A few studies have examined relationships between mercury exposures and adverse immunological reactions in humans, but there is little evidence of mercury-associated autoimmunity in humans.Mercury is an immunotoxic metal that induces autoimmune disease in rodents. Highly susceptible mouse strains such as SJL/N, A.SW, B10.S  were measured by indirect immunofluorescence. Anti-fibrillarin autoantibodies (AFA) were measured by immunoblotting.In a gold mining site, there was a high prevalence of ANA and ANoA: 40.8% with detectable ANoA at ≥1:10 serum dilution, and 54.1% with detectable ANA . In a riverine town, where the population is exposed to methylmercury by fish consumption, both prevalence and levels of autoantibodies were lower: 18% with detectable ANoA and 10.7% with detectable ANA. In a reference site with lower mercury exposures, both prevalence and levels of autoantibodies were much lower: only 2.0% detectable ANoA, and only 7.1% with detectable ANA. In the gold mining population, we also examined serum for AFA in those subjects with detectable ANoA (≥1:10). There was no evidence for mercury induction of this autoantibody.This is the first study to report immunologic changes, indicative of autoimmune dysfunction in persons exposed to mercury, which may also reflect interactions with infectious disease and other factors. Mercury has been recognized as a significant environmental and public health problem for more than 40 years, primarily for its effects on the developing nervous system, as expressed in tragic episodes of human poisoning in Japan and Iraq . Awarene2H5Hg+), the active compound in thimerosal and other medical compounds, induces in a dose-dependent pattern all the features of systemic autoimmunity that have been described after exposure to mercuric chloride (HgCl2) [0), through dermal or eye contact, as ethylmercury, and by absorption through the gastrointestinal track, primarily as methylmercury (CH3Hg+) through ingestion of contaminated fish [0 vapor easily crosses the pulmonary alveolar membranes to enter the circulatory system, where it is primarily bound to red blood cells, and is rapidly distributed to the central nervous system, and the kidneys [2+ by glutathione [In rodent models exposure to inorganic and organic mercury has a range of immunotoxic effects, functionally associated with decreased cell-mediated immunity and the induction of autoimmunity . These e (HgCl2) . Mercuryted fish . Inhaled kidneys . Mercurytathione . After etathione ,10.0 vapor may involve deposition of autoantibodies to basement membrane proteins in the glomerulus [+CD45RA), T-cell proliferation, and plasma IgM were decreased, while IgG levels were increased, relative to controls [In the literature, no cases of frank autoimmune disease have been reported in persons exposed to mercury, occupationally or environmentally . A few somerulus ,14. In aomerulus . No studomerulus ,16. In acontrols .The goal of this study was to test the hypothesis that exposures to methylmercury and/or inorganic mercury may have effects on specific markers of mercury-induced autoimmunity, that is, ANA and antinucleolar (ANoA) autoantibodies, and in a subset of subjects anti-fibrillarin (AFA) autoantibodies. ANoA autoantibodies, a marker found in some human autoimmune diseases , have beWe were able to conduct this study in collaboration with an ongoing epidemiological surveillance of mercury exposures in Amazonian Brazil, where populations are exposed to both inorganic and organic mercury associated with gold mining activities ,24. In AIn this study we analyzed autoantibodies and mercury exposures in three populations from the state of Pará, Brazil. These groups were exposed to different types of mercury in different settings, with exposure to other risk factors not all of which were determined. Therefore, these may contribute to the observed differences among communities, in addition to mercury exposures. We report here that exposures to mercury are associated with significant increases in the prevalence of elevated serum ANoA.Rio-Rato, a garimpo or gold mine site, most of the population was directly involved in gold extraction and refining, resulting in relatively high but often episodic exposures to inorganic mercury, similar to those described by us and others [Jacareacanga, a riverine community on the Tapajós River several hundred km downstream from the region of active gold mining in Pará, the inhabitants consume fish known to be contaminated with methylmercury [Tabatinga, located on the lower Amazon River east of the Tapajós, the population has no direct or indirect contact with gold mining, and fish collected in this village have levels of methylmercury [To test our hypothesis we examined three separate populations, selected from ongoing studies of mercury exposures and health status being conducted by FUNASA , under the leadership of Dr Santos of the Evandro Chagas Institute. The communities in our study were chosen from this surveillance database on the basis of differences in exposures to mercury and other risk factors in Pará, Brazil. At d others ,26. Thisd others . At Jacalmercury ,29,30. Tlmercury . Finallylmercury  within tlmercury . TabatinThe overall design of the mercury surveillance studies conducted by Dr Santos is a community based, cross sectional survey of Brazilian populations in Amazonia, focused on the states of Pará, Amazonas, Acre, and Rondônia, including gold mining sites, riverine communities, and populations without exposure to mercury. The study design is described in detail by Santos and colleagues ,32. In aSubjects' exposure to mercury was determined in two ways. First, information was gathered by questionnaire on occupational history (contact with and use of mercury in gold mining), and/or fish consumption (by weekly frequency and predominant types of fish consumed). Second, mercury concentrations were measured in biologic compartments. For persons in Tabatinga and Jacareacanga with chronic exposure via fish consumption, hair mercury (μg Hg/g of hair) was used as the exposure biomarker as recommended . Hair saBlood samples were collected by venipuncture and sera were separated on site by centrifuge, aliquoted and immediately frozen on liquid nitrogen for transport by air to the IEC in Belem (Pará). Aliquots of frozen serum were stored at -80°C and then transferred on dry ice to Baltimore via air transport accompanied by Dr Silbergeld. All analyses were done under blinded conditions.The serum samples were stored at -80°C until analysis. Each aliquot was thawed and 10 μL taken for analysis by indirect immunofluorescence (IIF) microscopy using commercially available slides prepared from human epithelial cells (HEp-2) as substrate (INOVA Diagnostics) following the methods of Burek and Rose . The sliThe antigen proteins were obtained from rat liver nuclei , and theThe concentration of serum autoantibodies is expressed in terms of the dilution factor at which fluorescence could still be detected. Detection of autoantibodies at a serum dilution of ≥1:40 is considered "positive" for most clinical uses . HoweverMeans for continuous variables (median for variables with skewed distributions) and percents for categorical variables were computed for the descriptive analysis in our data. Chi Square test was used to compare categorical variables and Student's t-test was used for continuous variables. In Jacareacanga we stratified hair mercury levels based on World Health Organization guidance (≤8 or ≥8 μg/g hair). In Tabatinga we stratified exposure by the observed median level (5.57 μg/g) since most hair mercury concentrations were below 8 μg/g. In Rio-Rato we used urine mercury levels based on WHO guidance (≤5 or ≥5 μg/L). We used the mean age for each population to stratify by age. Logistic regression modeling was used to evaluate the effect of mercury exposures on prevalence of ANA and ANoA (for 1:10 and 1:40 cutoffs), while controlling for age, sex, prevalent malaria, past history of malaria, and occupation. All data were analyzed using the SAS v.8.1 statistical package.Because of substantial differences among the populations and sites, we present the results for each site separately.Tabatinga is a typical riverine community in the lower Amazon. The community sample consisted of 98 adults, with 73% females, and a mean age of 44 years, Table . This coThe prevalence of detectable ANA and ANoA in the Tabatinga samples was very low Table . Most meJacareacanga is a riverine settlement of approximately 500 persons, located on the mid-Tapajós River. The 140 subjects consisted of 54% women and had a mean age of 25 years . Persons from Tabatinga, with the lowest range of mercury exposures, had lower prevalence of detectable ANA or ANoA, and in those few persons with detectable autoantibodies, the concentrations were low. Nonetheless, Tabatinga subjects had significantly elevated mercury levels, as compared to North America populations ,38, whicIt is possible that exposure to inorganic mercury may be more "autoimmunogenic" than exposure to methylmercury, as shown in mice models . In contWe examined the serum ANA and ANoA results at both 1:10 and 1:40 dilutions. The results in Jacareacanga and Rio-Rato subjects are clearly different from studies of healthy individuals in the US and Brazil . InterprIn Rio-Rato and Jacareacanga subjects, other risk factors were related to elevations in ANoA and ANA. In Rio-Rato, time spent at the site and in gold mining was positively correlated with likelihood of elevated ANoA. This variable, time spent at the site, may represent