Acacia tree species. This study investigated germination of Acacia karroo, A. luederitzii and Dichrostachys cinerea, thought to be increasing in density, as well as the historically successful encroaching woody species, A. nilotica, in savanna grassland, Hluhluwe-iMfolozi Park, South Africa. A. karroo is thought to be replacing A. nilotica as the dominant microphyllous species in the park. We tested the hypothesis that observed increases in certain woody plants in a savanna were related to seed germination and seedling establishment. Germination is compared among species for burnt and unburnt seeds on burnt and unburnt plots at three different locations for both hot and cool fires.While fire has been used in some instances to control the increase of woody plants, it has also been reported that fire may cause an increase in certain fire-tolerant Acacia karroo showed higher germination (A. karroo 5.1%, A. nilotica 1.5% and A. luederitzii 5.0%) levels and better establishment (A. karroo 4.9%, A. nilotica 0.4% and A. luederitzii 0.4%). Seeds of the shrub Dichrostachys cinerea did not germinate in the field after fire and it is thought that some other germination cue is needed. On average, burning of A. karroo, A. nilotica and A. luederitzii seeds did not affect germination. There was a significant difference in the germination of burnt seeds on burnt sites (4.5%) and burnt seeds on unburnt plots (2.5%). Similarly, unburnt seeds on unburnt sites germinated better (4.9%) than unburnt seeds on burnt sites (2.8%).A. karroo and that fires may not be hot enough or may occur at the wrong time of year to control A. karroo establishment in HiP. Although germination and establishment of A. karroo was higher than for A. nilotica a competitive advantage after fire could not be shown.We conclude that a combination of factors may be responsible for the success of  Acacia karroo Hayne [A. nilotica (L.) Willd. Ex Del. subsp. kraussiana (Benth.) Brenan, [Dichrostachys cinerea (L.) Wight & Arn. and A. luederitzii Engl. var. retinens (Sim) Ross & Brenan are also thought to contribute to this phenomenon.The increasing density in the woody component of savannas has been widely reported -5 with soo Hayne ,7 and A. Brenan, ,9. in soIn hard seeded legumes dormancy is broken by rupturing part of the seed coat. The rupturing of the seed coat may be induced by heat from fire  enablingWhile some studies report that a decrease in grass cover favours the establishment of woody seedlings due to reduced competition ,21, otheAcacia species are shade intolerant resulting in decreased seedling establishment in shady areas [Acacia species have been found to be tolerant of low light conditions and may even experience increased seedling survival [Some dy areas ,23,24. Osurvival .Acacia densities [The frequency of fires may affect the direction of change in woody plant density . While iensities , it is aensities . This coA. nilotica, A. karroo, A. luederitzii and Dichrostachys cinerea in Hluhluwe-iMfolozi Park (HiP), where an increase in woody plant density over the past 40 years has been reported [A. karroo is apparently replacing A. nilotica as the dominant microphyllous element [This study investigated the direct (heat) and indirect  effects of fire on seed germination and seedling establishment of reported -28. It h element ,28.This study reports on the effects of burning, fire intensity and burning of sites on germination; burning, fire intensity, burning of sites and grass length (shade) on seedling establishment and specific species responses to treatments (treatment species interactions).D. cinerea germinated in the field and it was therefore excluded from the model for the field experiment. Testing for differences among treatments was based on the maximum number of seedlings for each species at each location over the 31-week period  indicating that the model was a good fit. Location and species were the only main effects significantly affecting germination  indicating that the model fitted the data well. Location and species were the only main effects significantly affecting establishment in the field Table . Acacia  species .Interaction terms, location × burn status, location × grass length, location × site burn status, burn status × site burn status and grass length × site burn status had a significant effect on establishment Table  Figure .Acacia karroo was 16.2 times more likely to establish than A. nilotica. Similarly A. luederitzii was 1.4 times more likely to establish than A. nilotica while A. karroo had 11.2 times more chance of establishing than A. luederitzii. Species differences in establishment for the various treatments are given in Table The odds of establishment were 8046.2 times less at Le Dube than at Nombali and 5850.5 times less at Le Dube than at Seme. 1.4 times more seedlings were likely to establish at Nombali than at Seme.D. cinerea in the field suggests that some disturbance other than fire is needed to cause a release from dormancy and commence germination.The lack of germination of A. karroo seeds germinated, which was higher than the other two species. Story [A. karroo, with 6.6% of seeds germinating under natural conditions in the field. He also found that A. karroo germination was erratic, with germinations still being recorded after 423 days. This was similar to what was found in this study, with the number of A. karroo seedlings still increasing until the end of the experiment. Acacia nilotica also showed dormancy with sporadic germination events over the 31-week period. Acacia luederitzii did not show dormancy with most germinations taking place in the first 3 weeks of the experiment. Acacia nilotica has a thick seed coat, which could account for it's poor level of germination. One would predict increased germination of burnt seeds due to a breaking of dormancy [Acacia species are temperature specific, suggesting a temperature threshold for germination [et al. [A. nilotica seeds to be highly vulnerable to fire with a 80% mortality of seeds on the soil surface. The current study, however, found no difference in germination between burnt and unburnt seed or seeds burnt at different temperatures. This finding is inconsistent with the recent study by Kanz [Germination of all species in the field was low. As the seeds relied on cotyledons for food, soil moisture may have been a limiting factor. As rainfall was not recorded, this should be kept in mind when interpreting the results. Five point one percent of s. Story  found sidormancy , but thimination ,20. This [et al.  found A. by Kanz  had simiAcacia drepanolobium in Kenya.Location had a significant effect on germination with Le Dube having very low germination overall and Seme having the most germinations. Germination at Nombali and Seme were similar. Site-specific effects may be attributed to various factors such as microclimate or soil type. Sites may also have different water infiltration rates and runoff, which may result in differences in germination levels. Okello and Young , howeverThe current study did not find a difference in the number of seedlings in burnt and unburnt patches. While neither burning of seeds nor burning of sites had any effect on germination, the interaction factor proved significant with unburnt seeds showing increased germination in unburnt sites as did burnt seeds in burnt sites. Kanz  also fouCeratotherium simum) grazing lawn with very short grass, which may lead to seeds losing moisture through more direct sunlight. This suggests a similar pattern to the seed burn × site burn interaction. The tall grass site at Seme had higher germination than any of the other tall or short grass sites. This may be due to possible site-specific effects mentioned earlier.Whilst more seeds germinated in short grass at both Le Dube and Nombali, those at the short-grass site (Seme) had higher levels of germination in tall grass sites. The short grass site at Seme is a white rhinoceros  may be detrimental to the germination of seeds  while colability . Tall grNo species factor interactions were observed suggesting that though species had different germination levels, they did not respond differently to the treatments.The same factors and interactions found to be significant influences on germination were found to influence establishment. This was expected as increased germination for these treatments would result in better establishment. The interaction patterns for most of the treatments, however, were different to those of the germination model. Owing to the low levels of germination, interspecific and intraspecific competition was thought to play a minor role in seedling establishment.A. karroo seedlings. This species is known to be dependent on moisture availability for survival [Le Dube again had the least seedlings at 31 weeks while Nombali had the best establishment. Seme, which had the highest level of germination, had establishment levels somewhere between that of the other two sites. It is again suggested that this may be due to soil or rainfall factors. Forty-five out of forty-eight seedlings established at Nombali and thirty-three out of thirty-five at Seme were survival  and thesA. karroo seedlings had established as opposed to six of A. nilotica and three of A. luederitzii. The high germination, but poor survival of A. luederitzii suggests that the absence of this species in the Hluhluwe section of HiP is not due to seed limitation or germinability, but possibly due to environmental factors decreasing its ability to establish. The differences in seedling survival between species are consistent with those reported by Kanz [A. karroo than A. nilotica.At week 31, 87  by Kanz  who founThe location × grass length interaction revealed the same patterns as for germination with regards to Nombali and Seme with Seme showing better establishment in tall grass and Nombali showing better establishment in short grass. There was no difference between establishment on tall and short grass at Le Dube. The short grass site at Nombali had the highest number of seedlings surviving at week 31.The grass length × site burn interaction displayed the same patterns as for the germination model, but this was not the case for the seed burn status × site burn status interaction. While unburnt seeds still did well on unburnt sites, burnt and unburnt seeds showed decreased establishment on burnt sites suggesting that, as a result of increased irradiance, burnt (open) sites may not hold sufficient moisture for seedlings to survive.Acacia species have been reported elsewhere [The interaction effects found to be significant for establishment only, both suggest the importance of fire temperature. Location × seed burn status and location × site burn status could both relate to the different grass lengths, and thus specific fire temperatures, at the three sites. Temperature sensitivity in lsewhere ,14,17,20A. karroo during the first year. Similarly, there was no difference in establishment of A. karroo in burnt or unburnt and tall or short grass sites. Smith & Goodman [A. nilotica seedlings, however, almost exclusively occurred away from canopy cover, suggesting an inability to establish in shaded environments. Acacia tortilis also showed a greater proportion of established seedlings in open than shaded areas [A. nilotica in tall and short grass, but its establishment was so low that no real prediction can be made.Chirara, Frost & Gwarazimba  found th Goodman  reporteded areas . We did A. karroo is strongly moisture dependent [A. karroo is more likely to invade moist rather than semi-arid grassland. This suggests that Hluhluwe Game Reserve, being an area with moist grassland, would be more prone to invasion by A. karroo. It has also been reported that A. karroo has the ability to withstand fire [A. karroo in the field and may be the reason for A. karroo's success over A. nilotica as the most important encroaching Acacia species in HiP at present. The literature does, however, suggest that high intensity fires may result in seed mortality [A. karroo seedlings survive fires from as little as 12 months of age [A. karroo. It has been reported that spring burns are the most effective )A. karro2 fenced protected area comprising the former Hluhluwe and iMfolozi Game Reserves, and the corridor of land that links the areas. The park has a moderate coastal climate, ranges in altitude from 60 – 750 m above sea level [The study was done in HiP, KwaZulu-Natal, South Africa (28°00' – 28°26' S, 31°43' – 32°09'E). HiP