Myasthenic crisis is the most serious life-threatening event in generalized myasthenia gravis (MG) patients. The objective of this study was to assess the long-term impact of thymectomy on rate and severity of these attacks in Iranian patients.We reviewed the clinical records from 272 myasthenic patients diagnosed and treated in our neurology clinic during 1985 to 2002. Fifty-three patients were excluded because of unconfirmed diagnosis, ocular form of MG, contraindication to surgery, concomitant diseases and loss to follow-up. The Osserman classification was used to assess the initial severity of the disease. Frequency and severity of the attacks were compared between two groups with appropriate statistical tests according to the nature of variables. Multivariate logistic regression analysis was used to assess the predictors of myasthenic crisis in the group of patients without thymoma.110 patients were in thymectomy group and the other 109 patients were on medical therapy. These two groups had no significant differences with respect to age at onset, gender, Osserman score in baseline and follow up period. 62 patients  had reported 89 attacks of myasthenic crisis. 20 patients of 62 (32%) were in thymectomy group and 42 (68%) were in the other group. There was significant difference between the two groups in number of patients with crisis . In addition, these attacks were more severe in group of non-thymectomized patients as the duration of ICU admission was longer and they needed more ventilatory support during their attacks. Regression model showed thymectomy and lower age at onset as two predictors of decrement in myasthenic crisis rate in non-thymomatous MG patients.It is suggested that frequency and severity of myasthenic attacks as important endpoints in evaluation of MG patients. Thymectomy seems to have a preventive role on rate and severity of these attacks. Myasthenia Gravis (MG) is the mostly known autoimmune disease in human, mediated by autoantibodies against the nicotinic receptor of acetylcholine in neuromuscular junctions . Two carMyasthenic crisis refers to a rapid deterioration in neuromuscular function with respiratory compromise due to ventilatory muscle insufficiency or weakness of upper airway musculature or both . These aIn general, four methods of treatment are currently in use : antichoAlthough there is no convincing evidence regarding the use of thymectomy in non-thymomatous MG patients ,13, mostThymectomy effects on occurrence and intensity of respiratory crisis have not been studied yet. Concerning value of this life-threatening event in MG patients, we studied the role of thymectomy on long-term frequency and severity of these attacks.We reviewed the clinical records from 272 myasthenic patients diagnosed and treated in the neurology clinic of Shariati Hospital, Tehran, during 1985 to 2002. All included patients had positive history and physical examination, confirmed with positive Tensilon test and electromyography (with repetitive nerve stimulation test). Searching for thymomas, all patients had a chest computed tomography (CT) scan.The following patients were excluded from the study: 3 patients with doubtful test results and unconfirmed diagnosis; 25 patients with ocular form of MG (as these patients have no presentation of crisis); 2 patients with thymoma (and two episodes of crisis in the previous year), but contraindicated for surgery because of coronary artery disease; 6 patients with thyroid function abnormalities (because of their known effects on induction and reduction of myasthenic crises); 2 patients with concomitant rheumatoid arthritis (and consumption of high doses of corticosteroids and immunosuppressives); and 15 patients lost from follow-up with incomplete data. 219 MG patients enrolled in the study.Myasthenic crisis was defined as an attack that compels physicians to hospitalize the patient and carefully supervise patient's respiratory capacity . In mostAll included patients were eligible for surgery. Patients with CT confirmed thymoma underwent elective thymectomy as soon as possible. In the first visit after diagnosis confirmation in non-thymomatous patients, the advantages and drawbacks of operation (including risks of anesthesia in MG patients) had been explained to them and deciding between the two choices of thymectomy or conservative therapy had been left to the agreement between physicians and patients. No particular protocol had been established for these patients and physicians had only a consultant role for patients with no force on them.For clinical assessment of the initial severity of the disease, the classification of Osserman  had beent test and analysis of variances (ANOVA) as parametric tests, Mann-Whitney U test as non-parametric test, and Pearson's chi-square test for qualitative differences (SPSS software, version 10.0). P values less than 0.05 were taken to indicate statistical significance. Continuous variables are shown as the mean ± standard deviation (SD) and non-parametric variables with their median and range. Odds ratios and relative risks with 95% confidence intervals (95% CI) were calculated to assess the proportional risk of crisis between two groups.The participants' baseline characteristics and follow-up data were analyzed by Student's In the subgroup of patients without thymoma, multivariate logistic regression analysis was used to assess the relationship between frequency of myasthenic crisis and several risk factors such as sex, start age of the disease, Osserman score and thymectomy. According to its percentile, start age was divided into three categories as bottom one-third (under percentile 33), middle one-third (between percentiles 34 and 66), and top one-third (over percentile 67).110 patients underwent thymectomy  and 109 patients were on conservative therapy. Baseline characteristics and follow-up periods are shown in Table Totally, 62 patients in both groups experienced 89 attacks of respiratory failure during their follow-up, which accounts for 28.3% of the study population. As it is shown in Table To assess the severity of each attack, we considered duration of ICU admission (days), need to respiratory support and need to plasmapheresis or IVIG during crisis as indicating variables. As it is shown in Table U test revealed significant difference in this regard (P < 0.001). Severity indexes did not differ significantly between thymomatous and non-thymomatous patients Table Pathology reports in thymectomized patients revealed 16 cases of thymoma. Fifty percent of these patients (8 of 16) had experienced crisis, which accounts for 40% (8 of 20) of thymectomized patients with history of crisis. Mann-Whitney Totally, there were 203 patients without thymoma. Regression analysis in this subgroup of patients revealed that group of non-thymectomized patients as well as two third of patients with higher ages at the beginning of the disease were more prone to respiratory attacks Table option to increase the probability of remission or improvement in these patients [Persuasive evidence for thymectomy in non-thymomatous generalized myasthenic patients is not on hand. In an attempt to establish standards in this regard, American Academy of Neurology advised thymectomy as only an patients . In thiset al. in 1981, 15 of 28 thymectomized patients experienced 21 crises during follow-up period [Surprisingly, none of these studies has considered myasthenic crisis frequency as their study endpoint. For instance, in the study of Cohen p period . In anotp period . This stIn our experience, persistence on thymectomy for all myasthenic patients would not be so wisely. Response to thymectomy is highly variable among these patients. Method of gaining agreement between physicians and patients would bring some benefits. As it is shown in Table When myasthenic crisis occurrence is considered as the main endpoint of treatment in myasthenic patients, thymectomy has a great impact. Patients on conservative therapy had risk of about 3-fold of thymectomized patients to encounter respiratory attacks. However, the overall frequency of myasthenic crisis among our study population could be considered more than similar reports ,4, perhaSome previous studies have shown the benefit of thymectomy in the control of symptoms of MG. It has been revealed that delay in surgery could worsen the prognosis of MG  and the During myasthenic crisis, Weakness of the respiratory muscles may be out of proportion to that of other skeletal muscles. In rare cases, ventilatory failure is the only clinically apparent manifestation of the disease ,25. In aGenerally, infection is the most common trigger for myasthenic crisis . Higher The main limitation of this study was its retrospective design and the lack of randomization. Apparently, within a period of 17 years, many changes have occurred to diagnostic and therapeutic facilities as well as knowledge and attitude of physicians. These changes could have significant consequences on the outcome of myasthenic patients. Although various operative approaches seem not to differ drastically , variousIn conclusion, we suggest evaluation of various aspects of myasthenic crisis as an important endpoint in long-term assessment of generalized MG patients. Moreover, we showed that thymectomy has a preventive role on rate and severity of myasthenic attacks in age and disease severity matched groups of patients. However, this effect needs further evaluation in upcoming prospective studies.None declared.In advance, suggestion of the design of the study was from our professor AkS. Data extraction and initial analysis were done by AlS and HT. SA participated in the design and implementation of the study. AM performed additional analyses and wrote the first draft of the paper. MS and SA both had helpful and valuable comments in revising the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


