Following the tsunami, a detailed overview of the area specific transmission levels is essential in assessing the risk of malaria in Sri Lanka. Recent information on vector insecticide resistance, parasite drug resistance, and insights into the national policy for malaria diagnosis and treatment are important in assisting national and international agencies in their control efforts.Monthly records over the period January 1995 – October 2004 of confirmed malaria cases were used to perform an analysis of malaria distribution at district spatial resolution. Also, a focused review of published reports and routinely collected information was performed.The incidence of malaria was only 1 case per thousand population in the 10 months leading up to the disaster, in the districts with the highest transmission.Although relocated people may be more exposed to mosquito bites, and their capacity to handle diseases affected, the environmental changes caused by the tsunami are unlikely to enhance breeding of the principal vector, and, given the present low parasite reservoir, the likelihood of a malaria outbreak is low. However, close monitoring of the situation is necessary, especially as December – February is normally the peak transmission season. Despite some losses, the Sri Lanka public health system is capable of dealing with the possible threat of a malaria outbreak after the tsunami. The influx of foreign medical assistance, drugs, and insecticides may interfere with malaria surveillance, and the long term malaria control strategy of Sri Lanka, if not in accordance with government policy. After the tsunami hit Sri Lanka on 26 December 2004, news reports and public health agencies warned against the possibilities of an increase of vector borne diseases, in particular malaria and dengue. Immediately after the disaster, an estimated 860,000 people were displaced and more than 820 emergency camps established throughout the affected areas . By 14 JPlasmodium vivax and the remainder by Plasmodium falciparum [Malaria in Sri Lanka is of a highly unstable nature and has historically fluctuated greatly over the years and with significant seasonal differences. Sixty-five to eighty percent of the malaria cases are caused by lciparum . Recentllciparum . The preet al. [Malaria maps were based on monthly records over the period January 2004 – October 2004 (the most recent month for which data recording was complete at the time of writing) of microscopically confirmed malaria parasite positive blood smear readings, at district spatial resolution. These were collected by the Anti Malaria Campaign (AMC) Directorate of the Ministry of Health from aggregated disease records reported by governmental hospitals and mobile clinics. Additionally, in the temporal analysis, monthly data by district for the period 2001 – 2002, and data by sub district for 1995 – 2000 as described by Briët et al. . For allet al. . The natet al. . For eacet al. [A focused review of literature has been performed, identifying crucial information for the outbreak preparedness and control during the emergency phase. The intent was not to present a complete review of malaria in Sri Lanka but to provide information useful for an assessment of the current situation. A general review of malaria in Sri Lanka can be found in Konradsen, Amerasinghe et al. .P. falciparum and P. vivax and dormant stages of P. vivax normally provide the parasite reservoir for bridging periods of low seasonal transmission (with unsuitable conditions for mosquito vectors). Under the present policy of administering primaquine in addition to chloroquine (see section on diagnosis and treatment), the reservoir of dormant stages of P. vivax will be low and this will delay a possible outbreak. It must be emphasized that the low level of malaria transmission in the recent past does not guarantee that localized or even island wide epidemics will not occur. In the past, even after periods of very low levels of malaria transmission, outbreaks have occurred, often due to constraints placed on the public health system, by unusual rainfall patterns or by yet unexplained factors.The country-wide malaria incidence increased from January 1996 to January 2000, with the typical seasonality of high peaks around January and lower peaks around June – July, but it has decreased dramatically since January 2000 Figure  and 5. AAn important factor to consider in the current situation is the capacity of the existing health care service. Following the tsunami the Sri Lanka Ministry of Health reported 22 hospitals and nine administrative buildings damaged or completely destroyed, mostly in Ampara and Trincomalee districts . It has In some places, central stocks of medical supplies were destroyed, including the Regional Medical Supply Division in the Ampara District. However, sufficient drugs have been imported during the days and weeks following the disaster. The World Health Organization has drawn up plans for antimalarials, insecticides and spray equipment to be made available on request. Although the increased capacity at the district and provincial levels has improved the co-ordination, a risk remains that local needs for health care are not adequately covered in spite of the availability of significant resources. In some parts of the island, especially areas in the east, affected both by the destruction caused by the tsunami and by exceptionally heavy rainfall in the weeks following, distribution of drugs has been problematic and this has left certain communities vulnerable.Whereas the overall capacity to provide treatment and routine malaria control activities, in general, has not been severely hampered, the routine health information system will have been constrained by the large number of autonomous health camps set up, and their lack of integration with the established surveillance system. It is essential to establish a system for monitoring malaria in the affected areas. Many people are moving back to their old place of residence trying to rebuild livelihoods and it will be essential for the public health authorities to keep contact with these communities to prevent an increase in malaria going unnoticed.P. vivax as well as P. falciparum infection. Primaquine is not administered to children below one year, and those with known G-6PD enzyme deficiency, and for pregnant mothers.In Sri Lanka, microscopy on blood smears or use of rapid diagnostic test kits have been the standard diagnostic procedure, and precedes the prescription of drugs to the patient. In the current situation, with the many small health clinics established within emergency camps, it is likely that the use of rapid diagnostic kits would be the more feasible means of confirmation. The first line drugs recommended for malaria treatment in Sri Lanka is still a chloroquine and primaquine (PQ) combination for cases of P. vivax infections in Sri Lanka. The first chloroquine-resistant P. falciparum case was reported in 1984 [in vivo chloroquine resistance has been recorded in malarious areas [P. falciparum the government recommended drug is sulphadoxine-pyrimethamine (SP). However, SP is not recommended for the last trimester of pregnancy, first six weeks of lactation and for children below two months of age. The first SP-resistant case of P. falciparum was reported in 1992 in Polonnaruwa district. Up to 1999, five to six cases have been reported by the AMC. More recently (January – June 2002), SP resistant P. falciparum has been documented in the Northern Province [So far, there have been no reports of chloroquine-resistant  in 1984 . Up to 6us areas ,15-17. FIn the current emergency situation, with many (foreign) doctors working autonomously, the diagnosis and treatment practices may depart from the established government guidelines and new antimalarials are also likely to be brought in. Moreover, the current practice of restricting SP to government hospitals will be difficult to enforce. Similarly, introduction of low quality and obsolete drugs will be difficult to counter at community level at the current stage of supervisory capacity and co-ordination level. Drugs have been reported stolen from warehouses, allegedly finding their way to private trade establishments . OverallAnopheles subpictus sibling species B, which is a well-known coastal breeding species in Sri Lanka. However, it has not been directly incriminated as a field vector in Sri Lanka, despite its susceptibility to P. falciparum [An. subpictus sibling species B and the complete absence of Anopheles culicifacies , and suggested that this An. subpictus sibling may have a role in transmission. It is noteworthy, that freshwater An. subpictus , which breeds in muddy rain fed puddles, has been consistently incriminated in malaria transmission in many inland areas of Sri Lanka [Anopheles vagus. This species has been linked as a vector responsible for a malaria outbreak in southern Sri Lanka [An. subpictus nor An. vagus, are likely to cause major malaria epidemics but could, at high density, be responsible for focal outbreaks that need quick action. Thus, it is important that an entomological monitoring programme be set up in the period leading up to and during the south west monsoon that is expected during May to June 2005 in the tsunami affected western and southern Sri Lanka. It should be noted that the infamous Asian brackish water breeding malaria vector Anopheles sundaicus, which is a threat in the tsunami-affected areas in Indonesia, Myanmar, and the Andaman and Nicobar islands [The seawater brought inland by the tsunami has mixed with monsoon rainwater to form puddles of varying salinity. Also, thousands of muddy surface water puddles have been created as a result of destruction and rehabilitation activities that are already underway. The brackish puddles are expected to favour the breeding of lciparum . Neverthlciparum  found peri Lanka . Anotherri Lanka ,21. On p islands , does noAn. culicifacies type E [Anopheles culicifacies also breeds in abandoned gem mining pits, agricultural wells and to a lesser extent in pools in agricultural water reservoirs [An. culicifacies, unless it blocks waterways and creates pooling. Post-tsunami development activities may revive banned sand mining practices in rivers. If this happens, clear water pools created by these sand mining activities may serve as breeding sites for An. culicifacies [Aedes aegypti, does not breed in saline water [The main vector in Sri Lanka is s type E ,24, whicne water . HoweverThe Colombo based Head Office of the AMC gives the overall guidelines for island wide vector control, while each province works out a plan for control activities based on the distribution and level of malaria transmission. Several malaria vector control interventions are currently employed within the country. In all districts, residual insecticide spray activities are focused on areas where malaria transmission has been established by confirmed malaria cases. The control of anopheline larvae using mostly chemicals focuses on sites close to human habitation. Small-scale