Through major efforts to reduce costs and expand access to antiretroviral therapy worldwide, widespread delivery of effective treatment to people living with HIV/AIDS is now conceivable even in severely resource-constrained settings. However, the potential epidemiologic impact of treatment in the context of a broader strategy for HIV/AIDS control has not yet been examined. In this paper, we quantify the opportunities and potential risks of large-scale treatment roll-out.We used an epidemiologic model of HIV/AIDS, calibrated to sub-Saharan Africa, to investigate a range of possible positive and negative health outcomes under alternative scenarios that reflect varying implementation of prevention and treatment. In baseline projections, reflecting “business as usual,” the numbers of new infections and AIDS deaths are expected to continue rising. In two scenarios representing treatment-centered strategies, with different assumptions about the impact of treatment on transmissibility and behavior, the change in the total number of new infections through 2020 ranges from a 10% increase to a 6% reduction, while the number of AIDS deaths through 2020 declines by 9% to 13%. A prevention-centered strategy provides greater reductions in incidence (36%) and mortality reductions similar to those of the treatment-centered scenarios by 2020, but more modest mortality benefits over the next 5 to 10 years. If treatment enhances prevention in a combined response, the expected benefits are substantial—29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response are considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%). Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response scenario with positive treatment–prevention synergies.These analyses demonstrate the importance of integrating expanded care activities with prevention activities if there are to be long-term reductions in the number of new HIV infections and significant declines in AIDS mortality. Treatment can enable more effective prevention, and prevention makes treatment affordable. Sustained progress in the global fight against HIV/AIDS will be attained only through a comprehensive response. Combining the two approaches of prevention and treatment of HIV/AIDS could avert more than 29 million new HIV infections by 2020 In June 2001, heads of state and government convened a United Nations Special Session on HIV/AIDS and adopted unanimously the “Declaration of Commitment on HIV/AIDS” . Today, The theme of the 15th International AIDS Conference in Bangkok last summer was timely and relevant. “Access for All” calls for extending to all of those in need both sufficient resources and a set of proven interventions to prevent new infections and save lives through effective treatment. Recent developments in HIV treatment, with simple combination therapies priced at less than US$150 per year—unthinkable just a short time ago—were a major driver of discussions during the conference. Widespread access to effective antiretroviral therapy (ART) for people living with HIV/AIDS is now conceivable even in countries with severely limited resources.The World Health Organization and its partners in the Joint United Nations Programme on HIV/AIDS have defined an ambitious “3 by 5” target of 3 million people on ART—half of those in most urgent need—by the end of 2005. The potential epidemiologic impact of large-scale roll-out of treatment programs, however, remains uncertain. Experience to date is limited, and comes mostly from Western countries and Brazil. While declines in AIDS mortality in the industrialized world have been impressive ,7,8, man.In our previous analysis of the potential benefits of a comprehensive package of preventive interventions , we noteBaseline projections of HIV epidemics in sub-Saharan Africa have been developed by the Joint United Nations Programme on HIV/AIDS and the World Health Organization based on the most current data available, and in collaboration with epidemiologic experts and analysts within the countries assessed . These “To simulate the effects of prevention and treatment on HIV/AIDS incidence, prevalence, and mortality, we first adapted the analytic approach used in the previously described Goals model  to allowThe model includes underlying regional demography, acquisition of HIV and other sexually transmitted infections (STIs), progression from HIV to AIDS, and progression from AIDS to death. Annual risks of HIV infection in each risk group depend on the number of partnerships, the number of sex acts per partnership, HIV prevalence among partners and condom use. These risks are magnified by the presence of other STIs  and alsoThe regional models were calibrated as follows: first, plausible ranges were specified for model parameters governing sexual behavior and biological factors  based on review of published studies and survey results; second, multiple simulations were undertaken by sampling values from each of the ranges and recalculating the model for each set of sampled parameter values; third, model fit was assessed by comparing modeled prevalence for adult males and females separately to baseline projections through 2020; and fourth, the best-fitting parameter set in each regional model was selected for the purpose of scenario analysis see .Potential impacts of prevention efforts at a given coverage level were based on previously published estimates  for a coWe examined a range of alternative scenarios based on various levels and effectiveness of prevention interventions, with and without successful attainment of the 3 by 5 treatment target for sub-Saharan Africa:Risk behaviors are maintained at current levels, and no treatment scale-up occurs. This is simply the baseline scenario that produces a relatively stable prevalence rate over the duration of the projection, with the number of people living with HIV and the number of new infections rising slowly over time because of population growth.In two alternative scenarios, the 3 by 5 target of 50% coverage of those in need of treatment by the end of 2005 is attained, and scale-up continues to reach 80% ART coverage of those in need by 2010, maintained at 80% thereafter. In an “optimal ART effects” scenario, we assumed that treatment reduces transmissibility by 99%, and that those under treatment have 50% lower annual partnership numbers and two times higher condom use than other adults. With a response that focuses primarily on treatment, it is assumed that behavior in the general community of infected and uninfected adults is unchanged from the baseline. In an alternative “mixed ART effects” scenario, less optimistic assumptions were made: that treatment reduces transmissibility only to the same levels as in asymptomatic infected individuals (two-thirds reduction from no treatment), and that behavior in treated patients is the same as in other adults. To capture the possibility of behavioral disinhibition in response to treatment availability, we assumed that condom use declines by 10% in both treated patients and the general community, with other behaviors unchanged. The potential for disinhibition is suggested primarily by experience in some developed countries, where condom use increased dramatically in the populations at highest risk prior to the introduction of ART but then declined; the likelihood and magnitude of reductions in condom use in sub-Saharan Africa, where such prevention-induced changes generally are much less prominent today, might be questioned. We therefore considered in sensitivity analyses a variant of this scenario that excludes disinhibition but preserves all other assumptions.In the absence of wide availability of treatment, reflecting weaker political and social support for HIV control efforts, we modeled a scenario in which the comprehensive prevention package described previously  has onlyWe examined two scenarios combining treatment and prevention efforts, reflecting either optimistic or pessimistic possibilities. In the optimistic scenario, treatment strengthens prevention efforts. ART coverage is the same as in the two treatment-centered scenarios, with optimal assumptions about treatment impact on transmissibility and patient behavior. It is assumed that widespread availability of treatment enables the full impact of prevention efforts to be attained as described by Stover et al. . In a moAdditional scenarios could include pessimistic assumptions about limited ART scale-up levels and timing, emergence of large-scale drug resistance resulting from low adherence, or other possible unintended outcomes of wider treatment. Certainly, large-scale treatment efforts will demand close monitoring of adverse effects. However, experience with treatment programs in developing countries has been encouraging thus far, with reported adherence levels that are at least as high as those in developed countries ,19.In the baseline projections for sub-Saharan Africa, the annual number of new adult HIV infections rises from 2.4 to 3.7 million between 2004 and 2020, and adult AIDS mortality rises from 1.8 to 2.6 million . If scalWith less optimistic assumptions (treatment-centered response/mixed ART effects), the number of new infections rises, to 4.3 million per year by 2020 (a 14% increase); mortality trends are similar to the optimistic scenario in the short term, but worse in the long term, even compared to the baseline. Excluding the assumption of reduced condom use through disinhibition from the treatment-centered/mixed effects scenario has minimal effect on the results, lowering the number of new infections in 2020 by only 2% compared to the scenario that includes disinhibition.A prevention-centered response would have greater impact on the number of new infections, lowering annual incidence by more than half by 2020. The long-term mortality trend is more favorable in the prevention-centered scenario than in the treatment-centered scenario because of reduced incidence, but prevention would produce negligible mortality benefits in the near- and mid-term future in comparison to strategies that include ART. Alternative assumptions regarding overall effectiveness in a prevention-centered response produce results that scale as expected, with reductions in annual incidence of 34% to 64% and reductions in annual mortality of 20% to 42% by 2020.If treatment and effective prevention are scaled up jointly in a combined response, the benefits in terms of both infections