Phytophthora ramorum is the exotic pathogen, discovered in the late 1990s, that is responsible for sudden oak death in California forests and ramorum blight of common ornamentals. The nursery trade has moved this pathogen from source populations on the West Coast to locations across the United States, thus risking introduction to other native forests. We examined the genetic diversity of P. ramorum in United States nurseries by microsatellite genotyping 279 isolates collected from 19 states between 2004 and 2007. Of the three known P. ramorum clonal lineages, the most common and genetically diverse lineage in the sample was NA1. Two eastward migration pathways were revealed in the clustering of NA1 isolates into two groups, one containing isolates from Connecticut, Oregon, and Washington and the other isolates from California and the remaining states. This finding is consistent with trace forward analyses conducted by the US Department of Agriculture's Animal and Plant Health Inspection Service. At the same time, genetic diversities in several states equaled those observed in California, Oregon, and Washington and two-thirds of multilocus genotypes exhibited limited geographic distributions, indicating that mutation was common during or subsequent to migration. Together, these data suggest that migration, rapid mutation, and genetic drift all play a role in structuring the genetic diversity of P. ramorum in US nurseries. This work demonstrates that fast-evolving genetic markers can be used to examine the evolutionary processes acting on recently introduced pathogens and to infer their putative migration patterns, thus showing promise for the application of forensics to plant pathogens.Recently introduced, exotic plant pathogens may exhibit low genetic diversity and be limited to clonal reproduction. However, rapidly mutating molecular markers such as microsatellites can reveal genetic variation within these populations and be used to model putative migration patterns.  Phytophthora ramorum, has caused devastating levels of mortality of live oak and tanoak trees in coastal California forests and in urban and suburban landscapes in the San Francisco Bay Area. This pathogen also causes non-lethal disease on popular ornamental plants, including rhododendrons, viburnums, and camellias. P. ramorum was discovered in California in the late 1990s and is exotic to the United States. Recently, presence of the disease in wholesale nurseries in California, Oregon, and Washington has led to shipments of diseased plants across the US, thus risking the introduction of the pathogen to other vulnerable forests. We examined the genetic diversity of this pathogen in US nurseries in order to better understand its evolution in nurseries and movement between states. We found that California populations were genetically different enough from Oregon and Washington populations that infestations of the pathogen found in nurseries in other states could be distinguished as having originated from California or the Northwest. Our inferences were consistent with trace forward investigations by regulatory agencies.Sudden oak death, caused by the fungus-like pathogen  Phytophthora infestans, causal agent of potato and tomato late blight, is a well known example of a plant pathogen able to adapt while reproducing clonally, as observed by changing virulence on host cultivars Puccinia striiformis f.sp. tritici, in Australia and New Zealand Plant pathogens that have been introduced to a new environment may be characterized by low genetic diversity due to a genetic bottleneck experienced during the process of introduction and establishment, given that only one or a few genotypes are usually introduced Genetic markers are also beginning to be used for forensic purposes in human pathogens. Microbial forensics is “the detection of reliably measured molecular variations between related microbial strains and their use to infer the origin, relationships, or transmission route of a particular isolate” Phytophthora ramorum, the causal agent of sudden oak death, was recently introduced to North America and is responsible for the rapid decline of forest populations of tanoak (Lithocarpus densiflorus) and coast live oak (Quercus agrifolia) in northern California coastal forests and parts of coastal southern Oregon P. ramorum is also a foliar and twig pathogen on common ornamentals, such as Rhododendron, Viburnum, Pieris, and Camellia. Thus, P. ramorum has been found in nurseries in North America and Europe, and nursery shipments have been implicated in the movement of the pathogen. There is serious concern about the inadvertent transfer of P. ramorum to other susceptible ecosystems, such as the Appalachians P. ramorum has had significant economic and societal impacts P. ramorum is a diploid oomycete, located in the kingdom Stramenopila along with diatoms, golden-brown algae, and brown algae P. ramorum have confirmed the clonal reproduction of this pathogen and have proved valuable for examining its population structure P. ramorum have been found in nurseries P. ramorum research community P. ramorum is self-sterile; sexual reproduction requires contact between two different mating types. All tested NA1 and NA2 isolates have been mating type A2 and EU1 isolates mating type A1 with the exception of rare finds of A2 in Belgium P. ramorum-positive nurseries have been in California, Oregon, and Washington, where annual inspection and sampling is required for nurseries that ship interstate and contain host or associated host plants on the P. ramorum host lists per the Federal Interim Rule of 2007 (7 CFR 301.92). West Coast nurseries that ship non-host nursery stock interstate are also required to be inspected annually. When found, infected plants are quarantined and destroyed under the authority of the Plant Protection Act of 2000. P. ramorum has also been found in states that received shipments from infected West Coast nurseries. For example, shipments of millions of potentially infected plants were made from a large California nursery to over 1,200 nurseries in 39 states in 2004 P. ramorum and it has been determined that the nursery shipped potentially infected P. ramorum host or associated host plants, the nursery is required to provide to the US Department of Agriculture's Animal and Plant Health Inspection Service (USDA APHIS) a list of all host and associated host plants that were shipped from the nursery during the preceding 12 months. A trace forward protocol  is implemented to determine whether the receiving nurseries or landscapes have become infested. Similarly, a trace back protocol is implemented at the infested shipping nursery to investigate the potential source of P. ramorum.Most of the P. ramorum using mitochondrial DNA sequence, AFLP, or microsatellites have focused on the broad diversity of a worldwide sample of P. ramorum isolates Previous studies examining neutral genetic variation in nursery populations of P. ramorum in US nurseries using 279 isolates collected from infected nurseries from across the US between 2004 and 2007. There is interest in the P. ramorum community in using genetic markers to link new detections of P. ramorum in both nursery and wildland settings to possible sources; therefore, we typed microsatellite loci known to show variation within and between the P. ramorum clonal lineages to examine their utility in confirming or contributing to trace forward and trace back investigations and, more generally, the potential for forensic analysis of P. ramorum. We specifically address four major questions regarding nursery populations of P. ramorum: 1) Do nursery populations show genetic diversity and population structure or are they dominated by a single dominant or founding genotype? 2) Are West Coast infestations more genetically diverse than those in other states, as might be expected if infestations are older and effectively larger in Oregon, Washington, and California? 3) Have the populations of the West Coast states changed between 2004 and 2007 in a way that would indicate that eradication measures have or have not been effective? 4) Can we use these genetic markers to infer the major migration pathways and potential sources of recent migrants?Here we report on the population genetic analysis of P. ramorum lineages and no recombinant multilocus genotypes were observed that would be indicative of sexual reproduction between lineages. Thirty-four EU1 isolates and 17 NA2 isolates were identified in the sample , 3.0% (P = 0.13), and 4.9% (P<0.0001) of the total variation, respectively. When data were clone corrected there was significant variation among states but not among years within states . AMOVA confirmed significant variation between these groups, which accounted for 33% of the variation. Ten isolates could not be assigned to one or the other group with a probability greater than 0.75 and 31 isolates were not assigned with a probability greater than 0.95 . The optThe relative rates of immigration to mutation among West Coast states and from these states eastward were estimated using a coalescent-based approach, as implemented in the program Migrate. We used a migration model in which the three West Coast source populations could both send and receive migrants, but the combined population representing all other states could only receive immigrants. This migration model is consistent with nursery industry shipment patterns. The ratio of immigration rate to mutation rate (m/µ) tended to be higher for the non-West Coast sample, but with a large amount of uncertainty in the estimates . Many ofP. ramorum host and associated host plants out of state is confirmed to be infested with P. ramorum, the USDA APHIS