application of larvivorous fish and environmental modifications are also carried out. Since 1997, mosquito nets, which are biannually treated with insecticide, are distributed free of charge in malarious areas. During the last two years, the main control effort has been through these nets. Since January 2004, 80,000 nets with long lasting insecticide have been distributed. Also, nets are available for purchase from outlets in most parts of the country.An. culicifacies and a range of potential secondary vectors such as An. subpictus and An. vagus have shown high level of resistance to either organochlorines, organophosphates or to both groups of insecticides [An. culicifacies and An. subpictus has been found resistant. Currently, synthetic pyrethroids such as Cyfluthrin, Deltamethrin, Etofenprox, and Lambda-cyhalothrin are being used in the country. At present, Fenitrothion is the only organophosphate used for vector control. A study conducted by Abhayawardana from 1990 to 1992  on An. subpictus found 68% and 54% susceptibility to Malathion and Fenitrothion, respectively, for inland species (sibling species A), whereas for coastal species (primarily sibling species B) it was 100%. However, the latter was found resistant to permethrin [Studies in Sri Lanka over the 1990s on cticides ,26-28. Drmethrin . From seThe majority of the people initially affected by the disaster are still living in emergency camps or in other places close to the coast. At the time of writing, to the best of our knowledge, relatively few people have moved from areas of low or no malaria transmission to areas of high transmission. However, during the next phases, when people may be resettled in semi-permanent and later in permanent housing, communities may be relocated from areas where they have had no malaria experience to malarious areas. In these situations, the communities' capacity to cope with malaria infection will be low.An. culicifacies breeding, were at significantly higher risk for malaria than people living further away [Despite distribution of nets to many camps, and intensified vector control in some areas, people in the emergency camps  and those returning to damaged houses are more exposed to mosquito bites than in pre-disaster housing, due to the open nature of the shelter. Additionally, most families will have lost mosquito nets or other means to protect against mosquito bites. It is more difficult to assess the protective effect of tents that have been set up in most of the semi-permanent camps established. The location of semi permanent and permanent settlements may have a significant effect on the risk of infection. Epidemiological studies from other parts of Sri Lanka have shown that people living within 750 m of a stream with her away .P. vivax and P. falciparum malaria incidence distribution on the island of Sri Lanka at district resolution in the 10 months preceding the tsunami, and an analysis of monthly malaria incidence in the country since January 1995. The malaria incidence was historically low, which implies a limited parasite reservoir in the human population. In spite of the fact that the months of December - February are normally the peak period for transmission, given the transmission level in the months leading up to the disaster, the risk of a large-scale outbreak seems to be limited. However, the low transmission levels over the past years may also have made people less alert to possible outbreaks, and the population would have less protective immunity towards the disease. The environmental changes resulting from the tsunami do not create particular opportunities for breeding of the principal malaria vector An. culicifacies but potential does exist for less important species such as An. subpictus and An. vagus. People living in emergency camps or returning to pre-disaster areas of residence are at higher risk of mosquito bites than normal. In spite of the emergency, the capacity of the public health authorities to perform malaria preventive and curative interventions remains high and essential supplies and staff capacity is not a problem. However, co-ordination of assistance and maintaining a strong surveillance system remain significant areas of concern. Increased attention to the establishment of a monitoring system including both parasitological and entomological parameters is recommended. Likewise, the large inflow of donated drugs and insecticides outside government control will potentially have long term implications on malaria control and case management, and especially the quality of administered drugs and the development of drug resistance requires careful monitoring.This paper provides maps of both GNLG collected the malaria data. OJTB checked the data, calculated incidence, made the maps. FK, FPA and PHA performed the focused literature review. All authors helped write, read and approved the final manuscript.


In a sample of 32 chromosomes from a Nordic population, we attempted to determine the allele frequencies of 80 non-synonymous SNPs, and found 20 novel polymorphic markers. GPCR receptors of physiological and clinical importance were prioritized for statistical analysis. Natural variation and rare mutation information were merged and presented online in the Human GPCR-DB database The average number of SNPs per 1000 bases of exonic sequence was found to be twice the average number of SNPs per Kilobase of intronic regions (2.2 versus 1.0). Of the 338 genes, 111 were single exon genes, that is, were intronless. The average number of exonic-SNPs per single-exon gene was 3.5 (n = 395) while that for multi-exon genes was 0.8 (n = 1176). The average number of variations within the different protein domain  indicates a lower rate of variation in the trans-membrane region of Monoamine GPCRs, as compared to Chemokine- and Peptide-receptor sub-classes of GPCRs.Single-exon GPCRs on average have approximately three times the number of SNPs as compared to GPCRs with introns. Among various functional classes of GPCRs, Monoamine GPRCs have lower number of natural variations within the trans-membrane domain indicating evolutionary selection against non-synonymous changes within the membrane-localizing domain of this sub-class of GPCRs. From a pharmacogenetics viewpoint, the information about natural variations within GPCR transcripts and peptides, with allele frequency and validation data and disease association, is an important, yet currently unavailable, public resource. A database with functional promoter SNP, allele frequency, peptide variation information, population and haplotype information presented in a graphically accessible format would facilitate pharmacogenomics research related to GPCR proteins. HUMAN GPCR-DB aims to provide such a public resource. Also the rate of false positive SNPs determined experimentally in GPCR genes is reported to be relatively high [The 7TM (7 trans-membrane domain proteins) genes, also known as the hetero-trimeric GTP-binding protein (G protein)-coupled receptors (GPCRs), are members of a large family of genes with an estimated 700 members in the human genome . These rely high . For typSeveral recent studies have focused on the subset of nsSNPs that most likely influence phenotype -13. CompA total of 427 non-redundant Human GPCR peptides were obtained from Swissprot of which 338 had Swissprot ID and the remaining had only TrEMBL identifications. Also, 89 of the 427 GPCR were classified as olfactory receptors. While all of the 427 entries were included in the HUMAN GPCR-DB database, only non-olfactory (i.e. 338) were considered for further analysis of gene structure, alternative transcripts, SNPs, and protein variations. Although the TrEMBL entries have also been included, the data displayed for these entries would be more accurately presented in future updates of the database. The statistical calculations for the genomic SNPs are based on the 338 non-olfactory entries, while the data for nsSNPs encoding peptide variations is based on 222 entries for which there are documented evidence for a 7-TM domain structure.Transcript information for each gene was used for calculating average exonic and intronic SNPs. For genes with multiple transcript variants, the longest transcript was selected. Average number of SNPs per exon n = 1511) of the 338 genes was one, while the average number of SNPs per intron (n = 1174) was nine. However, the average number of SNPs per 1000 bases of exonic sequence was twice the average number of SNPs per kilobase of intronic regions (2.2 versus 1.0). Of the 338 genes, 111 were single exon genes, that is, were intronless. The average number of exonic-SNPs per single-exon gene was 3.5 (n = 395) while that for multi-exon genes was 0.8 (n = 1176). This observation is in agreement with earlier observation based on a smaller number of GPCRs, where, compared to intronless GPCRs, exons in genes with introns on average had fewer SNPs [11 of theOf the 1511 SNPs from the exons of 338 GPCR transcripts, 816 SNPs were coding SNPs, among which 392 were nsSNP; 211 of which had no validation information in either of the source databases, i.e. NCBI/dbSNP and HGVBase. The number of validated and non-validated SNPs is shown in Table Table , 101 GPCRs from 338 were categorized either as 'peptide receptors' (n = 47) or 'chemokine receptors' (n = 54) or 'monoamine receptors' (n = 36). The distribution of nsSNPs across the 5 structural and functional domains  of these 101 GPCRs was calculated  and the query gene. The exons are drawn in proportion to the largest exon, while the introns are of fixed length. The SNP list provides allele information, flanking sequences  and links for validation information and source databases. The promoter information is drawn in a similar manner, with mouse conserved regions indicated with green bars underneath and SNPs within conserved regions marked with a symbol 'M' in the SNP full-list. SNPs predicted to influence protein binding according to our prediction model are marked 'T' in the SNP full-list. The link for protein variations displays a window with SNP, marked in red arrows, and mutation distribution across 3 regions of the peptide; N-terminus, C-terminus and trans-membrane and loop regions. Association with diseases and the corresponding PubMed ID are displayed as a popup menu following the link under 'disease' column.The HUMAN GPCR-DB is currently online We have constructed a database, which combines mutation information with validated SNP information from publicly available sources. We have then attempted to validate and determine the frequencies of 80 of the 123 non-synonymous SNPs for which no validation information was available publicly. Proportion of true polymorphic loci was 20%, in agreement with reported expectations from several studies .The statistical approach for the analysis of distribution of natural variations in GPCRs, presented here is borrowed from two recent studies ,16. WhilAlthough HUMAN GPCR-DB database does obtain the bulk of the information and data from Ensembl and dbSNP, it is not merely a