Nicolas Menzies and colleagues investigate the potential impact and cost-effectiveness of implementing Xpert MTB/RIF for diagnosing tuberculosis in five southern African countries. The Xpert MTB/RIF test enables rapid detection of tuberculosis (TB) and rifampicin resistance. The World Health Organization recommends Xpert for initial diagnosis in individuals suspected of having multidrug-resistant TB (MDR-TB) or HIV-associated TB, and many countries are moving quickly toward adopting Xpert. As roll-out proceeds, it is essential to understand the potential health impact and cost-effectiveness of diagnostic strategies based on Xpert.We evaluated potential health and economic consequences of implementing Xpert in five southern African countries—Botswana, Lesotho, Namibia, South Africa, and Swaziland—where drug resistance and TB-HIV coinfection are prevalent. Using a calibrated, dynamic mathematical model, we compared the status quo diagnostic algorithm, emphasizing sputum smear, against an algorithm incorporating Xpert for initial diagnosis. Results were projected over 10- and 20-y time periods starting from 2012. Compared to status quo, implementation of Xpert would avert 132,000  TB cases and 182,000  TB deaths in southern Africa over the 10 y following introduction, and would reduce prevalence by 28% (14%–40%) by 2022, with more modest reductions in incidence. Health system costs are projected to increase substantially with Xpert, by US$460 million (294–699 million) over 10 y. Antiretroviral therapy for HIV represents a substantial fraction of these additional costs, because of improved survival in TB/HIV-infected populations through better TB case-finding and treatment. Costs for treating MDR-TB are also expected to rise significantly with Xpert scale-up. Relative to status quo, Xpert has an estimated cost-effectiveness of US$959  per disability-adjusted life-year averted over 10 y. Across countries, cost-effectiveness ratios ranged from US$792  in Swaziland to US$1,257  in Botswana. Assessing outcomes over a 10-y period focuses on the near-term consequences of Xpert adoption, but the cost-effectiveness results are conservative, with cost-effectiveness ratios assessed over a 20-y time horizon approximately 20% lower than the 10-y values.Introduction of Xpert could substantially change TB morbidity and mortality through improved case-finding and treatment, with more limited impact on long-term transmission dynamics. Despite extant uncertainty about TB natural history and intervention impact in southern Africa, adoption of Xpert evidently offers reasonable value for its cost, based on conventional benchmarks for cost-effectiveness. However, the additional financial burden would be substantial, including significant increases in costs for treating HIV and MDR-TB. Given the fundamental influence of HIV on TB dynamics and intervention costs, care should be taken when interpreting the results of this analysis outside of settings with high HIV prevalence.Please see later in the article for the Editors' Summary Mycobacterium tuberculosis, the bacterium that causes TB, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of TB are a persistent cough, weight loss, fever, and night sweats. Diagnostic tests for TB include sputum smear analysis (microscopic examination of mucus coughed up from the lungs for the presence of M. tuberculosis) and mycobacterial liquid culture (growth of M. tuberculosis from sputum and determination of its drug sensitivity). TB can be cured by taking several antibiotics daily for at least six months, although the recent emergence of multidrug-resistant TB (MDR-TB) is making the disease increasingly hard to treat.In 2010, about 9 million people developed tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—and about 1.5 million people died from the disease. Most of these deaths were in low- and middle-income countries, and a quarter were in HIV-positive individuals, who are particularly susceptible to TB. M. tuberculosis and DNA differences that make the bacteria resistant to the drug rifampicin (an indicator of MDR-TB) within two hours. Many countries are moving toward adopting Xpert for TB diagnosis, so it is essential to understand the population health impact and cost-effectiveness of diagnostic strategies based on this test. Here, the researchers use a calibrated, dynamic mathematical model of TB to investigate the consequences of Xpert MTB/RIF implementation in five southern African countries where both TB-HIV coinfection and MDR-TB are common.To reduce the global TB burden, active disease must be diagnosed quickly and accurately. In most high-burden settings, however, TB diagnosis relies on sputum smear analysis, which fails to identify some people  who have TB. Mycobacterial culture correctly identifies more infected people but is slow and costly, and many high-burden settings lack the infrastructure for high-volume culture diagnosis of TB. Faced with these diagnostic inadequacies, the World Health Organization (WHO) recently recommended the use of Xpert MTB/RIF for initial diagnosis in patients suspected of having MDR-TB or HIV-associated TB. This new, automated DNA test detects The researchers used their mathematical model, which simulates the movement of individuals through different stages of TB infection, to investigate the potential health and economic consequences of implementing Xpert for initial TB diagnosis in Botswana, Lesotho, Namibia, South Africa, and Swaziland. In the modeled scenarios, compared to an diagnostic approach based on sputum smear (the “status quo”), implementation of Xpert averted an estimated 132,000 TB cases and 182,000 TB deaths in southern Africa over the ten years following its introduction, reduced the proportion of the population with TB by 28%, and increased health service costs by US$460 million. Much of this cost increase reflected increased antiretroviral therapy costs for TB/HIV-infected individuals who survived TB infection because of better case-finding and treatment. Finally, relative to the status quo, over ten years, Xpert implementation in southern Africa cost US$959 for every DALY (disability-adjusted life-year) averted. Cost-effectiveness ratios in individual countries ranged from US$792 per DALY averted in Swaziland to US$1,257 per DALY averted in Botswana.These findings suggest that Xpert implementation in southern Africa could substantially reduce TB illness and death through improved case-finding and treatment, but that the impact of Xpert on long-term transmission dynamics may be more limited. Although the additional financial burden associated with Xpert roll-out is likely to be substantial, these findings suggest that using Xpert for TB diagnosis offers reasonable value given its cost. WHO considers any intervention with a cost-effectiveness ratio less than the per-capita gross domestic product (GDP) highly cost-effective—in 2010, the per-capita GDP ranged from US$7,000 in South Africa and Botswana to US$982 in Lesotho.These findings may not be generalizable to regions with different HIV infection rates, and their accuracy is likely to be affected by the quality of the data fed into the mathematical model and by the structure of the model. Thus, it is essential that the impact of Xpert-based TB diagnosis be carefully evaluated as the approach is rolled out, and that the information generated by these evaluations be used to improve the accuracy of model-based estimates of the long-term effects of this new strategy for TB diagnosis.http://dx.doi.org/10.1371/journal.pmed.1001347.Please access these websites via the online version of this summary at tuberculosis, including general information on tuberculosis diagnostics and specific information on the roll-out of the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report; WHO also provides information about tuberculosis and HIVWHO provides information  on all aspects of Stop TB Partnership is working towards tuberculosis elimination; patient stories about TB-HIV coinfection are availableThe tuberculosis, and about TB diagnosisThe US Centers for Disease Control and Prevention has information about tuberculosisThe US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosisThe tuberculosis (in English and Spanish)MedlinePlus has links to further information about  Tuberculosis (TB) remains a leading cause of global mortality and morbidity, with an estimated 9 million new TB cases and 1.5 million TB-related deaths in 2010 Recently, the Xpert MTB/RIF automated DNA test has been shown to provide rapid and sensitive detection of TB and rifampicin (RIF) resistance In December 2010, WHO recommended that Xpert be used for initial diagnosis in patients suspected of having multidrug-resistant TB (MDR-TB) or HIV-associated TB disease In this study we used a calibrated, dynamic mathematical model of TB to quantify the potential health and economic consequences of introducing Xpert in five southern African countries characterized by high prevalence of HIV infection and extant multidrug resistance. Comparing a diagnostic strategy based on Xpert to the status quo, we predicted changes in TB incidence, prevalence, mortality, and drug resistance; estimated health system costs; and assessed the incremental cost-effectiveness of Xpert adoption.We evaluated the population health outcomes and health system costs associated with two alternative strategies for diagnosing TB, the first based on current diagnostic algorithms and the second based on implementing Xpert in accordance with current WHO recommendations. Comparisons between these two strategies were made using a calibrated mathematical model of TB, reflecting key features of TB transmission dynamics and natural history, interactions with HIV infection, and patterns and trends in TB control interventions and treatment for HIV/AIDS. Model simulations were undertaken for five southern African countries: Botswana, Lesotho, Namibia, South Africa, and Swaziland. We assessed changes in epidemiological outcomes and health system costs over 10-y and 20-y time horizons, as well as the incremental cost-effectiveness ratio (ICER) of the Xpert strategy compared to the current algorithm.A “status quo scenario” was created to represent the current diagnostic approach. Under this approach, all patients with suspected TB receive an initial sputum smear, and those diagnosed as smear-positive are directed to treatment. Sputum culture is indicated for patients with suspected TB who test smear-negative but who have a history of TB treatment or in whom there is a strong suspicion of TB. Drug sensitivity testing (DST) is indicated for treatment-experienced patients diagnosed with TB. Those who receive DST are initiated on a treatment regimen appropriate to their drug resistance profile, while those who do not receive DST are initiated on the standard first-line regimen. In the main analysis we assumed that the coverage of culture testing would be 20% (range 10%–30%) among smear-negative, treatment-naïve