C. elegans and S. cerevisiae genomes were 25%–50% more constrained prior to duplication than the genes that failed to leave duplicates. We further show that conserved genes have been consistently prolific in generating duplicates for hundreds of millions of years in these two species. These findings reveal one way in which gene duplication shapes the content of eukaryotic genomes. Our finding that the set of duplicate genes is biased has important implications for genome-scale studies.A central goal in genome biology is to understand the origin and maintenance of genic diversity. Over evolutionary time, each gene's contribution to the genic content of an organism depends not only on its probability of long-term survival, but also on its propensity to generate duplicates that are themselves capable of long-term survival. In this study we investigate which types of genes are likely to generate functional and persistent duplicates. We demonstrate that genes that have generated duplicates in the  Gene duplication is a key process in genome evolution. These authors show that highly conserved genes duplicate more often and are therefore likely to contribute more to the content of eukaryotic genomes Gene duplication is the most important source of new genes and consequently a vital source of genetic novelty . RecentlSaccharomyces cerevisiae (Investigating this aspect of gene duplication will not only help answer questions about gene content—such as why certain proteins duplicate to generate multigene families while others remain in single copy—but will provide insight into the process of duplication itself. Each of the three steps leading to the generation of preserved gene duplicates, including their (1) mutational generation, (2) fixation in a population, and (3) preservation through a period when they may be functionally redundant, may favor some genes over others. For example, gene duplicates that lead to an advantageous increase in gene dosage will be preferentially fixed by positive selection, as has been observed in bacteria and revisiae . For othrevisiae , and whicis-regulatory regions, expressed in many tissues . This measure of protein evolution has been shown to be related to several important properties of genes, including dispensability, level of expression, and the number of protein–protein interactions (S. cerevisiae and Caenorhabditis elegans with those that have not.For these reasons we chose to investigate a bias in the molecular attributes of the genes that duplicate. One very informative gene attribute is the rate of protein evolution defined as the number of nonsynonymous substitutions per nonsynonymous site in a given time (ractions . We chosS. cerevisiae and C. elegans (the “study genes”), but instead measured evolutionary rates in two distantly related outgroup species, Drosophila melanogaster and Anopheles gambiae (such a pair of orthologs is referred to as a “representative pair”). Because evolutionary rates for a particular gene are highly correlated in diverse lineages (S. cerevisiae and C. elegans appear to be a biased set of slowly evolving genes and that slowly evolving genes have been consistently prolific in generating duplicates for hundreds of millions of years in these lineages.Such a comparison is not straightforward since gene duplication itself may affect the rate of molecular evolution . To avoilineages , we reaslineages . Our resS. cerevisiae and C. elegans and the number of representative pairs of these genes found in D. melanogaster and A. gambiae are provided in S. cerevisiae and C. elegans have much slower rates of evolution (Mann–Whitney U test, p < 0.001 for both) (S. cerevisiae evolve on average more than 50% slower than the representative pairs of singletons (0.192 versus 0.302), while in C. elegans the difference exceeds 25% (0.230 versus 0.296).The number of duplicate pairs and singleton genes identified in the genomes of or both) . The repA. gambiae and D. melanogaster. Results from this analysis echoed those of the KA comparisons: representative pairs of duplicate genes in both S. cerevisiae and C. elegans are much less likely to have accumulated insertions or deletions than representative pairs of singletons (Mann–Whitney U test, p < 0.0001 for both)  .KA and the indel rate. First, some of the orthologs identified in D. melanogaster and A. gambiae have undergone duplication in these lineages. This could both affect their rates of evolution, as discussed above, and also lead to the identification of the slowest evolving paralog in D. melanogaster and A. gambiae for the representative pairs of study genes. The latter effect can lead to an artificially low estimate of the evolutionary rates. To test for this possibility, we repeated our analysis using only representative pairs that have not duplicated in either D. melanogaster or A. gambiae. Although this analysis included substantially fewer genes (see p < 0.005 for both organisms). Second, we wanted to make sure that the bias is not due to the peculiarly slow evolution of duplicate genes in multigene families. A reanalysis for only those duplicated genes (in the study organisms) with no other paralogs in the genome revealed very similar results (data not shown). Third, it is possible that our conservative definition of “singleton” may have artificially biased the set of singletons towards rapidly evolving genes. This could be true if slowly evolving singleton genes tend to possess anciently conserved, widely shared protein domains. By generating homology to other genes, these domains may make these singletons fall below the conservative E-value cutoff that we used. To test this possibility, we relaxed our criteria for singleton genes to include all those genes with no E-value less than 10–10. The average rate of evolution for this group of singleton genes was no different than for the former set (data not shown).To further validate these conclusions, we wanted to test several potential sources of error in our analysis of both enes see , the resKS) for the representative pairs of the two classes of genes. This measure is customarily used as a proxy for mutation rate because substitutions at synonymous sites are generally thought to be selectively neutral. However, in many genes, especially those expressed at high levels, synonymous sites appear to be under selection, as evidenced by codon bias. For such genes, the rate of synonymous evolution will underestimate the rate of mutation , gene class (whether the representative pair was for a duplicate or singleton), and KS (between representative pairs). Our results, presented in S. cerevisiae duplicate genes have a lower KS than representative pairs of singleton genes (Spearman Correlation column), but that this correlation disappears when we control for codon bias . Thus, in the case of S. cerevisiae, the higher codon bias of the slowly evolving representative pairs completely accounts for the differences in KS between the two groups. For C. elegans, the KS of the representative pairs for duplicate genes is in fact marginally higher than that for singleton genes, and this slight trend remains when codon bias is taken into account. Thus, mutational differences cannot account for the differences in the rate of protein evolution in either S. cerevisiae or C. elegans.To overcome this complication, we computed the partial correlation coefficients between each of the three factors: codon bias (measured by the codon adaptation index [CAI]  in D. meKS above. D. melanogaster), and KA (of the representative pairs). This analysis revealed some important differences in how the duplication bias is generated in S. cerevisiae and C. elegans.We can also use the level of codon bias to gain additional insight into the potential reasons for the generation and maintenance of duplicate copies of conserved genes. Codon bias is a proxy for the level of expression , while tS. cerevisiae show that the CAI of the representative pairs of duplicates is greater than that of the representative pairs of singleton genes. This indicates that the genes leading to preserved duplicates in S. cerevisiae tend to be unusually highly expressed (p < 0.001). In contrast, for C. elegans, duplicate genes do not appear to be biased towards highly expressed genes (p > 0.1). This difference may reflect a disparity in the mutational generation, fixation, or preservation of duplicates in these two organisms. This analysis also reveals that when codon bias is held constant, the relationship between KA and gene class persists in both organisms. In the case of C. elegans, the correlation coefficient between gene class and KA remains nearly identical when CAI is held constant. For S. cerevisiae, the partial correlation coefficient between KA and gene class does decrease when CAI is held constant (but remains highly significant), implying that the slower evolution of representative pairs of the duplicated genes in S. cerevisiae is partly mediated by preferential duplication of highly expressed genes. To validate these conclusions, we repeated the same analysis using CAI values in the study organisms rather than in D. melanogaster. This analysis revealed very similar results (data not shown).First, both direct and partial correlations for S. cerevisiae and C. elegans lineages, we plotted the evolutionary rate of representative pairs and the average CAI (both in D. melanogaster and in the study organisms) for duplicate pairs of different age classes (where age is measured by KS between the duplicate study genes) , this analysis captures the uniformity of the bias in these lineages. For both organisms, slowly evolving genes appear to have led to the duplicate genes in all age classes (covering hundreds of millions of years). For C. elegans, both the evolutionary rates of the representative pairs and their CAI values remain virtually constant for duplicated genes of all ages. In addition, the CAI values for the duplicate pairs of different ages in C. elegans are very similar to the CAI values for singletons—the only exception is a slight elevation in the CAI for duplicate pairs in the KS range from 1 to 1.5. By contrast, the plot for S. cerevisiae reveals that young duplicate genes (KS < 2.0) tend to have representative pairs with a lower KA than those of older pairs, and this trend is paralleled by the elevated CAI of these young duplicate pairs.To determine whether conserved genes have been preferentially duplicated throughout the history of the y genes) . While lS. cerevisiae is that duplicate pairs with a high codon bias are expected to have a depressed value of KS, as discussed above, and thus will appear younger than they really are. To overcome this problem, we corrected KS estimates for S. cerevisiae genes based on their CAI using a simple approach recently developed for this species  (S. cerevisiae was biased towards highly expressed genes, this could explain the heterogeneity in both KA and CAI and could explain why duplicate genes in C. elegans, an organism that has likely not undergone a whole-genome duplication, were not enriched for genes with a high level of expression. With respect to this hypothesis, it is interesting to note that for young duplicate genes (KS < 2), KA estimates for representative pairs of duplicate genes in S. cerevisiae are much lower than for duplicate genes in C. elegans, whereas for older duplicate genes (KS > 2), the KA estimates are roughly equivalent in both S. cerevisiae and C. elegans.A problem for interpreting this trend in cies see  (A. Hirsslightly , but thears ago) . If the S. cerevisiae  many of the genes that are duplicated in a single individual are deleterious and thus are quickly removed from the population or (2) many of the duplicate genes that reach fixation in a population do so because of positive selection for the duplicate copy, rather than reaching fixation neutrally by genetic drift.For the first mechanism to work, increases in the dosage of slowly evolving genes must be less likely to have deleterious consequences to organismal fitness than increases in the dosage of more rapidly evolving genes. Recent empirical work, however, has shown that the opposite might be true. In particular, data from yeast have shown that less dispensable, slowly evolving genes are more likely to be haploinsufficient than dispensable genes . This imS. cerevisiae and bacteria  many of the duplicate gene copies that arise in a population are lost quickly to nonfunctionalizing mutations  and (2)  tissues , or of g tissues . Because tissues , these mS. cerevisiae or C. elegans  an increased rate of evolution for the duplicated genes immediately following duplication e.g.,  and (2) S. cerevisiae, C. elegans, D. melanogaster, and A. gambiae were downloaded from GenBank (Bethesda, Maryland, United States) at http://www.ncbi.nlm.nih.gov/Ftp/index.html. To identify duplicate and singleton genes, a reciprocal protein BLAST  and eliminated all representative pairs with paralogs with an E-value of less than 0.1 (leaving only 38 duplicates and 114 singletons and 29 duplicates and 318 singletons, respectively). Results from the reanalysis revealed significant trends similar to those found when using all representative pairs.To test whether including duplicate pairs and singleton genes with representative pairs possessing paralogs in the D. melanogaster, S. cerevisiae, and C. elegans genomes using the program CodonW . The table used to calculate CAI for S. cerevisiae is the standard table included in the package. We obtained the appropriate codon usage tables for C. elegans and D. melanogaster from studies by We obtained CAI values for genes in the S. cerevisiae, we used CAI values of each pair to help obtain a better relative estimate of their ages. This was necessary because duplicate pairs with a high codon bias effectively have fewer neutral synonymous sites, resulting in the gross underestimation of their age based on KS alone (S. cerevisiae is given by KS = rt(1 – c), where r is the rate of synonymous substitution in genes with no codon bias, t is time, and c is codon bias as measured by CAI . Rearranging this equation yields the formula KS′ = rt = KS/(1 – c), which we used to obtain corrected estimates of the age of duplicate pairs in S. cerevisiae. No such correction was made for C. elegans genes because they were not shown to have a significantly higher codon bias than singleton genes and because no simple means of correction is presently known.For duplicate genes in S alone ; Li 1997


