Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed.Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections.Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 . Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases.Considerable variation of The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance. With the emergence of artemisinin resistance in Western Cambodia and along its international border with Thailand as well as further afield in Vietnam and along Thailand’s western border with Myanmar, there is a public health mandate to characterize the parasite populations responsible for poor clinical outcomes, as well as better understand the dynamics of how these parasite clones co-exist -3. Gene Plasmodium falciparum parasites, has become well established both to describe variability in alleles within a population of parasites, as well as to distinguish recrudescence from new infection in cases of recurrent disease [msp1 and msp2) and the glutamate-rich protein (glurp) [msp2 gene can be similarly grouped into two allelic families, FC27 and IC3D7 [glurp gene [The use of polymerase chain reaction (PCR) genotyping around a limited number of polymorphic genes, which are known to be highly variable in  disease -8. This  (glurp) . This me (glurp) . The usend IC3D7 ,12. Onlyurp gene .P. falciparum malaria. The trial was conducted in a rural district of Western Cambodia, close to the international border with Thailand and where reports of AS treatment failures had already emerged [msp1, msp2 and glurp in this Cambodian study population, and (2) to assess whether conventional classification of recrudescence/re-infection based on samples collected only at baseline and day of failure (Df) remained valid once interim sampling was performed. There were two working hypotheses. Firstly, that AS-resistant patients were infected with novel subpopulations of relatively drug-resistant parasites not identified at baseline; as the majority of more sensitive parasites were killed, the resistant parasites would persist and become detectable during a seven-day course of AS therapy. Secondly, that new falciparum infections during follow-up had persistence of these intrinsically more resistant parasite subpopulations. Hence, these cases should be reclassified as recrudescent, making the true failure rate higher with longitudinal genotyping compared to the conventional time points of baseline and Df.The present analysis was conducted as part of a clinical trial of artesunate (AS) monotherapy designed to probe for and characterize clinical artemisinin resistance in adult patients with uncomplicated  emerged . The maiP. falciparum malaria were randomized to receive one of three AS monotherapy regimens given as a single daily oral dose: 2, 4 or 6 mg/kg/day  for seven days. Inclusion criteria were age 18 to 65 years, fever or history of fever within 48 hours of presentation, mono-infection with P. falciparum as determined on Giemsa-stained thick and thin blood films by light microscopy, and parasite density 1,000-200,000 asexual parasites/ml blood. Exclusion criteria were signs and symptoms of severe malaria and documented use of anti-malarial drugs within the preceding 30 days. All patients remained on the study ward for the first week until completion of AS administration and returned for weekly outpatient review until Day 42. Patient outcomes for the main treatment study were classified according to clinical and parasitological responses [The study was part of a randomized clinical trial conducted during 2008-2009 at Tasanh Health Center located in Battambang Province in Western Cambodia, a region of low malaria transmission, with just one third of the study population reporting one or more previous episodes of malaria. Full details of the study are described elsewhere . Brieflyesponses . PrimaryP. falciparum throughout the follow up period (n = 14); (Group C) “Recurrence” . Groups B and C when combined were termed “artemisinin resistant”. Group D constituted the remaining patients not included in A, B or C. For the purposes of the present analysis, enrolled patients developing Plasmodium vivax mono-infection during follow-up and confirmed by vivax-specific PCR were considered cured of P. falciparum infection.For the longitudinal genotyping analysis conducted over the first seven days (Days 0 to 6), three subgroups of patients were selected and defined as follows: (Group A) “artemisinin sensitive”  (n = 18); (Group B) “slow parasite clearance” (parasite clearance time >96 hours by microscopy and remained cured of f) if malaria recurrence occurred. All samples were transferred to cryovials, stored in liquid nitrogen at the study site for transfer to Bangkok, and then kept frozen at -80°C until used for DNA extraction.Prior to initiation of AS therapy (Day 0), blood for parasite density, species confirmation and genotyping was collected into EDTA. Further samples were collected daily on Days 1 to 6 and again on the day of treatment failure (Dmsp1 (block 2), msp2 (block 3), and glurp (R2 repeat region) were amplified by nested PCR. In the primary reaction, the oligonucleotide primers corresponded to conserved sequences within msp1 (block 2), msp2 (block 3) and glurp, and in the nested reaction, separate primer pairs targeted the respective allelic types of msp1  and msp2 (FC27 and IC3D7) for amplification [msp1, msp2 and glurp PCR products were loaded on 2% agarose gels, stained with ethidium bromide, separated by electrophoresis and visualized under UV trans-illumination . Samples from an individual patient were run in adjacent lanes. If there was no amplification for any allelic family, the PCR was repeated with three times the quantity of template DNA. If no amplification was detected after this second reaction, amplification was classified as unsuccessful. Analyses of number of genotypes and size polymorphism were digitalized using Quantity One® software . A minimum of ten base pairs size difference was required to define an additional genotype.Genomic DNA was extracted from 200 μl whole blood per patient using the Qiagen DNA extraction kit  according to manufacturer’s instructions. The polymorphic regions of the merozoite surface proteins fication . For conmsp1, msp2 and glurp at baseline and at Df was used to classify parasitological outcomes for LTF cases. Genotyping was done stepwise with an initial msp2 determination, followed by msp1 genotyping of the paired blood samples found to have at least one identical msp2 allele before and after treatment. Based on analysis of the 3D7 strain, alleles were considered the same if molecular weights were within ten base pairs (bp) [Comparison of genotyping patterns of irs (bp) . It was irs (bp) . If a suAllelic variants were analysed only with respect to the size of polymorphisms given the high level of discrimination afforded by the Quantity One® software computer analysis.Continuous data were expressed as medians with interquartile ranges or, in the case of parasite counts, as geometric means with 95% confidence intervals (95% CI). Continuous data from patient groups were compared using the non-parametric Kruskall-Wallis test. Numerical data were expressed as proportions and compared using Chi-square or Fisher’s exact tests as appropriate. All statistical tests were performed at the 5% significance level and corresponding 95% confidence intervals were estimated. Statistical analyses were performed using SPSS version 12.0  and Stata version 11 .msp1 125, msp2 137 and glurp 143). Four patients were subsequently determined not to meet all treatment study entry criteria, but genotype samples from these cases were retained for this molecular analysis. Some 139 patients were randomized to one of three oral AS monotherapy regimens; three were subsequently lost to follow-up while the remaining 136 were followed until a study endpoint was met. One case re-presented with recurrence 56 days after enrolment into the treatment study and was included in this analysis; another developed neutropenia on D3 and was withdrawn from the treatment study but retained for safety follow up and developed malaria recurrence at D42. Baseline characteristics and clinical/parasitological outcomes for patients are given in Table f).PCR genotyping was successful for one or more genes in baseline samples from 143 patients who had undergone screening procedures prior to study enrolment  than msp1  and glurp  , but not in the AS-resistant subgroup.Some 13 distinct patterns of genotype (haplotypes) were observed at baseline Tables . Recurremsp1, msp2 or glurp alleles present  msp2 and 8% (12/143) glurp samples, respectively. The maximum number of clones seen for a single gene was six (one case with six clear bands of msp2-FC27 in addition to two clones of msp1-K1); this case remained cured at 42 days but had a prolonged parasite clearance time of 102 hours. All other polyclonal infections had just two or three clones. In the three subgroups of artemisinin sensitive, slow clearers and recurrences, multiple P. falciparum alleles of msp-1, msp-2 and glurp, were detected in 5/18, 3/14 and 7/9 patients  . Five of nine cases were polyclonal at Df.Overall, 45/143 (31%) of the study population had evidence of a polyclonal infection at baseline with two or more Parasite genotype was evaluated daily on Days 0-6 of AS therapy for individual recurrence cases Figure . In all msp2-3D7) observed on Days 3 and 5, when asexual parasitaemia was still positive by light microscopy; this could represent a minority clone potentially relatively resistant to AS treatment. However, this allele was not detected again at Df. In the remaining eight recurrence cases, while fewer of the alleles present at baseline were detected as parasitaemia cleared, no new alleles emerged.Only one case of recurrence had a new allele  no minority alleles emerged during seven days of treatment.f samples, eight of nine (89%) Plasmodium falciparum recurrences were determined to be recrudescent and one of nine (11%) was a new infection. Longitudinal genotyping on days 1-6 revealed that in six of nine cases no new minority parasite populations were detected during treatment, thus supporting the classification of recrudescence, although not all alleles present at baseline were detected at the time of recurrence. In one case (participant 140) described above, minority alleles were observed on Days 3 and 5 of AS treatment but not on Df. and thus the classification remained as recrudescence. One case (88) was also classified as recrudescent based on the appearance of at least one allele present in the baseline sample, as well as novel alleles at Df. Case 112 was classified as a new infection based on the appearance of novel alleles at the time of recurrence, none of which were observed during the first week. Thus in all nine cases of malaria recurrence, additional genotyping during AS treatment on days 1-6 did not alter