Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit Acute Tubular Necrosis (ATN) is characterized by acute tubular cell injury and renal dysfunction In particular, several groups demonstrated that the injection of MSC could help the recovery of Acute Kidney Injury (AKI), when experimentally induced by ischemia-reperfusion injury pos stem cells derived from amniotic fluid (hAFSC) can be easily propagated in vitro, maintaining stem cell-like properties such as pluripotentiality as well as expression of Oct-4, SSEA-4, CD90 and CD105 in vitro and in vivo. They do not form teratomas, and they maintain their telomeric length over many population doublings as well as, normal karyotype nu/nu mice, hAFSC can integrate and differentiate and they express lung epithelial markers following injury. Atala, his group and we pos cells for kidney regeneration ex vivo culture system and were demonstrated to integrate into the developing organ and participate in all steps of nephrogenesis during development. Herein, we now evaluate the function of hAFSC to rescue damaged kidneys in vivo in a mouse model of ATN. In kidneys with ATN caused by glycerol-induced rhabdomyolysis, hAFSC showed the capability to modulate the immune response and restore physiological kidney function parameters to normal. Therefore, we conclude that AFSC represent a new, potentially pluripotential cell source for kidney tissue protection, repair and regeneration.Our group has also reported the use of amniotic, c-kitin vivo applications, to exclude chromosomal abnormalities that could compromise their pluripotential capability  did not reveal any bioluminescence, while the transfected cells exposed to luciferin continue to express the bioluminescent signal after 20 population doublings  of cells that exhibited a readily detectable optical signal. In 6 hAFSC after damage induction. The animal injected with luciferin only (as a negative control) did not show any signal (panel 1), while the lucierase fluorescent signal is clearly evident, and even spreads into multiple zones of the body, such as the lung over the first few hours (panels 2–3). The signal for hAFSC in the area of the kidney can be seen at 24 hours after injection (panel 4), was strongest at 48 hours and 72 hours, and persisted for up to 6 days (panels 5–6), after which the signal began to diminish over the next several days (panel 7–8). However, 21 days after injection, the signal was still evident in the area of the kidney (panel 9). Animals were sacrificed at 21 days after injections of hAFSC and DNA extraction and PCR were performed on injected and non-injected kidneys in order to determine the presence of luciferase gene. Both the luciferase gene and a human housekeeping gene ACTB (both expressed only in human cells) were present only in the injected kidney tissue, as confirmed by the absence of the both housekeeping ACTB and luciferase gene in non-injected kidneys  in normal nu/nu mice, which averaged 0.6 mg/dl and 27 mg/dl, respectively. After intramuscular injection of glycerol on day 0, creatinine levels increased to as high as 1.10 mg/dl, showing a peak between 48 and 72 hours after injection. Similarly, the level of BUN increased up to 70 mg/dl after glycerol injection and a peak was detected between 48 to 72 hours. The injected dose of glycerol chosen is a sub-lethal dose. Therefore, after the peak, levels of creatinine and BUN resolved back to normal after a period of 3 weeks.A control group of 10 Remarkably, animals subjected to damage induced with glycerol and receiving an intrarenal hAFSC injection demonstrated no increase in the levels of creatinine or BUN during the expected acute phase of injury, showing a statistically significant difference when compared with animals treated with PBS after ATN or with animals that underwent only ATN induction. Absence of increases in both creatinine and BUN levels was demonstrated also in animals that received only intra-renal injection of hAFSC without glycerol damage, demonstrating that hAFSC alone did not alter these kidney physiological parameters over time  did not show any cast formation at basal level (data not shown), and were therefore not included in the statistical analysis comparing ATN groups of animals.The proliferative activity and the apoptosis of tubular cells were compared between the experimental groups at the different time points .The analysis of the data showed no statistical difference in proliferation activity between the animals that were injected in the kidney with saline vehicle solution (PBS) after glycerol injection versus animals that underwent only glycerol injection over time. In contrast, animals