The importance of low high-density lipoprotein cholesterol (HDL-C), elevated non HDL-C (as part of the metabolic syndrome, prediabetes, and type 2 diabetes mellitus), and an isolated low HDL-C is rapidly emerging. The antiatherosclerotic roles of reverse cholesterol transport and the pleiotropic antioxidant – anti-inflammatory mechanistic effects of HDL-C are undergoing rapid exponential growth.In 1997 a 53-year-old Caucasian male presented with a lipoprotein profile of many years duration with an isolated low HDL-C and uric acid levels in the upper quintile of normal. He developed an acute myocardial infarction involving the right coronary artery and had percutaneous transluminal coronary angioplasty with stenting of this lesion. He also demonstrated a non-critical non-flow limiting lesion of the proximal left anterior descending coronary artery at the time of this evaluation.Following a program of global risk reduction this patient has done well over the past 7 years and remains free of any clinical signs and symptoms of atherosclerosis. His HDL-C and uric acid levels are currently in the normal physiological range.Low HDL-C and isolated low HDL-C constitute an important risk factor for atherosclerosis. Therapies that lead to a return to normal physiologic range of HDL-C may result in the delay of atherosclerotic progression. MRH, a 53-year-old Caucasian male (physician) developed an acute inferior myocardial infarction (MI) associated with bradycardia and occasional PVCs. Emergency medication included aspirin, nitroglycerin and a bolus of TPA.The cardiology team preformed PCTA at the site of near complete blockage of the right coronary artery with successful stent placement. At this time a non-critical 40% lesion located in the proximal left anterior descending coronary artery was noted, which was not manipulated. The patient was discharged following 24 hours of stable monitoring.Herpes Simplex encephalitis 1989 with full recovery.Relapsing fever 1971 full recovery, spontaneous left pneumothorax times two (1982–83), lumbar fusion back surgery 1985, and Mother with CVA (cerebellar) age 58 full recovery. Died of Hodgkin's lymphoma 64. Brother with type 1 diabetes mellitus with onset at age 29  died in sleep age 51.Father with CVA (vertebrobasilar) age 75 with full recovery, COPD, died in sleep while recovering from TIA and pneumonia age 84.Grandparents lived to their 80s and died of old age.High stress family physician who seldom drank alcohol and smoked a pipe occasionally. Blood pressure at times of high stress would elevate to 140/85–88 and return to 120–130s/ 70–75 at times of non-stress in the office. He was physically active with no dedicated exercise programFive months prior to MI and reflective of numerous metabolic profiles over the preceding decades.Total cholesterol 198 mg/dLTriglycerides 154 mg/dLHDL-C 34 mg/dL. HDL-C (1970–1973 32 mg/dL and 34 mg/dL)LDL-C calculated 120 mg/dLNon HDL-C = (198-34) = 164Total Chol/HDL ratio = 6.2 > than 5 and is highUric acid 6.5 mg/dLBlood sugar non-fasting 102 mg/dLHomocysteine first week post MI fasting: 28 mcmol/LLFTs, electrolytes, calcium and phosphorus, serum iron, renal function, and CBC were all in normal range.Patient started a program reflecting the global risk reduction approach described in the RAAS acronym table  and is cCurrent Laboratory Values 2004:Total cholesterol: 138 mg/dLTriglycerides: 94 mg/dLHDL-C: 45 mg/dLLDL-C calculated: 74 mg/dLNon HDL-C: (138-45) = 93Total Chol/HDL ratio = 3.0Uric acid: 6.5 mg/dLBlood sugar: Fasting 80 mg/dL, 2 hour post prandial 118 mg/dLHomocysteine: 7.2 mcmol/LLp(a): 4.2 mg/dL in normal range immediate post MI and again at this time: 4.3 mg/dL.hs-CRP: 0.7 mg/L.LFTs, electrolytes, calcium and phosphorus, serum iron, renal function, and CBC are all in normal range.This patient has done well over the past seven years and remains free of any clinical signs and symptoms of cardiovascular disease. While this patient will always remain a CHD risk, his current laboratory values remain in a normal physiological range. As noted above his HDL-C and uric acid levels are currently in the normal physiological range and his hs-CRP remains in the second quartile.According to Framingham risk scores associated with the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) guidelines , few wouIf we score this patient according to the estimate of 10-year risk for men he gets 6 points for age 53, 2 points for total cholesterol 160–199 age 53, 3 points for being a pipe smoker, 2 points for HDL being < 40 mg/dL, and 1 point for systolic blood pressure 140–159 untreated. This totals 14 points and results in an estimated 10-year risk for men of 16%, which is less than the 20% recommended for more aggressive therapy.Even if we apply the NCEP ATP III guidelines of having two plus risk factors: Male sex, hypertension, smoking, and low HDL-C with a 10 risk < or = to 20% we obtain the following recommendations: LDL-C goal < 130 mg/dL, initiation of TLC if LDL-C is = or > 130 mg/dL, consideration of drug therapy if LDL-C is > or = to 130 mg/dL after three months of TLC. It is important to note that our patient had a LDL-C of 120 mg/dL prior to his event. Even if we look at the non HDL-C levels, which are allowed to be 30 mg/dL higher than LDL-C goals we have a patient with a non HDL-C of only 164. MRH became a CHD risk patient within a short period of time of 5 months.The importance of low HDL-C and cardiovascular disease associated with the lipid triad  found in the metabolic syndrome (metS) and overt type 2 diabetes mellitus (T2DM) and a contributing factor to the elevated non HDL-C discussed in the current NCEP ATP III guidelines or the patients with isolated low HDL-C is rapidly evolving.The accelerated atherosclerosis (atheroscleropathy) associated with the metS and T2DM has been previously reviewed and is definitely a serious problem associated with the current epidemic of obesity – diabesity and T2DM .Both isolated low HDL-C and elevated non HDL-C  levels are difficult to get to known NCEP ATP III recommendations and this task usually requires combination therapy. These therapies consist of therapeutic life style changes and pharmacotherapy including statins, fibrates, selective cholesterol inhibitors such as ezetimibe, and niacin in addition to a global risk reduction of all non HDL-C existing risk factors table  5]..5].In this case report a focus on isolated low HDL-C is appropriate. This case report demonstrates a marked improvement of all lipid parameters including his low HDL-C. However, this marked improvement is not always as simple as this case and therefore, both the patient and the clinician need to be very patient, as well as, creative in order to achieve global risk reduction .In 1977 the Tromso Heart Study demonstrated that CAD patients have HDL-C levels 35% lower than controls and those patients with low HDL-C are three times more likely to develop CAD than those with elevated LDL-C . These eet al. were able to show an inverse association of high HDL-C and low coronary risk was as statically as strong as the direct association of high LDL-C and high coronary risk in a cohort of men and women age 40–82 followed for 12 years who were free from CAD at study entry. At any level of cholesterol low HDL-C increases the rate of CHD [The inverse relation of HDL-C to CHD events has been widely discussed since the original publication of data from the Framingham study 1986) ,8. Caste6 ,8. CasThe NCEP ATP III guidelines clearly defines a level < 40 mg/dL as an independent risk factor for CHD . RaisingMichael Miller has stated: "Low HDL-C is the most common lipoprotein abnormality in patients with CHD and is predictive of CHD events, even when total cholesterol levels are normal" .et al., found that the prevalence of isolated low HDL-C as a risk factor for CHD mortality to be present in one out of six or 16.6 % while studying a 21-year follow up of 8000 men [Goldbourt U 8000 men . FurtherThere are at least eight secondary causes for low HDL-C table  and at lHDL-C is synthesized in the intestine and liver and is extremely important in reverse cholesterol transport from the tissues to the liver for disposal. It works in conjunction with the ABCA1 cholesterol transporter within the intimal macrophages  associated with overt T2DM and multiple risk factors associated with accelerated atherosclerosis may result in a damaging effect to HDL-C and interfere with the ABCA1 transporter in reverse cholesterol transport via a mechanism of oxidation and nitration of tyrosine residues on the apo A-1 lipoprotein outer shell of HDL-C lipoprotein . This biLifestyle changes that are important in raising low HDL-C consist of smoking cessation, weight loss, exercise, and the use of alcohol in moderation.HDL-C has numerous positive effects on the endothelium and arterial vessel wall, which decrease non-diabetic atherosclerosis and the accelerated atherosclerosis – atheroscleropathy associated with metS and overt T2DM table .NCEP ATP III allows the clinician to factor in the additional risks associated with novel, emerging risk markers such as our patients elevated homocysteine. Other risk markers would be the lipid markers: Elevated triglyceride, remnant lipoproteins, lipoprotein (a), an abnormal TC/HDL-C ratio, small dense LDL particles, HDL subspecies, and apolipoprotein A and B. The non lipid markers would include: An elevated glucose, inflammatory markers (elevated hs-CRP and the emerging importance of the various interleukins and in particular IL-6, which is the driving force behind hs-CRP elevation), coagulation markers (elevated PAI-1, Lp(a), and fibrinogen), the emerging roles of matrix