subset of these major databases. While Ensembl provides sequence and genetic variation information, it provides SNP validation information obtained from public sources, which may include, as shown in several published studies, up to 50% false positives. NCBI's dbSNP provides validation-, submitter- and method-information, yet rates of false positives have proven to be high. These databases harbor information of genetic variations for all coding and non-coding regions of the human genome. The HUMAN GPCR-DB, in addition to providing this set of information, provides in-house validation and assay-information for non-validated nsSNPs. HUMAN GPCR-DB provides natural variation, mutation, promoter- and peptide-variation information along with gene structure and peptide 7-TM structure information and SNP validation information of a focused group of clinically important genes.Transcriptional regulatory regions on the 5'-FR of human genes encode short sequences which serve as targets for binding of transcription factors (TFs). Eukaryotic TFs tolerate considerable sequence variation in their target sites and recent works in bioinformatics -19 have The functional domains of human GPCRs have over the passed decade been dissected by systematically mutating the peptide sequence . A colleOf the 123 nsSNPs without validation information, we have currently validated 80 SNPs. Future updates would include information about the remaining nsSNPs, and any additional which are reported by public databases. We have also collected 120 published SNPs, which have as yet not been deposited at public databases, or are in the process of being deposited. We would have a complete update of SNP data for every new release of NCBI and HGVBase SNP tables. New GPCR identification and characterization, or changes in existing GPCR genes or proteins information would be updated once a year from Ensembl and SwissProt Databases. PubMed references would be updated monthly or as often as necessary to complete the mutation coverage of the 222 GPCR proteins. Haplotype information and genetic association studies for available SNPs along with published records on functional promoter SNPs would be added. A valuable addition would be to indicate variation frequencies in ethnically diverse populations. We are currently adding such information about the allele frequencies and populations in the database and would be provided in future updates.Single-exon GPCRs on average have approximately three times the number of SNPs as compared to GPCRs with introns. Among various functional classes of GPCRs, Monoamine GPRCs have lower number of natural variations within the trans-membrane domain indicating evolutionary selection against non-synonymous changes within membrane localizing domain. The HUMAN GPCR-DB compiles SNPs and mutations in one database. Using a recently developed method for identification of functionally important SNPs in the 5'-flanking regions of human, with approximately 70% success rate, the database highlights such SNPs to facilitate selection and study of functionally important promoter SNPs. was queried with the search term 'GO:0004930', which describes the GPCR group of genes. The list of Swiss-Prot and TrEMBL entries were fetched from GPCRDB [. All the lists were merged and redundancies removed and Ensembl gene numbers (ENSG) were obtained for all of the genes from Ensembl genome server. The final list consisted of total of 427 genes with unique ENSG numbers. Of these 427 genes, 89 genes belonged to the sub-family of olfactory genes. Also of the 427 genes, 222 had demonstrable or convincing evidence for GPCR domain structure and sequence information in Ensembl and SwissProt databases.The list of Human GPCR genes was compiled by collecting gene names from several different sources and subsequently the list was updated by removing duplicates and entries with incomplete information like peptide fragments, partial sequences and hypothetical proteins. The Ensembl MART genome server database was queried for GPCR family members. The Gene Ontology server Amigo m GPCRDB , ensemblThe gene and transcript map information were obtained from Ensembl databases 'homo_sapien_core_25_34d' and 'ensemble_mart_25_1', released in September 2004. The tables for gene mapping, SNP mapping and Human-Mouse alignment were obtained from ensemble, dbSNP, HGVBASE and UCSC and installed locally. For the chromosomal and genomic location of SNPs and validation information, NCBI's dbSNP tables 'snp', 'snpcontigloc', and 'snpcontiglocusid' and Ensembl's tables 'ContigHit' and 'locus' were used. Information about the location of the SNP within protein domains was obtained from Swissprot using bioperl modules for accessing protein features. HGVBase release version 14 was used for obtaining HGVBASE SNP identities and validation and frequency information. The flanking sequence information was obtained from both Ensembl's RefSNP table and by downloading sequence flat files from dSNP ;For mapping Transcription Factor Binding Sites, the TFBS perl programming system  was used7TM: 7 transmembrane; GPCR:G-protein Coupled Receptors; SNP: single Nucleotide Polymorphism; nsSNP : non-synonymous SNPs; cSNP : coding SNP; rSNP : regulatory SNPs; N-term : N terminus; C-term : C terminus; e-loop : external loops; i-loop : internal loops.SM-T did all the coding, analysis, manuscript preparation and reviewer correspondence. AJB contributed genotyping information and validated a number of SNPs. CW provided running costs and assistance with writing the manuscript. All authors read and approved the final manuscript.Table 4 A list of natural non-synonymous variations and mutations with references to articles describing the phenotype associated with the variation.Click here for file


Recent years have seen an expansion in the use of Geographic Information Systems (GIS) in environmental health research. In this field GIS can be used to detect disease clustering, to analyze access to hospital emergency care, to predict environmental outbreaks, and to estimate exposure to toxic compounds. Despite these advances the inability of GIS to properly handle temporal information is increasingly recognised as a significant constraint. The effective representation and visualization of both spatial and temporal dimensions therefore is expected to significantly enhance our ability to undertake environmental health research using time-referenced geospatial data. Especially for diseases with long latency periods (such as cancer) the ability to represent, quantify and model individual exposure through time is a critical component of risk estimation. In response to this need a STIS – a Space Time Information System has been developed to visualize and analyze objects simultaneously through space and time.In this paper we present a "first use" of a STIS in a case-control study of the relationship between arsenic exposure and bladder cancer in south eastern Michigan. Individual arsenic exposure is reconstructed by incorporating spatiotemporal data including residential mobility and drinking water habits. The unique contribution of the STIS is its ability to visualize and analyze residential histories over different temporal scales. Participant information is viewed and statistically analyzed using dynamic views in which values of an attribute change through time. These views include tables, graphs (such as histograms and scatterplots), and maps. In addition, these views can be linked and synchronized for complex data exploration using cartographic brushing, statistical brushing, and animation.The STIS provides new and powerful ways to visualize and analyze how individual exposure and associated environmental variables change through time. We expect to see innovative space-time methods being utilized in future environmental health research now that the successful "first use" of a STIS in exposure reconstruction has been accomplished. Geographic Information Systems are beneficial tools in modelling static representations of reality; however they fall short in their ability to handle time. The ability to store, visualize, and analyze both the temporal and spatial dimension of data continues to be a challenging task. Over the past decade, there have been several attempts to include time enabled capabilities into GIS.  and 2]  2] propoThe importance of GIS for medical research and epidemiology has long been recognized -7, and GThe ability to effectively represent, query, and model the temporal dimension is expected to significantly enhance researchers' abilities to undertake environmental health research with georeferenced data. Studying an individual's exposure over time is a key factor in determining risk, particularly for diseases with long latency periods such as cancer , because.In this research we applied a STIS to visualize and analyze data from a bladder cancer case-control study. The objective of the epidemiologic research project is to identify a range of factors that have contributed to bladder cancer incidence in Michigan, with the focus on spatial and spatiotemporal patterns of exposure to naturally occurring arsenic in drinking water. Cases are recruited from the Michigan State Cancer Registry and diagnosed in the years 2000–2003. Controls are frequency matched to cases by age (± 5 years), race, and gender, and recruited using a random digit dialing procedure from an age-weighted list. To be eligible for inclusion in the study, participants must have lived in the eleven county study area for at least the past five years and had no prior history of cancer (with the exception of non-melanoma skin cancer). The goal is to enroll 1400 participants in total. This is an ongoing five year project and only some preliminary spatiotemporal datasets, visualization tools, and results are shown here. Conclusive results will not be available for a few more years, until data has been collected and analyzed for all 1400 participants. The STIS is being developed at BioMedware, in Ann Arbor Michigan with funding from the National Institutes of Environmental Health Sciences and the National Cancer Institute. In this paper STIS is used to visualize and analyze data from a bladder cancer case-control study but it can also be used for health/environment interactions or marketplace sales trends. More information about the STIS and a free 30 day download can be evaluated at Data from a case-control study of bladder cancer in south eastern Michigan was used to evaluate the efficacy of the STIS for documenting and visualizing space-time relationships between cases, controls and putative risk factors. Lifetime exposure to arsenic in drinking water  was object, space-time coordinate, attributes} where object identifies the modelled entity (e.g. person X); space-time coordinate is a spatiotemporal location which may be a space-time point  or a space-time polygon ; and attributes are observations on objects (e.g. income).The motivation for this system comes from the idea that the 'what and where' of conventional GIS needs to be extended to the 'what, where, and