DRD4 7-repeat allele carriers take 25% more risk than individuals without the 7-repeat allele. These findings contribute to the emerging literature on the genetic determinants of economic behavior.Individuals vary in their willingness to take financial risks. Here we show that variants of two genes that regulate dopamine and serotonin neurotransmission and have been previously linked to emotional behavior, anxiety and addiction (5-HTTLPR and DRD4) are significant determinants of risk taking in investment decisions. We find that the 5-HTTLPR  Risk preferences describe individuals' willingness to take or avoid risk in a variety of settings, including financial choice, and are an essential component of any model of economic behavior. Individuals vary in the extent to which they are willing to take financial risks, which may be explained in part by individual differences in heritable traits. Classical twin design studies estimate that genetic effects account for 20% variation in risk taking in experimental lottery choices Recent findings in neuroscience suggest that the neurotransmitters dopamine 5-HTTLPR) and dopamine D4 receptor (DRD4) exon III polymorphism. The 5-HTTLPR consists of a 44-base pair insertion or deletion, generating either a long (l) or a short (s) allele. The short variant of the polymorphism reduces the transcriptional efficiency of the 5-HTT gene promoter and is associated with higher scores on neuroticism and harm avoidance DRD4) exon III polymorphism has been linked to novelty seeking and pathological gambling. Individuals with the 7-repeat allele have higher novelty seeking scores than those with other DRD4 variants Given the mounting evidence that the dopaminergic and serotonergic systems are involved in decision making, and that genetic variations have a significant effect on the physiology of these two systems 5-HTTLPR and DRD4, contribute to individual differences in financial risk taking preferences. Based on prior work, we hypothesized that individuals carrying two copies of the s allele of the 5-HTTLPR would be significantly more risk averse relative to individuals carrying one or two copies of the l allele. Additionally, we hypothesized that individuals with a 7-repeat allele of the DRD4 polymorphism would be significantly more risk seeking relative to those individuals without the 7-repeat allele.Here we investigated whether or not genetic variations in these two candidate functional polymorphisms, 5-HTTLPR and DRD4 functional polymorphisms (see Supplement).We elicited financial risk preferences in an experimental setting where participants made multiple investment decisions allocating funds between a risky and a riskless asset, and were compensated based on the performance of their chosen financial portfolio . We subs5-HTTLPR are significantly more risk averse relative to individuals carrying one or two copies of the long allele. Additionally, individuals with the 7-repeat allele of DRD4 are significantly more risk seeking relative to those individuals without the 7-repeat allele. These findings provide novel evidence of a genetic basis for financial choices.We found that individuals carrying two copies of the short allele of the M age = 22.4 yrs; SD age: 4.9 yrs) completed the investment task and were subsequently genotyped for the 5-HTTLPR and DRD4 functional polymorphisms. Genotyping was conducted by ACGT Inc.   using the software package E-Prime. Five to nine subjects were in the laboratory solving the investment task at the same time, with each individual in a cubicle separated from the rest of the participants. Participants gave informed consent prior to participating and the study was approved by the IRB committee at Northwestern University. The entire experiment took 1.5 hours to complete and the average pay per subject was $25.s allele, 44 carriers  with one or two copies of the l allele of the 5-HTTLPR polymorphism . Additionally, we conducted statistical analyses for HW equilibrium of DRD4 genotypes .The entire sample consisted of 21 carriers  homozygous for the Participants first completed the investment task and were then genotyped. On each of the 96 trials see  of the tAt the time of making a choice, subjects knew the actual rate of return of the risk-free asset and the two possible outcomes of the risky security, or, equivalently, the expected value and standard deviation of the risky return. These values differed from trial to trial, as did the amount of money available to the subject to invest. The actual rate of return for the risky asset on any trial was not revealed until the end of the experiment. At the end of the experiment, each subject selected a random number between 1 and 96 by picking a ball from an urn. That number determined the trial for which the subject would receive payment. If on any trial a subject chose to invest in the risky asset an amount larger that the maximum investment allowed ($T+$15), or if they did not respond, that trial was marked as invalid. If an invalid trial was selected from the urn, the final payment was only the show-up fee of $15. Subjects therefore had incentives to always enter their choice for the risky investment, and to treat each of the 96 trials as the one that would determine their pay. By deferring information about earnings until the end of the experiment we eliminate wealth effects that may change subjects' choices depending on past outcomes.In each trial subjects had six seconds to learn the information about the return distribution of the risky security and the return of the safe asset. They had six additional seconds to enter the dollar amount they wished to invest in the risky asset, which was an integer that could range from zero to the maximum investment of $T+$15. A 2-second fixation screen indicated a new trial was about to begin.M risky allocation: $9.78; SD risky allocation: $7.16) on each trial depended on the characteristics of the two investment choices, as would be predicted by standard models of economic choice where individuals have mean-variance preferences excess risky investment and it measures how risk seeking an individual is relative to the average person in the subject pool.The amount of money participants invested in the risky security  less in the risky asset than those carrying one or two copies of the long allele of the genotype (p<0.02 in a one-tailed mean comparison test), in excess of the benchmark model. Similarly, individuals who carry the 7-repeat allele in the DRD4 gene invest $2.46  more in the risky asset than those lacking the 7-repeat allele (p<0.04 in a one-tailed mean comparison test).Results demonstrate that financial risk seeking is correlated with the D4DR and the propensity to choose risky investment options using a similar experimental setup. The current work complements a growing body of work demonstrating the heritability of economic decision-making Our findings show that two functional polymorphisms known to regulate serotonergic and dopaminergic activity in the brain are associated with individual differences in financial risk-seeking behavior. Consistent with our results, a contemporaneous study Prior work in behavioral genetics suggests an evolutionary explanation for our findings. For instance, a seminal study Methods S1Supplementary Materials and Methods(0.03 MB DOC)Click here for additional data file.Table S1Allele and genotype frequencies for 5HTTLPR polymorphism(0.03 MB DOC)Click here for additional data file.Table S2Allele and genotype frequencies for DRD4 polymorphism(0.05 MB DOC)Click here for additional data file.