classification of the final parasitological outcome.Based on conventional genotyping methodology using only baseline and Df, so did not appear to be associated with subsequent treatment failure. Two of the 23 cases had novel alleles present at Df but these had not been detected at all during AS therapy despite the sensitivity of the molecular methods used. In this Cambodian setting, where most enrolled patients had persistence of asexual parasitaemia beyond 72 hours, the parasites responsible for slow parasite clearance and recurrent malaria are the dominant or co-dominant clones in an acute infection. Further, because they are also present in fast parasite clearers, they are well established in the Tasanh parasite population. These data also support recent reports of highly conserved, yet genotypically very distinct, populations of parasites from a number of sites in Cambodia including this study site [This study aimed to detect emerging drug-resistant minority parasite clones during slowly clearing malaria infections that could account for cases of unrecognized treatment failure in a setting of clinical AS resistance. In fact, of the 23 cases in the artemisinin-resistant subgroup in whom daily parasite genotyping was undertaken for seven days, including nine cases with recurrent disease, a minority clone was detected in only one; moreover this clone was not present at Dudy site .P. falciparum parasites was revealed in baseline samples from the symptomatic patient population. In keeping with surveys from West Africa and Thailand, these Cambodian isolates showed greater polymorphism of the msp2 allelic family (IC3D7 and FC27) compared to msp1 and glurp[msp1, msp2 and glurp in a relatively isolated area of Western Cambodia are similar to those from directly across the border in Trat Province, Thailand [In this setting of low malaria transmission and emerging drug resistance, considerable heterogeneity within the and glurp,16. ThesThailand . Comparamsp1, msp2 and glurp within a single sample, was found in just 31% of subjects overall at baseline . This suggests a relatively low diversity of the P. falciparum parasite population in this localized area of Western Cambodia. A study from Kampala, Uganda, also a relatively low transmission area, found 45/98 (46%) samples were polyclonal for msp2 at baseline [msp2 clones detected [msp1 32% of 45 symptomatic and 64% of 45 asymptomatic infections in a hyperendemic region of Gabon had polyclonality of msp1[Evidence of polyclonal infection, in the form of the presence of two or more alleles from detected . For mspy of msp1. An intrIn the present study, six clones were demonstrated in one baseline sample, although only two or three clones were detected in the remaining 44 patients with polyclonal infections. In general, studies from high transmission areas have shown that asymptomatic malaria patients harbour more parasite clones than those with symptomatic disease . The samP. falciparum parasitaemia (Df), which occurred between 21 and 58 days after commencing AS. Genotyping patterns from seven of nine treatment failure cases showed multiple parasite alleles of msp1 and/or msp2 at baseline, although only a minority were then detected during the following days of AS treatment. This may be due to greater sensitivity of some parasite strains to AS treatment, as well as to declining parasitaemia in the small volume of sampled blood, or to a combination of both. An alternative explanation for the disappearance of some parasite strains, which later re-appear at the time of treatment failure, is parasite dormancy triggered by the presence of AS [Additional daily samples were genotyped during seven days of AS treatment (Days 0 to 6) as well as on the day of recurrent ce of AS ,25.P. falciparum malaria in France revealed 19/20 (95%) cases harboured between two and five clones, with some cases having up to three dominant clones in addition to other minority clones [msp1 and msp2 detected at baseline [However, repeated genotyping of symptomatic travellers returning from Africa before and during 96 hours of quinine treatment for y clones . Moreovebaseline . This laP. falciparum populations undergoing selection or modification in the presence of drug. Parasite DNA is detectable in peripheral blood for less than 48 hours following the death of a parasite in response to drug or immune mechanisms and before being removed from the circulation by circulating phagocytes and spleen macrophages [versus 10 parasites/μL [f. Therefore the quantification results are unlikely to be influenced significantly by the presence of circulating gametocytes. Using current PCR strategies, gametocytes can only be detected differentially by reverse transcription-PCR (RT-PCR) [A striking feature in patients undergoing longitudinal sampling over seven days was the presence of parasite DNA up to seven days after the initiation of AS therapy; this may represent relatively drug-resistant rophages . Therefosites/μL ), an add(RT-PCR) . The abif has been made previously and may be particularly valuable in high transmission settings [P. falciparum genotyping recommends possible use of polyacrylamide gels, capillary electrophoresis, or use of dedicated fragment sizing software (used here), to increase discriminatory power and reduce inconsistencies between clinical trial methodologies [In this study, conventional genotyping assigned the outcome of six of nine samples as recrudescence; additional genotyping on Days 1-6 did not alter this assessment. In two further cases, while the final classification was not altered by additional genotyping the presence of additional bands does illustrate some of the dilemmas inherent in this genotyping technique. Mixed genotype results may profoundly impact estimates of drug resistance if they form a significant proportion of treatment failure cases in clinical trials, and depending on how they are interpreted . If circsettings ,31. Convdologies .P. falciparum would have enabled potential misclassification of outcome to be examined in more detail. Additional emergent minority populations may have been detected if sampling had occurred more frequently than once daily. Band size estimates, while measured by automated reader with as much precision as possible, may also have led to minor inaccuracies. Moreover band size variability between the same alleles can potentially occur, leading to false categorization as different alleles. Analysis of samples in this study was limited to once per day so minority parasite clones with only a sporadic and low-level presence in the peripheral circulation compared to the dominant clone(s) could have been missed.There are several limitations to this analysis. More cases of treatment failure, in particular of new infections with msp1, msp2 and glurp in malaria patients from a localized area of Western Cambodia revealed considerable variation in msp2 alleles but well-conserved msp1 and glurp. Longitudinal genotyping of cases with recurrent malaria during seven days of anti-malarial treatment showed homogeneous genotype dynamics and did not alter the outcome classification compared to conventional genotyping at baseline and Df; however it did serve to illustrate some of the inherent complexities when interpreting the parasitological basis of recurrent disease. In contrast to Africa, where malaria transmission rates are higher and artemisinin resistance does not currently appear problematic, lower polyclonality of infection was observed in malaria patients in Cambodia. The exception was the group of recurrent malaria infections in which polyclonality was much higher. The potential role of polyclonal infections in contributing to the spread of artemisinin resistance deserves further investigation.Limited genotyping of The authors declare that they have no competing interests.DB conceived the study; PG, SS, SW, KP, and ST performed the molecular genetic studies; YS, CL, SS, SD, MF, DS, DB conducted the clinical trial; PG, CL and DB analysed the data; PG, CL, ST, MS, CMC, DW, MF, JM, DS, and DB wrote the manuscript. All authors read and approved the final manuscript.


Strongylocentrotus franciscanus has vastly greater levels of phenotypic and genetic variation for larval size in future CO2 conditions compared to the mussel species Mytilus trossulus. Using these measures we demonstrate that S. franciscanus may have faster evolutionary responses within 50 years of the onset of predicted year-2100 CO2 conditions despite having lower population turnover rates. Our comparisons suggest that information on genetic variation, phenotypic variation, and key demographic parameters, may lend valuable insight into relative evolutionary potentials across a large number of species.The global acidification of the earth's oceans is predicted to impact biodiversity via physiological effects impacting growth, survival, reproduction, and immunology, leading to changes in species abundances and global distributions. However, the degree to which these changes will play out critically depends on the evolutionary rate at which populations will respond to natural selection imposed by ocean acidification, which remains largely unquantified. Here we measure the potential for an evolutionary response to ocean acidification in larval development rate in two coastal invertebrates using a full-factorial breeding design. We show that the sea urchin species  By the end of this century, changes in carbonate chemistry of the open ocean's surface waters are expected to exceed those experienced within the past 20 million years The oceans have absorbed approximately 30% of anthropogenic CO2 measured in present-day populations will apply to future populations, and by necessity overlook the potential for evolutionary change. Within-generation responses to global change may reflect the physiological acclimation capacity, or reaction norms, of individual organisms. However, cross-generational responses, and our understanding of the ecological impacts of climate change in general, depend critically on the extent to which populations will evolve in response to their changing environment Projections of future ecological change, however, hinge on the assumption that responses to elevated CO2. Artificial selection experiments have demonstrated evolutionary responses in a short-lived photosynthetic plant  Saccostrea glomerata resulted in selected lines that, serendipitously, suffered reduced effects of ocean acidification on shell growth compared to nonselected lines, indicating the existence of selectable genetic variation for CO2-dependent growth Some key studies have revealed a potential for adaptation to elevated CO2 conditions within a single generation of two animals of ecological and economic importance. The bay mussel, Mytilus trossulus, directly provides complex habitat for other members of temperate intertidal communities Strongylocentrotus franciscanus, is