when' of reality and spatiotemporal modelling. Based on similar spatiotemporal approaches , objectspace time coordinate, in addition to the well known descriptors , we also specify a movement model that defines how the object moves through space as a function of time. Among the simplest of movement models is an instantaneous displacement such that the object ceases to exist at one location and immediately reappears at another location. We use this simple model to describe residential histories.Within the Morphing describes how the shape of geographic features (such as lines and polygons) changes through time. Here an object is comprised of multiple vertices changing shape through time by the addition, deletion and movement of vertices. This is called network morphing (for lines) and polygon morphing (for polygons). Morphing can be gradual, in which case the change in the object's shape occurs over a defined time interval; or it can be abrupt. In our research we utilize this approach to model cadastral systems and the realignment of administrative and political boundaries. This allows us to track, for example, how municipal water districts change through time, and to then estimate arsenic exposure from drinking water for individuals on municipal water supplies.Attributes are observations on variables describing the modelled entity and its environment  Our data model assumes observations occur at discrete times at which the attributes of an object are quantified. Attribute change models describe how the values of attributes change between observation times. The simplest attribute change model is a step function that updates an attribute's value when a new observation is made on that attribute. More complex change functions that obtain values from nearby locations are used to interpolate values through space and time for both categorical and continuous data [ous data . These ious data . In thisous data ).We reconstructed individual exposures by incorporating spatiotemporal data on residential mobility (where people have lived throughout their lives), water supplies , and drinking water habits. Only locations in which the participants have lived or worked for longer than one year were collected and geocoded. Data about diet, smoking, and medical history were also collected by a phone interview or written questionnaire. A point file (where each point represents a participant) was then imported into the STIS along with associated database files containing attribute information such as address and primary source of drinking water. Table Other point files were imported including present and historical data on industries and contaminated sites in the study area. A township map and water supply boundary map were imported as polygons. In addition to temporal changes in attributes such as township population, source of community's water supply, and number of people served, town boundaries and water supply boundaries changed with time. New towns were incorporated, community systems expanded their borders, and occasionally, communities were combined and town boundaries dissolved. All of these temporal changes were handled using attribute change models and morphing.time slice (similar to a collection of GIS static maps) where changes take place at specified times for all objects in the dataset, or (2) a time series where data varies asynchronously and objects move or change attributes at different times. For example Census data are time slice data – attributes remain constant for a decade (1980–1990) and then all attributes are updated with the next decade's census information (1990–2000). On the other hand, data associated with tracking residential histories are time series data, with household moves occurring at different times for each individual. The system imports data at temporal granularities varying from seconds to years; and the data may then be analyzed at these different time scales.We imported shapefiles describing the above data using the STIS data import facility that allows the variables to be time stamped. The user is prompted to import vector information into a new geography or an existing geography . The user must tell the system whether the data is (1) a Being able to visualize changes in boundaries and attribute values over time is an effective approach to better understanding and exploring data. Because time is a dimension of the data rather than an attribute all views of the data are easily animated. Analogous to a static GIS, attributes of data are visualized by specifying colour, shape, and size of graphical elements (e.g. symbols). However, in contrast to a GIS, the STIS easily facilitates visualization of changing polygon shapes and attribute values over time by animating maps, histograms, and tables simultaneously. Valuable information that might be lost in an atemporal GIS is captured and can become the focus of analysis in the STIS. There are four major visualization views – maps, graphs , tables, and time plots.map view displays spatial data and the user interacts with the maps by zooming, panning, selecting, and querying. The added feature of the STIS is the animation toolbar. It is employed to show individuals changing place of residence through time; arsenic-emitting industries being founded, operating, and going out of business; municipal water supply districts growing and coalescing; and attribute values, such as arsenic concentrations, changing through time.(1) The histograms, scatter plots, and box plots are also animated over time. An individual or group of individuals (e.g. cases vs. controls) may be selected at one point in time and the user can explore how that selection's values change through time. For example, we used this feature to explore how individual arsenic exposure changed over a participant's lifetime. We also used it to compare estimated arsenic burdens for the cases to those of the control population.(2) In the STIS Table views also are animated, as the given value of a variable  will change through time. Tables thus show how data values change over time by updating a given objects value when it increases or decreases.(3) time plot graphs time on the x-axis and the value of a variable, such as estimated arsenic exposure, on the y-axis. Objects of interest, such as cases and controls, then map into this bivariate time plot to explore time dependencies in arsenic exposure. Unlike the other views, the time plot is not animated because it already shows the entire time range of the data on the x-axis.(4) The time-link visualization windows. Maps, statistical graphics, and tables may be time-linked so that all of the views are synchronized to the same point in time. Animating the time-linked windows then displays the views simultaneously changing through time. We use this feature to display the changing residential locations of the cases and controls along with the locations and emission volumes of arsenic-producing industries. All of this is done within the context of municipal water districts whose boundaries morph and whose arsenic concentrations are dynamic. While this map visualization is occurring we observe how the frequency distributions of modelled arsenic exposure are changing for the cases relative to that of the controls. Participants (cases or controls) are thus easily evaluated and compared to other participants in terms of their residential histories, and population-level characteristics, such as the mean and dispersion for arsenic exposure estimates, may be compared statistically as they evolve over time.A novel feature of the STIS is the ability to Statistical and Cartographic Brushing is employed to link together the views associated with a given dataset. This is made possible by using unique identifiers  to link together corresponding values on the maps and statistical graphics. Statistical brushing is used to select objects (such as the points on a scatter plot) and to then highlight the corresponding objects on maps and other statistical graphics. Cartographic brushing occurs when objects are selected on a map, and their corresponding values on the statistical graphics are highlighted. We used statistical brushing to select participants with high arsenic exposures, and to then identify their locations on maps of their residential histories. We use cartographic brushing to explore possible associations between proximity to arsenic emitting industries and the local densities of cases relative to the controls.We first investigate changes in the water supply systems Figure . It is cVisualizing the movement of bladder cancer cases and controls through time is crucial in our analysis of arsenic exposure and how it relates to the incidence of bladder cancer. Figure In this analysis we are interested in the temporal variability in arsenic exposure in cases versus controls as well as clusters of high arsenic values. Arsenic exposure was calculated by multiplying arsenic concentration (μg/L) by home consumption of water and beverages made with water (L/day) at each residence and for each change in water consumption. Data regarding water and beverage consumption was obtained via survey . We utilThe time plot is another visualization method and provides information over the entire time range  arsenic values in participants' drinking water. Data regarding arsenic in drinking water was collected at the kitchen tap of each participant from their present residence. Water samples were stored on ice, acidified with 0.2% trace metal grade nitric acid, and refrigerated until analysis. Water samples were subsequently analyzed for arsenic using an inductively coupled plasma mass spectrometer  . A map oIn this paper we presented a novel application of a space time information system to analyze some preliminary data in an ongoing case-control bladder cancer study. This approach is significant in that it not only visualizes the movement and attribute changes of spatial objects  but also allows the user to compare values of these objects over time by time-linking windows. This ability to handle high temporal resolution data is enabling new approaches to exposure assessment. In the near future the STIS will be able to integrate exposure assessment models using an Application Programmers Interface (API). Users will have the flexibility to program specific models outside the software and then visualize their outcome in the STIS using the API. For less technically sophisticated users, a methods toolbar will be included, where common modelling algorithms will be made available using a simple calculator-type interface. Other plans for the software include importing and supporting raster files, exporting animated maps as movies (for presentations), visualizing geospatial lifelines ,19 in a Some authors are also affiliated with BioMedware a research company that also develops software for the exploratory spatial and temporal analysis of health and environmental data. With funding from the National Cancer Institute, GMJ, AMK, and GAA developed STIS, which is a commercial product of  Terraseer.