Patients who experienced a virologic breakthrough and had a baseline mutation at codon 135 were more likely to evolve a single NNRTI resistance mutation  but less likely to evolve multiple NNRTI resistance mutations . Mutations at codon 135 do not affect response rates, but affect the pattern of development of NNRTI resistance mutations. This has important implications for the subsequent use of newer NNRTIs such as etravirine in salvage therapy.To evaluate the impact of mutations at reverse transcriptase codon 135 on treatment outcomes in patients receiving NNRTI-based antiretroviral therapy, a total of 68 patients (30 with and 38 without baseline mutations at codon 135) were evaluated. Median increases in CD4 counts were 135 and 90 cells/mm The use of highly active antiretroviral therapy (HAART) has proven remarkably effective in controlling the progression of HIV/AIDS and prolonging survival, but these benefits can be compromised by the development of drug resistance. Recent estimates of the prevalence of drug resistance during the first years of widespread use of potent antiretroviral therapy have indicated that about 70% of treated adults with detectable viremia have isolates with drug resistance mutations , 2. ClinIn vitro, a 2.5-fold increase in IC50 to nevirapine or delavirdine  while 38 patients  had no mutations at codon 135. All patients carried subtype B virus. Median baseline CD4 cell counts and plasma viral loads were 190 cells/mm3 and +90 cells/mm3 (p=0.32) while the CD4 cell counts were 300 cells/mm3 and 345 cells/mm3 (p=0.84) in patients with and without baseline mutations at codon 135, respectively  or V (21%) mutation at position 135 suggesting that non-synonymous mutations at position 135 of RT may lead to viral escape from host CTL responses [The virologic impact of mutations at codon 135 is not clear despite the fact that such mutations are extremely common. It is likely that mutations at RT codon 135 affect NNRTI susceptibility by virtue of their proximity, in the p51 component of the RT dimer, to the NNRTI binding site . The β7-esponses . Other sesponses . Thus, tet al. found that the amino acid site 135 of RT was associated with reduced susceptibility to both nevirapine and delavirdine [et al. reported that a decreased susceptibility to efavirenz and nevirapine at baseline was seen in viruses with mutations at codon 135 in antiretroviral naïve patients [et al.; however, found no relationship between the presence of mutations at codon 135 and virologic response [vs 4/22, p=0.029) which is in agreement with results from previous studies [vs 12/22, p=0.033) and possibly some NRTI mutations such as the M184V . This contrasts with previous reports which suggested that the presence of baseline mutations at codon 135 may accelerate the accumulation of more classical NNRTI resistance mutations. It should be emphasized that much of these data were generated with non-subtype B strains of HIV. This may explain why, in populations infected with subtype B virus, very different results may be obtained [Mutations at RT codon 135 are not widely recognized as associated with more classical NNRTI-resistance mutations and their clinical implications are unclear. Brown patients . In addipatients . Harrigaresponse . In agre studies . Howeverobtained .et al. identified 24 accessory RT codon mutations, including mutations at codon 135, as significantly associated with the accumulation of TAMs [Although it is difficult to attribute all the mutations we observed to NNRTI treatment, it is generally agreed that NRTIs do not affect the evolution of NNRTI-associated mutations or mutations selected by non-reverse transcriptase inhibitors. However, one recent publication reports some intriguing associations between NNRTI and NRTI mutations. Cane  of TAMs . In our One important implication of our results relates to the possible sequencing of NNRTIs in clinical practice, with the availability of second generation agents in this class. It may be that in patients with baseline mutations at codon 135, NNRTI resistance may be associated with the development of a single additional mutation, increasing the likelihood of agents such as etravirine remaining effective. This would not be the case if multiple NNRTI mutations were to develop, a situation that may be more likely with subtype B virus wild type at the 135 codon of RT.et al., this can lead to further decrease in susceptibility to NNRTIs in the context of a pre-existing 135 mutation [The study has several limitations. First, it was conducted in a relatively small number of patients. Second, the study was done on IDUs enrolled in a DOT program and thus, the results might not be generalized to non-IDU populations or non-DOT settings. Third, resistance mutations considered where only those based on the IAS-USA guidelines and some other crucial or unknown mutations might not have been properly evaluated including minor mutations such as L283I. According to Brown In conclusion, our results indicate no significant differences in CD4 cell counts and HIV plasma viral load responses to NNRTI-based regimens as a function of baseline 135 genotype. However, in patients with baseline mutations at codon 135 and experiencing virologic breakthrough, there were more evolution of single and less evolution of multiple NNRTI resistance mutations. This may have important implications with respect to the initial selection of patients to receive NNRTI-based therapy at baseline with a view to sequencing NNRTIs using newer agents in this class in subsequent courses of therapy.


Linagliptin is a novel member of the dipeptidyl peptidase-4 (DPP-4) inhibitor class of antidiabetic drugs. DPP-4 inhibitors increase incretin (glucagon-like peptide-1 and gastric inhibitory polypeptide) levels, inhibit glucagon release and, more importantly, increase insulin secretion and inhibit gastric emptying. Currently, phase III clinical studies with linagliptin are underway to evaluate its clinical efficacy and safety. Linagliptin is expected to be one of the most appropriate therapies for Japanese patients with DM, as deficient insulin secretion is a greater concern than insulin resistance in this population. The number of patients with DM in Japan is increasing and this trend is predicted to continue. Several antidiabetic drugs are currently marketed in Japan; however there is no information describing the effective dose of linagliptin for Japanese patients with DM.Many patients with diabetes mellitus (DM) require a combination of antidiabetic drugs with complementary mechanisms of action to lower their hemoglobin AThis prospective, randomized, double-blind study will compare linagliptin with placebo over a 12-week period. The study has also been designed to evaluate the safety and efficacy of linagliptin by comparing it with another antidiabetic, voglibose, over a 26-week treatment period. Four treatment groups have been established for these comparisons. A phase IIb/III combined study design has been utilized for this purpose and the approach for calculating sample size is described.This is the first phase IIb/III study to examine the long-term safety and efficacy of linagliptin in diabetes patients in the Japanese population.Clinicaltrials.gov (NCT00654381). Diabetes mellitus affects around 171 million people worldwide and this figure is expected to double by the year 2030. Type 2 diabetes mellitus (T2DM) accounts for between 90 and 95% of all cases of diabetes [1c). Although no superior marker to HbA1c has yet been proposed, its use raises a number of methodological and ethical challenges in terms of conducting clinical trials. In general, antidiabetic agents effect changes in HbA1c levels by only a few percent . In contrast, patients vary widely both in their individual HbA1c levels (7-14%) and in disease response to various treatments. The inherent variability of HbA1c levels within the diabetic population and the relatively small changes caused by treatment dictate that clinical studies attempting to establish the efficacy of a new drug for T2DM need to involve relatively large numbers of subjects to demonstrate any statistically significant results confirming efficacy. Where studies compare the efficacy of one drug