length of exposure to both mercury and malaria infection. In Jacareacanga, there was a positive relationship between malaria (any past reported cases) and likelihood of elevated ANoA. We examined these potential biological interactions among mercury, malaria, and autoimmune biomarkers further, because of studies demonstrating that repeated malaria infections are associated with increased levels of autoantibodies, including ANA, presumably due to cytotoxic damage and exposure of intracellular epitopes ,45. OtheIt is difficult to draw any firm conclusions from these analyses, since the malaria data in Jacareacanga were based upon unconfirmed self-reports. We could not test this hypothesis in the Rio-Rato group, since almost all subjects had prevalent malaria and extensive histories of past infection.Plasmodium yoelii) and impairs acquisition of immunity to murine malaria in the Nussenzweig model [We have reported a suggested correlation between mercury exposures and number of past malaria infections among gold miners in another gold mine settlement, in Brazil, at Piranha . We haveig model . Mercuryig model ,49.Finally, we may speculate as to why we have been able to observe associations between mercury exposures and these biomarkers of autoimmune dysfunction in these populations, while most clinical or epidemiological studies of mercury and immunotoxicity have been negative or only weakly positive -13. In tOur study is the first to report a correlation between biomarkers (ANA and ANoA) and mercury exposure in humans. In addition, co-exposures to mercury and infectious diseases, including malaria, may set the stage for eliciting discernible alterations in immune function. Whether such co-exposures increase the risks of autoimmune disease will require further studies, which are underway.ANA – antinuclear autoantibodiesANoA – antinucleolar autoantibodiesAFA – anti-fibrillarin autoantibodiesIIF – indirect immunofluorescenceThe authors declare that they have no competing interests.IA Silva carried out the analysis of autoantibodies, participated in the statistical analysis, and drafted the manuscript. JF Nyland helped in autoantibody analysis and technical editing of the manuscript; A Gorman helped in autoantibody analysis; AM Ventura, JM de Sousa, and ECO Santos carried out the field studies, malaria assessments, and mercury analysis. CL Burek and NR Rose guided the antibody analysis. EK Silbergeld participated in the design and coordinated the study; she also participated in the Jacareacanga field study, and in the draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Why is that many infectious diseases, like cholera, malaria, and meningococcal meningitis, show seasonal patterns? And how can we accurately determine these patterns? Meningococcal meningitis in western Africa shows recurrent seasonal patterns every year. Epidemics typically start at the beginning of February and last until May. We can try to explain the observed patterns on the basis of some seasonally varying environmental factor that favors disease transmission. Air dryness produced by strong dust winds is the most likely candidate.PLoS Medicine is a remarkable exception But while there are qualitative “stories” of this kind in the literature for many seasonal phenomena, convincing quantitative evidence to support them remains largely elusive. Instead, we tend to see weak associations between environmental and transmission variables when measured by simple, linear correlations. The study of meningococcal meningitis in Mali by Sultan and colleagues in this issue of Why is a focus on the seasonality of infectious diseases and its variation from year to year so important? Isn't it more important for us to instead understand the effects of long-term climate change on human health?At first sight, understanding seasonal patterns seems disconnected from understanding the impact of long-term climate change. However, seasonal patterns are one major pathway for the subtle but potentially drastic effects of climate change on disease dynamics. Long-term climate change affects seasonal patterns through the lengthening of the transmission season and the crossing of environmental and demographic thresholds that underlie seasonal outbreaks One important difficulty in uncovering seasonal drivers of infectious diseases is to identify the appropriate scale of analysis. The relationship between disease and climate described by Sultan and colleagues only becomes apparent at large spatial scales. The authors argue that these large scales are necessary to eliminate “idiosyncratic” variability in the relationship between cases and climate at the local level. In other words, there are only weak correlations between seasonal variations and climate variables at small scales because of the multiple other factors that play a local role and act as noise.But we should be cautious about the suggestion that appropriate larger scales will always resolve the problem of local variability and present strong linear associations between climate and disease. Public health measures might require predictions not only at national and regional scales, but also at a variety of smaller scales.Moreover, one important source of variation in how infectious diseases respond to climate is the fraction of susceptible individuals in the population. This fraction varies over time as the result of immunity acquired by previous infection, and by the input of births and migrants into the pool of susceptible people. The constant waxing and waning of this pool of hosts underlies the intrinsic potential of the population dynamics of infectious diseases to oscillate and create epidemic outbreaks. The tendency of these intrinsic cycles to go up and down in synchrony at different locations in space will determine whether susceptibility levels act as noise at small scales or, alternatively, whether their effect must be considered in conjunction with climate at larger scales. Because the number of susceptible individuals is a hidden variable in most epidemiological analyses, recently proposed methods for its reconstruction from data on cases must be combined with studies on climate variation if we are to understand the interaction between susceptibility levels and climate variation ,4,5.The problem of scale also arises when we need to identify the appropriate timing  to detect strong associations between disease outbreaks and environmental covariates. This is particularly important when strong couplings between environment and transmission occur only transiently. This seems to be the case for cholera in Bangladesh, where couplings are strong during El Niños, but considerably weaker the rest of the time Besides scale, specific seasonal drivers are often elusive because of the simpler reason that in nature seasonality is ubiquitous. Multiple and covarying drivers have been proposed for the seasonal nature of cholera, including temperature, rainfall, and plankton blooms Ecologists have considered seasonality in mathematical models of the population dynamics of infectious diseases. Models of populations with seasonally forced, dynamic interactions (births, deaths, aggregation, or disease transmission) reveal an array of possible responses, from simple yearly cycles, through cycles that repeat with longer periods, to irregular chaotic fluctuations. Some models also predict intermittent switching between different dynamic infectious disease behaviors. But typical models consider only simple seasonal forcing functions . There are some important exceptions to this—some models do incorporate more complicated seasonal forcing functions that describe the actual processes underlying the seasonal drivers of transmission. Examples are models of childhood diseases that describe the regular stopping and starting of school terms ,10,11, aThe explicit way in which models treat seasonal environmental drivers may be critical in addressing the links between within-year seasonal cycles and those of longer period that are observed in many infectious diseases. For meningococcal meningitis, we still need to examine the connection between the seasonal association described by Sultan and colleagues and the previously proposed role of humidity in inter-annual cycles Sultan and colleagues' study is exceptional in that it illustrates a clear relationship between an external environmental variable and the initiation of disease outbreaks. In contrast, many studies seeking environmental drivers are plagued by the many confounding factors, particularly the impact that other components of global change have on the transmission dynamics of infectious diseases. Thus, when we examine datasets for malaria, we must also consider the evolution of drug resistance and a growing human population that is increasingly forced to live in areas that are marginal for agricultural production but optimal for malaria transmission.Given this complexity, a serious limiting factor to quantitative analyses and predictive models of ecological and disease patterns is the lack of long-term disease records with similar data collected over a network of spatial locations. The handful of extremely valuable records that have allowed progress in understanding long-term patterns in disease dynamics pale in comparison to the spatiotemporal coverage available for climate studies and modeling. The need to resolve these issues of scale and confounding variability only underscores the urgency and importance of maintaining and developing systematic surveillance programs for infectious diseases around the world.