Clinicians at Haydom Lutheran Hospital, a rural hospital in northern Tanzania noted an unusually high case-fatality rate of pediatric meningitis and suspected an outbreak of an unknown agent or an organism resistant to the empirical therapy.We established a provisional microbiology laboratory to investigate the suspected outbreak. Blood and spinal fluid specimens were taken from children below the age of seven years with suspected meningitis. The blood and spinal fluid specimens were inoculated in commercial blood culture bottles and locally prepared Thayer-Martin medium in slanted tubes, respectively. The bacterial isolates were sent to Norway for further investigation, including susceptibility testing and pulsed-field gel-electrophoresis (PFGE).Salmonella enterica serotype Enteritidis, all of whom died. Two children had S. Enteritidis septicemia without meningitis and both survived. Genotyping with PFGE suggested a clonal outbreak. The salmonella strain was resistant to ampicillin and sensitive to gentamicin, the two drugs commonly used to treat neonatal meningitis at the hospital.Among 24 children with suspected meningitis and/or septicemia, five neonates had meningitis caused by The investigation reminds us that nontyphoidal salmonellae can cause meningitis associated with very high case-fatality rates. Resistance to multiple antimicrobial agents increases the risk of treatment failure and may have contributed to the fatal outcome in all of the five patients with salmonella meningitis. The investigation indicated that the outbreak was nosocomial and the outbreak subsided after hygienic measures were instituted. Establishing a provisional microbiological laboratory is a valuable and affordable tool to investigate and control outbreaks even in remote rural areas. Nontyphoidal salmonellae are a common cause of food-borne illnesses. In Africa, nontyphoidal salmonellae are the most common cause of bloodstream infections in children younger than five years . While mAt Haydom Lutheran Hospital, a rural hospital in northern Tanzania, clinicians noted an extraordinarily high case-fatality rate (>60%) from pediatric meningitis in the period January 1998 to April 2000. It was suspected that the high case-fatality rate of meningitis was due to an outbreak of an unusual etiological agent, or an organism resistant to the hospital's standard empirical treatment, which was ampicillin and gentamicin for infants (<2 months) and penicillin and chloramphenicol for older children. Thus, we established a provisional microbiology laboratory to identify the causative agents and to facilitate the implementation of effective preventive measures.From a total of 360 children admitted from July to August 2000, blood and/or spinal fluid specimens were collected from 24 children aged one day to six years (median age 23.5 days) with suspected meningitis and/or septicaemia, after careful evaluation by the attending pediatrician. Blood and spinal fluid specimens were inoculated in BBL SeptiChek blood-culture bottles  and on locally prepared non-selective Thayer-Martin medium in slanted tubes, respectively. All cultures were incubated at 35°C for 5 days and inspected daily for bacterial growth. Positive bacterial specimens were shipped to Institute of Medicine, Haukeland University Hospital, Norway, for further study. The total cost of the laboratory reagents used on site was $475. Positive specimens were identified using standard laboratory methods [Salmonella enterica serotype Enteritidis was isolated from seven patients, of whom four had positive cultures from both blood and spinal fluid and three from blood only. One of the three patients, who had S. Enteritidis isolated only from blood culture, had pus in the spinal fluid, and was therefore considered a case of S. Enteritidis meningitis, resulting in a total of five cases of S. Enteritidis meningitis. One isolate each of Staphylococcus aureus and Streptococcus pyogenes were isolated from spinal fluid and three isolates of coagulase-negative staphylococci were isolated from blood. Antimicrobial susceptibility testing showed that all the S. Enteritidis isolates were resistant to chloramphenicol and ampicillin, intermediate resistant to cefuroxime, and sensitive to gentamicin, cefotaxime and ciprofloxacin. As shown in Figure S. Enteritidis isolates except one, which differed by only one band , whereas those with other infections or negative cultures, on average, were older . All children with S. Enteritidis infections had been delivered at Haydom Lutheran Hospital, and five out of the seven never left the hospital before they became ill. Table S. Enteritidis infection. All five children with S. Enteritidis meningitis died. The two children with S. Enteritidis sepsis survived. Five (29%) of the 17 children without verified S. Enteritidis infection also died. Among the 24 children investigated, S. Enteritidis meningitis was associated with a relative risk of 3.8  for fatal outcome.On clinical grounds, twenty-four children were considered to have possible sepsis or meningitis. Blood culture was taken from 23 children, but 13 had received prior antibiotic treatment. Spinal fluid culture was taken from 16 children, of whom ten had received prior antibiotic treatment. Both blood culture and spinal fluid culture was obtained from a total of 15 children. S. Enteritidis was not anticipated. Genotyping with PFGE suggested a clonal outbreak. This genotyping information, the susceptibility patterns and the clinical information that all children with S. Enteritidis infections were born at the hospital and that the majority never left the hospital before they became ill, strongly suggest that the outbreak was nosocomial. Nontyphoidal salmonella infections are frequently associated with animal reservoirs and infection usually originates from food products. Nosocomial spread of nontyphoidal salmonellae, particularly in neonatal wards, is known from the literature [The finding of an outbreak of bacteremia and meningitis caused by terature . Neonateterature . In prevterature ,9. The eS. Enteritidis strain responsible for the outbreak was resistant to two of the first-line drugs, ampicillin and chloramphenicol, but sensitive to gentamicin. This finding is not surprising, since multi-drug-resistant S. Enteritidis has been reported from this region for decades [de facto monotherapy with gentamicin may be suboptimal as treatment for S. Enteritidis meningitis considering that gentamicin traverses the blood-brain-barrier poorly, is bound to proteins and inhibited by the acidity in infected cerebrospinal fluid. However, it also reflects the poor prognosis of neonatal S. Enteritidis meningitis regardless of therapy. Molyneux reported fatal outcome for 58% of children with meningitis caused by nontyphoidal salmonellae despite routine treatment with chloramphenicol, to which all bacterial isolates were sensitive in vitro [The  decades . The chiin vitro . Many auin vitro , not onlin vitro . The staInfections caused by nontyphoidal salmonellae in children in Africa are more common during the rainy season and have been associated with malaria, anemia and malnutrition . HoweverS. Enteritidis can cause meningitis, which carries a very high case-fatality rate [S. Enteritidis can easily spread by nosocomial transmission, particularly in neonatal wards. Resistance to multiple antimicrobial agents increases the risk that empirical therapy will fail, especially in settings where modern cephalosporins are not affordable. Without adequate laboratory facilities, correct diagnosis and treatment of bacterial meningitis and septicemia in children remains a challenge. However, the report also shows that establishing a provisional microbiology laboratory can be a valuable and affordable tool to investigate and curb epidemics even in the setting of remote rural Africa, provided there is a proficient laboratory willing to assist.There are a number of lessons to be learned from this investigation. We are reminded that ity rate ,3,4. OurNone declared.HV was the principal investigator, participated in the planning and execution of the work, including performing the on-site microbiological procedures, and analysis of data, and was the main responsible author. BB participated in planning, microbiological investigations in Norway, data analysis and writing. CK and NN undertook clinical investigation and sample collection and participated in the writing. RJ was responsible for the microbiological investigations in Norway and participated in the data analysis and writing. NL was the project coordinator and participated in planning, data analysis and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1. CYP19 (aromatase) [NAT1 gene. The increasing number of detected mutations in the NAT1, makes genotyping