metalloproteinases (MMPs) and of course the established risk marker of an elevated homocysteine. It is interesting to note that Qujeq D et al., noted a negative correlation between total homocysteine and HDL-C levels (p < 0.05, r = 0.93) in a study evaluating 126 patients  with unequivocal changes of acute myocardial infarction in the electrocardiogram as compared to 135 normal healthy controls, while noting a positive correlation between total homocysteine and LDL-C levels (p < 0.05, r = 0.98) [The reader may note that the patients' uric acid level was quite elevated prior to his acute coronary event and that this level returned to a very normal level following global risk reduction and aggressive therapy for his multiple risk factors in addition to his isolated low HDL-C. Although not a considered a risk factor or even an emerging, novel risk marker, uric acid may be a quite sensitive marker of underlying redox and oxidative stress. Uric acid levels greater than 4 mg/dL may be considered a red flag in those patients, such as our case report, with high risk for CHD .When viewing the sources for atherosclerosis it is important to note that there are two routes for accumulation of atherogenic lipoproteins (input and outflow) within the arterial intima and subsequent remodeling of the arterial vessel wall.L-A avw = L-in - L-out.The atherosclerosis equation: Lipoprotein Accumulation (retention) in the arterial vessel wall = Lipoprotein in - lipoprotein out. non HDL-C = total Cholesterol – HDL-C .L-in, would equal the net lipoproteins derived from the GI tract (absorption) plus that synthesized by the liver. Lipoprotein out is strictly via reverse cholesterol transport to the liver and secretion via bile into the gut. L-in is primarily the beta lipoproteins or apolipoprotein B containing lipoprotein particles, whereas L-out depends primarily on the alpha lipoproteins, apolipoprotein A or HDL-C. The beta lipoproteins are atherogenic and the alpha lipoproteins are antiatherogenic. From this analogy one can see can see why non HDL-C was so important in the recent NCEP ATPIII guidelines: This is also why the recent global (52 countries) INTERHART study found the ApoB/ApoA-1 ratio (the ratio of atherogenic lipoproteins to non atherogenic lipoproteins) to be the best predictor of CHD (odds ratio of 3.25 for top verses lowest quintile) as compared to the other eight other risk factors (table L-Aavw = ApoB/ApoA-1 ratio of the INTERHART studyL-in, would be comparable to the faucet (GI tract and Liver) delivering the atherogenic apoB lipoproteins. While the kitchen sink would represent the accumulation of atherogenic lipoproteins within the arterial vessel wall or L-Aavw.In a like manner, the DRAIN would represent L-out or HDL-C or apoA-1 lipoproteins. From this analogy it can easily be seen that if there is inadequate HDL-C or apoA-1 the atherogenic kitchen sink will overflow and result in acute coronary syndromes as happened in our case report figure .While the treatment of isolated HDL-C may seem overwhelming at times, it will be rewarding for both the clinician and the patient as demonstrated by the our case study. This patient has done well for seven years and it is anticipated he will continue to do well with his laboratory values now in a sustained, normal physiologic range.Additional tests by nuclear magnetic resonance spectroscopy (NMR LipoProfile) would assist us in knowing the LDL particle number (LDL-P) and would assist us in even more aggressive therapy. In addition to his current goals he has met, he should have an LDL-P under 1000 micromol/L and small LDL-P under 700 micromol/L.Apo A-1Milano and Apo A-1Paris have resulted in a marked increase in research interest for the HDL-C lipoprotein particle and its future manipulation [Even though we have discussed LDL-C from a quantity perspective, due to an isolated low HDL-C, we should additionally be aware that there exists and equally important role for the quality of HDL-C . Recentlpulation . In the pulation . Recentlpulation . Additiopulation .Recently John Snow, M.D. (1813–1858), a legendary figure in the field of epidemiology, of London, England was honored . He hypoCould low HDL-C be the "pump handle" of atherosclerosis and CHD?ABCA-1: ATP binding cassette transporter A-1BMI: body mass indexCHD: coronary heart diseaseCAD: coronary artery diseasehs-CRP: highly sensitive C reactive proteinT2DM: type 2 diabetes mellitusmetS: metabolic syndromeHDL-C: high density lipoprotein cholesterolLDL-C: low density lipoprotein cholesterolLFTs: liver function testsPCTA: percutaneous transluminal coronary angioplastyVLDL-C: very low density lipoprotein cholesterolTC: total cholesterolTLC: therapeutic lifestyle changesTPA: tissue plasminogen activatorThe author(s) declare that they have no competing interests.MRH conceived the idea to write this manuscript. MRH and SCT wrote, and edited this manuscript together.


A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low MgFollowing unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs.Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through periods of receptor blockade. The similarity to key features of long-term depression and long-term potentiation suggests a possible relation to these phenomena. Additionally, a short term potentiation and decay (<5 min) were observed during sudden start of NMDA receptor activation supporting the idea that NMDA receptor mediated responses are highly plastic. In the present study, we will further develop the concept of gradually decaying responses.Hippocampal synapses display a variety of activity dependent changes that may represent basic elements of memory. Of foremost interest are long-term potentiation (LTP) and depression (LTD), especially forms that depend on N-methyl-D-aspartate (NMDA) receptor activation and therefore can attain "associative" properties -3. The schannels . Dependieceptors . Severaleceptors . We have of time . FollowiOne critical issue regarding LTP, LTD as well as other forms of glutamatergic synaptic plasticity, is the relative contribution of different glutamate receptor subtypes in creating the synaptic modification. Knowledge about this matter may be helpful in elucidating the underlying modification. While a selective change of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been cherished -10, espe2+ solution in the presence of NMDA receptor antagonist AP5 to allow for pathway equalization (see Methods) without evoking NMDA EPSPs. Synaptic transmission was then entirely blocked by adding AMPA receptor antagonist CNQX, followed by unblocking of NMDA receptors by wash out of AP5. During this time, only one pathway was stimulated, keeping the other one silent for later use. As illustrated in Fig. 2+ remained stable for several hours (changing to 96 ± 5 % of baseline after 2 h, n = 5, not illustrated).AMPA EPSPs were initially recorded in low MgTo exclude the possibility that the observed decay of NMDA responses were due to deterioration of slices, implying a general decrease of essential physiological processes, the experiment was repeated for the other pathway, i.e. the one that had not previously expressed NMDA responses (continuation in the same set of 8 slices). As seen in Fig. The same experiment was used to address the question of persistency as well as input specificity of the slowly developing depression of NMDA EPSPs. As can be seen in Fig. The above results suggest that the progressive decay observed in a single pathway as an instant event actually represents a long-term, pathway-specific change that can be assessed long later by comparison across pathways. To further examine the relation between the instantaneously recorded depression and the one measured about 90 min later, the relation between the two was plotted as illustrated in Fig. To pinpoint the induction mechanism, in terms of a pre- versus postsynaptic location, experiments were carried out in a similar way as above except for keeping AP5 in the solution during the first session see Fig. . Hence, Depending on the type of synaptic modification, EPSP waveforms may change in different ways, and previous work in our lab has demonstrated that NMDA EPSPs are more prone than AMPA EPSPs to show these changes . Taking While our data show that the time window matters for measuring NMDA EPSPs, all the above results were qualitatively similar regardless of which window was used. It can be noted, however, that the depression of NMDA EPSPs by the end of a recording session was less pronounced using early measurements than late ones .In 12 out of the 20 experiments, the fiber volley was well separated from the stimulus artifact allowing it to be properly measured. No significant change was detected, the end-of session value amounting 103 ± 2 % of the value at the EPSP peak (p > 0.05, not illustrated).In the above, NMDA receptor activation occurred gradually while the antagonist AP5 was washed out. This is in line with the experimental protocol used in our previous work on composite EPSPs containing both AMPA and NMDA components . HoweverIn order to determine whether the transient potentiation had any obvious relation to the slow depression of NMDA EPSPs, the relation between the two was examined. Thus, the degree of initial potentiation was calculated by comparing the pathways just after stimulation was started and the depression was determined, as before, by comparing the end-of-session value with the peak value  activation of afferents. The decline was found to be a form of long-term synaptic depression with an induction linked to NMDA receptor activation and with an expression that was maintained through periods without such activation. Several of its basic characteristics were similar to those of conventional LTP and LTD, suggesting a possible relation to these phenomena.in vitro due to declining viability of biological tissue or other experimental imperfections. Such unspecific "run down" can not account for the present findings since the gradual depression of responses could be repeated in the same slice, using a previously undepressed pathway. On the other hand, if the experiment was repeated twice in the same pathway, the second occasion revealed a diminished NMDA EPSP that showed little further decay. Together, these results show that the depression is input specific and long lasting and that it can saturate. Moreover, the lack of associated changes of the fiber volley speaks against a failure of axon conductance [Decaying responses is a potential side effect in long-term, electric recording ductance , favorinWhile both pre- and postsynaptic expression mechanisms appear feasible, certain mechanisms of induction can be excluded. For instance, a decrease in probability of glutamate release due to a direct depletion of the vesicle pool is unlikely since AMPA EPSPs could be evoked for several hours without significant decay . EveThe input specificity and NMDA dependent induction of the current depression conform with basic properties of conventional LTP and LTD . The depGradually decaying, NMDA receptor mediated responses were observed previously in our lab during recording of composite AMPA-NMDA EPSPs for several hours ,23. AtteWhile we observed that isolated NMDA EPSPs decay "spontaneously", most prior studies employing such EPSPs did not report a decay. It might be that limitations of