subjected to damage induced with glycerol and that received intrarenal hAFSC injection demonstrated a significant increase in cell proliferation between 48 hours and 72 hours  after glycerol injection as well as in animals that underwent only glycerol injection, as measured during the acute phase of injury. In contrast, in animals that received intrarenal injection of hAFSC a decrease of apoptosis was noted, as shown in 1. Interleukins; 2. Activators of B Lymphocytes; 3. Activators of Natural Killers; 4. Chemotactic attractors of Granulocytes and Macrophages; 5. Multiple biological effectors. We did not include a specific category for activators of T Lymphocytes, since in this mouse model activated T Lymphocytes are not expressed. We analyzed the expression of cytokines in all the experimental groups at different time points up to two weeks. The mouse specific cytokine assay does not cross react with the human cytokine assay. This was confirmed by incubating digested kidney extracts with membranes specific for human cytokines and conversely incubating hAFSC with membranes specific for mouse cytokines (data not shown).Since the salutary effect of hAFSC injection occurred during the acute phase of ATN, we postulated that this protective effect might involve acute changes in the kidney's cytokine expression. Cytokines expression arrays were performed using Cytokine Array Membranes . There was no significant difference, with very few exceptions, between the ATN mice treated with vehicle solution (PBS) versus just ATN induction, as shown in Table S2. In addition, the levels of cytokines in the hAFSC treated ATN mice, compared with basal nu/nu mouse kidney levels showed that there was an increase of anti-inflammatory cytokines such as IL-10 and IL-1Ra (Table S1). Moreover, there was no significant increase in pro inflammatory cytokines such as IL 27, IL 12p70, IL 2, IL 23 (Table S1). Indeed the hAFSC injected ATN mice showed a decrease in JE, M-CSF, MIP-2, MPC-5 and KC expression, when versus the ATN mice treated with vehicle solution (PBS) or just ATN induction (Table S2).Mice treated with intrarenal hAFSC after induction of ATN showed statistically significant comparisons, mostly at 7 and 14 days, in mouse cytokine levels, when compared with mice treated with either intrarenal vehicle saline solution (PBS) or just with ATN induction alone, or when compared with Table S1 and in vitro culture) as reported in Table S3 especially during the first days after injection, while most of them returned to normal at 7 days and were not detectable at 14 days.Mice that were injected with hAFSC, only without inducing ATN, underwent a limited tissue reaction in terms of production of different cytokines, but for most of them this expression was very early after the injection (within the first few days) and the levels returned to normal by 14 days as shown in nu/nu mouse kidneys with glycerol-induced ATN. This model of ATN involves a complex sequence of events wherein myoglobin, released from damaged muscle, damages the epithelial cells of the proximal tubules, producing cast formation, vasoconstriction and decreased glomerular filtration. Peak of the damage represented by high levels of apoptotic cells, increased levels of creatinine and BUN and by histological analysis was confirmed to be between 48–72 after induction of ATN.In this paper we demonstrate a protective role of hAFSC when injected directly into nu/nu mice, wherein the acute injury was performed in the lung instead of the kidney, decreased over time, showing a strong signal in the lung at the beginning that fades over the next few days. Nevertheless herein following intrarenal injection after ATN kidney damage, the bioluminescent signal is still present in the kidney after 21 days and hAFSC were found specifically located among the tubules. In order to determine if the injected cells can differentiate into tubular epithelial cells we performed both immunohistochemistry and RT-PCR using human specific antibodies and human primers. Genes such as PAX2, NPHS1 and lectins like Dolicholus Biflorus and Peanut Agglutinin are expressed by injected cells, indicating that at least some of the integrated cells are able to commit toward renal differentiation. Furthermore, in some rare instances, injected hAFSC cells could express human Glial Derived Neurotrophic Factor (GDNF), which is expressed during very early kidney development; GDNF is not usually detectable in the adult kidney, indicating that injected hAFSC can express also embryonic renal markers, retaining the ability of going through the nephrogenesis. We have previously shown that hAFSC have