using conventional restriction fragment length polymorphism (RFLP) or allele-specific amplification complicated. We developed a rapid and universal strategy based on single-track DNA sequencing analysis of a unique PCR product encompassing the entire NAT1 coding region (contains no introns) along with the flanking 5' and 3' untranslated regions. It allows a rapid and economic characterization of NAT1 alleles. Our method brings reproducible results on both Alf Express™ (Pharmacia) and ABI310 PRISM sequencing instruments and may be adopted for majority of epidemiological studies with relevance of NAT1 in environmentally related diseases.We have previously described the single sequencing reaction (SSR) protocol for assessment of a known polymorphism in the 3'utr region of omatase) . Here weNAT1 gene is highly polymorphic . Expression of NAT1*16 but not NAT1*10 and NAT1*11 caused a 2-fold decrease in the amount and catalytic activity of NAT1 in COS-1 cell cytosol [NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein [N-acetyltransferases  are implicated in the biotransformation of primary arylamines (e.g. 2-naphthylamine and aminobiphenyls), heterocyclic amines, hydrazines, and their N-hydroxylated metabolites present in tobacco smoke and food . An incr cytosol -14. All  protein .NAT1 and NAT2 acetylation polymorphisms modify the risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers [NAT2*4 and NAT1*10 were suggested by the increased frequency of the NAT2*4/NAT1*10 haplotype [NAT1 genotypes in the population, the attributable cancer risk may be high. Large-scale molecular epidemiological studies that investigate the role of NAT1 genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 acetylation polymorphisms in cancer risk [Epidemiological studies suggest that the  cancers -18. Inteaplotype -21. The cer risk .NAT1 with two sequencing tracks: A and G and 11 variants in the 3' flanking area with two tracks: A and T . Despite the different chemistry, Figures A-track sequencing of the 3'-untranslated region of e et al. . This chNAT1 gene by single-track sequencing reactions in 192 control individuals. Previously, we have shown that single-track sequencing reactions performed on PCR products, with subsequent analysis on an Alf Express™ DNA Sequencer, can be as informative, sensitive, and accurate as complete sequencing reactions but more economical option for the genotyping of known polymorphisms in the human aromatase gene [NAT1.A modified automated DNA sequencing with a fluorescent label was used to genotype PCR products spanning through the whole This paper shows that single-track sequencing reactions can be used as a tool for screening for all types of known polymorphisms in field laboratories with limited or overloaded sequence capacity. Compared to standard sequencing, this single track sequencing may have better signal to noise ratio. This approach is time and cost effective and can be accommodated for high-throughput analyses in epidemiology studies.Chemicals for PCR and sequencing were purchased from ABI  and Amersham Biosciences .The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available.NAT1 (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH2O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl2 (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase  and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling  included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels.A single PCR amplification of the entire coding region and the 3' flanking area of NAT1, respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH2O, 0.5 μl of 10-times concentrated FS polymerase buffer 2SO4, 150 mM MgCl2), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of NAT1 PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH2O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer , 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl NAT1 PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.The SNPs studied by this assay are summarized in Table ABPs – aminobiphenyls, NAT1 – N-acetyltransferase 1, SSR (Single Sequencing Reaction)PS performed the SSR analysis on an Alf Express™ and prepared the first draft of the paper, CFS and MS optimized the SSR analysis for an ABI system in the lab of EHK, TK designed primers and contributed with reagents, materials, and advice, VNK brought the idea, organized the study and was responsible for the revisions of the paper.


EGFR and KRAS appear to be mutually exclusive.Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug.We sought to determine whether mutations in EGFR and KRAS.Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both  KRAS gene in lung cancer patients treated with two different kinase inhibitors suggests that tumors with KRAS mutations do not respond to these drugsMutational analysis of the  ERBB family encode receptor tyrosine kinases that mediate cellular responses to growth signals. Somatic mutations in the tyrosine kinase domains of two ERBB genes, epidermal growth factor receptor (EGFR) and HER2, have been found in a proportion of lung adenocarcinomas [EGFR, mutations are associated with sensitivity to the small-molecule kinase inhibitors gefitinib (Iressa) [Genes of the rcinomas ,2,3,4. F(Iressa) ,2,3 and RAS genes. Some 15%–30% of lung adenocarcinomas contain activating mutations in the RAS family member KRAS. These mutations are most frequently found in codons 12 and 13 in exon 2 [EGFR and KRAS mutations are rarely found in the same tumors, suggesting that they have functionally equivalent roles in lung tumorigenesis ([EGFR mutations are common in tumors from patients who have smoked less than 100 cigarettes in their lifetimes (“never smokers”) [KRAS mutations more commonly occur in individuals with a history of substantial cigarette use [ERBB signaling pathways include downstream GTPases encoded by n exon 2 , and mayn exon 2 . Interesgenesis  and KRAS2 (exon 2). Here, we show that mutations in KRAS are associated with primary resistance to single-agent gefitinib or erlotinib. Our results suggest that a determination of mutational status for both EGFR and KRAS may help define which patients are likely to benefit from receiving gefitinib or erlotinib.We sought to determine whether n = 24), and prospectively for patients on erlotinib, who received 150 mg orally once daily (n = 36). The latter cohort of patients was part of a clinical trial of erlotinib for patients with bronchioloalveolar carcinoma. The analysis presented here includes specimens we previously reported on (n = 17 for gefitinib and n = 17 for erlotinib) [Tumor specimens were obtained through protocols approved by the institutional review board of Memorial Sloan-Kettering Cancer Center, as previously described  see Pro. Tumor maily n = 6. The laAll specimens were reviewed by a single reference pathologist (M. F. Z.). Imaging studies were assessed by a single reference radiologist (R. T. H.), who graded responses according to Response Evaluation Criteria in Solid Tumors (RECIST) . Both obn = 26) or stable disease (n = 12) as defined by RECIST. No patients had a complete response.Eight of nine patients with tumors sensitive to gefitinib had objective partial responses as defined by RECIST, i.e., at least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the sum measured at baseline. The ninth patient had marked clinical improvement, as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, this individual had radiographic lesions  that were deemed nonmeasurable by RECIST criteria. As erlotinib-treated patients were all in a clinical trial, all had disease measurable using RECIST guidelines. For both drugs in this study, tumors were considered refractory if they did not undergo sufficient shrinkage to qualify for partial response. This definition includes patients whose “best overall response” was either progression of disease  were as published [KRAS analyses, the following nested primer sets for exon 2 were used: huKRAS2 ex2F, 5′-GAATGGTCCTGCACCAGTAA-3′; huKRAS2 ex2R, 5′-GTGTGACATGTTCTAATATAGTCA-3′; huKRAS2 ex2Fint, 5′-GTCCTGCACCAGTAATATGC-3′; and huKRAS2 ex2Rint, 5′-ATGTTCTAATATAGTCACATTTTC-3′.Genomic DNA was extracted from tumors embedded in paraffin blocks, except for tumor 109T, which was a fresh-frozen tumor specimen. Primers for ublished . For KRAEGFR and KRAS, PCR was performed using the HotStarTaq Master Mix Kit , as per manufacturer's instructions. Use of this method often obviated the need for nested PCR sets. All sequencing reactions were performed in both forward and reverse directions, and all mutations were confirmed by PCR amplification of an independent DNA isolate.For both EGFR-Ex19-FWD1, 5′-GCACCATCTCACAATTGCCAGTTA-3′, and EGFR-Ex19-REV1, 5′-Fam-AAAAGGTGGGCCTGAGGTTCA-3′. Using serial dilutions of DNA from the