The Tropical Disease Initiative will be a Web-based, community- wide effort where scientists from the public and private sectors join together to discover new treatments Only about 1% of newly developed drugs are for tropical diseases, such as African sleeping sickness, dengue fever, and leishmaniasis It is easy and correct to say that Western governments could solve this problem by paying existing institutions to focus on cures for tropical diseases. But sadly, there does not appear to be enough political will for this to happen. In any case, grants and patent incentives were never designed with tropical diseases in mind.www.tropicaldisease.org).Two main kinds of proposals have been suggested for tackling the problem. The first is to ask sponsors—governments and charities—to subsidize developing-country purchases at a guaranteed price ,3,4. TheThe idea behind asking sponsors to subsidize developing country purchases at a guaranteed price is that this will prop up drug prices and restore incentives for developing new drugs ,3,4. In www.iowh.org), a not-for-profit pharmaceutical company funded mainly through private sources and the Gates Foundation, and the Drugs for Neglected Diseases Initiative (www.dndi.org), a public sector not-for-profit organization designed to mobilize resources for R&D on new drugs for neglected diseases.In the “Virtual Pharma” approach, governments and philanthropies fund organizations that identify and help support the most promising private and academic research. Examples include the Institute for One World Health  that permit anyone to use the information for any purpose, provided proper attribution is given.Licenses similar to the Creative Commons Attribution License (www.opensource.org/licenses/gpl-license.php) that prohibit users from seeking intellectual property rights.Licenses such as the General Public License . Importantly, TDI would be a great boost to the efforts of Virtual Pharmas, because it would help to contain the costs of discovering, developing, and manufacturing drugs.TDI would contain costs in three important ways. First, TDI would ask volunteers to donate their time (and any patentable discoveries) to the collaboration. Instead of financial incentives, TDI would offer volunteers non-monetary rewards, such as ideological satisfaction, the acquisition of new skills, enhancement of professional reputation, and the ability to advertise one's skills to potential employers. Software collaborations have demonstrated that these incentives are a good way to attract and motivate programmers Second, we have already pointed out that existing proposals have difficulty containing costs. The root cause is patents. Normally, society relies on competition to keep prices low. Patents—by design—short-circuit competition by giving the owners the legal right to prevent others from using (or even developing) their invention. TDI, on the other hand, would restore competition by making drug candidates available to anyone who wanted to develop them. We expect sponsors to exploit this advantage by signing development contracts with whichever company offers the lowest bid. Such competitive bidding is a powerful way to contain costs, and is also a good way to develop drugs. Virtual Pharma has extensive experience supervising contract research.Third, the absence of patents would continue to keep prices low once drugs reached the market. The generic drug industry shows what happens once drug makers are allowed to compete. US drugs frequently fall to about one-third their original price when patents expire Would universities and corporations really let their people volunteer? Won't they insist on intellectual property rights? The practical answer is that sensible managers do not care about intellectual property rights unless they expect to earn a profit. This explains why sophisticated university licensing offices seldom bother to interfere with open-source software projects that are not commercially valuable www.marchofdimes.com) developed polio vaccines without any patents at all Finally, there are precedents for private companies developing drugs off patent. During the 1950s, March of Dimes Open-source drug discovery is feasible—that is, no known scientific or economic barrier bars the way. But what are the risks? Experience with software collaborations highlights the main social and economic challenges. First, the project will have to find and motivate volunteers. Based on existing software collaborations, we estimate a required minimum “critical mass” of a few dozen active members. Second, modest chemistry and biology experiments will be needed to increase the chances for success. Resources of several hundred thousand dollars per year—mostly in the form of in-kind donations of databases, laboratory access, and computing time—would make open-source drug discovery much more powerful. By most standards, such risks are real but acceptable.The largest uncertainties are scientific. Can a volunteer effort based on computational biology and modest experiments produce the high-quality drug candidates that Virtual Pharma needs? A successful program must (1) make a significant contribution toward supplying the genomic insights that tropical disease research needs to move forward, and (2) make useful drug candidates available for development and production under open-source licenses. Open-source drug discovery looks feasible. The only way to be sure is to do the experiment—and we invite you to join us.http://www.tropicaldisease.orgTo learn more about TDI or to volunteer, go to


Malaria continues to claim one to two million lives a year, mainly those of children in sub-Saharan Africa. Reduction in mortality depends, in part, on improving the quality of hospital care, the training of healthcare workers and improvements in public health. This study examined the prognostic indicators of severe falciparum malaria in Gabonese children.Plasmodium falciparum infection and one or more features of severe malaria were enrolled.An observational study examining the clinical presentations and laboratory features of severe malaria was conducted at the Centre Hospitalier de Libreville, Gabon over two years. Febrile children aged from 0 to 10 years with Most children presenting with severe falciparum malaria were less than 5 years (92.3% of 583 cases). Anaemia was the most frequent feature of severe malaria (67.8% of cases), followed by respiratory distress (31%), cerebral malaria (24%) hyperlactataemia (16%) and then hypoglycaemia (10%). Anaemia was more common in children under 18 months old, while cerebral malaria usually occurred in those over 18 months. The overall case fatality rate was 9%. The prognostic indicators with the highest case fatality rates were coma/seizures, hyperlactataemia and hypoglycaemia, and the highest case fatality rate was in children with all three of these features.Prompt and appropriate, classification and treatment of malaria helps identify the most severely ill children and aids early and appropriate management of the severely ill child. Plasmodium falciparum infection is very rare after the age of 5 years in highly endemic areas. Presentations of severe malaria are different at different ages and in areas with different levels of transmission. In Gabon, a country of about 1.2 million people, malaria is the main cause of neurological, haematological and infectious emergencies at the Centre Hospitalier de Libreville (CHL), the country's tertiary referral centre [Each year 500 million infections and up to 2.7 millions deaths are attributable to malaria , about 9l centre . Previoul centre ,5,9. Thel centre . In Afril centre . Better This study was carried out in the CHL between 1st August 2000 and 31st July 2002. The CHL is the largest public hospital in the country, situated in Libreville, the capital city . Services at the CHL include an Emergency Department (20 beds), a Paediatrics Unit (80 beds) and an Intensive Care Unit (12 beds). Ethical permission for the study was granted by the Gabonese Ministry of Health.pro-forma sheet. A blood sample (2 ml) was drawn (anti-coagulated with EDTA) for quantitative examination of a blood film for malarial parasites using the Lambaréné method [P. falciparum in 100 high power fields of a thick film. The schematic process of the inclusion is shown in Figure Febrile children were referred the study team and seen on admission by a Malaria Clinical Research Unit (MCRU) clinician, summary data were recorded on a é method , measureé method . Blood fP. falciparum seen on blood film) and had one or more of the following features of severity: [Febrile children (or those with history of fever in the last 48 hours) were considered for inclusion in the study if they were: aged 0 to 10 years of age (inclusive), had malaria  without a loading dose, followed by oral quinine when tolerated. Pyrexial children received paracetamol suppositories . Seizures were controlled with diazepam . Severe anaemia was corrected by transfusion of packed red cells (15 ml/kg over 4 hours) screened for blood borne infections. Hypoglycaemia was treated with a slow intravenous injection of hypertonic glucose 40 % at a dose of 1 ml/kg. Nasal oxygen at 6 1/minute was given to children with respiratory distress.An MCRU clinician performed a full physical examination daily until discharge for each child. Laboratory assessments including parasitaemia, blood glucose and lactate were performed during hospitalization as necessary. The outcome  was recorded.2 tests with Yates' correction or Fisher's exact test. ANOVA test were used for multiple comparisons of variances, with Tukey's post hoc test. Assessments of prognostic factors were conducted with logistic regression model. A p value < 0.05 was considered as significant. Specific prevalence for each subgroup has been defined as the ratio of the number of cases observed in this sub-group over the population of this same sub-group. Hyperlactataemia is usually defined as a blood lactate concentration of ≥ 5 mmol/L. As the Accusport has been found to have poor agreement with the gold standard YSI 2300 [Statistical analysis was carried out with Epi info 6.04  and Stata Statistical Software . Normality of data