patients, and 80% (range 70%–90%) among smear-negative, treatment-experienced patients. We assumed further that 80% (range 70%–90%) of treatment-experienced patients diagnosed with TB would go on to receive DST. Given limited empirical data on country-specific coverage of culture and DST, these values were all varied across wide ranges in sensitivity analyses.An “Xpert scenario” was constructed based on the diagnostic algorithms suggested for high HIV prevalence settings in the May 2011 WHO recommendations for Xpert implementation We developed a dynamic compartmental model of TB following the conventions of earlier models The core TB model simulates the movements of individuals between states that capture important features of TB transmission, natural history, and treatment. Individuals enter the model in the susceptible state, where they face a risk of TB infection. The risk of infection is modeled as a time-dependent variable that reflects contact rates between infected and uninfected individuals, and transmission probabilities that allow for varying infectivity across different categories of active disease. Upon infection, individuals progress either directly to active disease or to latent infection. Individuals with latent infection may subsequently progress to active TB or be superinfected by a different TB strain. Active disease is categorized as smear-positive or smear-negative. Smear-negative cases may progress to smear-positive, and all individuals with active disease may spontaneously self-cure, which returns them to the latent/recovered state. An individual with active disease can be diagnosed as a TB case, according to the characteristics of the diagnostic algorithm, and initiated on treatment (as described in detail below). All individuals in the model are subject to a background mortality rate and to TB-related mortality specific to each active disease state.HIV coinfection can alter the natural history of TB, with HIV-infected individuals having a higher probability of primary progressive TB upon initial infection Model states are further subdivided to account for differences in drug resistance among circulating TB strains, including (1) pan-sensitive TB, (2) isoniazid (INH) mono-resistant TB, (3) RIF mono-resistant TB, (4) TB resistant to both INH and RIF (MDR-TB), and (5) TB resistant to INH and RIF plus one or more second-line drugs (MDR+/XDR-TB). An individual in the susceptible state who is newly infected with TB transitions to the subdivision of the infecting strain. An individual with latent TB who is superinfected by a different strain transitions to the subdivision of the superinfecting strain. Individuals may also develop acquired drug resistance as a result of TB treatment, transitioning to subdivisions with broader resistance profiles.A final subdivision of model states distinguishes treatment-naïve from treatment-experienced individuals, as diagnostic algorithms may dictate different confirmatory tests depending on an individual's history of prior treatment. Individuals enter the model in the treatment-naïve subdivision, and all individuals exiting their first course of TB treatment  transition to the treatment-experienced subdivision.The model is implemented as a series of difference equations with a monthly time step. A full description of model structure and equations is given in The model allows for TB diagnosis and treatment through the national TB DOTS program, or through non-DOTS providers functioning outside the national program. Uptake into treatment programs requires that individuals (1) present to a health facility and are identified as patients with suspected TB, (2) are diagnosed as active cases, and (3) are initiated on regimens determined by their background characteristics and information on drug sensitivity, if available. The model accounts for differences in test performance and information provided by each diagnostic algorithm, and for attrition between diagnosis and treatment, which varies depending on the delay to test results Individuals on TB treatment may successfully complete treatment, fail, default (become lost to follow-up), or die. Those who successfully complete treatment return to the latent/recovered state. A percentage of individuals failing therapy are identified as failures by the treatment program and reinitiate treatment, while all others return to active disease. Individuals who fail or default from treatment may acquire resistance to the drugs they have received. The model allows individuals with pan-sensitive TB to develop mono-INH-resistant TB, mono-RIF-resistant TB, or MDR-TB directly. Individuals with mono-INH- or mono-RIF-resistant TB can develop MDR-TB, and individuals with MDR-TB can develop MDR+/XDR-TB, with the rates of acquiring drug resistance dependent on a patient's TB drug regimen and current drug resistance profile , the latter incorporating disability weights from the Global Burden of Disease study The sensitivity of the model to changes in individual parameters was investigated through traditional one-way sensitivity analyses as well as by computing partial rank correlation coefficients across the set of simulation results produced by the Bayesian uncertainty analysis Finally, we conducted a probabilistic sensitivity analysis to assess the uncertainty around the optimal choice of diagnostic strategy resulting from the joint effects of uncertainty around all input parameters simultaneously, and these results are presented as posterior intervals around key model outcomes and as cost-effectiveness acceptability curves.Based on our model simulations, the positive predictive value for Xpert diagnosis of active TB, at full coverage by 2014, would be 96.9% (95% CI: 93.4–98.7), compared to 88.4% (81.5–93.1) for the status quo algorithm. The negative predictive values for Xpert and the status quo would be 93.9% (88.8–97.2) and 79.3% (67.6–87.9), respectively. We estimate the positive predictive value for the diagnosis of RIF resistance by Xpert to be 67.3% (51.3–82.0) and the negative predictive value 99.9% (99.8–100.0). The relatively low positive predictive value indicates that Xpert is expected to produce a number of false positive diagnoses of RIF resistance, with relatively modest implications for treatment outcomes, as we assume that a subsequent DST is required before individuals receive an MDR-TB diagnosis. Under the Xpert algorithm, 5.8 (95% CI: 3.8–9.2) patients are tested for TB for each active case starting treatment, compared to 7.5 (4.9–12.1) under the status quo, a consequence of improved sensitivity in the Xpert algorithm. The average duration of infectiousness is 9.9 mo (95% CI: 6.7–14.0) under the Xpert algorithm compared to 12.8 mo (9.6–14.0) under the status quo. The benefit of the reduced duration of infectiousness is primarily accrued among individuals with smear-negative TB, for whom the duration of infectiousness is reduced from 19.3 mo (13.8–24.6) under the status quo to 12.1 mo (7.8–18.0) under the Xpert scenario. Results for those with smear-positive disease are comparable under both scenarios. Treatment effectiveness  rises only marginally under the Xpert scenario, with the probability of cure 2.7 (95% CI: 1.6–4.4) percentage points higher than in the status quo scenario. Introduction of Xpert is projected to produce immediate and sustained changes in TB epidemiology . Within Summing the health effects of Xpert introduction over the first 10 y of implementation, this strategy is estimated to prevent 132,000  of the estimated 2.6 million (1.7–4.3 million) new TB cases and 182,000  of the estimated 1.2 million (0.6–2.0 million) TB deaths projected for southern Africa under the status quo.We conducted one-way sensitivity analyses for all model inputs. The cost-effectiveness ratios presented in Further sensitivity analyses  of Xpert-determined TB-negative individuals with HIV-positive or unknown status, as described in recent South African Xpert guidelines Finally, we constructed cost-effectiveness acceptability curves to consider the likelihood that Xpert would be cost-effective under different thresholds for societal willingness to pay for an additional year of healthy life . If sociIn this study, we used a dynamic, calibrated mathematical model of TB to evaluate the potential health and economic consequences associated with scaling up the new Xpert MTB/RIF test in settings with high TB burden, prevalent MDR-TB, and high concurrent prevalence of HIV. Our modeling approach enables quantification of the population-level health effects of alternative diagnostic strategies, projections of impact over the short term and longer time horizons, and assessment of the economic impact and cost-effectiveness of scaling up Xpert compared to continuation of the status quo diagnostic approach.Our results indicate that the introduction of the Xpert MTB/RIF diagnostic has the potential to produce a substantial reduction in TB morbidity and mortality in southern Africa. For individuals with smear-negative TB, the benefits of Xpert implementation would be immediate, leading to the diagnosis and early treatment of many individuals who would be missed by the conventional diagnostic algorithm. Over a longer time frame, the introduction of Xpert would reduce transmission and reduce the reservoir of latent TB infection in the population, but these secondary effects are smaller than might have been anticipated. Even accounting for indirect transmission benefits, we project that TB incidence will remain substantial after three decades of Xpert use, in the absence of other modifications to the status quo TB control strategy. This is due to the large existing pool of latently infected individuals whose progression to active disease would be unmitigated by improved diagnostics, and to the fact that a substantial fraction of the additional cases diagnosed using Xpert will be smear-negative cases, who are less likely to transmit infection than smear-positive cases.Along with the projected health benefits of scaling up Xpert will come significantly increased demands on healthcare resources. The large increase in funding required under the Xpert scenario raises the question of affordability. Although our cost-effectiveness results suggest that the introduction of Xpert represents good value for money according to typical international benchmarks, it does not automatically follow that TB program budgets will be able to absorb these changes. Whereas current debate about the costs of Xpert roll-out focuses largely on equipment and consumables connected directly to the assay, our results show that the indirect cost consequences associated with improved case-finding overshadow the direct costs of diagnosis. If current guidelines are followed, the adoption of Xpert places three key demands on a health system that are additional to the direct costs of diagnosis: providing first-line TB treatment to the large number of additional pan-sensitive TB cases that will be identified, providing