a grazer of habitat-forming kelps 2 conditions on larval development (under the SRES A2 scenario 2 conditions and (iii) simulate the response of each species to multiple generations of selection at year-2100 CO2 in order to compare the potentials for adaptive evolution in larval traits in either species.In the present study, we use a full-factorial breeding design to estimate the additive genetic component of phenotypic variation in growth under present-day and near-future COM. trossulus were fed with organic particles in the seawater system, and S. franciscanus were fed with macrocystis kelp. Animals were kept and handled in accordance with the animal care guidelines of the center . See Male and female individuals were collected from sites in Barkeley Sound, British Columbia, and transported in vessels containing ambient seawater to the Bamfield Marine Sciences Center within 30 minutes of collection. Animals were maintained in large, covered tanks with flow-through, unfiltered seawater  for 1–2 weeks before use. M. trossulus gametes were obtained from four females and ten males by warm-water spawning induction S. franciscanus, gametes from 10 females and 10 males were obtained by spawning inductions using 0.5 M KCl injection through the peristome M. trossulus from 4 dams and 10 sires, and 100 families of S. franciscanus from 10 dams and 10 sires. In order to ensure that we sampled the effects of both paternal and maternal genetic variation on offspring phenotypes, we cultured larvae through the onset of obligate zygotic transcription in ambient seawater: for 24 hours in M. trossulus or 48 hours in S. franciscanus−1 for M. trossulus, and 1.8 larvae ml−1 for S. franciscanus) with treatment or control seawater (see below). All cultures for each experiment were kept in an incubator at 12°C with fluctuation below 1.5°C, and a 12 h day/night light cycle, and were rotated twice daily within the incubator.Male and female gametes of each species were combined in a factorial manner  and CO2 gas  with Smart-Trak® mass flow controllers  to desired CO2 concentrations. Actual CO2 gas concentrations were verified with a Qubit S151 CO2 Analyzer. Experimental gases were vigorously bubbled into 20 L polyethylene carboys filled with 0.5 µm filtered seawater and allowed to equilibrate for at least 24 h. Carboys were kept at 11–13°C before use. We monitored the pH  of treatment and control water immediately before, during, and at the end of each experiment, using an Omega PHH-830 pH meter. For the M. trossulus experiment, high-CO2 water was equilibrated directly, using a gas mixture of 1000 ppm CO2, whereas for the S. franciscanus experiment, two batches of seawater that had been equilibrated with different CO2-enriched mixed air (800 and 1800 ppm) were combined equally to obtain water with approximately the same pH as that resulting from the 1000 ppm CO2 gas mixture (pH = 7.9). For both experiments, low CO2 treatments were obtained by running outdoor ambient air through the mass flow controllers and into a carboy to equilibrate with filtered seawater, in exactly the same way as the enriched gas (pH = 8.3). The resultant pH values, and differences between treatment and control, were consistent across species . Extra cultures and seawater samples were placed in each box to monitor larval development and pH levels. The effect of common treatment box was minor and accounted for statistically in all analyses of CO2 and parental effects  ambient air bubble in each culture. Five replicate cultures of each CO2 treatment were made for all 100 families . Extra cultures and seawater samples were made to monitor development and pH of treatments. For both M. trossulus and S. franciscanus, the effect of culture jars  was accounted for statistically, thus accounting for differential variation introduced by this difference in experimental technique , and size was measured as the maximum anterior-posterior length parallel to the valve hinge , and size was measured as post-oral arm length, body length, and body width. We used two metrics of size in S. franciscanus: overall larval length  reflect differences in morphogenesis, but not an increase in total organic content.We cultured larvae through to the end of their obligate nonfeeding period of morphogenesis, before the usual onset of feeding. We focused on heritability of growth variation in this early period of development in order to avoid potentially large artifacts of laboratory culture and feeding conditions on phenotypic variation in larval morphogenesis ve hinge . For S. S. franciscanus female were found to be highly affected by CO2 treatment, however this maternal family was also the first to be placed into control and treatment pCO2 conditions. As it was not possible to determine whether the strong effect of CO2 on this female was due to timing of treatment or to maternal effects specific to this female, all families with this female were discarded from further analyses.Families of one 2 and the contribution of parents to the overall variance in larval size, we fitted a mixed-effects linear model with CO2 treatment as a fixed effect, and sire, dam, sire*dam interaction, replicate culture, and treatment box (for M. trossulus), as crossed random effects, using the LME4 package in R To determine the overall effect of CO2 conditions using ASReml ijklL is the size of the lth sampled larva, within the kth incubation jar, from the ith dam and the jth sire; and sire (s), dam (d), sire-dam interaction (i), and culture (c) are random effects on the overall mean size (u). For M. trossulus, treatment box was also included as a random effect.Variance components of sire, dam, and sire*dam interaction were estimated separately for present-day and future CO2) was calculated as the ratio between sire-based genetic variation and total phenotypic variation.Following the sire model, we estimated sire-based additive genetic variance as four times the sire variance component, and phenotypic variance as the sum of sire, dam, sire*dam interaction, and residual variance components (hence culture and treatment box effects were excluded from phenotypic variance) 2), as four times the difference between the dam and the sire variance components, divided by phenotypic variance The full-factorial cross also allows calculation of maternal effects  to obtain an expected mean and variation in larval duration for each species. Alternatively, there could be little or no change in the time to metamorphic competency, and the processes leading to smaller larval sizes in our experiments could result in smaller, less developed, or nutritionally compromised larvae at the time of metamorphosis To estimate the selection differential (S), we assumed that variation in larval size observed within treatments reflects variation in individuals' developmental rates, and is directly related to variation in larval duration required for individuals to become competent to metamorphose in situ observations have been estimated at 13–28% day−1 for bivalves and 6–26% day−1 for sea urchins 2 (30 days for M. trossulus and 50 days for S. franciscanus). The resultant fitness functions are shown in We produced a fitness function for larval development time based on per-day mortality rates in the plankton due to predation, advection from suitable habitats, starvation, and disease We computed a selection differential (S) from the difference in mean development time before and after the fitness function was applied on a simulated population of larvae.M. trossulus to contribute 30, 50, or 90% of individuals to the next years' breeders, based on previous demographic observations in the focal region S. franciscanus to contribute 10, 30, and 50% of individuals to the breeders 4 years post-metamorphosis, based on estimated demographic parameters In a species with overlapping generations, new recruits become sexually mature and join an existing population of surviving mature adults from previous years. To account for overlapping generations in our simulations, we included a population turnover rate, defined as the mean proportion of a breeding population made up of new recruits in a given year. Hence, the response to selection for each year was a weighted average of phenotypes from the existing population of surviving mature adults and the new recruits. To explore the sensitivity of model outcomes to different breeder turnover rates expected for each species, we allowed surviving larvae of M. trossulus (generation time of 1 year), and 45 overlapping generations in S. franciscanus (generation time of 5 years). We explored results using the range of heritabilities (h2) corresponding to maternal effects between zero and 100% heritable.To characterize the rate of adaptation, we iterated the breeder's equation with this breeder turnover rate for 50 years of evolution. This allowed 49 overlapping generations in 2 levels projected for year 2100 (under a SRES A2 scenario Mytilus trossulus and Strongylocentrotus franciscanus larvae (2 was similar between species: a 2.0% (S. franciscanus) to 3.2% (M. trossulus) decrease in larval body length (2 treatment (Atmospheric COs larvae . The effy length . Includireatment .2) of larval size at age were low but non-zero under low pCO2, while at high pCO2 heritability was zero in M. trossulus, and low but non-zero in S. franciscanus (2) were high for both species under both conditions  were similar under the two treatments , two species with the same heritability can have different responses if one species has greater absolute amounts of phenotypic variation on which selection can act.While the maternal effects tend to reduce the precision of heritability estimates and of the absolute rate of evolution, they do not greatly confound the comparison of relative rates of evolution between the two species. M. trossulus tend to form ephemeral populations in disturbed habitat patches associated with its larger conspecific, M. californianus, and a large proportion of breeders are thought to be replaced every year S. franciscanus have longer life-spans of many decades, with a much smaller proportion of adults replaced annually by new recruits M. trossulus (0.9) did both species have a similar rate of evolution. Accurate estimation of breeder replacement is therefore an important aspect of predicting relative rates of evolution under climate change.Evolution can occur more rapidly if more offspring surviving selection become incorporated into a population every year. In our simulations, population turnover rate had a direct effect on the rate of evolution, so uncertainty about the magnitude of this parameter in nature will lead to uncertainty in predictions about relative rates of evolution in natural populations. Population turnover rates are likely to differ greatly among marine species with vastly different reproductive modes, life histories, and demographic processes. In the northeast