distribution was checked using either Shapiro-Wilks or Kolmogrov -Smirnov test. Normally distributed data were analysed by two-tailed Student's T test and non-normally distributed data with the Mann-Whitney U statistic. Proportions were compared with χYSI 2300  or YSI 1YSI 2300 , a definP. falciparum. A lower prevalence of malaria was seen in children aged< 6 months . Specific prevalence of malaria rises after the 6 first months of life until it reaches a maximum at 47 months (47.5%), after which it declines again. Severe anaemia was most frequent in children less than 24 months old with 68 % of the cases of severe malarial anaemia occurring before this age. In contrast, the highest specific prevalence of cerebral malaria was found in children aged > 12 months. Sixty five percent of cases of cerebral malaria occurred in children aged between 12–48 months.During the study period (1st August 2000-31st July 2002), 8,036 febrile children were screened for malaria at the hospital. The data for 7,980 of these children were analysed: 4,368 male (54.5%) and 3,612 female (45.5%). Seventy-five percent these were less than 5 years of age. 3,156 (39.3%) of the 8,036 febrile children screened in the MCRU had a positive blood film for The admission clinical, laboratory and parasitological characteristics of the 583 children with severe malaria are shown in the Figure Severe anaemia was the commonest feature of severe malaria present in 395  of the children. Neurological presentations (either coma or repeated convulsions) were present in 228 (39.2%) of the children. Respiratory distress occurred in 181 (31%) of the children ; hyperlactataemia in 73 (15.7%) and hypoglycaemia in 33 (6.2%) of the children with severe malaria. Hyperparasitaemia (> = 20% of circulating infected red blood cells) was relatively rare, occurring in only 24 (4.1%) of the children with severe malaria.Renal failure, acute pulmonary oedema and spontaneous bleeding are uncommon complications of childhood malaria ,9,10 andFigure Of 583 patients, 52 died , giving an overall case fatality rate of 8.9%. Seven children were lost to follow-up. Forty seven deaths (90%) occurred within the first 24 hours after admission.Neurological sequelae were present in 27 (5 %) of the 531 survivors. The case fatality rate was significantly higher in females than males . Children who died were older than those who survived (mean (SD) = 35.4 (41.9) vs. 25.0 (18.2) months, p = 0.0009).Of the 52 deaths in the course of the study, 32 (61.5%) presented with cerebral malaria, 32 (61.5%) presented with respiratory distress, and 20 (50% of the 40 with measurements) had hyperlactataemia.Thirty (60%) of the children who died presented with convulsions, and 13 (25%) with hypoglycaemia. Twenty four (46.2%) of those who died had severe anaemia. Mortalities in each sub-group demonstrated that hyperparasitaemia  and severe anaemia  had a better prognosis than cerebral malaria  and hypoglycaemia . Thirty-two (17.7%) of the 181 patients with RD died, as did 30 (11%) of the 264 patients with convulsions and 20 (27.4 %) of the 73 patients with hyperlactataemia.A multiple logistic regression model identified coma , hyperlactataemia , respiratory distress  and hypoglycaemia  as independent predictors of a fatal outcome. Severe anaemia, hyperparasitaemia and thrombocytopaenia were not shown to be predictors of death  with severe malaria were afebrile on admission as observed elsewhere [Fever is a characteristic feature of lsewhere . Self-meSevere anaemia was the most frequent feature of severity in this study, but was associated with decreased mortality. A similar observation in a recent Ghanaian study showed a better outcome in children with severe anaemia . These fDespite the increasing toll of HIV infection, and the continuing burden of diarrhoeal disease, malnutrition and respiratory tract infections, malaria remains a major cause of childhood death in endemic regions . The oveThe independent prognostic indicators in this study were cerebral malaria, respiratory distress, hypoglycaemia and hyperlactataemia. These observations are entirely consistent with a large number of studies where the independent predictors of a fatal outcome in malaria are impaired consciousness and metabolic dysfunction  ,14,16-18Estimates of the prevalence of hypoglycaemia have been reported in Africa, ranging from 8% to 34% ,29. In sThe definition of hyperlactataemia used in this study was a blood lactate concentration higher than the conventional cut-off (≥ 5 mmol/L). This was necessary because of the limitations of the analyser used, but probably means that the frequency of true hyperlactataemia was underestimated. The Accusport™ analyser used has been shown to have poor agreement with "gold standard" machines ,15,30.Hyperlactataemia is a frequent and serious complication of severe malaria in childhood ,9,10, whThese differences may partly be explained by low inter-observer agreement for this variable, geographical variations in disease pattern as well as differing definitions of severe malaria. Results from many studies consistently show that respiratory distress is a life-threatening syndrome in childhood malaria ,33. RespThe clinical and laboratory presentations of severe malaria are described in a hospitalized population of children in Gabon. The severe cases are likely to be only the "tip of the iceberg", many children living far from health care units may die whilst travelling to the nearest hospital. Most deaths from malaria occurred in the first 24 hours of admission, which highlights the need for early recognition of the most severely ill children. Early diagnosis and classification of severe malaria would allow appropriate management, including basic adjunctive therapy such as to prevent hypoglycaemia, and better use of scarce healthcare resources. Together these improvements could contribute to a reduction in the intractably high mortality due to the disease.AD is a MCRU clinician. He participated to the study and wrote the article. PN conducted the study for his MD thesis. RT and MB are paediatricians who participated in the study. TP is a UK collaborator who participated in the study and helped write the article. MM was involved with the intensive care of the children. UMR participated in the study as a MCRU clinician. JJ helped to write the article. EK did all the statistical analysis. ENM, PK, SK and MK coordinated the realization of the study and edited the final version approved by all authors.


Pneumonia remains the leading cause of death in young children. The poor specificity of chest radiographs (CXRs) to diagnose pneumococcal pneumonia may underestimate the efficacy of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.The efficacy of nine-valent pneumococcal conjugate vaccine among children not infected with HIV  increased when CXR-confirmed pneumonia was associated with serum C-reactive protein of 120 mg/l (12mg/dl) or more and procalcitonin of 5.0 ng/ml or more . Similar results were observed in children infected with HIV.C-reactive protein and procalcitonin improve the specificity of CXR to diagnose pneumococcal pneumonia and may be useful for the future evaluation of the effectiveness of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia. Adding two blood measurements to the standard X-ray exam might lead to more specific diagnoses for pneumococcal pneumonia and allow more accurate estimate of the efficacy of pneumococcal vaccines While pneumonia remains the leading cause of death in children, the absence of sensitive and specific tools to make an etiological diagnosis is a major limitation to our understanding of the efficacy of vaccines against pneumonia. Blood cultures lack sensitivity, and cultures from lung aspirates may be influenced by delayed presentation, antecedent antibiotic therapy, difficulties finding an accessible site to aspirate, lack of skills in performing the procedure, and the perception of both parents and clinicians that the procedure is too invasive ,2.Procalcitonin, at a low threshold ≥0.25 ng/ml), has been shown to be useful in directing the use of antibiotics in adults with pneumonia , and a r ng/ml, hBased on the postulate that the most likely chest radiograph (CXR) manifestation of pneumococcal pneumonia is alveolar consolidation, we reported the efficacy of a nine-valent pneumococcal conjugate vaccine (PnCV) in reducing CXR-confirmed pneumonia based on definitions recommended by a World Health Organization working group . The obsWe thus evaluated the usefulness of procalcitonin and CRP to improve the specificity of CXR-confirmed pneumonia as an endpoint in vaccine efficacy trials. This analysis was not a primary objective of the study, and is therefore an hypothesis-generating analysis, which should be tested as an a priori hypothesis in other study settings.197  or a placebo at 6, 10, and 14 wk of age. The nine-valent PnCV included serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F   × 100, where ARU is attack rate in unvaccinated individuals and ARV is attack rate in vaccinated indivduals. The vaccine-attributable reduction in disease (VAR) was estimated by measuring the difference in incidence rate between vaccine and placebo recipients and expressed per 100,000 child years of observation. A S. pneumoniae of any serotype from blood.All analyses were performed on an intent-to-treat basis, which included the first event of CXR-confirmed pneumonia in any child who had received at least a single dose of study vaccine. Vaccine efficacy calculations were also performed for bacteremic pneumococcal pneumonia that included all first episodes of pneumonia associated with growth of The efficacy study and subsequent study evaluating the role of procalcitonin and CRP in children with CXR-confirmed pneumonia were approved by the Ethics Committee for Research on Human Subjects, University of the Witwatersrand, South Africa . Guardiap = 0.88). The point estimate of vaccine efficacy in this cohort of children for whom serum was available was 21% , not significantly different from that determined in the original study . At CRP levels of 120 mg/l or more in conjunction with the primary CXR-confirmed outcome measure, the estimate of