Mention greenhouse gases to most people and they're apt to think of carbon dioxide, fossil fuels, and big cars. Though carbon dioxide emissions are the major source of greenhouse gases, methane is far more effective at trapping heat in the atmosphere. Like increasing carbon dioxide levels, rising levels of atmospheric methane have been attributed to human activity, mostly in the form of landfills, natural gas and oil processing (about 60%), domesticated livestock (cattle account for about 75% of livestock contributions), and rice fields .Ruminants—from cows and water buffalo to llamas and vicunas—emit methane gas as a natural by-product of their digestive process, which confers a unique ability to digest cellulose. Ruminants don't digest cellulose directly, but depend on a variety of microbes living in their rumen (main stomach) to do it for them. These microbes ferment cellulose, breaking it down into products the ruminant can digest. During this process, some microbes—bacteria called methanogens—produce methane, which ruminants expel by eructation (otherwise known as belching).PLoS Biology, a multidisciplinary team spanning the fields of genomics, bioinformatics, microbiology, evolutionary biology, and molecular biology report the complete genome sequence of Methylococcus capsulatus and shed light on the metabolism and biology of this ubiquitous microbe.Luckily, there are microbes, called methanotrophs, that consume methane. A type of aerobic bacteria, methanotrophs oxidize methane as an energy and carbon source using the enzyme methane monooxygenase. They've been found in soils, landfills, sediments, hotsprings, and peat bogs, among other environments. Methanotrophs have been the subject of increasing interest because they can use methane as a sole source of carbon and energy—which means they play a major role in global carbon cycles—and could dramatically reduce biologically generated methane emissions. They've also been the focus of bioremediation efforts aimed at environmental decontamination. And now, with the publication of the first complete genome sequence of a methanotroph, such efforts will be all the easier. In this issue of M. capsulatus genome comprises about 3.3 million base pairs—which is about average for a free-living bacterium—with an estimated 3,120 genes. The genome appears well-equipped to meet the specialized needs of this methanotroph, with what appear to be multiple pathways involved in the metabolism of methane and duplications of genes that code for methane monooxygenases, which are essential for the first step of methane oxidation.Contained in a single, circular molecule, the M. capsulatus employs different pathways depending on the availability of molecules needed to sustain cellular activities. Most surprising, the researchers note, was evidence that this methane specialist can turn into a sort of metabolic generalist—with a capacity to use sugars, hydrogen, and sulfur—and appears able to survive reduced oxygen levels. These genome-based hypotheses will require experimental validation, the authors note, but could have important implications for M. capsulatus ecology—including what environments might be amenable to methanotroph-mediated bioremediation.Ward et al. also found evidence of “genomic redundancy” in methane oxidation pathways, suggesting that M. capsulatus genome provides a platform for investigating the details of methanotroph biology and its potential as a biotech workhorse. It may also guide efforts to harness this bacterium's penchant for methane to reduce global greenhouse gas emissions, to degrade chlorinated hydrocarbons and other persistent pollutants, and to produce protein for animal feed.The genomes of important microbial players in the carbon cycle—including microbes involved in photosynthesis and methanogenesis (methane production)—have already been sequenced. With the addition of a sequenced methanotroph genome, scientists can systematically investigate different genes and regulatory elements to better understand how these methane consumers fit into this global cycle. The


Large-scale sequence comparison is a powerful tool for biological inference in modern molecular biology. Comparing new sequences to those in annotated databases is a useful source of functional and structural information about these sequences. Using software such as the basic local alignment search tool (BLAST) or HMMPFAM to identify statistically significant matches between newly sequenced segments of genetic material and those in databases is an important task for most molecular biologists. Searching algorithms are intrinsically slow and data-intensive, especially in light of the rapid growth of biological sequence databases due to the emergence of high throughput DNA sequencing techniques. Thus, traditional bioinformatics tools are impractical on PCs and even on dedicated UNIX servers. To take advantage of larger databases and more reliable methods, high performance computation becomes necessary.We describe the implementation of SS-Wrapper (Similarity Search Wrapper), a package of wrapper applications that can parallelize similarity search applications on a Linux cluster. Our wrapper utilizes a query segmentation-search (QS-search) approach to parallelize sequence database search applications. It takes into consideration load balancing between each node on the cluster to maximize resource usage. QS-search is designed to wrap many different search tools, such as BLAST and HMMPFAM using the same interface. This implementation does not alter the original program, so newly obtained programs and program updates should be accommodated easily. Benchmark experiments using QS-search to optimize BLAST and HMMPFAM showed that QS-search accelerated the performance of these programs almost linearly in proportion to the number of CPUs used. We have also implemented a wrapper that utilizes a database segmentation approach (DS-BLAST) that provides a complementary solution for BLAST searches when the database is too large to fit into the memory of a single node.Used together, QS-search and DS-BLAST provide a flexible solution to adapt sequential similarity searching applications in high performance computing environments. Their ease of use and their ability to wrap a variety of database search programs provide an analytical architecture to assist both the seasoned bioinformaticist and the wet-bench biologist. Evolution can be measured and studied on a number of different scales, one of which is through the determination and comparison of genetic sequence information. Current-day gene sequences in living organisms have arisen through modifications of an array of ancestral sequences. Duplication with modification is the central paradigm of protein evolution, wherein new proteins and/or new biological functions are fashioned from earlier ones. . To deteThe basic local alignment search tool (BLAST) ,4 is by While BLAST provides a balance between search sensitivity and speed, in many cases a researcher would like to detect more distant sequence similarities by employing search strategies that maximize the sensitivity of the search. A profile-based comparison which, for example, compares a sequence to a hidden Markov model (HMM) representing an empirically derived estimate of all possible evolutionary changes for a protein of a particular function, generally permits identification of a much higher proportion of distantly related sequences . There aTaking advantage of larger databases and more sensitive searching methods necessitates the use of high performance computing (HPC) platforms. Traditionally, HPC has been synonymous with high-priced vector or parallel supercomputers, but rapid advances in microprocessor and network bandwidth technologies are changing the definition . ClusterThe objective of this study was to implement a generic wrapper application that could deploy similarity searching applications on a Linux cluster. Design criteria for the wrapper included the ability to deploy applications without the need to alter the original application and the ability to increase the speed of the underlying application in a linear manner dependent on the number of cluster nodes available. To meet these objectives, tools in the SS-Wrapper package were written in C/C++ using the message passing interface (MPI) . Tools iUsing multiple processors is practical only when the computational task is too large to complete on a single processor in a reasonable amount of time. In the case of database searching, the database to be searched may be too large or the set of query sequences used in the search may be too large to accomplish the search on a workstation with a single or even double or quad processor architecture. Since the search must compare every query sequence to every database sequence, parallelizing the process can be accomplished by three different methods. The first method is to split the query sequence file into smaller subsets and apply each subset to one particular node of the cluster in a search against the entire database. The second method calls for the database to be split into a series of smaller files, one of which is distributed to each node of the cluster; then the entire file of query sequences is searched against each of the database segments. Finally, it is possible to use a combination of query sequence splitting and database splitting to accomplish the search.Using the query splitting approach does not require any modification to the output generated by search programs such as BLAST. When utilizing the database splitting approach, however, a correction must be made to the reported E-value for any particular hit. Most similarity comparison tools provide a statistical measure  that gives an indication of the statistical significance of a match between the query sequence and a particular database hit. This statistical measure is generally influenced by the size of the search space  and therefore the E-value needs to be recalculated when only a portion of the database is being searched. An added complication is that some search tools require that the database be intact. For example, PSI-BLAST  is a varIn contrast to database splitting, query splitting offers greater flexibility in that the query file segments can more easily be adaptively distributed to the nodes of the cluster according to the load on each particular node during the search process. As any one node becomes available, another segment of the query file can be distributed to that node during the search process, which improves performance. It is difficult to predict the workload on any one node before the search begins, and the time required to complete a program running on a cluster depends on the processor that finishes last. Adaptive distribution of the workload maximizes resource utilization. Optimizing resource utilization is dependent on finding a balance between having larger numbers of smaller tasks versus increased startup and communication overhead due to distribution of the required query and database files to each node. For this reason, for our query splitting wrapper (QS-search), we adopted a hybrid strategy wherein approximately 90% of the total workload is evenly divided and distributed to each node at the beginning of the search using a modified bucket algorithm (described below). Then the remaining 10% of the workload is divided and distributed to each node as it becomes available after completing its previous task. This strategy is accomplished using a master-slave model, where one node is set aside to act as the master, which is responsible for distributing the workload and supervising the slave nodes, which perform the computations.In general, the query splitting approach seems to be superior to the database splitting approach due to higher performance and fewer post-search processing tasks. Database splitting does provide a distinct advantage when the database is too large to fit into the physical memory of a single node ,14. NCBIAs indicated above, a modified bucket algorithm is used to split up the query sequences for QS-search, and a similar algorithm is used to split up the database sequences for DS-BLAST. The modified bucket algorithm works as follows: First, the sequences are sorted according to length. In the first cycle, sequences are placed one at a time into each bucket in descending order of length. In the next two cycles, individual sequences continue to be placed into each bucket after first reversing the order of the buckets. Bucket order is reversed every two cycles and the process continues until all sequences have been placed into a bucket. At the end of this process, each bucket contains nearly the same number of sequences, and the total length of all sequences in any one bucket is also approximately the same as the total sequence length of any other bucket. In the end, therefore, the file of query sequences or the file of database sequences is evenly divided in both length and number.The BLAST E-value is a function of the size of the effective search space, which is dependent on three factors: the number of sequences in the database, the total combined length of all sequences in the database, and the length of the query sequence ,18. FiguThe QS-search executable (qssearch) provides the same interface for all search tools. The command line is as follows:qssearch -c <command> -q <query> -d <database> -o <output> -l <local scratch> -x <database files>DS-BLAST uses two executables: dsblast and dsformatdb. dsformatdb is responsible for splitting the database into fragments according to the modified bucket algorithm and then formats these fragments using the NCBI formatdb executable.The command line for dsformatdb is as follows:dsformatdb -n <number> -c <command> -d <database> -p <path>The command line for dsblast is as follows:dsblast -o <output> -c <command> -l <local scratch> -d <database> -q <query>The command-line variables are as follows:• -c command: normal command line used for the underlying application including all desired options• -q query: query filename in fasta format• -d database: database filename• -o output: output filename• -l local scratch: temporary directory on each node• -x database files: a space-delimited list of the database file names generated by the search program's formatting utility (formatdb for BLAST)• -n number: desired number of database fragments• -p path: directory to store database fragments.All benchmark experiments were performed on a Linux cluster in the Department of Engineering at the University of Alabama at Birmingham . The cluFigure Figure We also compared the performance of DS-BLAST to that of mpiBLAST  version To increase the speed and efficiency of sequence similarity search programs, we have developed the SS-Wrapper package, a series of wrapper applications that supports the deployment of sequence similarity searches on high-performance computing clusters. QS-search implements a query sequence splitting approach for the deployment of NCBI BLAST and HMMPFAM. It also will support other similarity search programs, including all variants of NCBI BLAST (blastn, blastp, blastx, tblastn, and tblastx) as well as all options provided by the blastall executable. Because this implementation does not alter the original program, program updates and new programs should be easily accommodated. The output from QS-search is effectively identical to that produced by the underlying program. QS-search is designed to provide optimal load balancing and maximize resource usage when using computer clusters. The performance gain approaches linearity in proportion to the number of processors employed.When the database is too large to fit into the physical memory of a single node in the cluster, a database splitting approach should outperform the query splitting approach used by QS-search ,14. TherSS-Wrapper provides a suite of tools that makes large sequence similarity searches feasible by deploying the search on a Linux cluster. These tools permit the bioinformatics community to take advantage of the power of high-performance cluster computing. Other tools such as Disperse  and Turbelliotl@uab.edu for information on obtaining the software. All tools have been tested on an IBM Intel® processor-based Linux cluster with LAM/MPI [The SS-Wrapper package is freely available under the Artistic License described in the Open Source Initiative. The source code can be downloaded via ftp . Contact LAM/MPI  and shouCW was responsible for the conception, design, implementation, and testing of the SS-Wrapper package. EJL contributed to its conception and testing and provided overall project coordination. Both authors have read and approved the final manuscript.