additional HIV treatment to coinfected individuals who will live longer as a result of better TB care, and providing second-line TB treatment to the limited number of individuals diagnosed with drug-resistant TB. While our analysis accounts for all three demands, we recognize that response to each of these demands could be evaluated as a separate policy question. Such analyses are beyond the scope of our present study, but it is nevertheless important to note how the economics of Xpert are dependent on the additional interventions triggered by Xpert introduction—which are sensitive to both epidemiologic context and policy decisions. It is likely that existing resources and infrastructure will be called upon to support the introduction of Xpert and the cascade of complementary services this will trigger, and our findings underscore the concern raised by other commentators regarding the possible pitfalls of introducing Xpert into health systems that are already facing capacity constraints An important observation in this study is that substantial increases in HIV treatment costs are expected following introduction of Xpert. This critical insight has a large influence on the cost-effectiveness of Xpert that would be missed in simpler models that do not capture the concurrent dynamics of TB and HIV, and is consistent with other analyses pointing to the importance of HIV and ART access for TB outcomes in this setting Providing treatment to additional cases diagnosed with MDR-TB represents another major component of the incremental costs of Xpert adoption. In our base-case analysis, we assumed that second-line TB treatment would be available for diagnosed MDR-TB cases, which resulted in an estimated 2- to 3-fold increase in the volume of MDR-TB treatment under an Xpert scale-up scenario. If second-line therapy were less available than we assumed, the cost-effectiveness of Xpert would actually improve in the short term (at the cost of faster growth in drug resistance), as the reduction in treatment costs would outweigh the reduction in survival among MDR-TB patients receiving ineffective first-line regimens. Recent empirical cost analyses suggest that MDR-TB care costs may be even higher than estimated in our analysis, with a South African study estimating per-patient costs of over US$17,000 during the inpatient phase of therapy alone, more than 40 times the cost of treating drug-sensitive TB Although the scenarios considered in this analysis assumed that DST would be used prior to the initiation of patients on second-line regimens, the availability of DST remains limited in some settings. Of note, the 67% positive predictive value of the Xpert test for RIF resistance in this setting suggests that a positive result on the Xpert RIF test would be insufficient evidence to initiate individuals on second-line regimens, and further screening would be necessary. Further, the benefits achieved through better detection and treatment of drug-resistant TB would be offset by increases in the number of cases developing resistance, resulting from Xpert's better case detection and the resulting increase in treatment volume. Consequently, the percentage of all TB cases with MDR-TB after 10 and 20 y is projected to be higher under the Xpert scenario, although this result is not statistically significant, and—given the overall reduction in TB prevalence produced by Xpert—the absolute number of MDR-TB cases would be lower than under the status quo.A recent modeling study on Xpert introduction in three countries Our analysis has several limitations. The application of any mathematical model of TB is inevitably limited by uncertainty regarding the true values of epidemiologic and programmatic parameters. Our approach aims to reduce this parameter uncertainty through calibration, and to provide a valid quantitative expression of what parameter uncertainty remains based on Bayesian statistical inference; however, the uncertainty associated with model structure is impossible to quantify without building and assessing the whole range of possible model structures that might be adopted. For example, the results of this analysis would be different if the interdependency of TB and HIV epidemics were not considered, or if the indirect effect of Xpert on TB transmission were not captured. It will therefore be important to undertake continued empirical research evaluating the impact of Xpert as it is rolled out in practice, with the information generated by these evaluation efforts used to progressively refine the mathematical models used to estimate long-term intervention effects.In the results reported here, we constrained estimates on costs and health outcomes to account only for those that would accrue during either the first 10 y or the first 20 y following introduction of Xpert. While the choice of a limited time horizon acknowledges our increasing uncertainty about the distant future and reflects the immediacy of policy decisions, it also makes our results somewhat conservative. This is particularly true for the 10-y results, which truncate the full streams of future benefits that will be enjoyed by those patients who avert TB mortality or infection during the 10-y analysis period. Likewise, we observe that cost-effectiveness ratios are more attractive over the 20-y horizon than the 10-y horizon, reflecting the compounding benefits of interrupting transmission dynamics through better diagnosis and treatment. Moreover, the restriction of our study to adult populations will underestimate the total burden of disease that might be averted, with Xpert adoption likely to reduce pediatric TB through reduced exposure to actively infected adults as well as the direct application of the test for pediatric diagnosis Finally, we note that the results of the present analysis emphasize the importance of interactions between TB and HIV epidemiology in settings where both are highly prevalent, but we caution against generalizing these results to regions where HIV rates are meaningfully different from those in southern Africa. Additional analyses are urgently needed to assess the consequences of introducing Xpert elsewhere, particularly regions of low HIV prevalence or with different TB drug resistance patterns. Similarly, this study focused on the relative benefits of the status quo algorithm and the Xpert algorithm suggested by WHO for diagnosis of patients with suspected TB in settings with high HIV burden. While this is an important comparison to make, there is abundant scope for considering a wide array of alternatives, for example, considering different potential roles for sputum smear microscopy or chest X-ray within diagnostic algorithms designed around Xpert, or use of Xpert for different purposes, such as prior to provision of INH preventive therapy for individuals with HIV, or as part of active case-finding efforts Figure S1Status quo and Xpert diagnostic algorithms.(PDF)Click here for additional data file.Figure S2Incremental difference in epidemiologic outcomes between Xpert and status quo scenarios, 2012–2032.(PDF)Click here for additional data file.Figure S3Partial rank correlation coefficients for ten parameters with greatest influence on the cost-effectiveness of Xpert compared to status quo, South Africa, 10-y time horizon.(PDF)Click here for additional data file.Figure S4Three-way sensitivity analyses showing effects of changes in culture and DST coverage on major study outcomes, by country. (A) Botswana; (B) Lesotho; (C) Namibia; (D) South Africa; (E) Swaziland. Costs, DALYs, and ICERs assessed over a 10-y analytic horizon with a US$30 Xpert unit cost. All other parameters held at their mean posterior values.(PDF)Click here for additional data file.Figure S5Cost-effectiveness acceptability curves showing probability that Xpert strategy is cost-effective as a function of willingness to pay for health benefits.(PDF)Click here for additional data file.Text S1Technical appendix. Figures S1, S2, S3, S4, S5 are available as individual files, but are also included here for ease of access.(PDF)Click here for additional data file.


Both injury and aging of the central nervous system reportedly produce profound changes in gene expression. Therefore, aging may interfere with the success of therapeutic interventions which were tailored for young patients. Using genome-scale transcriptional profiling, we identified distinct age-dependent expression profiles in rat sensorimotor cortex during acute, subacute and chronic phases of spinal cord injury (SCI). Aging affects the cortical transcriptomes triggered by transection of the corticospinal tract as there was only a small overlap between the significantly lesion-regulated genes in both age groups. Over-representation analysis of the lesion-regulated genes revealed that, in addition to biological processes in common, such as lipid metabolism, others, such as activation of complement cascade, were specific for aged animals. When a recently developed treatment to suppress fibrotic scarring (anti-scarring treatment AST) was applied to the injured spinal cord of aged (22 months) and young (2 months) rats, we found that the cortical gene expression in old rats was modulated to resemble regeneration-associated profiles of young animals including the up-regulation of known repair promoting growth and transcription factors at 35 dpo. In combination with recent immunohistochemical findings demonstrating regenerative axon growth upon AST in aged animals, the present investigation on the level of gene expression strongly supports the feasibility of a successful AST therapy in elderly patients. There is a growing incidence of spinal cord injury (SCI) among older individuals. The percentage of patients older than 60 years at the time of injury has increased from 4% to 11% since 2000, and the average age has increased from 28.7 years in the 1970's to the current age of 40.7 years Aging has a profound effect on gene expression SCI elicits massive changes in gene expression in the spinal cord In this study, we investigated the extent and nature of the difference between the dynamic cortical gene expression profiles of aged (22-months-old) and young (2-months-old) rats following thoracic corticospinal tract (CST) transection, and whether the AST-induced regeneration program can be activated in aged animals. GeneChip analyses were performed on layers V/VI of the rat sensorimotor cortex at 1, 7 and 35 dpo (days post-operation), which represented acute, subacute and chronic stages of SCI, respectively.All animal experiments were conducted in agreement with national and international guidelines for animal safety and comfort. All of the surgical interventions and pre- and post-surgical animal care were provided in compliance with the German Animal Protection law and approved by the Animal Study and Ethics Committee of the State Office, Environmental and Consumer Protection of Northrhein Westfalia, LANUV NRW (Az: 8.87-50.10.34.09.081). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.2 and N2O at a ratio of 1∶2). Dorsal spinal cord hemisection, which included transection of the corticospinal tract with a Scouten wire knife at thoracic level eight Spinal cord surgery was performed under isoflurane inhalation anaesthesia  and decapitated. Snap frozen left hemispheres were cut (Bregma 0.96 to −1.96) into forty 50-µm-thick coronal cryosections, which were mounted on Superfrost microscopy slides in the cryostat cabinet. Cortical layers V/VI were dissected as previously described Quantile normalization, summarization with the RMA16 algorithm and baseline transformation of the data to medians of all samples, among other analyses, were performed with GeneSpring GX software . No arrays failed the quality-control analysis, and the correlation coefficients between biological replicate arrays were high; the lowest value was 0.95. Clustering analysis included building a global cluster of all 27,342 genes using self-organizing maps (SOM) and condition trees using average-linkage hierarchical clustering.2-transformed fold-change of +/−0.25 and by performing t-tests  with the Benjamini-Hochberg correction. These settings delivered a sufficiently high number of significantly regulated genes for further evaluation using pathway analysis tools. Classification according to gene ontology was performed using the online tool DAVID The total dataset was analyzed with a 3-way ANOVA . Comparisons between differentially expressed lesion vs. sham and between AST vs. lesion genes in young and aged animals were made by filtering for the log5′-gtccgagaggcttgcataactt-3′, 3′-agaatgccaacgtatacacatcgt-5′; Itgb7, 5′-agtgccctccaagcttaacca-3′, 3′- acagtccgtgggaagtcgata-5′; Hbb, 5′-catggcaagaaggtgataaacg-3′, 3′-tcacttgaggtgacactgttcgac-5′; C1qb, 5′-ttctcaccttctgcgactatgc-3′, 3′- agaacttcgacctcgtccttc-5′. qRT-PCR was performed using SYBR green chemistry (AppliedBiosystems) and relative changes in gene expression were determined using the ΔΔCt method. Ornithine decarboxylase1 (odc1) served as reference gene. Expression levels for each sample were normalized to the corresponding sham young control group, and were calculated relative to reference gene expression.Quantitative real-time RT-PCR (qRT-PCR) was used to measure the expression levels of klf7, intb7, c1qb and hbb mRNA in young animals at 35 dpo. Amplification primers for qRT-PCR analysis of the transcripts of were designed by using PrimerExpress2.0 Software  and subsequently tested for efficiency and specificity. The primer sequences were: Klf7, When the gene expression profiles in cortical layer V/VI of both young (2 months old) and aged (22 months old) groups of spinal injured rats were compared to the respective sham animals , we founFurther, we analyzed the biological processes exerted by significantly lesion-regulated genes in aged and young animals using three different tools: over-representation analysis of biological processes by DAVID and Ingenuity Pathway analysis (IPA) and gene set enrichment analysis by ErmineJ with receiver-operator curves (ROC) and correlation resampling (COR) methods. COR method does not require a list of significantly regulated genes but analyzes expression of all genes to rank the representative biological processes. ROC takes into account in which order the genes appear in the significance list (without cut-off). Despite of the small overlap among the significant lesion-regulated genes in aged and young animals , the anaMultifactorial analysis  revealed that depending on the time point, age or treatment 7470, 2617 or 1274 genes from a total of 21,798 probe sets were significantly regulated, respectively . The majA substantial number of genes  were differentially expressed in an age-specific manner when the genes showing treatment- and time point-specific regulation were pooled. We wanted to know, which biological processes may regulate age-specific genes. Biological processes, such as axonogenesis/axon guidance, myelination, synaptic transmission, cell adhesion and activation of immune responses, were enriched in this age-specific gene group. Although not regulated by injury, these functional gene groups are very interesting in the context of SCI as they might render the aged CNS more vulnerable to injury. For example, the expression of necdin, neurofascin and plasticity related gene 1 was lower in aged animals, whereas the expression of bone morphogenic factor 7 and protein tyrosine phosphatase non-receptor type 11 was upregulated. Importantly, ANOVA analysis also revealed that there was no interrelation between the age and treatment effects, thereby demonstrating the absence of a converse regulation in aged and young animals during different treatment conditions.Condition trees displaying the relationships between groups of animals were built at each time point because, as appeared from multifactorial analysis, the genes have very strong temporal regulation. . These trees  demonstrAs we found significant effects of the anti-scarring treatment at 35 dpo, we further analysed this time point to identify distinct gene regulation profiles. In the most prominent cluster of co-regulated genes  using quUsing a systematic microarray approach, we demonstrate that the local anti-scarring treatment to the injured spinal cord in aged and young animals appears to elicit a similar gene expression profile at the chronic stage of traumatic SCI.The cortical transcriptomes following SCI were initially largely age-specific, because there was little overlap of significantly SCI-regulated genes between aged and young animals. However, on the pathway level, there were common lesion-induced age-independent gene regulations found in processes such as glycolysis, RNA splicing and proteolysis. On the other hand, several of the biological processes exerted by regulated genes were predominant either in young or aged animals. For example, complement activation was upregulated in aged animals. This particular pathway might be especially unfavorable for regeneration, as it has been shown that the inhibition of complement pathways promotes functional recovery and reduces tissue damage Lesion-regulated genes were enriched for lipid metabolism, which in the context of SCI has thus far only received little attention. Fatty acids play an important role in neurite outgrowth as structural building blocks for extensive membrane biosynthesis. Polyunsaturated fatty acids (PUFA) such as arachidonic acid (AA) and docosahexaenoic acid (DHA), which are taken up by neurons, provide the necessary flexibility and fluidity for membranes http://www.gensat.org/imagenavigator.jsp?imageID=13651) suggesting a function of this cathepsin in SCI that needs further investigation.According to ErmineJ and IPA analyses proteolysis was one of the significant biological processes regulated in young animals at 1 dpo. Cathepsins, cysteine proteases, are a good example of this group of potent proteases that degrade intracellular proteins engulfed by lysosomes, but also extracellular elements such as elastin, fibronectin, laminin and collagens. Cathepsin S was upregulated in young lesioned animals, whereas cathepsin K transcripts were enhanced in aged animals at 1 dpo. Cathepsin S was proposed to play a role in the migration and activation of microglia to protect facial motoneurons from axotomy-induced injury Mitochondria-associated genes which are involved in energy production were enriched in lesioned animals indicating the importance of energy supply for cortical motor neurons (motor cortex layer V) after axotomy. These cells have a higher energy demand than the rest of the cortex It is interesting to note, that at 7 dpo no overlap of regulated genes could be observed between young and aged rats in cortical layer V/VI after SCI . Genes cNeurofascin, a cell surface protein which belongs to the immunoglobulin superfamily, was expressed at lower levels in aged animals than in the young. This protein has been shown to regulate mechanisms of plasticity including neurite outgrowth, the formation of postsynaptic components and the stabilization of neural structures Plasticity related gene 1 (Prg1), neuron-specific membrane-associated lipid phosphate phosphatase, was expressed at lower levels in aged animals. Prg1 is associated with axon growth during development and regenerative sprouting following entorhinal cortex lesion as well as in synaptic plasticity and maintenance of the differentiated state throughout adulthood in vivo via anti-apoptotic mechanisms Some aging-specific alterations in gene expression might even be positive and/or reflect the coping of the CNS with cellular stress at higher age. For example, we found bone morphogenetic protein 7 (BMP7) expressed at higher levels in aged animals. BMP7 has neuroprotective capacity as it can enhance dendritic growth and protect cultured neurons from oxidative stress Anti-scarring treatment, which was locally applied immediately after the lesion, elicited massive changes in gene expression in the cortex at the chronic stage of SCI. As early as 1 and 7 dpo, the variation in gene expression was much less in AST-treated animals relative to lesion-only animals. At the chronic stage (35 dpo) of SCI upon AST the profiles in aged and young animals were more similar to each other than at earlier time points. The profiles included the up-regulation of many lesion-dependent down-regulated genes  to the sham-level or higher. This finding is consistent with recent reports demonstrating that an activated regeneration program underlies the increased axonal regrowth of CST in young animals upon AST Interestingly, the cell adhesion molecule ALCAM was found upregulated in our investigation and also in the study by Li et al. From the present findings we conclude, that age cannot be neglected as a factor in therapeutic responses. As was shown here for SCI and as others have reported for stroke, age-related changes produce distinct transcriptomes. However, despite the initial age-specific differences in gene expression, the local therapeutic intervention in the injured spinal cord by AST activated similar molecular programs in both aged and young animals. Thus AST overruns the age-specific differences in transcriptomic lesion responses. Together with previous immunohistological observations regarding axonal regrowth upon AST in aged animals Figure S1Unsupervised hierarchical clustering of all 72 chips. The underlying colored bars point to different clusters of animals. The red bar to the left indicates the clustering of young and old sham and AST animals at 5 weeks post-operation (wpo). This cluster represents at least half of the sham and AST animals of both ages at 5 wpo . The red bar on the right end represents the clustering of half of the lesioned young and old animals at 5 wpo. Moreover, the clustering results support the age-specific clustering at 1 dpo (green bars) and 7 dpo (blue bars) similar to the clustering of groups as shown in (TIF)Click here for additional data file.


Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively.TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC).We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone.The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'. Prostate cancer is the most common malignancy in men. In Europe each year an estimated number of 2.6 million new cases is diagnosed . The widHowever, acquiring tumor tissue from patients with metastatic prostate cancer often represents a challenge. Due to the propensity of metastatic prostate cancer to spread to bone biopsies can be technically challenging and limit repeated sampling of tumor deposits. As a consequence, considerably less is understood about the later acquired genetic alterations that emerge in the context of the selection pressure of an androgen-deprived milieu .TMPRSS2-ERG fusion in approximately 50%, 8p loss in approximately 30% to 50%, 8q gain in approximately 20% to 40% of cases, and the androgen receptor (AR) amplification in approximately 33% of CRPC cases [Consistent and frequent findings from recent genomic profiling studies in clinical metastatic prostate tumors include the PC cases -10. SevePC cases -14.The difficulties in acquiring tumor tissue can partly be addressed by elaborate procedures such as rapid autopsy programs to obtain high-quality metastatic tissue for analysis . HoweverAnother option represents the analysis of plasma DNA . One appHowever, the costs of the aforementioned plasma sequencing studies necessary for detection of rearrangements were prohibitive for routine clinical implementation ,38. In aThis study was conducted among men with prostate cancer , conducted according to the Declaration of Helsinki, and written informed consent was obtained from all patients and healthy blood donors. Blood from prostate cancer patients and from male controls without malignant disease was obtained from the Department of Urology or the Division of Clinical Oncology, Department of Internal Medicine, at the Medical University of Graz. From prostate cancer patients we obtained a buccal swab in addition. Blood samples from pregnant females and from female controls without malignant disease were collected at the Department of Obstetrics and Gynecology, Medical University of Graz. The blood samples from the pregnant females were taken prior to an invasive prenatal diagnostic procedure.Plasma DNA was prepared using the QIAamp DNA Blood Mini Kit  as previously described . SamplesWe performed CTC enumeration using the automated and FDA approved CellSearch assay. Blood samples (7.5 mL each) were collected into CellSave tubes . The Epithelial Cell Kit (Veridex) was applied for CTC enrichment and enumeration with the CellSearch system as described previously ,43.Array-CGH was carried out using a genome-wide oligonucleotide microarray platform , following the manufacturer's instructions (protocol version 6.0) as described . EvaluatSensitivity of our plasma-Seq approach was determined using serial dilutions of DNA from HT29 cell line  in the background of normal DNA . Since quantification using absorption or fluorescence absorption is often not reliable we used quantitative PCR to determine the amount of amplifiable DNA and normalized the samples to a standard concentration using the Type-it CNV SYBR Green PCR Kits . Dilution samples were then fragmented using the Covaris S220 System  to a maximum of 150-250 bp and 10 ng of each dilution were used for library preparation to simulate plasma DNA condition.Shotgun libraries were prepared using the TruSeq DNA LT Sample preparation Kit  following the manufacturer´s instructions with three exceptions. First, due to limited amounts of plasma DNA samples we used 5-10 ng of input DNA. Second, we omitted the fragmentation step since the size distribution of the plasma DNA samples was analyzed on a Bioanalyzer High Sensitivity Chip  and all samples showed an enrichment of fragments in the range of 160 to 340 bp. Third, for selective amplification of the library fragments that have adapter molecules on both ends we used 20-25 PCR cycles. Four libraries were pooled equimolarily and sequenced on an Illumina MiSeq .The MiSeq instrument was prepared following routine procedures. The run was initiated for 1x150 bases plus 1x25 bases of SBS sequencing, including on-board clustering and paired-end preparation, the sequencing of the respective barcode indices and analysis. On the completion of the run, data were base called and demultiplexed on the instrument . FASTQ format files in Illumina 1.8 format were considered for downstream analysis.We employed a previously published algorithm  to creat2 ratios.Low-coverage whole-genome sequencing reads were mapped to the PAR-masked genome and reads in different windows were counted and normalized by the total amount of reads. We further normalized read counts according to the GC content using LOWESS-statistics. In order to avoid position effects we normalized the sequencing data with GC-normalized read counts of plasma DNA of our healthy controls and calculated logResulting normalized ratios were segmented using circular binary segmentation (CBS)  and GLADIn order to check for the copy-number status of genes previously implicated in prostate-cancer initiation or progression we applied z-score statistics for each region focusing on specific targets (mainly genes) of variable length within the genome. At first we counted high-quality alignments against the PAR-masked hg19 genome within genes for each sample and normalized by expected read counts.Here expected reads are calculated asThen we subtracted the mean ratio of a group of controls and divided it by the standard deviation of that group.In order to establish a genome-wide z-score to detect aberrant genomic content in plasma, we divided the genome into equally-sized regions of 1 Mbp length and calculated z-scores therein.t-distribution with n-1 degrees of freedom. For controls, z-scores were calculated using cross-validation. In brief, z-score calculation of one control is based on means and standard deviation of the remaining controls. This prevents controls from serving as their own controls.Under the condition that all ratios were drawn from the same normal distribution, z-scores are distributed proportionally to Student's The variance of these cross-validated z-scores of controls is slightly higher than the variance of z-scores of tumor patients. Thus ROC performance is underestimated. This was confirmed in the simulation experiment described below.In order to summarize the information about high or low z-score that was observed in many tumor patients squared z-scores were summed up.hclust function of R using Manhattan distance that summed up the distance of each window.Genome-wide z-scores were calculated from S-scores. Other methods of aggregation of z-score information, such as sums of absolute values or PA scores , performin silico, artificial cases and controls were simulated from mean and standard deviations of ratios from 10 healthy controls according to a normal distribution. Simulated tumor cases were obtained through multiplication of the mean by the empirical copy number ratio of 204 prostate cancer cases [In order to validate the diagnostic performance of the genome-wide z-score er cases . Segmenter cases .in-silico dilutions of simulated tumor data. To this end we decreased the tumor signal using the formula below, where λ is the ratio of tumor DNA to normal DNA:To test the specificity of our approach at varying tumor DNA content, we performed We performed ROC analyses of 500 simulated controls and 102 published prostate tumor data and their respective dilutions using the pROC R-package . The proWe enriched 1.3 Mbp of seven plasma DNAs  including exonic sequences of 55 cancer genes and 38 introns of 18 genes, where fusion breakpoints have been described using Sure Select Custom DNA Kit  following the manufacturer's recommendations. Since we had very low amounts of input DNA we increased the number of cycles in the enrichment PCR to 20. Six libraries were pooled equimolarily and sequenced on an Illumina MiSeq .We generated a mean of 7.78 million reads , 150 bp paired-end reads on an Illumina MiSeq . Sequences were aligned using BWA  and duplWe further annotated resulting SNPs by employing annovar  and reduWe set very stringent criteria to reduce false positives according to previously published values : a mutatSince plasma DNA is fragmented the read pair method is not suitable for identification of structural rearrangements  and therAll sequencing raw data were deposited at the European Genome-phenome Archive (EGA) , which iPreviously, we demonstrated that tumor-specific, somatic chromosomal alterations can be detected from plasma of patients with cancer using array-CGH . In orden=2,909 for men; n=2,895 for women) throughout the genome and calculated for each 1-Mbp window the z-score by cross-validating each window against the other control samples from the same sex . We defined a significant change in the regional representation of plasma DNA as >3 SDs from the mean representation of the other healthy controls for the corresponding 1-Mbp window. A mean of 98.5% of the sequenced 1-Mbp windows from the 19 normal plasma samples showed normal representations in plasma   Figure .2 ratios [2 ratios and accordingly also increased z-scores . The main purpose of the genome-wide z-score is to distinguish between aneuploid and euploid plasma samples. The genome-wide z-score from the plasma of male individuals ranged from -1.10 to 2.78 and for female individuals from -0.48 to 2.64. We performed receiver operating characteristic (ROC) analyses of simulated next-generation sequencing data from 102 published prostate cancer data and 500 simulated controls based on the data from our healthy individuals. Using the equivalent of one-quarter MiSeq run, these analyses suggested that using the genome-wide z-score tumor DNA concentrations at levels ≥10% could be detected in the circulation of patients with prostate cancers with a sensitivity of >80% and specificity of >80% Figure .To test these estimates with actual data we fragmented DNA from the colorectal cancer cell line HT29 to sizes of approximately 150-250 bp to reflect the degree of fragmented DNA in plasma and performed serial dilution experiments with the fragmented DNA . We established the copy-number status of this cell line with undiluted, that is, 100%, DNA using both array-CGH and our next-generation sequencing approach  In addition, we sequenced with high coverage (approximately 50x) 55 genes frequently mutated in cancer according to the COSMIC [TMPRSS2-ERG-positive prostate cancers (herein referred to as GB-panel (Gene-Breakpoint panel)). In a further step identified mutations were verified by Sanger sequencing from both plasma DNA and constitutional DNA  to distinguish between somatic and germline mutations. If needed, somatic mutations can then be used to estimate by deep sequencing the fraction of mutated tumor DNA in the plasma.Our analysis of plasma samples from patients with cancer is two-fold Figure : (a) we e COSMIC  and Cance COSMIC  databaseWe then obtained 13 plasma samples from nine patients with metastatic prostate cancer . Accordingly, the genome-wide z-score was elevated for all prostate cancer patients and ranged from 125.14 (CRPC4) to 1155.77 (CSPC2)  to 77.6% (CSPC2))   had high-level gains in a region on chromosome × including the ) Figure .As control we performed array-CGH analyses of all plasma cases as described  in paralTMPRSS2-ERG rearrangement results in a well-defined 3-Mbp interstitial deletion on chromosome 21 [TMPRSS2-ERG-positive from TMPRSS2-ERG-negative prostate cancers.The fusion through deletion osome 21 ,70 and oosome 