vaccine efficacy increased to 38% (p = 0.05); at procalcitonin levels of 5 ng/ml or more, the estimated efficacy increased to 46% (p = 0.04); and if both conditions were met, the estimated efficacy increased to 64% (p = 0.006)  of 169 vaccine recipients and 167 (78.8%) of 212 placebo recipients (= 0.006) .p = 0.47). Whereas vaccine efficacy was not significant for CXR-confirmed pneumonia , vaccine efficacy was significant when measured against CXR-confirmed pneumonia in conjunction with a CRP level of 120 mg/l or more  or a procalcitonin level of 5 ng/ml or more . As observed in children not infected with HIV, the point efficacy estimate in children infected with HIV with CXR-confirmed pneumonia was greatest if both CRP and procalcitonin were at or above 120 mg/l and 5 ng/ml, respectively  of 182 vaccine recipients and 153 (73.2%) of 209 placebo recipients  identified 5.3-fold more cases of pneumonia that were prevented by vaccination than was identified by bacteremic pneumococcal pneumonia (VAR = 7) and 4.1-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 9). Similarly, CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml in children infected with HIV (VAR = 772) identified 1.6-fold more cases of pneumonia that were prevented by vaccination than was identified by pneumococcal bacteremic pneumonia (VAR = 483) and 2.2-fold more cases than was identified by vaccine-serotype-specific bacteremic pneumococcal pneumonia (VAR = 344).p = 0.027). This was true also among children infected with HIV .Among the children not infected with HIV with CXR-confirmed pneumonia, the likelihood of a difference between placebo and vaccine recipients was significantly increased by the presence of a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more , which are optimised when vaccine sensitivity is maximised. Our data show that the VAR is greater for CXR-confirmed pneumonia plus CRP ≥120 mg/l plus procalcitonin ≥5 ng/ml than that calculated using the very highly specific endpoints of serotype-specific or all-serotype pneumococcal bacteremic pneumonia. The best estimates of VAR will, however, be provided by endpoints that sacrifice some specificity for maximal sensitivity. Such studies include less specific clinical endpoints and are outside of the scope of the current analysis .Our findings have implications for the planning of future studies aimed at evaluating the effectiveness of pneumococcal vaccines against bacterial pneumonia. Our data suggest that the outcome measure of CXR-confirmed pneumonia together with elevated CRP and procalcitonin levels may be more accurate as a surrogate of pneumococcal pneumonia than CXRs on their own. The increased specificity of this endpoint may allow smaller sample sizes for future vaccine efficacy/effectiveness studies. Based on the incidence of CXR-confirmed pneumonia in this report , a sample size of 80,058 children were required to detect the 20% reduction in CXR-confirmed pneumonia with 80% power and an alpha of 0.05. The actual power for this outcome in this study was thus only 46.8%. Using the more specific outcome measure of CXR-confirmed pneumonia with a CRP level of 120 mg/l or more and a procalcitonin level of 5 ng/ml or more, the sample size required to detect the observed 64% reduction in outcome was 44,734, i.e., 56% of the sample size required when measuring vaccine efficacy against CXR-confirmed pneumonia alone, and the power of the current study would have been increased from 46.8% to 71.5%.Our findings therefore suggest that CXR-confirmed pneumonia coupled with serological markers of CRP and procalcitonin is a more specific marker of pneumococcal pneumonia and may therefore provide a closer estimate of the efficacy of the PnCV against pneumococcal pneumonia.Protocol S1(250 KB DOC).Click here for additional data file.Protocol S2(378 KB DOC).Click here for additional data file.Protocol S3(24 KB DOC).Click here for additional data file.Pneumonia is the leading cause of death in children worldwide. Pneumonia can be caused by different bacteria and viruses, and there are no easy diagnostic tests to find out which bacterium or virus has caused the disease in a particular patient. This not only causes problems with prescribing the best treatment, but also makes it hard to evaluate vaccines that might protect against some causes of the disease but not others.Pneumococcus) that is the leading cause of pneumonia in children. They have begun to test these vaccines in children, but were looking for more specific ways to distinguish cases of pneumonia caused by this particular bacterium from those caused by other bacteria or viruses.The researchers involved in this study have evaluated vaccines against a particular bacterium  is better at assessing whether the pneumonia vaccine works than just a chest X-ray alone.Because of the way this study was done—adding a specific analysis to a clinical trial after it was completed, rather than planning to test a hypothesis from the outset—it cannot be considered as proof of the idea tested. The results suggest that it is worth testing whether the combined diagnosis is more specific for pneumococcal pneumonia than chest X-rays alone, but new studies are needed to resolve the issue.http://www.vaccinealliance.org/The Global Alliance for Vaccines and Immunization (GAVI): http://www.vaccinealliance.org/Resources_Documents/Immunization_Focus/Download/update.phpGAVI Web page “Call for Intensified Research after Pneumococcus Trial Surprises”: http://www.who.int/vaccines/World Health Organization (WHO) Web site on vaccines: http://www.who.int/vaccines/en/olddocs/pneumococcus.shtml)WHO Web page “Pneumococcal Vaccines”:


Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian  and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia.Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades.Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent. Two mtDNA macro-haplogroups (M and N) that arose from the African haplogroup L3 encompass virtually all mtDNAs outside Africa -4. The pThe majority of Indian mtDNAs belong to macro-haplogroup M ,12-21. WThe overwhelming majority of the Iranian mtDNAs have been shown to lie in the West Eurasian domain of the global human mtDNA pool ,28. HereIndia congregates four linguistic domains  that occupy non-random spheres of the geographic distribution of its populations. The majority of the recent studies based on mtDNA variation have, in contrast to some , provideIn this study, we have analyzed the mtDNA variation in a sample of 796 Indians and 436 Iranians Table , and comWe found haplogroup M ubiquitous at almost 58% among the caste, and 72% among the tribal populations Table .With the exception of the diverse set of largely Indian-specific R lineages, the most frequent mtDNA haplogroup in India that derives from the phylogenetic node N is haplogroup W . The freAt 15% among the caste and 8% among the tribal populations haplogroup U is the most frequent sub-clade of R in India Table 12, see , see 2. Over 90% of the mtDNAs found in Iran belong to haplogroups HV, TJ, U, N1, N2 and X, commonly found in West Eurasia Table . This diCompared to India, haplogroup M frequency in Iran is marginally low (5.3%) and there are no distinguished Iranian-specific sub-clades of haplogroup M. All Iranian haplogroup M lineages can be seen as derived from other regional variants of the haplogroup: eleven show affiliation to haplogroup M lineages found in India, twelve in East and Central Asia  and one in northeast Africa (M1).Indian-specific (R5 and Indian-specific M and U2 variants) and East Asian-specific  mtDNAs, both, make up less than 4% of the Iranian mtDNA pool. We used Turkey (88.8 ± 4.0%) as the third parental population for evaluating the relative proportions of admixture from India (2.2 ± 1.7%) and China (9.1 ± 4.1%) into Iran. Therefore we can conclude that historic gene flow from India to Iran has been very limited.Approximately one tenth of the Indian haplogroup M mtDNAs fall into its major sub-clade M2, which is defined by the motif 477G-1780-8502-16319 . M2 can We found that R5, which is defined by transitions at nps 8594 , 16266 aTogether with the Indian-specific sub-clades of haplogroup U2 ,27, haplThe geographic distribution of the M2, R5 and U2i package seems to be rather uniform in the context of the Indian-specific maternal lineages (SAA p > 0.05). When excluding the mtDNAs that are likely to have arrived more recently from West or East Eurasia, the share of the package among the caste populations in northern and southern India is roughly similar . However, in accordance with expectations from the individual haplogroup distributions, the tribal groups speaking Austro-Asiatic and Tibeto-Burman languages are characterized by considerably lower values .versus [Based on the high frequency and diversity of haplogroup M in India and elsewhere in Asia, some authors have suggested (versus ) that M versus ,17,31. Fversus , this stversus  and [ourIn contrast to haplogroup M, ancient sub-clades of haplogroup N are spread both east and west of India as well as within India itself. Several migration scenarios involving multiple "out of Africa" events punctuated by space, time or both, could be invoked to explain the phylogeography of these mtDNA haplogroups. Yet, using the parsimony criterion it can be argued that only a single early migration that brought ancestral lineages, M and N , to South Asia could account for the extant mtDNA phylogeography in Eurasia . The finNearly a third of Indian mtDNAs belonging to haplogroup M could be assigned to its existing boughs and limbs with the current knowledge of the mtDNA coding region polymorphisms  of the mtDNA phylogeny ,36. QuinCoalescence estimates for these Indian-specific mtDNA haplogroups  fall largely between 20,000 and 30,000 ybp. These estimates overlap with those of many West Eurasian-specific  and Several Indian-specific mtDNA clades demonstrate a similar spread-pattern in southern India. We found haplogroups M4a, M6a and M18 in southeastern Tamil Nadu and Andhra Pradesh while they were absent from neighboring Karnataka and Kerala  for difRecently, Basu et al. (2003) supported the conclusions of ,38 by reAlthough two out of the four M2 