trace forward protocol is implemented by the receiving state(s). Shipping records are obtained for all host and associated host plants that were shipped in the preceding 12 months. These shipping records are used to conduct inspections to determine whether receiving nurseries or landscapes have become infested. Trace forward shipments from P. ramorum infested nurseries in CA, OR and WA to non-West Coast states resulted in the detection of P. ramorum in 12 states from 2004 to 2007 . Two of these, Connecticut and North Carolina, produced isolates with the second and third most common NA1 genotype, respectively. This suggests that only a few genotypes may be responsible for initiating P. ramorum infestations across the US. This is again consistent with USDA APHIS analysis, which indicated that shipments of infected plant material occurred only a few times. P. ramorum is also present in nurseries in British Columbia, Canada The most genetically variable populations were on the West Coast, as expected based on the large number of infected nurseries that have been found in these states , yet we P. ramorum forest populations (<5 yrs old) were observed to be as diverse as older forest populations (>10 yrs) and the genetic distance among new populations was greater than that observed among older populations Providing an interesting contrast to the single genotype shared among many states, we identified 36 NA1 multilocus genotypes that were unique to a state. Destruction of infected plants should ensure that populations in individual nurseries do not have the opportunity to grow large and small populations are subject to genetic drift. The observed genetic diversity and number of private genotypes suggests that there is also rapid mutation following the founding of a new nursery population and little to no gene flow following initial introduction. Interestingly, in California, several recently established P. ramorum-positive nurseries in 2007 than in previous years  was found in 2004, 2005, and 2006 but not 2007.Prospero et al.California nursery populations were dominated by a single genotype (NA1 MG 1), comprising 20 of the 36 isolates from the state. Mascheretti P. infestans allozymes P. cinnamomi microsatellites P. sojaeGiven the levels of heterozygosity observed at most of the microsatellite loci Phytophthora populations in the absence of sexual reproduction and unmask recessive traits or advantageous new mutations Mitotic recombination may purge deleterious mutations from The EU1 clonal lineage has now been found in all three west coast states P. ramorum when mutation rates are as high as appears to be the case for the PrMS43a and PrMS43b loci in the NA1 lineage. For example, an isolate of interest may differ from a suspected source population at one of these loci, thus raising doubts about their connection. Alternatively, convergence through repeat or reverse mutations may also have caused some Washington isolates to cluster with isolates from California and other states, which could falsely imply a direct connection between states where there is none. On the other hand, the relative homogeneity of the EU1 and NA2 lineages in US nurseries may hinder genetic-based tracing of isolates in these lineages. Nevertheless, our results were consistent with trace forward analyses and thus these microsatellites should be informative when used in conjunction with other data. The identification of more microsatellite loci that exhibit variation within the clonal lineages would strengthen these inferences The rapid mutation rates of these microsatellite loci has proven valuable for population genetic analyses, but poses a challenge for forensic tracing of P. ramorum from nurseries will be necessary to track the movement and diversification of the lineages and to identify new dominant genotypes, newly introduced lineages, or recombinant genotypes. As part of our efforts, the clonal lineage of each P. ramorum isolate genotyped, with permission from the provider of the isolate, is posted to a public website along with its county and state of origin at http://oregonstate.edu/~grunwaln/index.htm. Ongoing genotyping will also be valuable in evaluating how effective eradication efforts are in restricting migration, lowering effective population size, and increasing the effect of genetic drift.Continued genotyping of P. ramorum were obtained from scientists with State Departments of Agriculture, the US Department of Agriculture's Animal and Plant Health Inspection Service, universities and research institutions as new or recurring findings of infected nurseries occurred. Newly infected sites are subject to federal quarantine and could not be systematically sampled. Thus sampling intensity likely varied by state. For example, isolates from non-West Coast states may each represent one infested nursery, whereas recent samples from OR and WA include multiple isolates per nursery. Isolates for which we had detailed host information came from Camellia japonica, C. sasanqua, C. bonsai, Kalmia latifolia, Laurus noblis, Osmanthus heterophyllus, O. fragrans, Pieris japonica, Rhododendron spp., Viburnum tinus, and from soil and water baits. The 2004 shipments from CA to 39 states contained Camellia species. We do not know how many nurseries with recurrent infestations that were sampled over 2 or more years are represented in our dataset.Isolates of 3; 30 mg/L β-sitosterol ; 15 g agar; 800 ml deionized water) and stored at 20°C in the dark. All isolates were handled following the standard operating procedures associated with corresponding USDA APHIS permits and an exemption from the Director of the Oregon Department of Agriculture for work with P. ramorum under containment conditions.Upon receipt, isolates were transferred to cleared 20% V8 agar medium , 0.2 µM dNTPs, 3–6 µM of primer pairs, 0.5 U GenScript Taq DNA polymerase , and 0.5 µL (∼50 ng) DNA template. Loci PrMS43a and b were amplified using the following PCR program: 1 cycle of 92°C for 2 min, 35 cycles of 92°C for 30 s, 52°C for 30 s, and 72°C for 1 min, and 1 cycle of 72°C for 45 min. The final concentrations of the reaction mixture for PrMS43 (10 µL volume) were 1× PCR Buffer, 0.4 µM dNTPs, 0.3 µM forward and reverse primers, 1.0 U DNA polymerase, and 0.5 µL DNA template. Loci 18 and 64 were amplified with the PCR program: 1 cycle of 94°C for 2 min, 30 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s min, and 1 cycle of 72°C for 10 min. The final concentrations of the 10 µL reaction mixture for 18 and 64 were 1× PCR Buffer, 0.2 µM dNTPs, 0.2 µM forward and reverse primers, 0.5 U DNA polymerase, and 0.5 µL DNA template.Six microsatellite loci were genotyped that had previously shown variation among isolates within the Three isolates were used as positive controls in identification of the three clonal lineages and to ensure consistency among runs: PR-04-001 , PR-04-020 , and PR-04-015 . PCR products were sized using capillary electrophoresis on an 3100 Avant Genetic Analyzer  using the internal size-standard LIZ 500 (Applied Biosystems). Results were analyzed using GeneMapper 3.7 software packages (Applied Biosystems). Genotyping was replicated for a subset of isolates with independent DNA extractions, PCR, and sizing of fragments. Reproducibility of novel allele sizes was confirmed.Genetic distances among all identified multilocus genotypes were calculated over eight of the nine loci using Wright's modification of Roger's genetic distance GE5For the NA1 lineage and states and years with sample sizes of at least five isolates, we estimated multilocus genotypic diversity using Stoddart and Taylor's index Analysis of molecular variance (AMOVA) In order to examine genetic distances among isolates as measured by mutational differences, rather than mutation plus mitotic recombination, we collapsed the data to the haploid state. Three loci, PrMS39b, PrMS43a, and PrMS43b, were consistently homozygous. Loci 18 and 64 had two distinct size classes of alleles and only the larger of the two was variable among isolates for both loci. Thus when collapsed to haploid, the larger allele was retained. Three additional isolates appeared to exhibit mitotic recombination rather than mutation at otherwise uniformly heterozygous loci. These were two WA 2004 isolates (locus PrMS45) and a SC 2004 isolate (locus 64), multilocus genotypes 49, 53, and 35, respectively  and S2. et al.To examine the relationships among isolates, minimum spanning networks were constructed using the genetic distance of Bruvo To examine genetic structure in the NA1 sample, the clustering programs Structure 2.2 Maximum likelihood estimates of the ratio of immigration rate to mutation rate (m/µ) for West Coast states compared to the non-West Coast sample were obtained using the program Migrate version 2.4.3 Table S1Allele sizes at eight loci for each multilocus genotype (MG) observed within clonal lineages EU1, NA1, and NA2.(0.07 MB PDF)Click here for additional data file.Table S2Distribution of multilocus genotypes belonging to the NA1 clonal lineage.(0.06 MB PDF)Click here for additional data file.Table S3P. ramorum-positive nursery-related sites  by state and year (http://www.suddenoakdeath.org).Confirmed (0.06 MB PDF)Click here for additional data file.Figure S1Relationship between sample size and number of multilocus genotypes in the sample. Samples are isolates from one state in one year. The upper graph shows this relationship when multilocus genotypes from all three clonal lineages are considered. The lower graph shows the number of multilocus genotypes in the NA1 lineage only. Sample year is shown for the largest sample sizes to the right of the marker.(0.29 MB TIF)Click here for additional data file.