relative to another, the number of subjects required needs to be greater still. Pressure to recruit sufficient numbers of patients in a timely fashion makes it impractical to conduct such studies using treatment-naïve patients alone, where recruitment ideally should occur at, or around the time of diagnosis. The design of studies must therefore consider using patients already receiving treatment for diabetes. This introduces a further complicating factor, as previous exposure to an antidiabetic treatment may affect patient response to new treatment.The purpose of a drug development program is to establish through clinical trials whether a drug displays acceptable levels of safety and tolerability, and to establish whether the drug's actions are both beneficial and clinically relevant. In T2DM, the gold standard measure of efficacy is the impact of the antidiabetic agent on levels of glycosylated haemoglobin . It is possible that these patients will respond differently than Caucasian patients due to genetic differences. Differences in the pharmacokinetic and/or pharmacodynamic behaviour of drugs between Japanese subjects and subjects from other ethnic backgrounds have been observed, for example for the oral contraceptive Org 30659 [Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new and promising class of agents for the treatment of T2DM. Linagliptin is a novel member of the DPP-4 inhibitor class that may offer added clinical benefits over similar agents. These include a long half-life consistent with once-daily dosing, non-renal excretion that will not require dose adjustment following loss of renal function and excellent safety and tolerability profiles -6. As DPrg 30659  and the 5 mg and  mg. It i1c in a 12-week study conducted in Japanese patients with T2DM, and to examine the efficacy of linagliptin versus the antidiabetic agent voglibose. The study will also evaluate the long-term safety profile of linagliptin for up to 52 weeks. The study design, primary endpoints, patient inclusion/exclusion criteria and statistical methods used to calculate the study sample size are described.The primary objectives of the proposed study are to compare the efficacy of linagliptin versus placebo in lowering HbA1c levels of 7.0-10.0%). Patients taking oral anti-hyperglycemic drugs at screening are eligible to participate if they have a mean HbA1c value of > 7 - < 9% during the washout period. Other inclusion criteria include a stable HbA1c value (less than 10% variation during the last 2 weeks of the 4-week washout period) and a body mass index less than 40 kg/m2 at randomization. Patients are excluded from the study if they violate these inclusion criteria or if they meet the exclusion criteria specified in the protocol.This is a randomized, double-blind study with both a placebo- and an active-controlled parallel group. It is conducted in 47 centers in Japan. To be eligible for study enrollment, male or female subjects are required to be Japanese patients aged 20-80 years with T2DM .1c levels in patients receiving linagliptin with those receiving placebo at 12 weeks. The objective of Stage 2 is to compare changes in HbA1c levels in patients receiving linagliptin with those receiving voglibose after 26 weeks of treatment. The objective of Stage 3 is to determine the long-term safety and tolerability of linagliptin.This study includes three different stages, each with specific objectives, and will utilize four treatment arms (A-D) . Patients randomized to Group A will receive a placebo for 12 weeks after which they will receive linagliptin for 40 weeks; patients in the Group B (linagliptin 5 mg) and Group C (linagliptin 10 mg) treatment arms will receive the study drug for up to 52 weeks. Patients randomized to Group D (voglibose 0.6 mg) will receive the treatment for 26 weeks after which they will be switched to linagliptin for 26 weeks  and voglibose 0.6 mg (26 weeks).The proposed comparison of efficacy between linagliptin (5 and 10 mg) and both placebo and voglibose is based on changes in HbAOther parameters of interest measured will include inhibition of plasma DPP-4 activity, the proinsulin/insulin ratio, the homeostasis model assessment (HOMA) indices for insulin resistance and insulin secretion, body weight, waist circumference and plasma concentrations of linagliptin. Adverse event, safety, laboratory and hematology data will be collected during the study and will form part of the secondary endpoint data for determining the long-term safety profile of linagliptin.Patient reported outcomes will be assessed using the Diabetes Treatment Satisfaction Questionnaire (DTSQ) modified by Ishii , consist1c , the number of pre-study use of antidiabetic drugs, gender and study site.Four treatment arms are used to investigate the effective dose and safety profile of linagliptin in the study population. Randomization will be based on the Zelen rule  and follTreatment allocation is conducted by a sponsor-independent contractor. Following informed consent, the investigator completes a registration form that is sent to the allocation center. The registration form includes data on the subjects' inclusion and exclusion criteria. After receiving the registration form, the allocation center sends a registration report to the investigator. The investigator confirms that the patient satisfies the study criteria and sends a second registration form to the allocation center once the 4-week washout period is completed. The allocation center randomizes the patient according to the pre-defined criteria above and this information is provided to the investigator. Randomization is done once at the enrollment of study. At the randomization after the enrollment, patients are assigned, to 6 groups: placebo - linagliptin 5 mg, placebo - linagliptin 10 mg, linagliptin 5 mg, linagliptin 10 mg, voglibose - linagliptin 5 mg, and voglibose - linagliptin 10 mg. Therefore all patients ultimately take either linagliptin 5 mg or 10 mg, unless they withdraw or drop out the study.The statistical analyses are performed on an intention-to-treat basis. The full analysis dataset (FAS) is described in the ICH E9 guidelines , and is Baseline glycosylated hemoglobin levels and the number of antidiabetic drugs used before study enrollment are included in the model as covariates.1c level from baseline to 12 weeks of linagliptin treatment are analyzed in accordance with the model below, in order to compare with placebo in the double-blind treatment phase:Changes in HbAWhere:yijk : change in HbA1c at 12 weeks from baselineBHbA1ci : HbA1c of i-th patient at baseline Dosej : j-th dose group Diabtreatk : number of previously used antidiabetic drugs 1c following treatment with linagliptin are to be compared with those for voglibose after 26 weeks if linagliptin treatment fails to achieve a statistically significant difference in HbA1c levels relative to placebo (primary comparison dose at 12 weeks). The dosing schedule for linagliptin increases from 5 mg to 10 mg during study if linagliptin 5 mg fails to demonstrate superiority to placebo at 12 weeks. In addition, the study stops if neither of the linagliptin arms shows statistically significant differences from placebo in HbA1c levels at 12 weeks.Comparisons conducted at 12 weeks are between linagliptin and placebo. Patients who received placebo for 12 weeks and are then randomized to receive either linagliptin 5 mg or 10 mg are excluded from this analysis. Values of HbA1c at 26 weeks is:The model for HbAWhere:yijk : Change in HbA1c at 26 weeks from baselineHbA1ci : HbA1c of i-th patient at baseline Drugj: j-th treatment group Diabtreatk : number of previously used antidiabetic drugs 1c between groups are performed weekly by the closed testing procedure [Comparisons for HbArocedure . No form® 6.0  software on a Windows-based personal computer. On the basis of clinical studies conducted in Japan and other countries, it is estimated that the overall mean change in HbA1c levels after treatment with linagliptin 5 mg QD for 12 weeks will be approximately 0.5% compared with 0% in placebo. This will provide an overall mean between-groups treatment difference of 0.5%. Experience also suggests that the standard deviation of this difference