21 . We testTMPRSS2-ERG location on chromosome 21 in five patients  .Plasma-Seq identified a 3-Mbp deletion at the  reports . We thenWe had the opportunity to perform serial plasma analyses from two patients: CRPC1 and CSPC1. CRPC1 had his primary tumor completely resected in 1999, . Since the primary tumor appeared to be very heterogeneous . At the time of our plasma collections the patient was castration resistant and had stable disease under ongoing ADT and chemotherapy. Plasma-Seq identified again multiple prostate cancer-associated chromosomal alterations, such as 8p loss, gain of 8q regions, the 3-Mbp  Figures  and 5. TTMPRSS2-ERG deletion . Only a biopsy had been taken from the primary tumor to confirm diagnosis. The patient was clinically responding to castration therapy. We observed again a number of copy number changes, many of those characteristic of prostate cancer .AR-locus on chromosome Xq12, which is amplified in approximately 33% of patients with CRPC [AR, whereas AR amplification was present in four of the five CRPC cases. In order to validate the plasma-Seq gene-specific copy number estimates with another approach we selected a subset of samples  for validation of the AR copy-number status with qPCR. In fact, we observed a very close correlation between the plasma-Seq and the qPCR values . Whether such a value may indicate the beginning of ADT resistance remains presently unclear, as sufficient follow-up data were not available.For example, in prostate cancer, one of the most interesting regions is the ith CRPC . As expeAR and NCOA2 amplifications on 8q13.3 in early prostate cancer was reported [NCOA2 amplification along with broad amplifications on chromosome 8q [NCOA2 gene amplifications in five patients , thus, two CRPC and three CSPC cases, which may support the notion that NCOA2 amplifications may occur prior to AR amplification [We also tested our approach for some other genes, which have frequently been implicated in prostate cancer. For example, evidence for cooperation between reported . Howeverosome 8q . Our genfication .PTEN on 10q23.31 occurs in approximately 40% of prostate cancers [PTEN loss in five patients ; that is, in three CRPC and two CSPC cases. The AKT-inactivating phosphatase PHLPP1 on 18q21.33 has recently been identified as a prostate tumor suppressor [TMPRSS2-ERG fusion is associated with a deletion at chromosome 3p14 that includes the FOXP1 gene [TMPRSS2-ERG fusion, confirming the association between these two loci.Loss of  cancers ,74. We oppressor . We founXP1 gene . In factIn summary, our results suggest that gene-specific information can be derived from plasma-Seq, which may facilitate the evaluation of pathways potentially comprised in prostate cancer.This study represents the first whole-genome sequencing analysis from plasma DNA of patients with prostate cancer. Usually the identification of tumor genotypes that inform selection of targeted therapies is performed on the initial diagnostic specimen. However, these may not be readily available or in case of fine needle aspirates not sufficient for molecular analyses, as was the case for our patients who presented with metastatic disease. The only exception in our cohort was CRPC1, who had recurrent disease many years after initial operative treatment. We could demonstrate that the initial primary tumor specimen represented multifocal disease and none of the analyzed sectors was representative of the metastatic clone, which arose 13 years later. Thus, molecular analysis of plasma may provide a non-invasive approach for tumor cell genotyping, which can easily be repeated during the course of therapy.ERG and TMPRSS2 on chromosome 21 [Multiple lines of evidence support the copy number changes observed. First, the observation of known prostate cancer alterations in our dataset indicates successful performance of our assay. Second, our previously published array-based plasma method  was applosome 21 ,70 and cosome 21 . Finally2 ratios, approximately 2 h; calculation of z-scores, 30 min) and the costs (approximately €300) with which the aneuploidy scoring by plasma-Seq can be performed. In contrast, completion of the GB-panel analysis, done at 50x coverage, will normally require at least 7 days  not including verifications of mutations by Sanger sequencing or estimation of the fractional load of tumor fragments by deep sequencing.Tests for sensitivity and specificity of our approach suggested that tumor DNA concentrations at levels ≥10% can be detected with a sensitivity of >80% and specificity of 80%. Furthermore, our simulations and HT29 dilution experiments suggested that the genome-wide z-score detects aneuploidy even at tumor DNA concentrations of only 1%. In general, the resolution of non-invasive tumor genome-wide scans from plasma is limited by the depth of the sequencing and the percentage of tumor fragments in the plasma. Therefore, previously published similar studies ,38 emploAR, however no single gene other than AR had frequent mutations and even common, broadly mutated oncogenes such as PIK3CA, KRAS, and BRAF are not commonly mutated in prostate cancer [A disadvantage of low coverage whole-genome sequencing is that structural inter- and intrachromosomal rearrangements cannot be identified with high confidence. This is because plasma DNA fragments, whose paired-end reads map to different chromosomes or to the same chromosome but at large distances  apart, will likely not be detected in multiple reads. Another disadvantage is the reduced resolution for identification of mutations. However, several large scale whole-exome or whole-genome sequencing studies consistently reported low overall mutation rates even in heavily treated CRPCs ranging from 0.9 to 2.00 mutations per mega base -14. ThesAnother potential short-coming is that we do not know whether the changes observed in the plasma are related to the primary tumor or to any of the metastatic sites. In fact, it is currently unknown whether all tumor cells contribute to the plasma DNA equally and which factors influence the release of tumor DNA into the circulation. Further studies are needed to determine whether changes observed by plasma-Seq represent an average of the DNA alterations from all malignant sites or whether they show characteristic changes of the dominant tumor cell clone at the time of the blood collection.At present we do not know how our plasma DNA signatures perform compared with other emerging candidate markers, for example, CTC analysis . Howeverin vivo, thus providing information that may be useful in guiding therapeutic decisions.Whether these blood copy-number signatures will be true game changers for the management of prostate cancer has to be further evaluated. Drug development for castration-resistant prostate cancer is an area of intensive research and several new agents are currently being tested in phase 3 clinical trials. Interrogation of the genomic signature may reveal whether those targeted therapies are effectively hitting their target Our strategy may contribute to a better definition of the evolution towards a castration-resistant disease and could potentially aid in identifying patients more or less likely respond to AR-targeted therapies. The simplicity and the costs of our test are attractive and might ease the clinical translation. However, the extent to which these signatures contribute independent prognostic or predictive value beyond clinicopathological variables must be explored in more depth.ADT: Androgen deprivation therapy; AR: Androgen receptor; BLAT: Basic Local Alignment Search Tool (BLAST)-like alignment tool; BWA: Burrows-Wheeler Aligner; CGH: Comparative genomic hybridization; CRPC: Castration-resistant prostate cancer; CSPC: Castration-sensitive prostate cancer; CTC: Circulating tumor cell; EBI: European Bioinformatics Institute; EGA: European Genome-Phenome Archive; ESP: Exome Sequencing Project; GATK: Genome Analysis Toolkit; GB-panel: Gene-breakpoint panel; ROC: Receiver-operating characteristic; SD: Standard-deviation.The authors declare that they have no competing interests.EH, PU, JB, TB, MA, and CP carried out the molecular genetic studies, sequencing and deep sequencing, and participated in the sequence alignment. SG, KF, MP, FE, MH, and HS contributed patient material and reviewed the clinical data. FQ, PU, and EH performed the statistical analysis. SM and GH reviewed the histology. SR and KP performed CTC identification and enumeration. EH, PU, JBG, and MRS participated in the design and coordination of the study, project planning, and drafted the manuscript. All authors read and approved the final manuscript.Supplementary tables 1-4.Click here for filePlasma DNA analyses from pregnant women. Plasma DNA analyses from maternal blood with pregnancies with a trisomy 21 fetus (first panel), a trisomy 13 fetus (second panel), a trisomy 18 fetus (third panel), and an euploid fetus (fourth panel) .Click here for fileCopy-number status of the HT29 cell line. The upper panel illustrates the array-CGH profile, the lower panel the profile obtained with our next-generation sequencing approach. Both panels illustrate the copy number profile with undiluted, that is, 100%, DNA. In the array-CGH profile the multicolor bar codes at the top or bottom of the ratio profiles illustrate the results obtained during the iterative calculations with various window sizes, the single green and red bars summarize the regions which were gained or lost based on all calculations (for details see [ails see ). Black Click here for fileArray-CGH evaluations as control for our plasma-Seq approach: Array-CGH profiles of plasma samples CRPC2, CRPC3, CRPC5, CSPC2, and CSPC4. For all array-CGH profiles the multicolor bar codes at the top or bottom of the ratio profiles illustrate the results obtained during the iterative calculations with various window sizes, the single green and red bars summarize the regions which were gained or lost based on all calculations (for details see [ails see ). Black ails see . The arrClick here for fileHistology samples from the primary tumor of patient CRPC1. The six different samples are arranged according to the hierarchical clustering  from Figure Click here for fileAR copy number status with qPCR for plasma samples CRPC1, CRPC2, CRPC5, CSPC1, CSPC1_2, and CSPC2 showing a very close correlation between the plasma-Seq and the qPCR values.Validation of the Click here for file