Epidemiologic studies suggest a temporal trend of earlier onset and longer duration of puberty, raising concerns regarding the potential impact of environmental factors on pubertal development. Lead exposure has been associated with delayed pubertal onset in girls; however, epidemiologic data in boys are limited.We used multivariable logistic regression models to explore the cross-sectional association of blood lead levels with growth and pubertal onset based on physician-assessed testicular volume (TV) and pubertal staging in 489 boys 8–9 years of age from Chapaevsk, Russia. We used multivariable linear regression models to assess associations of blood lead levels with somatic growth at the study entry visit.p < 0.001) and weight (p = 0.06) after adjustment for birth weight, gestational age, and age at examination. In multivariable adjusted analyses, boys with blood lead levels ≥ 5 μg/dL had 43% reduced odds of having entered G2 compared with those with lower levels .The median (25th–75th percentile) blood lead level was 3 μg/dL (2–5 μg/dL). Height, weight, body mass index, birth weight, and gestational age were predictive of the onset of puberty as assessed either by TV (> 3 mL), genitalia stage (G2), or both. Blood lead level was inversely associated with height (Relatively low environmental blood lead levels were associated with decreased growth and differences in pubertal onset in periadolescent Russian boys. Future analyses of this prospective cohort will address pubertal onset and progression in relation to lead and other environmental chemicals. Puberty, the period of transition from childhood to attainment of mature reproductive function, is characterized by accelerated growth, development of secondary sexual characteristics, and psychological changes. Activation of the hypothalamic–pituitary–gonadal axis, manifest as increasing pulsatile secretion of gonadotropin-releasing hormone (GnRH), initiates puberty. Although the precise mechanisms responsible for pubertal onset are not fully understood, body mass and nutritional status , psychosEpidemiologic studies suggest a temporal trend of earlier pubertal onset in girls , with leThe present study was conducted among boys from Chapaevsk, a town of 72,000 residents, in central Russia. Several large industries in Chapaevsk previously manufactured chemical warfare agents but presently produce industrial and agricultural chemicals . These pThe study was approved by the Human Studies Institutional Review Boards of the Chapaevsk Medical Association, Harvard School of Public Health, University of Massachusetts Medical School, and Brigham and Women’s Hospital. The parent or guardian signed an informed consent, and the boy signed an assent form.a) children without legal guardians , because birth or family history was unavailable; b) children of Azerbaijanian nationality, because they were generally born elsewhere and likely to relocate; and c) children with chronic illnesses that might affect growth and puberty, such as severe asthma, renal failure, diabetes, and congenital cardiac defects.All boys 8–9 years of age who were residents of Chapaevsk, Russia, between May 2003 and May 2005 were identified, using health insurance records and the centralized clinic system servicing all Chapaevsk children. The following were exclusion criteria: The primary caregiver  completed nurse-administered health, lifestyle, and dietary questionnaires. The health and lifestyle questionnaires were developed through pilot work in Chapaevsk  and wereWith a nurse present, a trained physician (O.S.) performed a standardized examination including height, weight, and pubertal staging without knowledge of the boys’ lead levels. Height (to the nearest 0.1 cm) was measured in stocking feet using a stadiometer. Weight (to the nearest 0.1 kg) was measured with the boys in undergarments using a balance scale with moveable weights and level platform.Pubertal status was staged from 1 to 5 by visual inspection and compared with published photographs according to internationally accepted criteria . PubarchAt the physical examination visit, 3.0 mL of venous blood was collected in trace metal–free Vacutainer tubes  after cleaning the site serially with three alcohol pads. Whole blood samples were diluted with a matrix modifier solution and analyzed by Zeeman background-corrected flameless graphite furnace atomic absorption . All measured values were reported. The detection limit was 1.0 μg/dL.Although the data are from a prospective cohort study on the growth and development of Russian children, for this study only the baseline measurements for exposure were available. Therefore, the statistical analyses explored the cross-sectional association of blood lead levels with measures of growth and pubertal onset at baseline. Blood lead levels were modeled as either a continuous measure based on a natural logarithmic transformation to approximate a normal distribution or a dichotomous indicator of high lead defined as ≥ 5 μg/dL. We evaluated the association of log-transformed lead levels with somatic growth , penile length, and birth characteristics  using both univariate and multivariable linear regression models adjusted for potential confounders . All statistical models used the exact age of each boy in days based on the actual birth date. Study visits were timed to be close to the birthday, and the majority were conducted within 1 month of each boy’s birth date. Standard diagnostic methods were used to evaluate the appropriateness of the linear regression model  and to identify potential outliers.To evaluate the association of both log-transformed lead and high lead (≥ 5 μg/dL) with pubertal onset, we used logistic regression models for the binary outcomes of TV > 3 mL and ≥ stage 2 for genitalia and pubarche, considered separately. We also fit ordinal logistic regression models under a proportional odds assumption for TV . A test of the proportional odds assumption was conducted for each model, and there was no evidence that this assumption was violated. Similar to the above linear regression models, the logistic regression models were first fit to evaluate univariate effects of log-transformed lead or high lead, and then adjusted for birth weight, gestational age, height, BMI, and age at examination. Selection of covariates in the multivariable models were based on the association of birth history and somatic characteristics with puberty onset in the absence of lead. In addition, an assessment of potential confounding by nutritional status of the associations between lead and pubertal onset was conducted based on preliminary dietary information. Multivariate regression models were further adjusted for macronutrients  as a percentage of caloric intake, using the nutrient density method . A sensitivity analysis of the influence of high blood levels on associations with pubertal onset was conducted by excluding 16 (3%) subjects with blood lead values of ≥ 10 μg/dL.Demographic data and birth characteristics of the 489 boys enrolled in the study with blood lead levels, physical examinations, and questionnaire information are presented in The median (25th–75th percentile) blood lead levels of the boys were 3 μg/dL (2–5 μg/dL), with slightly higher levels among 9-year-old boys compared with 8-year-olds . Twenty-p < 0.001 and p = 0.06, respectively).Height and weight, but not BMI or penile length, were inversely associated with blood lead concentrations . For eacPubertal onset defined as TV > 3 mL was present in 14% of the boys, including 12% of 8-year-olds and 18% of 9-year-olds . Pubertap = 0.01] and a 13% increase in the odds of being in G2 per one-unit increase in BMI . Similar magnitudes of associations were found for these predictors and pubertal onset using TV categorized as six levels (1–6 mL) and two levels (≤ 3 mL and > 3 mL). Gestational age was inversely associated with pubertal onset based on TV > 3 mL .Height, weight, BMI, birth weight, and gestational age were predictive of pubertal onset as defined by either TV > 3 mL or G2, or both. In multivariable models with adjustment for exact age at examination and other growth and birth history measures, there were almost twice the odds of being in G2 per kilogram increase in birth weight [odds ratio (OR) = 1.91; 95% CI, 1.15–3.17; p = 0.10) (p = 0.03). In unadjusted models, there was an association between TV (which ranged from 1 to 6 mL) with dichotomized blood lead . However, after adjustment for covariates, the association became only marginally significant . No association of blood lead levels with either TV > 3 mL or stage P2 was identified, despite trends in the same direction as G2.After adjustment for confounders, when pubertal onset was defined using G2, each log unit increase in blood lead concentration was associated with a marginally significant reduction in odds of being in G2  . When blp = 0.06) and high blood lead . In addition, the association of TV with high lead (≥ 5 μg/dL) became significant after adjustment for dietary macronutrients .Several sensitivity analyses were conducted to evaluate the impact of nutritional status, socioeconomic status, a higher blood lead cut point (≥ 10 μg/dL), and elimination of outliers on the robustness of the associations between blood lead and pubertal onset. These sensitivity analyses helped to confirm and support the previous results. Associations of blood lead with G2 had similar ORs but narrower CIs after further adjustment for dietary macronutrients using the nutrient density approach in models with both log lead  and with high lead .Because shorter boys may have higher lead levels than taller boys and are less likely to have puberty onset, we also conducted several analyses to specifically assess the impact of stature on associations between blood lead levels and puberty. The adjusted models in p = 0.06). Similarly, exclusion of two boys with lead levels ≥ 30 μg/dL had essentially no effect on the results shown in After excluding 16 subjects with blood lead values ≥ 10 μg/dL, the significant association of pubertal onset reflected by G2 with high versus low blood lead persisted  . In addiOur findings suggest that lead is unlikely to be playing a role in the observed secular trend of earlier pubertal onset. However, independent of lead exposure, the pattern of pubertal development among the Chapaevsk boys  parallelAmong these Russian boys, there was a higher prevalence of pubertal onset as defined by ≥ G2 (30%) compared with that defined by TV > 3 mL (14%). Of the boys in G2, 60% had a prepubertal TV, whereas of the boys with TV > 3 mL, all but 12 boys (17%) were in G2. Although changes in genital staging and TV are thought to occur in parallel, few studies have specifically assessed both of these signs of pubertal onset. A similar discordance has been reported in a large East German study in which the 3rd percentile for G2 occurred at 8.5 years in contrast to 11.8 years for achieving a testicular length ≥ 30 mm  . Our incAnimal models suggest that both the timing and duration of exposure may be important for lead’s reproductive toxicity. For example, male rats exposed to lead during gestation had decreased birth weights and pubertal growth rates due to disrupted growth hormone (GH) secretion and decreased plasma insulin-like growth factor 1 (IGF-1) concentrations during puberty , 1998a. 210Pb accumulation in the median eminence , suggesting that dioxin exposure is unlikely to confound our findings. In future work, we will complete dioxin analyses for the remaining study participants and explore the interrelationships of lead and dioxin with growth and development.In addition to lead, dioxin exposure is of concern for the Russian boys studied. In preliminary analyses on data from 125 boys, serum dioxin had no correlation with blood lead levels . Although the lead-associated changes in pubertal onset of the boys in the study do not constitute clinically defined pubertal delay, modest changes in the mean age of pubertal onset can result in substantial increases in the occurrence of clinically abnormal onset in a population, and thereby have important public health consequences . Because


Acyrthosiphon pisum, is an emerging genomic model system for studies of polyphenisms, bacterial symbioses, host-plant specialization, and the vectoring of plant viruses. Here we provide estimates of nucleotide diversity and linkage disequilibrium (LD) in native (European) and introduced (United States) populations of the pea aphid. Because introductions can cause population bottlenecks, we hypothesized that U.S. populations harbor lower levels of nucleotide diversity and higher levels of LD than native populations.The pea aphid, We sampled four non-coding loci from 24 unique aphid clones from the U. S.  and 24 clones from Europe . For each locus, we sequenced approximately 1 kb from two amplicons spaced ~10 kb apart to estimate both short range and longer range LD. We sequenced over 250 kb in total. Nucleotide diversity averaged 0.6% across all loci and all populations. LD decayed slowly within ~1 kb but reached much lower levels over ~10 kb. Contrary to our expectations, neither LD nor nucleotide diversity were significantly different between native and introduced populations.Both introduced and native populations of pea aphids exhibit low levels of nucleotide diversity and moderate levels of LD. The introduction of pea aphids to North America has not led to a detectable reduction of nucleotide diversity or increase in LD relative to native populations. One important goal of evolutionary biology is to identify the genetic causes of phenotypic variation in wild populations. In theory, in large, randomly mating populations where all loci are in linkage equilibrium, genotype can be connected to phenotype by assaying every DNA polymorphism and associating those polymorphisms with phenotypic variation . In pracArabidopsis thaliana [Ficedula albicollis), significant LD extends over 400 to 500 kb [Caenorhabditis elegans LD generally decays to insignificant levels within a kilobase [Drosophila melanogaster within even shorter distances [Systematic studies of LD have been performed for few species and mainly for plants. Among plants, LD varies widely, with higher levels observed in predominately selfing organisms such as thaliana ,4 and sothaliana . Selfingthaliana  and maizthaliana ,8, LD dethaliana ,9,10. Ino 500 kb . In Caenkilobase ,13 and iistances .Acyrthosiphon pisum). Pea aphids, like most aphids, are cyclical parthenogens [Here we characterize levels of LD and nucleotide diversity in the pea aphid  and clover (genus Trifolium). Although Eurasian in origin, the pea aphid has been introduced to North America, presumably via agriculture, probably many times over the last 200 years [Pea aphids infest field crops of the pea family such as alfalfa  or red clover (Trifolium pratense) [Trifolium repens), but no alfalfa specialists have been reported. Clones found on alfalfa are more generalized in that they are able to feed on either clover or alfalfa [Aphid clones display increased survivorship and fecundity on their preferred host plants. In New York, pea aphids specialize on alfalfa (ratense) ,30 and tratense) . Specialratense) , Sweden ratense) , and theratense) . In Cali alfalfa . We sequTwelve pea aphid clones were collected from alfalfa fields in Yolo and Galt counties of central California by T. Leonardo and S. Nuzhdin . Twelve We assembled four contiguous segments of 10 to 20 kb of genomic DNA using sequences from the NCBI trace archive. Two of these loci, CAA and 380, are located on the X chromosome while the other two, 870 and 41, are on autosomes. Pea aphids have three autosomes and one large X chromosome that appears to contain about a third of the nuclear genome content . Small s2O. All PCR reactions were done with negative controls under the following conditions: an initial 95°C anneal, 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute, with a final 10 minute extension at 72°C. PCR products were purified using the Invitrogen  Purelink PCR purification kit and sequenced directly from both ends as well as with the internal primers listed in Table We wanted to assay LD within short distances (less than 1 kb) and longer distances (over about 10 kb), so we designed two sets of PCR primers for each of the four loci. The resulting amplicons from each set were spaced by about 10 kb. Because we had two amplicons per locus and four loci, in total we utilized eight amplicons (from each of the 48 clones). Specifically, our amplicons (sized between 500 and 1000 bp) were separated by 9249 bp for locus 380, by 11371 bp for locus 870, by 9163 bp for locus 41, and by 10684 bp for locus CAA. After release of the first assembly of the pea aphid genome, Acyr 1.0, we confirmed these distances, with locus 380 on contig 7746 in scaffold 5539, 870 on contig 14350 in scaffold 14350, 41 on contig 6316 in scaffold 12009 and CAA on contig 18522 in scaffold 15751. We also confirmed that all four of the loci are in intergenic regions. Primers are listed in Table FJ825706–FJ825751 and FJ858381–FJ858708.Sequences were assembled using CodonCode Aligner . Because these were natural pea aphid isolates, all loci had heterozygous positions and some had heterozygous insertions or deletions (indels). The software identified heterozygous positions that were confirmed by visual inspection of the sequence trace file. In some cases we did not sequence across the entire amplicon due to the presence of heterozygous indels. These areas were treated as unknown data. There were also instances where we were unable to amplify a locus in a given genotype. Figure AB, in this package is estimated via maximum likelihood, and then used to compute the difference in expected versus observed allele pairs, D, where D = pAB - pApB and the correlation coefficient between the pairs, r, where r = -D/(√(pApapBpb)). We thus report LD values between pairs of sites using the squared allele frequency correlation measure, r2. Rare sites  were excluded prior to computations. Significance of LD between SNPs was assessed using Fisher's exact test, also implemented using the Genetics package. To estimate the decay of LD over distance, we used nonlinear regression of LD between polymorphic sites versus physical distance [2 using a drift-recombination model is E(r2) = 1/(1 + C), where C equals the population recombination parameter (C = 4Ner). Adding mutation to the model, the expectation becomes:Analyses of LD were performed using the Genetics package  in the Rdistance . The exp2) = ((10 + C)/(2+C)(11+C))/(1+(3+C)(12+12C+C2)/(n(2+C)(11+C)), [n is the number of sequences sampled. We fit this model after replacing C by C*distance in base pairs between sites using PROC NLIN in SAS [E(r(11+C)),  where n N in SAS .st) for each of the four loci were estimated using Genepop [We calculated π, θ, and Tajima's D using R. π measures nucleotide diversity as the average number of nucleotide differences per site between all pairwise comparisons of sequences and θ is the level of heterozygosity determined by the number of variable positions ,42. Taji Genepop , which u Genepop . We also Genepop .To measure levels of sequence diversity and LD decay over short distances (less than 1 kb) as well as over longer distances (about 10 kb), we sequenced two amplicons spaced by about 10 kb at four different genomic regions for 12 clones from New York, 12 clones from California, and 24 clones from Europe (22 clones from France and two clones from Germany). The European clones consisted of 12 alfalfa and 12 clover specialists. Two of the genomic regions were from autosomes (41 and 870) and two were from the X chromosome (380 and CAA). A total of approximately 5452 bp was sequenced for each individual, with over 250 kb sequenced in total. None of the regions included coding sequence and all were in intergenic regions as determined by querying the "nr" (non-redundant) database with a blastX (translated versus protein database) search of their associated genomic contigs. We excluded all indel information from our analyses because we used diploid individuals and could not precisely assay the number or size of indels in some of our individuals. Heterozygous indels in some cases led to missing sequence data . Further, measures of nucleotide diversity were not significantly different between North American and European populations .Table All loci possessed indels. We observed two 1 bp indels in locus 41. Locus 870 had a 1 bp, a 2 bp, a 3 bp and a 5 bp indel. Locus CAA showed two 1 bp, a 4 bp and a 6 bp indel. Finally, locus 380 had five 1 bp indels, a 4 bp indel, and a 14 bp indel. There were additional indels that we did not directly observe: multiple individuals had sequencing problems at the same region likely due to a heterozygous indel. These can be inferred from Figure Under neutrality, the values of π  and θ (based on the number of segregating sites) should be equal ,47. Taji2 = 94.2, P = 0.001, California: X2 = 84.2, P = 0.014). Both deviations at locus 870 were towards a deficiency of heterozygotes. All four loci were in Hardy-Weinberg equilibrium within the two European populations.New York and California populations were in Hardy-Weinberg equilibrium for all loci except the autosomal locus 870 , separately for each population and each locus. The average correlation at both short-range (less than 1 kb) and long-range (about 10 kb) distances is listed in Table 2 within a kilobase for all loci pooled is displayed in Figure 2 values were low relative to the shorter spatial scale with an average of 0.20, although some of these comparisons were still significant , the average rWe have investigated the levels of nucleotide diversity and LD across four genomic regions in two introduced pea aphid populations, New York and California, and two host races from native populations in Europe. This is the first estimate of short and longer range LD decay for this species. Previous studies using primarily mtDNA sequence variation, RFLPs, and allozymes have reported low diversity in pea aphid populations ,48-50. LThe two loci on the X chromosome exhibit roughly half the diversity of the two loci on the autosomes. These lower X chromosome values may result, at least in part, from the lower effective population size for the X chromosome. In a population with equal numbers of males and females there are three X chromosomes for every four autosomes because females are XX and males are XO. Under neutrality, nucleotide diversity is directly proportional to effective population size . HoweverIn a previous study , we sequDrosophila simulans [Aedes aegypti [The levels of noncoding DNA diversity in the pea aphid are two and a half times lower than the diversity estimates of 0.0153 for intergenic regions of simulans  and 0.01 aegypti . Althoug aegypti , high va aegypti  and low We also examined levels of LD at two scales: within a kilobase, and across approximately 10 kb. Generally, LD is high and decays slowly within a kilobase , providing no evidence for the source of the New York populations. Both of these values are higher than for the comparison of European clover and alfalfa specialists (Fst = 0.155). This value is only slightly higher than the level of Fst reported by Simon et al. [st = 0.098) or microsatellites (Fst = 0.104).Pea aphids are specialized to a variety of host plants in Europe, including alfalfa and clover ,33. Simin et al.  for compst = 0.228), while lower in comparison with the European alfalfa clones (Fst = 0.168). Although this difference is not statistically significant  the trend of lower differentiation between California and the alfalfa specialists leads us to hypothesize that the host plant generalist aphids found in California are derived from European alfalfa specialists, but that subsequently there has been interbreeding with other introduced populations. Further tests of this hypothesis will require more extensive population sampling and marker development but will provide insight into the interplay of invasion of a new habitat and the gain or loss of host-plant specialization.The California pea aphids examined here were sampled from alfalfa, but all are generalists in that they can feed on both hosts . Interestingly, population differentiation is high between California and the European clover clones , finding low levels of nucleotide diversity and moderate levels of LD in all of the populations sampled.JAB and DLS conceived of the study, JAB carried out the experiments, JAB and SVN performed the analyses, and all three authors participated in the writing of the manuscript. All authors read and approved the final manuscript.


Recent treatment options to correct these abnormalities in patients with sepsis and organ dysfunction have yielded conflicting results. Using thromboelastometry . Hemostasis was analyzed with routine clotting tests as well as thromboelastometry every 12 hours for the first 48 hours, and at discharge from the ICU. Organ dysfunction was quantified using the Sequential Organ Failure Assessment (SOFA) score.P = 0.021) and clot formation time (CFT) from 97 ± 63 seconds to 63 ± 31 seconds (P = 0.017), whereas maximal clot firmness (MCF) increased from 62 ± 11 mm to 67 ± 9 mm (P = 0.035). Classification by SOFA score revealed that CFT was slower (P = 0.017) and MCF weaker (P = 0.005) in patients with more severe organ failure  as compared with patients who had lower SOFA scores . Along with increasing coagulation factor activity, the initially increased International Normalized Ratio (INR) and prolonged activated partial thromboplastin time (aPTT) corrected over time.Simplified Acute Physiology Score II and SOFA scores at ICU admission were 52 ± 15 and 9 ± 4, respectively. During the ICU stay the clotting time decreased from 65 ± 8 seconds to 57 ± 5 seconds (® remained within the reference ranges during the phase of critical illness in this cohort of patients with severe sepsis and septic shock without bleeding complications. Improved organ dysfunction upon discharge from the ICU was associated with shortened coagulation time, accelerated clot formation, and increased firmness of the formed blood clot when compared with values on admission. With increased severity of illness, changes of ROTEM® variables were more pronounced.Key variables of ROTEM Organ failure contributes cumulatively to mortality in patients with sepsis . One of 1 and F2 [In clinical practice, routinely performed blood coagulation tests only incompletely mirror sepsis-induced coagulation abnormalities, and hypercoagulation in particular is not detected. More advanced laboratory analyses or experimental methods to monitor coagulation in critically ill patients, including tissue factor levels , prothro1 and F2 , thrombi1 and F2 ,17, and 1 and F2 , have noEnd-points of routine coagulation tests occur early in the hemostatic process, whereas thromboelastography measures the viscoelastic characteristics of blood clot formation in a whole blood assay, and it may therefore provide additional information on coagulation. Thromboelastography assesses the influence of plasmatic factors and platelets during all phases of the coagulation process. Thus, it permits one to evaluate the initiation of coagulation, the propagation of clot formation, and the final firmness of the blood clot.in vitro models of endotoxemia or at a single time point in patients with sepsis [Thromboelastography has gained importance in the management of bleeding disorders in trauma and surgical patients -21. It hh sepsis -25. Howe® thromboelastometry; Pentapharm, Munich, Germany) in parallel with routine coagulation tests during the early phase of severe sepsis and septic shock, and to relate these abnormalities to organ dysfunction. We hypothesized that changes in ROTEM® variables may be related to the evolution of organ dysfunction. ROTEM® is a point-of-care device with limited susceptibility to shock and vibrations.The aim of this study was to evaluate the evolution of coagulation abnormalities using thromboelastometry . Written informed consent was obtained from the patients or their relatives. Thirty patients admitted to a 30-bed multidisciplinary intensive care unit (ICU) with a diagnosis of sepsis  were enAll patients with sepsis were monitored with a radial arterial line and a central venous line, and with a pulmonary artery catheter if they were unresponsive to initial volume loading. Treatment protocols were used for hemodynamic management, weaning from mechanical ventilation, analgesia and sedation, and insulin therapy. Thromboprophylaxis in all patients was achieved with graduated compression stockings or intermittent pneumatic compression devices in addition to low-dose unfractionated heparin 10,000 units/day, in accordance with the guidelines of the American College of Chest Physicians for critically ill patients . The hep®; Fresenius Kabi, Stans, Switzerland). Patients in the low Sequential Organ Failure Assessment (SOFA) group were administered a mean of 1,373 ± 1,929 ml over the whole study period; those in the high SOFA group received 2,523 ± 1,914 ml.Volume resuscitation with colloids in our patients was achieved with 6% hydroxyethyl starch 130/0.4 .® Analyzer and corresponding reagents .Routine coagulation screening, including platelet count, International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), levels of coagulation factors II, V, VII and X, and fibrinogen, were measured daily for the first 3 days and upon discharge. Routine coagulation laboratory measurements and analysis of coagulation factor activity were performed using the BCS® analyzer  every 12 hours for 48 hours and at ICU discharge. The method, technique and variables of thromboelastometry were described previously [® measures viscoelastic properties of clot formation and fibrinolysis. Because of the use of a ball-bearing system for power transduction, it is less susceptible to movement and vibration.Thromboelastometry was performed using a ROTEMeviously . Briefly® device was tested regularly for correct function using quality control serum .Tests were performed using ROTEM cups and pins. The ROTEM2 0.2 mol/l, 20 μl thromboplastin-phospholipid, 300 μl blood; EXTEM test: 20 μl CaCL2 0.2 mol/l, 20 μl tissue factor, 300 μl blood). In order to assess a possible effect of low-dose heparin administration to our patients, we also analyzed the effects of addition of heparinase to the blood samples (HEPTEM test) and compared the results with those of the INTEM test.We conducted intrinsically and extrinsically activated tests (INTEM and EXTEM), in accordance with the manufacturer's recommendations . Chemicals and reagents were purchased from Pentapharm GmbH.The influence of thrombocytes on clot firmness was estimated with a platelet-inactivating test (FIBTEM test: 20 μl CaCL® thromboelastometry are the following. The clotting time (CT), the equivalent to the reaction time (r time) of conventional TEG® , represents the initiation of coagulation. The propagation of clot formation, reflecting thrombin generation and early fibrin polymerization, is characterized by clot formation time (CFT), comparable to the clotting time (k time) and alpha angle of conventional TEG®. CFT is thereby defined as the time necessary to attain a clot firmness of 20 mm. The maximal clot firmness (MCF), corresponding to the maximal amplitude of conventional TEG®, which describes the final strength of the clot, is influenced by the fibrinogen concentration and the platelet