Pacific, individuals of 2 reflects variation in individual developmental rates, and (ii) that slower rates of development have a direct fitness cost due to greater mortality in the plankton. Support for the slower-rate assumption comes from accumulating observations of longer developmental times in planktotrophic larvae in acidified conditions Mytilus edulis2 will benefit from directly measuring fitness traits , or measuring entire larval durations Another important parameter is the fitness function associated with variation in the traits affected by ocean acidification. We based our general fitness function on two assumptions: (i) that variation in size-at-day at high CO2 responses has been found in the three previous studies in which it has been sought S. franciscanus and M. trossulus was apparent despite the imprecision of heritability estimates, because of the large differences in phenotypic variation. Given the need to anticipate the impacts of global change rapidly on a wide range of species, estimates of phenotypic variation, particularly of fitness traits, may prove valuable for identifying species with greater or less potential for adaptation. While estimates of genetic variation require complex mating or multiple-generational designs, we suggest that biologists report the inter-individual variation in ocean acidification impact studies, so that at least phenotypic variation can be estimated across a broad range of species.Taken together, our comparisons suggest that knowledge of among-species differences in (i) the scale of phenotypic variation, (ii) population turnover rates, and (iii) the fitness cost associated with trait variation may be as important as knowledge of heritability itself in predicting the evolutionary responses to ocean acidification. A genetic basis for variation in COWe have demonstrated how variation in individual responses to ocean acidification can be used to understand species-specific differences in evolutionary potentials. The larvae generated for use in ocean acidification experiments lend themselves to such analyses, and require  that larvae of different parents be raised separately, that parental information be retained for every individual, and that adequate replication is made within families to estimate the environmental source of variation. Steps to reduce the signal of maternal effects will be valuable, such as quantification of egg traits prior to fertilization, and raising larvae for longer time periods to reduce the effects of variation in egg provisioning. Improved estimates of evolutionary rates will benefit not only from further empirical support for fitness functions and demographic population turnover rates, but also consideration of (i) the role of genetic correlations between responses to different variables (particularly with regards to pH and temperature) or between life stages Methods S1(DOC)Click here for additional data file.Table S12 treatment on length variation in Effect of COM. trossulus and S. franciscanus. Akaike Information Criterion (AIC) comparison indicates that top models include maternal and paternal sources as random effects.(DOCX)Click here for additional data file.Table S2Phenotypic variance, additive genetic variance, heritability and maternal effects for larval size in M. trossulus and S. franciscanus2 treatments (± SEM). within CO(DOCX)Click here for additional data file.Figure S12 conditions in Variation in size-at-day under low and high COS. franciscanus, using the first principle component (PC1) of three skeletal rod lengths. (A) S. franciscanus larva at 7 days of development. Lines indicate length measurements for body length (l), post-oral arm (a), and transvers body rod (t) used in principle components analysis. Scale bar is 150 µm.  Variation in S. franciscanus PC1 after 5 days in ambient CO2 (light grey bars) and elevated CO2 (dark gray bars). Vertical bars indicate ±1 standard deviation from the mean. The same data are shown partitioned among (B) sires and (C) dams. Horizontal lines represent means for ambient (dashed), and high (solid) CO2 treatments. Heritability calculated from sire-based additive genetic variance, and maternal effects indicating variance attributed to dams over-and-above sire-based heritability, are given for each treatment . PC1 loadings were: a = 0.51, b = 0.58, c = 0.63, all loading with the same sign, and representing 0.53 of the variance.(TIF)Click here for additional data file.Figure S2Hypothesized fitness functions for development time used in evolutionary simulations of (A) Mytilus trossulus and (B) Strongylocentrotus franciscanus. Red lines represent the loss of fitness with greater development time due to a constant daily mortality rate in the plankton of 15% day−1, blue lines represent the loss of fitness expected with extremely rapid (short) development times, and black lines represent the resulting fitness function, the product of the red and blue fitness functions. The shape of the blue fitness function was arbitrarily chosen such that the equilibrium of the resultant fitness function (black) would center on the development time under low CO2 for each species.(TIF)Click here for additional data file.Figure S32 treatments during experiments, for (A) Separations in pH values between low (blue) and high (red) COM. trossulus and (B) S. franciscanus. Error bars indicate standard deviation. Although among-culture variance in pH was greater in the S. franciscanus experiment, variance in developmental responses attributed to culture jar was not included in calculation of genetic or phenotypic variance Click here for additional data file.Figure S42 treatment on Effect of COMytilus trossulus (A) and Strongylocentrotus franciscanus (B) larval size. Mean size from each culture is shown in grey, and data aggregated by sire and dam combination (family mean) are shown in black. Segments connect family means at high and low CO2 treatments.(TIF)Click here for additional data file.


To decrease the risk of postoperative complication, improving general and pulmonary conditioning preoperatively should be considered essential for patients scheduled to undergo lung surgery.The aim of this study is to develop a short-term beneficial program of preoperative pulmonary rehabilitation for lung cancer patients.From June 2009, comprehensive preoperative pulmonary rehabilitation (CHPR) including intensive nutritional support was performed prospectively using a multidisciplinary team-based approach. Postoperative complication rate and the transitions of pulmonary function in CHPR were compared with historical data of conventional preoperative pulmonary rehabilitation (CVPR) conducted since June 2006. The study population was limited to patients who underwent standard lobectomy.Postoperative complication rate in the CVPR (n = 29) and CHPR (n = 21) were 48.3% and 28.6% (p = 0.2428), respectively. Those in patients with Charlson Comorbidity Index scores ≥2 were 68.8% (n = 16) and 27.3% (n = 11), respectively (p = 0.0341) and those in patients with preoperative risk score in Estimation of Physiologic Ability and Surgical Stress scores >0.3 were 57.9% (n = 19) and 21.4% (n = 14), respectively (p = 0.0362). Vital capacities of pre- and post intervention before surgery in the CHPR group were 2.63±0.65 L and 2.75±0.63 L (p = 0.0043), respectively; however, their transition in the CVPR group was not statistically significant (p = 0.6815). Forced expiratory volumes in one second of pre- and post intervention before surgery in the CHPR group were 1.73±0.46 L and 1.87±0.46 L (p = 0.0012), respectively; however, their transition in the CVPR group was not statistically significant (p = 0.6424).CHPR appeared to be a beneficial and effective short-term preoperative rehabilitation protocol, especially in patients with poor preoperative conditions. Notwithstanding advances in management, complete anatomical resection remains the most effective treatment in patients with early stage lung cancer. According to various guidelines, acceptable mortality following resection varies between 4% and 7% for lobectomy and between 8% and 14% for pneumonectomy TM; Tsumura Co., Tokyo, Japan) have been recognized as beneficial supplements, and the effects of such medications on COPD have also been evaluated in terms of quality of life Branched-chain amino acids (BCAAs) are among the nine essential amino acids for humans, accounting for 35% of the essential amino acids in muscle proteins and 40% of the preformed amino acids required by mammals Although several previous studies demonstrated that well-planned short-term preoperative physiotherapy decreased the incidence of postoperative pulmonary complications, we hypothesized that pulmonary rehabilitation with intensive nutritional support could be a beneficial tool in improving the general conditions of lung cancer patients undergoing surgery.To establish a more enforceable and beneficial program for outpatients, we developed a comprehensive pulmonary rehabilitation (CHPR) protocol, which consists of physical exercise and intensive nutritional support with BCAAs and herbal medication, performed using multidisciplinary team approach. In this study, we analyzed the clinical effect of the CHPR protocol on the outcome of lung cancer patients who underwent standard lobectomy after pulmonary rehabilitation in our hospital.We started conventional preoperative pulmonary rehabilitation (CVPR) in 2006. CHPR using a multidisciplinary team approach was conducted prospectively from June 2009. Spirometric pulmonary function tests  were used to preoperatively assess the baseline severity of the underlying lung conditions. The inclusion criteria for this study were as follows: (1) age >70 years; (2) Vital Capacity (VC)/ideal VC (%VC) <80%; and (3) forced expiratory volume in one second (FEV1)/forced VC (FEV1%) <70%. Patients that met at least one of these criteria were registered in this study from June 2006 to June 2011. Other eligibility requirements included an Eastern Cooperative Oncology Group performance status of 0–1. Exclusion criteria included the presence of an actively treated malignancy or treatment of another malignancy within the past 1 year, presence of a metastatic disease, unstable cardiac disease, and cognitive impairment. All registered patients underwent pulmonary resection after preoperative pulmonary rehabilitation. In CVPR, 48 patients underwent lung resection, and 29 of them underwent standard lobectomy. Nineteen patients in CVPR were treated with limited resection (wedge resection or segmentectomy) mainly because their predicted postoperative FEV1 was less than 800 ml or they had peripheral small-sized ground-glass opacities on computed tomography imaging. On the other hand, 46 patients underwent surgery after CHPR, and 21 of them underwent standard lobectomy for their lung tumors. Twenty-five were treated by limited resection for the abovementioned reasons