recording time concealed the effect and cell dialysis during whole cell recording could also be a limiting factor. Actually, a recent study, recording "novel" responses under whole cell conditions, reported on decaying AMPA EPSPs but constant NMDA EPSPs . The posStandard LTP/LTD experiments compare relatively stable periods of recording before and after induction of the synaptic modification. This was not possible in the present case, since merely test stimulation evoked the decay. Therefore, comparisons were generally made between synaptic pathways subjected to different stimulus paradigms. The induction of depression in a single pathway during an initial 2 h period caused a subsequent difference between NMDA responses of the two pathways throughout a subsequent test period. The degree of initial decay was closely related to the later difference between pathways, suggesting that once depression occurred it could be maintained through periods of receptor blockade until testing was performed. Our data suggest a duration of the depression of more than 3 h after the initial induction period. This is in the range commonly referred to as "late", which is believed to involve special biochemistry such as gene expression and protein synthesis ,26. WhetThe gradual depression of NMDA EPSPs was found to saturate after 2–3 h as evidenced by both single and double session experiments. This is in line with several other forms of NMDA-dependent plasticity, including LTP, LTD and chemically induced variants, which are shown to be saturable -29. WhetPrevious work on conventionally induced LTD revealed an essential role for protein phosphatases in mediating the synaptic modification ,31. ConsNMDA receptors also have a number of other regulatory sites, allowing for modulation by glycine, polyamines, calcium, and redox agents  and theyAlthough a postsynaptic modification appears to be the primary choice, a presynaptic one that is initiated postsynaptically is also conceivable. In previous attempts to distinguish between pre- and postsynaptic mechanisms, LTD was compared with depression caused by various pharmacological agents with respect to the ability to influence the waveform of EPSPs . While LWhile the main line of experiments employed a smooth start of NMDA receptor activation following the gradual washout of AP5, another set of experiments made use of sudden activation by awaiting full washout until stimulation was started. Compared to smooth activation, there was an additional, transient potentiation that largely decayed within 20–30 stimuli. This is in accord with a previous study in hippocampal slices showing that stopping stimulation of composite AMPA-NMDA EPSPs for 10–60 min (and one case of isolated NMDA EPSP for 10 min) resulted in a transient potentiation when stimulation was resumed .2+ via the NMDA channels. Similar mechanisms of receptor desensitization/inactivation might be responsible in the present case in forming the transient phase after starting stimulation. Some details remain unexplained by this simple model, such as the biphasic character of the transient phase in terms of initial growth and subsequent decay. One can speculate that a minor LTP, or short-term potentiation, might be induced by the sudden activation of NMDA receptors and so would contribute to the initial growth, although the underlying cause is not addressed in this kind of explanation.Several other studies describe decaying NMDA responses in relation to inactivation or desensitization of receptors ,45. Acco2+ solution was used in our case to unblock NMDA receptors, similar unblocking may occur naturally in response to depolarization. Several important issues are still not settled. Is the saturation of the NMDA EPSP depression an absolute matter or can it be overcome, leading to further down regulation and possibly silencing of synapses? Conversely, is it possible to reverse, i.e. de-depress, the change by LTP or similar processes, allowing for bidirectional control? Further research is needed to resolve these questions.The above results emphasize that NMDA receptor mediated responses are highly plastic and that mere test stimulation can induce a short-term potentiation as well as a slowly developing depression that persists for several hours. The depression was input specific and saturable, and its induction required NMDA but not AMPA receptor activation in conformity with conventionally induced LTP and LTD, suggesting a relation to these phenomena. While a low Mg2 2, MgCl2 2, NaHCO3 26, NaH2PO4 1 and glucose 10, oxygenated by 95% O2, 5% CO2. The hippocampus was dissected out and transverse 400 μm thick slices were prepared by a vibratome or tissue chopper. The slices were initially kept in the same solution at room temperature for at least 60 min. As required, slices were then transferred to one or several "submerged type" recording chambers. During the experiment, slices were perfused at 30°C by a solution similar to that above except that the concentration of Mg2+ was 0.1 mM. The usage of low Mg2+ allowed for expression of NMDA receptor mediated responses.Experiments were performed on 12 to 18 day old Sprague-Dawley rats. The animals were decapitated after isoflurane (Forene) anesthesia in accordance with the guidelines of the Swedish Council for Laboratory Animals. All animal procedures were approved by the Local Ethics Committee at Göteborg University. The brain was removed and placed in an ice-cold artificial cerebrospinal fluid solution containing (in mM) NaCl 119, KCl 2.5, CaClStimulation was delivered as 0.1 ms negative constant current pulses via monopolar tungsten electrodes. For each slice, two stimulating electrodes were placed in the apical dendritic layer of CA1 pyramidal cells on either side of the recording electrode to provide for stimulation of two separate sets of afferents. Field EPSPs were recorded by using a glass micropipette filled with 3 M NaCl (4–10 MΩ resistance). The basal test stimulus frequency was 0.1 Hz with stimuli delivered alternately to the two electrodes, successive stimuli being separated by 5 s. To test the effect of stimulus interruption, one of the two electrodes was given no stimulation during a certain time, the other one remaining stimulated at 0.1 Hz.Recording commenced by monitoring isolated AMPA EPSPs in the presence of AP5 (50 μM) to block NMDA responses. A low concentration of CNQX (1 μM) was used to partially suppress the AMPA responses. In this way, somewhat larger stimulus strengths could be applied, suitable for evoking isolated NMDA EPSPs in the later part of the experiment. During the time of AMPA EPSP recording, the stimulus strengths were adjusted for each slice to equalize the synaptic inputs of the two pathways. This was essential for later comparison of NMDA EPSP across pathways. After obtaining a baseline of equal AMPA responses, the concentration of CNQX was raised to 10 μM which entirely blocked synaptic responces. The remaining non-synaptic response, consisting of stimulus artifact and presynaptic volley, was used to define "true zero".To study NMDA receptor mediated responses, CNQX (10 μM) was maintained in the solution while AP5 was washed out for one or several 2 h periods, referred to as sessions in the following. In between the sessions as well as afterwards, synaptic transmission was again blocked by applying AP5 (50 μM), framing in the sessions by periods of recording non-synaptic responses. Under the sessions, various tests were made depending on the purpose of investigation. Usually one input remained silent during the first session and stimulation was not resumed until after synaptic transmission was reblocked. In another kind of experiment, the initially silent pathway was reactivated in the early part of the first session after NMDA receptors were unblocked, providing a means for sudden start of NMDA receptor activation.Signals were amplified, filtered and transferred to a PC clone computer for on-line and off-line analysis by specially designed electronic equipment (based on an Eagle Instruments multifunction board) and own developed computer software. AMPA EPSPs were measured using an early time window (first 1.5 ms after the fiber volley) while NMDA EPSPs were measured using both an early (first 5 ms after volley) and a late (35–45 ms after artifact) time window. The late measurement was used in presenting most of the results, allowing easy comparison with previous work in our lab that estimated the NMDA component of composite EPSPs via a late measurement . SimilarMeasurements were calculated by integrating the curve along the specified time window after substraction of the prestimulus baseline. All values were corrected by substracting the corresponding measurements of the non-synaptic potential obtained after total blockage of the EPSPs (except when measuring the fiber volley). The final data were quantified as relative values compared to a reference level defining 100 %. While the initial baseline formed a natural reference for AMPA responses, the choice was less obvious for NMDA responses, leading us to use the highest level of responses for one of the pathways in one of the experimental sessions (selected to make sense). Results are expressed as mean ± S.E.M. Statistical comparisons were made using Student's t-test.Drugs were obtained from Tocris Cookson, UK; prefabricated stimulating electrodes were obtained from World Precision Instruments, FL USA, type TM33B.AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;AP5, D(-)-2-amino-5-phosphonopentanoic acid;CA, cornu ammonis;CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;EPSP, excitatory postsynaptic potential;LTD, long-term depression;LTP, long-term potentiation;NMDA, N-methyl-D-aspartate;MD planned and carried out most of the experiments including data analysis, and compiled the manuscript. RL carried out experiments, participated in the planning process and helped in shaping the final manuscript. HPX carried out the initial experiments establishing the effect of NMDA EPSP depression. BJ was responsible for logistics planning and participated in experiments. HW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