count.The variables that were measured using ROTEMpost hoc comparisons or, where appropriate, Friedman analysis and the Dunn test. In order to compare coagulation profiles in groups of patients with different organ failure severity, patients were divided into two groups using median SOFA score. Differences between the two groups were analyzed using analysis of variance for repeated measurements with one grouping factor (high versus low SOFA score) and one within-subject factor (time). The correlation between routine coagulation and ROTEM® variables with the severity of organ dysfunction defined by the SOFA score was studied using the nonparametric Spearman correlation (r) with pooled data from the continuous time points. Comparison of the clotting times between the INTEM and HEPTEM assays was analyzed with a two-sided t-test. For all statistical tests, significance was assumed at P < 0.05. Values are expressed as mean ± standard deviation or median (interquartile range) where appropriate.SigmaStat version 3.5  was used for statistical analysis. After testing for normal distribution (Kolmogorov-Smirnov test), data were analyzed using analysis of variance for repeated measurements and the Student-Newman-Keuls test for The characteristics of the included patients are shown in Table r = 0.39 and 0.51, respectively; P < 0.001 each).Coagulation factor levels, routine coagulation tests, hemoglobin, platelets, and white blood cell count are shown in Table Thromboelastometric variables in the entire study cohort are presented in Table In patients with higher scores for organ failure, MCF differed from the low SOFA group during the first 48 hours. This difference had resolved by the time of discharge Figure . CT did Platelet inhibition with cytochalasin revealed stronger impairment of MCF in the high SOFA group Figure . The rel® thromboelastometry CFT, alpha angle, and MCF correlated moderately with disease severity defined by the SOFA score  [The results of ROTEMl ranges ,34-37. Tl ranges , who destography ,23. Spietography  administhy [1+2) . IncubatIn critically ill patients with severe sepsis and septic shock, the disease has reached a more advanced stage – with advanced inflammatory response and sustained exposure to the infectious pathogen – by the time they present at the ICU. In the clinical setting, assessment of activation of coagulation by thromboelastography may thus not present with shortening of CT and CFT but rather with a development in the opposite direction, in which coagulation factors are consumed. A concomitant decrease in the production of clotting factors due to hepatic dysfunction in sepsis may also play a role, however .Our data are supported by an analysis of the placebo group in the PROWESS  study, which demonstrated that increased severity of sepsis is accompanied by prolonged coagulation times, suggesting decreased activity of coagulation factors as a result of increased consumption .Activation of coagulation is a well-known pathophysiological process in sepsis ,14-17,31There is concern that the administration of colloids may have affected the coagulation system. Although the doses of fluid resuscitation with 6% hydroxethyl starch 130/0.4 differed between the two groups, the administered volumes have reportedly only minor effects on parameters of thromboelastography ,42.® HEPTEM) we analyzed clotting time as the variable of interest in this context and excluded such an effect [A low-dose regimen of unfractionated heparin was administered to the patients in the present study in order to prevent thromboembolic complications. This could have influenced thromboelastography analysis. However, in a heparinase assay (ROTEMn effect . These r® parameters had to be interpreted on the basis of external reference values. Future investigations in a controlled study may reveal clearer relationships between organ dysfunction and the coagulation system assessed by thromboelastography.A limitation of our study is that the reported changes of ROTEM® remained within the reference ranges during the phase of critical illness in this cohort of patients with severe sepsis and septic shock without bleeding complications. Although average thromboelastometry variables did not provide additional information to standard coagulation tests, certain dynamics of ROTEM® variables were noted within the reference ranges. Improved organ dysfunction upon discharge from the ICU was associated with shortened coagulation time, accelerated clot formation, and increased firmness of the formed blood clot when compared with values on admission. With increased severity of illness, changes of ROTEM® variables were more pronounced.Key variables for ROTEMThromboelastography performed in patients with severe sepsis cannot reliably detect activation of coagulation in the sense of a hypercoagulable state. Further studies in patients with sepsis are warranted to investigate the role of thromboelastography in relation to bleeding and thromboembolic complications as end-points.• Key thromboelastometric variables remained within reference ranges during the course of critically illness in patients with sepsis without adverse bleeding events.• After resolution of the critical illness in patients with severe sepsis/septic shock, thromboelastometric variables exhibited shortened coagulation time, accelerated clot formation, and increased firmness of the formed blood clot when compared with values on admission. With increased severity of illness, these changes were more pronounced.in vitro or experimental studies.• Thromboelastometry, when performed in patients with established severe sepsis and septic shock, cannot reliably detect activation of coagulation in the sense of a hypercoagulable state, as suggested by aPTT: activated partial thromboplastin time; CFT: clot formation time; CT: clotting time; ICU: intensive care unit; INR: International Normalized Ratio; MCF: maximal clot firmness; SOFA: Sequential Organ Failure Assessment.The authors declare that they have no competing interests.FD made substantial contributions to the design of the study, analysis and interpretation of the data, and was involved in drafting the manuscript. UK, HF, and JSL made substantial contributions to the thromboelastometric measurements and to the acquisition of the data, and critically revised the manuscript for important intellectual content. JT critically revised the manuscript for important intellectual content. SMJ made substantial contributions to the concept and design of the study, and to the analysis and interpretation of the data, and was involved in drafting the manuscript. All authors read and approved the final manuscript.


Postoperative mortality (2 vs 2%) and morbidity  was similar between groups. More NC patients developed septic (2.4%) or respiratory (10.3%) complications compared to controls , with significantly more surgical complications if the interval between stopping NC and undergoing surgery was ⩽4 weeks (11%), compared to 5–8 (5.5%) or 9–12 (2.6%) weeks (P=0.009). The data suggest that liver resection for CRM is safe following NC. Early hepatobiliary involvement in multidisciplinary cancer care may lead to avoidance of potential perioperative adverse events.Neoadjuvant chemotherapy (NC) can improve the resectability of hepatic colorectal metastases (CRM). However, there is concern regarding its impact on operative risk. We reviewed 750 consecutive liver resections performed for CRM in a single unit (1996–2005) to evaluate whether NC affected morbidity and mortality. Redo hepatic resections or patients receiving adjuvant chemotherapy following primary resection were excluded. A total of 245 resections were performed in patients not requiring NC (control group)  and 252 in patients who had NC . The mean (s.d.) duration of surgery was less in the control group (241(64)  Colorectal cancer is the third most common malignancy in the UK, with an incidence of over 34 000 . Fifty pAdvances in surgical and anaesthetic technique have expanded the indications for liver resection in the past 20 years. However, in a landmark paper in 1996, The aim of the study was to determine the impact of NC on postoperative morbidity and mortality following liver resection for colorectal metastases (CRM).n=179).The records of 750 consecutive patients undergoing liver resections for CRM with a curative intent in a single institution between May 1997 and October 2005 were reviewed. Data were prospectively recorded on an Access®  database consisting of 268 data fields compiled contemporaneously from a standardised proforma. Data validation was performed by requesting duplicate information from patient charts by a dedicated database officer. Details of the chemotherapy regimens prospectively recorded included drug types, duration of chemotherapy and the length of time (in weeks) between the cessation of chemotherapy and liver resection. The time period 1997–2005 was chosen to include all patients given NC before liver resection and a group of contemporaneous controls who had never been exposed to chemotherapy. Patients undergoing resections before May 1997 were excluded from the study to reduce potential bias from improved surgical and anaesthetic technique (n=181) and patients undergoing redo hepatic resections (n=72) were excluded from the study. The remaining 497 patients were divided into two groups: a control group (n=245) who had surgery alone and a chemotherapy group (n=252) who received NC before liver resection.Those patients who had received neoadjuvant or adjuvant chemotherapy prior or following resection of the primary tumour  and postoperative mortality and morbidity. Postoperative mortality was defined as death during the same hospital admission or within 30 days of the operation date, if the patient was discharged earlier. Postoperative complications were documented prospectively and classified as minor, relevant (delayed patient discharge) or serious . The standard follow-up protocol included clinical examination, estimation of serum carcinoembryonic antigen level and abdominal ultrasonography or contrast-enhanced CT at 6 months, 1 year and annually thereafter until death.