will be approximately 0.9%. Employing an accepted one-sided statistical significance threshold of 2.5% and taking into account the weighting for placebo and linagliptin group randomization ratios (1:2), it is possible to predict a 90% power for the study with sample sizes of no less than 52 patients in the placebo group and no less than 104 patients in the linagliptin groups.Sample size calculations are performed using nQuery Advisor1c levels for the linagliptin treatment groups will continue to fall with ongoing therapy, estimates suggest that the mean change in HbA1c levels from baseline after a 26 week treatment with linagliptin 5 mg, will be approximately 0.7%, compared with 0.25% for voglibose 0.6 mg. It is expected that this difference between treatments will be reflected in a mean difference in HbA1c levels from baseline of 0.45% [Assuming that HbAof 0.45% . With a Experience derived from previous clinical trials in patients with T2DM suggests that a discontinuation/dropout rate of approximately 15% can be expected during the current 52-week trial. Assuming that the present study will experience a similar level of subject discontinuation, it is possible to adjust the recruitment targets to maintain study power in anticipation of similar attrition levels. A target sample size of 63 patients in the placebo group and 126 patients each in the linagliptin and voglibose treatment groups  is therefore required to power this study appropriately to achieve its primary objectives.The sample size in the linagliptin group also appears to satisfy the requirements for evaluation of safety in the clinical development of new drugs expected for long-term use in non-fatal diseases .The design of the present study is adopted as a means of addressing some of the ethical and methodological challenges in studying new drugs in T2DM populations.1c, particularly its 2 - 3 month period for turnover, tends to dictate the time course of T2DM clinical trials. Similarly the inter-patient variability in terms of the levels of HbA1c and its response to various treatments dictate that studies must recruit relatively large numbers of patients to demonstrate statistically significant changes in HbA1c compared with placebo [1c to return to baseline levels it would be necessary for the washout period to last 2-3 months, and if the study design involves a placebo arm, this would mean a significant number of patients going without treatment for an extended period.Glycosylated hemoglobin has emerged as the accepted marker of glycemic control and clinical efficacy in studies of diabetes . The nat placebo . The cha1c levels to return to the baseline level before initiation of the previous treatment. However, by adopting this approach it is hoped that we may address some of the concerns frequently raised about prolonged treatment withdrawal in clinical trials [1c levels at treatment initiation will most likely incorporate an aspect of the pre-study treatment response, the antidiabetic drug used and the previous extent of disease control per patient.Withholding treatment from patients for a prolonged period might be considered unethical. Therefore, the present study uses a 4-week washout period. Clearly, 4 weeks is insufficient for HbAl trials . The sta1c levels in the placebo group will not be included in any comparison, but instead will be viewed as an indication of the study population's baseline HbA1c values in the absence of the potentially confounding residual effects of previous therapies. A similar number of patients are needed to demonstrate the non-inferiority or superiority of linagliptin to placebo under imbalanced randomization and to voglibose. The study design can also be sufficiently powered for detecting statistically significant differences between linagliptin and placebo or voglibose. Using the methods described, we have calculated that this can be achieved without markedly increasing the number of subjects needed.The study design was selected to reduce the number of patients required to undergo a prolonged placebo exposure by employing an imbalanced randomization (placebo:linagliptin 5 mg:linagliptin 10 mg:voglibose = 1:2:2:2). Using this biased treatment allocation approach, it is possible to minimize the potentially unethical exposure of patients to placebo while maintaining sufficient power to complete the primary statistical comparison. Data after 16 weeks of placebo treatment can be useful for determining a true baseline (treatment naïvety) because any underlying glycemic controlling effects of prior antidiabetic treatment should have had sufficient time to reverse. The 16 week data for glycosylated HbAThe efficacy of short term linagliptin treatment in Japanese T2DM patients was reported by Kanada et al . We set Our study design may also facilitate recruitment relative to other methods. In clinical studies of antidiabetic drugs as monotherapy, all patients randomized to placebo groups receive only placebo; therefore, there are no perceived benefits for these patients. The use of placebo-only groups in clinical studies may cause patients to hesitate when asked to participate, as they are aware that they have a chance of receiving inadequate treatment during the trial. In the present study, patients receive placebo for a maximum of 16 weeks after which they are given active treatment. This design may serve to aid patient recruitment in clinical studies. Similarly, study designs which incorporate multiple objectives within a single study may reduce the burden of recruitment on the development program by maximizing the information derived from every study. This study investigates the efficacy of two different doses of linagliptin versus placebo (at 12 weeks), compares the relative efficacy of linagliptin versus voglibose (at 26 weeks) and provides long-term (52 week) safety data. Combining the objectives into a single study effectively cuts the pre-study clinical workup that would be needed if these investigations were conducted as three separate studies, thus reducing the patient recruitment burden. Other ongoing Phase III trials are evaluating linagliptin as both monotherapy and as add-on therapy to commonly used diabetes medications. It is anticipated that these trials combined with the results of the present study will support the suitability of linagliptin as a further treatment option for type 2 diabetes patients in Japan.In conclusion, a continuous flow study design has been applied to investigate three distinct areas of interest related to the novel DPP-4 inhibitor linagliptin in Japanese diabetes patients. The design described offers several logistical and ethical advantages, while remaining adequately powered to generate informative results for this patient population. Full results from this trial will be published in 2010.This trial was supported by Boehringer Ingelheim. With the exception of MT, all of the authors are full-time employees of Boehringer Ingelheim. Involvement in this trial does not conflict with the professional activities of MT.All authors read and approved the final manuscript. YH as an investigator in the study, drafted the manuscript and has made a substantial contribution to acquisition of data.


Three hundred and seventy-two patients were randomized between 3 regimens of chemotherapy: cyclophosphamide, intermittent melphalan, and melphalan with prednisone, and were followed up to death or for at least 5 years. There was no difference in survival between the treatments, either overall or in any subgroup of patients. Therefore, the choice among these 3 treatments should be guided by the patient's comfort and convenience. The most important prognostic feature at presentation was the quality of renal function. It was possible to define good, intermediate and poor renal-function groups which were highly correlated with prognosis (X2 for trend = 62.6). The haemoglobin level at presentation was strongly correlated with prognosis among patients in the good renal-function group. Among 107 patients who presented with good renal function and with haemoglobin above 100 g/l, the 5-year survival was 43%. Other prognostic features were much less important when account was taken of renal function and haemoglobin level.