As an alternative to direct DNA sequencing of PCR products, random PCR-RFLP is an efficient technique to discriminate between species. The PCR-RFLP-method is an inexpensive tool in forensic science, even if the template is degraded or contains only traces of DNA from various species.Interspecies-specific DNA sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed using PCR-RFLP technology to determine the source (i.e., species) of blood traces obtained from a leaf.Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).The method presented can be used for the discrimination of cattle ( Determination of the species from which traces of source material, such as blood stains on a leaf, originate can sometimes be a difficult task in forensic DNA analysis. For instance, insurance claims that involve car accidents with animals require authentication. Species identification is also essential in food quality control-procedures or for the detection and identification of animal material in food samples. Numerous analytical methods that rely on protein analysis have been developed for species identification, such as electrophoresis techniques ,2, immunHowever, before applicable for routine analysis, species-specific diagnostic polymorphisms or mutations must be determined.If these mutations affect restriction enzyme sites, a simple PCR-RFLP can be used as a "sceening tool" for detection.One area in which the PCR-RFLP screening tool would be useful is in training hunting hounds. Before hounds are accepted and approved for hunting, they have to be evaluated by different tests. These tests include an examination of the dog's ability for winding and trailing. To examine these abilities normally a track is prepared consisting of minute amounts of blood from game animals, e. g., wild boar, spotted on a few leaves that are distributed in the forest or field. A trained hound should be capable of winding and trailing without any problems. However, in a case that came to our attention, hounds were unable to wind and it was assumed by the owners that the examiner had used blood from domestic animals. Hence, the question as to the source of the track  was open.Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).To solve this problem we established genetic test (PCR-RFLP) that focused on the use of diagnostic polymorphisms in the mitochondrial cytochrome b gene: Our method can be used for the discrimination between the following mammal species: cattle (Bos taurus, Ovis aries, Capra hircus, Capreolus capreolus and Cervus elaphus) and the pattern of bands from the blood sample of one of the dry leaves. As controls, DNA-samples from known species were used. Specific RFLP patterns that distinguish between 5 species are analyzed here. To estimate the exact size of fragments, DNA sequence information was used. C. hircus shows a single fragment of 182 bp, whereas with C. capreolus a fragment of 162 bp was obtained. The RFLP pattern of C. elaphus consisted of 2 fragments of 108 bp and 54 bp respectively. The blood sample on the leaf shows four fragments: 114 bp and 68 bp-fragments, which are characteristic for B. taurus, and a 105 bp and 75 bp fragment which can be assigned to O. aries. This result indicates that the blood sample on the leaf represents a mixture from two species (B. taurus and O. aries). The results were obtained from two independent DNA extractions and confirmed several times by independent PCRs, as well as by DNA sequencing. Amplifications were repeated and species identification was verified by diagnostic Dloop sequencing [B. taurus and three nucleotide differences were observed.For the analysis, DNA was prepared from blood remains on different leaves. The DNA was amplified as described in Methods. Figure quencing . The DloB. taurus and O. aries is still possible for a ratio of 59:1 can lead to false results, but with the designed primer pair this problem was circumvented. For this reason we designed maximally discriminatory primers and the mismatches in each primer are sufficient to exclude human DNA under stringent PCR conditions. The primers were tested with different amounts of added DNA. This method can be used for analysis of mixed samples, since up to three species in different proportions can be determined. The RFLP test using other tissues, e.g., muscle, hair with roots, bones, and saliva, yielded reproducible results. Faeces have not been tested so far.However, when minimizing target DNA, the bands tend to fade away on the agarose gel. The presence of a specific PCR-RFLP for the species analysed, a fragment length of less than 200 bp and the exclusion of contaminating sequences improved methods for existing species determination. The principle of RFLP is often used in food analysis . HoweverThe speed and the efficiency of current nucleotide technology, such as automatic sequencing, will permit the identification of additional taxa. However, the establishment of a PCR-RFLP test would likely need further extensive experimentation.B. taurus, O. aries, C. hircus, C. capreolus and C. elaphus. However, after further development, the tested mitochondrial nucleotide sequences may allow the forensic identification of other animal species.In summary, we were able to show that a simple PCR-RFLP is efficient in differentiating between ® Tissue Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturers' handbook. Isolated DNA was diluted in 50 μL HPLC-H2O and used for further analyses.DNA was extracted from blood and tissue samples using QIAampBone samples were roughly ground with a pestle and mortar, then finely powdered in a Retsch mill. Bone powder (0.3 g) was incubated in 1.5 mL of 0.5 M EDTA (pH 8.3) for 20 h while rotating. The suspension was centrifuged for 4 min at 4000 rpm. The supernatant was transferred to a fresh tube or to an automated nucleic acids extraction system (Nucleic Acid Extractor 341A, Applied Biosystems) and 1.6 mL sterile distilled water (Ampuwa, Fresenius) was added. As the extraction procedure was automated the volumes of reagents dispensed may have varied between runs. Five hundred microliters of Proteinase K was added and the mixture incubated for 1 h at 58°C with shaking. Three milliliters of phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.5–8.0) were added and the mixture was further incubated at room temperature for 6 min while shaking. The phases were allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Chloroform (4.5 mL, 100%) was added to the aqueous phase and the mixture incubated for 6 min at room temperature while shaking. The phases were again allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Ninety microliters of sodium acetate (pH 4.5) and 3.2 mL of 100% isopropanol were added followed by incubation for 2 min with shaking. Five microliters of Glasmilk (Dianova) were added and the suspension was shaken for another 10 min. To obtain a pellet, the solution was filtered through Precipitette filters (Applied Biosystems) or centrifuged for 3 min at 5000 rpm. The pellet was washed with 80% ethanol and eluted into 50 μL sterile distilled water (Ampuwa, Fresenius). Five to ten microliters of extract were used for PCR amplification or the extract was stored at -20°C. Glasmilk was not removed prior to amplification.For RFLP analysis, a 195 bp long PCR fragment was amplified from mitochondrial cytochrome b region. The following primer pair was used for amplification, CB7u (5'-GCGTACGCAATCTTACGATCAA-3') and CB7l (5'-CTGGCCTCCAATTCATGTGAG-3').2; 150 μM dNTPs; 0,18 μM of each Primer and 2 U AmpliTaq Gold (PE Applied Biosystems). Cycling conditions included a denaturation step at 95°C for 3 minutes, followed by 32 cycles of 95°C for 1 minute, primer annealing at 54°C for 1 minute and elongation at 72°C for 1 minute in a thermocycler (Hybaid).The PCR was carried out in a total volume of 50 μL consisting of 10 ng DNA, 60 mM KCl; 12 mM Tris-HCl; 2.5 mM MgClThe 195 bp fragment was digested with TSP509 (New England Biolabs) for two hours at 65°C. The resulting fragments were separated by gelelectrophoresis in a 2.5 % agarose gel.A single fragment of the mitochondrial DNA Dloop region was amplified using primers Dloopu (5'-AAATGTAAAACGACGACGGCCAGTAATCCCAATAACTCAACAC-3') and Dloopll (5'-AAACAGGAAACAGCTATGACCACTCATCTAGGCATTTTC-3').2, pH 8.3) and Q-solution, 100 μM for each dNTP, with 1 M Taq Polymerase and 10 pmol of each primer. Four microlitres of the DNA-extract were added to the PCR mix. The amplification was carried out with initial denaturation at 95°C for 10 min, followed by 35 cycles of one denaturation step at 94°C for 40 sec, primer annealing at 52°C for 40 sec and primer extension at 72°C for 45 sec in a Hybaid thermocycler. PCR-products were purified using the QIAEX II Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturers' instructions. Sequencing was performed using ABI-Prism™ Dye Kit V3 (Applied Biosystems) in a 10 μL volume containing 2 μL purified PCR-product and 5 pmol of primer. Sequencing reactions underwent 27 cycles of 30 sec at 94°C, 30 sec at 50°C and 3 min at 60°C in a Techne thermocycler. The dye terminators were removed by sephadex-G45 column purification (Millipore). Sequencing reactions were electrophoresed for 2 h on an ABI Prism® 3100 genetic analyzer (Applied Biosystems) according to the manufacturers' instructions.Amplifications were performed in a final volume of 20 μL in 10 × PCR buffer (15 mM MgClIn order to test the specifity of the technique the following numbers of specimens were tested:7 unrelated samples from cattle: Holstein Frisian, Charolais, Limousin, Angus7 unrelated samples from sheep7 unrelated samples from goat7 unrelated samples from deer collected from Lower Saxony and North Hesse7 unrelated samples from roe deer collected from Lower SaxonyOvis aries musimon) and observed approximately 100% sequence identity compared with Ovis aries. Furthermore, we investigated different cattle breeds and we could not find any sequence differences within the tested cytochrome b DNA-fragment.Regarding closely related species, we analyzed mouflon DNA (IP performed the DNA extractions, PCR-RFLP analysis and mtDNA Dloop DNA sequencing.JB developed and provided the PCR-RFLP protocol for species identification.BB was responsible for funding, supervision of the research project, manuscript writing and editing as well as scientific correspondence.


As such, Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates.in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals.By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures.These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells,  In vivo gene targeting of highly specific cell subsets remains the main challenge for gene therapy of a broad range of conditions associated with acquired diseases, including infectious disorders, cancer and failure of the hematopoietic system [In vivo gene transfection is more appealing than in vitro transfection of an aliquot of cells or tissue that would be then reinfused to the patients, because it potentially concerns the total population of targeted cells disseminated in the whole body; this is particularly relevant to patients with primary or secondary failures of the hematopoietic system, since, in most instances, residual foci of hematopoiesis exist that cannot be easily located and cannot be collected by a marrow harvest procedure. In vivo targeted transfection of specific subsets of hematopoietic stem cells (HSC) might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors.c system ,2. In viin vivo targeted gene transfer cannot use current viral vectors. Although cationic liposomes have been promising systems in transfecting cells in tissue culture, it has been recognised that their in vitro efficiency does not correlate with their ability to deliver DNA after in vivo administration [Systemic gene delivery systems are needed for therapeutic applications in which the target cells are not directly accessible . Howeverstration -10.Tissue-specific targeting can be achieved through ligand receptor interactions ,12. We hin vitro by the transfer of G418 resistance (neor) into human T-cell lines [in vivo by the transfer of β-galactosidase activity into mouse splenocytes [in vivo, we have shown a specific tumor targeting after a single intravenous injection in mice bearing tumour expressing the renal carcinoma – related G250 tumor associated antigen [In previous studies, antibodies directed against internalised cell surface antigens such as the T lymphocyte-related CD3 molecule or the B lymphocyte-related surface IgD were chemically coupled to purified plasmid DNA encoding various reporter genes. This approach was validated both ll lines  or humanll lines  and in venocytes . We haveenocytes . More re antigen .in vitro biological effect on hematopoiesis [in vitro, this study was focused on specific in vivo targeting of hematopoietic tissues.We have previously reported that the method is suitable for the production of a functional growth factor in specifically CD117+ targeted cells, mediating an opoiesis . As our IL-3-encoding plasmid DNA. CD117 antigen is expressed on a CD34+ hematopoietic subpopulation and is readily internalised upon binding to its ligand [in vivo targeting of hematopoietic immature cells via a systemic route, mediating an efficient in vivo transgene expression.In the present study, we used anti-CD117 (c-kit) mAb covalently coupled to human The human IL-3 coding sequence (R&D Systems, Minneapolis, Minnesota) was ligated to synthetic fragments containing the natural leader sequence of human IL-3 and was subcloned into pCEP4 vector (Invitrogen Corporation). Transgene expression was controlled by the cytomegalovirus (CMV) enhancer-promoter sequence. The Epstein-Barr Virus replication (oriP) and nuclear antigen (encoded by the EBNA-1 gene) were carried by this plasmid to permit extrachromosomal replication in human, primate and canine cells . pCEP4 aIgG mAbs were chemically coupled to plasmid DNA as previously described . Brieflyin vitro transfection of HSC may be observed in a dose-dependent effect for up to 100 μg of conjugate [We have previously shown that onjugate .BalbC mice (6 weeks) were intravenously injected with a dose of up to 400μg of monoclonal 2B8 (BD Biosciences Pharmingen) covalently coupled to the pIL3 plasmid (named conjugate) and as negative control the monoclonal 2B8 and plasmid DNA uncoupled (named unconjugate) or irrelevant human monoclonal antibody (G250) covalently coupled to the pIL3 plasmid (named control conjugate) or physiological serum (named control serum).enhanced green fluorescent protein encoding plasmid pEGFP-1 provided from Clontech and was named eGFP conjugate.In a set of experiments, two intraperitoneal injections of chloroquine (32.5 mg/kg) were performed 2 hours and just a few minutes before intravenous injection of conjugates. The tolerance of chloroquine (used to prevent the degradation of the plasmid for transfection assays, 20) was in the range reported in mice for the study of malaria treatment . MonocloMice were intravenously injected twice (day 0 and day 2) and euthanasied 5, 7 or 10 days after the first injection of the conjugate, after proper anaesthesia.Human IL-3 production in serum was assayed by High Sensitivity ELISA (R & D Systems). Controls were sera or cell culture supernatants of control mice (unconjugate, control conjugate, control serum).After euthanasia, the presence of the transgene was investigated in blood, brain, lungs, liver, spleen, kidneys, adrenal glands and bone marrow. In order, to observe toxicity the weight of mice and their organs were measured (brain, lungs, liver, spleen, kidneys).In mice injected with eGFP conjugate, a MACS magnetic cell separation systems  was used to enrich cells expressing CD117 and Sca1 from mononuclear bone marrow cells. Negative and positive cells were collected for experimental use. To achieve a purity greater than 50%, it was necessary to perform two sequential passes through magnetic columns. The overall recovery of CD117 was about 30% and enrichment 40 fold, as assessed by the fraction of CD117/Sca1 positive population before and after separation. Cells were analysed by flow cytometry to determine the purity of cell fractions. Then the presence of eGFP positive cells was investigated by flow cytometry into negative fraction (CD117/Sca1 negative populations) and positive cell fractions (CD117/Sca1 positive populations). All experiments were conducted according to French regulation for animal experimentation (Ministry of agriculture Act No.87848, 1987).Long-term cultures of bone marrow cells were performed, as previously described . At one 5 cells from bone marrow were assayed for clonogenic hematopoietic immature cells [5 cells/ml in complete methylcellulose M3434 from Stem Cell Technologies (West Broadway, Vancouver, Canada). Cultures were incubated at 37°C in 5% CO2 and removed at 14 days. Colonies were defined as containing more than 40 cells using an inverted microscope. Cells were then harvested and studied for IL-3 gene expression. Two weeks post-transfection, semi-solid colonies were removed from methylcellulose culture for PCR analysis of the presence of the pIL3 plasmid.5 × 10re cells . Briefly33P-5'-end labeled oligo-probe specific of human IL3 coding sequence (ACGGCCGCACCCACGCGACA), in Southern blot analysis as previously described [The simultaneous isolation of total cellular RNA and DNA from tissues or cells was performed using TriPure Isolation Reagent Kit (Roche Diagnostics) . Total cescribed . To deteescribed .Balb/C mice were intravenously injected twice (day 0 and day 3), with the anti-mouse CD117 (c-kit) 2B8 mAb conjugated to pIL3 expression vector. Control animals received unconjugated pIL3 expression vector and 2B8 mAb (named control unconjugate) or irrelevant G250 mouse mAb covalently coupled to the pIL3 plasmid (named control conjugate) or physiological serum (named control serum). To increase the transgene processing into cells, mice were injected with the conjugate up to a dose of 400μg in the presence or not of chloroquine known to diminish endosomal DNA degradation . Mice weGene targeting was then evaluated by injecting mice with eGFP conjugated or unconjugated to either 2B8 mAb or to G250 control mAb. At day 5, the presence of transfected cells into bone marrow mononucleated cells was analysed into the purified CD117- and CD117+ subpopulations, by flow cytometry using anti-CD117 and anti-Sca1 Abs. As shown in Table IL3 transgene DNA in the spleen of transfected animals up to day 7 (not shown). In control animals (control serum, unconjugate, control conjugate), pIL3 DNA but no RNA was detected in peripheral blood but not in serum until day 5 after the first injection and then disappeared .To assess the tissue specificity of the targeting, presence of pIL3 plasmid was investigated in bone marrow, blood cells, liver, spleen, lungs, kidneys, adrenal glands, and brain. PCR analysis of genomic DNA and RNA isolated from bone marrow and blood (or serum) was performed using primer specific pIL3 sequences. Specificity of the PCR and RT-PCR products was assessed by a Southern blot hybridised with a specific radiolabelled human IL3 probe. The expected 542 bp band of the PCR product corresponding to the IL3-transgene presence (both DNA and RNA) were was specifically detected in the bone marrow of transfected mice up to 7 days for RNA and 10 days for DNA, post transfection , did not reveal any change, suggesting the lack of toxicity detected in mice receiving the conjugate (data not shown). Furthermore, since no IL3 transgene was evidenced in these organs, further investigation of potential toxicity of conjugate might not be relevant.Finally, clonogenic assay hematopoietic immature cells were performed on cells removed from sacrificed animals. As shown in Table Long-term cultures of bone marrow cells from mice receiving the conjugate or the controls were performed. After 1 week of selection in hygromycin-containing medium (plasmid conferred hygromycin resistance), cells were cultured for another 2 weeks and then viable cells were quantified using trypan blue exclusion assay. As illustrated on Figure vitro can be especially useful for studying the biological effects of various growth factors [in vitro biological effect on hematopoiesis [in vitro, the present study focus on specific targeting of hematopoietic tissues, in vivo.Although much progress has been accomplished in the field of gene therapy over the last years, there is still a need to develop more effective vectors and new strategies . Using a factors . Our conopoiesis . Since oWe first demonstrated the efficacy of our approach since the transgene and its product (RNA and circulating human IL3) were found in mice injected with anti-CD117/pIL3 conjugate. It is of note that although human IL3 was only detected in plasma of chloroquine-treated mice injected with high quantity of conjugate (400μg); human IL3 encoding RNA were evidenced in treated mice injected with lower quantity of conjugate (100μg). These results were in accordance with the design of these experiments aiming at observing even a transitory and local effect (within the bone marrow).PCR analyses of tissues evidenced the specific targeting of the hematopoietic system since brain, liver and lungs were negative. Only the spleen of mice transfected with the conjugate and kidneys of control animals (transfected with unconjugate mixture of Ab and DNA or with the control conjugate) displayed a positive PCR signal. Observed shortly after the last plasmid injection in blood, the presence of plasmid might be due to the intravenous administration route used and in kidney, to a progressive elimination of the plasmid in this organ of refinement. These results correspond to kinetic of plasmid availability when not using the specific vector (conjugate) to carry plasmid into progenitor cells. In the latter case, CD117+ cells were specifically transfected, and among them, Sca1+ cells were positive, suggesting a targeting of hematopietic progenitor cells via the systemic route.Several parameters contribute to the efficiency and specificity of our system such as the internalisation of the antigen targeted, the choice of the transgene used, the tissues targeted, the conformation of the conjugate. Bone marrow was a good candidate for gene targeting as it is a highly proliferative tissue, as opposed to tissues which possess terminally differentiated cells such as hepatocytes or adipocytes, which are more resistant to transfection .in vivo performance. These include avid interaction with serum components, resulting in colloidal instability, including both aggregation and dissociation of the conjugates and rapid elimination from blood circulation [in vivo administration. Protamine has been shown to cause condensation of DNA, which promotes cellular entry [Factors affecting the bioavailabilty of the administered conjugates strongly determine their culation ,32. Therar entry ,34. Our gene delivery system is specific and leads to transient gene delivery and expression. It may prove useful and safe for numerous clinical applications of gene transfer in hemato-oncology and radiopathology, whereby a stable genetic modification is not required, in contrast to the gene therapy approaches for genetic diseases. For example, it may be of interest to facilitate the long-term reconstitution of hematopoiesis through transient gene delivery into progenitor cells of patients after therapeutic and /or accidental exposure to chemo/radiotherapy. Whether our approach could be used to potentate hematopoietic reconstitution following irradiation remains to be studied.Our HSC Hematopoietic Stem CellsThe author(s) declare that they have no competing interests.AC, OD, AD, MB, SF, MM, PP carried out the studies. FH, DT participated to the designed of the study and its coordination. All authors read and approved the final manuscript.