χ2 test or Fisher's Exact test as appropriate. Trends over time were assesses using the χ2 test for trend (γ-correction). P-values of <0.05 were considered to represent statistical significance. Data were analysed using SPSS software (version 11.0) .Continuous variables were compared using the analysis of variance and categorical variables were analysed using the The patient demographics in the control and chemotherapy groups are detailed in P=0.014) by a means of 14 min. In addition, the mean blood loss was higher in the chemotherapy group (449 ml) compared to the controls (390 ml), but this failed to reach statistical significance (P=0.067). Further examination of the largest subgroup of patients in the chemotherapy group, the 127 patients (50.4%) who received oxaliplatin-based chemotherapy was performed, with their operative details compared with those of the control patients. Again, there was no difference in Pringle clamp time  and a significantly longer mean operating time . However, in this subgroup analysis, a significantly greater mean (s.d.) blood loss in the patients receiving neoadjuvant oxaliplatin (473 (530) ml), compared to the control patients (390 (264) ml, P=0.046), was shown.The extent of liver resection performed was similar between the control and NC groups , but wheP=1.0, Fisher's Exact test). The cause of death is detailed in P=0.34, Fisher's Exact test). The incidence of specific complications is shown in P=0.027) or sepsis/abscesses (P=0.016) in the chemotherapy group. The six cases complicated by sepsis/abscess were two cases of sepsis/multiorgan failure, one epidural abscess, that led to paraplegia, one patient with MRSA cellulitis, one infected Hickman line and one patient who developed peritonitis of unknown cause. There was no difference in the incidence of hepatic insufficiency in the control (6.1%) or chemotherapy (7.5%) groups (There were 10-postoperative deaths in the series (2%), five in each of the control and chemotherapy groups  groups . This wa) groups . One patP=0.96, χ2 test for trend). However, P=0.009).The time interval (in weeks) between finishing chemotherapy and undergoing surgery  did not n=18) to generate adequate statistical power.The effect of the duration of chemotherapy on postoperative morbidity and mortality was then considered. Patients were grouped into those who had chemotherapy for less than 3 months, 3–6 months and more than 6 months . Althougin vivo assessment of tumour chemoresponsiveness, allowing clinicians to continue treatment for responders or change treatment for nonresponders. Furthermore, there is evidence that the response to chemotherapy is a prognostic indicator of survival even when subsequent liver resection is performed with curative intent , biliary fistula (5.5 vs 1.6%) and intra-abdominal infections (4.5 vs 0.8%) were all higher in those patients who had received NC compared to those undergoing surgery alone. P=0.03). Although the present results showed a higher incidence of hepatic insufficiency in those patients who had NC, this difference failed to reach statistical significance, possibly reflecting the low overall incidence of this complication in this series. A significant increase in respiratory and septic complications in the patients receiving NC, compared to the controls was, however, demonstrated.This study shows that NC before liver resection for CRM does not increase postoperative morbidity and mortality. This finding is supported by early data from the MD Anderson Cancer Centre on 108 patients who underwent major liver resection for CRM, of whom 61 had preoperative chemotherapy (27 5-FUFA and 34 5-FUFA+irinotecan) with no adverse effect on clinical outcome  who had chemotherapy for more than 6 months duration. These differences failed to reach statistical significance, possibly due to the small numbers involved (a Type II error). This is in contrast to an analysis of our early experience (1987–2002) where we found significantly increased morbidity in those patients who had had chemotherapy for more than 6 months (The present data demonstrate how the postoperative morbidity, life-threatening complications and surgical complications were all greater in the small number of patients (In this study, hepatic resection was performed in 12 patients who had had a complete response to chemotherapy. The authors believe it is important to resect all documented sites of disease within the liver, even if some lesions have shown a complete response to chemotherapy, as there is evidence that these metastases will eventually recur. In conclusion, this is the largest study to date, which has addressed the question as to whether NC increases the risk of liver resection for CRM. The present study confirmed that chemotherapy before resection of colorectal liver metastases is safe, despite the performance status of the patients receiving chemotherapy being lower than that of the controls, and the surgery taking longer, with more associated blood loss. However, whereas NC has become an integral part of the quest to improve resection rates and survival, no treatment is without risk, as highlighted by the increased respiratory and septic complications in the chemotherapy group. The present study suggests that avoiding giving chemotherapy for a prolonged duration and carefully timing subsequent liver surgery to each individual patient's recovery from chemotherapy may result in immediate outcomes comparable to those patients who have not received chemotherapy. We would advocate the involvement of hepatobiliary surgeons at an early stage in the multidisciplinary management of these patients, allowing appropriate planning of surgical and chemotherapeutic strategies.


After simple geometric and atmospheric correction, we found a significant relationship  between in situ measured TSM concentrations and MODIS band normalization difference of band 3 and band 1. From this, we retrieved TSM concentrations in eight regions of Lake Taihu in 2007 and analyzed the characteristic distribution and variation of TSM. Synoptic maps of model-estimated TSM of 2007 showed clear geographical and seasonal variations. TSM in Central Lake and Southern Lakeshore were consistently higher than in other regions, while TSM in East Taihu was generally the lowest among the regions throughout the year. Furthermore, a wide range of TSM concentrations appeared from winter to summer. TSM in winter could be several times that in summer.Although there has been considerable effort to use remotely sensed images to provide synoptic maps of total suspended matter (TSM), there are limited studies on universal TSM retrieval models. In this paper, we have developed a TSM retrieval model for Lake Taihu using TSM concentrations measured  It determines the transparency of water and can also serve as a transport medium for various pollutants , e.g., hWith the development of remote sensing technology, satellite estimates of water quality significantly complement conventional monitoring techniques and have found widespread applications. Remotely sensed monitoring of TSM started in 1974 using ERTSA data . Since tFor water quality management of large lakes like Lake Taihu, a severely eutrophic lake, it is essential to quickly and accurately understand the dynamic variations in water quality. TSM monitoring from remote sensing in Lake Taihu has been studied for many years, and the methodologies used can be divided into three types: (1) analytical methods, also referred to as bio-optical algorithms, in which reflectance spectra are simulated by theoretical formulas based on the specific inherent optical properties of substances in the water ,9; (2) eIn this paper, Lake Taihu was selected as the study area, and a common yearly-scaled TSM retrieval model is developed. Synoptic maps of TSM in 2007 are obtained using this model, and the characteristic temporal and spatial variability of TSM is analyzed. In addition, considering that atmospheric correction is essential, especially for multi-temporal MODIS images, a simple but effective image-based relative radiometric normalization method is presented. The significance of this study is that it is the first attempt to establish a common TSM retrieval model for practical application in Lake Taihu.2.2. It is a shallow lake, with mean depth 1.9 m, and maximum depth 2.6 m at a water surface elevation of 3.0 m above sea level. The Lake Taihu drainage basin (Microcystis spp.) have occurred frequently, which badly affects the function of the lake as a drinking water supply. More efficient water quality monitoring of the lake is urgently needed [Lake Taihu is located in eastern China between 30° 55′ 40″ N and 31° 32′ 58″ N, and 119° 52′ 32″ E and 120° 36′ 10″ E. It is the third largest freshwater lake in China, with a total water area of about 2,338.11 kmge basin  is the my needed .3.3.1.in situ measurements were carried out once a month in 2005 by the Taihu Ecosystem Research and Field Observation Station, Chinese Academy of Sciences . Twenty-eight sampling sites were selected, which were considered representative of surface distributions of TSM encompassing the whole of Lake Taihu. February, May, August and November water samples were collected at all 28 sampling sites, while in the other eight months the field survey was only conducted at 13 sites, mainly located in the north part of Lake Taihu. The distribution of the 28 sampling stations in Lake Taihu is shown in TSM TSM concentrations were measured by filtering the water samples (taken from 0.5 m below the water surface) on Whatman Grade GF/F filters, which were dried and weighed on an electronic balance.3.2.MODIS images provide high radiometric sensitivity (12 bit) in 36 spectral bands from 0.4 μm to 14.4 μm in wavelength. The spatial resolution is 250 m for bands 1–2, 500 m for bands 3–7, and 1,000 m for bands 8–36. As mentioned above, numerous studies have demonstrated that MODIS has the potential for use in water quality monitoring. In particular, the MODIS 250 m and 500 m bands, centered at 469 nm (b3), 555 nm (b4), 645 nm (b1) and 858 nm (b2), can provide sufficient sensitivity for water applications [MODIS-Terra level 1B calibrated radiance products, with spatial resolutions of 250 m and 500 m, covering the region of interest in the middle November of 2003 and the whole of 2005 and 2007 were downloaded via the web interface of the Level 1 and Atmosphere Archive and Distribution System (LAADS) of the National Aeronautics and Space Administration (NASA). MODIS images from 2005 were obtained for days corresponding to field measurements of TSM, and the time difference between the field measurements and MODIS images was no more than three days. Because of cloud coverage over the target no appropriate images were available for March and June of 2005. Two cloud-free MODIS images from every month (except June) in 2007, were downloaded to analyze the distribution variation of TSM in Lake Taihu.