To determine which agents cause neurologic disease in horses, we conducted reverse transcription PCR on isolates from of a horse with encephalitis and 111 other horses with acute disease. Shuni virus was found in 7 horses, 5 of which had neurologic signs. Testing for lesser known viruses should be considered for horses with unexplained illness. Several mosquito-borne alphaviruses, flaviviruses, and orthobunyaviruses, including West Nile, Rift Valley fever, and chikungunya viruses, with zoonotic potential have emerged from Africa to cause major outbreaks in previously unaffected areas , a member of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu. SHUV-specific primers were designed and used to perform reverse transcription PCRs (RT-PCRs) on specimens from an additional 111 horses with fever and nervous disease that had been screened for the more common pathogens over 18 months. The study was conducted in accordance with the recommendations of the Faculty of Health Sciences Ethics Committee of the University of Pretoria under protocols 129/2006 and H016-09.In January 2009, a crossbreed yearling horse (case SAE 18/09) was found wandering aimlessly in its paddock in the Vaalwater District of Limpopo Province, South Africa. The horse became progressively ataxic and, when recumbent, was referred to the hospital at the Faculty of Veterinary Science, University of Pretoria. When examined, the horse was unaware of its surroundings and paddled constantly (front legs swinging inward in their trajectory). Sedation, including the use of ketamine as a last resort, failed to calm the animal. The yearling experienced several episodes of muscle spasm interspersed with tremors and was euthanized when its condition was deemed terminal. Cytologic examination of a cerebrospinal fluid sample taken at euthanasia found pleocytosis with 98% lymphocytes, suggestive of viral infection. An autopsy was performed, and various specimens were submitted for histopathologic and virologic examination. A cytopathic agent isolated from the brain of the horse could not be identified as one of the common horse pathogens, but electron microscopic examination of negative-stained preparations of culture fluid and resin sections of infected cells showed 80-nm to100-nm particles resembling bunyaviruses .http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the fragment showed that the amplicon was related to members of the Simbu serogroup of the genus Orthobunyavirus, and neighbor-joining phylogenetic analysis indicated high bootstrap support (>91%) for placement of SAE 18/09 within the Shuni, Aino, and Kaikular branches of the serogroup. The isolate shared 95.9% identity with SHUV and 91.1% identity with Aino virus (results not shown).RNA was extracted from infected cultures by using the QIAamp Viral RNA Mini Kit . RT-PCRs were performed by using the Titan One Tube RT-PCR Kit  with published primers Bunya 1 and 2, which amplify a 550-bp fragment of the N gene of the S RNA segment of orthobunyaviruses , which forms part of the Bioedit program (www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenetic trees were generated by using maximum-likelihood estimation in PhyML (http://code.google.com/p/phyml/) with 100 bootstrap replicates. P-distance analyses were carried out for nucleotide and amino acid sequences by using MEGA4 www.megasoftware.net).SHUV-specific amplification was then performed by using SHUV-specific primers designed for the present study, i.e., SHUVS111+ (5′-CGA TAC CGT TAG AGT CTT CTT CC-3′) and SHUVS688- (5′-CGA ATT GGG CAA GGA AAG T-3′). Nested PCRs were performed by using primers SHUVS178+ (5′-CCG AGT GTT GAT CTT ACA TTT GGT-3′) and SHUVS611- (5′-GCT GCA CGG ACA GCA TCT A-3′) and the Expand High FidelityPLUS PCR System  to produce 430-bp amplicons. Sequences were edited by using Sequencher version 4.6  and aligned by using the ClustalW subroutine  of 26 with unexplained fever and 5 (6%) of 86 with nervous disease . In 1 ofMaximum-likelihood analysis of 330-nt fragments of the amplicons with corresponding sequences of representatives of the Simbu serogroup of orthobunyaviruses confirmed that all strains clustered with SHUV virus . The nucCuilicoides spp. midges tested as part of arbovirus surveys and in 1 instance from a febrile child in Nigeria) (–Culex theileri mosquitoes caught near Johannesburg and from cattle and a goat in KwaZulu-Natal Province, South Africa (,,SHUV was first isolated in the 1960s from cattle and sheep in abattoirs (,Over 18 months we identified 7 cases of SHUV infection, 5 of which were associated with neurologic signs. Our findings suggest that the role of SHUV as a pathogen may be underestimated and that it should be investigated routinely as a possible cause of unexplained nervous disease in humans and other animals in Africa. Most cases were identified in the autumn and winter months, which overlap with African horse sickeness, equine encephalosis, and West Nile virus outbreaks in South Africa (The discovery of a co-infection with Middelburg virus in 1 of the horses implies that broad screening for arbovirus infections in unexplained illnesses is warranted, and consideration should be given to inclusion of generic RT-PCRs for alphaviruses, flaviviruses, othobunyaviruses, and vesiculoviruses, in addition to African horse sickness and equine encephalosis viruses in future studies. Moreover, the inclusion of tests for immune response would improve the success rate for establishing diagnoses because viremia is fleeting in most arbovirus infections.


The risk of falling is associated with cognitive dysfunction. Older adults with mild cognitive impairment (MCI) exhibit an accelerated reduction of brain volume, and face an increased risk of falling. The current study examined the relationship between baseline physical performance, baseline gray matter volume and falls during a 12-month follow-up period among community-dwelling older adults with MCI.Forty-two older adults with MCI  underwent structural magnetic resonance imaging and baseline physical performance assessment, including knee-extension strength, one-legged standing time, and walking speed with normal pace. ‘Fallers’ were defined as people who had one or more falls during the 12-month follow-up period.p < .01). One-legged standing time (sec)  was associated with a significantly lower rate of falls during the 12-month follow-up after adjusting for age, sex, body mass index, and history of falling in the past year at baseline. Voxel-based morphometry was used to examine differences in baseline gray matter volume between fallers and non-fallers, revealing that fallers exhibited a significantly greater reduction in the bilateral middle frontal gyrus and superior frontal gyrus.Of the 42 participants, 26.2% (n = 11) experienced at least one fall during the 12-month follow-up period. Fallers exhibited slower walking speed and shorter one-legged standing time compared with non-fallers (both Poor balance predicts falls over 12 months, and baseline lower gray matter densities in the middle frontal gyrus and superior frontal gyrus were associated with falls in older adults with MCI. Maintaining physical function, especially balance, and brain structural changes through many sorts of prevention strategies in the early stage of cognitive decline may contribute to decreasing the risk of falls in older adults with MCI. Falls and fall-related injuries are a common healthcare problem, and represent important causes of morbidity and mortality in older populations. One-third of all community-dwelling adults age 65 years and older experience at least one fall annually . Many diSeveral studies have examined falling in older adults with dementia, such as Alzheimer’s disease ,12. HoweAlthough prospective evidence suggests that WMH are an important risk factor for falls in community-based older populations ,18, it rThe sample for this longitudinal study consisted of 42 community-dwelling older adults with MCI who completed a randomized controlled trial (RCT)  evaluating the effects of multicomponent exercise on cognitive function. The Ethics Committee of the National Center for Geriatrics and Gerontology approved the study protocol. The study design and the primary results of the RCT have been described previously . All parAt baseline, all participants underwent an extensive assessment of measures by licensed and well-trained physical therapists.Isometric knee extension strength was tested twice using a dynamometer  from the dominant leg . Knee extension was measured while the participant was sitting on a chair with a backrest and the knee flexed to 90°. A testing pad was attached to the front lower leg of the participant and strapped to the leg of the chair. The participant was instructed to push the pad with maximal strength. Licensed and well-trained physical therapists confirmed compensatory movement and assessed muscle strength. Participants practiced several times before data collection. Two trials were conducted, and the maximal isometric strength was determined as the peak torque (Nm) in the data analysis.The OLS test is a commonly used balance assessment of postural stability. For the OLS test, we asked participants to look straight ahead at a dot 50 cm in front of them, then to stand on their preferred leg with their eyes open and hands down alongside the trunk. OLS balance was measured as the length of time (0–60 s) participants were able to stand on one leg. The better of the two trials was used for statistical analysis.WS was measured using a 5-m walking test. The participants’ usual WS was measured over an 11-m straight and level path. The time taken (in seconds) to pass the 5-m mark on the path was used as the participant’s score. A 3-m approach was allowed before the starting marker, and an additional 3 m of space was provided after the end marker of the 5-m path to ensure a usual walking pace throughout the task. Participants were instructed to walk the 11-m path at their usual walking pace. The time to complete the 5-m walking test was measured once and was used to calculate walking speed (m/min).Fall frequency during the 12-month follow-up period was measured with two face-to-face interviews at 6 months and 12 months after baseline measurements. A fall was defined as “an unexpected event in which the person comes to rest on the ground, floor, or lower level” . In thishttp://dbm.neuro.uni-jena.de/vbm/), which is incorporated in the SPM8 software (http://www.fil.ion.ucl.ac.uk/spm/), running on MATLAB R2010a (Mathworks). Diffeomorphic Anatomical Registration using Exponentiated Lie algebra (DARTEL) [Magnetic resonance imaging (MRI) was performed using a 1.5-T system . Three-dimensional volumetric acquisition of a T1-weighted gradient-echo sequence was then used to produce a gapless series of thin sagittal sections using a magnetization preparation rapid-acquisition gradient-echo sequence . Tissue segmentation, regulation, registration, and normalization were conducted in the VBM8 toolbox ((DARTEL)  was condt-tests. Chi-square tests for differences in proportions were used to compare differences in sex and history of falling in the past year at baseline between the faller and non-faller groups. To describe variations in different physical performance factors related to falls, multivariate logistic regression analyses were performed to reveal the physical performance factors independently related to falls during the 12-month follow-up after adjusting for age, sex, body mass index (kg/m2), and history of falling in the past year at baseline. We calculated the odds ratios (OR) with 95% confidence intervals (CI). These statistical analyses were calculated using SPSS for Windows version 19.0 .For baseline comparisons, basic characteristics and physical performance tests including knee-extension strength, OLS, and WS were compared between fallers and non-fallers using P < .001 (uncorrected), with an extent threshold of 100 voxels.In the voxel-based morphometry (VBM) analysis, data preprocessing and analysis was performed with the VBM8 toolbox, which is incorporated in the SPM8 software. VBM  was usedp < .01) and slower walking speed (p < .01) compared with non-fallers. In addition, the faller group had a significantly higher rate of fall history at baseline compared with the non-faller group (p < .01). In the multivariate logistic regression, OLS time (sec)  was associated with a significantly lower rate of falls during the 12-month follow-up after adjusting for age, sex, body mass index, and history of falling in the past year at baseline. There was no statistical evidence of associations between falls and knee-extension strength (Nm)  and walking speed (m/min)  (Table The characteristics and physical performance tests at baseline are presented in Table The gray matter density profiles used for examining differences between fallers and non-fallers at baseline are shown in Figure The present study examined whether baseline physical performance and gray matter volume are related to falls during a 12-month follow-up period in