and deaths averted could be substantially higher. In an optimistic scenario in which treatment programs support expanded prevention, the annual number of new infections would be 74% lower and annual mortality would be 47% lower by 2020, compared to baseline. It is worth noting that the long-term decline in AIDS deaths is driven more by prevention of new infections than by direct survivorship benefits from ART. In a pessimistic scenario in which a more narrow treatment focus limits effective prevention, the overall benefits are much more modest, with 26% and 16% reductions, respectively, in new infections and mortality by 2020 compared to the baseline.Prevalence rises by 7% in the optimal and by 27% in the mixed treatment-centered scenarios by 2020, as longer survival for treated patients offsets reductions in new infections through reduced transmissibility (and risk reductions among treated patients in the more optimistic scenario) . In scenThe total number of infections averted through a combined response would be 29 million over the period 2004 to 2020 if treatment enhances prevention, a benefit that is ten times greater than that of a strategy which focuses on treatment only, even with optimal assumptions, and 51% greater than that of a strategy which focuses on (less effective) prevention alone . If a trCombining treatment with prevention efforts will reduce the resource needs for treatment substantially in the long term . In the In this paper, we have examined the potential epidemiologic impact of global HIV/AIDS control efforts under a range of alternative scenarios reflecting varying implementation of strategies for prevention and treatment. Although we focus in particular on population health outcomes and epidemiologic trends, we recognize that there are numerous other social, economic, and individual health effects of interventions including ART that are beyond the scope of this analysis. We also restrict our focus in this paper to sub-Saharan Africa, where the overwhelming majority of people living with and dying from HIV/AIDS reside; however, our findings have broader applicability and more general implications in the worldwide fight against HIV/AIDS, which we highlight here.Effective prevention requires more than having sufficient funds to offer information and services. It also requires an environment that encourages people to internalize messages about risky behavior and to adopt actual behavior change, and allows people to utilize services such as testing and counseling without fear of stigma or discrimination. Stoneburner and Low-Beer have argued that the supportive social and political environment in Uganda allowed people to discuss AIDS with family members and close friends, which led to greater behavior change than in Kenya or Zambia where most people received information from mass media only . People Involving communities and family members in the delivery of treatment—for example, as treatment monitors—offers unique entry points for effective prevention activities and a lever for population-wide behavior change. Experience with community roll-out of treatment programs has shown, for example, that uptake of voluntary counseling and testing increased by 300% in one year of roll-out in Haiti, and by a factor of 12 in Khayelitsha, South Africa, after treatment introduction ,22. A stDuring most of the past 15 years, efforts to address the AIDS epidemic in sub-Saharan Africa have focused on prevention. There have been successes in some countries, but overall these efforts have not achieved their goals. The advent of vastly expanding treatment programs in the coming years, if opportunities to capitalize on broadened political support and community mobilization can be seized, offers the potential to enhance prevention effectiveness and avert many new infections and deaths.While prevention programs are unlikely to achieve full impact in the absence of treatment, so too is the impact of treatment programs reduced if vigorous prevention efforts are absent. Without effective prevention, the number of people requiring care and treatment will grow each year. As more and more people are kept alive with ART the treatment burden will become enormous unless effective prevention reduces the number of people becoming newly infected.Without effective prevention programs, we project that the number of people receiving treatment will grow to 6.3 million by 2010 and up to 9.2 million by 2020 in Africa alone to achieve 80% coverage of those in urgent need. Meeting this need would require a tremendous increase in financing, human capacity and infrastructure that might not be attainable.If effective prevention programs are combined with treatment programs, the same level of 80% ART coverage would be achieved by treating 5.8 million in 2010 and 4.2 million in 2020. In other words, the same goal could be attained at a far lower treatment cost and with a much greater chance of sustainability.Over the long term, it is effective prevention that will reduce the burden of illness due to AIDS and the number of people in need of ART. The lessons learned in the industrialized world have to be taken on board. The availability of treatment and the shift in focus away from very effective prevention programs has led to increases in unsafe sexual behavior, STIs, and HIV transmission in some settings ,10,11. TCountries in sub-Saharan Africa are faced with the most devastating epidemic of our times. We now have the unique opportunity to derive the maximum impact from available resources. The results from our analyses show how potential synergies between prevention and treatment could be translated into considerable health benefits at the population level. But synergies do not mean simply that prevention and treatment are pursued in parallel. When whole communities become involved in the scale-up of treatment access—as will be necessary to achieve the ambitious treatment targets defined by the 3 by 5 campaign—crucial opportunities can be created for increasing their involvement in prevention activities. Only if interactions with patients, family, and community members occasioned by the provision of treatment are also used to reinforce prevention, and only if prevention workers have an opportunity to refer those in need to care and treatment, will we move at last from slogans to impact.Figure S1(80 KB EPS).Click here for additional data file.Protocol S1(172 KB PDF).Click here for additional data file.Infections from HIV continue to increase, especially in sub-Saharan Africa. The World Health Organization has a plan to get more than 3 million people on treatment by 2005 (the “3 by 5” initiative); however, the overall effect of this plan on the population's health is uncertain, and will depend on the balance between treatment and prevention efforts.They tried to predict the number of new infections and deaths each year in sub-Saharan Africa from now until 2020 depending on whether control efforts focused on prevention, treatment, or both. What they found was that by far the most effective way of decreasing new infections and deaths was to combine the two approaches, and that by doing so more than 29 million new infections and 10 million deaths might be prevented compared with continuing at current levels of prevention and care.Despite the huge amounts of money directed at HIV/AIDS, because the problem is so vast, the resources are not enough. Hence it is important to target these resources effectively. Policy makers around the world could use information like this to decide where best to direct attention and funding to combat HIV/AIDS.http://www.unaids.org/wad2004/report.htmlJoint United Nations Programme on HIV/AIDS, AIDS epidemic update, December 2004: http://www.who.int/3by5/World Heath Organization, 3 by 5 Initiative: http://www.kff.org/hivaids/hivghpwgpackage.cfmGlobal HIV Prevention Working Group:


Vascular endothelial growth factor (VEGF)-C is implicated in lymphangiogenesis, however the exact role of VEGF-C in promoting lymphatic spread of cancer cells remains largely unknown.The expression of VEGF-C was immunohistochemically determined in 97 endoscopic biopsy specimens from 46 patients with submucosal gastric carcinoma (SGC). Nodal metastases including micrometastasis and isolated tumor cells (ITC) were evaluated by immunohistochemical staining for cytokeratin in 1650 lymph nodes, and tumor cells in these metastatic nodes were also examined for VEGF-C expression.In biopsy samples, VEGF-C was positively detected in 21 (47%) patients. Metastases were identified in 46 (2.8%) nodes from 15 (33%) patients. Metastases were detected in 39 nodes by hematoxylin-eosin (H&E) staining and in additional 7 nodes as ITC by immunohistochemical staining. The rate of lymph node metastases was significantly correlated with VEGF-C expression in biopsy samples (p < 0.05). The positive and negative predictive values of VEGF-C in biopsy specimens for nodal metastasis were 44 %(10/21) and 80% (20/25), respectively. Among the 46 metastatic nodes, tumor cells in 29 (63%) nodes positive patients expressed VEGF-C, whereas those in 17 (37%) nodes did not. VEGF-C expression was high in macronodular foci in medullary areas, whereas more than half of ITC or micrometastasis located in peripheral sinus lacked the expression of VEGF-C.Despite the significant correlation, immunodetcetion of VEGF-C in endoscopic biopsy specimens could not accurately predict the nodal status, and thus cannot be applied for the decision of the treatment for SGC. VEGF-C may not be essential for lymphatic transport, but rather important to develop the macronodular lesion in metastatic nodes. The incidence of early gastric carcinoma defined as being confined to the mucosa or submucosal layer has increased. In Japan, endoscopic mucosal resection (EMR) is now generally accepted for intramucosal cancers that are associated with a minimal risk of regional lymph node (LN) metastasis -4. For sVECF-C is known to bind VEGFR-3, which is specifically expressed on lymphatic vessels and stimulates lymphangiogenesis ,10. ManyRecently, small metastatic lesions have been detected genetically or immunohistochemically in various cancers, even though they were diagnosed as negative by conventional examination with H&E staining. Such lesions are designated as micrometastasis or isolated tumor cells (ITC). The biological and clinical significance of such minute nodal invasion of carcinoma cells