for the patients in CVPR. Since the amount of resected lung parenchyma itself strongly influenced postoperative outcomes, eligibility for analysis in this study was limited to patients who underwent standard lobectomy for histologically confirmed stage I–IIIA NSCLC. Therefore, the study population consisted of 29 patients in the CVPR group and 21 patients in the CHPR group . Data frThe demographic data and clinical features of the enrolled patients were collected from the institutional database. Written informed consent was obtained from all enrolled patients, and the protocol of this study was approved by National Hospital Organization Kure Medical Center and Chugoku Cancer Center Institutional Review Board Ethics Committee .TM, which is a herbal medicine and composed of 10 nature remedies, were administered daily from the initiation of physical training to discharge after surgery. The daily amount of BCAA supplementation was 6.2 g, which consisted of two packs of supplement . Registered dieticians chose one of these supplements mainly on the basis of the status of dietary intake, because the amount of total calorie in each of them was different. Multidisciplinary team approach was also accomplished by discussing each patient's conditioning during periodical conferences attended by surgeons, physical therapists, dieticians, and nurses. CVPR consisted only of conventional physical training directed by physical therapists at least once a week preoperatively. Basically, conventional physiotherapy mainly focused on muscle training exercises for improving the activity of daily life. However, there were no apparent differences in the CHPR and CVPR physical therapy programs, except for the minimal required times of hospital appointments. Although the intensity level was usually determined using the Borg scale and the perceived exertion of 13 was the goal intensity level, this level and the quality of physical therapy seemed to have gradually changed over the time period of this study. With the exception of the preoperative pulmonary rehabilitation protocol, standard perioperative management and medications did not changed throughout the study period. Five patients in CHPR were diagnosed with COPD concomitant with lung cancer, and 3 of 5 patients initiated inhalation therapy  simultaneously with the CHPR protocol. In the CHPR program, assessment and planning were performed through team conferences and multiple sessions of counseling conducted for each patient. The participants in both groups received uniform, standard postoperative respiratory care and physical therapy until discharge.In the CHPR group, the patients were trained to master adequate breathing and coughing techniques, instructed on incentive respiratory exercise, and practiced peripheral muscle exercise training including a cycle ergometer, under the direction of registered physical therapists taking hospital appointments at least twice weekly for 2–5 weeks. The differences in durations between the patients were because of various reasons, such as the choice of date of surgery that was decided by the patients' or for institutional convenience. Registered dieticians assessed the daily diet and directed optimized diet therapy for patients at least twice preoperatively. BCAAs and HochuekkitoThe preoperative conditions of the patients were assessed by the Charlson Comorbidity Index (CCI) and Estimation of Physiologic Ability and Surgical Stress (E-PASS) scores  and 3 thIn the literature, various rates of postoperative complication have been presented even in patients with equivalent preoperative conditions. These differences appear to have been because of the varying definitions of postoperative complication in each investigation. In this study, the definition of postoperative complication was clearly established , and medComparisons were performed using JMP for Windows (version 9.0) statistical software package . The results are expressed as means ± standard errors for parameters. Differences in characteristics between CVPR and CHPR were determined using the Mann – Whitney U-test. The transition of pulmonary function after preoperative pulmonary rehabilitation was assessed using a paired t-test. The difference in postoperative complication rate between the two groups was analyzed using Fisher's exact test. Associations among risk factors, postoperative complication, and patient groups were assessed by means of univariate and multivariate logistic regression analyses. Multiple logistic regression analysis was performed using variables found to be p<0.25 by univariate analysis. Differences were considered statistically significant for p<0.05.The mean duration and number of sections in each group were 29.1±8.9 days and 8.4±3.4 times in CHPR, 27.9±7.8 days and 6.8±4.5 in CVPR, respectively. The patient characteristics and clinicopathological features are shown in A statistically significant beneficial effect on pulmonary function (VC and FEV1) was observed in the CHPR group ; however, the transition of those in CVPR was not statistically significant .The impact of preoperative pulmonary rehabilitation on lung cancer patients planning to undergo lung resection needs to be more clearly investigated, with special emphasis on postoperative complication The most frequently advocated obstacle in scheduling preoperative pulmonary rehabilitation for lung cancer patients is the necessity to perform surgical treatment without delay, taking into consideration the potential for malignancies. Recent literature demonstrated that a 10-session preoperative pulmonary rehabilitation may improve postoperative lung re-expansion and decrease the length of hospital stay; however, a 4-week preoperative program proved to be very difficult to apply Nutritional support for improving exercise performance has been recognized as an important intervention, especially for physically deconditioned individuals including those with poor pulmonary function There were several limitations of this study that should be noted when assessing the results. First of all, although the CHPR protocol was adopted prospectively, most of the CVPR data were collected retrospectively. The number of patients included in each cohort was relatively small, and the assignment of the patients to the two groups was not random. It is important to note that data of CVPR and CHPR were collected at predominantly different time periods, although 11 patients in CVPR were registered after starting the CHPR program. In addition, other various factors including exercise capacity, QOL, and nutritional variants need to be evaluated for elucidating potential benefit of the CHPR protocol. Moreover, this study was based on the data of patients at a single institution. Taken together, we demonstrated the clinical benefit of the CHPR protocol with short-term preoperative pulmonary rehabilitation in this study; however, prospective randomized studies should be conducted before such treatment can be recommended in routine clinical practice. At present, a multicenter, prospective feasibility study is being conducted by the study group of national hospital organizations in Japan for evaluating the clinical effect of CHPR via a multidisciplinary team approach.


The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis.RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples.LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution. The intestine is a dose limiting organ during radiation therapy of abdominal cancers and a critical determinant of survival in radiological emergency situations. This is mainly due to the radiosensitivity of its rapidly proliferating crypt epithelium. The intestinal mucosa invariably sustains the most severe injury and undergoes the most change after exposure to ionizing radiation in situ hybridization and immunohistochemistry have provided substantial information about the molecular and cellular events that occur in the bowel mucosa in response to ionizing irradiation. However, for analysis of differential gene expression patterns and pathways regulated during the development of radiation toxicity, more powerful methods such as expression arrays must be used.Methods such as The isolation of RNA from whole tissue is relatively quick, easy, and may reveal important information about differential gene expression patterns. However, the utility of analysis performed on whole gut RNA extracts is questionable because it is hampered both by the structural complexity of the intestine, as well as by the relative change in structure that the mucosa undergoes during the process of development of radiation toxicity. In contrast, RNA isolated by laser capture microdissection (LCM) can be used to isolate relatively pure cell populations or tissue segments of interest from fixed tissue sections, thereby avoiding both of the problems associated with whole tissue RNA extracts LCM is widely used for gene expression analysis in both basic research and clinical applications Here, a well-defined mouse model of total body radiation was used to compare the transcriptional profiles of LCM-separated mucosal and whole gut specimens obtained in parallel. Our results clearly revealed that it is important to use LCM for analysis of pathways related to cellular injury, cell cycle control, and DNA damage repair pathways, all pathways that are central in the radiation response. Only genes where there are substantial differences in expression levels, such as certain apoptotic and cytokine signaling pathways, can be safely analyzed in whole gut RNA extracts.The experimental protocol was reviewed and approved by the University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System's (CAVHS) Institutional Animal Care and Use Committees (IACUC).Experiments were carried out in a total of 40 random-bred male CD2F1 mice , 6–7 weeks old on arrival and with an initial body weight of 22–25 g. Animals were housed in conventional cages under standardized conditions with controlled temperature and humidity and a 12–12-h day-night light cycle. Animals had free access to water and chow .Total body γ irradiation was performed as described before Collection of intestinal mucosa was performed by LCM as shown in Mucosal RNA was extracted from the LCM-dissected jejunum using the RNAqueous-Micro kit  specifically designed to recover small amounts of RNA from LCM samples.Whole gut RNA was extracted from jejunum collected at different time points following irradiation. Tissues were snap frozen in liquid nitrogen and stored at −80°C. Frozen tissue samples were homogenized in Ultraspec RNA reagent , according to the manufacturer's instructions. Two µg of RNA were kept for microarray analysis  and 2 µg were used for real time PCR.DNase-treated RNA was quantitated using the Rediplate 96 Ribogreen RNA quantitation kit .RNA samples from LCM dissected mucosa and whole gut were screened by checking their integrity and