is a 960 kmea level  and has ea level . Periodiea level . The ranea level .Most of Hluhluwe Game Reserve is found on rocks of the Ecca and Beaufort series with some basalt in the east . King 3 identifiThe main soils types associated with the Ecca and Beaufort series are Swartland and Sterkspruit, while areas of Shortlands, Milkwood and Bonheim series are found in association with the dolerite regions . They alThe vegetation in the park has been described as bushveld – savannah comprising five broad vegetation types . The thiAcacia luederitzii occurs in large numbers in certain areas of the iMfolozi part of the reserve but is mostly absent from the Hluhluwe and Corridor sections. Acacia nilotica, A. karroo and D. cinerea are found throughout the park. As opposed to the scattered trees found in iMfolozi, A. nilotica covers extensive areas of Hluhluwe and the Corridor and is usually found below the 300 m contour [A. karroo – D. cinerea induced thicket throughout the area, but particularly in the Corridor and Hluhluwe Reserves. Acacia luederitzii seeds used in this study were therefore collected in iMfolozi Game Reserve while those of the other species were collected in Hluhluwe.The field experiment took place in the Hluhluwe and Corridor sections of the HiP.  contour . Whatele contour  describeA. nilotica, A. karroo, A. leuderitzii and D. cinerea was tested in a field experiment. Seeds of all species were collected between May and August 2000. Parasitized seeds were extracted. Prior to planned management burns, six groups of seeds were placed in tall grass  and six in short grass (shorter than 0.10 m) at three locations . Tall grass produces hotter fires than short grass due to increased fuel load, which increases available heat energy [Dichrostachys cinerea seeds were only put out at Seme and Nombali. Each group contained 22 A. nilotica, 25 A. karroo, 10 A. leuderitzii and 10 D. cinerea seeds. Seeds were placed on the soil surface a day before each of the burns . This is considered the natural situation for the seeds with soil stored seed banks being virtually non-existent [The effect of fire, fire intensity and burning of sites on the germination of seeds of t energy . Sites wexistent . Seme anWe thus applied 96 possible seed treatment combinations for investigating factors affecting germination in the field (4 species × 2 burn treatments × 3 locations × 2 location burn treatments × 2 fire intensities).A. nilotica, A. karroo, A. leuderitzii and D. cinerea, data as on week 31 of the field experiment described above were used. Seedlings were considered to be established when they were rooted in the ground and the cotyledons replaced with leaves. Establishment was based on the total number of seeds.To test the effect of fire, fire intensity, burning of sites and grass length (shade) on seedling establishment of ®" [The "STATISTICA®"  Generali®" . In bothwhere = the log of variable 1 and 2 at different levels of the factors as given belowλ' = the overall mean effect of the categories = the effect of the jth species (j = A. karroo, A. luederitzii, A. nilotica, D. cinerea) = the effect of the kth location  = the effect of the lth seed burn status (l = burnt, unburnt) = the effect of the mth grass length  = the effect of the nth site burn status (n = burnt, unburnt) = the interaction effect between the jth species and the kth location = the interaction effect between the mth grass length and the nth site burn status.The logit model may be written as a generalized linear model as follows:, , , , , , , , ,  and  are parameters to be estimated from the data and B, C, D, E and F refer to the explanatory variables species, location, burn status, grass length and site burnt status respectively. The estimated parameters for the GLZ were used to obtain the estimated parameters for the logit model. The estimated parameters of the odds were calculated for each factor or combination of factors (including the intercept) as the exponent of the estimated parameters of the logit model. The estimated odds of germination under any condition were then calculated as the product of the estimated parameter of the odds of the intercept (estimated geometric mean odds) and the factor or combination of factors in question. The odds of germination for significant treatment combinations were compared.where The predicted number of seeds germinating and seedlings establishing as calculated with the model based on presence/absence data, were seen as being appropriate for interpretation as summaries of the data. Thus, differences in the predicted mean number of seeds germinating and seedlings establishing  were illustrated graphically for each significant treatment combination.MW designed the experiment, participated in fieldwork, performed the statistical analysis and drafted the document. MJS participated in fieldwork, the coordination of the study and drafting of the document. JJM supervised the work and assisted in the drafting of the document. All authors read and approved the final manuscript.Acacia seeds in HiP. Gives parameters of the logit model and estimated odds of germination for the various levels of the factors used.The parameters of the logit model and odds, estimated odds of germination and the ratio of germination to non-germination for the factors included in the model for germination of certain Click here for fileAcacia species in HiP. Gives parameters of the logit model and estimated odds of establishment for the various levels of the factors used.The parameters of the logit model and odds, estimated odds of establishment and the ratio of establishment to non-establishment for the factors included in the model for establishment of certain Click here for fileOdds ratios for all significant interactions of the establishment model. Compares the odds of establishment between different levels of the factors used.Click here for file


Our purpose was to determine whether AFI<5 cm after preterm premature rupture of the membranes (PPROM) is associated with an increased risk of perinatal morbidity.We performed a prospective cohort study of 95 singleton pregnancies complicated by preterm premature rupture of the membranes (PPROM) with delivery between 26 and 34 weeks gestation.2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test.Patients were categorized in two groups on the basis of amniotic fluid index<5, (AFI<5 cm)(n = 26) or AFI ≥ 5 cm (n = 69). Categorical data were tested for significance with the χAll 2-sided p values < 0.05 were considered significant.Both groups were similar with respect to selected demographics, gestational age at rupture of the membranes, gestational age at the delivery, birth weight. Both groups were similar with respect to selected variable, latency until delivery, early onset neonatal sepsis, RDS and neonatal death. Patients with AFI<5 cm demonstrated greater frequency of C/S delivery for non reassuring fetal tests (23%vs 2.8%) (p = 0.001). Our study demonstrated that patients in group I had a significant increase in the frequency of clinical chorioamnionitis (P < 0/001). Post partum infections were not seen in 2 groups.An AFI<5 cm after PPROM between 26 and 34 weeks gestation is associated with an increased risk of maternal infections and frequency of C/S. Preterm premature rupture of the membranes (PPROM) is one of the most common complications of the pregnancy.Preterm PROM is an important cause of perinatal morbidity and mortality, particularly because it is associated with brief latency from membrane rupture to deliver, perinatal infection, and umbilical cord compression due to oligohydramnios. PPROM is multifactor in nature. In any given patient, one or more path physiologic processes may be evident. Choriodecidual infection or inflammation appears to play an important role in etiology of preterm PROM, especially at early gestational ages.It has also been proposed that amniotic fluid posse's certain bacteriostatic properties that protect against potential infections processes and that a decrease in amniotic fluid volume may impair the gravid women ability to combat such infections, this latter hypothesis was tested by Vintziuleos, et al .These investigations demonstrated that patients with olighhydramnios (AFI<5), were at greater risk of having chorioamnionitis and subsequent sepsis in the neonate ,8. Our pWe performed a prospective cohort study of infants delivered between 26 weeks – 34 weeks gestation after preterm premature rupture of the membranes.This study was performed at Vali-e-Asr hospital at the medical university of Tehran between October 2000 and February 2002.All patients were at between 26 and 34 of weeks of pregnancy as best estimated by LMP, and confirmed by ultrasonography. In all patients rupture of the membrane was diagnosed by sterile speculum examination using pooled fluid, fern test and Nitrazine paper test.Patients with clinical chorioamnionitis, nonreassuring fetal status, obstetrical indication for immediate delivery, major congenital anomalies and advanced labor , and a growth-restricted fetus at initial admission were excluded. 95 singleton pregnancies have observed until 34 weeks of gestation. At admission, all patients had an ultrasonographic examination, which included confirmation of the estimated gestational age, and cumulative 4 guardant AFI measurements, as previously described by Phelan et al. [All patients received antibiotic prophylaxis at admission consisting of Ampicillin plus Erythromycin for 7 days following. In addition all patients received a single course of Betamethasone, consisting of two 12 mg Betamethasone injections during the first 24 hours after admission. Fetal surveillance incorporated daily non-stress testing. For fetuses with non-reassuring, non stress test results, biophysical profile assessments were performed. Indications for delivery included: labor, the diagnosis of clinical chorioamnionitis or non-reassuring fetal test results. Eligible patients were subsequently categorized into 2 groups on the basis of the admission AFI measurement. Patients in group 1 were those with an AFI<5 cm, whereas those in group 2 had AFI ≥ 5 cm. The 2 groups were compared for demographic characteristics, the estimated gestation age at both rupture of the membranes and delivery, latency until delivery, mode of delivery, birth weight, the development of clinical chorioamnionitis, postpartum endometritis, early onset neonatal sepsis and respiratory distress syndrome.Neonatal sepsis was diagnosed by positive blood, urine, or cerebrospinal fluid cultures. Possible neonatal sepsis was diagnosed when two or more of the following criteria were present: white blood cell count less than 5000/mm3, polymorphonuclear counts less than 1800/mm3, ratio of bands to total neutrophil counts greater than 0.2. Early onset neonatal sepsis was defined as sepsis in a neonate with positive culture results or possible sepsis within the first 48 hours of life and prior to the antibiotic administration. The clinical diagnosis of chorioamnionitis was made in presence of two or more of the following criteria: maternal fever greater than 38 C, maternal tachycardia (120 beats per minute or more), leukocytosis , fetal tachycardia (greater than 160 beats per minute), uterine tenderness, and foul-smelling amniotic fluid.2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test.Categorical data were tested for significance with the χAll 2-sided p values <.05 were considered significant.A total of 95 patients with preterm premature rupture of membranes were included in the study;26 were included in group I (AFI<5 cm) and 69 in group II (AFI ≥ 5 cm). The 2 groups were similar with respect to maternal age, Parity, and gestational age at admission . Post partum infections were not seen in 2 groups.Early onset neonatal sepsis, respiratory distress syndrome (RDS), neonatal deaths were not significantly different between the 2 groups. Table Complications of preterm premature rupture of membranes count for approximately 25% to 33% of all preterm deliveries.Approximately, 75% of women will be delivered within 1 weeks of presentation ,11.Expectant management with antenatal antibiotics and corticosteroid administration are recommended the standard of care in the setting of PPROM at gestational ages of ≤ 34 ,4,9,10. Amniocentesis and amniotic fluid volume have been advocated as a useful adjunct for identifying these patients Several sOther investigators have demonstrated an association between the development of chorioamnionitis and a shorter latency in patients with PPROM -8. Post Our study demonstrated that the patients with oligohydramnios were more likely to undergo cesarean delivery because of non-reassuring fetal heart rate patterns and is consistent with the findings of these other studies ,6,8.This study didn't show an increased frequency of early onset sepsis in the group I (AFI<5 cm), because all newborn infants in the study were treated possible sepsis with clinical symptom and laboratory evidence. 