After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis.Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with  Although therapies (such as surgery or androgen ablation) have improved the survival rate of the patients, lack of success with hormone-refractory prostate cancer remains. The advanced tumors are often resistant to chemotherapeutic drugs. Although the mechanisms for the resistance are unclear, it has been postulated that the development of tumors, such as refractory prostate cancer, is related to the accumulation of genetic or epigenetic alterations. In Asian, PL is one of well-established medicinal mushrooms that have been using to treat various human malignancies. Studies showed that the water-soluble fraction of PL is biologically active, which probably is polysaccharide A potential of a drug to induce apoptosis has been widely used as a strategy for developing new cancer therapy. It is known that apoptosis can be triggered by a variety of internal or external signals. Caspases, a highly conserved family of cysteine proteases, are key apoptotic effectors in cells and play critical roles in the regulation of apoptosis through a chain cleavage reaction Previously, we have reported that PL is able to sensitize cultured prostate cancer cells to apoptosis, in which caspases were activated Phellinus Linteus powder was purchased from Panbio-Tech  and dissolved in water. After boiled for 5 min, PL solution was centrifuged and supernatant was collected for the study. DMEM (Dulbecco's modified Eagle's medium); antibiotics (penicillin and streptomycin) and trypsin-EDTA were purchased from Invitrogen. Human prostate cancer PC3 cells (American Type Culture Collection) were cultured in DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 unites/ml of penicillin, 100 µg/ml of streptomycin.Male nude mice  (Taconic) at ages of 4–6 weeks were used. Human prostate cancer PC3 cells (5×106) in 100 µl PBS were inoculated into the right flank area of each mouse. Three mice were served as a positive control. For the inhibition of tumor formation, 6 mice were treated with PL (30 mg/kg in 100 µl PBS) right after the inoculation and subsequently administrated the same amount of PL every 2 days. Another 3 mice were treated with equal amount of control Hitachi mushroom solution following the same time course as that used for PL. The sizes of the tumors were measured every 4 days with a caliper and calculated according to Tamayko's formula Isolated tumors were fixed in 4% phosphate-buffered neutral paraformaldehyde solution (pH 7.4). The histological specimens were sectioned and stained with hematoxylin/eosin (H&E), TUNEL or corresponding antibodies (Santa Crutz Biotec.).p value of <0.05 was considered significant.Means and standard deviations of the results of the experiments were computed. Standard deviations are displayed as error bars in the figures. A Student's T test was used and a 1 arrest in normal prostate epithelial cells Studies have suggested that PL possess an anti-tumor property We also measured the diameters of the inoculated tumors, one week later after the inoculation when all the mice started to show the sign of the formation of solid tumor masses. The measurements were taken every four days for 12 days Fig. 2A We then tested the effect of PL on the existing tumors. When the inoculated tumors reached to the size of 1.0 cm in diameter, PL was injected into the mice every two days for total ten days. The tumors were isolated and prepared for histopathological or immunohistochemitry analysis. The HE staining revealed that the tumors isolated from PL- or control mouse were well-circumscribed with connective tissues Fig. 3A.in vitro data is consistent with that obtained from the animal experiments.To further test the ability of PL to induce apoptosis, DU145 and PC3 cells were treated with PL at 1 mg/ml or 2 mg/ml for 48 h. Annexin V analysis were conducted Fig. 4. Ain vivo to block the growth of the tumors formed by the inoculation of human prostate cancer DU145 or PC3 cells. The histochemistry staining analysis showed that the majority of the cells in the tumors isolated from PL-treated mice are apoptotic, manifested by positively stained with TUNEL and reacted with an anti-caspase 3 antibody. The occurrence of apoptosis was also demonstrated in cultured DU145 or PC3 cells after the treatment with PL for 48 in a dose dependent manner. Thus, our study, using in vivo as well in vitro assays, inidcates that PL treatment initiates caspase cascade to induce apoptosis in prostate cancer cells, which provide a strong evidence for the potential of PL as an anti-prostate cancer remedy.Previously, we demonstrated that PL induces cultured prostate cancer cells to undergo apoptosis 1 phase of the cell cycle by blocking the expression of cyclin D1 and its interaction with cell cycle-dependent kinases 4 and 6 in vitro experiments, treatment with PL elicits cell checkpoint controls in normal tissues or organs, which is not harmful to the mice. In the inoculated tumors, the same treatment appears to be very much cytotoxic.It is known that PL is non-toxic in general in vivo study indicates that PL treatment is also able to initiate apoptosis via activating caspase 3 that are the effector in caspase chain reaction. The investigation of whether other caspases are involved in the induction of apoptosis in vivo is under way.Programmed cell death is important machinery for excluding abnormal or cancerous cells. Activation of caspase family members is at the core of apoptosis, representing a point of intersection of various apoptotic pathways in various types of cancer cells, including prostate cancer cells. TNF or Fas/CD95 receptors, mitochondrial proteins (such as cytochrome c) and granzyme are able to induce cell death through activation of the caspase cascade PL is one of well-established medicinal mushrooms that have been taken orally as a common health-promoting dietary supplement or an adjuvant to treat malignancies in Asia for many decades. The extraction from water-soluble PL fraction shows a relatively homogeneous molecular weight distribution on gel permeation HPLC and is estimated to be around 150–180 kD from the retention time on HPLC pullulan molecular markers in vivo data support the notion that PL, by switching on a tumor suppression-related machinery, can be developed as an efficient therapy to treat human malignancies, such as refractory prostate cancer.In summary, the successful use of PL to treat prostate cancer requires the fully understanding of the mechanisms for how PL mediates anti-tumor activities in tumors. Our previous study demonstrated that PL, through activation of caspases, is able to induce apoptosis in cultured cancer cells. In the present investigation, using xenograft assay, we further demonstrate that the injection of PL into nude mice with formed subcutaneous tumors by inoculated with prostate cancer DU145 or PC3 cells induces majority of inoculated tumor cells to lose their viabilities. Thus, our


Kip1 and is associated with poor prognosis in early breast cancer. The purpose of the present study was to examine the role of these proteins as predictors of clinical outcome and response to chemotherapy in locally advanced breast cancer.Preoperative chemotherapy is often used in patients with locally advanced breast cancer. However, commonly used clinical and pathological parameters are poor predictors of response to this type of therapy. Recent studies have suggested that altered regulation of the cell cycle in cancer may be involved in resistance to chemotherapy. Over-expression of the ubiquitin ligase Skp2 results in loss of the cell cycle inhibitor p27Kip1 were determined by immunohistochemistry both before and after preoperative chemotherapy in 40 patients with locally advanced breast cancer. All patients were treated with cyclophosphamide/doxorubicin (adriamycin)/5-fluorouracil (CAF) and some patients received additional treatment with docetaxel. Expression data were compared with patients' clinical and pathological features, clinical outcome, and response to chemotherapy.The expression levels of Skp2 and p27Kip1 levels, tumor grade, and expression of estrogen and progesterone receptors. Both Skp2 and p27Kip1 were found to be accurate prognostic markers for disease-free and overall survival. High preoperative expression of Skp2 was associated with resistance to CAF therapy in 94% of patients (P < 0.0001) but not with resistance to docetaxel.Skp2 expression before preoperative chemotherapy was inversely related to p27Skp2 expression may be a useful marker for predicting response to doxorubicin-based preoperative chemotherapy and clinical outcome in patients with locally advanced breast cancer. Preoperative chemotherapy is widely used in the management of primary breast cancer, and particularly in patients who present with locally advanced disease -3. StudiThe mechanisms responsible for resistance of cancer cells to chemotherapy, whether inherent or acquired, are poorly understood. Because cell cycle regulation is a key mechanism by which most chemotherapy agents exert their cytotoxic effect, alterations that may occur in cell cycle regulation in cancer may contribute to lack of response to some agents . Under nWAF1 (where the definition of WAF1 is 'wild-type p53 activated fragment 1') is a Cdk inhibitor that blocks the cell cycle at the G1/S transition by inhibiting the activity of Cdk2 and Cdk4/6 . Moreover, in these experiments changes in Skp2 levels also did not translate into changes in p27Kip1 levels. It is noteworthy, however, that despite the lack of changes in p27Kip1, Skp2 levels were independently associated with a significantly better disease-free survival. This supports the concept that Skp2 has other important oncogenic effects and that reduction in Skp2 levels is a rational therapeutic objective.First, similar to previous reports in early breast cancer, in locally advanced disease we found that high expression of Skp2 or low expression of p27ctively) . This suP > 0.001; odds ratio = 13.73). Although the statistical significance for the ability of Skp2 to predict response to CAF is high, the number of patients in this study is rather small. Therefore, confirmatory evidence from larger studies is required before we may propose that this marker be used to decide whether to administer doxorubicin-based preoperative chemotherapy to patients with breast cancer. It is also important to bear in mind that two additional drugs are included in this regimen, which might have influenced the findings of the present study to some degree. In contrast, the relationship between Skp2 expression and docetaxel resistance is difficult to interpret. The patients who received docetaxel were a selected and relatively small group of patients who did not respond to anthracycline-based chemotherapy. Although in this group of patients the expression of Skp2 was not associated with docetaxel resistance, larger studies conducted in patients receiving taxane-based regimens are needed to determine the role of Skp2 as a predictor in taxane-based chemotherapy.Finally, we found that Skp2 is an accurate predictor of response to doxorubicin-based chemotherapy. Skp2 levels were high in 80% of the poor responders; more specifically, when Skp2 levels were high, 94% of the patients did not respond sufficiently to doxorubicin-based therapy. Multivariate analysis, including all available clinical and pathological parameters, revealed that Skp2 is an extremely accurate predictor for response to doxorubicin-based /5-fluorouracil; Cdk = cyclin-dependent kinase; ER = estrogen receptor; KIP = kinase inhibitor protein; PgR = progesterone receptor; Skp2 = S-phase kinase protein 2; WAF1 = wild-type p53 activated fragment 1.The authors declare that they have no competing interests.SD carried out the immunohistochemical studies, performed the statistical analysis and participated in drafting the manuscript. OBI carried out the pathological analysis and scoring of the immunohistochemical results. MS carried out part of the immunohistochemical studies and study design. BS performed the statistical analysis. DDH conceived of the study, participated in the design of the study, participated in the sequence alliance and helped to draft the manuscript. All authors read and approved the final version.