In acute renal failure, a pronounced net protein catabolism occurs that has long been associated with corticoid action. By competitively blocking the glucocorticoid receptor with the potent antiglucocorticoid RU 38486, the present study addressed the question to what extent does corticoid action specific to uremia cause the observed muscle degradation, and does inhibition of glucocorticoid action reduce the protein wasting?RU 38486 was administered in a dose of 50 mg/kg/24 h for 48 h after operation to fasted bilaterally nephrectomized (BNX) male adult Wistar rats and sham operated (SHAM) controls. Protein turnover was evaluated by high performance liquid chromatography (HPLC) of amino acid efflux in sera from isolated perfused hindquarters of animals treated with RU 38486 versus untreated controls.Administration of RU 38486 reduces the total amino acid efflux (TAAE) by 18.6% in SHAM and 15.6% in BNX and efflux of the indicator of net protein turnover, phenylalanine (Phe) by 33.3% in SHAM and 13% in BNX animals as compared to the equally operated, but untreated animals. However, the significantly higher protein degradation observed in BNX (0.6 ± 0.2 nmol/min/g muscle) versus SHAM (0.2 ± 0.1 nmol/min/g muscle) rats, as demonstrated by the marker of myofribrillar proteolytic rate, 3-Methylhistidine (3 MH) remains unaffected by administration of RU 38486 (0.5 ± 0.1 v. 0.2 ± 0.1 nmol/min/g muscle in BNX v. SHAM).RU 38486 does not act on changes of muscular protein turnover specific to uremia but reduces the effect of stress- stimulated elevated corticosterone secretion arising from surgery and fasting. A potentially beneficial effect against stress- induced catabolism in severe illness can be postulated that merits further study. Persike and Addis[Bondy et al. [Schäfer et al. [Schäfer et. al used the experimental approach of inhibiting activation of the glucocorticoid receptor by enteral application of the potent glucocorticoid antagonist RU 38486 in acutely uremic rats [As part of the complex uremic metabolic syndrome, pronounced disturbances of carbohydrate and lipid metabolism are commonly observed, as are pathologic changes of amino acid and protein turnover . An incrand Addis, and in y et al.  showed tr et al. ,5 who pomic rats ,8, so famic rats , in whicWith this experimental design and by comparing sham-operated and bilaterally nephrectomized animals, the present study looked at the question to what extent does corticoid action specific to uremia cause the observed muscle degradation, and does inhibition of glucocorticoid action reduce the protein wasting?Animal experimentation was carried out on male Wistar rats, aged 11–15 weeks, weighing 217–225 g, from the animal experimentation facilities of Heinrich Heine University, Düsseldorf. Permission to use animals for experimentation was given by Regierungspräsident Düsseldorf, file nr. 26.4203.1-217/87 according to German federal law. Surgery for nephrectomy, sham nephrectomy and preparation for perfusion was carried out under narcosis with hexobarbital (EVIPAN- Na: 15–20 mg/ 100 g BW).Bilateral nephrectomy was performed using a dorsal access, ligation of renal vessel string, and excision of the kidney, leaving the adrenal glands in place. Sham operated animals underwent the same manipulations except for the ligation and excision. After surgery, animals were fasted for 48 h until perfusion. Nephrectomized animals had access to drinking water on the day of surgery for 8 hours, then were deprived of liquid to avoid lung edema. Sham animals had free access to drinking water throughout.Animals were randomly assigned to one of four groups: bilaterally nephrectomized (BNX) and sham operated (SHAM) treated with RU38486 and untreated BNX and SHAM animals. For treatment, RU38486 was dissolved in phenylmethanol, then mixed with sesame oil to form a milky suspension which was injected into subcutaneously into the lateral abdomen in three subdoses within 24 h, adding up to a total dose of 5 mg/100 g/BW.48 h after initial surgery, animals were narcotized and prepared for perfusion as previously described.The hindquarter was linked to the recirculation system after full passage of 70 ml of pre- perfusion medium, as shown in illustration 1. The pre- perfusion medium was discarded and not used for the recirculation experiment.-6 mmol/l phentolamine were added to avoid vessel contractions.The perfusion was carried out with a half- synthetic medium on the basis of Krebs- Ringer- bicarbonate buffer (KRB), pH 7.38 . Oxygen During perfusion, the arterial pH and perfusate oxygenation were monitored using a pH- meter and a total oxygen content analyzer (LEX- O2- CON, Lexington Instr., Mass., USA). At the beginning and end of perfusion, plasma samples were frozen for amino acid analysis. Amino acid analysis by HPLC was carried out using 25 μl of deproteinized perfusate sample, mixed with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid to form OPA- adducts that were separated on a reversed phase column and measured by fluometry. Quantification was done by comparison with a standard amino acid mix including 3-methylhistidine. Of the 20 proteinogenic amino acids, cysteine, proline, and asparic acid were not included in HPLC analysis.Statistical analysis was done using the "Student" t- test for ungrouped, non- paired data with f = n1 + n2-2 and a significance level of p < 0.05.During the 48 h fasting period between operation and perfusion, animals had a pronounced loss of BW table . In SHAMOxygen utilization in the perfused muscle tissues is roughly the same in all four groups (data not shown) and equal to in vivo data previously reported  from ratTotal amount and spectrum of amino acids released during reperfusion: During perfusion, amino acids are released in varying amounts as shown in ill. 2. Nephrectomized animals (BNX) showed a general increase in amino acid release. This, however, is significant only in a few individual amino acids. The total amino acid efflux increases by 10,4 % (p = 0.05) without qualitative change. Notable exception is glycine, which is released to a lesser amount in nephrectomized animals. Amino acid release after treatment with RU 38486: Nephrectomy equally increases the amino acid efflux in animals treated with RU 38486 by 14.4% (p < 0.05) without change in spectrum. The total efflux of amino acids, however, is significantly reduced in the comparison SHAM/ SHAM+RU (-18.6%, p < 0.05) and BNX/ BNX+RU (-15.6%, p < 0.001). Release of 3-methylhistidine: 3-methylhistidine, a derivate of histidin mainly from actin and myosin in sceletal muscle and intestinal mucosa [l mucosa , is not Acute renal failure is a catabolic state, and unfortunately the inherent acceleration of protein breakdown cannot be suppressed effectively by provision of exogenous nutritional substrates . The sitOne factor that has for long been associated with the disturbances of protein metabolism is glucocorticoid action. The first observations date back to the nineteen-forties, when Persike and Addis  reportedIn order to evaluate the relative importance of glucocorticoid action on protein metabolism in acute renal failure, an experimental setting was chosen that allowed to study glucocorticoid action indirectly by selective blockade of the glucocorticoid receptor with the potent antiglucocorticoid RU38486, a substance that binds to the receptor without activating the further process of transcription . ParenteThe isolated perfusion procedure introduced by Ruderman is well In the given experimental setting, it is difficult to account for the in detail contribution of protein degradation, amino acid intermediate metabolism, and protein synthesis. Factors that modify the efflux are transport systems in the cell membrane -28, whicin ultima causa remain still unclear[Sham- operated animals are catabolic at the time of perfusion, having lost about 36 g BW (see results), which is approximately 16% of initial BW. Rats of this age are still growing, with an increase of approx. 5 g/ day (2–3% BW). The weil unclear. Nephrecl unclear, of whicl unclear. At firsBy contrast, in the comparison of the two groups treated with RU38486, the amino acid release remains increased in nephrectomized animals, albeit to a lesser extent, stressing the very point that glucocorticoids are only one of several factors that contribute to the net protein wasting. Chronically uremic rats with increased corticosterone- secretion , and a pLowell et al. found no reduction of the efflux of 3-methylhistidin after adrenalectomy in the perfused hindquarter of fasted animals [Teschner [This presumption is supported by the finding that RU 38486 had no effect on the efflux of 3-methylhistidin. While this result is in contrast to Schäfer et al. [ animals , and in Teschner . As respTeschner  and actiTeschner  comparedTeschner , it seemThe presented data fail to show that RU38486 inhibits glucocorticoid action in the specific uremic setting while it clearly reduces the elevated net protein catabolism compared to non- operated animals. This suggests that glucocorticoid mediated protein wasting in acute uremia is rather a by- product of the overall stress, in the present experimental setting caused by surgery and fasting, than due to an independent action specific to uremia.While this finding abolishes hopes to counteract muscle wasting in uremia by administration of an anticorticoid drug and indirectly rather stresses the well described clinicalBoth sham- operated and nephrectomized animals show an increase in net protein catabolism. RU38486 clearly reduces the net protein wasting in both groups, but the increase in net protein catabolism observed over sham- operated animals remains unchanged in nephrectomized rats. The effect of antiglucocorticoid RU38486 may be attributed to an inhibition of fasting and operative stress- induced cortisol action which, even when within the physiological range, promotes increased protein turnover , and to The author(s) declare that they have no competing interests.A.M. carried out animal experimentation, sample analysis, statistical analysis and wrote the manuscript.The pre-publication history for this paper can be accessed here:


The incidence of reported adolescent and adult pertussis continues to rise in the United States. Acellular pertussis vaccines for adolescents and adults have been developed and may be available soon for use in the U.S. Our objectives were: (1) to describe patient valuations of pertussis disease and vaccination; and (2) to compare valuations for short-term and long-term health states associated with pertussis.We conducted telephone surveys with 515 adult patients and parents of adolescent patients with pertussis in Massachusetts to determine valuations of pertussis-related health states for disease and vaccination using time trade-off (TTO) and contingent valuation (CV) techniques. Respondents were randomized to complete either a short-term or long-term TTO exercise. Discrimination between health states for each valuation technique was assessed using Tukey's method, and valuations for short-term vs. long-term health states were compared using the Wilcoxon rank-sum test.Three hundred three (59%) and 309 (60%) respondents completed and understood the TTO and CV exercises, respectively. Overall, respondents gave lower valuations  to avoid a given state for adolescent/adult disease compared to vaccine adverse events. Infant complications due to pertussis were considered worse than adolescent/adult disease, regardless of the method of valuation. The short-term TTO resulted in lower mean valuations and larger mean differences between health states than the long-term TTO exercise.Pertussis was considered worse than adverse events due to vaccination. Short-term health-state valuation is better able to discriminate among health states, which is useful for cost-utility analysis. The incidence of reported pertussis continues to rise in the United States despite high levels of childhood vaccination ,2. WaninRecently completed clinical trials suggest that the booster may prevent cough illness related to pertussis among adolescents and adults ,9. ThougMethods commonly used for measuring health-state valuations include contingent valuation (CV) and time trade-off (TTO) . ContingAnother common approach to measuring the benefits and harms of health interventions relies on health-state utilities. Utilities measure a person's preferences for specific outcomes on a scale of 0 to 1, on which 0 typically represents a state equivalent to death while 1 represents the best imaginable health. The time trade-off method is one of several approaches used to assess health utilities. Using the TTO method, respondents are asked how much longevity they would be willing to give up, if any, to avoid living with a particular health outcome. Traditionally, TTO questions have been framed as giving up time to avoid a long-term or chronic health state . HoweverWe conducted a survey using TTO and CV methods to determine the health-state valuations of adult patients and parents of adolescent patients diagnosed with pertussis. We compared two alternative approaches to framing TTO questions, based on either short-term or long-term health states. We hypothesized that framing questions as short-term rather than as long-term health outcomes would allow for better discrimination between states.Structured telephone interviews were conducted with adult patients (≥ 18 years) and parents of adolescent patients 11–17 years) diagnosed with confirmed pertussis in Massachusetts from December 1, 2001 to January 31, 2003 [–17 yearsDescriptions of the health states were derived with input from 3 pertussis experts Table . Adults For TTO questions, respondents were asked the maximum amount of time they would be willing to trade from the end of their lives to avoid a particular health outcome now. Adults were asked how much time they would give up from the end of their lives to avoid living in a particular health state themselves, while parents were asked how much time they would give up from the end of their own lives to avert the health state in the adolescent ,20. ThisAlthough TTO questions traditionally have been framed using permanent health states for valuations of chronic disease , the heaCV questions elicited the amount of money that a respondent would be willing to pay to avoid living in a particular health state for 8 weeks. We chose to frame the CV questions as short-term health states for both versions of the survey based on prior work ,22. RespTelephone interviews were conducted in English or Spanish using standardized forms. Some respondents either were unable to complete or refused to answer the entire set of TTO questions or CV questions. If any answers were missing within a set of questions, respondents were excluded from analyses (of that set) in order to assess population means. If the respondent completed either set of questions, trained interviewers judged how well the respondent understood the TTO or CV questions separately based on a 3-point scale (good understanding, some understanding, limited understanding). Respondents were excluded from further analyses if they were thought to have either some or limited understanding of the tasks presented .We calculated utilities based on the TTO exercise, under alternative assumptions about discounting of future health outcomes. For long-term health states, the utility was based on the proportion of time that the respondent would be willing to give up to avoid a lifetime health state for themselves, for their adolescent child, or for a hypothetical infant Figure . Life exIt is most straightforward computationally to start with the disutility, rather than the utility, of a particular health state, computed as the ratio of the duration of life that would be given up  to the expected duration of time lived in the health state. In the absence of discounting, the disutility was calculated simply by dividing the amount of time traded from the end of the respondent's life by the LE of the beneficiary. The utility was then calculated by subtracting this result from one. With discounting, we assumed that individuals compared the present values of the two different streams of life in the trade-off, in a way that reflects declining relative weight for future consequences, and we computed utilities based on a discount rate (r) of 3% per year . As empiUsing the formula for discounting a continuous stream of life , we obta-r (LE of respondent)) - (1/r) * (1 - e-r (LE of respondent - years of life traded))(1/r) *  was-r (LE of beneficiary))(1/r) * ) - (1/r) * (1 - e-r (LE of respondent - years of life traded))(1/r) * )(1/r) *  and medians (with interquartile ranges). We assumed that the maximum amount of discounted time traded from the end of the respondent's life could not exceed the duration of the present health state; thus, any utilities that would be negative based on the computations described above were instead set to 0. For parent respondents who were asked questions about how much time they would trade to avoid long-term health states in their adolescents, we used interval regression with left censoring to calculate mean utilities . In inteTo compare demographic characteristics of respondents for the short-term vs. long-term TTO surveys, we used the chi-squared test for categorical variables and the t-test for continuous variables. To determine if mean health state utilities and WTP values were significantly different from one another, we used Tukey's method, which is a non-parametric test that allows for multiple pairwise comparisons assuming all sample sizes are equal  ComparisFive hundred fifteen adult pertussis patients and parents of adolescent pertussis patients were eligible and participated in the survey Figure . CharactWe compared demographic characteristics of respondents who completed and understood the survey with those who did not. Parents of adolescents with higher household incomes (p = 0.022) and higher educational levels (p= 0.017) were more likely to complete and