7 of the included neonates had positive blood cultures or spinal fluid cultures; as a result, there was not sensitive mechanism for appropriately determining the diagnosis of early sepsis. The negative cultures in the neonates with possible sepsis may be related to inadequate culturing techniques or the inherent difficulty encountered by most laboratories in isolating anaerobic bacteria.Perhaps, Diagnosis of early onset neonatal sepsis and close observation for early signs of sepsis and more aggressive evaluation and early treatment for neonatal sepsis have decreased early onset sepsis in 2 groups. Preterm premature rupture of the membranes is associated with a significant decrease in the frequency of neonatal respiratory distress syndrome. In the Sims EJ study (2002), The frequency of respiratory distress syndrome in the neonate complicated with PPROM was (17%).11 This study evaluate the effect of AFI on the frequency of respiratory distress syndrome among two group that are complicated with PPROM. The frequency of respiratory distress syndrome in the neonate was not significantly lower in the group (II) than in the group I. (11/8% vs26/1%) (P < .01). The identification of oligohydramnios, defined as an AFI<5 cm, patients with preterm PPROM appear to indicate a significant risk of chorioamnionitis and early onset neonatal sepsis. These finding can aid in the counseling of patients with PPROM and may have several clinical application.Management of PPROM requires an accurate diagnosis as well as evaluation of costs and the risks and the benefits of continued pregnancy or expeditious delivery. It is important that the patient be well informed regarding the potential for subsequent maternal, fetal, and neonatal complications regardless of the management approach.The pre-publication history for this paper can be accessed here:


Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.Sus scrofa that encode cytochrome P450 aromatases–enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems. The emergence of complete genomes for many organisms, including humans, has created the need for hypotheses concerning the "function" of specific genes that encode specific proteins. While "function" is interpreted by different workers in different ways , DarwiniSystems hypotheses can be supported by information from many areas. Geology, paleontology, and genomics, for example, provide three records that capture the natural history of past life on Earth. At the same time, structural biology, genetics, and organic chemistry describe the structures, behaviors and reactivities of proteins that allow them to support present life. It has been appreciated that a combination of these six types of analysis provides insights into functional behavior of proteins that cannot be provided by any of these alone . Over thThe aromatase family of proteins offers an interesting system to illustrate the power of this combination as a way to create hypotheses regarding protein function within a system . These hAromatases are cytochrome P450-dependent enzymes that use dioxygen to catalyze a multistep transformation of an androgenic steroid (such as testosterone) to an estrogenic steroid (such as estradiol) Figure . The proDifferent numbers of aromatase genes are found in different vertebrates. Two aromatase genes are known in teleost fish ,7. Only Sus scrofa). Three different mRNA molecules had been reported in different tissues from pig [A still different phenomenology is observed in the pig  and ox (Bos taurus) were constructed for transitions at the silent sites of two-fold redundant codon systems where the encoded amino acid was conserved between the species [f2 = 0.87, corresponding to a TREx distance of kt = 0.332. As the fossil record constrains the date of divergence of these two lineages to be 60 ± 5 Ma [Sus scrofa and Bos taurus project an equilibrium value for f2 = 0.54 [-9 transitions/silent site/year during the time interval that separates these lineages.To calibrate the silent TREx clock, inter-taxa histograms relating pig , birds versus mammals (f2 = 0.612), primates versus ungulates (f2 = 0.823), and horses versus artiodactyls (f2 = 0.828). Assuming a time-invariant single lineage first order rate constant of 3.6 × 10-9 changes/site/year and an equilibrium f2 of 0.54, the corresponding dates of divergence are calculated to be 435, 258, 67, and 65 Ma respectively, with the oldest dates being the least precise. The last three of these dates of divergence are similar to those suggested by the paleontological record [f2 values. A tree for the artiodactyl lineage was constructed from the corresponding TREx distances . Briefly, the rate constants for transitions and transversions are more different than the two rate constants for purine-purine and pyrimidine-pyrimidine transitions. Further, nucleotide frequencies can be used to calibrate the end equilibrium points for two-fold redundant codon systems directly, and this permits an "approach to equilibrium" formalism, well known in chemical kinetics, to be applied [The TREx clock is not widely used. It may, however, provide more accurate dates in regions where synonymous transitions have not equilibrated than conventional clocks that combine data from synonymous transitions and synonymous transversions, or from non-synonymous changes. A comparison of different clocks will be provided in detail elsewhere , corresponding to a range in the time of divergence of 26–38 Ma. The TREx distances from the divergence of all of the porcine aromatases and the modern forms ranges from 0.082–0.116, corresponding to dates of divergence in the range of 27–39 Ma. This suggests that the three aromatase paralogs diverged in the late Eocene to mid Oligocene.Sus scrofa. Both peccary and babirusa seminal plasma  was probed by PCR (Polymerase Chain Reaction) amplification using exon 4-specific primers [To further correlate the duplication of the genes with the fossil record, genomic DNA was analyzed from relatives of  primers . Bands h primers ,34.Diacodexis, from the early Eocene ca. 55 Ma) [These data are consistent with an evolutionary model that holds that the ancestor of pig and oxen (approximated in the fossil record by . 55 Ma)  containeSus). Note that there has long been the tacit assumption that large litters in suoids represent the primitive artiodactyl condition. Large litters are primitive for mammals in general, and because suoids are plesiomorphic in some anatomical conditions relative to other artiodactyls (e.g., short legs, retention of four digits, bunodont cheek teeth), they have been assumed to be plesiomorphic in other respects.This does, however, suggest that the emergence of the aromatase paralogs was approximately contemporaneous with the emergence of a litter in the Suoidea larger than that found in the ancestral artiodactyl condition. While ruminant and camelid artiodactyls have only one-two young per litter, suoids in general have at least two young per litter (as seen in peccaries) and most suines (true pigs) routinely have three-four young  shows that they also only had one-two young [Other data suggest that small litters are in fact the primitive artiodactyl condition. Tragulids (mouse deer or chevrotains) are surviving small, primitive ruminants that are not too dissimilar from wo young ,37. A clA/KS value), strongly suggests functional change when the value is significantly greater than unity [A/KS values were reconstructed for individual branches of the evolutionary tree derived from the Darwin bioinformatics workbench (see Methods) using a distance matrix and ancestral states constructed by the method of Messier and Stewart [A/KS value of 0.35. A higher KA/KS value of 0.85 is found in the episodes of evolution near when the pig aromatases diverged. While a KA/KS value of 0.85 does not require the conclusion that positive selection occurred during the emergence of these aromatase paralogs, an inference based on the magnitude of KA/KS in one branch, relative to the KA/KS value for typical branches [The approximate correlation in time of the aromatase divergence in Suoidea with the enlargement of litters in Suoidea suggests, as a hypothesis, that the two are functionally related. To expand on this hypothesis, we sought genomic signatures of functional change within the aromatase paralogs. The number of non-synonymous changes in the gene divided by the number of the synonymous changes, normalized for the number of non-synonymous and synonymous sites  was again found in the divergence of the swine isoforms on the branch leading to the ancestor of the placental and embryonic enzymes following their divergence from the pig ovarian enzyme. The distribution of substitutions along this branch is consistent with altered functional constraints for the placental and embryonic enzymes compared with their extinct and extant counterparts  , a homolA/KS values  involving pig paralogs was not random over the protein surface. Rather, the sites are clustered near the substrate binding pocket, and in a region of the surface believed to contact the co-reductant protein, as identified by mutagenesis experiments in the homolog [In contrast, the distribution of sites accepting amino acid replacements during the episode of rapid sequence evolution of branch 1 . The events of interest (such as the extinction of dinosaurs) are frequently distant in time, or require a passing of time (as for speciation), making them difficult to reproduce in a laboratory. The scale of the concepts involved (species, environments, planets) also does not lend these concepts to laboratory models and laboratory-controlled tests. Further, a reductionist approach, even when available, will not necessarily generate data that are relevant to the big issue that concerns the natural historian. The emphasis on data and proof has ameliorated the worst excesses of storytelling in natural history, with enormous positive impact.In part to make this connection, natural historians have sought to change the research paradigm in their field to favor quantitative data directed towards the "proof" of hypotheses over "story telling". Proving hypotheses is difficult in natural history  for a protein that had been selected for functional reasons. Today, the typical researcher knows the structure of many proteins, and wishes to select one for expression and study based on a hypothesis about its potential function.Here, the fact that any definition of function, which must make reference to fitness, requires some systems, ecological, or planetary context, makes the natural historian a natural source of hypotheses. Their full reductionist armamentarium is available in the laboratory to test and explore any hypothesis that the natural historian might provide. The biomedical researchers may like some guidance from the natural historian to narrow the broad selection, or to shorten the random walk, if only slightly.For this purpose, the forswearing by natural historians of storytelling has come at a most inopportune time. To the modern natural historian, creating hypothesis can easily be regarded as "storytelling". They are reluctant to do so, and may criticize as atavistic colleagues who do.This has created a vacuum in the scientific community. Very few laboratories exist that can draw upon an expertise in natural history to generate stories that create hypotheses for the researcher working in experimental biochemistry and molecular biology.This article is designed in part to illustrate how this vacuum might be filled. Here, we do not just tell a story based on natural history, or even a story based on natural history supplemented with physiology and molecular sequence data. Rather, we show how the addition of other data, including data from X-ray crystallography, can make a story sufficiently rich that it can be viewed as being internally consistent with a wide range of independent data drawn from independent sources. This creates a hypothesis that is more than a story, even if it is less than