In silico experiments reveal how evolution can work--without missing links. What can biologists learn from them? Rich Lenski decided he was onto a good thing from his very first encounter with digital evolution. It all began when he used the technology in which artificial organisms in the form of computer code evolve independently by self-replicating, mutating, and competing to re-examine an earlier study with bacteria. The original study had contradicted ‘some influential theory’ suggesting that random mutations show a systematic tendency towards synergistic interactions. His digital results, he discovered, matched his organic ones.‘It's great when these two powerful experimental systems agree, because it suggests some generality about the evolution of genetic architectures', recalls Lenski, professor of microbial ecology at Michigan State University (MSU). ‘But even if the digital and biological realms sometimes come into scientific conflict, it would only lead one to ask why and then probe the relevant factors more deeply’.He can hardly contain himself. ‘It's a win–win situation, leading towards increased generality, on the one hand, and further experiments to better understand specific outcomes, on the other’. For his part, Lenski has since gone much further with the technology  and alsoRichard Lenski is using digital organisms to do ‘impossible’ evolutionary experiments. In one, he says, ‘we test every incipient mutation before it occurs in a population and then allow it or disallow it, depending on its fitness effect, to see how important neutral and deleterious mutations are for long-term adaptation’.Lenski, professor of microbial ecology at Michigan State University, says his mind boggles at how digital evolution opens up so many avenues for research. ‘I sometimes feel like a kid in a candy store who might starve because he can't make up his mind what he wants’.exactly is the hypothesis I want to test, and what exactly must I measure to test that hypothesis?’These opportunities and, at the other end, the prospect of having too much data to analyse, which Lenski admits is a strange thing for an evolutionary biologist to complain about, enforce a discipline to prioritise and define objectives: ‘What Such enthusiasm for the technology makes it difficult for him to understand why some biologists might dismiss digital evolution as ‘very interesting but with no value’ or turn their backs on it altogether. ‘My own view’, says Lenski, ‘is that something that is very interesting is also worth thinking about and exploring more fully, especially when it offers the opportunity to examine complex problems in greater depth and with more precision than is otherwise possible’.But he cautions against mistaking his enthusiasm for studying digital organisms as a call to abandon other lines of research. ‘There's obviously much of value for understanding evolution that comes from many different empirical and theoretical perspectives’, he says. ‘That's one reason that evolutionary biology is such a vibrant field right now’.Lenski still spends as much research time on bacteria as he does on digital organisms. ‘Although it's sometimes frustrating not to be able to devote 100% to each system, each one is so interesting to me that I couldn't bear to drop either of them’. The two systems have different strengths and limitations, which Lenski tries to exploit in his research, he says.E. coli populations, he and his colleagues showed earlier this year how they used gene-expression arrays to work backwards to a set of key mutations in a global regulatory gene. More recent work, currently being written up, ‘has led us to some adaptive mutations in several other key loci’, he notes.From his laboratory's studies on long-term As for his digital research using the Avida software system, Lenski acknowledges that speed is an obvious advantage, but not the most significant one. ‘An even more important advantage is the ability to observe the dynamics and dissect the outcomes of evolution with absolute precision. For example, there are no missing links in the digital world’.Nevertheless, he wryly highlights one shortcoming of Avida: ‘We'll know that we have been successful once the Avidians have evolved the ability to design their own experiments and write the papers without us’.This is a world where time-scales contract and, above all, where other constraints of ‘wet’ biology have no place.Earlier this year, he and Chris Adami, who heads the Digital Life Laboratory at the California Institute of Technology , published some breathtaking findings from the field. Their collaboration brings together biologists and computer scientists, physicists and philosophers in an artificial world on a quest to understand how evolution works. Though they may still be some way from reaching that objective, their latest advance suggests that they are on the right track.The research confronts evolutionary theory's long-standing challenge to explain how an organism can develop complex features simply as a result of random mutation and natural selection. The challenge remains a controversial one, too. Supporters of intelligent design, a branch of the creationist movement, promote the notion of ‘irreducible complexity’ as evidence that Darwinian evolution is a flawed theory. The notion purports that a complex feature cannot evolve sequentially from its elements, and must have been designed in one step by some higher intelligence.Traditional investigations, based on molecular biology and palaeontology, have yielded much evidence about the incremental evolution of the eye or the brain, for instance. But continuing ignorance about many developmental processes and the absence of key fossil records mean that accounts without missing links, to endorse the theory, may never be realised.Which is what tempted Lenski and Adami to examine the challenge in their virtual world. This is a world where timescales contract and, above all, where other constraints of ‘wet’ biology have no place. ‘It's not just the speed, by any means’, says Lenski. ‘It's also the power to manipulate almost any variable one can imagine, to measure variables with absolute precision, to store information that then allows one to trace back a complex chain of events, and to take evolved organisms and subject them to new sorts of analyses that one might not even have anticipated when first collecting the data’.It is a place where virtue is made of simplicity. ‘The worlds we're dealing with here are extraordinarily simple compared with the real world’, says Adami. ‘Any of the biochemistry associated with transcription and translation, for example, anything more complex than relatively short viral types of genomes, that's out of our league’, he notes. ‘We can't see transcription and translation because we don't have transcription and translation–-we go right from sequence to function’.But the principles of evolutionary theory make such restrictions unimportant, he says. ‘Many of the [theory's] predictions don't depend on these little details of molecular biology’, notes Adami. ‘The principles are very, very general, and very simple, and in the end they are mostly responsible for the overall dynamics that you see in these simple systems’. Lenski goes further. These virtual realities, he says, ‘offer us a window into an alternative world, and perhaps even a part of the future of our own, where the fundamental evolutionary mechanisms of mutation and natural selection play out in a novel physical realm’.Escherichia coli. ‘We're making great strides elucidating the precise genetic bases of the adaptation that has occurred during tens of thousands of generations in our long-term E. coli populations’, he reports. ‘Even after more than 30,000 generations in a constant environment, we're still seeing some major phenotypic evolutionary changes’, he adds.Lenski is interested in watching evolution as it happens and has a track record in the study of evolving organic systems, primarily using http://dllab.caltech.edu/avida/) earlier this year, a decade after work began.Adami, who also works in theoretical physics at the Jet Propulsion Laboratory at Caltech, has developed a software platform, known as Avida, for research on evolving computer programs, the digital organisms that he terms ‘Avidians’. The second version, Avida 2.0, became available for free public use  transitional forms, even when the transitional forms are favoured for performing different functions from those that evolve later’.For many other biologists, however, digital evolution seems to have very little relevance. One eminent British evolutionary biologist dismissed the research in just eight words, according to the field's godfather, Tom Ray. ‘His comment: “It's just not biology. Period. End of discussion”. That's the whole story right there’, recalls Ray.Less strident reservations concern the limits on complexity that the virtual world imposes and suspicions about the ability of digital processing to mirror evolutionary principles accurately. For Francisco Ayala, professor of biological sciences at the University of California, Irvine, it appears to be simply a question of trust in the natural world. ‘Computers can give you only what you put in’, he says. ‘With natural models, you're not putting anything in—you're segregating a small region as an aspect of reality’.There are also more mundane worries over the technical skills needed for the computational operations, a fear acknowledged by Lenski. ‘Computational skills are certainly opening up some exciting new directions [in evolutionary biology]’, he says, ‘but there are of course many other useful skills and fascinating directions’. At Caltech, meanwhile, Adami's team is trying to make Avida easier to use, backed by the National Institutes of Health's first-ever funding for digital-life work.Misunderstandings about the technology arise over whether the research is an ‘instance’ or a ‘model’ of evolution, suggests Ray, who now divides his time between the Advanced Telecommunications Research Laboratories in Kyoto and the University of Oklahoma, where he holds chairs in zoology and computer science. ‘I never intended [Tierra] as a model, but that's the way a lot of people saw it because they weren't really prepared for this new idea, this different perspective of another instance of life’, he says. ‘They had a more traditional view of what you do with a computer, which is that you send e-mail, you process things, and you make models’.Levels of veracity determine limits of extrapolation, says Ray. ‘Digital evolution is an abstraction, and it's not going to be able to tell us what humans will evolve into or why dinosaurs went extinct or what will be the next emerging disease…. You need the whole planet to do that kind of modelling’. But once you appreciate the constraints, ‘it's a phenomenally good tool, because it's evolution in a bottle. You can instrument it 100%’, he notes. ‘I think Lenski and Adami have done a very good job of developing it that way’. Ray himself is now more interested in genomics and pharmacology and their application in a biologically inspired engineering project to design software agents, or ‘virtual creatures’, as he terms them.For Lenski, experiments with Avida provide ‘both an “instance” and a “model” of evolution’. He says that ‘populations of the digital organisms really do evolve and adapt, albeit in an unfamiliar physical realm. At the same time, they provide a sort of experimental model for testing and understanding the general principles of evolution’.And he agrees with Ray that digital evolution is not intended to explain how we got where we are today, ‘in the sense of unravelling which species are more related to which other species, or what organismal features are adaptive for what purposes, and so forth’. The goal, says Lenski, is to examine evolutionary processes and dynamics in greater depth and detail than are otherwise possible. ‘Watching a process as it occurs and being able to probe genetic details and manipulate environmental variables can provide new insights and evidence that one cannot get by comparative studies that typically require one to infer historical processes from present-day patterns’.Pseudomonas fluorescens, which grow from single genotypes in pristine tubes, to test long-standing hypotheses about the causes of ecological diversification. ‘The bottom line is that we're reducing the complexity we see in the real world to a much more manageable level’, he says. ‘The nice thing about bacterial populations is that ecological and evolutionary timescales coincide, so that you can actually see the ecological context of evolutionary change’.Such developments fascinate and enthral Paul Rainey, an evolutionary ecologist, even though he rarely needs any computing power for his research and recognises that digital evolution still lacks an ecological dimension. Rainey, who earlier this year moved from Oxford to become professor of ecology and evolution at the University of Auckland, uses bacterial populations of ‘It's a phenomenally good tool, because it's evolution in a bottle.’Rainey, a friend and colleague of Lenski's, would welcome the chance to take advantage of the speed, robustness, and flexibility of digital evolution to further his research, but doubts whether the technology will ever be able to match the performance of his ‘wet’ laboratory. Though his natural model is simple, it remains far too complex to program, he suspects. ‘We try to understand how selection is working in this very complex ecological context, which includes interactions between genotypes and within genotypes and interactions with an environment that is constantly changing’, he says. ‘This sets the scene for selection, and the selective forces are constantly changing…. None of that complexity is really captured in Avida’.But Rainey is in for a surprise, according to Adami. ‘The pace of development of Avida has accelerated’, he says. ‘More people are working on it because we have bigger grants. And Charles Ofria [who helped to design the software as a postgraduate at Caltech] is doing much of the development at Michigan State [University, where he is now assistant professor of computer science and engineering] with his students’. The result is that Avidians have made their first steps towards sexual freedom within ecologically diverse environments or, more accurately, code recombinations in a multi-niche virtual world.For almost a decade, says Adami, Avida has been a single-niche world in which every organism in the population sees exactly the same world and only a single species inhabits that world. But Avida has now been expanded, he continues, ‘in such a manner that populations can see different types of worlds and they can adapt independently to different resources’. A research paper is being finalised on how the software is making its first steps towards incorporating the notion of evolutionary ecology. ‘We show what pressures are necessary to make a population that is homogenous branch out and speciate into a stable system’, notes Adami. ‘Now we want to explore recombination, which we've always shied away from.’ With asexual reproduction virtually understood, the researchers are ready to tackle sexual reproduction in the digital world, says Adami. ‘Some people are furiously working at implementing that.’‘Our goal is not to mimic natural systems in detail, but rather to expand Avida to give digital organisms access to more of the basic processes of life’, says Lenski. ‘Our goal is not so much to endow the ancestral organisms with additional capabilities, but rather we want to see how digital organisms will evolve if they are placed in an altered world where such things as sex and communication are physically possible. I see many years of interesting research along these lines’.Reflecting on future applications for the research, Lenski suggests it highlights how the traffic in computational biology is now becoming a significant and little recognised twoway exchange. Computer scientists are not only helping biologists to organise and analyse their vast datasets, says Lenski, but ‘biological principles, from evolution and genetics to neurobiology and ecology, are informing computer scientists and engineers in designing software and hardware … and that holds tremendous promise for the future’.