-5, granulocyte-macrophage colony-stimulating factor), as crucial molecular regulators, responsible for chronic inflammation in the middle ear and the chronic condition of OME. | [
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Cognitive dysfunction is common in critically ill patients, not only during the acute illness but also long after its resolution. A large number of pathophysiologic mechanisms are thought to underlie critical illness-associated cognitive dysfunction, including neuro-transmitter abnormalities and occult diffuse brain injury. Markers that could be used to evaluate the influence of specific mechanisms in individual patients include serum anticholinergic activity, certain brain proteins, and tissue sodium concentration determination via high-resolution three-dimensional magnetic resonance imaging. Although recent therapeutic advances in this area are exciting, they are still too immature to influence patient care. Additional research is needed if we are to understand better the relative contributions of specific mechanisms to the development of critical illness-associated cognitive dysfunction and to determine whether these mechanisms might be amenable to treatment or prevention. | [
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] |
Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approach for analyzing many types of protein microarrays. | [
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Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A γ-aminobutyric acid receptor (GABA(A)R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA(A)R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system. | [
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BACKGROUND: With the increased occurrence of outbreaks of H5N1 worldwide there is concern that the virus could enter commercial poultry farms with severe economic consequences. METHODOLOGY/PRINCIPAL FINDINGS: We analyse data from four recent outbreaks of highly pathogenic avian influenza (HPAI) in commercial poultry to estimate the farm-to-farm reproductive number for HPAI. The reproductive number is a key measure of the transmissibility of HPAI at the farm level because it can be used to evaluate the effectiveness of the control measures. In these outbreaks the mean farm-to-farm reproductive number prior to controls ranged from 1.1 to 2.4, with the maximum farm-based reproductive number in the range 2.2 to 3.2. Enhanced bio-security, movement restrictions and prompt isolation of the infected farms in all four outbreaks substantially reduced the reproductive number, but it remained close to the threshold value 1 necessary to ensure the disease will be eradicated. CONCLUSIONS/SIGNIFICANCE: Our results show that depending on the particular situation in which an outbreak of avian influenza occurs, current controls might not be enough to eradicate the disease, and therefore a close monitoring of the outbreak is required. The method we used for estimating the reproductive number is straightforward to implement and can be used in real-time. It therefore can be a useful tool to inform policy decisions. | [
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] |
BACKGROUND: Planning public health responses against pandemic influenza relies on predictive models by which the impact of different intervention strategies can be evaluated. Research has to date rather focused on producing predictions for certain localities or under specific conditions, than on designing a publicly available planning tool which can be applied by public health administrations. Here, we provide such a tool which is reproducible by an explicitly formulated structure and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. RESULTS: InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java, operates platform independent and can be executed on regular desktop computers. CONCLUSION: InfluSim is an online available software which efficiently assists public health planners in designing optimal interventions against pandemic influenza. It can reproduce the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability. | [
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Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens. | [
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] |
BACKGROUND: Ubiquitin and ubiquitin-like proteins (Ubl) are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. FINDINGS: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs). Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. CONCLUSION: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan. | [
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A common overlapping site on the N-terminal IgV-like domain of human carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) is targeted by several important human respiratory pathogens. These include Neisseria meningitidis (Nm) and Haemophilus influenzae (Hi) that can cause disseminated or persistent localized infections. To define the precise structural features that determine the binding of distinct pathogens with CEACAMs, we have undertaken molecular modelling and mutation of the receptor molecules at previously implicated key target residues required for bacterial binding. These include Ser-32, Tyr-34, Val-39, Gln-44 and Gln-89, in addition to Ile-91, the primary docking site for the pathogens. Most, but not all, of these residues located adjacent to each other in a previous N-domain model of human CEACAM1, which was based on REI, CD2 and CD4. In the current studies, we have refined this model based on the mouse CEACAM1 crystal structure, and observe that all of the above residues form an exposed continuous binding region on the N-domain. Examination of the model also suggested that substitution of two of these residues 34 and 89 could affect the accessibility of Ile-91 for ligand binding. By introducing selected mutations at the positions 91, 34 and 89, we confirmed the primary importance of Ile-91 in all bacterial binding to CEACAM1 despite the inter- and intraspecies structural differences between the bacterial CEACAM-binding ligands. The studies further indicated that the efficiency of binding was significantly enhanced for specific strains by mutations such as Y34F and Q89N, which also altered the hierarchy of Nm versus Hi strain binding. These studies imply that distinct polymorphisms in human epithelial CEACAMs have the potential to decrease or increase the risk of infection by the receptor-targeting pathogens. | [
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] |