proven.Sus. Steroid production by the porcine embryo is tightly controlled by the transient expression of aromatase and 17-hydroxylase (P450C17) between days 10 and 13 [With aromatase, the congruence of our different analyses makes a compelling suggestion that the three aromatase paralogs in pigs arose by two duplication events in the late Eocene or early Oligocene. The emergence of the aromatase paralogs corresponded approximately in time to the emergence of larger litter size in suines. This implies that the two duplication events are functionally related to the larger litter sizes. This inference is consistent with the physiological impact of estrogen synthesis by these paralogs in 0 and 13 ,21,52. I0 and 13 . AdequatThis is consistent with a structural biological analysis that correlates specific amino acid replacements with specific changes in the substrate and product specificity of the protein . InteresA/KS ratio of 0.93 would not, by itself, compel any particular interpretation. Its implications are greater given the relatively low KA/KS ratios of other branches of the tree. But the addition of crystallographic information, itself not compelling, makes a combination that is more compelling.It should be noted that the hypothesis is supported by the combination of data that individually would not have strength past storytelling. Thus, the KA/KS ratio  compel the inference of an episode of adaptive change. Intriguingly, these changes are also clustered in the same regions of the structure as those changing along branch 1 leading to the stem fetal/placental enzyme, near the substrate and co-reductant binding sites. This implies that the substrate/product specificity of the ancestral aromatase protein was not like that of either the human or the pig placental forms, but rather reflects features that arose convergently in these two species [Further, this hypothesis generation itself generates discoveries that might lead to their own hypotheses. An analysis of the evolutionary branches separating pigs and humans suggests an additional episode of adaptive change. The branch leading to the ancestor of human aromatase (branch 3) has a remarkably high K species .Notably, four of the sites (positions 47, 153, 219, 269) that undergo replacement during the emergence of pig placental aromatase from the last common ancestor are the same as four that arose in the emergence of the human aromatase from its last common ancestor. Of these, the amino acid replacements are identical at two sites . The probability associated with randomly observing this pattern is extremely low (0.000021) . An addiIt is important to point out that even an analysis this broad is likely to cover only a small part of a complicated reproductive endocrinology that must be associated with larger litter sizes. For example, the exact nature of the products produced by individual aromatases remains controversial, and may be different in laboratory studies depending on the conditions where they are studied ,60-62. Tet al. [Further, an elegant recent study by Corbin et al.  identifiet al. [In fact, some data suggest that uterine exposure to androgens severely decreases litter size and embryonic survival during the time of maternal recognition of pregnancy . This iset al.  that theDiacodexis, see Figure It goes without saying that still more factors might be associated with an increase in litter size from one-two  young, a reproductive strategy apparently not available to larger, cursorial (running-adapted) ungulates, which give birth to precocial (i.e., well developed) young that are fully locomotory at birth [Nevertheless, the first aromatase duplication, shared by pigs and peccaries, appears to have happened in the late Eocene (recognizing the error associated with these dates), around 35 Ma Figure . This waat birth .The second aromatase duplication, with the ensuing capacity to produce multiple young, probably occurred within the family Suidae, some time during the Oligocene. The molecular data suggest dates of divergence between porcine fetal and placental aromatases as between 27–38 Ma, and the earliest known suid is of early Oligocene age , around b places this data at ca. 18 Ma (data not shown). Clearly, further study is warranted.This type of speculation opens questions. For example, the babirusa (an Indonesian pig) is reported to have average litters of one-two individuals ,69. WhilThe aromatase family offers an example where a combination of phylogenetic analysis, molecular evolutionary analysis, and chemical analysis set within the context of the paleontological and geological records, and supported by contemporary bioinformatics and molecular modeling tools, permits a higher order level of hypothesis generation concerning the function of proteins. Rather than simply an Enzyme Commission number (E.C. 1.14.14.1 for aromatase), a description of catalytic activity (the enzyme oxidizes testosterone), or a description of the regulatory pattern (the protein expressed between day 10 and 13), this type of analysis can generate a truly functional hypothesis: that the embryonic enzyme oxidizes testosterone as a way of managing the larger litter sizes that emerged in the Suoidea during a time of dramatic planetary cooling (ca. 35 Ma).Such hypotheses set a higher bar, and a more useful standard, for the field of systems biology. Evolutionary theory holds that the only mechanism for obtaining functional behavior in a biological system is natural selection. Selection, based on a frequently poorly defined concept of "fitness", is determined by a context that not only includes the cell and tissue, but also the organism, the ecosystem, and a changing planet . One caninter alia, from a study of Indo-European languages. Proto-Indo-European (PIE) language had words for "pig"  following their domestication. Evidence for ancient domestication of pigs comes, ah, etc. ), indicaah, etc. . With thcbrg@inf.ethz.ch.Calculations were done under the RedHat Linux 6.3 operating system on an Intel-Pentium III instrument using Blackdown's Java-SDK 1.1.8. PAML calculations were done on an IBM PC using the Unix operating system. Sequence analyses were aided by the DARWIN bioinformatics package . The DARInitially, pairwise alignments were constructed for the aromatase protein sequences available in the database. An evolutionary distance in PAM units was calculated for each pair by applying the PamEstimator-package from DARWIN using an empirical log-odds matrix. From this, a preliminary evolutionary tree was built for the mammalian sequences, with branch lengths along internal nodes calculated to minimize a least-squares distance. The sequences of the ancestral genes and proteins at branch points in the tree were then reconstructed. From there, mutations  at both the DNA level and protein level were assigned to individual branches in the tree using the method of Fitch .n) of two-fold redundant amino acids (Cys, Asp, Glu, Phe, His, Lys, Asn, Gln, and Tyr) that are conserved in the aligned pairs was determined. The number of those amino acids that are encoded by the same codon (c) was determined, and the fraction (f2 = c/n) of the codons that are the same were then tabulated /(1-Equil)), where Equil is the f2 value expected after a large number of nucleotide substitutions have occurred at the synonymous sites [The evolutionary history of the aromatase family was then analyzed using the transition redundant exchange (TREx) metric based on an analysis of two-fold redundant codon systems ,78. Thesable see ). The TRus sites .The DNA sequences for aromatase were phylogenetically analyzed using a maximum likelihood framework in PAUP 4.0* (beta 10) , with thf2 value characteristic of orthologs determined [For inter-taxon analyses, families in the MasterCatalog (EraGen Biosciences, Madison WI) were identified that contained at least one representative protein from both of the taxa of interest. For these families, all inter-taxa pairs of genes were extracted, together with the pairwise protein sequence alignment. A pairwise alignment of the DNA sequences was then generated to follow the protein sequence alignment. If a family contained more than one sequence of a species belonging to one of the taxa analyzed, then those sequences were checked to determine whether they were duplicate entries into the database. If this was the case, only one of the duplicate sequences was retained in the analysis. A histogram of inter-taxa pairs was constructed, and the termined . This waCodon biases were obtained from the CUTG (Codon Usage Tabulated from GenBank) made available by the Kazusa DNA Research Institute Foundation, Japan .A/KS values to individual branches. Tests of parallel evolution were conducted using Converge [Pairwise TREx distances were used to generate lengths for the branches connecting the swine paralogs using the minimum evolution criterion in PAUP. This preliminary analysis was followed by a maximum likelihood analysis for the complete dataset using the PAML program . This inConverge , implemeSecondary structural data based on homology modeling for aromatases were generated using the DARWIN bioinformatics package, and in agreement with previous studies ,84. RendEAG performed the evolutionary, statistical and structural analyses, and prepared the manuscript. LGG cloned genes as part of his Masters work, and called the evolutionary problem to the attention of SAB. TL provided computational infrastructure. RCMS and FAS initiated the work with suid reproductive endocrinology, and supervised LGG. DRS and DAL did the initial bioinformatics analysis. CMJ provided paleontological expertise, constructed the cladogram, and helped prepare the manuscript. SAB has developed planetary biological analysis as a paradigm for generating hypotheses about the biological function of proteins, and prepared the manuscript.Illustration of planetary biology. This figure illustrates the concepts of planetary biology as they relate to combining genomic, paleontological, chemical and ecological records to understand the history of the biosphere.Click here for fileAn analysis of silent nucleotide substitutions in vertebrate aromatases. The first five columns from the left indicate the index number of sequence 1 compared with sequence 2, the fraction of sites at conserved two-fold redundant coding systems that are identical (f2), the number of such sites that are conserved (c2), and the number of such sites overall (n2). The remaining columns report analogous data: for silent sites in codon systems where a change at the third nucleotide is silent only if the change is a pyrimidine-pyrimidine transition (f2y, c2y, n2y); in silent sites where a change at the third nucleotide is silent only if the change is a purine-purine transition (f2r, c2r, n2r); for the silent sites at three-fold redundant codon systems (f3, c3, n3); and for the silent sites at four-fold redundant codon systems (f4, c4, n4).Click here for file