sequences reported by Basu et al. (2003) among the Lodha, Mundas and Santals  do harbLanguage families present today in India, such as Indo-European, Dravidic and Austro-Asiatic, are all much younger than the majority of indigenous mtDNA lineages found among their present-day speakers at high frequencies see . It woulAdditionally, we note that some recent linguistic and archaeological evidence place the spread of the Austro-Asiatic languages in the Neolithic, in conjunction with the dispersal of rice cultivation from the Yangtze River basin . If thisBroadly, the average proportion of mtDNAs from West Eurasia among Indian caste populations is 17% Table . In the In comparison to an overall frequency of 17% CR 15.1–19.3%) among the caste populations, only 7% (CR 6.2 – 9.0%) of the mtDNAs from the tribal groups show affiliation to the West Eurasian haplogroups. The observed difference could be caused by differences in the extent of gene flow from the west to different social layers of the Indian society [.3% amongThis western Eurasian contribution into the Indian maternal gene pool can be broadly divided into two different components. Over two thirds of the West Eurasian-specific mtDNAs found in India are made up by haplogroups HV, TJ, N1 and West Eurasian-specific branches of haplogroup U. It is likely that these mtDNA haplogroups have been carried to western India both by relatively low-intensity long-lasting admixture at the border regions as well as a consequence of numerous but probably limited migrations during the last 10, 000 ybp . The remSimilarly to HV, TJ, N1, the spread of U7, W and R2 in India is geographically uneven – the three haplogroups are much more frequent in the northwestern states  of the 1136 mtDNA haplotypes found among continental Indians (including Pakistan and Bangladesh) are singletons and 41% of those that occur more than once are restricted to a single population. Only a few haplotypes are shared among five or more populations.The number of shared haplotypes between pairs of social, linguistic and geographic groups of Indian populations is slightly (but in most cases insignificantly) lower than that between random groups of Indian populations taken for reference . Where the decline of shared haplotypes is significant relative to the reference, it is most probably caused by large differences in the sample sizes of the groups under comparison Table .An alternative method that assesses the degree of haplotype sharing between populations is to investigate the combined frequency of the shared haplotypes in two population groups. Thus, amongst the northern and the southern population groups the combined frequency of the haplotypes present also in the other group is significantly lower than that which we observed in the case of random groups. This is not surprising because West Eurasian-specific mtDNA haplogroups are rather frequent in northwest India. Because the Indo-European and the Dravidic speakers of India are largely concentrated to the northern and southern parts of the subcontinent, respectively, the differences arising from geographic division of the Indian populations also correspond to these linguistic groupings Table .Three Indian-specific haplogroups, M2, U2i and R5, which encompass about 15% of the Indian mtDNA pool, exhibit equally deep coalescence ages of about 50,000 – 70,000 years. Thus, their spread can be associated with the initial peopling of South Asia.Haplogroups U7, W and R2 harbor a number of similar traits. Their overlapping geographic distributions and coalescence times suggest some degree of genetic continuum in the area spanning from the Near and Middle East through northwest India and reaching north into Central Asia. The coalescence estimates for these haplogroups are equally deep  in these different regions. That may be a result of either relatively more recent albeit large in scale migrations that brought along most of the diversity or may indeed reflect the region-specific expansions of these haplogroups. The former explanation could be ruled out since it is impossible to envisage a substantial movement of mtDNAs from South Asia that would not include haplogroup M. The same is true for the opposite – the share of U7, W and R2 within the West Eurasian-specific mtDNA haplogroups is two-fold higher in India than it is in Iran. Moreover, the South- and West Asian-specific sub-branches of haplogroup U7 predate the last glacial maximum. Therefore, deep autochthonous history of these haplogroups in the region remains to be the most parsimonious explanation.Through the use of mtDNA coding region markers, we were able to classify altogether a quarter of the Indian M and R mtDNAs into a number of Indian-specific mtDNA haplogroups, four of which we newly identified. Several of these are characterized by clear patterns in their geographic distribution and/or different frequencies among different socio-cultural groups of India. Additional efforts should be undertaken to identify new coding region markers in order to further improve the classification of Indian mtDNAs. Indeed, much of the information is still hidden by the poor resolution of the Indian mtDNA tree and further piling of HVS-I datasets would add little to deepen our understanding of the demographic history of South and Southwest Asia.in situ by major demographic episodes in the past and garnished by relatively minor events of gene flow both from the West and the East during more recent chapters of the demographic history in the region.We found that haplogroup M frequency drops abruptly from about 60% in India to about 5% in Iran, marking the western border of the haplogroup M distribution. A similarly sharp border cuts the distribution of Indian-specific mtDNA haplogroups to the east and to the north of the subcontinent. We therefore propose that the initial mtDNA pool established upon the peopling of South Asia has not been replaced but has rather been reshaped MtDNA sequence variation in a total of 796 Indian samples most of which are held in a collection at Newcastle University  was analTribal populations constitute 15% of the total sample size. The Lodha (n = 56) live mostly in the western part of Midnapore district of West Bengal where they are also known as Kheria or Kharia. Their total population size was ~59000 according to the 1981 census. Their language belongs to the Mundari branch of the Austro-Asiatic language family . The KanThe non-tribal Indian samples analyzed contained 105 West Bengalis of different caste rank, 58 Konkanastha Brahmins from Bombay, 53 Gujaratis, 50 Moors and 82 Sinhalese from Sri Lanka, 109 Punjabis of different caste rank from the Punjab, 25 Brahmins from Uttar Pradesh, 35 Rajputs from Rajasthan, 55 Parsees from Maharashtra and 100 subjects from Cochin, Kerala (including 45 Jews who have moved to Israel)   and KyrgDNA was extracted using standard phenol-chloroform methods . The hyp2) were calculated following [Median networks ,61 were ollowing . CoalescThe software kindly provided by Vincent Macaulay was adopted in order to calculate the 95% credible regions (CR) from the posterior distribution of the proportion of a haplogroup/sub-haplogroup in the population.Haplotype diversity was estimated asi number of sequences with one distinct haplotype.where n is the number of sequences, k the number of distinct haplotypes, and nHaplogroup isofrequency maps were generated using the Kriging method in Surfer 7 program of Golden Software. Haplogroup frequencies were averaged over populations from the same state in India, provinces in China and Thailand and geographic divisions in Iran. The data points for Kriging are shown as black dots, while the sample size applicable to the data point is given adjacent to the dot. In relatively small and isolated groups  random genetic drift might seriously affect the haplogroup frequencies, which may become uninformative when a whole region (e.g. state) is considered . Therefore, the tribal data were excluded from the haplogroup isofrequency maps calculation. When illustrating the spread of mtDNA haplogroups native to West Eurasia, East Eurasia and India Figure , panel DSpatial autocorrelation analysis was done using the PASSAGE software packet ,66. The Haplogroup frequencies based admixture proportions were calculated using the ADMIX 2.0 software .Haplotypes were defined as the HVS-I motif combined with the haplogroup label for haplotype analysis. In this way it was possible to discriminate between mtDNAs with identical HVS-I motifs but otherwise known to belong to different haplogroups (e.g. sequences with the CRS motif in HVS-I belonging to haplogroups H or R). In order to assess the results of haplotype sharing analysis between population groups  we divided the Indian populations ten times randomly into two sets and analyzed the level of haplotype sharing between these sets.SM and SSP collected most of the Indian samples (in 1970's) and have been keeping the collection in Newcastle University, United Kingdom. MM, TK, GH and KK carried out the RFLP analysis and mtDNA sequencing of the majority of the Indian samples. EM, JP, PS and MK carried out the RFLP analysis and mtDNA sequencing of the Iranian samples. DMB collected the DNA and analyzed mtDNA variation amongst the Indian samples from Cochin and the Cochin Jews collected (the latter) in Israel under supervision of KS, who also contributed to the editing of the manuscript. AT provided some critical new information and, in addition to KS, MTPG and PE, engaged into valuable discussions during manuscript preparation. MM did most of the phylogenetic analysis and was in charge of manuscript writing while TK and RV contributed significantly during both stages and were responsible for conceiving and designing the study. All authors read and approved the final manuscript.Table 8. Excel spreadsheet. MtDNA haplogroup frequencies among tribal populations. Frequencies of mtDNA haplogroups amongst the tribal populations of India, China and Thailand as averaged over states of India and provinces in China and Thailand.Click here for fileTable 12. Excel spreadsheet. Comparison of the mtDNA haplogroup frequencies amongst the Indian caste and tribal populations.Click here for fileTable 9. Excel spreadsheet. MtDNA haplogroup frequencies in India and Iran.Click here for fileTable 11. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian caste populations as averaged over states of India.Click here for fileFigure 12. Image file in PNG format. Network of HVS-I haplotypes belonging to haplogroup R5. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in  minus 16,000 and shown along links between haplotypes. The diagnostic R5 coding region marker 8594 is shown in bold and numbered as in . Character changes are specified only for transversions. Underlined samples are those in which the marker 8594 was assayed by either RFLP analysis (-8592 MboI) or direct sequencing. Sample codes are as in Table 6 . Coalescence estimates marked with an asterisk are calculated excluding tribal populations .Click here for fileTable 10. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian populations.Click here for filein silico and by typing for additional mtDNA coding region markers.Table 7. Excel spreadsheet. MtDNA variation in the studied populations (raw data). MtDNA control and coding region variation in the populations that were used in the study. The database includes both the newly obtained datasets and the previously published datasets. The latter were in many cases reanalyzed both Click here for fileTable 6. Excel spreadsheet. The list of studied populations. List and details of the populations whose mtDNA were used in the study. This includes both newly obtained datasets and previously published datasetsClick here for fileTable 13. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst different linguistic groupings of Indian populations.Click here for file


Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. This effect seems to be due to aromatic glucosinolate content. Glucosinolates are known for have both antiproliferative and proapoptotic actions.Maca is a cruciferous cultivated in the highlands of Peru. The absolute content of glucosinolates in Maca hypocotyls is relatively higher than that reported in other cruciferous crops. Therefore, Maca may have proapoptotic and anti-proliferative effects in the prostate.Male rats treated with or without aqueous extracts of three ecotypes of Maca  were analyzed to determine the effect on ventral prostate weight, epithelial height and duct luminal area. Effects on serum testosterone (T) and estradiol (E2) levels were also assessed. Besides, the effect of Red Maca on prostate was analyzed in rats treated with testosterone enanthate (TE).Red Maca but neither Yellow nor Black Maca reduced significantly ventral prostate size in rats. Serum T or E2 levels were not affected by any of the ecotypes of Maca assessed. Red Maca also prevented the prostate weight increase induced by TE treatment. Red Maca administered for 42 days reduced ventral prostatic epithelial height. TE increased ventral prostatic epithelial height and duct luminal area. These increases by TE were reduced after treatment with Red Maca for 42 days. Histology pictures in rats treated with Red Maca plus TE were similar to controls. Phytochemical screening showed that aqueous extract of Red Maca has alkaloids, steroids, tannins, saponins, and cardiotonic glycosides. The IR spectra of the three ecotypes of Maca in 3800-650 cm (-1) region had 7 peaks representing 7 functional chemical groups. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl glucosinolate.Red Maca, a cruciferous plant from the highland of Peru, reduced ventral prostate size in normal and TE treated rats. Lepidium meyenii, a traditional Peruvian cruciferous vegetable known as Maca, grows exclusively at altitudes over 4000 m. The hypocotyl, the edible part of the plant, is used as a nutritional supplement and for its enhancing sperm production properties [operties . Maca isoperties .Lepidium [Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer -5. CruciLepidium ,6, incluIn the many hundreds of cruciferous species investigated, all are able to synthesize glucosinolates . GlucosiUpon ingestion by humans  or rats Almost all of the mammalian chemoprotective activity from cruciferous is due to these isothiocyanates ,14. TherLepidium meyenii (Maca) may have important effects on prostate. More recently, it has been demonstrated that an integral suspension of Lepidium latifolium significantly reduced prostate size and volume in castrated rats where the hyperplasia was induced by steroid treatment [The absolute content of glucosinolates in fresh Maca hypocotyls is relatively higher than reported in other cruciferous crops . The mosreatment . For sucad libitum. All animal experiments were conducted in compliance with "Guide for the care and use of laboratory animals" of the National Institutes of Health from the USA [Adult Holtzman rats were obtained from our Animal House at the Universidad Peruana Cayetano Heredia and used for the present study. The rats were maintained 4–6 per cage at environmental temperature 22°C) with a 12:12 h light/dark cycle. Also they were fed with Purina laboratory chow and tap water °C with aRats were treated with vehicle, Yellow Maca, Red Maca or Black Maca for 7 days in dose of 2 g dried Maca hypocotyls/Kg BW. This dose was selected from a previous dose response study . Each MaRats were injected (i.m) with 0.1 ml (25 mg) of testosterone enanthate (TE) on day 1 and day 7. Control rats received 0.1 ml oil (im) at day 1 and at day 7. A group treated with TE received also Red Maca (2 g/Kg) for 14 days and another group treated with TE received Red Maca for 42 days. Control rats received vehicle by oral route for 14 or 42 days. Oral treatment (Maca or vehicle) and intramuscular treatment (TE or vehicle) both started on day 1.Lepidium meyenii were obtained from Carhuamayo, Junin at 4000 m altitude. The ages of different Maca plants were similar. All hypocotyls were obtained at the same time. Irma Fernandez, a Botanist of the Department of Pharmaceutical Sciences at Universidad Peruana Cayetano Heredia, authenticated the identity of the plant by visual inspection. The biological activity of the plant is located in the hypocotyls, which are consumed by natives after natural drying. Traditionally, the dried hypocotyls of Maca are boiled and served as juice.The dried hypocotyls of For the present study, the aqueous extract of the hypocotyls was prepared according to the traditional method. First, 500 g of the dried hypocotyls were pulverized and placed in a container with 1500 ml of water, and boiled for 120 minutes. Next, the preparation was left standing to cool, and then it was filtered. Finally, the filtrate containing 333 mg of dry Maca hypocotyls in 1 ml was placed in small vials and kept in 4°C refrigerator.One day after the last treatment, rats were sacrificed by decapitation. Blood sample was obtained from cervical trunk from rats treated for 7 days with different ecotypes of Maca. Blood samples were centrifuged at 1,000 g, and sera were separated, placed in vials and kept frozen until assayed for sex hormone levels. Ventral prostate was used for histological study.After animals were sacrificed several organs  were collected, dissected free of fat, and weighed.Serum estradiol and testosterone concentrations were measured by radioimmunoassay using commercial kits . The hormone labeled with iodine-125 was used as radioactive marker. Samples were run in the same assay to avoid inter-assay variation. The intra-assay variation was 6.42% for estradiol, and 5.5% for testosterone. Sensitivity of testosterone assay was 4 ng/dl and for estradiol assay was 8 pg/ml.Ventral prostate lobes obtained from experiment 2 were excised and dissected free of fat. Ventral prostates (VP) were immersion-fixed in Bouin's fixative. After their dehydration, VP were embedded in paraffin. The tissue blocks were sectioned into 5 um thickness and stained with hematoxylin and eosine (H&E), and then observed under a light microscope.2) were measured by sampling 10 random sections per slide in the peripheral region of the ventral prostate. In each duct, 20 cells were measured for epithelial height. All assessments were performed using an axiostar plus microscope . The images were captured by a Moticam2000  coupled to a personal computer. Motic image plus 2.0 software (Motic Instruments Inc.) was used for measurements of prostatic epithelial height and duct luminal area and calculated by statistic ANOVA test. Pictures at 50× and 400× magnifications are included.Epithelial height (um) and duct lumen area . An overhead-attenuated total refraction (ATR) accessory was equipped as the sample stage for solid samples. All spectral measurements were done at 1 cm-1 resolutions. Data are presented as absorbance units. Each peak represents the presence of a functional chemical group. Differences in the height of absorbance peaks reflect differences in amount of functional groups.IR spectra of lyophilized aqueous extracts of Red, Yellow and Black Maca were measured from 3800 cmData were analyzed using the statistical package STATA (version 8.0) for personal computer .Data are presented as mean ± standard error of the mean (SEM). Homogeneity of variances was assessed by the Bartlett test. If variances were homogeneous, differences between groups were assessed by analysis of variance (ANOVA). If F value in the ANOVA test was significant, the differences between pair of means were assessed by the Scheffeé test. If variances were non homogeneous, non parametric tests were used.P < 0.05 was considered statistically significant.A value of Ventral prostate weight was significantly reduced in rats treated for 7 days with Red Maca (P < 0.05). Black Maca and Yellow Maca did not modify ventral prostate weight Figure . SeminalMeans of serum testosterone and estradiol levels were similar between control group and groups treated for 7 days Figure  with RedTreatment with TE increased significantly ventral prostate weight almost to double value of the control group (P < 0.05) Figures  and 4a. Red Maca administered for 14 days also reduced ventral prostate weight in rats treated with TE (P < 0.05). The effect was more noticeable after 42 days of treatment with Red Maca (P < 0.05). After 42 days of treatment with Red Maca, the ventral