genomic DNA contamination. Selected RNA samples were processed for microarray analysis using Illumina one-color MouseWG-6_V2 chip. The sample size for LCM and for whole gut microarray was N = 4/time point.Microarray data was analyzed by Genespring GX 11.0 . Raw data were log2 transformed and then normalized to the 75th percentile of all values on a chip. Student's T-test was used to examine for differentially expressed genes. A list of genes with ≥2.0-fold change was generated first and tested by the Benjamini-Hochberg Multiple Testing Correction procedure. Significant genes were selected by a cut-off of p<0.05 and fold change > = 2.0.The selected genes were subsequently analyzed using IPA 5.0 . Pathways which were predicted to be influenced by the differentially expressed genes were ranked in order of significance and further analyzed by overlaying with function, disease and canonical pathways. An overlap p-value<0.05 indicates a statistically significant, non-random association between a set of genes in the dataset and a related function. The p-value of overlap is calculated by the Fisher's Exact Test.Verification of gene expression was carried out using different sets of total RNA samples from those that were sent for microarray. For each RNA sample, 1 µg was used as the template for cDNA synthesis by using the High Capacity cDNA reverse transcription kit  according to the protocol provided by the manufacturer. Steady-state mRNA levels were measured with real-time quantitative PCR using the following Applied Biosystems predesigned Taqman gene expression assays: Casp14, Mbl2, Mcpt1, Eda2r, CCL-9 and IL-33. PCR amplification and detection were carried out on an ABI prism 7000 Detection System (Applied Biosystems). Transcript levels were normalized to eukaryotic 18S rRNA and calculated relative to control, unirradiated jejunum, using the standard ΔΔCt method.5 µm sections were mounted on glass slides. TUNEL assay was performed using the Dead End fluorometric TUNEL kit  following the manufacturer's instruction. Image acquisition was performed on a Zeiss Imager Z1 Epifluorescence Microscope .5 µm sections were mounted on slides, deparaffinized in xylene, and rehydrated in graded solutions of ethanol. After blocking with 5% normal serum, sections were incubated with primary antibody overnight at 4°C, followed with Alexa Fluor conjugated secondary antibody incubation  for 1 h at room temperature. Slides were mounted with Vectashield Mounting Medium with DAPI for nuclear staining . Image acquisition and intensity measurements were performed on a Zeiss Imager Z1 Epifluorescence Microscope .Data are presented as mean ± standard deviation. Statistical analysis was performed by t-test. p<0.05 was considered to be significant. For the microarray, a list of genes with > = 2-fold change was generated by t-test and filtered by the “Benjamini and Hochberg false discovery test”. Genes with a false discovery of less than 0.05 were used to for the final list of differentially expressed genes. For IPA, the p-value of overlap is calculated by the Fisher's Exact Test.To verify radiation-induced cellular injury/death, TUNEL staining was applied on the intestinal tissue at each time point. The gene list with a cut-off of 2-fold and p-value<0.05 was generated at each time point. In LCM samples, the number of genes that were altered ≥2-fold was 615 at 4 h, 461 at 24 h, and 613 at 3.5 d post radiation. In the whole gut, the number of genes that altered ≥2-fold was 799 at 4 h, 784 at 24 h, and 1624 at 3.5 d post radiation. Venn diagrams  demonstrThe change of selected genes of Casp14, Mbl2, Mcpt1, CCL-9, IL-33 and Eda2r at 4 different time points were validated by real-time RT-PCR using Applied Biosystems predesigned Taqman gene expression assays . The lefThe lists of altered genes were imported into IPA for pathway analysis. In the whole gut, there were no significantly activated cellular injury pathways at 4 and 24 h. At 3.5 d, up-regulation of DNA damage repair was not significant. Instead, there was a down-regulated HIF1α signaling.Corresponding to cellular injury and DNA damage repair, In the whole gut, there was no significantly activated pathway at 4 and 24 h. At 3.5 d, among the highly up-regulated pathways in the mucosa, only BTG was significantly changed .As a rapidly renewing tissue, the intestine undergoes extensive cell death in response to radiation. To study the global impact of gene expression profiles on cell death in mucosa and whole gut, regulation of apoptosis pathways was analyzed by IPA. In whole gut, p53 signaling was significantly up-regulated at both 4 h and 24 h. At 3.5 day, there was wide down regulation of various genes involved in apoptosis such as Calcium-induced T lymphocyte apoptosis signaling, Aryl hydrocarbon receptor signaling, Nur77 signaling in lymphocytes, and CD27 signaling in lymphocytes.Gene microarray revealed that radiation induced a striking increase of casp-14, both in LCM samples from the intestinal mucosa and in samples from the whole gut. For example, while expression of casp14 mRNA was very low under normal conditions in mucosa, it increased 67-fold at 4 h, 33-fold at 24 h and then dropped to baseline levels at 3.5 d . IHC verin vivo cell alterations during disease development. As the intestinal mucosa manifests the most severe injury among the compartments of the small intestinal wall, studying changes in gene expression in the mucosa can provide important information about the mechanisms of radiation injury. Unfortunately, because of the extremely unstable nature of intestinal RNA, preparing RNA from mucosal LCM samples is cumbersome, expensive and the preparation of tissues and process of LCM rather time-consuming. This study used a well-defined mouse model of total body irradiation to perform a rigorous comparison of the transcriptional profiles of parallel LCM-separated mucosal and whole gut specimens at various times after irradiation. We found that radiation induced strong activation of pathways mediating cellular injury, lymphocyte apoptosis, tight junction, cell cycle control and DNA damage repair at different time points in mucosa but, with exception of the p53 pathway, not in the whole gut. These results, which to our knowledge are the first from a systematic, rigorous comparison of LCM and whole tissue RNA extracts, suggest that obtaining mucosal samples is essential for the study of these critical signaling pathways. In addition, we report that the newly identified genes of Casp14 and Eda2r could provide novel research objectives and offer new therapeutic strategies for intestinal radiation injury.LCM is a technique by which pure cell populations or tissue segments of interest can be procured from frozen or fixed tissue sections. Since the first development of LCM in 1996, it has become widely used in the basic research and clinical areas for the study of A single dose (8.0 Gy) of total body γ radiation to CD2F1 mice did not cause significant 30-day lethality but considerable histological damage and, as shown by TUNEL staining coincident with upregulation of CD27 signaling, a wave of apoptosis in mucosa At 24 h post radiation, the most significantly changed pathways were tight junction and p53 signaling pathways. The disrupted tight junction signaling in mucosa could possibly explain a response to the breakdown of the integrity of epithelial tight junction. There was significant up-regulation of p53 signaling both in the mucosa and the whole gut. The up-regulated p53 signaling in mucosa and whole gut at 24 h while TUNEL positive nuclei were significantly decreased at this time could repudiate the direct link of p53 to cell death in the intestine, as documented in other models A broad up-regulation of cell cycle control and DNA damage repair pathways occurred at 3.5 d in mucosa coinciding with downregulation of pathways that control apoptosis in both mucosa and whole gut. As expected, up-regulated DNA damage repair and cell cycle control signaling were mainly evident in the mucosa, i.e., the compartment where most DNA damage occurs. ATM, a key regulator of multiple signaling cascades in response to radiation-induced DNA strand breaks It is interesting to note that up-regulation of pathways that control the coagulation system, lymphocyte apoptosis, tight junction, cell cycle control, and DNA damage repair was largely only found in the LCM-isolated mucosa but not in the whole gut extract. The sequential activation of these pathways after radiation exposure provides important information for understanding the mechanism of the injury. Thus, inclusion of the non-mucosal layers may seriously dilute the altered mucosal genes and disguise activation of relevant pathways.A novel and unexpected finding was the marked increase of Casp14 and Eda2r expression, present in both mucosa and whole gut samples. The sharp and robust increase of these genes at the early time points suggests their important roles in the regulation of acute intestinal radiation injury.Casp14 belongs to a conserved family of aspartate-specific proteinases. It is expressed in the suprabasal layers of the epidermis and is associated with protection against UVB-induced apoptosis and water loss EDA2R is a type III transmembrane protein of the TNFR (tumor necrosis factor receptor) superfamily and has been identified as a p53 target which regulates p53-mediated anoikis One concern of this study is the comparability of the gene expression profiles from LCM and whole gut. To address this problem, RNA samples for microarray processing were screened by checking their integrity and genomic contamination. The parallel expression pattern of Casp14 at both mucosa and the whole gut revealed the comparability of the expression profiles from LCM and whole gut. The analysis result of activated cellular injury, apoptosis, and DNA damage repair pathways in mucosa but not in the whole gut is consistent to the pattern of cellular injury demonstrated by TUNEL staining. Taken together, these data demonstrate the reliability of the analysis in this paper.In conclusion, the present investigation revealed that alterations of many of the pathways known to undergo changes in response to radiation exposure are obscured if RNA extracts are obtained from whole gut samples as opposed to by LCM of the mucosa only. Hence, our data strongly suggest that RNA for analysis of pathways related to the coagulation system, lymphocyte apoptosis signaling, tight junction signaling, cell cycle control, and DNA damage repair signaling should be procured by LCM. This study also suggests that the newly identified genes Casp14 and Eda2r could provide novel research objectives and potential therapeutic targets to protect against or mitigate intestinal radiation injury. Future study will investigate RNA expression related to the position or differentiation state of the cells in the mucosa during intestinal radiation injury.