Most self-pollinating plants are annuals. According to the 'time-limitation' hypothesis, this association between selfing and the annual life cycle has evolved as a consequence of strong r-selection, involving severe time-limitation for completing the life cycle. Under this model, selection from frequent density-independent mortality in ephemeral habitats minimizes time to flower maturation, with selfing as a trade-off, and / or selection minimizes the time between flower maturation and ovule fertilization, in which case selfing has a direct fitness benefit. Predictions arising from this hypothesis were evaluated using phylogenetically-independent contrasts of several life history traits in predominantly selfing versus outcrossing annuals from a data base of 118 species distributed across 14 families. Data for life history traits specifically related to maturation and pollination times were obtained by monitoring the start and completion of different stages of reproductive development in a greenhouse study of selfing and outcrossing annuals from an unbiased sample of 25 species involving five pair-wise family comparisons and four pair-wise genus comparisons.Selfing annuals in general had significantly shorter plant heights, smaller flowers, shorter bud development times, shorter flower longevity and smaller seed sizes compared with their outcrossing annual relatives. Age at first flower did not differ significantly between selfing and outcrossing annuals.This is the first multi-species study to report these general life-history differences between selfers and outcrossers among annuals exclusively. The results are all explained more parsimoniously by selection associated with time-limitation than by selection associated with pollinator/mate limitation. The shorter bud development time reported here for selfing annuals is predicted explicitly by the time-limitation hypothesis for the fitness benefit of selfing . Support for the time-limitation hypothesis is also evident from published surveys: whereas selfers and outcrossers are about equally represented among annual species as a whole, selfers occur in much higher frequencies among the annual species found in two of the most severely time-limited habitats where flowering plants grow – deserts and cultivated habitats. Inter-limited ,4-8. , versus time-limitation . Accordingly, these two hypotheses for selfing involve very different assumptions and predictions.A recent third hypothesis, the 'time-limitation' hypothesis, predicts that both selfing and the annual life cycle are concurrent products of strong 'r-selection' associated with high density-independent mortality risk in ephemeral habitats with a severely limited period of time available to complete the life cycle . Both thdirect fitness benefit; abbreviating the time between anthesis and ovule fertilization may ensure that there is enough remaining time in the growing season (after ovule fertilization) to allow complete seed and fruit maturation [The time-limitation hypothesis has direct and indirect components. The indirect component predicts higher selfing rates in annuals as a trade-off of selection for earlier reproductive maturity in annuals ,13 Figu. More raturation  Fig 1b)direct fiHowever, the two components of time-limitation cannot be separated clearly, as they operate simultaneously; i.e., earlier onset of flowering, shorter flower development time, smaller flowers and selfing can all be interpreted to have direct fitness benefits because they may all contribute directly to accelerating the life cycle . Indeed,The time-limitation hypothesis remains untested. Some recent studies have explored the rapid growth and maturation time of annuals in terms of bud development rates and ontogeny -15. HoweThe objective of the present study was to compare, for annuals exclusively, life history traits associated with selfing versus outcrossing using several species from a wide range of plant families. Phylogenetically-independent contrasts (PIC) were used to control for confounding effects due to common ancestry among species . Using aData on the timing of life history stages (i.e. age at first flower, bud development time, and flower longevity) were also obtained from a greenhouse study of 25 annual species involving 5 families. The time-limitation hypothesis predicts that selfers should produce mature flowers more quickly and should have shorter flowering times.Based on phylogenetically-independent contrasts, selfing annuals had significantly shorter plant heights  or flower longevity (P = 0.328). Age at first flower was not significantly related to mating system for any of the family or genus comparisons .Bud development time Figure  and flowThere is a rich body of theory and empirical work on the evolution of selfing in flowering plants [e.g. ,4-7,9,10for annuals the time-limitation hypothesis provides a more parsimonious explanation for the differences in traits between selfers and outcrossers. We consider each of these traits in turn below.At the same time, these results are not inconsistent with the predictions of selection resulting from pollinator/mate-limitation associated with the traditional reproductive assurance hypothesis. Just as with many situations where two different mechanisms can potentially produce the same outcome/pattern, it is not easy here to clearly distinguish between the roles of "pollinator/mate-limitation" and "time-limitation". Nevertheless there are two important contributions from our study: First, in reporting significant life history differences between selfers and outcrossers, our multi-species study is unique in its comparison of monocarpic annual species exclusively. All previous multi-species studies of trait comparisons between selfers and outcrossers have involved variable mixes of monocarpic and longer-lived polycarpic species. Second, by comparing annuals exclusively, our results provide indirect support for the time-limitation hypothesis, not by rejecting the role of pollinator/mate-limitation, but rather by representing a system in which it is more plausible to argue for the role of time-limitation; i.e., compared with pollinator/mate-limitation, time-limitation as a selection factor favoring selfing is likely to have been much stronger, more persistent and more widespread. The strength of this argument lies in the fact that the annual life history is unequivocally a product of some type of time-limitation favoring an abbreviated life cycle, which is promoted by (among other things) selfing (as opposed to outcrossing) Fig. . It is mCrepis tectorum. Arroyo [Limnanthes floccosa flowered earlier than the outcrossing L. alba, as predicted by the time-limitation hypothesis. The results for flowering times in the present study may be confounded by the controlled greenhouse environment of constant day-length, temperature and moisture regime. In the field, flowering times may be triggered by environmental cues. L. floccosa, for example, uses soil moisture to trigger the early onset of flowering, thus escaping the detrimental effects of soil desiccation during seed development [Taller plants may attract more pollinators and, hence, experience greater outcrossing rates ,21. The . Arroyo , howeverelopment . Note alOne of the most well established trends of predominantly self-fertilizing species is their reduced flower sizes compared with outcrossing species ,17. The Under the time-limitation hypothesis, smaller flowers and selfing may be tradeoffs of selection for precocity Figure , or smalMimulus guttatus [Clarkia xantiana [C. tembloriensis [Arenaria uniflora develop over a longer period of time than observed in outcrossing populations. In the field, selfing populations of A. uniflora were also observed to flower at the same time or even later than outcrossing populations [A. uniflora may have arisen through reproductive assurance in response to competition for pollinators [vis-à-vis the fitness benefits of selfing.Selfers had significantly shorter bud development times in all but one of the independent family contrasts Figure  and all guttatus  and in Cxantiana . Howeveroriensis . Hill, Loriensis  reportedulations , suggestlinators . The evolinators ,3,14, eaTrifolium hirtum; Fabaceae), with a large proportion of flowers open for only one day, which is common amongst self-fertilizing species [The families and genera in which selfers had shorter bud development times also had significantly shorter flower longevities Figure . In fact species . The pre species . This fi species . If timemaximum flower longevity, since there were no pollinators in the greenhouse, nor was hand pollination conducted. Pollination has been shown to induce floral senescence in numerous species [It is important to note that our data measure  species . This efStrong r-selection associated with the annual life form presumably favors wide dispersal mechanisms (for colonizing new and distant sites) which may be conferred by small seed sizes . The repCrepis tectorum took an average of 16 days for fruit maturation, whereas outcrossing individuals of the same species required 43.3 days. Small seed size may also be simply a trade-off of selection for high fecundity, also favored by strong r-selection [An alternative explanation, however, is offered by an extension of the time-limitation hypothesis: strong r-selection favors an acceleration of all stages of the life cycle Figure , includielection .While most selfers are annuals, it is not the case that most annuals are selfers. An unbiased literature survey  suggestsClarkia xantiana [Limnanthes alba and its selfing relative L. floccosa [L. alba was reduced by one sixth, whereas the seed set of L. floccosa found in the same area was virtually unaffected by the identical drought [While rigorous tests of these predictions have yet to be explored, some preliminary support is available from published surveys. From a representative sample of Mediterranean annuals , we findxantiana  and betwfloccosa . Since t drought .The association between 'weediness' and self-fertilization has also been noted ,30. An eBotanists have long known that selfing is particularly associated with the annual life cycle in flowering plants . The preThe effect of time-limitation under strong r-selection is to minimize the duration of the life cycle, with selfing favored directly Figure  and/or ivis-à-vis Figure Designing empirical studies that clearly distinguish between mechanisms involving time-limitation versus pollinator/mate limitation remain a challenge but we anticipate that our results and our discussion of these issues may help to inspire further research along these lines. Future studies may be designed to test more directly the role of limited pollination time (The literature was surveyed to obtain breeding information (i.e. selfing versus outcrossing) for as many annuals species as possible. For each species, data on plant height, flower size, and seed size were obtained where possible from standard floras and other published literature. A complete database was assembled for 118 species from both Europe and North America, involving 14 families  was known and could also be obtained as germinable seeds. Seeds were obtained from a variety of sources; Herbiseed, Rancho Santa Ana Botanic Gardens, Chiltern Seeds, S&S Seeds, and the National Plant Germplasm System. Our search lead to an unbiased sample of 25 candidate species, allowing five pair-wise family comparisons and four pair-wise genus comparisons (Table ©). Pots were placed in a greenhouse and watered daily until the appearance of their first true leaves. Subsequently, they were watered uniformly every second day to ensure that the soil was kept moist. Some species were germinated in a petri-dish in a growth chamber (23°C, 12 hour cycles of light and dark), after which they were transplanted into pots and placed in the greenhouse. Each species was replicated five times, with one plant per pot. Pots were arranged randomly on benches at a density of 1 pot per 0.093 m2.Most species were germinated in 15 cm pots filled to 3 cm below the top with standard potting soil  were used to supplement the light exposure to 16 hours of light per day. The greenhouse was kept at an average temperature of 23.1°C during the day and dropped to 20.0°C at night. The plants were fertilized every 2 weeks with 200 ml per pot of a 2g/L concentration of 20–20–20 N-P-K fertilizer.For each plant, age at first flower, bud development time, and flower longevity were measured. Emergence of the first pair of true leaves, after the cotyledons, was considered day 1 of the plant's life. Age at first flower was measured in days from day 1 to when the first flower opened on each plant. Bud development time (n = 3 buds per plant) was calculated as the number of days from the first appearance of a new bud until the flower opened. The same three buds on each plant were then monitored every day after opening, and the number of days until the flower senesced (flower longevity) was recorded for each. A flower was considered to be senesced when the corolla wilted, fell apart, or became discolored, as designated by Primack . Any floFor bud development time and flower longevity, the three replicate measurements for each plant were averaged, and then these values for the five replicate plants were averaged to obtain a mean value for the species. The data were analyzed with a standard least squares one-way analysis of variance (ANOVA) model, with a post-hoc contrast between selfers and outcrossers. These analyses were done for each family and genus separately in order to control for phylogeny at these levels. In cases where the data were non-normal, a log-transformation was applied which corrected the distribution.RS collected the data, performed most of the analyses, participated in the design of the study, and wrote the first draft as a B.Sc.(Hons) thesis. LWA conceived of the study, participated in its design and coordination, and wrote the final draft for submission to BMC. Both authors read and approved the final manuscript.


Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology. Noroviruses -- which cause epidemic gastroenteritis -- can now be grown in cells of the innate immune system, providing a tool to examine this pathogen while offering insights into norovirus biology Norovirus  of the family Caliciviridae are major agents of acute gastroenteritis (−/−) mice (Viruses within the genus nteritis . Norovirnteritis . Most nonteritis . Our recnteritis . This pr/−) mice  that areHere we show for the first time that MNV-1 grows in macrophages (MΦ) and dendritic cells (DCs) and provide the first tissue culture model for a norovirus. Using this model we demonstrate that MNV-1 growth in vitro was inhibited by the IFN-αβ receptor and STAT-1. In addition, we isolated the first three-times plaque-purified strain of MNV-1 (MNV-1.CW1) and characterized it in vitro and in vivo. Sequencing of serial passages of MNV-1.CW1 indicated remarkable sequence stability over time and indicated that an amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with a loss of virulence in vivo.−/− mice were infected with MNV-1 by the oral route and tissue sections analyzed by immunohistochemistry for the presence of MNV-1 protein. MNV-1-specific staining was observed in spleen and liver 2 d postinfection (As part of our ongoing investigation into MNV-1 pathogenesis, STAT1nfection . Interesnfection A and 1B.nfection C and 1D.nfection . Furthernfection C.−/− (IFNαβγR−/−) mice (−/− BMMΦ and BMDCs, but not STAT1−/− murine embryonic fibroblasts (MEFs)  and bone-marrow-derived DCs (BMDCs) were inoculated with a MNV-1 stock derived from the brain of infected IFNαβγ receptor/−) mice . Cytopats (MEFs) A. While We used this information to screen available MΦ cell lines for growth of MNV-1, including the murine lines RAW 264.7  and J774−/− BMMΦ, STAT1−/− and wt BMDCs, and RAW 264.7 cells consistently supported the growth of MNV-1, while wt BMMΦ varied in their ability to support virus growth  with three rounds of plaque purification in RAW 264.7 cells to generate the MNV-1.CW1 strain. This strain was amplified in RAW 264.7 cells, after which virus particles were concentrated and subjected to purification by isopycnic centrifugation in CsCl. A distinct band was observed in CsCl gradients at a density of 1.35 ± 0.01 g/cmoviruses . Examinaoviruses A. Furtheoviruses B,C. Westoviruses B and masoviruses , did notoviruses D. MAb A6oviruses E. TogethTo compare MNV-1 replication in cells with that of other caliciviruses, we analyzed viral RNA and protein synthesis in MNV-1.CW1-infected RAW 264.7 cells. Northern blot analysis using a probe specific for the positive strand of the MNV-1 genome showed an increase in the accumulation of full-length (7.4 kb) and subgenomic-length (2.3 kb) MNV-1 genome over time F. RadiolPositive-strand RNA viruses , includi−/− MΦ and MEFs (−/− cells compared to wt cells, and MNV-1.CW1 (P3) growth was consistently observed in wt BMMΦ. These data demonstrated that our plaque purification and serial passage in RAW 264.7 cells had not changed the tropism of the virus for primary DCs and MΦ and confirmed the importance of STAT-1 in controlling MNV-1 growth at the cellular level.To determine whether the plaque purification and sequential amplification of MNV-1 in RAW 264.7 cells had altered its growth characteristics, different cell types were infected with passage (P) 3 of MNV-1.CW1. In general, the growth of MNV-1.CW1 (P3) in wt or STAT1and MEFs A as welland MEFs B and 5A.−/−, or protein kinase R (PKR)−/− are not susceptible . Therefore, we determined whether molecules other than STAT-1 exhibited antiviral effects at the level of the infected cell. Primary BMMΦ from wt mice or mouse strains deficient in STAT-1, IFNαβR, IFNγR, IFNαβγR, iNOS, or PKR were directly compared for their ability to support virus replication at two different multiplicities of infection (MOIs) (−/− mice supported MNV-1 virus replication, with higher titers observed in cells deficient in STAT-1. Cells obtained from mice lacking both Type I and II IFNR (IFNαβγR−/−) or Type I IFNR alone (IFNαβR−/−) supported replication of virus as efficiently as STAT1−/− cells. In addition, wt BMMΦ and wt BMDCs secrete IFNα after MNV-1-infection, as determined by IFNα enzyme-linked immunosorbent assay (ELISA) (data not shown). This is consistent with a direct role for IFN signaling in MNV-1 growth but does not rule out the possibility that effects of STAT-1 and IFNαβR occur in vivo prior to explantation of the bone marrow. Absence of IFNγR, iNOS, or PKR did not have a statistically significant effect on MNV-1 growth in BMMΦ. Together, these data demonstrate that the antiviral molecules STAT-1 and IFNαβ are part of a cellular response that limits norovirus growth.Previous studies demonstrated that a lack of STAT-1 or both IFNαβR and IFNγR increase susceptability to MNV-1 infection. Mice lacking individual IFNR, inducible nitric oxide (iNOS)n (MOIs) B. Again,−/− mice were infected orally with MNV-1.CW1 from three successive passages (P1, P2, and P3) , 5,000 times greater than the lethal dose for P1. In addition, immunohistochemical analysis of sectioned spleen and liver from STAT1−/− mice infected orally with 1.5 × 106 pfu of MNV-1.CW1 (P3) did not reveal any MNV-1-specific staining, unlike the parental virus (see To address the effects of cell culture adaptation on virulence, STAT1 and P3) A. Oral a and P3) . These d and P3) . In contTo examine the molecular basis for this attenuation, consensus sequence analysis was performed on the RNA genome of MNV-1 present in the original brain tissue stock , and in viruses from each subsequent cell culture passage of MNV-1.CW1 (P1 through P3) . Three n−/− mice led to our finding that MNV-1 grows in these cell types in vitro. This provides the first tissue culture model for a norovirus. In addition, the antiviral Type I IFN response with signaling through STAT-1 is crucial for resistance to murine norovirus infection in vivo and in vitro. Taken together the previous in vivo data . IFNαβR−/−, IFNγR−/−, and IFNαβγR−/−  or brain homogenate from uninfected mice. Organs were collected into 10% buffered formalin and embedded in paraffin for sectioning by standard methods. Immunohistochemistry was performed as described previously  per well. Plates were incubated at 37 °C and 5% CO2 for 2 d. To visualize plaques, cells were stained with 2 ml of 56 °C 1.5% SeaKem agarose in complete MEM containing 1% neutral red per well for 6–8 h.RAW 264.7 cells were seeded into six-well plates at a density of 2 × 10For plaque neutralization assays, differing concentrations of purified mAb  were incubated with equal plaque-forming units of either MNV-1.CW1 or MNV-1 brain homogenate for 30 min at 37 °C prior to performing the MNV-1 plaque assay.g) in a SW32 rotor. Cell pellets were then resuspended in PBS and mixed with CsCl to a final density of 1.335 g/cm3 and centrifuged for at least 18 h at 35,000 rpm (115,000 g) in a SW55 rotor. A wide lower band (1.35 ± 0.01g/cm3) and narrow upper band (1.31 ± 0.01g/cm3) were typically seen in the gradient. Each band was collected by puncturing the side of the tube with a needle before overnight dialysis against PBS at 4 °C.RAW 264.7 cells were infected with MNV-1.CW1 for 2 d at an MOI of 0.05. Cellular debris was removed from freeze/thaw lysates by low-speed centrifugation for 20 min at 3,000 rpm. Supernatants were layered on top of a 5-ml 30% sucrose cushion and centrifuged at 4 °C for 2.5 h at 27,000 rpm  according to the manufacturer's recommendations. One microgram of total RNA from MNV-1- or mock-infected cells was subjected to electrophoresis on a 1% formaldehyde gel. RNA Millennium Size Markers (Ambion, Austin, Texas, United States) were used as size markers. Northern blotting was performed using standard protocols (The region of the MNV-1 genome from nt 5,617 to 7,039 was amplified by RT-PCR and cloned into the pGEM-T Easy (Promega, Madison, Wisconsin, United States) vector between the T7 and SP6 promoters. The resulting plasmid was linearized with Bsu361 and in vitro transcribed with SP6 RNA polymerase (Roche, Indianapolis, Indiana, United States) to generate RNA transcript probes for detection of positive-sense viral RNA, or with T7 polymerase (Roche) to generate transcripts for detection of negative-sense viral RNA. To label probes, the transcription reaction was carried out in the presence of [Protocols . Probes The ELISA was performed as described previously  with theNegative staining electron microscopy of CsCl-purified virions was performed as described previously . For thiRNA was extracted from brain tissue or cell culture material with Trizol (Invitrogen) and reverse transcribed with Superscript II enzyme (Invitrogen). Genome-specific sequences were PCR-amplified with Elongase enzyme (Invitrogen) to produce seven overlapping fragments. The DNA fragments were gel-purifed and sequenced directly with reagents in the BigDye Terminator version 3.1 Cycle Sequencing Kit  on a 3100 DNA sequencer (Applied Biosystems). Data were analyzed with the Sequencher software package (Gene Codes Corporation, Ann Arbor, Michigan, United States). Oligonucleotide primer sequences are available upon request.