prostate weight was reduced more than 50% Figure . After 1Ventral prostate weight related to body weight was lower in the group treated for 14 days with Red Maca (0.11 ± 0.01 g/100 g BW) than in controls (0.15 ± 0.006 g/100 g BW) (P < 0.05). Rats treated with TE plus Red Maca had lower prostate weight related to BW  than rats treated with TE (0.29 ± 0.01) but prostate weight relative to BW was still higher in rats treated with TE plus Red Maca than in controls (P < 0.05). The same pattern was observed when rats were treated for 42 days with Red Maca (Data not shown).Rats treated with two TE injections showed lower body weight at day 42 after the first injection (P < 0.05) compared to controls (290.63 ± 30.48 g and 383.60 ± 11.31 g in TE and vehicle treated groups). This low body weight was also observed in the group treated with TE plus Red Maca (319.30 ± 3.08 g) (P < 0.05).Weights of testes, epididymis, kidneys, liver, spleen, lungs and heart were not affected by treatment with Red Maca for 7, 14 or 42 days (Data not shown).Sections of ventral prostate in the peripheral region of the ductal system after 14 and 42 days of treatment with vehicle (control), Red Maca, ET, or ET plus Red Maca are shown in Figures At 50× magnification, the number of ducts per field from rats treated with Red Maca for 14 days was slightly higher than in controls Figures  and 5c. At 50× magnification, compared to control Figure , the treQuantitative analyses of epithelial height and luminal area in rats treated for 42 days are presented in Figure Rats treated during 2 weeks with injections of TE once a week showed higher prostatic epithelial height (P < 0.05) and duct luminal area (P < 0.05) at day 42 compared to controls Figure . Rats trThe phytochemical analysis of the ethanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, saponins and cardiotonic glycosides, and the absence of flavonoids, anthraquinones, tannins, sesquiterpene lactones and coumarins Table .The phytochemical analysis of the methanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, tannins and saponins, and the absence of flavonoids, coumarins, anthraquinones, sesquiterpene lactones and cardenolides Table . The pos-1 region had 7 peaks, which were at 3291 cm-1, 2927 cm-1, 1614 cm-1, 1406 cm-1, 1022 cm-1, 924 cm-1 and 862 cm-1. These peaks are due to C-H, OH, amides, amines, carboxylic acids, aromatic, and alkyls groups, respectively. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl glucosinolate , a cruciferous plant that grows exclusively over 4000 m in Peruvian Andes, affect ventral prostate size. It was of great interest to demonstrate that Red Maca reduced significantly ventral prostate weight. This effect was not observed after treatment with Yellow or Black Maca. The effect of Red Maca was specific for prostate, since other organs as testes, epididymis, seminal vesicles, kidneys, spleen, liver, lungs and heart were not affected.The present study was designed to determine if different ecotypes of It was also demonstrated an effect of Red Maca on rats in which ventral prostate size was enlarged by two injections of testosterone enanthate. In fact, Red Maca administered for 14 or 42 days reduced the effect of TE. At 42 days, the ventral prostate size of rats treated with TE plus Red Maca was similar to that of control rats treated only with vehicle. Epithelial height and luminal areas were proved to be sensitive parameters for the evaluation of androgen effects on prostates . The preGrowth of the prostate is a hormone-mediated phenomenon regulated by both androgens and estrogens . HoweverLepidium could be an important alternative for treatment of prostate diseases. Other Brassica from the genus Lepidium, as Lepidium latifolium reduced prostate weight [Lepidium may have important anti-proliferative and proapoptotic effects. In Red Maca treated rats, cell size has decreased and membrane blebbing and nuclear distortion are apparent suggesting a pro-apoptotic effect.Recently, increasing evidence has been presented suggesting that cruciferous (Brassicas) vegetables may reduce the risk of prostate cancer development ,4. The ge weight  suggestiIt is still unknown the active principle for the effect of Red Maca on ventral prostate and why the action is specific since any other organ was affected. Moreover, the different effects among ecotypes seem to be due to different amount of active metabolites.Lepidium meyenii. In the aqueous extract is possible to find glucosinolates [This study used aqueous extract of dried hypocotyls of inolates  and anthinolates . Both coinolates -18,30. Ainolates . This alinolates . Beta cainolates  and theyinolates . FurtherIndeed, the data presented here show that Red Maca reduced ventral prostate size in normal adult rats and also in rats treated with testosterone enanthate. Hence, it is proposed that Red Maca may have important implications under pathological conditions of the prostate.GFG conceived of the study participating in its design, coordination, and drafting the manuscript.SM participated in the design of the study and the study of different ecotypes of Maca.JN participated in the biological study with different ecotypes of Maca.GF participated in the phytochemical screeningJR participated in the statistical analysisSY participated in the histological studyPY participated in the histological studyMG participated in the design and analysis of results, and its interpretation.All authors read and approved the final manuscript.


The growing library of sequenced genomes is challenging scientists to extract new biological meaning from DNA sequences. Comparative analysis of the mouse and human genome, for example, has already revealed that mutation rates in the 3 billion base pairs of the human genome vary considerably. What accounts for this regional disparity, however, is unclear. Mutations—substitutions in the nucleotide bases of DNA—produce variation in the genome. In classical evolutionary theory, natural selection drives evolutionary change by determining which of these mutations live on in the next generation or die with the organism. Mutations can be neutral, harmful, or beneficial, though the neutral theory of molecular evolution predicts that most mutations are “nearly” neutral or only slightly deleterious, while beneficial mutations—which confer a survival advantage on an organism and, if it reproduces, on its progeny—are quite rare. As a whole, mutations occur at the rate of approximately five substitutions per billion nucleotide sites per year.There are many types of neutral mutations—that is, mutations that have no effect on function. DNA base substitutions that lie outside of gene-coding regions or occur within introns (regions that are excised before being translated into a protein sequence) can fall into this category. Neutral mutations can also occur within gene-coding regions. For example, there are many instances where more than one codon—say, CUU, CUC, CUA, CUG—specify the same amino acid—in this case, leucine. Since these mutations can be used to gauge the neutral mutation rate of a region in the genome, they can be used to analyze the relationship between local mutation rates and gene location. Correlating gene mutation rates with their location in the genome, Jeffrey Chuang and Hao Li not only confirm that regional mutation rates indeed exist, but also calculate the size of these regions. Strikingly, certain classes of genes tend to congregate in mutational “hot spots”—regions with high mutation rates—while other types of genes gravitate toward “cold spots”—regions with relatively low mutation rates.Chuang and Li first determined whether mutation rates have regional biases—that is, whether the frequency and distribution of mutations follow a distinct pattern along the genome. The researchers calculated the substitution rates of neutral mutations in nearly 15,000 orthologous mouse and human genes—orthologous genes are genes that have evolved from a common ancestor without diverging in biological function—and found that mutation rates were in fact skewed toward either high or low rates. Mutation rate analysis of the orthologs' neighbors revealed rates similarly skewed toward high or low substitutions, indicating that the region itself, rather than a particular gene, is prone to these differential rates. These regions, Chuang and Li report, were either one megabase or ten megabases long, affecting up to roughly 100 genes.But the question remained: Does the organism take advantage of these mutational hot and cold spots? If there is an adaptive advantage, gene families should occur in an appropriate mutational zone. In mutational hot spots, for example, one would expect to find genes that would benefit from high rates of mutation, which would in turn facilitate flexible responses to constantly changing environmental stimuli. Likewise, one would expect genes in cold regions to need protection from potentially deleterious mutations. And that's just what Chuang and Li found. Overall, genes in hot regions code for proteins involved in cell signaling, such as olfactory receptors, G-protein coupled receptors, membrane proteins, and immune response proteins—being in an area subject to high mutation rates means these genes can evolve quickly enough to adapt to constantly changing stimuli. Cold-region genes code for “housekeeping” proteins involved in core cellular processes, like transcription regulation and protein modification—these genes tend to be highly conserved, changing very little since they first evolved.Thus, it appears that natural selection may also operate at the level of gene location, relegating genes to different mutational genomic niches according to their function. While Chuang and Li explore possible mechanisms to account for these genomic niches—such as gene duplication and gene transposition—they argue that the selective pressures that influence gene location are the same that influence mutations in genes. By calculating the sizes of these mutational hot and cold regions, the researchers lay the groundwork for investigating genetic mechanisms that operate on these scales. And by showing that location matters, they have revealed a new force in genome evolution.