The Au NT labeled MSCs, injected intramuscularly in the lower limb of the Lewis rat, were detected and spatially resolved. Furthermore, our quantitative in vitro cell studies indicate that US/PA imaging is capable of high detection sensitivity (1×104 cells/mL) of the Au NT labeled MSCs. Finally, Au NT labeled MSCs captured in the PEGylated fibrin gel system were imaged in vivo, as well as in vitro, over a one week time period, suggesting that longitudinal cell tracking using US/PA imaging is possible. Overall, Au NT labeling of MSCs and US/PA imaging can be an alternative approach in stem cell imaging capable of noninvasive, sensitive, quantitative, longitudinal assessment of stem cell behaviors with high spatial and temporal resolutions at sufficient depths.Longitudinal monitoring of cells is required in order to understand the role of delivered stem cells in therapeutic neovascularization. However, there is not an imaging technique that is capable of quantitative, longitudinal assessment of stem cell behaviors with high spatial resolution and sufficient penetration depth. In this study,  Ischemic heart disease is one of the most common killers of American men and women, accounting for about half a million deaths every year Unfortunately, none of the current stem cell imaging modalities can satisfy all requirements simultaneously. Magnetic resonance imaging (MRI), with contrast agents based on superparamagnetic iron oxide, has been widely researched for stem cell monitoring. Although MRI can achieve relatively high resolution and long-term cell tracking, it has low sensitivity with the current contrast agents in vivo monitoring of MSC behavior and neovascularization promoted by MSCs In this study, we explored the feasibility of imaging stem cells using ultrasound-guided photoacoustic (US/PA) imaging in vivo as well as in vitro experiments were performed to evaluate in vivo detectability, sensitivity, quantification, and long-term MSC tracking ability with the suggested method. To demonstrate the feasibility of noninvasive monitoring of MSCs labeled with Au NTs, in vivo US/PA imaging of the nanotracer labeled MSCs injected intramuscularly in the lower limb of the rat was conducted. Following in vivo studies, sensitivity as well as quantification of the ultrasound and photoacoustic imaging method were assessed using a tissue mimicking gelatin phantom with inclusions of Au NT labeled and non-labeled MSCs. Furthermore, Au NT labeled MSCs cultured in vitro in a PEGylated fibrin gel system were imaged over a one week time period to verify the possibility of longitudinal cell tracking using photoacoustic imaging. Finally, longitudinal in vivo monitoring of spatial distribution of the PEGylated fibrin gel containing the Au NT labeled MSCs was demonstrated at the three different time points .Unfortunately, MSCs do not have sufficient optical absorption and cannot be directly visualized using US/PA imaging. However, as depicted in 5 nanotracers per MSC.Gold nanoparticles are one of the most widely used nanotracers for imaging cells of various types due to their excellent biocompatibility, as well as tunable optical properties in vivo monitoring of MSCs, the Au NT labeled MSCs were injected intramuscularly in the hind limb of the rat and visualized using US/PA imaging. A PEGylated fibrin gel with Au NT loaded MSCs (1×105 cells/mL) was injected intramuscularly in the lateral gastrocnemius (LGAS) of an anesthetized Lewis rat and immediately imaged at a range of wavelengths from 650 nm to 920 nm with a fluence of 11 mJ/cm2, which was verified to be safe  optical window in biological tissue, allow noninvasive photoacoustic imaging as well as spectral analysis to distinguish MSCs from background tissue . To demo be safe  and lowe be safe  can be a be safe . Therefoin vivo detectability of the Au NT labeled stem cells, accurate quantification of stem cells with high sensitivity is one of the essential requirements for effective stem cell tracking methods. To evaluate the sensitivity and cell quantification ability of photoacoustic imaging, an in vitro experiment using a tissue mimicking gelatin phantom was performed. 2, is shown in 2 value of 0.984) of the mean values of the photoacoustic signal amplitudes as a function of the nanotracer concentration on a linear scale. The results indicate that the amplitude of the photoacoustic signal is proportional to the concentration of optical absorbers, which are the Au NTs loaded inside MSCs. The results also indicate that with a fluence of 5.1 mJ/cm2, a photoacoustic signal can be detected for a nanotracer concentration as low as 4.53×109 NTs/mL, which corresponds to 1×104 cells/mL and 200 cells. Therefore, the photoacoustic imaging method can precisely quantify MSC concentrations with excellent sensitivity better than other noninvasive stem cell tracking methods In addition to the 2 . As shown in 10 NTs/mL, [1.71±0.27]×1010 NTs/mL, and [1.37±0.18]×1010 NTs/mL for day 1, 4, and 7, respectively. Furthermore, sensitivity of the photoacoustic imaging was enough to detect the labeled MSCs in spite of the decrease in Au NT loading. Hence, this result implies that photoacoustic imaging can be used to assess temporal behaviors of the Au NT labeled MSCs.Although we can achieve high sensitivity and cell quantification ability using the photoacoustic imaging, continuous cell tracking is essential to monitor MSC migration and proliferation. To demonstrate this, longitudinal (up to 7 days) photoacoustic images of MSCs loaded with Au NTs, or MSCs only, in PEGylated fibrin gels were acquired at a wavelength of 750 nm with a fluence of 11 mJ/cm2 . The PEGin vitro study, the Au NT labeled MSCs were longitudinally monitored in vivo. After injection of the PEGylated fibrin gel with Au NT loaded MSCs  into the LGAS of the Lewis rat, the labeled MSCs were imaged at the three different time points  using the ultrasound guided spectroscopic imaging. As demonstrated above, spectral analysis of the photoacoustic signals acquired at a range of wavelengths from 650 nm to 920 nm differentiated the Au NT labeled MSCs from other tissue components. in vivo imaging result. The photoacoustic signals obtained from the spatial positions of the labeled MSCs determined by the spectral analysis were summed and displayed in green color in the graph. Moreover, the photoacoustic signals from the background tissue were also analyzed and added to the graph with yellow color for the control. The Au NT concentration at day 3, 7, and 10 were able to be quantified to 2.27×1010 NTs/mL, 2.06×1010 NTs/mL, and 1.59×1010 NTs/mL respectively. Furthermore, this result also implies that the Au NT labeled MSCs can be detected if photoacoustic signals from the labeled cells are reasonably stronger than those from the background tissue, which are equivalent to [2.11±0.37]×103 MSCs and [3.18±5.56]×109 NTs/mL. Therefore, noninvasive in vivo longitudinal monitoring of MSCs labeled with nanotracers is capable with quantification and high sensitivity using US/PA imaging.Based on the longitudinal in vivo monitoring of MSCs labeled with nanotracers. Once Au NT labeled MSCs are implanted into tissue, photoacoustic imaging can noninvasively detect the presence of MSCs with sufficient penetration depth (∼10–50 mm) and spatial resolution (∼20–300 µm), which are scalable depending on the specification of the transducer, such as the center frequency 2–103 cells) and good cell viability over a long-term period (∼2 weeks), as demonstrated with our in vitro and in vivo photoacoustic imaging results with the PEGylated fibrin gel system. As mentioned in the introduction of this paper, other stem cell imaging modalities have their own drawbacks, such as sensitivity and spatial resolution (MRI), safety and tracking period (PET and SPECT), and penetration depth  The results of this study demonstrate the feasibility of longitudinal in vivo tracking using US/PA imaging. Our in vivo work consisted of animals which did not undergo any injury , and thus neovascularization was not sufficiently promoted because the PEGylated fibrin gels with MSCs were injected into normal muscles. However, based on our previous research, we believe that the PEGylated fibrin can promote neovascularization in an ischemic muscle due to the upregulation of certain growth factors and paracrine signals as a result of the wound healing response In addition to monitoring the spatial distribution of the stem cells, neovascularization promoted from stem cells needs to be monitored to estimate the efficacy of the stem cell treatment. However, this work was an initial study to demonstrate the feasibility of longitudinal in vivo US/PA imaging can be a promising alternative for visualizing and tracking MSCs within tissue engineered constructs.US/PA imaging can be a valuable tool for tissue engineering because it allows noninvasive and longitudinal tracking of MSCs with penetration depths up to several centimeters and reasonable spatial resolution. While optical imaging is widely being used in the tissue engineering field, such as fluorescence and multiphoton imaging, it suffers from limited penetration depth (∼0.5 mm) and photobleaching in vivo US/PA imaging, which indicates the ability of noninvasive monitoring of MSCs. Furthermore, the Au NT labeled MSCs were identified with high detection sensitivity in vitro (4.53×109 NTs/mL corresponding to 1×104 cells/mL and 200 cells) using ultrasound and photoacoustic imaging. In addition, Au NT labeled MSCs captured in the PEGylated fibrin gel system were able to be imaged in vivo, as well as in vitro, over a one week time period, which implies the possibility of longitudinal cell tracking using photoacoustic imaging. Accordingly, US/PA imaging using Au NT labeling of MSCs has great potential to be an alternative imaging method to monitor stem cell distribution and better understand the process of neovasularization and the wound healing response. Future work for this study includes in vivo monitoring of long-term MSC behaviors following an injury in order to assess and monitor the participation of MSCs in the process of neovascularization.In conclusion, the presented work demonstrates the ability of the US/PA imaging to noninvasively monitor MSCs labeled with Au NTs. Au NT labeled MSCs injected intramuscularly in the lower limb of the Lewis rat were capable of being visualized using 4) under reflux, as described elsewhere Gold nanotracers, which are 20 nm gold nanospheres, were synthesized via citrate reduction of tetrachloroauric (III) acid (HAuCl5 cells/mL) were combined with the PEGylated fibrinogen mixture in a 1∶1 volume ratio. The reaction underwent gelation by adding an equal volume solution of thrombin . The unbound free PEG was rinsed. The final concentrations in the gel were 10 mg/mL fibrinogen; 1 mg/mL of SG-PEG; 5×104 cells/mL; and 12.5 U/mL of thrombin. The gels were then incubated at 37°C in MSC growth medium.The PEGylated fibrin gels were prepared by adding difunctional succinimidyl glutarate (SG)-PEG  to fibrinogen  in a 1∶1 volume ratio. The reaction was allowed to take place at room temperature for 3–5 minute. MSCs (2×105 cells/mL. Thrombin (25 U/mL in 40 mM CaCl2) was loaded into a 23 G needle. An equal volume of the PEGylated fibrin solution containing cells was then loaded into the syringe and the solution was mixed within the syringe by shaking the syringe. The final concentration of the PEGylated fibrin gel