H1650 non-small-cell lung cancer cell line , this apositive ), this ap-values, and confidence intervals were calculated using Statistics with Confidence software [Fisher's Exact Test was used to calculate software .EGFR and KRAS. Collectively, nine of 38 (24%) tumors refractory to either kinase inhibitor had KRAS mutations, while zero of 21 (0%) drug-sensitive tumors had such mutations (p = 0.02)  drug-resistant tumors (p = 6.8 × 10−11). The 95% CIs for these observed response rates are 57%–90% and 0%–9%, respectively. All 17 tumors with EGFR mutations responded to gefitinib or erlotinib, while all nine tumors with KRAS mutations did not (p = 3.2 × 10−7).We identified 60 lung adenocarcinomas from individual patients with tumors shown to be sensitive or refractory to single-agent gefitinib or erlotinib and evaluated these tumors for mutations in  = 0.02) . The 95%EGFR and KRAS mutational status with drug and treatment response is detailed in KRAS mutations is shown in KRAS mutations in the patients treated with erlotinib was low, probably because of the fact that all patients treated with this drug had bronchioloalveolar carcinoma, which rarely has RAS mutations [KRAS2 may have missed some RAS mutations. However, in our analysis of the exonic regions encoding the first 100 amino acids of KRAS in 110 surgically resected early-stage non-small-cell lung cancers, we have found 18 mutations, and all were in either codon 12 or codon 13, encoded by exon 2 . Another possibility is that the erlotinib-treated tumors have mutations in other RAS genes, since a minority of RAS mutations in lung cancer have been reported to occur in N- or HRAS [Correlation of utations . Alterna or HRAS ,6.EGFR mutations are associated with sensitivity to gefitinib or erlotinib . Second, these data show that tumors with KRAS exon 2 mutations (n = 9) are associated with a lack of response to these kinase inhibitors . Third, no drug-sensitive tumors had KRAS exon 2 mutations (n = 21). Whether KRAS mutational status can be used to predict responses to gefitinib or erlotinib in patients whose tumors have wild-type EGFR sequence is still under investigation: our analysis comparing response rates for tumors with neither EGFR nor KRAS mutations versus tumors with wild-type EGFR but mutated KRAS does not reach statistical significance . Nevertheless, these findings suggest that patients whose lung adenocarcinomas have KRAS mutations will not experience significant tumor regression with either drug.These results have important clinical implications. First, they extend previous data from our group and others showing that lung adenocarcinomas containing EGFR mutations in tumors responsive to EGFR kinase inhibitors has varied from 71% to 100% .Click here for additional data file.Protocol S2(1.9 MB PDF).Click here for additional data file.Protocol S3(60 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) accession number for the KRAS2 sequence discussed in this paper is 3845; the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the KRAS2 sequence discussed in this paper is NT_009714.16.The LocusLink  and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the EGFR mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the KRAS gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs.Unfortunately, KRAS.They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded  or not, and tested whether the tumors had normal or abnormal KRAS gene. Most of these tumors had EGFR mutations. Conversely, tumors that had abnormal KRAS  did not shrink while treated with gefitinib or erlotinib.Tumors that got significantly smaller while treated with gefitinib or erlotinib  had a normal EGFR and KRAS mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs.Both gefitinib and erlotinib are expensive and have side effects. Testing for EGFR and KRAS tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up.Before doing http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htmUS Food and Drug Administration information page on Iressa: http://www.cancerhelp.org.uk/help/default.asp?page=10296Cancer Research UK information page about erlotinib:


X) have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs) and apoptosis in cultured tumor cell lines.Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates , and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay.Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds.Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when developing NOX-based chemotherapeutic agents.Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NO Vastly increasing the functional consequences of NO• production is its interrelationship between the nitroxyl anion (NO-) and the nitrosonium cation (NO+) depending upon the redox environment in which NO• is being produced. Each of these interrelated redox species demonstrates its own biological consequences. Collectively, nitric oxide biology attributable to the integrated actions of these three species (referred to as NOX) demonstrates broad reaching consequences.Nitric oxide  the capacity to trigger mutagenesis, 2) enhance growth, invasion, angiogenesis and metastasis of tumors, 3) select for increasingly virulent tumor cell clones, and 4) suppress the host anti-tumor immune response  which donate NO• its pure form into the aqueous environment. The other two compounds were thiol-based nitric oxide donors , which preferentially donate NO+-equivalents to other thiols. Additionally, these donors differed in their stability. Based upon our spectrophotometric analysis at 37°C and a pH of 7.4, Spermine-NONOate has t1/2 = 5 hours, DETA-NONOate a t1/2 ≈ 24 hours, SNAP a t1/2 = 10 hours, and glyco-SNAP a t1/2 = 28 hours [data not shown]. Furthermore, NONOate-based donors donate two molar equivalents of NOX per mole of donor compound, whereas the thiol donors deliver only one mole of NOX per mole of donor compound. These donor compounds were chosen to assess whether differences in the concentration, mode, or the rate of NOX delivery alter the genotoxicity of this radical species.Cells were incubated with one of four NO3PO4. Then 50 μl of 0.1% napthylethylenediamine dihydrochloride in 2.5% H3PO4 was added in a 96-well microtiter plate. After incubation at room temperature for 30 minutes, the absorbance was measured on a microplate reader  at 540 nm. The concentration of nitrite in the media was quantified as derived from standard curves created by adding known concentrations of NaNO2 from 50 to 300 μM sodium nitrite. NOX delivery kinetics was confirmed by determining changes in λ maximum of each compound over time on a spectrophotometer .Nitric oxide donors were added to media without cells or to cell supernatants at increasing concentrations from 75 to 600 μM. After 24 hours of incubation the amount of nitrite produced in the media was assayed by the Greiss reaction as previously described ,13. Brie+2 and Mg+2-free phosphate buffer at 56°C were quickly layered onto a fully frosted slide and immediately covered with a cover-slip. The slides were kept at 4°C to allow the agarose to solidify. After gently removing the cover-slip a 50 μl aliquot of cell suspension was mixed with an equal volume of 1% low melting point agarose  at 37°C and quickly pipetted onto the first agarose layer in the same manner. Finally, 70 μl of 0.5% LMP agarose was added to cover the cell layer. The slide sandwiches without cover-slips were immersed in freshly prepared, cold lysing buffer  and kept at 4°C for 45 min to 1 hour.The COMET assay was performed according to the procedure of Singh et al. with a few modifications . Briefly2EDTA] for 8 to 20 minutes in the dark at 4°C to allow DNA unwinding and expression of the alkali-labile sites. The timing for lysis and unwinding was determined empirically for each cell line. Electrophoresis was conducted at 4°C in the dark for 20 min at 25 V and 300 mA. The slides were then rinsed gently twice with neutralizing buffer . Each slide was stained with 120 μl of propidium iodine (Sigma) at a concentration of 5 μg/ml and covered with a cover-slip.The slides were placed on a horizontal gel electrophoresis platform and covered with cold alkaline buffer .We then confirmed that the published half-lives of our donor compounds were consistent with the kinetic release of NOX resulting from the addition of our NOX donors, we examined immunostaining for nitrotyrosine .We then sought to determine whether NOBecause TUNEL positivity represents a late event in the apoptotic cascade, we wanted to ensure that the assay time frame did not represent assessment prior to detectable apoptosis. Therefore, we confirmed our apoptosis results via Annexin-V/propidium iodide FACS sorting. After exposure of our cell lines to 600 μM of SNAP or Spermine-NONOate, we confirmed the absence of any