There have been few reports of pharmacokinetic models that have been linked to models of the cardiovascular system. Such models could predict the cardiovascular effects of a drug under a variety of circumstances. Limiting factors may be the lack of a suitably simple cardiovascular model, the difficulty in managing extensive cardiovascular data sets, and the lack of physiologically based pharmacokinetic models that can account for blood flow changes that may be caused by a drug. An approach for addressing these limitations is proposed, and illustrated using data on the cardiovascular effects of magnesium given intravenously to sheep.The cardiovascular model was based on compartments for venous and arterial blood. Blood flowed from arterial to venous compartments via a passive flow through a systemic vascular resistance. Blood flowed from venous to arterial via a pump (the heart-lung system), the pumping rate was governed by the venous pressure (Frank-Starling mechanism). Heart rate was controlled via the difference between arterial blood pressure and a set point (Baroreceptor control). Constraints were made to pressure-volume relationships, pressure-stroke volume relationships, and physical limits were imposed to produce plausible cardiac function curves and baseline cardiovascular variables. "Cardiovascular radar plots" were developed for concisely displaying the cardiovascular status. A recirculatory kinetic model of magnesium was developed that could account for the large changes in cardiac output caused by this drug. Arterial concentrations predicted by the kinetic model were linked to the systemic vascular resistance and venous compliance terms of the cardiovascular model. The kinetic-dynamic model based on a training data set (30 mmol over 2 min) was used to predict the results for a separate validation data set (30 mmol over 5 min).-1) described the distribution of magnesium into a deeper (probably intracellular) compartment. The final kinetic-dynamic model was able to predict the validation data set. The mean prediction error for the arterial magnesium concentrations, cardiac output and mean arterial blood pressure for the validation data set were 0.02, 3.0 and 6.1%, respectively.The kinetic-dynamic model was able to describe the training data set. A recirculatory kinetic model was a good description of the acute kinetics of magnesium in sheep. The volume of distribution of magnesium in the lungs was 0.89 L, and in the body was 4.02 L. A permeability term (0.59 L minThe combination of a recirculatory model and a simple two-compartment cardiovascular model was able to describe and predict the kinetics and cardiovascular effects of magnesium in sheep. The effective use of some drugs can be limited by their adverse effects on the cardiovascular system, particularly when they are used intravenously in relatively high doses. There have been many studies documenting the cardio-vascular effects of drugs. Similarly, many mathematical models of the cardiovascular system, of varying complexity, have been presented in the literature ,2. In pia priori the cardiovascular consequences of, for example, a change in dose regimen of a drug.In contrast, for traditional ("non-volatile") drugs, there have been very few instances in which kinetic models of a drug have been linked to cardiovascular pharmacodynamic models. The work of Francheteau et al. is an important exception , but evemax model). Rather, a number of cardiovascular variables may be altered simultaneously in a manner that is complex and interrelated. Thus, any dynamic model used must account for these intrinsic relationships between cardiovascular variables. Another problem is that changes in the cardiovascular system (in particular blood flow distribution) invariably alter the kinetics of the drug under study. Therefore, the kinetic model of the drug must be able to account for the effects of blood flow changes on the disposition of the drug. This requires the kinetic model to have a physiological basis, and importantly excludes the widely used mamillary compartmental pharmacokinetic model.There are a number of difficulties in implementing this approach for traditional drugs. One problem is that most drugs do not cause changes in one single cardiovascular variable (such as blood pressure) that can be described in the usual manner using a simple semi-empirical dynamic model  are important. This depends on the drug and the intended use of the model, but it is proposed that there is a minimum set of variables that is needed for a basic description of cardiovascular status.2. Devising a way of conveniently presenting the output of the dynamic model for a range of cardiovascular variables for comparison with data.3. Identifying a cardiovascular model of the appropriate complexity. Ideally the model must be of the minimum complexity that includes the cardiovascular variables identified above, and the major sites of action of the drugs.4. Identifying which parameters of the cardiovascular model can be estimated by curve-fitting, and which require prior estimates or measurements of physiological values. Most cardiovascular models are stiff numerical systems with many parameters, and only a small number can be estimated by curve-fitting the data in the traditional way.With respect to the pharmacokinetic model, there remains one crucial step:5. Constructing a kinetic model with a physiological basis that is sufficiently realistic to describe and predict the concentration of the drug in the key target organs controlling the cardiovascular system. On first principles, these could be expected to include:a. the myocardial concentrations when the drug has a direct myocardial effect ;b. the CNS concentrations when the drug affects the cardio-respiratory control centre of the brain;c. the arterial blood concentration when the drug affects baro-receptors or smooth muscle in the walls of the arterial vascular system;d. the venous blood concentration when the drug affects smooth muscle in the wall of the capacitance vessels of the venous vascular system.It is known that these concentrations can follow different time-courses, particularly after bolus administration or a change of infusion rate ,11. HoweThe data used to construct the model were collected in the same laboratory using a conscious chronically instrumented sheep preparation and have been published previously ,9. This Data set 1  , predominantly for curve-fitting. The R language, Version 1.9.0,  was usedi is a weighting term, p is the number of parameters and n is the number of data points:For the least squares curve-fitting, the best fit was determined as that with the highest Model Selection Criteria (MSC) and without non-identifiable parameters. The MSC is essentially an inverse Akaike Information Criterion scaled to compensate for data sets of different magnitudes , and is calculated as follows where wAll data points were weighted equally. A parameter was arbitrarily defined as non-identifiable if the standard deviation of the parameter returned by the fitting program was greater than the parameter estimate (i.e. the coefficient of variation was greater than 100%). A model with non-identifiable parameters means that the data do not contain sufficient information to estimate the parameter with precision.The symbols used throughout have been based on standards for the pharmacokinetic literature. Unfortunately, the use of C for both concentration (in pharmacokinetics) and compliance (in cardiovascular physiology) creates of conflict for this paper. To avoid confusion, CPL will be used for compliance here.-1), Stroke volume (SV, L), Heart rate (HR, min-1), Cardiac output (CO, L min-1), Systemic vascular resistance (SVR, Resistance units, RU) and mean arterial blood pressure (MAP, mmHg).The choice of the cardiovascular variables used in the model is clearly dependent on the drug under study and the intended purpose of the model. However, we propose that a minimum of 7 fundamental cardiovascular variables is sufficient for most pharmacological purposes. These variables are shown with their default unit of measurement in the model: Central venous pressure (CVP, mmHg), Myocardial contractility .That the function of the left and right side of the heart is the same, and there are no abnormalities in the pulmonary vasculature so that the heart-lung system can be treated as one pump.Long time-scale events such as fluid shifts and renal mechanisms controlling blood pressure are ignored.Furthermore, this choice of variables is dictated by several fundamental relationships between the variables. Firstly, that myocardial contractility is a proportionality constant between CVP pressure and stroke volume (the volume of blood pumped with each beat of the heart).CVP * CNT ≈ SV    ...(2)Mathematically, CNT must therefore have the units of volume / pressure. However, contractility is difficult to measure in vivo, and that there are a number of surrogate measures including dp/dt. These can also be used with appropriate scaling factors.That there are no factors affecting the relationship between myocardial fibre length (the true determinant of stroke volume) and central venous pressure . CVP is therefore used as an easily measured index for myocardial fibre length – the assumption is that the two are related using a scaling factor. Left ventricular end diastolic pressure is also as an alternative index when data are presented as percent change from baseline.The second relationship is that between stroke volume and heart rate to give the cardiac output (the volume of blood pumped by the heart per unit time):SV * HR = CO    ...(3)The last relationship is that between cardiac output and systemic vascular resistance to approximate the mean arterial blood pressure.CO/SVR ≈ MAP    ...(4)This is because MAP usually greatly exceeds the CVP:CO/SVR = MAP-CVP    ...(5)-1 min.SVR therefore has the units of pressure over flow. In this paper, the resistance units (RU) are therefore mmHg LThe effect of a drug on one or two variables can usually be summarised on a plot of the variable (drug effect) against time. However, it is more difficult to summarise the dynamic effect of a drug on the cardiovascular system for the following reasons: First, the large number of variables required in the summary, where the seven described above could be considered a minimum. Second, the fixed inter-relationships (e.g. Eqns. 2–4) between these variables that should be revealed by the summary ). Third, usually an analysis requires comparing one cardiovascular state (e.g. pre-drug) with another (e.g. post-drug), or examining the time-course of drug effects.It is proposed that a "cardiovascular radar plot" with a modified logarithmic, normalised scale is an efficient means of limiting these problems. An example radar plot is shown and described in Fig. There are many published models of the cardiovascular system of various levels of complexity and intended for various tasks . HoweverIn vivo, the cardiovascular system has two major control systems; control of cardiac output via the Frank-Starling mechanism, and control of blood pressure via baroreceptor control of heart rate. These were added progressively to the model.A simple model of the Frank-Starling mechanism was developed Fig.  assumingPressure = Volume/Compliance    ...