HLA locus on chromosome 6 as a susceptibility locus. Other regions of interest include loci on chromosomes 11, 2, and 12.We have used the genome-wide marker genotypes from Genetic Analysis Workshop 15 Problem 2 to explore joint evidence for genetic linkage to rheumatoid arthritis across several samples. The data consisted of four high-density genome scans on samples selected for rheumatoid arthritis. We cleaned the data, removed intermarker linkage disequilibrium, and assembled the samples onto a common genetic map using genome sequence positions as a reference for map interpolation. The individual studies were combined first at the genotype level  prior to a multipoint linkage analysis on the combined sample, and second using the genome scan meta-analysis method after linkage analysis of each sample. The two approaches were compared, and give strong support to the  Problem 2 of Genetic Analysis Workshop 15 (GAW15) includes genome-wide genotyping of marker sets for linkage studies in rheumatoid arthritis (RA). Four research groups contributed sets of markers across the genome genotyped in four independent pedigree samples. NARAC (North American Rheumatoid Arthritis Consortium) held by far the largest sample, consisting of multiplex families genotyped for 10-cM linkage mapping set and a panel of single-nucleotide polymorphisms (SNPs) genotyped by Illumina. A Canadian group provided pedigrees genotyped for the same Illumina marker panel as well as a dense 100 k Affymetrix SNP map. ECRAF (European Consortium on Rheumatoid Arthritis Families) genotyped a dense microsatellite panel. There was also a United Kingdom (UK) data set, comprising both microsatellite and SNP markers with a two-stage design.p-value), with a concomitant loss of spatial accuracy. The GSMA statistic is calculated for each bin as the mean rank across studies, with significance levels determined by permutation of the observed ranks within each study.The diversity of these samples and marker maps presents a complex problem to anyone seeking to merge them in order to achieve the greater power of a combined sample. In particular, we were interested in judging whether there is a "best" way to merge marker maps, or if available information would force one particular solution. We combined the data by placing markers on a common genetic map and performing linkage analysis on the whole sample jointly , which We were also interested in determining the evidence for linkage across the genome in each of the four samples compared with mega-analysis and meta-analysis. The NARAC sample is considerably larger than the others , and might be expected to outweigh the others in any joint analysis.Four data sets were analyzed for autosomal genetic linkage to RA Table . The CanWe assembled all markers from all studies onto a common centimorgan (cM) genetic map using a procedure described in Hamshere et al. . MarkersThe UK microsatellite positions were judged to be on the same map as NARAC, on the basis of 19 (out of 20) microsatellites common to both sets, which had identical centimorgan positions. Because the UK SNP marker map was of unknown provenance and the marker names cryptic, it was assumed to be comparable if not identical to the UK microsatellite map.r > 0.99). Of note are three markers which differed considerably in genetic position between the two maps: D9S144, D12S43, and D16S289 were originally positioned at 0 cM, and shifted by between 20 and 100 cM in the standard map, due to their current position in the sequence database. These instances were excluded from our analysis, and should be followed up in order to be sure of the identity and location of the typed marker.Because all other marker sets could be positioned relative to the NARAC/UK genetic map with a minimum number of assumptions, this was used as a standard map, and the NARAC microsatellites were used as reference markers (RMs). NCBI base-pair positioning permitted the interpolation of all other markers into each RM interval. Markers positioned outside of this standard map on each chromosome were removed from the analyses to prevent the possibility of negative map positions or overinflated chromosomal lengths. A total of 517 markers were removed from the NARAC and Canada SNP maps, and 74 markers from the ECRAF microsatellite map. A high level of correlation was observed between the original map and our standard map (interpolated on the basis of sequence positions) for the ECRAF microsatellites  was eliminated from each data set in order to prevent artificial inflation of the multipoint linkage statistics arising from incorrect allele frequency estimation in cases of missing parental data . In the Multipoint inkage analysis of the RA binary trait was performed on a 2-cM grid using MERLIN , with thA GSMA was performed as described previously ,9. The rThe maximum LOD score on each chromosome from the four individual samples are presented in Table High linkage scores were also detected on chromosomes 11 , 2 , and 4 . Loci showing low or modest linkage with some concordance across studies included chromosomes 9 (136–146 cM), 12 (104–136 cM), and 21 (30–52 cM). Five chromosomes  showed no LOD score > 1.The results of the meta-analysis Table  implicatThree of these samples  have been used in previous meta-analyses ,12. As iHLA-DRB1 locus in bin 6.2. CTLA, which may be a susceptibility locus for RA [PTPN22 locus maps to bin 1.6, which was the 25th highest ranked bin in the meta-analysis, and was not significant. This locus did not feature in any of the individual analyses, although the mega-analysis shows a small peak over the gene locus at 156 cM, with a maximum score of 0.99 .Several of these bins span candidate gene loci, including the s for RA  is locatThe conduct of our study was largely focused on the details of combining samples for mega-analysis in a rigorous fashion, and we conclude that the process adopted will often be dictated by the characteristics of the samples, and the information available on each. Researchers should follow common sense in including the data that provide the maximum information for each sample. The use of commercial high-density SNP maps, and the full-genome physical maps have made this process easier and probably more accurate than in the recent past. Meta-analysis techniques such as GSMA are more straightforward to perform than a mega-analysis, since they do not require access to the genotype data. Both the meta- and mega-analyses detect the strong linkage to chromosome 6. In addition, regions on chromosomes 2 (194 cM) and 12 (42 cM) are highlighted by both analyses. However, the mega-analysis showed evidence for linkage to chromosomes 10q and 11q, which was not observed in the GSMA. This is because chromosomes 2 and 12 show consistent linkage evidence across the four samples, whereas the linkages of chromosomes 10q and 11q are due almost entirely to high LOD scores from the NARAC sample, with negligible linkage evidence in the other three samples. The GSMA assigns significance to regions where linkage statistics are consistently highly-ranked across studies, but does not take into account the magnitude of the linkage statistics. Thus, it will not detect linkages based on a very high linkage statistic from one sample.This is the largest combined sample analyzed so far for linkage to RA. Both meta- and mega-analysis detected a highly significant linkage to chromosome 6p, with weaker linkages to chromosomes 6q, 2q, and 12p. In each of these regions there was consistent linkage evidence across the four samples. The mega-analysis also detected linkage to chromosomes 10q and 11q. In these regions, the linkage evidence came almost entirely from the NARAC sample, so they were not picked up by the meta-analysis. When performing an analysis of combined samples, it is important to ensure that marker maps are compatible, and that as many genotyping errors as possible are removed. It is also important to keep inter-marker LD to a minimum.The author(s) declare that they have no competing interests.