is still controversial -38. In tForty-six patients with SGC diagnosed and treated by curative gastrectomy with standard lymph node dissection at the First Department of Surgery, Tokyo University Hospital, Tokyo, between 1994 and 2002 were included in this study. These patients were examined endoscopically prior to surgery; several pieces of tissue specimens were then sampled with routine biopsy forceps from various portions of the tumor. Formalin-fixed and paraffin-embedded sections of 97 biopsy specimens and 1650 dissected lymph nodes derived from these 46 patients were evaluated in this study. Additionally, all the resected primary tumors were histologically examined with H&E staining according to the Japanese Classification of Gastric Carcinoma . Tumors 2O2 and 0.05% diaminobenzidine tetrahydrochloride for 3 minute. Light counterstaining with Mayer's hematoxylin was performed. The 46 lymph nodes that showed the presence of carcinoma were also evaluated for the expression of VEGF-C with the same immunostaining method.The expression of VEGF-C was investigated with immunohistochemical staining using affinity purified goat polyclonal antibodies against VEGF-C . Sections (3-μm thick) of biopsy samples were deparaffinized in xylene, hydrated through a graded series of ethanol, and then immersed in 3% hydrogen peroxide in 100% methanol for 30 min to inhibit endogenous peroxidase activity. To activate the antigens, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 minutes. After being rinsed in phosphate-buffered saline (PBS), the sections were incubated with normal rabbit serum for 10 min, and then incubated overnight at 4°C in humid chambers with the primary antibody to VEGF-C at 1/30 dilution. After three washes with PBS, the sections were incubated with biotinylated rabbit anti-goat immunoglobulin for 20 minute. After washing again with PBS, the slides were treated with peroxidase-conjugated streptavidin for 20 minutes, and developed by immersion in 0.01% HThe dissected lymph nodes were fixed in 10% formalin and embedded in paraffin. From each node, one 3-μm-thick section was prepared for H&E staining, and another three serial 5-μm sections were prepared for immunohistochemical staining with CAM 5.2 , a mouse monoclonal antibody that reacts with human cytokeratin numbers 8 and 18 . The strMetastasis was defined as the presence of tumor cells, whether single or in small clusters detected by H&E or immunohistochemical staining. Metastatic lesions that were more than 2.0 mm in diameter were defined as macrometastasis, while micrometastasis was defined as a tumor deposit larger than 0.2 mm but less than 2.0 mm, and ITC was defined as a tumor deposit less than 0.2 mm in maximum diameter.All statistical calculations were carried out using StatView-J 5.0 statistical software . The relationship between clinical and pathological characteristics of patients and the expression of VEGF-C was examined by Fisher's exact test. Differences with a p value of less than 0.05 were considered to be statistically significant.Metastases were observed in 12 patients (26.1%) and 39 lymph nodes (2.4%) by H&E examination Fig. . Among tTable The biopsy specimens were divided into two categories by the staining pattern of VEGF-C, diffuse or focal staining of carcinoma cells as described previously . When diAmong the 97 biopsy samples from 46 patients, carcinoma cells were contained in only one biopsy sample in 14 patients, and in 2, 3 and 4 biopsy samples in 16, 13 and 3 patients, respectively. In all of the latter cases, carcinoma cells in biopsy samples derived from different places showed exactly the same staining pattern of VEGF-C, and thus VEGF-C-positive and -negative tumors could be clearly distinguished.In biopsy specimens, VEGF-C was positively detected in 21 (46%) cases. As shown in Table Nodal metastasis was detected in 10 (48%) of 21 VEGF-C-positive tumors, and the rate was significantly higher than that in VEGF-C-negative tumors as evaluated by biopsy specimens  (p = 0.047). However, the positive and negative predictive values of VEGF-C in biopsy for nodal status were 44% (10/21) and 75% (20/25), respectively, and 5 (20%) of 25 VEGF-C-negative tumors were accompanied with lymph node metastasis.Table Among 36 nodes from 10 patients who were determined as VEGF-C-positive in biopsy samples, tumor cells located in 10 (29%) nodes from 4 (40%) patients totally lacked the expression of VEGF-C. This finding clearly indicates that carcinoma cells that highly express VEGF-C are not always preferentially transported to the regional lymph nodes, even though the expression of this lymphangiogenic factor had a positive correlation with lymph node metastasis.More interestingly, the expression of VEGF-C was strongly correlated with the size of metastatic foci and the location of carcinoma cells in metastatic nodes Table . When caMany cancers metastasize to regional lymph nodes, and a positive nodal status often correlates with a poor prognosis of patients. However, the mechanisms of lymphatic metastasis have not been investigated in detail. Recent studies have demonstrated that the expression of VEGF-C is enhanced in various solid tumors, suggesting the possible contribution of VEGF-C to nodal metastasis, possibly through lymphangiogenesis ,42. NumbIn this study, therefore, we evaluated the expression of VEGF-C in biopsy samples in SGC. Our initial hypothesis was that VEGF-C expression can predict the accurate nodal status including micrometastasis/ITC, and thus may be useful to avoid the unnecessary gastrectomy in some SGC. Our results suggest that VEGF-C expression in biopsy specimens correlate with lymph node metastasis in SGC. However, the positive and negative predictive values were 44% and 80% respectively, and 20% of VEGF-C-negative tumors were node positive. This suggests that the immunodetection of VEGF-C in biopsy samples can not be used as clinical indicator to decide the treatment of SGC.Present study provides some interesting findings on the possible role of VEGF-C in nodal metastasis. Biopsy samples were obtained at preoperative endoscopy, fixed with formalin immediately after biopsy, and thus appear to reflect the in situ expression level of VEGF-C more precisely than surgically resected specimens. In our results, VEGF-C expression in biopsy samples showed a significant correlation with that in surgical specimens with 57% sensitivity and 91% specificity. However, more than half (60%) of the tumors categorized as VEGF-C negative in biopsy specimens were positive in surgical specimens, although VEGF-C-positive tumors in biopsy samples showed a good consistency with those in surgical specimens. This raises the possibility that VEGF-C expression may be somewhat upregulated by surgical manipulation.VEGF gene expression is regulated by a variety of stimuli, and hypoxia is known to be one of the most potent inducers of VEGF-A ,44. VEGFin vivo experiments using VEGF-C-transfected tumors have shown the same histological findings [Nonetheless, our data showed a significant correlation of VEGF-C expression in biopsy specimens with nodal metastasis, supporting a possible role of VEGF-C in lymphatic metastasis. As with hematogeneous metastasis, lymphatic metastasis of cancer cells is considered to be divided into several steps: invasion to lymphatic capillaries, movement into the lymphatic lumen with the lymphatic stream, attachment to the subcapsular sinus of lymph nodes, and invasion into the cortex. Lymphangiogenesis means the development and proliferation of new lymphatics from host vessels, but the ability of tumor cells to induce lymphangiogenesis and the presence of intratumoral lymphatic vessels are controversial. However, most malignant tumors are known to be associated with an increased number of lymphatic vessels in the peripheral area . In factfindings ,50. SincIn regional lymph nodes, tumor cells are thought to reach the peripheral sinus from afferent lymphatics. In fact, many metastatic cells were detected around the sinus area unless they developed into a macronodular lesion. Recently, small lesions have been divided into two categories; micrometastasis and isolated tumor cells (ITC), which are distinguished based on their size . MicromeIn our study, the metastatic lesions did not always express VEGF-C, and such small metastatic foci often lacked the expression of VEGF-C. Especially, ITC identified only with immunohistochemical staining are totally negative for VEGF-C. In addition, in 4 tumors with lymphatic invasion, none of the tumor cells located in the lymphatic vessels in the primary tumor expressed VEGF-C (data not presented). These unexpected results suggest that expression of VEGF-C in tumor cells is not relevant to the transportation to regional nodes once they enter lymphatic vessels.In contrast, most of the macrometastases or cancer cells invading the medullary area of metastatic nodes highly expressed VEGF-C. This phenomenon was quite interesting, though not fully explained by today's knowledge. This may suggest that proliferation and invasion in the internal area of metastatic nodes may partially require VEGF-C expression in tumor cells. Thus far, there is no definite report on the effects of VEGF-C on tumor cells. The VEGF-receptor 3 (VEGFR-3), a specific ligand of VEGF-C, was expressed only on certain tumor cells -54 and nIn summary, our retrospective study demonstrated that VEGF-C expression in tumor cells in biopsy specimens was significantly correlated with lymphatic metastasis in SGC, although the accuracy was not high enough to be used for clinical indicator. Metastatic tumor cells in micrometastasis or ITC located in marginal sinus often lacked the expression of VEGF-C, whereas macrometastasis located in the medullary area in metastatic nodes highly express VEGF-C. This suggests a possibility that expression of VEGF-C is not essential for lymphatic transport from primary tumor, but rather important to develop the macronodular lesion in metastatic lymph nodes.The author(s) declare that they have no competing interests.MI. Conceived of the study and wrote the original version of the manuscript.JK. Carried out the literature search and helped in drafting the manuscript.SK. Collected clinical and pathologic data and participated in manuscript preparationHN Helped to shape the idea for the study coordinated the study and edited the manuscript.All authors have read and approved the final manuscript.