(1) Geometric correctionEach image was geo-referenced based on the WGS 84 UTM zone 51 North coordinate system. The 500 m resolution images were resized to 250 m using the nearest neighbor resampling method during geo-referencing.(2) Atmospheric correctionBesides the changes in target reflectance, changes in satellite sensor calibration, differences in illumination and observation angles, and variations in atmospheric effects can induce variation in a sensor’s response to a given target over time. Therefore, to detect the actual changes in ground target from the total signal measured at the spacecraft sensor, atmospheric correction is necessary. Two types of atmospheric corrections, absolute correction and relative correction, are commonly employed to normalize remotely sensed images for time-series inter-comparison –17. Absoet al. [band1R and band2R are the radiance of band 1 and band 2 respectively. These PIFs are mainly situated in the new industrial zone around Taihu Lake, such as Changzhou National Hi-tech District, Wuxi Economic District and Suzhou National Hi-tech District. The other set of PIFs is located in clean sea, where water quality is good and little influenced by TSM. Following the normalization procedure of Du, these two screened PIFs sets were achieved. After identifying the PIFs centers in the density map, the normalization regression line is obtained as that intersecting these two PIFs centers. The last step is to recalculate the target image pixel values based on the normalization regression function. Now, the target image has been normalized to the scale of the base image [A variety of relative radiometric correction techniques have been developed , most ofet al.  developeet al.  developeet al.  proposedet al. , and theet al. . Assumptet al. ,24. Ther3.3.in situ measured TSM concentration data in 2005. All 166 data pairs were used for the development of the TSM retrieval model, and another 16 data pairs from 2003 were used for model validation.In total, there were 166 pairs of radiometric normalized MODIS radiance data and −1) to atmospherically corrected MODIS band data. This included single band (band 1–4) and band combinations . The best fitting model was selected to map the temporal and spatial variability of TSM in 2007.Instead of using a sophisticated optical model, simple linear and non-linear models  were used to correlate TSM concentration (mg L4.−1) and MODIS single band, band difference, band ratio and band normalized difference, showing that the last two models performed better than the first two models in terms of R and standard error. Comparison between in situ measured TSM and model-estimated TSM from the last two models using both the 166 data pairs for model development and the 16 data pairs for model validation is shown in Finally, the synoptic maps of TSM concentrations in 2007 were obtained see , which r−1; Meiliang Bay, Western and Eastern Lakeshore also show relatively high TSM concentrations; while there is good water quality in East Taihu and Zhushan Bay, with the maximum TSM concentrations being <100 mg L−1. Moreover, the variation ranges of TSM concentration in the open areas are much larger than in the bays, and the minimum variation range is located in East Taihu due to the sheltered environment and dense hydrophytes. Furthermore, obvious seasonal variations in TSM are also characterized in the lake. The water body of Lake Taihu is divided into eight water regions. These include Meiliang Bay (MB), Zhushan Bay (ZB), Gonghu Bay (GB), Central Lake (CL), Western Lakeshore (WL), Southern Lakeshore (SL), East Taihu (ET) and Eastern Lakeshore (EL) . Figure 5.5.1.−1) and relatively corrected MODIS Terra band 1 data used in this study  and TSM [a concentration is >10 μg L−1, there are an absorption peak and a reflectance peak in 672 nm and 695 nm respectively [a strongly absorb spectrum in the blue region (440 nm). Hence MODIS band 3 data (blue band reflectance) could assist band 1 data (red band reflectance) to weaken the influence from chlorophyll a and CDOM.A significant  relationship was observed between TSM concentration  measured in situ and relatively corrected MODIS Terra band 1 data, which is consistent with the conclusions of other studies. The model performance analysis indicated that this empirical model could map TSM concentrations successfully except when TSM concentration is higher than 200 mg L−1. The temporal and spatial distribution of TSM concentrations in Lake Taihu from January to December 2007 revealed that Central Lake and Southern Lakeshore are characterized by extremely high TSM concentrations, and there is good water quality in East Taihu. The variation range of TSM concentration in the open area is much larger than that in the bays, affected by higher wind velocities in the open area. The minimum variation range is located in East Taihu due to the sheltered environment and dense hydrophytes. On the whole maximum TSM concentrations appear in winter and the minimum appear in summer. This pattern is notable in the Central Lake, but very weak in East Taihu.The common model retrieving TSM concentration showed that a significant  relationship was observed between TSM concentrations (mg Li.e., the slope of the regression line, It should be noted that the approach developed in this study could be applied to other inland regions, but the specific relationship between TSM concentration and MODIS data (


Climates are changing rapidly, producing warm climate conditions globally not previously observed in modern history. Malaria is of great concern as a cause of human mortality and morbidity, particularly across Africa, thanks in large part to the presence there of a particularly competent suite of mosquito vector species.Anopheles gambiae vector complex species in Africa over the coming 50 years, based on ecological niche model projections over two global climate models, two scenarios of climate change, and detailed spatial summaries of human population distributions.I derive spatially explicit estimates of human populations living in regions newly suitable climatically for populations of two key For both species, under all scenarios, given the changing spatial distribution of appropriate conditions and the current population distribution, the models predict a reduction of 11.3–30.2% in the percentage of the overall population living in areas climatically suitable for these vector species in coming decades, but reductions and increases are focused in different regions: malaria vector suitability is likely to decrease in West Africa, but increase in eastern and southern Africa.Climate change effects on African malaria vectors shift their distributional potential from west to east and south, which has implications for overall numbers of people exposed to these vector species. Although the total is reduced, malaria is likely to pose novel public health problems in areas where it has not previously been common. Anopheles mosquitoes, that endangers more than 2.5 × 109 humans annually [Malaria is a vector-borne anthroponosis, transmitted in large part by annually , but appannually ,5, givenannually -8 withinImplications of climate change for malaria transmission across Africa have been the subject of numerous commentaries -12 and aEcological niche modeling uses nonrandom associations between known occurrences of species and digital geospatial data layers summarizing relevant environmental parameters to reconstruct the suite of environmental conditions under which the species is able to maintain populations without immigrational subsidy . ModeledAnopheles gambiae complex: A. gambiae sensu stricto and A. arabiensis, for which 581 and 501 occurrence records were available, respectively. These data are based on sampling that was intense in some countries, and negligible or absent in others – as such, an extrapolative approach to their analysis becomes key. As such, I related the occurrence data to a suite of environmental data including dimensions of climate  and topon; from ). The reEcological niche models were generated using the Genetic Algorithm for Rule-Set Prediction (GARP) , a nicheThe first rule is created by applying a method chosen randomly from a set of inferential tools . The geFollowing recent recommendations , for eacFuture climate conditions were reconstructed based on two general circulation models (GCMs) that have been used to simulate future climates: those of the Hadley Centre (HadCM3)  and the Finally, I calculated human populations living within the potential distributions of these competent malaria vectors across sub-Saharan Africa based on the LandScan database ,30, a 1 Anopheles gambiae and A. arabiensis, the two most important malaria vectors in Africa, have broad distributions across Africa [versus unsuitable. Projections of the ecological niche model onto likely future climate conditions indicated that both mosquito species are likely to see less suitable conditions for their populations across portions of West Africa, where temperature increases of 1.5–2.7°C are likely to be manifested Anopheless Africa  – sufficA. gambiae and 520,289,130 for A. arabiensis over 10.5 × 106 and 14.5 × 106 km2, respectively. Under the two greenhouse gas scenarios, >30 million people are living in areas projected to see increasing exposure to A. gambiae, and >14 million people are living in areas projected to see increased exposure to A. arabiensis. At the same time, however, 78–111 million people are living in areas projected to see reduced exposure to A. gambiae, and 135–171 million people are living in areas projected to see reduced exposure to A. arabiensis  A2 scenario is associated with 20–30% Scientific results regarding implications of climate change are usually pessimistic in nature. This analysis departs somewhat from that theme – malaria vector species are expected to undergo distributional shifts in the face of changing climates that will leave fewer people overall in areas suitable for the important vector species. The net reduction nonetheless includes increasing malaria vector presence in areas of eastern and southern Africa that are not presently exposed to these species. As such, the picture is one of complex rearrangement of malaria transmission across Africa with climate change.The author declares that they have no competing interests.ATP developed entire manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/59/prepub