Deletions and apparent transcriptional abnormalities of the FHIT gene at 3p14.2 have recently been reported in a wide variety of solid tumours. To determine whether lesions of this gene also occur in leukaemia, we have analysed a total of 97 patients  and 16 normal individuals. Intact FHIT transcripts from all cases were amplified using RT-PCR. In addition, smaller size bands that were less intense than the full-length products were amplified from several samples from patients with leukaemia and also from normal leucocytes. Sequencing of the small products revealed that they were derived from FHIT transcripts lacking whole exons. Using single-strand conformation polymorphism analysis, no mutations in the coding sequence were detected in any patient. Furthermore, loss of heterozygosity was not seen in any of 36 informative patients at D3S1300 or D3S1481, markers located within the FHIT locus. We conclude that the FHIT gene and other uncharacterized tumour-suppressor genes at 3p14.2 are unlikely to be involved in the pathogenesis of acute leukaemia or progression of CML from chronic phase to blast crisis. Moreover, low-abundance FHIT transcripts that lack whole exons are not specific to malignant cells and should not be taken as evidence of an abnormality in the absence of demonstrable genomic DNA lesions.


During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous.in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen with a catalytic RNA. Extensive genotypic sampling from two replicate lineages was gathered and phylogenetic networks were constructed to elucidate the structure of the evolving RNA populations. A common distribution was found in which a mutant sequence was present at high frequency, surrounded by a cloud of mutant with lower frequencies. This is a typical distribution of quasispecies. Most of the mutants in these clouds were connected by short Hamming distance values, indicating their close relatedness.Using continuous The quasispecies nature of mutant RNA clouds facilitates the recovery of genotypes under pressure of being removed from the population by random drift. The empirical populations therefore evolved a genotypic resiliency despite a high mutation rate by adopting the characteristics of quasispecies, implying that primordial RNA pools could have used this strategy to avoid extinction. During the origins and early evolution of life on the Earth, the contemporary notion of well-delineated species was not yet realized. Instead, the adaptive fate of populations of naked molecules would have been more accurately described by the quasispecies model of mutant distributions, which was introduced in the 1970's by Eigen . Under aA quasispecies is basically a steady-state dynamic of mutant molecules distributed around a parental genotype, the so-called master sequence, which occupies a central position in the genotypic network space. This dynamic occurs after a sufficient amount of time at high mutational rates, such that the progeny of an individual genotype (the mutant cloud) can be rapidly produced. Importantly, the target of selection is the genotypic distribution as a whole, not single genotypes. Eigen realized that simple genetic entities would gain an evolutionary advantage by a primitive form of group selection in which replication rate deficiencies could be offset by the heightened production of new mutant types . DiffereAlthough the quasispecies concept was originally intended as a description of molecular replicators cooperating and competing for survival prior to their historical encapsulation in membranes that inexorably linked genotype with phenotype ,7, it soin vitro evolution [To date however, the use of a catalytic RNA in a system capable of forming quasispecies has not been reported. Currently available data are restricted to genomic or genome-fragment RNAs that can carry genetic information but not perform a catalytic function. The aim of the current work is to extend the quasispecies concept back at least as far as to a system in which the catalytic function of an RNA molecule is integral to its own replication process, even if the actual polymerization of nucleotides is carried out by an exogenous polymerase. Here, the continuous evolution (CE) volution  of classvolution  of smallvolution . Now, wi2 was added to the reaction vessel at a final concentration of 40 μM to increase the mutational rate. The in vitro per nucleotide error rate of MMLV-RT has been estimated at about 1/30,000 [2+ ions lower the substrate specificity of RNA- and DNA-dependent DNA polymerases both in vivo and in vitro, resulting in a 6-30-fold higher error rate [2, in which a meltdown was observed at an average of 24.3 bursts [Four clonal 100-molecule populations of the RNA class I ligase ribozyme B16-19 Figure  were eac1/30,000 , but Mn2ror rate -30. All 3 bursts . In fact3 bursts .2 (the 600-molecule lineage 3D) were genotyped for comparison.To investigate the cause of the extended time to extinction, a preliminary inspection of the populational genetic variability using RFLP was performed. In general, the cDNA in a population at any burst can be amplified via PCR and then genotyped by either RFLP or nucleotide sequence analysis. The fixation, or nearly so, of mutant forms was evidenced in all the lineages at selected bursts. Based on RFLP assays [Additional file Alignment of the nucleotide sequencing data for lineages 6H and 6L showed a trend in the population dynamics in which a majority of the clones have the same genotype, while a minority have slightly different ones. This observation was the first clue that quasispecies behavior may be present in these ligase populations. Phylogenetic networks were drawn Figures  and 3 to2 were genotyped. Unlike lineages 6H and 6L, the network diagrams of these bursts failed to exhibit a dominant master sequence .For comparison, 33 and 48 clones, respectively, from bursts 10 and 50 of the smallest lineage  that sue Figure . Also thThe observed clouds also showed that there existed a fluctuating dynamic in these populations, as the shape of the clouds -- and gross amount of mutant sequences -- changed from one burst to another Figures  and 3E. H = -Σpi(lnpi) where pi is the frequency of the ith clone). Deeper examination of the bursts in this lineage ; burst 35, MS1 (91%); burst 50, MS2 (48%). This dynamic of master sequences being displaced by one another resembles that of clonal interference, in which advantageous mutants have to compete for resources and some get displaced -33. A neIn lineage 6L, four bursts were evaluated Figure . These s2, was not seen in the current study, although short insertions, deletions, and rearrangements were common in the last 10-12 nt at the 3' end of the ligase , the genotypes that appeared in lineages 6H and 6L were not identical to the dominant genotypes that appeared in lineages evolved without added mutational pressure . Some spin vitro, although a formal test of this will require a comparison of fitness values.In general, from all the networks drawn, it was observed that the number of mutations that appeared in more than one burst and/or lineage constituted 61% of the total number of sequences explored, and the great majority of these mutants became part of master sequences during the lineages' evolutionary history  method [2 was added to the reaction vessel to increase the error rate of protein enzymes. Populations evolved under these conditions did not show a shortened extinction time, as was observed previously when mutational meltdown conditions prevailed under