Isoprenylcysteine carboxyl methyltransferase (Icmt) is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes.In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet) provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC). Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA). Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy) and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME). This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and that BFC and AFCME bind to different forms of the enzyme.These studies establish that catalysis by human Icmt proceeds through an ordered sequential mechanism and provide a kinetic framework for analysis of specific inhibitors of this key enzyme. Posttranslational modification of eukaryotic proteins with lipids is a prevalent mechanism for controlling the subcellular localization and activity of these proteins . Most prWhile isoprenoid modification of CaaX proteins is the principal determinant of their membrane targeting, the subsequent steps of proteolysis and methylation are clearly important. Studies performed using small molecule prenylcysteines such as N-acetyl-S-farnesyl-L-cysteine (AFC) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC)  showed tAlthough Icmt is a protein methyltransferase, the enzyme can efficiently modify simple prenylcysteines ,20-22, aMethods). BFC was characterized in terms of its ability to serve as a substrate for recombinant human Icmt using a standard in vitro methylation assay . Hence, BFC is a comparable to K-Ras as an Icmt substrate and has the added advantages of being a stable small molecule that can be readily captured on avidin resins.To overcome the inherent problems associated with use of S-prenylated peptides and proteins as Icmt substrates , we synthesized a small molecule substrate for the enzyme that contains an appended biotin moiety Fig.  to facilsay Fig. . The appDetailed knowledge of the kinetic mechanism of an enzyme is very important in providing the foundation for analysis of structure-function studies and in inhibitor design studies. For a two substrate enzyme reaction such as that catalyzed by Icmt, there are three basic mechanisms: i) random sequential, ii) ordered sequential, and iii) ping-pong ,29. Initi of 1.2 ± 0.2 μM  and AdoHcy. To determine the order of the dissociation of these two products, we performed product inhibition using the prenylcysteine methylester, AFCME, and AdoHcy. Examination of the initial rates of product formation at fixed BFC and varying AdoMet concentrations in presence of three concentrations of AdoHcy  essentially identical to that of an authentic substrate, farnesylated, Rce1-proteolyzed, K-Ras protein, whereas the apparent Km for AFC is 10-fold higher at 20 μM -S-adenosyl-L-methionine was purchased from Perkin Elmer Life Sciences, farnesylthioacetic acid (FTA) was purchased from Biomol, S-(5'-adenosyl)-L-homocysteine was purchased from Fluka. The N-acetyl-S-farnesylcysteine methyl ester (AFCME) and N-acetyl-S-farnesylcysteine methyl amide (AFCMA) were synthesized as previously described . Rec. Rectranscribed ; the butanol phase was then evaporated under reduce pressure and the FC product washed several times with hexane to remove residual farnesyl bromide.S-Farnesyl L-cysteine (FC) was prepared by reaction of farnesyl bromide with L-cysteine in methanol/ammoniac solvent as described . The res2O (1:1). The butanol phase was dried and the residue dissolved in 100 μl of methanol; this solution was then diluted to 1 ml by addition of 0.1% trifluoroacetic acid (TFA). The addition of the TFA resulted in the appearance of a white precipitate of essentially pure BFC as judged by chromatography and NMR analysis.Two coupling procedures were used to attach the biotin moiety to the amino group of FC; both involved the use of biotin N-hydroxysuccinimide methylester (biotin-NHS) as the biotinylation agent. In the first method, an excess of biotin-NHS (75 μmol) to FC (7.5 μmol) was used. The two compounds were dissolved in 2.3 ml of DMSO and 200 μl of 1M Hepes, pH 12, was added. Following a 2 h incubation at room temperature, the DMSO was evaporated under reduced pressure and the resulting residue extracted with a solution of butanol/H2O (1:1), and the butanol phase dried and the residue dissolved in methanol. Because of the excess of FC present with the BFC product, specific precipitation of the BFC product was unsuccessful. Instead, the BFC was purified by preparative HPLC on a C18 column developed in CH3CN/H2O (2:3). On this matrix, BFC was well-resolved from FC. The peak fractions were collected and solvent evaporated under reduced pressure; the resulting product was white solid that was >95% pure as judged by analytical reverse-phase HPLC methyl incorporation into the small molecule substrate BFC. For the standard assay, reactions were initiated by addition of Sf9 membranes containing Icmt (0.5 μg protein) to an assay mixture containing BFC (4 μM) and [3H]AdoMet  in100 mM Hepes, pH 7.4 and 5 mM MgCl2 in a total volume of 45 μl. Reactions were carried out for 20 min at 37°C, whereupon they were terminated by addition of 5 μl of 10% Tween 20. Following termination, streptavidin beads  were added, and the mixtures mixed by gentle agitation overnight at 4°C. The beads were harvested by centrifugation in a tabletop microcentrifuge at 10,000 rpm for 5 min and washed 3 times with 500 μl of 20 mM NaH2PO4, pH 7.4, containing 150 mM NaCl. The beads were then suspended in 100 μl of the same buffer, transferred to scintillation vials, and radioactivity determined. For the kinetic analyses, the concentrations of substrates  or additional ligands  were varied as detailed in the legends to the appropriate figures.The assay developed for Icmt activity involved quantitation of [Icmt, Isoprenylcysteine carboxyl methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; AFC, N-acetyl-L-farnesylcysteine; AGGC, N-acetyl-S-geranylgeranyl-L-cysteine; TFA, trifluoroacetic acid; BFC, biotin-S-farnesyl-L-cysteine; FTA, S-farnesylthioacetic acid; AFCME, N-acetyl-S-farnesyl-L-cysteine methylester; AFCMA, N-acetyl-S-farnesyl-L-cysteine methyl amideRAB carried out all of the studies reported. PJC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Both authors read and approved the final manuscript.