(6)The solution to the simple Frank-Starling model can be found algebraically, but for consistency is shown in a, CPLv) were chosen to duplicate the following behaviour  as the ratio of arterial compliance to venous compliance (Cratio).Central to the Frank-Starling model is the concept of cardiac function curves – usually given as the pressure in each pool as cardiac function (contractility) is increased from zero to a normal value. These curves are useful for finding appropriate initial estimates for blood volume, arterial and venous compliance, and systemic vascular resistance. To achieve the physiologically plausible cardiac function curve shown in Fig. our Fig.  which isThe control of arterial blood pressure via baroreceptor control of heart rate was added to the simple Frank-Starling model, as shown in Fig. v0 and Va0, see Fig. The final version of the model introduced a number of constraints to increase its physiological plausibility. These were: 1) Assuming that under baseline conditions that approximately 1/3 of the total blood pool is in the arterial system. 2) That the intercepts of the pressure-volume "curves" for the venous and arterial compartments were linear such that both curves gave the mean circulatory pressure (MCP) at the unstressed volumes (VHR = MAPset + MAPdelta*(HRgain*S1)    ...(7)The resultant cardiac function curves for this model are shown in Fig. For convenience, the target cardiovascular variables of the final constrained model discussed above are summarised in Table The sensitivity of the baseline cardio-vascular model to changes in parameter values was determined via Monte-Carlo simulation . Multi-vv, CPLratio, CNT, S1 and S2. Vblood could also be considered a primary variable, but it was considered unlikely that magnesium could change the blood volume. The remaining cardiovascular variables were considered secondary in that they would change in response to changes in the primary variables as given by Eqns 2 to 4.Changes in cardiovascular variables with the administration of magnesium were analysed as percentage change from baseline. This removed the contribution of inter-animal variability in baseline cardiovascular variables (which was nevertheless minor ,9) to vaInitially, the only primary parameter fitted to the data for each time point was SVR while the other parameters were held constant. This was based on the prior knowledge that this was the primary mechanism of action of magnesium. If the MSC was low and the cardiovascular radar plot showed a poor fit between model predictions and the data, an additional parameter was fitted one at a time from the remaining parameters listed above. A parameter was removed from the fit if it produced an undefined estimate. The parameter was kept in the fit if it improved the MSC and the fidelity of observed vs. predicted plots on the cardiovascular radar. By this process, the values of the primary cardiovascular parameter at each key time point required to describe the observed data were determined.Conventional mamillary pharmacokinetic models are essentially empirical and do not include parameters (other than clearance) that represent defined physiological processes. This is problematic when drugs affect the cardiovascular system, or it is necessary to predict the kinetics of the drug when the underlying physiology has changed. This was the case for magnesium, which affected cardiac output significantly Figs.  &10. FulThe magnesium concentration data from Data set 1 were used to develop a recirculatory model of magnesium kinetics that could account for the observed cardiac output changes. The processed used was similar to that described by the authors for other drugs . The finKey points during the model development process were: 1) The representation of the lungs as a single compartment. 2) The representation of the cardiac output change as an empirical forcing function  and the concentrations of magnesium in arterial and coronary sinus blood were examined using hysteresis plots (effect vs. concentration). A concentration-effect relationship was considered plausible if produced a predictable relationship with minimal hysteresis that was consistent with the known mechanisms of action of the drug.By these criteria, it was found that the arterial concentrations were the better predictor of the fitted cardiovascular parameters shown in Table art < 2.66 thenif CSVR = 17elseart + 21.68    ...(9)SVR = -1.759*Cv). This was related to the arterial concentration using a simple threshold CPL-1 . This suggests that specifying the value for one of these parameters significantly constrains the value that can be taken for the other, as would be expected on physiological grounds. It can be concluded that each parameter had an important role to play in the model, and that each could only take a limited range of values to be consistent with the required baseline physiology. By extension, the assumptions regarding the values of these parameters are likely to be appropriate. Furthermore, the changes in these parameters observed following magnesium administration therefore reflect the effects of this drug rather than uncertainty in the parameter space of the model.The baseline cardiovascular variables and the parameters that produced them are summarised in Tables The method of estimating cardiovascular model parameters from cardiovascular data for individual time points was effective. Thus, it was possible to find a parameter set at each time point Table  that proThe recirculatory pharmacokinetic model was able to fit the observed concentrations with adequate fidelity Fig. , middle A feature of recirculatory pharmacokinetic models is that their initial kinetics are governed by first-pass passage of drug through the lungs, and the dilution of the injected drug with the cardiac output . The carRelating the estimated cardiovascular parameters in Table The recirculatory model of magnesium disposition was able to accurately predict the time-course of the arterial magnesium concentrations observed for the validation Data set 2, despite the large change in cardiac output produced by magnesium Fig. . The meaThe final pharmacodynamic model was able to accurately predict the time-course of the cardiac output changes observed for the validation Data set 2 Fig. . The meaThe dynamic model captured the general trend of the mean arterial blood pressure for the validation data Fig. , but somIn this paper, all cardiovascular effects were related to the arterial concentration of magnesium. As covered in the introduction, there may be other sites in the body that have a theoretical claim to being the most appropriate link concentration for certain cardiovascular dynamic effects. For example, the reductions in myocardial contractility caused by thiopental have been shown to have a better temporal relationship to the thiopental concentrations in the myocardium itself rather than in arterial blood . This coIn recirculatory models, it is possible to add a "target organ" to represent organs such as the heart . The facThere are a number of limitations of this modelling approach, many of which are inherent in the assumptions made in the construction of the model. Other limitations may become apparent if the model is used outside of the range of the data used to develop the model. For example, the CL term in the kinetic model was very low Table . This maThe cardiovascular model also assumes an instantaneous baroreceptor response. While it is relatively easy (in modelling terms) to add a delay to this response, this was not supported by the data. However, if the model is extended to situations with very rapid blood pressure changes (e.g. orthostatic hypotension) this deficiency may become significant.Constructing physiologically based models, even of the simplicity presented here, requires crossing many decisions points where a choice must be made from multiple options – sometimes the choices are data driven, sometimes theory driven, sometimes the subjective experience of the model maker must be called upon. While a "wrong" model is evident because it does no match the data, there is clearly no "right" model of the cardiovascular system. It is anticipated that more limitations of the cardiovascular dynamic model will become apparent when model is rigorously compared to data for other drugs, and for other cardiovascular scenarios. It is should be expected that the model will continue to evolve as these data are collected and analysed.The combination of the recirculatory kinetic model and the simple cardiovascular dynamic model was able to describe and predict the concentrations and cardiovascular effects of magnesium in sheep. It is proposed that the general methods used here could be applied to other drugs with cardiovascular effects. The authors are currently applying the method to intravenous anaesthetics.Cardiovascular term DescriptionFrank-Starling modelblood Blood volumeVv Central venous pressureCVP = Pa Mean arterial pressureMAP = PMCP Mean circulatory pressurea Volume of blood in arterial compartmentVv Volume of blood in venous compartmentVa0 Volume of blood in arterial compartment at MCPVv0 Volume of blood in venous compartment at MCPVa Arterial complianceCPLv Venous complianceCPLratio Ratio of venous over arterial complianceCPLSVR Systemic vascular resistanceCO Cardiac outputL Cardiac output (left side)COR Cardiac output (right side)COHR Heart rateSV Stroke volumekc unit conversion factor – contractilityCNT Contractilityadditional for Frank-Starling-Baroreceptor modelMAPset Mean arterial pressure set pointHRgain Gain for heart rate controladditional for Constrained-Frank-Starling-Baroreceptor modelaS Pressure in arterial compartment when stressedPvS Pressure in venous compartment when stressedPaS Volume in arterial compartment when stressedVvS Volume in venous compartment when stressedVslopeMAP slope for arterial pressure-volume relationshipintMAP intercept for arterial pressure-volume relationshipslopeCVP slope for venous pressure-volume relationshipintCVP intercept for venous pressure-volume relationshipS1 Sympathetic tone coefficient – ChronotropyS2 Sympathetic tone coefficient – Contractilitymax maximum for stroke volume-CVP relationshipSV50 half-volume for stroke volume-CVP relationshipSVnSV "Hill factor" for stroke volume-CVP relationshipPharmacokinetic term Description DescriptionRecirculatory model0 doserate of zero order infusionRtau duration of zero order infusionart Arterial magnesium concentration Cven Venous magnesium concentration Cart,e Arterial magnesium concentration (endogenous)Cven,e Venous magnesium concentration (endogenous)Cart,x Arterial magnesium concentration (exogenous)Cven,x Venous magnesium concentration (exogenous)Clung Apparent distribution volume of the lungVCL Clearancebody Apparent distribution volume of the body compartmentVPS Permeability-surface area product of deep compartmentdeep Apparent distribution volume of the deep compartmentVRNU participated in the original magnesium studies and performed the modelling. GLL participated in the original magnesium studies and acted as a resource for cardiovascular theory. Both authors contributed to the manuscript.The simple Frank-Starling model. The simple Frank-Starling model written in pseudo-code to generate cardiac function curves. The code is intended to run in the "Scientist" differential equation solving program.Click here for fileThe Frank-Starling-Baroreceptor model. The equations for the Frank-Starling-Baroreceptor model with baroreceptor control written in pseudo-code to generate cardiac function curves.Click here for fileThe Constrained Frank-Starling-Baroreceptor model. The equations used for the final cardiovascular model written in pseudo-code to generate cardiac function curves.Click here for fileThe final pharmacokinetic-pharmacodynamic model. The equations used for the final model written in pseudo-code.Click here for file