only a weak mutational pressure of no added MnCl2 ["What is a quasispecies?" is the exact title of at least two papers ,38. At t) method  and the ed MnCl2 . The datin vitro evolution experiments with catalytic RNA. Other in vitro experiments with ribozymes have shown either convergence on a phenotype or recurrence of a genotype or motif, but not the type of dynamic of quasispecies that we are documenting here. For example, Yingfu Li and colleagues studied how the composition of a population of RNA-cleaving DNAzymes changed over time in response to selective pressures acting on the phenotype [2+ mutagenesis experiments presented here, the mutagen has a direct effect on the RNA structure, and therefore selection favored variants that mutated away the EthBr-binding sites, and a single "winner" emerged. Examples of experiments in which there is a convergent to a relatively more efficient solution for the population have been reviewed [in vitro evolution studies have been initiated from pools of limited diversity but in which a recurrent solution would appear upon independent trials. This was the evolution dynamic that gave rise to the class I ligase [Quasispecies behavior has never before been demonstrated during henotype ,40. Simihenotype . In thisreviewed ,42-45. OI ligase , and theI ligase , but qua2+ to the reaction vessels increases the mutation rate of the replication process and consequently it can alter the equilibrium distribution of the population [The current study employed the ribozyme B16-19, which is highly proficient in catalyzing the ligation reaction necessary for the CE to occur ,48, and pulation .The shift in the equilibrium distribution is possible because of two main reasons, first stated and then explained.(1) Catalytic RNA sequences such as ligase ribozymes posses the property of buffering mutations through epistatic interactions between secondary structure arrangements ,49. Thes(2) The fitness of each genotype in the population is normalized with the total number of genotypes in the system (assuming single locus theory applies). Thus, the proportional contribution of each genotype to the total fitness decreases as the number of genotypes increases .e.g., low Hamming distance values). The genetic load generated in higher-class mutants will likely disrupt secondary interactions and the stability of the individual ligases. Oddly enough, an increase in the mutational rate does not cause a proportional increase in the genetic load, and therefore the population does not become extinct at a faster pace. What could be happening in this case is that mutants of lower class emerge quickly, generating a wide low-mutant class in the early evolutionary pathway of the population. These mutants have a short Hamming distances and thus probably similar fitness values. Wilke [The high degeneracy observed in ribozyme genotype-to-phenotype maps insures that the majority of point mutations are neutral ,35. In ss. Wilke  observede.g., in a few generations) be regenerated from one another in the eventual case of being removed from the population by random drift. In contrast, poorly connected genotypes  will have a slow recovery into the population, if at all. In this scenario, because mutants with short Hamming distance may have close fitness values, individual sequences are not essential for the survival of the population, rather the group of close-connected individuals with mutational robustness [e.g., the strength that random drift has in populations of small effective sizes). This genotypic malleability allows the population to avoid a mutational meltdown, and stay extant.The level of connection in the matrix is determined by the Hamming distance values of the mutants in the network. Genotypes that are closely connected by short Hamming distances are closely related in the sense that they can rapidly  is an advantageous feature to ensure survival of populations during the RNA world [e.g., autocatalytic function). In contrast, less interconnected elements can serve as a reservoir of mutations without a detrimental effect on this information. Thus, during the ancient acellular times at the biogenesis on the Earth, the assemblage of information cores, perhaps in the form of quasispecies clouds, may have provided the necessary route to increase population sizes, and allow enough time for information to mature into more sophisticated functions necessary for a cellular type of life.These results -- of ligase RNA molecules capable of forming population structures in which cooperation is more beneficial than competition -- suggest that altruistic behavior  were incubated with 64 pmol of the substrate oligo S-163 , 50 pmol of TAS 1.23 primer for reverse transcription (5'-GCTGAGCCTGCGATTGG-3'), 250 units of MMLV reverse transcriptase , 50 units T7 RNA polymerase , 5 nmol each dNTP, 50 nmol each rNTP, 25 mM MgCl2, and 40 μM MnCl2, in a reaction buffer with 50 mM KCl, 30 mM 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS), pH 8.3. The 25 μL reaction mix was incubated at 37°C for exactly 22 minutes, at the completion of which the reaction was stopped by the addition of 981 μL of water. An 8.2 μL aliquot was taken from the dilution tube and used to seed the next reaction cycle of the continuous evolution process, thereby preserving a constant harmonic mean of the population size [Ligase ribozyme populations were evolved using the continuous hodology ,25,26,61(1) The survival of the evolved populations was surveyed through PCR amplifications of all the bursts used to seed a reaction cycle. The PCR products were electrophoresed through 2% agarose gels containing ethidium bromide. Visualization of the gels by trans-illumination allowed the identification of a correctly band size when the population remained alive.(2) Preliminary genetic variability was evaluated by RFLP using the restriction enzymes employed in the previous publication . In the E. coli competent cells . Between 60-120 colonies per burst were chosen completely at random for genotyping. Colony PCR was used to isolate the insert from single clones and further sequencing was done with BDT v3.1 chemistry. The sequences were aligned with ClustalX 2.0.11 software; the alignments were edited with BioEdit sequence alignment editor v7.0.9.0  and the chromatogram viewer FinchTV v1.4 .Bursts were chosen for in-depth sequence analysis based on whether they displayed variability by RFLP and/or on a general desire to examine early, middle, and later bursts of 50-burst lineages. In one case (lineage 6L), two adjacent bursts were analyzed because the RFLP analysis suggested a rapid genotypic shift at that time. Specific bursts of the evolving populations with genotypic diversity were cloned using the CloneJet™ PCR Cloning Kit  and To estimate if the number of clones sampled in each selected burst contained all the unique genotypes present in the population, rarefaction plots were constructed. Here, the pool of known genotypes is entered into the computer and then drawn in a random order by a computer  and plotted together using the median-joining method  implemenThe authors declare that they have no competing interests.NL designed the project; CDA helped in design details, performed the experiments, and processed the data; NL and CDA discussed the data implications and wrote the manuscript.Figure S1 - Comparison of two lineages of ligases evolved at different environmental chemistries; Figure S2 - Mutants detected with TaqαI restriction enzyme; Table S1 - Summary data gathered from the rarefaction plots.Click here for file