Tools performing such analyses are often restricted to a category score based on a cutoff in the ranked list and a significance calculation based on random gene permutations as null hypothesis.Ranked gene lists from microarray experiments are usually analysed by assigning significance to predefined gene categories, ), to compare different scores and null hypotheses in gene category analysis, using Gene Ontology annotations for category definition. When a cutoff-based score was used, results depended strongly on the choice of cutoff, introducing an arbitrariness in the analysis. Comparing results using random gene permutations and random sample permutations, respectively, we found that the assigned significance of a category depended strongly on the choice of null hypothesis. Compared to sample label permutations, gene permutations gave much smaller p-values for large categories with many coexpressed genes.We analysed three publicly available data sets, in each of which samples were divided in two classes and genes ranked according to their correlation to class labels. We developed a program, Catmap (available for download at In gene category analyses of ranked gene lists, a cutoff independent score is preferable. The choice of null hypothesis is very important; random gene permutations does not work well as an approximation to sample label permutations. These gene annotation analyses can unravel new information about pathways and cellular functions responsible for different phenotypes. Computational tools aiding in this process have recently been developed -8, most p-value [In microarray analyses such as clustering, which provide defined subsets of genes with no internal ranking, it is natural to base the score on the number of category genes in the relevant subset. However, ranking of genes appear in many techniques for microarray analysis, such as correlation of gene expression to target profiles  and scorp-value ,8, whichp-value , investip-value for the assigned score, a set of gene lists, ranked according to a chosen null hypothesis, are needed. The simplest choice of null hypothesis is just random gene permutations, and for some rank-based scores, the p-value can then be calculated analytically, without explicitly performing the permutations. However, the random gene permutations null hypothesis assumes independence of gene expression over biological samples, and the p-value is thus a combination of the p-value of how important the category is and the p-value for the genes of the category being coexpressed. When category genes behave similarly over a wide range of experimental conditions, the coexpression does not indicate relevance of the category for the question under study. In many analyses, a more appropriate null hypothesis is therefore sample label permutations, in which a set of ranked gene lists are generated based on the gene expression correlations to randomly permuted target values of the samples. This approach accounts for correlations between category genes and gives p-values that are bounded from below by the number of possible permutations of the samples in the data set. The latter is particularly important in data sets with few samples. Despite this, publicly available tools for gene annotation analysis are restricted to gene permutations [To calculate a utations -8.p-values, that would otherwise have been computationally infeasible. The input to the program is two files and some arguments. The first file contains the biologically relevant ranked list of genes and, if needed, additional ranked gene lists drawn from the null hypothesis. The second file contains the categories and their corresponding genes. The input arguments can either be specified on the command line or in a settings file, and are as follows: 1) a choice between the cutoff score the Wilcoxon rank sum score; 2) a choice of null hypothesis, which can be either the above mentioned user-supplied ranked lists or random gene permutations; 3) the number of permutations used in multiple category testing. If zero, no multiple category testing is performed.We present a program, Catmap, for gene category analysis based on ranked gene lists. The program uses either the number of genes above a cutoff or the Wilcoxon rank sum as score, and the significance of the score can be calculated from a user supplied set of ranked lists, thus allowing for sample label permutations. Furthermore, the program calculates corrections for multiple category testing, using permutation results to assess an effective number of independent categories, which enables Catmap to estimate very small multiple category p-value, the multiple comparison p-value, the false discovery rate, the ROC area , the number of genes in the category, and the 25th, 50th, and 75th percentiles of the ranks. The other output file, the companion file, contains all the categories, with all the genes and their ranks listed below. Each line contains a gene and its rank. The program can be downloaded at [The output of Catmap is two files. The main output file contains all the categories, one on each line ordered according to their significance. The line of a category contains the oaded at , where fet al. [p-values from cutoff independent scoring, showing that the p-value depends strongly on the choice of cutoff. This is further illustrated by the very different cutoffs at which the minimized cutoff-based p-value was obtained. A table with all categories is provided as a supplement [see We analysed the breast cancer data set of van 't Veer et al.  with a cment see .p-values, using the Wilcoxon rank sum and the minimized cutoff-based p-value, respectively. The p-value based on the Wilcoxon rank sum was most often larger than the minimal cutoff-based p-value. Since the latter is biased by a minimization process, it must be interpreted as a score, rather than a p-value, thus requiring additional analyses to find statistical significance [Compared to the variations between the cutoff-based alternatives, the results shown in Table ificance ,8.p-values based on gene permutations tend to be lower than those based on sample label permutations. For categories with small p-values, there are remarkable differences, in particular for large categories with more than 20 genes. Since the gene permutation null hypothesis assumes independent genes, we expect a GO category whose genes are uncorrelated to have roughly the same p-value under the two different null hypotheses, whereas a significant category whose genes are highly correlated will get a lower p-value using the gene permutation null hypothesis. To illustrate this coexpression effect, we picked two large categories, "carboxylic acid metabolism" and "M phase", which are encircled in Figure et al. [p-values for the two null hypotheses, while "M phase" has a p-value of 10-7using gene permutations but the much higher p-value of 3 · 10-2 using sample label permutations. As seen in Figure Using the Wilcoxon rank sum, we compared the results of different null hypotheses. Three publicly available data sets were examined ,13,20. AIn Table p-value used in FuncAssociate [individual p-values for categories, but ranks categories based on the chosen score. Nevertheless, they give results similar to those obtained with the Wilcoxon rank sum and gene permutation. This is expected, since the minimized p-value is calculated with gene permutations, and the score adopted in GSEA [p-value, based on gene permutations, would do. It should be noted that GSEA, FuncAssociate, and iGA calculate multiple hypotheses corrected p-values, but these do not change the ranking of categories.Table ssociate  and iGA ssociate . These t in GSEA  ranks cap-value score on one hand, and the Wilcoxon rank sum on the other, in the treatment of categories for which only a subset of genes have expressions correlating significantly with the question under study. The important genes being in the top of the ranked list will give the category a good score with all three score functions, provided the remaining, seemingly insignificant, genes are distributed in the ranked list as expected by random. However, if these less important genes lie higher in the list than expected by random (though not high enough to affect the Kolmogorov-Smirnov or min-p scores), the category will be considered more important by the Wilcoxon rank sum. Reversely, if the less important category genes prevail in the bottom of the list, the Wilcoxon rank sum score function will deem the category as unimportant, while the other two scores will give the category a high significance, based on the top ranked genes alone. Whether seemingly insignificant genes being ranked better or poorer than explainable by random expectations should be observed or ignored is of course a matter of taste, and a possibility is to use several score functions, that may complement each other. The differences are, however, much smaller than those related to choice of null hypothesis, as revealed in Table There is a possible difference . Furthermore, for all data sets and ontologies studied, Neff was approximately half of the total number of categories. If this is a general feature for GO categories, the simple Bonferroni correction would not be totally unreasonable for small p-values.For all 3 sub-ontologies, the effective number of categories, r et al.  the numbp-values were found for 1000 random gene lists.Figure et al. [It should be noted that whenever several ranked lists are examined as part of a project, this additional source of multiple hypotheses testing should also be corrected for. An example of such a correction, for cutoff-based score functions, is presented by Corà et al. .p-values of biologically relevant categories depend strongly on the choice of cutoff. The cutoff independent Wilcoxon rank sum score overcomes the problem, representing an alternative to the Kolmogorov-Smirnov score [p-value [We developed a computer program for calculating the significance of gene categories in a ranked list of genes. Corrections for multiple category testing can be performed by the program. To investigate the properties of different scores and null hypotheses, we analyzed three publicly