significant increase in apoptosis Figure .X in the modulation of caspase enzyme function via an in vitro caspase activity assay , when compared to control cells. This inhibition appears to be attributable to basal S-nitrosation of caspase-3, since the addition of 1 mM DTT also significantly increased caspase-3 activity. With the addition of exogenous NOX via our thiol-based donor compound, SNAP, a significant decrease in the activity of both caspases was found. In contrast, our NONOate-based NOX donor, Spermine, was unable to decrease either caspase-3 or -9 activities. The inhibition of both caspase's activity by SNAP was reversed with exposure to 1 mM DTT, suggesting that transnitrosation of these caspase enzymes may contribute to the inhibition of enzymatic activity. These data suggest that thiol-based NOX-mediated inhibition of caspase activity in A549 cells may cause a relative tolerance to ongoing DNA damage by inhibiting apoptosis.In an attempt to explain these seemingly contradictory data, we examined the role of NOy figure . Using tX genotoxicity is highly dependent upon the concentration and kinetics of delivery of NOX. In addition, tumor cells appear to have increased genomic instability but increased resistance to ongoing NOX-mediated genotoxicity when compared to more normal transformed cells which demonstrate less genomic instability but increased susceptibility to ongoing DNA damage. Moreover, the mechanism of NOX delivery appears to have profound consequences upon the function of downstream effector molecules such as caspases.We have demonstrated that NOX. The seemingly paradoxic nature of NOX in cancer has made investigations in this area quite enigmatic.Over the past 20–30 years key proteins have been increasingly implicated as targets for nitric oxide signaling. With respect to its role in cancer, both carcino-protective  and carcX's role in the induction and promotion of cancer [X as a result of cigarette smoking may predispose individuals to the development of this type of cancer.Increasing evidence supports NOf cancer ,20. Chrof cancer ,22. WithX has been hypothesized to be carcinogenic is through oxidative/nitrosative DNA damage and genotoxicity [X can induce mutational events via DNA oxidation, deamination, point-mutations, and strand-breaks, thus contributing to the multi-step process of carcinogenesis. A number of papers have documented that NOX can cause DNA strand breaks in areas of chronic nitrosative stress [X donors increases DNA strand-breaks in a concentration dependent fashion. Moreover, the kinetics of NOX exposure also affects its genotoxicity. It is hypothesized that this ongoing DNA damage can lead to cancer development. In support of this notion, in a mouse model of inflammatory mediated lung carcinogenesis, studies have demonstrated that genetic ablation of the inducible isoform of nitric oxide synthase decreased the lung tumor multiplicity after exposure to the carcinogen, urethane [One mechanism by which NOtoxicity -25. NOX e stress -28. Our urethane .X-dependent DNA strand breaks in A549 cells is in stark contrast to NOX's inability to induce DNA fragmentation in our control fibroblast cell line, WI38. Normally, DNA damage is repaired by a complex series of DNA repair mechanisms. Defects in repair mechanisms can predispose people to cancer development [X. Research is ongoing to determine whether tumor elicited NOX production may contribute to tumor progression.The data presented herein of NOelopment . An increlopment . As a reelopment , and theX on cell viability and apoptosis. Certain studies have shown that NOX by itself does not cause A549 cells to die, but the addition of hyperoxia induces rapid cell death [X donor SNAP decreased cell viability via apoptosis in a concentration dependent fashion [If DNA damage overwhelms repair mechanisms, normal cells are triggered to undergo apoptosis. Redundant mechanisms for the induction of apoptosis exist, but the most common pathway involves the induction of p53. With the induction of p53, apoptosis is triggered through the release of cytochrome c and caspase activation. Despite possessing wild-type p53, our data demonstrated that in A549 cells we were unable to detect a decrease in cell viability as measured by the MTT assay until very high concentrations of donors, or an increase in cell apoptosis via either the TUNEL assay or Annexin V/propidium iodide FACS. There are conflicting results in the literature regarding the effects of exogenous NOll death , perhaps fashion . This stFurther confusing the interrelationship between DNA fragmentation and apoptosis is the fact that the prevailing opinion in the literature is that there exists a direct correlation between the fragmentation seen in the COMET assay and the fragmentation seen in apoptosis. With triggering of apoptosis, DNA strands are cleaved or nicked by nucleases, exposing 3'-hydroxyl ends. Nick-end cleavage may not necessarily equate with strand breaks seen with various mutagens. A growing body of evidence suggests that the fragmentation found in the COMET assay is not necessarily related to apoptotic fragmentation ,36. StudX's possible carcinogenic role is the implication from several lines of study that NOX can influence enzymatic function of such enzymes as caspases [X may increase the threshold by which cells that have undergone a level of genotoxicity would be triggered to undergo apoptosis. If any member of the apoptotic pathway is lost or inhibited, the threshold for apoptosis would theoretically be increased. NOX has demonstrated a capability of inhibiting caspase activity through S-nitrosation of key thiols [X-mediated inhibition of both caspase-3 and -9 in A549 cells with ongoing DNA mutational events. In fact, prior data has demonstrated that basal inhibition of mitochondrial caspases by cysteine nitrosation must be removed in order to activate the caspase-mediated apoptotic pathways [X donor appeared to inhibit caspase activity more efficiently that the NONOate donor further supports this notion, since it is well known that nitroso-thiols will preferentially transnitrosate other thiols [X-donor compounds as cytotoxic cancer therapy, a careful consideration of potential downstream effects of the mode of NOX delivery should be undertaken. Caspases can be inhibited not only by direct protein S-nitrosation, but also indirectly by a cGMP-mediated pathways [Lending credence to NOcaspases . In thisy thiols ,39. Our pathways . Our datpathways . The facr thiols . In lighX-based chemotherapeutic strategies. It is well established that tumors demonstrate a high rate of cellular turnover, with the vast majority of cells undergoing cell death resulting from ongoing genomic instability. Only those clones possessing a survival advantage will be able to repopulate the tumor and thus contribute to increasing aggressiveness, the ability to invade and metastasize, and resistance to further therapeutic interventions. Chronic nitrosative stress may be contributing to this process by selecting for more virulent clones via inducing DNA mutational events and perpetuating these mutations through a relative inhibition of apoptosis. Moreover, as more investigations explore the potential use of NOX delivery as a possible cytotoxic chemotherapy, the issues of concentration, kinetics, and mechanism of delivery must be carefully considered in order to avoid untoward pro-carcinogenic side effects.The implications of these data can be applied both to the understanding of tumor progression as well as the design of NOX. Taken together, these data support the role of NOX in nitrosative genomic instability as well as inhibiting apoptosis, implicating it in cancer promotion. The demonstration of these same findings in rodent cell lines would establish the foundation for animal models with which to fully elucidate the role of NOX in tumor development and growth.In conclusion, we demonstrate that tumor cells experience an increase in DNA strand breaks with increasing nitrosative stress relative to the concentration and kinetics of delivery. This nitrosative stress does not correlate with increased cell death or apoptosis in established tumor cell lines, which is in stark contrast to non-tumor immortalized cells. The lack of apoptosis associated with increased DNA fragmentation may in part be explained by the inhibition of caspase-3 and -9 activity by thiol-based delivery but not NONOate-based delivery of NOBB conceived of the study, participated in the design of the study, and performed the statistical analysis. GH interpreted the results of the FACS and immunoassays. NH carried out the majority of the COMET assays, as well as performing the caspase assay. JR participated in the study design and coordination. All authors read and approved the final manuscript.