understand the CV survey. Also, parents who were white (p = 0.011) with higher educational levels (p = 0.010) were more likely to complete and understand the TTO survey. Adult respondents who completed and understood the CV and TTO survey were significantly younger  and had higher educational levels  than those who did not.CV and TTO responses for short-term and long-term health states for adolescents are described in Table Short-term and long-term TTO responses for adult respondents are described in Table TTO and CV responses for infant health states are described in Table Overall, mean utilities were higher for long-term health states than for short-term health states. These differences were significant for adolescents with mild cough (p = 0.045), severe cough (p = 0.001), and pneumonia (p = 0.001). No significant differences were found between short-term and long-term health states for adults. For infants, we also found significant differences with higher mean utilities reported for long-term health states compared to short-term health states .We evaluated associations between demographic characteristics such as age, race/ethnicity, education, and household income and estimates provided by respondents for the TTO or CV exercise. Older age was associated with lower utilities for short-term health states for adolescent/adult disease (i.e. mild cough, severe cough, pneumonia) and infant disease (i.e. respiratory and neurologic complications) (p < 0.05). Older age was also associated with lower utilities for long-term health states such as systemic reaction, adolescent/adult disease, and infant disease (p < 0.05). Higher income was associated with lower utilities for long-term mild cough and short-term respiratory complications, although older respondents were more likely to report higher household incomes (p < 0.001).Higher income was significantly associated with higher WTP values for the following health states: systemic reaction, adolescent/adult disease, and infant disease (p < 0.05). We also found an association between higher respondent education and higher WTP estimates for pneumonia, although we note that education and income were themselves positively correlated (ρ = 0.358, p < 0.0001).Tables Vaccination programs in the US have traditionally been life-saving and cost-saving ,32. Howeet al. is an alternative approach for assessing process utility [We explored differences in utilities for short-term and long-term health states using open-ended TTO questions. Other methods for short-term health state valuation described in the literature include chained TTO, sleep tradeoff (STO), and waiting tradeoff (WTO). The chained TTO has been shown to have good consistency and reliability ,33,34. H utility . While tWe found that the rankings of health states based on mean utilities were essentially the same using either short-term or long-term health states. However, the short-term TTO resulted in lower mean utility estimates and larger mean differences between health states than the long-term TTO exercise, thus allowing for better discrimination of health states in cost-utility analyses. These results suggest that responses may not fulfill the constant proportional trade-off assumption, which requires that the TTO utility be independent of the duration of the specified health state. While previous studies using TTO and other valuation methods have also found that the constant proportional trade-off assumption does not always hold, the direction of the discrepancy has been mixed -40.The short-term health state approach may have violated the constant proportional trade-off assumption because respondents were less averse to giving up small amounts of time from the ends of their lives (days or weeks) compared to large amounts of time (months or years) in the long-term approach. In other words, asking respondents to give up a few days or weeks from the ends of their lives may not be considered a significant loss, even to avoid a short-duration health state lasting only 8 weeks. However, giving up months or years of life is considerably more difficult for individuals, even to avoid an intermediate- or long-term health state. It may be that a threshold exists whereby individuals are more willing to give up a very small portion of their lives for perfect health, but as the duration of health states increase, they are less willing to give up time per health unit gained, resulting in failure to behave according to the constant proportional trade-off assumption. This aversion to giving up larger amounts of time may play an important role in measuring utilities, particularly since the short duration of these health states over the lifetime of individuals would otherwise lead to nearly imperceptible, but arguably important differences in terms of quality-adjusted life years.The impact of discounting is important to consider since assuming no discounting can lead to a downward bias, while a high discount rate can lead to an upward bias, which may strongly devalue the benefits of any preventive intervention ,41-43. WThe CV exercise resulted in similar mean health state rankings to our TTO exercise, and these estimates were positively correlated with income, which is not surprising since individuals were asked to respond in consideration of their actual household income . For theAs always, there are limitations to our study. First, our respondents were either adult pertussis patients or parents of adolescents with pertussis. We elicited patient and caregiver valuations as part of a larger study to determine societal costs of pertussis in adolescents and adults. While the U.S. Panel on Cost-effectiveness in Health and Medicine suggests that community preferences be used where possible, there iSecond, selection bias might arise from our survey completion rate of around 60%, although this is comparable to other published valuation studies ,23. In oBecause of the complexity of the task required, it is not surprising that respondents with higher educational levels were more likely to complete and understand the preferences exercise. In addition, most respondents were white, well educated, and had relatively high household incomes. Household income was associated with utilities as well as willingness-to-pay to avoid pertussis. To address this limitation, economic analyses of pertussis should vary willingness-to-pay and utilities over wide ranges that would reflect the preferences of a general population. Further research in more socioeconomically diverse populations should also be considered.et al. found that a mother's WTP to prevent a cold is approximately twice as large for the child as for the mother[Another issue that should be explored more thoroughly is the impact of parents as surrogate respondents for children and the method of preference elicitation. We asked parents of adolescents to serve as proxy respondents for their child. Interestingly, parents of adolescents were less likely to provide answers to the long-term TTO exercise than adult respondents, which may suggest that parents had difficulty answering the TTO question for long-term illnesses in their children. Also, trading time from the parent's life to avoid illness in a child may result in preferences that incorporate other aspects of their relationship such as altruism. Previous work in the CV literature has suggested that altruism may significantly affect valuations. For example, Liu he mother. While pIn this study, we estimated health-state valuations regarding pertussis disease and vaccination among adult patients and parents of adolescent patients. Patient preferences in conjunction with health outcomes will be key factors in deciding whether or not to implement a universal vaccination policy for adolescents or adults. The results from our study suggest that short-term health-state valuation may provide a reasonable approach to assessing preferences given its superior ability to discriminate between states, which may be particularly useful for cost-utility analyses for future vaccination programs.Dr. Lee had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Lee, Salomon, LieuAcquisition of data: Lee, LieuAnalysis and interpretation of data: Lee, Salomon, LeBaron, LieuDrafting of the manuscript: LeeCritical revision of the manuscript for important intellectual content: Lee, Salomon, LeBaron, LieuStatistical analysis: Lee, SalomonObtained funding: LieuAdministrative, technical, or material support: LieuStudy supervision: Salomon, LieuThis study, part of the Joint Initiative on Vaccine Economics Project, was supported by the National Immunization Program, Centers for Disease Control and Prevention, via cooperative agreement with the Association of Teachers of Preventive Medicine, Task order #TS-0675. Dr. Lee's work was also supported in part by the grants T32 HS00063 and K08 HS013908-01A1 from the Agency for Healthcare Research and Quality, U.S. Department of Health and Human Services. Dr. Salomon was supported by the National Institute on Aging (Grant P01 AG17625).


MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a  A transgenic mouse model demonstrates that developmental context may be the reason why the spectrum of tumors differs in children and adults The frequency of cancer development varies depending on the age of the host. In humans, the most common childhood cancers include tumors of the hematopoietic system, nervous system, and skeletal muscle system. In contrast, in the adult population, solid tumors of the lung, colon, breast, and prostate are more common. Differences in types of cancers in hosts of different ages may reflect the abundance of cells in a differentiative state susceptible to tumorigenesis . Indeed,C-MYC oncogene to induce tumorigenesis in mice of different ages using a novel conditional transgenic model system for C-MYC–induced hepatocellular carcinoma (HCC).We recently reported that upon oncogene inactivation tumor cells can differentiate into mature cells, and in this new differentiative context the reactivation of an oncogene fails to restore tumorigenesis . Based oC-MYC (now referred to as MYC) is a member of a family of proto-oncogenes comprising C-MYC, N-MYC, and L-MYC. MYC encodes a transcription factor that, as part of a heterodimeric complex with MAX, regulates the expression of a multitude of genes involved in regulating cellular proliferation and growth  and other oncogenes (e.g., RAS, T antigen) in murine hepatocytes results in HCC  . We founMYC in murine hepatocytes  transgenic for both TRE-r masses B. The nor masses C. Tumorsr masses A–2C. Thescid mice. Tumors formed in the inoculated mice after an 8–10-week latency  succumbed to neoplasia within 8 weeks. Mice in which MYC was induced at 4 weeks or 6–12 weeks of age developed tumors after a mean latency of 15 and 35 weeks, respectively . We did not observe any histological changes in the liver after 10 d of MYC activation . Thus, there may be an absolute limit to the ability of MYC to induce liver growth. MYC activation in adult hepatocytes was not associated with a change in overall liver weight . However, this latency of tumorigenesis in adult mice after partial hepatectomy was still up to two magnitudes longer than what was observed when MYC was activated in embryonic and neonatal mice (<10 d and 4 weeks, respectively)   and 3A. i of HCC B and 11CMYC's ability to induce cellular growth versus proliferation and tumorigenesis depends on the differentiative context of a cellular lineage. Our results have general implications for the mechanisms by which MYC and other oncogenes induce and are restrained from causing tumorigenesis.Here we demonstrate that MYC during embryonic development and at birth caused gross liver enlargement and rapid emergence of neoplasia. This increased liver size resulted from increased cell number or cellular hyperplasia. In contrast, in adult murine hepatocytes, MYC activation induced cell growth, resulting in cellular hypertrophy and endoreduplication. MYC overexpression caused HCC in adult mice less frequently than in neonates, and only after a prolonged latency. We conclude that the ability of MYC to induce cellular proliferation and tumorigenesis appears to be determined by the relative developmental maturation of a cellular lineage.We found that the initial consequences of MYC overexpression, as well as its ability to induce tumorigenesis in murine hepatocytes, depend on the host age. The activation of MYC induction. We were able to address this possibility directly by examining the consequences of MYC overexpression in adult hepatocytes after partial hepatectomy, which resulted in the immediate induction of adult hepatocyte proliferation associated with complete liver regeneration within 2 weeks. Adult mice only exhibited a modest increased susceptibility to MYC-induced tumorigenesis observed when MYC was induced in embryonic mice. This is despite the fact that after partial hepatectomy the total number of proliferating hepatocytes would be at least two magnitudes greater than in a neonatal or embryonic liver, whose total mass is much smaller. We conclude that developmentally specific parameters other than proliferation are more likely to play a causative role in the differential susceptibility of embryonic and neonatal hepatocytes to MYC-induced tumorigenesis.We recognized that an alternative explanation for our results is that only hepatocytes that are actively proliferating are susceptible to neoplastic transformation upon Several different development-related factors could account for our observations. The relative ability of MYC to induce tumorigenesis in developmentally immature hosts may reflect the relative increased abundance of immature hepatocytes. Homeostatic mechanisms that regulate liver size and hepatocyte proliferation may permit MYC-induced proliferative expansion in the developing livers of young hosts and restrain proliferation in the mature liver of adult hosts. Differences in rates of tumorigenesis may be related to telomerase function that monitors the distance from senescence, as recently described . An unliMYC, from inducing cancer in adult somatic cells. MYC activation alone is capable of enforcing DNA replication, but not cell division  from the drinking water of mice transgenic for both TRE-MYC and LAP-tTA.The TRE-eviously . The LT-eviously . The p53MYC was activated in the liver by removing doxycycline treatment from the water. MYC was activated during embryonic development by removing doxycycline treatment from the mating cage before conception. MYC was activated at birth by removing doxycycline from the mating cage immediately after the birth of the litter. Mice were monitored daily and were sacrificed when moribund. During necropsy, liver tissues were saved by fixation in 10% buffered formalin or by being snap frozen in liquid nitrogen. For transplantation, liver tumor specimens were sliced into small pieces, incubated first in calcium-free Hank's Buffered Saline Solution (HBSS) on a stirring plate at 37 °C for 20 min, then washed and resuspended in 1× digestion buffer with 1.5 mg/ml collagenase, and incubated on a stirring plate at 37 °C for an additional 40 min. The solution was then filtered through a 100-μm filter, washed, and resuspended in PBS twice. After the first wash, the solution was resuspended in 10 ml of PBS. After the second filtering and wash, the solution was resuspended in 500 μl of PBS. A quantity of 106 cells was injected subcutaneously into scid mice (250 μl per injection) using a 1-ml syringe and 27-gauge needle. Mice showed signs of tumorigenesis within 2–3 weeks of inoculation. Mice were sacrificed when tumors reached 2 cm in size. To prepare 5× digestion buffer, 1.12 g of KCl, 37.94 g of NaCl, 0.69 g of NaH2PO4*H2O, and 9.9 g of dextrose monohydrate were dissolved in 1 liter of H2O.The median and left lateral lobes comprise about 70% of the liver and their removal is recognized classically as a partial hepatectomy. We performed a one-third partial hepatectomy, removing only the median lobe of the liver. Mice were anesthetized with 16 mg/kg ketamine/xylazine. An incision was made through the midline ventral abdominal skin and abdominal muscles, extending from just above the xiphoid cartilage to about halfway towards the base of the tail. A small bolster was placed under the thorax, causing the liver to fall forwards away from the diaphragm. The liver was pushed out and the suspensory ligaments were cut with blunt-end scissors. The median lobe was raised and a ligature was tied around it with the blood vessels at the base. The liver lobe was removed by cutting close to the ligature with sharp scissors. The bolster was removed and the muscle and skin incisions were closed. The mice were monitored hourly for pain and dehydration after the procedure.Liver tissues were fixed in 10% buffered formalin for 24 h and then transferred to 70% ethanol until embedding in paraffin. Tissue sections 4 μm thick were cut from paraffin-embedded blocks and placed on glass slides. Hematoxylin and eosin (H&E) staining was performed using standard procedures. The Stanford Histology Core laboratory prepared paraffin sections and performed H&E staining. We measured Ki67 expression by immunofluorescence using a mouse anti-human Ki67 monoclonal antibody . We used the Vector M.O.M. Basic Kit . Sections were deparaffinized with xylene and rehydrated through graded alcohol washes, followed by antigen retrieval in a microwave for 15 min in Vector Antigen Unmasking solution (H-3300). The slides were then incubated in 100 mM glycine for 2 × 8 min to reduce fluorescent background. Slides were blocked by incubation in avidin for 10 min followed by biotin for 10 min using the Dako biotin blocking system  and subsequently incubated for 1 h in M.O.M. IgG-blocking reagent diluted 1:4 in PBS. Slides were then incubated for 1 h in mouse anti-human Ki67 monoclonal antibody diluted 1:100 in M.O.M. diluent. Slides were washed in TBST for 3 × 5 min to reduce background and were then treated with M.O.M. biotin-labeled anti-mouse IgG, diluted 1:250 in M.O.M. diluent. Following another 3 × 5 min of TBST washes, slides were incubated for exactly 45 min in Cy3-conjugated streptavidin diluted 1:800 in PBS (Amersham Biosciences, Piscataway, New Jersey, United States) in the dark. To visualize nuclei, slides were counterstained with 0.2 μg/ml DAPI. Ki67-positive cells were visualized by fluorescence microscopy.Western analysis was performed using conventional techniques. Liver tissues were disrupted and protein was isolated using a pestle and tube homogenizer in NP-40 lysis buffer. Equal protein was loaded in each lane, as quantitated by the Bicinchoninic Acid (BCA) Protein Assay (Pierce, Rockford, Illinois, United States). Proteins were electrophoresed on 10% Tris-HCl polyacrylamide gels at 100 V for 60 min and transferred on PVDF membranes at 100 V for 60 min. The membrane was blocked in 5% nonfat dry milk solution in TBS at 4 °C overnight. MYC protein expression was detected using the C-19 rabbit polyclonal antibody that recognizes mouse and human MYC . p53 protein expression was detected using the NCL-p53-CM5p rabbit polyclonal antibody (Vector Laboratories). As a positive control, we used a hematopoietic tumor previously shown to overexpress p53 that was generously provided by Dr. Kevin Smith.Images of H&E-stained liver sections were made with a Nikon Eclipse E800 microscope utilizing a Spot RT Slider digital camera (Diagnostic Instruments, Sterling Heights, Michigan, United States) and Spot Advanced Software (version 3.2.4, Diagnostic Instruments). To estimate cellular and nuclear volume, the radii of at least five hepatocytes per field were measured in at least three fields.Hepatocytes were collected by a two-step in situ perfusion technique . First, Cell size was determined by FACS forward versus light scatter of isolated hepatocytes utilizing a Becton Dickinson FACSCaliber (BD Biosciences). Data were analyzed using Cellquest v3.3 Software (BD Biosciences).Nuclei were prepared for staining by touching a cut piece of liver to superfrost slides. The smears were then air dried, fixed in formalin for 5 min, and stored in 70% ethanol. The nuclei were stained for DNA content analysis according to the Feulgen technique .Apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, using the In Situ Death Detection Kit (Boehringer Mannheim, Indianapolis, Indiana, United States). In order to visualize the nuclei, cells were counterstained with DAPI (0.2 μg/ml). TUNEL-positive cells were visualized by fluorescence microscopy.p21, MDM2, and glyceraldehye-3-phosphate dehydrogenase (GAPDH) were used as probes  product specifications. cDNAs corresponding to s probes . The proTo evaluate loss of heterozygosity of liver tumors derived from mice heterozygous for p53 deletion, PCR analysis was performed as previously described .260 measurements. The quality of RNA was measured on a formaldehyde, 1% agarose gel.Total cellular RNA was isolated from snap-frozen liver tissue using the Invitrogen Micro-to-Midi Total RNA Purification System according to the manufacturer's instructions. The amount of total RNA isolated from tissues was quantified using spectrophotometric ODMYC and rodent GAPDH were measured by real-time quantitative RT-PCR using the 5′ nuclease technology on an ABI PRISM 7900HT Sequence Detection System . The probe sequences for human c-MYC were forward primer 5′-CCCCTGGTGCTCCATGAG-3′ and reverse primer 5′-GCCTGCCTCTTTCCACAGA-3′. The human c-MYC probe, 5′-TCCTCCTCAGAGTCGC-3′, was labeled with FAM dye-MGB. A VIC TaqMan rodent GAPDH control reagents kit (Applied Biosystems) was used to measure mouse GAPDH. The RNA was reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's protocol with a minor modification, the addition of RNase inhibitor (Applied Biosystems) at a final concentration of 1 U/μl. Samples were incubated at 25 °C for 10 min and 37 °C for 180 min. PCR reactions were prepared in a final volume of 20 μl, with final concentrations of 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and cDNA derived from 20 ng of input RNA as determined by spectrophotometric OD260 measurements. Thermal cycling conditions comprised an initial UNG incubation at 50 °C for 2 min, AmpliTaqGold DNA polymerase activation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. Each measurement was performed in triplicate and the threshold cycle t), and cDNA prepared from Universal Mouse Reference RNA (Stratagene). Universal Human Reference RNA was used for human c-MYC and Universal Mouse Reference RNA was used for rodent GAPDH.The RNA of human c-http://us.expasy.org/sprot/) for the loci discussed in this paper are the following: Cdkn1a (mouse), P39689; Mdm2 (mouse), P23804; Myc (human), P01106; Myc (mouse), P01108; and Tp53 (mouse), P02340.Swiss-Prot accession numbers (


Many studies associated nosocomial infections with increased hospital costs due to extra days in hospital, staff time, extra investigations and drug treatment. The cost of antibiotic treatment for these infections represents a significant part of hospital expenditure. This prospective observational study was designed to determine the daily antibiotic cost of nosocomial infections per infected adult patient in Akdeniz University Hospital.All adult patients admitted to the ICUs between January 1, 2000, and June 30, 2003 who had only one nosocomial infection during their stay were included in the study. Infection sites and pathogens, antimicrobial treatment of patient and it's cost were recorded. Daily antibiotic costs were calculated per infected patient.Pseudomonas aeruginosa infections was the most expensive infection treated. Piperacillin-tazobactam and amikacin were the most prescribed antibiotics, and meropenem was the most expensive drug for treatment of the nosocomial infections in the ICU.Among the 8460 study patients, 817 (16.6%) developed 1407 episodes of nosocomial infection. Two hundred thirty three (2.7%) presented with only one nosocomial infection. Mean daily antibiotic cost was $89.64. Daily antibiotic cost was $99.02 for pneumonia, $94.32 for bloodstream infection, $94.31 for surgical site infection, $52.37 for urinary tract infection, and $162.35 for the other infections per patient. The treatment of Daily antibiotic cost of nosocomial infections is an important part of extra costs that should be reduced providing rational antibiotic usage in hospitals. Nosocomial infections are frequent complications of hospitalization and also an important public health problem in developing countries, as well as in developed ones. The socioeconomic impact, ie, prolongation of hospitalization, mortality, and cost of these infections adversely effects patients and nations' economic well-being . The cosThe aim of our study was to determine daily antibiotic cost of nosocomial infection per infected patient in a university hospital.Akdeniz University Hospital is a 600-bed tertiary referral centre in Antalya, Turkey, treating 27 000 patients per year. The study was conducted in six adult medical and surgical intensive care units (ICUs) with a total of 51 ICU beds. Neonatal ICU was not included in the study. Since 1993, the institutional policies of hospital infection control have been implemented by infection control team.In our hospital, routine prospective, active surveillance of nosocomial infections in all ICUs is performed by one infection control nurse, supervised by an infection control physician. Nosocomial infections are defined using the Centers for Disease Control and Prevention criteria ,6. We doBetween January 1, 2000 and June 30, 2003, all inpatients hospitalized in one of the adult ICUs were included in this study. Data on antimicrobial treatment were recorded for patients aged 15 or above presenting with only one nosocomial infection. For all patients included in the study, the following were recorded: age, sex, infection site, microbiologic data, antimicrobial therapy and antibiotic cost.In our ICUs, all antimicrobial prescriptions are recommended by an infectious disease consultant. Antimicrobial agents prescribed only for therapeutic indications were recorded. The daily antibiotic cost was calculated in US dollars based on June 2003 prices of antimicrobial agents provided by the hospital pharmacy. The daily antibiotic cost per infected patient was calculated by the multiplication of box price and number of daily doses that was used for that infection. Two costs were calculated for each antimicrobial; the minimal cost (min) was based on the lowest recommended parenteral daily dose and the maximal cost (max) was based on the highest recommended parenteral daily dose.Between January 1, 2000, and June 30, 2003, a total of 8460 patients were admitted to the adult ICUs. Overall, 817 patients developed 1407 episodes of nosocomial infections, accounting for an infection rate of 16,6%. Among them, 233 patients  had only one nosocomial infection. Mean daily antibiotic cost was found $89,64 per infected patient ($8,56 to $359,28).Among the sites of nosocomial infections, urinary tract infections had the lowest daily antibiotic cost per infected patient Table . The meaPseudomonas aeruginosa was the most prevalent bacteria followed by Klebsiella spp. and Acinetobacter spp.. P. aeruginosa infections had the highest overall daily antibiotic cost per infected patient than other pathogens. Among 21 Staphylococcus aureus strains 15 (72%) were resistant to methicillin. The overall daily antibiotic cost of methicillin resistant S. aureus (MRSA) infections was two to three times higher than infections with susceptible strains. However, median daily cost per infected patient for MRSA infections were lower than susceptible strain infections.Out of 233 patients, 206 patients had microbiologically documented infections, 177 patients were infected by a single pathogen while 29 patients were diagnosed as having polymicrobial infections  declare that they have no competing interests.DI collated and analyzed the data, participated in the study design and was principal writer of manuscript. ANY conceived the study. GO carried out the laboratory studies. RS, FG, OT and LM participated in the patient management. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


PLoS Biology, biochemist Jeremy Brockes and his colleagues at University College London tie this muscle cell de-differentiation to expression of a single gene, Msx1, a player in limb development and regeneration across species.Cell and molecular biologists have a good start at understanding the adult salamander's enviable ability to completely regrow a lost limb or jaw.  Happily, mammals share many of the required cellular skills—though in an untapped form. In response to injury, fully differentiated salamander muscle cells can produce less specialized cells that are capable of multiplying and recreating lost tissue. In this month's Msx1 appears at the borders of maturing tissues in salamanders, chickens, and mice, and represses differentiation of muscle cells  during development. Msx1 promotes amphibian limb and tail regeneration, zebrafish fin repair, and regrowth of the tips of digits in mice. Here, Anoop Kumar et al. clarify the gene's role in muscle repair by demonstrating that Msx1 expression is required for de-differentiation of salamander myofibers—mature muscle tissue without the ability to produce new cells—into cells with single nuclei (mononucleate cells) that are able to multiply.The protein encoded by Large, elongated cells, myofibers are distinguished by a striped pattern of actin and myosin, the proteins that produce muscle contractions. They develop from precursor cells, which proliferate and fuse together into multinucleate intermediates called myotubes. As myotubes bulk up on actin and myosin, pushing their many nuclei to the cell periphery, they mature into myofibers. Once differentiated, myofibers are committed to the life of a muscle workhorse. They cannot divide; mature muscle in mammals adds strength and repairs injury by accumulating more actin and myosin, and by fusing single-nucleus “satellite” cells into new or existing myofibers.Msx1. A fraction of mouse myotubes made to express Msx1 break off mononucleate cells that can be induced to express markers for bone, cartilage, fat, or muscle.Unlike their mammalian counterparts, salamander and newt myofibers respond to injury by splitting into several multinucleated fragments, or by budding off several of their nuclei to create individual cells. These mononucleate progeny multiply and develop to replace lost tissue. Kumar et al. found that more than half of salamander myofibers spontaneously fragment and bud when dissected and placed in cell culture. Prior study has shown that mouse myofibers don't normally behave this way, although cultured mouse myotubes can imitate amphibian myofibers if given a push, in the form of Msx1 RNA and protein appeared in actively fragmenting and budding myofibers, especially in and around their nuclei. When Kumar et al. blocked the translation of Msx1 mRNA, the myofibers did not generate new cells.In the salamander cells, Msx1 as a pivotal regulator of muscle cell re-entry into the cycle of cell division and tissue growth. In this context, it doesn't drive cell division—on the contrary, the authors showed that Msx1 induced cell splitting without DNA replication. The gene drove committed muscle cells to donate nuclei, creating a pool of new cells to divide, differentiate, and repair damage. Given what it can do for mouse cells in culture, the question remains whether Msx1 might help awaken a latent capacity for regeneration in living mammals.The results confirm