available data sets ,13,20. Cov score -17 and t[p-value . The ranp-values and the ranking of categories depend strongly on the choice of null hypothesis. Compared to sample label permutations, gene permutations gave much smaller p-values for large categories with many coexpressed genes.Though sample label permutations in many situations represent a better null hypothesis than gene permutations, available gene annotation analysis tools are restricted to the latter. Our implementation allows for both null hypotheses, and we find that both the The implemented algorithm treats the categories sequentially and independently. As score function for category relevance, the program uses either the Wilcoxon rank sum or the number of genes above a given cutoff in the ranked list. The latter is implemented for method comparison and for the case of a defined subset of relevant genes, without internal ranking.For the case of the Wilcoxon rank sum, the user can supply a set of ranked lists distributed according to an appropriate null hypothesis, or request random permutations of genes as the null hypothesis. In the latter case, the significance of the score is calculated analytically by the program, using either an exact calculation by an iterative method, a Gaussian approximation, or a continuous volume approximation. The program chooses method based on a balance between accuracy and computation time. Details are presented in supplementary information see .p-value of category relevance is determined with Fisher's exact test [For the case of the cutoff-based score function, the act test , correspN independent categories are tested simultaneously, family-wise error rate simply means calculating the probability,When pmultiple (q) = 1 - (1 - q)N,     (1)p-value below any given number q by chance. For correlated categories, we make the assumption that the same functional form describes pmultiple (q), with N replaced by an effective number of independent categories Neff. We find Neff by generating a number, K, of ordered lists under the null hypothesis and calculating the p-values of all categories. We fit Neff using the maximum likelihood estimationthat at least one category has a pk is the minimal p-value for the k'th ordered list.where j highest ranked categories is found by counting the number of p-values from K permuted lists lower than the p-value of the j:th category and divide this number with K · j. For the case of sample label permutations, when a user supplied set of ranked gene lists are used to represent the null hypothesis, the first K lists are used to find Neff and false discovery rates, and the remaining lists are used to calculate p-values for each of the K lists.The false discovery rate for the The algorithm is implemented in the Perl program Catmap.pl and is released under the GNU General Public License (GPL). Catmap.pl, together with user instructions, is available for download at .Using Catmap, we analysed three publicly available data sets with gene annotations from the Gene Ontology.et al. [The data set of van 't Veer et al.  consistset al. , and renet al. .et al. [The data set of Golub et al.  consistset al. [The data set of Alon et al.  consistset al. [All genes were first mapped to corresponding UniGene clusters . For theet al.  this mapet al. ,20, the et al. , and comTB and MK implemented the algorithms in Catmap. All authors participated in the design of the study, prepared, read, and approved the final manuscript.supplement to Table 1.Click here for filesupplement to Table 2.Click here for filep-values for the Wilcoxon rank sum score.Click here for file


A great deal of research on the prefrontal cortex (PF), especially in nonhuman primates, has focused on the theory that it functions predominantly in the maintenance of short-term memories, and neurophysiologists have often interpreted PF's delay-period activity in the context of this theory. Neuroimaging results, however, suggest that PF's function extends beyond the maintenance of memories to include aspects of attention, such as the monitoring and selection of information. To explore alternative interpretations of PF's delay-period activity, we investigated the discharge rates of single PF neurons as monkeys attended to a stimulus marking one location while remembering a different, unmarked location. Both locations served as potential targets of a saccadic eye movement. Although the task made intensive demands on short-term memory, the largest proportion of PF neurons represented attended locations, not remembered ones. The present findings show that short-term memory functions cannot account for all, or even most, delay-period activity in the part of PF explored. Instead, PF's delay-period activity probably contributes more to the process of attentional selection. Persistent activity of neurons in an area of the frontal lobe - the prefrontal cortex - is often proposed to underlie short- term memory. Mikhail Lebedev and colleagues provide an alternative explanation Note, however, that when we use the phrase maintenance memory, many authorities would use “working memory” instead.Once the concept of working memory  became edelay-period activity applies to neuronal activity that follows the transient presentation of an instruction cue and persists until a subsequent “go” or “trigger” signal. The description of delay-period activity in PFdl appeared very early in the history of behavioral neurophysiology . Dimming of the circle signaled that the monkeys should make an eye movement to its current location , for the following reasons. As a key feature of the experimental design, the circle's brightness changed only subtly and remained visible in its new form only briefly. Because the monkeys could not predict whether the circle would brighten or dim and because that subtle, short-lived event provided essential information about the time and target of the response, the monkeys had to attend to the circle intently during the period preceding the trigger signal. As a result of the central fixation requirement, this attention was necessarily covert, although it seems likely that the monkeys would have attended overtly to the circle , had they been allowed to do so. Indeed, the monkeys did so during training. The Discussion takes up the issues of divided attention, multiple motor plans, default motor plans, and other interpretational issues.Brightening of the circle indicated that the monkeys should make a saccade to the circle's initial location on that trial, which the monkeys had to remember in order to perform the task correctly . We can only speculate about the cause of this difference, but reaction times on Rem-trials may have been longer because attention had to be disengaged from the circle's location and reoriented to the remembered one prior to the response. For the “no-memory” condition (not given in p < 0.001). These data are consistent with the idea that each of the two marked locations attracted attention in the no-memory condition, whereas the monkeys directed most of their covert attentional resources to the attended location in the standard version of the task. We acknowledge, however, that there are other interpretations of these data. On control trials, for example, when the saccade was always toward the circle, saccade initiation was approximately 18 ms slower when the circle brightened  than on trials when it dimmed . Thus, factors other than the orientation of attention probably contributed to reaction-time differences.preferred location. The lowest firing rate occurred when the monkey attended to the 270° location, termed the least preferred location.Att),attended-location index remembered-location index  see . A neuroAtt)(I and remembered (RemI) locations. Each data point on the scatter plot represents a single spatially tuned neuron (both monkeys combined). Tuning for the remembered location (red symbols) was both weaker and less frequent than tuning for the attended location (blue symbols). Note that hybrid cells (green symbols) fill most of the space between the other two classes and that relatively few cells represent a single location exclusively. For example, many of the neurons classed as memory cells show some sensitivity to the attended location, albeit not a statistically significant one by the test that we employed. For the entire group of spatially tuned neurons , the mean selectivity indexes (± SEM) for the attended and remembered locations were AttI = 1.84 ± 0.08  and RemI = 1.21 ± 0.02 , which differed significantly at the p < 0.001 level (Wilcoxon matched-pairs test). p < 0.001).early period) and during the last 800 ms of the delay period, immediately prior to the trigger signal (the late period). . This measure is devoid of any bias caused by a cell's tuning properties in one task period or the other, but it includes the contribution of the spatially untuned cells. When we restricted the comparison to neurons that had any type of significant spatial tuning, in either the early or late periods, the late tuning index (1.76 ± 0.07) continued to exceed the early one (1.42 ± 0.05) significantly (p < 0.001). Most important, we obtained similar results for neurons with significant tuning to the circle's location, which characterizes attention and hybrid cells . We examined whether these results merely reflected the presence of a stimulus in the monkey's visual field and found strong evidence to the contrary. We compared tuning for the circle's location during the 800 ms before the circle started moving  were predominantly attention cells . Neurons dorsomedial to the fundus (n = 412) fell into the three cell classes approximately equally . These regional differences within PFdl were highly significant for each monkey . Cells with significant memory signals  composed 70% of the spatially tuned population in dorsomedial PFdl, but only 20% in ventrolateral PFdl.Based on a cytoarchitectonic analysis conducted on two of the three hemispheres, all of the cells situated ventrolateral to the fundus of the principal sulcus were located within area 46 and none were located in area 12. The area 46/12 architectonic boundary was first described by The right side of Population