community-dwelling older adults with MCI. Our results indicated that older adults with MCI exhibiting poor balance had a greater risk of falls during the 12-month follow-up period, while adjusting for age, sex, body mass index, and history of falling at baseline. In addition, baseline lower gray matter volume in the middle frontal gyrus and superior frontal gyrus was associated with the occurrence of subsequent falls. To our knowledge, this is the first study to examine the association between lower gray matter density and risk of falls in older adults with MCI.Problems with gait and balance have been reported to have the strongest association with falling ,31. SlowUnlike previous investigations, the current study included MRI scanning and a follow-up assessment of falls in community-dwelling older adults with MCI. The results provide the first evidence that lower gray matter volume in the middle and superior frontal gyrus is related to the occurrence of subsequent falls among older adults with MCI. Age-related changes in the brain may contribute to the subtle onset of motor disturbances in older people. Previous brain-imaging studies of older adults have reported that age-related changes in the brain, such as lower global brain volume, WMH, and microbleeds, are associated with clinical measures of poor balance and slow gait -43. The Growing evidence suggests that brain function is associated with physical function, as confirmed by neuroimaging techniques. Structural changes of the brain in older people are reported to be related to physical performance, such as gait dysfunction ,45, postSeveral limitations of the current study should be noted. First, fall experience during the 12-month follow-up period were confirmed with two face-to-face interviews at 6-months and 12-months after baseline, while previous studies have reported that monthly fall diaries and follow-up telephone calls provide more accurate measures of fall frequency ,57. SecoThe current findings indicate that poor balance predicts falls over a 12-month period, and that lower gray matter volume in the middle frontal gyrus and superior frontal gyrus was associated with falls in older adults with MCI. Maintaining physical function, especially balance, and brain structural changes through many sorts of prevention strategies in the early stage of cognitive decline may contribute to decreasing the risk of falls in older adults with MCI.The authors declare that they have no competing interests.HM has made substantial contributions to conception and design, subject recruitment, analysis and interpretation of data, and writing the manuscript. HS has made substantial contributions to conception and design, subject recruitment, interpretation of data, and writing the manuscript. TD has made substantial contributions to subject recruitment, acquisition of data, interpretation of data, and manuscript preparation. HP has made substantial contributions to conception and design, interpretation of data, and writing the manuscript. DY contributed subject recruitment and manuscript preparation. KU and KT contributed subject recruitment and acquisition of data. TLA has been involved in drafting the manuscript or revising it critically for important intellectual content. TS has made substantial contributions to conception and design and writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/102/prepub


Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a central role in the innate immune systems of plants and vertebrates. In plants, NLR proteins function as intracellular receptors that detect pathogen effector proteins directly, or indirectly by recognizing effector-induced modifications to other host proteins. NLR activation triggers a suite of defense responses associated with programed cell death (PCD). The molecular mechanisms underlying NLR activation, and how activation is translated into defense responses, have been particularly challenging to elucidate in plants. Recent reports, however, are beginning to shed some light. It is becoming clear that plant NLR proteins are targeted to diverse sub-cellular locations, likely depending on the locations where the effectors are detected. These reports also indicate that some NLRs re-localize following effector detection, while others do not, and such relocalization may reflect differences in signaling pathways. There have also been recent advances in understanding the structure of plant NLR proteins, with crystal structures now available for the N-terminal domains of two well-studied NLRs, a coiled-coil (CC) domain and a Toll-interleukin Receptor (TIR). Significant improvements in molecular modeling have enabled more informed structure-function studies, illuminating roles of intra- and inter-molecular interactions in NLR activation regulation. Several independent studies also suggest that intracellular trafficking is involved in NLR-mediated resistance. Lastly, progress is being made on identifying transcriptional regulatory complexes activated by NLRs. Current models for how plant NLR proteins are activated and how they induce defenses are discussed, with an emphasis on what remains to be determined. Plants do not have an adaptive immune system like that found in vertebrate animals. Instead, plants depend solely on an innate immune system that bears intriguing similarities to animal innate immune systems, but is likely independently evolved [see review by Jacob et al. ]. Plant Effector triggered immunity is mostly mediated by nucleotide-binding leucine-rich repeat (NLR) proteins. Plant NLR proteins usually contain a C-terminal LRR domain and a central NB-ARC domain  .The NB-AHere, we focus on the advances made in the last 2 years toward understanding how plant NLRs are activated and how signaling is initiated and transduced. We highlight the compartmentalization of plant NLRs, intra-/inter-molecular interactions before and after activation, and structural and genetic insights into NLR downstream signaling.Arabidopsis TNL, RPS4, is required for RPS4-mediated resistance in the presence of its cognate effector AvrRps4 , localizes to both the nucleus and cytosol , 18. SeqArabidopsis require PM localization to function  but not an unrecognized derivative AVR3a(EM)  . MoreoveArabidopsis RPP1 LRR domain and the oomycete ATR1 effector have been demonstrated using co-immunoprecipitation analysis  containing 4] provides additional insights into the physical interactions between the NB-ARC domain and the LRR domain that function to inhibit NLR auto-activation . NLRC4 dAn open question in plant NLR studies is which domain(s) of plant NLRs is/are directly responsible for downstream signaling. In mammalian cells, the NLR activation usually results in the recruitment and activation of pro-caspase-1 through homotypic interaction with the N-terminal CARD . This leArabidopsis RPS5 proteins. In the case of Rx, overexpression of the NB subdomain alone was found to be sufficient for inducing cell death, suggesting that this domain plays a roll in engaging downstream signaling components  by Arabidopsis RPS2. In this example, RPS2 is activated by the degradation of RIN4 following cleavage by AvrRpt2 . NB-ARC domains form a deep nucleotide-binding pocket. In the “off” state, the NB-ARC domain adopts a “closed” structure where ADP is preferentially bound and coordinates intramolecular interactions to stabilize this structure , 50, 51.Apaf-1 and CED-4 are known to form oligomers through NB-ARC-mediated interaction , 56. TheArabidopsis RPS5 self-associates prior to activation  from the fungus Colletotrichum higginsianum. It is not yet known whether RRS1 and RPS4 physically associate, but both at least partially localize to the nucleus in the presence of effectors  family, which contributes to defense responses activated by Arabidopsis RPS2 (a CNL), RPP2, and RPP4 (TNLs)  . There accessful . In addilic acid . TogetheAquilegia caerulea and the dicotyledonous order Lamiales, as well as from the grass family (Poaceae), suggesting that the TNL family may be dependent on ADR1 family members for activating resistance  . Consistsistance . Since gfunction .Arabidopsis, which is required for resistance mediated by TNLs but not CNLs  in not CNLs , 71. EDSand SNC1 . These cbidopsis , 74. Furbidopsis . This stbidopsis . This fiSaccharomyces cerevisiae and Caenorhabditis elegans , was shown to interact with the WRKY45 transcription factor in the nucleus  interacts with SNC1, a TNL protein, and knocking out TPR1 compromises immunity mediated by SNC1 . Signifiesponses . Most re nucleus . This in2+, as cell death induced by RPS2 and RPM1 can be eliminated by the calcium channel blocker LaCl3 ; Nicotiana benthamiana and Solanum lycopersicum (tomato) (Solanaceae), and Cucumis sativus  (N. benthamiana and/or N. tabacum (tobacco) by transient expression of several different TNL and CNL proteins from diverse plant species, including Arabidopsis, flax, and barley  and Arabidopsis (a dicot and member of the mustard family).Although it is clear that different plant NLRs employ different signaling pathways, these signaling pathways appear to be broadly conserved across plant species, as evidenced by functional transfer of NLRs between species. For example, the itaceae) . In addid barley , 86–88. . hordei . InteresP. syringae  protein, has recently been shown to be required for RPS2- and RPS5-mediated resistance, but not for HR cell death  and N-ethylmaleimide-sensitive factor vesicle fusing ATPase, and soluble N-ethylmaleimide-sensitive factor adaptor protein 33 (SNAP33). RPS2 has also been shown to upregulate production of miR393b, a microRNA that targets at least three different genes likely involved in endomembrane trafficking  is the dual role of the LRR domain in keeping the NLR in the “off state” in the absence of pathogen, and promoting the switch to the “on state” in the presence of pathogen (via binding to effectors or effector-modified host proteins). We have very little insight, however, into how the LRR domain accomplishes either of these roles. The recent structure of mouse NLRC4 indicates that in animal NLRs, the LRR folds back across NB-ARC domain with the N-terminal portion of the LRR making multiple contacts with the NB and ARC3 domains, effectively placing a lid over the ADP/ATP binding pocket. The absence of the ARC3 domain in plant NLRs makes it a certainty that the contacts between the LRR and NB-ARC will differ from NLRC4, but based on the locations of autoactivating mutations and on deletion analyses, the general structure is likely to be similar, with just the N-terminal portion of the LRR (approximately four repeats) required to form the lid . The C-tA second holy grail is identifying the immediate downstream interacting proteins for PM associated CNLs. Although several transcription factors have now been identified that interact with nuclear-localized TNLs and CNLs, we still lack good candidates for downstream signaling proteins for NLRs that signal from the PM such as RPM1, RPS2, and RPS5. Proteomic approaches hold some promise for shedding light on this unknown , 97, butP. syringae, the HR is still a hallmark of NLR activation. One study has implicated fusion of the vacuolar and PM as the primary event leading to cell death (A third holy grail is a better understanding of how cell death is executed during NLR-mediated HR. Although cell death is apparently not required for resistance, at least to ll death . If thisIn summary, although much has been learned in the nearly 20 years since the first NLR was identified, major questions remain. Providing answers to these questions will require both creativity and improvements in technology, but will no doubt come.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.