injection was 10 mg/mL fibrinogen; 1 mg/mL SG-PEG; 1×105 cells/mL; and 12.5 U/mL thrombin. A total volume of 300 µL was injected in the lateral gastrocnemius of the rat. Injection depth was about 5 mm under the skin. Prior to the injection, the rat was anesthetized using 0.5–2.0% isoflurane with 1 L/min of oxygen and the hind limb of the rat was shaved.Animal handling and care followed the recommendations of the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. All protocols were approved by the Animal Care Committee of the University of Texas at Austin. Lewis rats (11 weeks old) weighing 200–250 g were used for the intramuscular injection and subsequent US/PA imaging. PEGylated fibrin injections were prepared by combining fibrinogen (40 mg/mL) and difunctional SG-PEG (4 mg/mL) in a 1∶1 volume ratio and allowing the reaction to take place at 37°C for 20 minutes. An equal volume of MSCs labeled with Au NTs were mixed with the PEGylated fibrin solution in a 1∶1 volume ratio at a concentration of 5×103 cells/mL, 1×104 cells/mL, 5×104 cells/mL, and 2×105 cells/mL). A gelatin solution with the same concentration of gelatin and silica particles as the bottom layer was used to cover the inclusions after solidification. The absorbance spectrums of all cell solutions in the eight inclusions were measured using a UV-vis spectrophotometer. The ICP-MS were used to calculate the concentrations of Au NTs for inclusions with the labeled MSCs .The tissue mimicking phantom with non-labeled or labeled MSC inclusions was prepared for combined ultrasound and photoacoustic (US/PA) imaging. The bottom layer of the phantom was composed of gelatin  with 15 µm diameter silica particles  for ultrasonic scattering. The eight inclusions (20 µL each) were placed on top of the bottom layer and were composed of gelatin solutions mixed with the non-labeled or labeled MSCs suspended in cell culture medium at four different concentrations of cells  or a 25 MHz single element, focused transducer  with a data acquisition board in a computer . A tunable OPO laser beam  pumped by a pulsed Nd:YAG laser  was delivered through an optical fiber bundle . The fiber bundle was combined with the ultrasound transducer and aligned such that the transmitted light would focus at the focal point of the transducer. The trigger signal from the laser system was synchronized with the computer or the Vevo 2100 imaging system to capture the photoacoustic signal when the pulsed laser irradiated. Once the photoacoustic transient and ultrasound pulse-echo signal was captured, the off-line image processing was performed with the acquired imaging data. The ultrasound and photoacoustic signals were bandpass filtered (5–45 MHz) to reduce noise. The magnitudes of the ultrasound and photoacoustic signals were calculated by taking the absolute values of analytic signals obtained using the Hilbert transform. The photoacoustic signal magnitude was then compensated for wavelength dependent laser fluence. The compensated ultrasound and photoacoustic signals were interpolated to 2D images. The combined ultrasound and photoacoustic images were created by overlaying photoacoustic intensities higher than a user-defined threshold on the grayscale ultrasound images.Figure S1Optical selectivity of photoacoustic imaging for spectral analysis. (A) The normalized absorbance spectra of Au NTs (top) and the Au NT labeled MSCs (middle). The green shaded region shows the optical window in biological tissue, which is the optimal wavelength ranges for in vivo imaging. Comparison within the optical window of the normalized absorbance spectra of labeled MSCs, oxygenated and deoxygenated hemoglobin (bottom). (B) Photoacoustic signal amplitude from the inclusions with 5,000 MSCs at multiple wavelengths .(TIFF)Click here for additional data file.Figure S2Cell viability after laser irradiation. Diagram of the experimental setup for laser irradiation (left) and fluorescent images of LIVE/DEAD stained MSCs loaded with nanotracers (right). MSCs were irradiated with 50 laser pulses at 532 nm wavelength. Cells remain viable after irradiation at laser fluencies reaching 17 mJ/cm2, but cell death was observed for laser fluence higher than 26 mJ/cm2.(TIFF)Click here for additional data file.


Studies have shown that podocyturia, i.e., urinary loss of viable podocytes , is associated with proteinuria in preeclampsia. We postulated that urinary podocyte loss may persist after preeclamptic pregnancies, thus resulting in renal injury. This may lead to future chronic renal injury. In addition, we compared the postpartum levels of the angiogenic factors, which previously have been associated with preeclampsia, between normotensive versus preeclamptic pregnancies.The diagnosis of preeclampsia was confirmed using standard clinical criteria. Random blood and urine samples were obtained within 24 hours prior to delivery and 5 to 8 weeks postpartum. Urine sediments were cultured for 24 hours to select for viable cells and staining for podocin was used to identify podocytes. Serum samples were analyzed for the levels of angiogenic markers using ELISA (enzyme-linked immunosorbent assay) methodology.At delivery, preeclamptic patients (n = 10) had significantly higher proteinuria (p = 0.006) and podocyturia (p<0.001) than normotensive pregnant patients (n = 18). Postpartum proteinuria was similar between these two groups (p = 0.37), while podocyturia was present in 3 of 10 women with preeclampsia and in none of the normotensive controls (p = 0.037). Angiogenic marker levels, including placental growth factor, soluble vascular endothelial growth factor receptor fms-like tyrosine kinase receptor-1 and endoglin, were not significantly different between women with preeclampsia and women with a normotensive pregnancy, either at delivery or postpartum.Persistent urinary podocyte loss after preeclamptic pregnancies may constitute a marker of ongoing, subclinical renal injury. Preeclampsia is a pregnancy-specific disorder clinically characterized by hypertension and proteinuria that occurs after 20 weeks of gestation To-date, several studies have demonstrated that proteinuria in preeclampsia is associated with podocyturia, i.e., urinary excretion and loss of glomerular epithelial cells, also known as podocytes The first aim of this study was to examine whether podocyturia, a marker of glomerular injury, persists after delivery. We postulate that ongoing urinary podocyte loss postpartum may contribute to chronic kidney injury, which has been documented years after the affected pregnancies This study was approved by the Mayo Clinic Institutional Review Board. Written consent was obtained from all women prior to enrollment in the study. The diagnosis of preeclampsia was established based on the presence of the following criteria Our initial cohort consisted of 273 women who were enrolled during their first prenatal visits, and then followed prospectively during their pregnancies. Pregnancy outcomes were ascertained at the time of hospitalization for delivery. Fifteen women developed preeclampsia, two of whom demonstrated additional characteristic findings of HELLP syndrome; 15 developed gestational hypertension and 204 had normotensive pregnancies. Six women had uncomplicated twin pregnancies and 33 women had one or more of the following pregnancy complications other than preeclampsia and gestational hypertension: gestational diabetes (n = 16), intrauterine growth retardation (n = 5), and pre-term delivery (n = 17). Our study cohort consisted of a total of 28 patients, 10 patients with preeclampsia/HELLP and 18 normotensive controls for whom blood and random urine samples were available both within 24-hours prior to delivery and at 5–8 weeks postpartum.Serum samples were analyzed for the levels of angiogenic markers  using Quantikine ELISA (enzyme-linked immunosorbent assay) kits . Aliquots of the midstream urine samples (50–100 mL) were collected in sterile containers and processed as previously described th, 75th percentile. Rank of values between groups (normotensive and preeclamptic pregnancies), at delivery and postpartum, were each compared using the Kruskal-Wallis rank sum test. Variables with normal distribution were expressed as mean ± standard deviation (SD), and compared between the groups using a two sample t-test. Counts of the number of subjects positive for podocyturia between the preeclampsia and normotensive groups, at either delivery or postpartum, were compared using Fisher’s exact test.The amount of 24-hour proteinuria was estimated from a P/Cr ratio and was expressed in gram protein/gram Cr. Podocyturia was expressed as a ratio of the number of podocytes per visual field to the milligrams of Cr of the respective urine sample. All continuous laboratory measurements were reported as median+interquartile range (IQR), 25Urine and plasma samples within 24 hours of delivery and 5–8 weeks postpartum were available for 10 patients with preeclampsia/HELLP and 18 normotensive controls. The women with normotensive pregnancies did not differ significantly from those with preeclampsia/HELLP with respect to demographic parameters, such as age, number of gestations, parity, or nulliparous status .At the time of delivery, women with preeclampsia/HELLP had significantly higher systolic blood pressures , diastolic blood pressures  (p<0.001) , and P/Cp<0.001) . Podocytp<0.001) .Postpartum samples were obtained at 44±7 days and 43±4 days after delivery for cases and controls, respectively. Postpartum systolic and diastolic blood pressures were similar between the women with preeclampsia/HELLP, p = 0.96 and p = 0.08, respectively  and preeclamptic pregnancies . Among the 3 women who demonstrated podocyturia postpartum, one went on to develop 2+ proteinuria (measured by the dipstick method) and hypertension; one was diagnosed with an autoimmune connective tissue disorder, but had no urinalysis/renal function follow up; and the third patient had no documented renal or other events. Among the women with normotensive pregnancies, none of them developed hypertension or renal disease for the duration of follow-up. Interestingly, the one woman classified as normotensive and with podocyturia at delivery had two diastolic blood pressures >90 mm Hg, five hours apart, but delivered before an official clinical diagnosis of preeclampsia could be made.This pilot study demonstrates that podocyturia may persist postpartum in 30% of women with preeclampsia/HELLP, despite the resolution of proteinuria. We and others have reported previously that podocyturia is present in patients during the acute phase of preeclampsia and commonly is absent in normotensive controls Viable urinary podocytes have been documented in a variety of glomerular diseases, and particularly, during the active phases of disease The primary limiting factor of our study is the relatively small sample size. Nevertheless, we feel that podocyte injury and subsequent shedding in the urine: (1) is inextricably linked to preeclampsia, (2) confirms the diagnosis at the time of acute presentation with high sensitivity and specificity, and (3) may serve as a biomarker for future renal and cardiovascular risk in the postpartum period. The podocyte culturing technique is technically complex, lengthy, and requires a high level of training and expertise, thus making it unsuitable for large scale studies. Newly developed technologies, based on either mass spectrometry