representations of the attended and remembered locations were further analyzed using a neuron-dropping analysis. Neuron-dropping curves express the strength of spatial tuning as the ability to estimate a spatial variable from the activity of a neuronal ensemble, as a function of ensemble size. We randomly selected an ensemble from the population of recorded PFdl neurons and used a single trial of activity from each cell to estimate both the attended and remembered locations. The findings of the neuron-dropping analysis agree with those from the analysis of single-cell activity and the population histograms and thus provide independent support. However, neuron-dropping analysis offers several advantages over the population histograms, in addition to providing confirmation of those results. In neuron-dropping, the estimation of either an attended or remembered location does not depend on any assumptions about the nature of the spatial tuning curve or the relative importance of very active cells versus those showing less activity. It does not ascribe any special significance to increases in activity relative to baseline (excitation) versus decreases (inhibition) or to the most preferred and least preferred locations. Each cell's activity contributes to the population estimation for all locations regardless of the direction of its modulation relative to baseline and whether that modulation significantly differs from baseline levels. Furthermore, the computation makes no assumption about any relationship between tuning for attended locations and remembered ones. This analysis also has the advantage that its results are expressed as a percentage of correct estimations by the neuronal ensemble, thereby facilitating comparison with the monkeys' performance, which in this experiment always exceeded 75% correct and sometimes approached 100% .RemI > 1.0). The same analysis was applied to the ventromedial and dorsolateral regions within the PFdl, described in the section entitled Histological Analysis, above (not shown). The ventrolateral subpopulation of PFdl neurons see A–4C overWe also used a neuron-dropping analysis to examine the ensemble's properties during response selection and execution. On Att-trials, the estimation of the attended location , while attending covertly to a stimulus located in peripheral visual space, make it unlikely that attention was further divided . Accordinot remembering some location. However, there is no basis for assuming a “memory” of a currently visible stimulus. It seems especially unlikely that the monkeys “remembered” the attended location in the context of the requirement that they centrally fixate while attending somewhere and remembering somewhere else.Second, the monkeys were required to remember the place where the circle first appeared on each trial, and their performance shows that they did so. Did they also “remember” the attended location? There is ample precedent for skepticism about the proposition that monkeys are Third, we cannot rule out the participation of neurons we class as attention or memory cells in a variety of processes involved in preparing or planning the movement or selecting the response target. Prior to the trigger signal, the monkeys may have prepared to make a movement to the remembered location, to the attended location, to both, or to neither. Fourth, we need to consider the possibility that the neural signals we observed reflect the prediction or anticipation of reward. attention has been used to cover many disparate concepts, including the effects of attention on sensory processing and the mechanisms that mediate those influences. We emphasize that the present finding differs from previous ones describing effects of attention on phasic, sensory-like responses. Often called the enhancement effect, the finding that sensory responses are larger when a stimulus or location is more attended was first described for the superior colliculus  in the orientation of spatial attention. In the context of the present results, the finding that inactivation of parts of PFdl  producedThe present results agree better with those of The present study reexamined the interpretation of PFdl's delay-period activity in terms of the maintenance-memory theory. We found that other factors are more important than mnemonic ones. The present results do not argue against a short-term memory function for PF, as one among many contributions to behavior. Nor should they lead to the dismissal of interpretations of some delay-period activity in PF, or some neuroimaging signals from that region, in terms of short-term memory. However, spatial memory signals occur less frequently in PFdl than the maintenance-memory theory predicts. Our data thus accord better with neuroimaging and neuropsychological studies indicating that PF plays a major role in attentional selection, including the monitoring of information and actions .How do our findings mesh with the fact that damage to PF appears to produce deficits in short-term memory, as Although attention could account for many findings about PF, we do not aim to replace one monolithic theory of PF function—the maintenance-memory theory—with an equally monolithic “attention theory.” Delay-period activity appears to reflect the learning and implementation of behavior-guiding rules , categor(Macaca mulatta) to perform the task. Each monkey sat in a primate chair in front of a computer monitor placed 57 cm from the monkey's eyes. We recorded eye position with an infrared oculometer and sampled at 250 Hz. The monkeys pressed a waist-high button with their right hand to start each trial and did not release the button until the end of the trial. Once the monkeys pressed the button, a 0.2° fixation point appeared at the center of the screen. After they had fixated this stimulus for 1.0–1.5 s, a 2° solid, gray circle appeared 8° from the center of the screen in one of four places. We trained two rhesus monkeys After the monkeys learned the task, we implanted recording chambers over the left (monkeys 1 and 2) and right (monkey 1) PFdl. For monkey 1, we used a single-electrode microdrive to obtain single-neuron activity records; for monkey 2, we used a microdrive that independently moved up to seven electrodes. During recordings in monkey 1, we intentionally biased the selection of task-related neurons toward those with delay-period activity. In monkey 2, we recorded the activity of all isolated neurons, regardless of whether they were task related.For histological reconstruction of recording sites, we examined Nissl-stained sections of 40 μm thickness from the right hemisphere in monkey 1 and the left hemisphere in monkey 2.ij,F with rows (i) corresponding to the remembered location and columns (j) to the attended location F is the firing rate on the thl trial for which position i was the remembered location and position j was the attended location, and 1N and 2N are total number of elements in the respective sums. Control trials were excluded from the calculation by not considering the diagonal elements of ijF .where We evaluated tuning to the attended location similarly by comparing across-column variability with within-column variability, one row at a time. The strength of representation of the attended location, was quantified as:ij (l).F To classify neurons into those representing remembered versus attended location, we used an 800-ms period preceding the trigger signal. We also evaluated spatial tuning (I) during the final 800-ms period before the circle started to move. .We used two task periods to compute the single trial firing rates We computed the population histograms of n neurons from a population. Then, for a given condition , we selected one trial of that condition randomly from each neuron . All the other trials for that neuron contributed to a look-up table of firing rates. This look-up table consisted of a matrix of average firing rates ij><F for remembered locations, i, and attended locations, j. The differences between firing rates in the look-up table and the rate on the selected trial were rank ordered, with a smaller rank signifying a closer match. We then summed the ranks ijr across individual neurons and took the remembered and attended locations associated with the lowest combined rank as the population estimation. The estimated remembered location either agreed or disagreed with the actual remembered location of the selected trial, as did the estimated attended location in a separate computation. Repeating this procedure for a given number of neurons, n, more than 2,400 times—each time starting with a randomly selected set of test trials —yielded a percentage of correct estimations of the attended and remembered locations. We then calculated neuron-dropping curves for ensembles of size one to the total number of neurons, but typically the range 1–100 sufficed to capture the main features of the population estimation.Neuron-dropping curves D and S7 To assess the representation of attended and remembered locations during the delays see A–7D, we Figure S1The activity matrix is the same as in (103 KB PPT).Click here for additional data file.Figure S2The activity matrix is the same as in (95 KB PPT).Click here for additional data file.Figure S3The activity matrix in (A) is the same as in (151 KB PPT).Click here for additional data file.Figure S4A table of tuning indexes is given at the top for each of the cell classes (plotted in the bottom part of the figure), combinations of those classes, and other groups of cells as described in the left column. These population averages are divided into two groups of columns, those on the left showing data for the period before the circle began rotating (early) and those on the right showing data for the period after it had stopped and the monkey awaited the trigger signal (late). In the plot, the dashed line shows the median values, the dotted line shows the upper IQR.(56 KB PPT).Click here for additional data file.Figure S5Same PFdl neuron as in (188 KB PPT).Click here for additional data file.Figure S6Same PFdl neuron as in (156 KB PPT).Click here for additional data file.Figure S7Format as in (46 KB PPT).Click here for additional data file.