The immune reconstitution inflammatory syndrome (IRIS) in HIV-infected patients initiating antiretroviral therapy (ART) results from restored immunity to specific infectious or non-infectious antigens. A paradoxical clinical worsening of a known condition or the appearance of a new condition after initiating therapy characterizes the syndrome. Potential mechanisms for the syndrome include a partial recovery of the immune system or exuberant host immunological responses to antigenic stimuli. The overall incidence of IRIS is unknown, but is dependent on the population studied and its underlying opportunistic infectious burden. The infectious pathogens most frequently implicated in the syndrome are mycobacteria, varicella zoster, herpesviruses, and cytomegalovirus (CMV). No single treatment option exists and depends on the underlying infectious agent and its clinical presentation. Prospective cohort studies addressing the optimal screening and treatment of opportunistic infections in patients eligible for ART are currently being conducted. These studies will provide evidence for the development of treatment guidelines in order to reduce the burden of IRIS. We review the available literature on the pathogenesis and epidemiology of IRIS, and present treatment options for the more common infectious manifestations of this diverse syndrome and for manifestations associated with a high morbidity. | [
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BACKGROUND: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. METHODS AND PRINCIPAL FINDINGS: Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. CONCLUSION/SIGNIFICANCE: Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance. | [
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] |
Despite intensive ongoing research, key aspects of the spatial-temporal evolution of the 2001 foot and mouth disease (FMD) epidemic in Great Britain (GB) remain unexplained. Here we develop a Markov Chain Monte Carlo (MCMC) method for estimating epidemiological parameters of the 2001 outbreak for a range of simple transmission models. We make the simplifying assumption that infectious farms were completely observed in 2001, equivalent to assuming that farms that were proactively culled but not diagnosed with FMD were not infectious, even if some were infected. We estimate how transmission parameters varied through time, highlighting the impact of the control measures on the progression of the epidemic. We demonstrate statistically significant evidence for assortative contact patterns between animals of the same species. Predictive risk maps of the transmission potential in different geographic areas of GB are presented for the fitted models. | [
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Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems. | [
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BACKGROUND: Since 2001, state and local health departments in the United States (US) have accelerated efforts to prepare for high-impact public health emergencies. One component of these activities has been the development and conduct of exercise programs to assess capabilities, train staff and build relationships. This paper summarizes lessons learned from tabletop exercises about public health emergency preparedness and about the process of developing, conducting, and evaluating them. METHODS: We developed, conducted, and evaluated 31 tabletop exercises in partnership with state and local health departments throughout the US from 2003 to 2006. Participant self evaluations, after action reports, and tabletop exercise evaluation forms were used to identify aspects of the exercises themselves, as well as public health emergency responses that participants found more or less challenging, and to highlight lessons learned about tabletop exercise design. RESULTS: Designing the exercises involved substantial collaboration with representatives from participating health departments to assure that the scenarios were credible, focused attention on local preparedness needs and priorities, and were logistically feasible to implement. During execution of the exercises, nearly all health departments struggled with a common set of challenges relating to disease surveillance, epidemiologic investigations, communications, command and control, and health care surge capacity. In contrast, performance strengths were more varied across participating sites, reflecting specific attributes of individual health departments or communities, experience with actual public health emergencies, or the emphasis of prior preparedness efforts. CONCLUSION: The design, conduct, and evaluation of the tabletop exercises described in this report benefited from collaborative planning that involved stakeholders from participating health departments and exercise developers and facilitators from outside the participating agencies. While these exercises identified both strengths and vulnerabilities in emergency preparedness, additional work is needed to develop reliable metrics to gauge exercise performance, inform follow-up action steps, and to develop re-evaluation exercise designs that assess the impact of post-exercise interventions. | [
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] |
BACKGROUND: The wide use of Affymetrix microarray in broadened fields of biological research has made the probeset annotation an important issue. Standard Affymetrix probeset annotation is at gene level, i.e. a probeset is precisely linked to a gene, and probeset intensity is interpreted as gene expression. The increased knowledge that one gene may have multiple transcript variants clearly brings up the necessity of updating this gene-level annotation to a refined transcript-level. RESULTS: Through performing rigorous alignments of the Affymetrix probe sequences against a comprehensive pool of currently available transcript sequences, and further linking the probesets to the International Protein Index, we generated transcript-level or protein-level annotation tables for two popular Affymetrix expression arrays, Mouse Genome 430A 2.0 Array and Human Genome U133A Array. Application of our new annotations in re-examining existing expression data sets shows increased expression consistency among synonymous probesets and strengthened expression correlation between interacting proteins. CONCLUSION: By refining the standard Affymetrix annotation of microarray probesets from the gene level to the transcript level and protein level, one can achieve a more reliable interpretation of their experimental data, which may lead to discovery of more profound regulatory mechanism. | [
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