The Pharmaceutical Benefits Scheme (PBS) grew by 8% in 2003–04; a slower rate than the 12.0% pa average growth over the last decade. Nevertheless, the sustainability of the Scheme remained an ongoing concern given an aging population and the continued introduction of useful (but increasingly expensive) new medicines. There was also concern that the Australia-United States Free Trade Agreement could place further pressure on the Scheme. In 2003, as in 2002, the government proposed a 27% increase in PBS patient co-payments and safety-net thresholds in order to transfer more of the cost of the PBS from the government to consumers. While this measure was initially blocked by the Senate, the forthcoming election resulted in the Labor Party eventually supporting this policy. Recommendations of the Pharmaceutical Benefits Advisory Committee to list, not list or defer a decision to list a medicine on the PBS were made publicly available for the first time and the full cost of PBS medicines appeared on medicine labels if the price was greater than the co-payment. Pharmaceutical reform in Victorian public hospitals designed to minimise PBS cost-shifting was evaluated and extended to other States and Territories. Programs promoting the quality use of medicines were further developed coordinated by the National Prescribing Service, Australian Divisions of General Practice and the Pharmacy Guild of Australia. The extensive uptake of computerised prescribing software by GPs produced benefits but also problems. The latter included pharmaceutical promotion occurring at the time of prescribing, failure to incorporate key sources of objective therapeutic information in the software and gross variation in the ability of various programs to detect important drug-drug interactions. These issues remain to be tackled. This paper reviews the growth of the Pharmaceutical Benefits Scheme (PBS) during 2002–03; concerns about the sustainability of the Scheme, the government's response, a potential new threat that emerged and issues that remain to be tackled.From March 2003 to March 2004, a total of $5.8 billion was spent on prescription medicines subsidised under the PBS. Of this, $4.89 billion (84%) was paid by the Commonwealth, the remaining $0.91 billion through patient co-payments [In 2003, as in 2002, the government proposed a 27% increase in PBS patient co-payments and safety-net thresholds in order to transfer more of the cost of the PBS from the government to consumers. Once again this measure was rejected by Labor and other opposition parties in the Senate because of concern that such increases would impact on equitable access to necessary medicines . RegardlBy mid 2004, the Labor party was faced with an impending Federal election and had serious trouble costing its tax and spending promises. As a consequence, Labor abandoned their previous principled stand in the Senate of blocking the government's proposed increase in PBS copayments and safety-net thresholds  arguing Several other measures were introduced by the government in order to improve the community's understanding of PBS processes and costs. From June 2003, all recommendations of the Pharmaceutical Benefits Advisory Committee (PBAC) to list, not list or defer a decision to list a medicine on the PBS were made publicly available on the PBS website . UnfortuFrom 1 August 2003 the full cost of PBS medicines appeared on medicine labels if the price was greater than the co-payment. The full cost included what the consumer has paid and the amount that is paid through the PBS. The aim was to help people understand what medicines really cost and how the PBS helps make medicines affordable for all. In addition, the government commissioned a $24 million advertising campaign that emphasised that patient responsibility was, "the prescription for a healthy PBS". Critics noted that by neglecting to inform the public that pharmaceutical marketing and inappropriate prescribing habits of doctors also produced pressures on the PBS, the campaign missed an opportunity to initiate a more balanced and constructive debate about the viability of the PBS .During the year under review, pharmaceutical reforms designed to stop PBS cost-shifting in Victorian public hospitals were evaluated . The refPrograms promoting the quality use of medicines (QUM) were further developed throughout 2002–03. Specific programs were coordinated by the National Prescribing Service (NPS), Australian Divisions of General Practice and the Pharmacy Guild of Australia.The NPS was set up in 1999 with government funding but with an independent Board of Directors in order to provide unbiased educative activities to assist health practitioners (and more recently consumers) to use medicines wisely. Evaluation of NPS activities has consistently shown that spending money on targeted QUM interventions can save considerably more money on the PBS by reducing inappropriate prescribing. It was estimated that NPS activities during the period 1 July 2000 to 30 June 2002 generated PBS savings in the range of $55.6 million to $83.9 million through the following prescribing intervention programs: antibiotics in primary care; peptic ulcer management; management of dyspepsia; COX-2 selective NSAIDs; managing hypertension; and managing dyslipidaemia .In 2003, the NPS received an additional allocation of government money to provide educational material about drugs newly listed on the PBS (the RADAR project). The latter was in response to considerable evidence that intensive pharmaceutical promotion at the time of PBS listing was associated with drugs being prescribed for broader indications than those indicated in the PBS listing  . HoweverThe Enhanced Divisional Quality Use of Medicines (EDQUM) program was a 1999–2000 Federal Budget initiative, originally announced as the Incentives for Quality Prescribing (IQP) program. The program offered Divisions of General Practice (Divisions) a proportion of monies saved if Divisional QUM activities improved prescribing and lowered PBS costs. The Divisions were allowed to use any savings made for a range of primary health care activities. The program evolved significantly due to feedback from the medical profession. It was implemented on a pilot basis in thirteen Divisions on 1 July 2002. Under the program, Divisions were encouraged to invest their own resources in a range of drug utilisation data collection and/or education related activities. Activities were implemented in close consultation with the NPS and focused on one or more of the following target drug groups: antibiotics, peptic ulcer drugs and cardiovascular drugs.An evaluation of the pilot EDQUM project was undertaken in early 2004 . BarrierThe Third Community Pharmacy Agreement between the Government and the Pharmacy Guild of Australia (1 July 2000 to 30 June 2005) also provided a range of QUM activities over the year in question including medication reviews of problem patients (conducted at the request of GPs), quality care pharmacy programs and the provision of consumer medicine information .In 2003–2004 the PBS became caught up in negotiations concerning the Australia-United States Free Trade Agreement (AUSFTA). This saga has been extensively reported elsewhere . The govThe EDQUM project highlighted the needs for software standards in order to extract comparable drug utilisation data from different prescribing systems. The need for prescribing software standards has also been raised in connection with three other issues of relevance to the PBS: pharmaceutical promotion, independent therapeutic information and drug-drug interaction checking.The uptake of computers by Australian general practitioners (GPs) was stimulated by the Australian government in 1999. A one-off grant of around $10,000 was offered to those practices that purchased a computer, acquired internet connectivity (an E-mail address) and promised to use computer prescribing software to write the majority of their prescriptions. This increased the numbers of GPs writing prescriptions with the aid of a computer from around 50% in 1999 to more than 90% in 2004 . LegibleOne software vendor  became the dominant market leader because its business model relied on pharmaceutical promotion to heavily subsidise the cost of GPs purchasing and updating its prescribing software . This business model facilitated software uptake but also resulted in advertisements for the latest and most expensive drugs appearing on the computer screen at the time of prescribing (and elsewhere). GPs using this software package were shown to prescribe more antibiotics per patient than those who wrote 'scripts manually. It was suggested that this may have been due to default settings in the software automatically writing in the maximum number of repeat prescriptions allowed. Another However, the issue of pharmaceutical promotion in prescribing software has yet to be tackled. Pharmaceutical promotion has never been allowed on government supplied 'script pads. It is hard to understand why it was allowed on the computerised equivalent. Pharmaceutical promotion distorts the information flow to physicians by selectively promoting the benefits of the latest and most expensive drugs. It provides minimal information about drug side-effects, contra-indications and opportunity costs. Cost-effective generic drugs are rarely promoted and non-drug solutions usually not at all. Pharmaceutical promotion has clearly been shown to influence physician's prescribing  and has Government intervention is also required to ensure that key national resources of objective therapeutic information, such as the Australian Medicines Handbook and Therapeutic Guidelines, are incorporated in prescribing software. The provision of objective therapeutic information is an important strategy of the QUM component of Australian National Medicinal Drug Policy . IronicaMore recently, the NPS RADAR project has shown the way forward. RADAR provides independent information to health professionals about medicines that have a new or a changed listing on the PBS. RADAR drug monographs have recently been incorporated in four leading GP prescribing packages using an open standard interface. This project has moved ahead because the Australian government provided financial support to both the NPS and software vendors to enable the RADAR integration to take place.Following a workshop on electronic decision support, HL7 Australia has presented a work plan to the Australian Health Information Council Electronic Decision Support Steering Committee that would build on the RADAR project by incorporating the Australian Medicines Handbook and Therapeutic Guidelines into clinical software in a standard manner . HoweverThe third area of prescribing software requiring government intervention is standards for drug-drug interaction checking. The NPS tested four popular GP software packages by entering a common set of elderly patients on multiple medications . This reThe PBS remained in the media and policy spotlight during 2003–04. While the growth rate of the PBS has slowed during the year under review the sustainability of the Scheme remains an ongoing concern. One strategy adopted by the government was to transfer more of the cost of medicines to consumers through higher PBS co-payments and increased safety-net thresholds. However, such measures can result in higher costs elsewhere if poorer patients forgo necessary medicines and end up being hospitalised with uncontrolled disease.Cost-shifting (and patient inconvenience) was reduced by allowing State and Territory public hospitals limited access to the PBS but these reforms also showed the need for changes in the PBS to make it more suitable for hospital practice and the desirability of further integrating health funding systems.Educational strategies focusing on the quality use of PBS medicines were successfully pursued but would benefit from increased funding. In addition, there was an exploratory attempt to focus the attention of Divisions on PBS costs by rewarding them with a moiety of any money saved by their members through more cost-effective prescribing. However, the difficulties experienced by the EDQUM project in extracting useful drug utilisation data from computerised prescribing systems highlighted the need for prescribing software standards as did other problems with such software.Information communication technology and information management (ICT/IM) has the potential to allow individual health practitioners, Divisions and governments to compare what is being done with what is recommended best-practice, highlight major discrepancies, and provide targeted education and appropriate incentives to reduce the gap. However, as the events of 2003–04 show, this potential is unlikely to be realised if the development of clinical computer systems is left solely to market forces.Dr. Harvey is a past Board member of Therapeutic Guidelines Limited, Co-Chair of HL7 Australia's Decision Support Technical Committee, a Councillor of the Australia Consumer's Association and a member of the Australian Labor Party.