Drosophila, identifying two mutants. A new reverse-genetics mutagenesis method that uses the crosslinking drug hexamethylphosphoramide to introduce small deletions has been used to generate and screen pools of mutagenized  Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism  Drosophila melanogaster has been the prime genetic model organism for almost a century. This success story is mainly founded on countless so-called forward genetic screens designed to elucidate gene functions on the basis of their mutant phenotypes. Many of those screens reached a scale that has been termed 'saturating' as they identify all nonredundant genes involved in a certain phenotypic trait. However, forward genetic screens are limited in that they are only capable of uncovering functions that are easily measurable or visible. Furthermore, genes having a redundant or nonessential role are less likely to be found by forward genetics.The fruitfly The reverse genetic approach to unravel gene function starts with the DNA sequence. Mutations within the gene are induced and identified by various techniques and only subsequently is the mutant phenotype analyzed . ReverseDrosophila genome by insertion mutagenesis is far from being achieved [Both undirected and directed reverse genetic techniques have certain advantages and drawbacks. Transposon-based mutagenesis tends to be nonrandom because of the occurrence of hotspots for transposon integration. The use of transposable elements of different origin, such as P-elements and piggyBac, which exhibit a different insertion bias, can partly circumvent this problem. However despite large-scale efforts, the ultimate goal of covering the whole achieved ,11. Moreachieved .C. elegans [Drosophila is not feasible.RNAi and small interfering RNA (siRNA) screens provide a powerful tool to dissect the function of genes at a genome-wide scale -14, but  elegans  and for Drosophila allows for generation of both null as well as hypomorphic mutations [Targeted gene knockout in utations . HoweverN-ethyl-N-nitrosourea (ENU) [Random mutagenesis in reverse genetics generally relies on well-established techniques and commonly used mutagens, such as ethylmethansulfonate (EMS) ,17 and Nea (ENU) . Those cea (ENU) , by denaea (ENU) ,17, or bea (ENU) ,20-22. MFast neutrons have also been used to introduce small DNA lesions, which can simply be resolved by agarose electrophoresis after PCR amplification . This kiWe reasoned that it would be worthwhile to establish a generally applicable reverse genetic technique based on an unbiased and practicable random mutagenesis and an efficient mutation-detection performed on standard laboratory equipment. Here we introduce a novel mutagenesis protocol utilizing the cross-linking drug hexamethylphosphoramide (HMPA) , streamlThere are two ways to handle mutagenized progeny. Either large collections are established and maintained, which then are systematically and continuously screened for mutations of interest, or mutagenized progeny are screened directly and only animals exhibiting a desired trait are kept. The first method is in practice an F3 screen, which requires balancing of mutagenized chromosomes and maintenance of many stocks. This approach is far more labor-intensive than a simple F1 screen of progeny and thus is more suited to stock centers. Moreover, balancer chromosomes have many DNA sequence polymorphisms to wild-type chromosomes (our unpublished data), which will interfere with detection of mutagen-induced sequence polymorphisms.To circumvent the inherent problems with balancers, we devised an alternative genetic strategy, which had to fulfill the following criteria. First, mutagenized chromosomes have to be passed on in an unrecombined form such that mutations cannot be lost. Second, the mutagenized chromosomes should be brought into an isogenic background for mutation detection. Third, for economic reasons stock-keeping should be kept at an absolute minimum.yw wild-type laboratory strain but containing the same dominant marker on the two major autosomes. Both chromosome 2 and chromosome 3 are carrying white+ marked P-element insertions, which were chosen because white+ expression is restricted to different subregions of the eye  isogenic to our e Figure . Chromose Figure . Thus, tyw strain background. Thus, the marked autosomes remained unrecombined and could be unambiguously assigned because of the dominant character of the white+ transgenes . However, analysis of X-chromosomal loci would require additional handling of F1 females or mutagenesis of F0 females and hence we did not carry out X-chromosomal screens.The crossing scheme and analysis procedure illustrated was optimized for autosomal genetics. We have generated another strain, KNF307, which in addition carries X chromosomes marked by a characteristic enhancer trap insertion at the et al. [et al. [EMS has been used as a deletion-inducing chemical in large-scale screens , but unbet al.  ranged i [et al. . The cro [et al. . As our N,N-dimethylbenzylamine, which in our hands potentiated the sterilizing activity of HMPA.We modified the original HMPA mutagenesis protocol to administer a shorter, but more intense pulse of HMPA . As HMPA is reported to induce deletions as small as 2 bp and as a mutated allele is diluted 10-fold as a result of our pooling of five flies, we decided to analyze PCR fragments on a sequencer offering maximal resolution and high sensitivity. We have also evaluated the 'poison-primer technique' which is reported to preferentially amplify alleles with a deletion at the poison-primer binding site from large pools . HoweverPCR products were analyzed on either a gel-based or a capillary sequencer . To increase efficiency of mutation detection on gels, we pooled up to three PCR products. These were labeled with different fluorescent tags, partly because they were of similar size Figure .white- mutations at the transgenes on chromosomes 2 and 3. Overall, we found 24 mutations in about 62,700 male and female flies. Two flies were mosaic for the mutations. Given that mosaicism can only be scored in eyes and there only in nonoverlapping expression domains, the mutation rates discussed below may be slightly underestimated , because we did not recover any white- mutation. As sperm development takes up to 10 days [-4 mutations at the white+ loci, which are about twice as large as the vermilion locus. Our mutagenesis procedure involving an overnight incubation with HMPA rather than a 3-day incubation with HMPA and N,N-dimethylbenzylamine is therefore not much less efficient than the original protocol.Aguirrezabalaga et al.  reported 10 days , we alsowhite- mutations between brood 2 and brood 3  of 2,064 bp. We assume that any deletion within the ORF would generate a null phenotype. Only 14 out of 31 HMPA-induced deletions selected at the vermilion locus would have been scoreable by our PCR approach, because the remaining 17 mutations were caused by large deletions affecting both primer-binding sites [The following additional parameters can be utilized to estimate mutant recovery. The ng sites .-4 × 14/31 × (710 - 2 × 20)/2,064)) or one mutation in 6,063 pools, respectively. Taking into account the fact that two mosaic flies may not have transmitted (reducing the mutation rate to 2.0 × 10-4), the estimate would be one mutation in 33,883 flies or one in 6,777 pools.We designed PCR primers for each gene to be scored such that they encompass the first coding exon and the PCR products are between 450 and 807 bp in size. The average weighted length of our PCR fragments was 710 bp (including two primers of 20 nucleotides each). We thus expect one mutation in 30,317 flies . According to the estimate we would have expected three.CG15000, which during the course of this study turned out to be the second exon of the dNAB locus .The first mutation detected was a 41-bp deletion in the first exon of CG17367 on the capillary sequencer , but we have not systematically investigated all of the white- mutations.This study focused on implementing HMPA mutagenesis for reverse genetics. As discussed above, HMPA efficacy has been assessed from mutations at the -3 nucleotide substitutions at the 1 kb awd locus [-4 deletions per 2 kb white+ locus for HMPA), but mutagen dose cannot be increased further because of the concomitant increase in male sterility.While the analysis of PCR fragment-length polymorphisms on our sequencers was very efficient, HMPA mutagenesis turned out to be the limiting parameter. It is about 28-fold less efficient than EMS mutagenesis when it is assumed that all HMPA hits are deleterious . HMPA induced mutations can be detected by fragment-length analysis of primary PCR products on standard sequencers. Hence, screening for small deletions reduces PCR costs by a factor of 2 and spares the effort of secondary assays.Mutant handling is independent of the mutagenesis protocol and may be combined with either EMS or HMPA mutagenesis. For example, TILLING can be performed both on large mutant collections and on a continuous supply of freshly generated mutants.Finally, given the genotoxic properties of HMPA in both prokaryotes and higher eukaryotes ,32, bothy, w; CG31666-white+; CG32111-white+) males were starved for 4 to 6 hours in a plastic bottle containing three layers of water-soaked LS14 filter papers (Schleicher & Schüll). A 1.1 ml sample of HMPA solution  was carefully applied to the filters using a syringe with a long needle (21G2) inserted through the foam stopper. The starved males were exposed to the HMPA solution overnight. Bromophenol blue does not affect mutagenicity detectably, but stains the guts of the flies blue and thus enables mutagen uptake to be monitored and controlled. Freshly eclosed flies do not ingest enough mutagen. HMPA-contaminated plasticware must be disposed of by thermal waste treatment.About 150 1-3-day-old F0 KNF306 . After 2 days males were taken out and crossed to yw virgins in new bottles (brood 2A) and this cross was transferred after 3 days (brood 2B). After another 2 days F0 males were recovered and mated to fresh yw virgins (brood 3A). F1 males of broods 2A, 2B and 3A were collected and mated individually to three yw virgins in about 650 separate crosses per week. Five hundred non-sterile males were removed after 4 to 5 days and five males were pooled for DNA extraction. Fertilized females were returned, and unsuccessful crosses were discarded. If analysis of PCR fragments indicated a primary positive pool, crosses were traced back and kept for further analysis; the other crosses were discarded. From each of the five crosses of primary positive pools a single F2 male or female containing the chromosome of interest as manifested by the typical eye-color pattern was collected for DNA extraction. If PCR analysis yielded a secondary positive result in one of the five F2 flies, a single F2 male containing the chromosome of interest was taken out from the respective cross for balancing .2O) the beads were virtually free of contaminating DNA.) Debris was allowed to settle for about 5 min and each 50 to 100 μl of supernatant were transferred into a 96-well PCR plate. The reactions were incubated in a thermocycler for 30 min at 37°C, and finally for 5 min at 95°C to heat-inactivate proteinase K.DNA was extracted in bulk by squishing pools of each five flies through mechanic force in a vibration mill (Retsch MM30) programmed to shake for 20 sec at 20 strokes per second. Flies were placed into wells of a 96-well deep-well plate. Each well was then filled with 500 μl squishing buffer  and one tungsten carbide bead (Qiagen). The deep-well plate was then sealed with a rubber mat (Eppendorf) and clamped into the vibration mill. , 0.6 μl dNTPs (10 mM), 0.1 μl fluorescently labeled primer 1 (100 μM), 0.1 μl primer 2 (100 μM), 0.12 μl hot-start Taq polymerase , 3 μl 10× buffer containing MgCl2 (Qiagen). Cycling conditions were 95°C 15 min, 35 × , 72°C 2 min, 4°C.A Tecan pipeting robot was used for PCR setup. To 5 μl of template DNA, master-mix was added and PCR was performed on an MJR thermocycler that was integrated into the robot. The master-mix per reaction was composed of 20.48 μl ddHThree differently labeled PCR reactions  were then pooled. To facilitate sizing of fragments we also added ROX1000 size marker (Applied Biosystems) to five DNA pools. Samples of 1.5 μl pooled DNA were mixed with 1.5 μl loading buffer (consisting of one part 25 mM EDTA pH 8.0 with 50 mg/ml blue dextran and five parts HiDi formamide (Applied Biosystems)). The reactions were incubated for 3 min at 95°C, cooled down, and 1.5 μl each were loaded onto a 96-lane ABI 377 sequencer. Run conditions were as follows: 1 h pre-run at 1,000 V, 35 mA, 51°C and 10 h run at 2,400 V, 50 mA, 51°C. Gel images recorded at four different color channels by the GeneScan software were analyzed visually.Slight modifications to this protocol were introduced for analysis performed on an ABI 3730 capillary sequencer. First, DNA was diluted 20-fold before PCR. Second, after PCR, reactions were diluted 100-fold and 2 μl of diluted PCR products were added to each 15 μl HiDi formamide (Applied Biosystems). PCR product was diluted on a Tecan pipeting robot. Diluted DNA was denatured for 2 min at 95°C before analysis. Sample injection (10 sec) and analysis  was done according to standard protocols. Identification of deletion fragments was then performed by visual inspection of gel-images generated by the Data Collection Software . No internal size standard was used, as deletion fragments were identified relative to wild-type PCR product.The following additional files are available with the online version of this paper. Additional data file The time schedule of mutagenesis, fly work, and screeningClick here for additional data fileInformation on the 10 other genes scoredClick here for additional data file


Microstimulation, a technique that activates a cluster of nerve cells by zapping them with a weak electrical current, has helped make causal links between neurons and behavior. For instance, when neurons in an area of the visual cortex that are “tuned” to a particular direction of motion are microstimulated, the way monkeys perceive moving dots on a video screen changes. Microstimulation seems to change what they see. Similar work has also been done for neurons that respond to binocular disparity—the depth-of-field information you gain because each eye has a slightly different view of the world.But many neurons respond, or are tuned, to more than one dimension, leading scientists to wonder how information from these multidimensional neurons contributes to perception—especially when some of that information is irrelevant to a given task. As they report in this issue, Gregory DeAngelis and William Newsome find that neurons tuned to both direction and binocular disparity contribute little to monkeys' perception of motion.The researchers asked three rhesus monkeys to determine the direction a group of dots was moving on a TV screen—a task that can be done regardless of the perceived depth of the dots. The authors had already located two different types of neurons in each of the monkey's brains: sites tuned strongly to direction and multidimensional sites tuned to both direction and binocular disparity. They then determined each site's exact preference: the direction of motion and degree of binocular disparity (if present) that triggered maximum neural activity. The researchers then showed the monkeys several sets of video displays, some with the dots moving in the “preferred” direction and some not. The microstimulation acts somewhat like adding dots in the preferred direction—which confuses monkeys when the real dots are moving against preference and aids them in trials of preferred moving dots. If the behavioral effect of microstimulation—whether it be a help or a hindrance—was significant, it meant that the monkeys were monitoring the activated neurons to perform the task at hand; if there was no change, the stimulated neurons were not being recruited.DeAngelis and Newsome hypothesized that multidimensional neurons  might be ignored during pure motion perception tasks. For two of the three monkeys, this was true. Microstimulation of multidimensional sites had no effect on their behavior, compared to the significant effect of microstimulation of direction-only sites. But for the third monkey, called monkey R, microstimulation of both types of sites had significant effects on his performance. He didn't seem to be ignoring anything. The authors proposed that the monkeys could be using different neural strategies to complete the same task. This conclusion is supported by the fact that monkey R performed better on the task than the other monkeys; he appeared to be recruiting any neuron with applicable information, unlike the others, who seemed to rely on neurons tuned solely to direction of motion. Furthermore, for the few multidimensional sites that affected behavior, their contribution was tempered by how well the depth, or disparity, of the video matched the preference of the stimulated neurons.The results of this paper show that even if neurons carry information that can aid in perceptual decision making, they may not participate, depending on how they are tuned along other (irrelevant) stimulus dimensions. All directional neurons are not created equal—some are more useful than others for a particular task. Whether neurons that respond to a particular stimulus contribute to the task at hand depends on how closely that stimulus hews to the neurons' preference as well as on the subject's learned strategy for performing the task. This neural flexibility, the authors point out, suggests that the brain uses complex, variable strategies to respond to changing environmental stimuli. Techniques like microstimulation will be helpful in drawing the connections between neural activity and behavior.