the potential to integrate into embryonic kidneys and can participate in key steps of nephrogenesis, indicating that hAFSC can be induced toward to a renal fate when placed in an appropriate environment The ability for hAFSC to home to injured organs was confirmed using luciferase positive hAFSC detected their bioluminescence after injection. As shown in our previous paper We determined that hAFSC have the ability to modulate kidney function during ATN, as reflected in serum creatinine and BUN levels. However, the timing of hAFSC is critical: hAFSC injected during the acute phase of ATN (between 48–72 hours after the IM injection of glycerol), had no effect on creatinine and BUN levels (data not shown), and implies that if injury is already established the damage is not attenuated. In contrast, hAFSC injected into the kidney on the same day of glycerol injection resulted in no observed peaks in creatinine or BUN. This beneficial effect with hAFSC was also correlated with significant increases in proliferative activity of tubular epithelial cells, decreased cast formation, and decreased apoptosis of tubular epithelial cells.We therefore speculate that hAFSC can, when injected early enough (in this study contemporaneously with the time of injury) attenuate acute renal damage, underscoring the potential protective effects of hAFSC. Moreover, even 14 days after injury hAFSC injection into the kidney still correlated with increased tubular cell proliferation and less tubular damage. Thus, we hypothesize that hAFSC might accelerate the proliferation of epithelial tubular cells that were only partially damaged, while in addition preventing apoptosis. This mechanism of protection therefore appears to lead to better maintenance of tubular structure, as seen in PAS staining, thus avoiding the increase in BUN and creatinine typically seen in IM-glycerol-induced ATN.et al.et al.During acute renal injury the immune response plays a key role. Damaged kidney endothelial cells attract leukocytes, vasomediators are released with injury, and epithelial cells of the tubule produce pro-inflammatory and chemotactic cytokines nuFoxn1) was chosen in order to be able inject human derived stem cells and evaluate their effect over time, avoiding outright rejection. Nu/nu mice are immunodeficient, lacking activated T Lymphocytes but not their precursors. They have normal B Lymphocytes and they have evidence of increase numbers of NK cells. Thus, even though they are immunodeficient, they do possess some ability to mount and modulate a partial immune response when stimulated nu/nu mice with glycerol induced ATN to human stem cells after kidney injection has never been demonstrated before, and the response could diverge significantly from wild type mice injected with mouse-specific stem cells.The animal model used in these experiments  are significantly increased early in the course of ATN  had returned to normal by 14 days, particularly when compared with ATN mice injected with PBS or with just ATN, i.e., in the latter mice, inflammation persists longer. This is also in accordance with the presence of tubular damage in the latter groups at 14 days. This suggests that an important function of hAFSC may be to actually prevent the acute injury. We further speculate that, in this particular as shown , while tnu/nu mice treated with hAFSC, there is over all improvement in the maintenance of tissue homeostasis, which prevented progression of the acute phase in glycerol-induced ATN, most likely through stimulating proliferation of tubular epithelial cells and by cytokine-mediated paracrine mechanisms. Furthermore, after the acute phase, the decrease of creatinine levels, as well as less cast formation and the increase of tubular cell proliferation is still statistically significant at two weeks, at which time damage still persists in the ATN mice treated only with PBS or not treated at all. This is also in accordance with the data on cytokine levels that are still significantly elevated at 1 and 2 weeks in ATN mice treated with PBS only or not treated at all, as compared to ATN mice treated with hAFSC in which cytokine levels had resolved by 1–2 weeks.With a few exceptions, most human cytokines are also active on mouse cells in vivo, we injected them into nu/nu mice without ATN induction. In these mice, injected only with hAFSC there was no alteration of creatinine or BUN, no increase in apoptosis or proliferation or tubular damage; thus indicating that hAFSC per se do not alter the normal physiology and morphology of the kidney. However injection of hAFSC alone did stimulate the production of some cytokines by nu/nu mouse kidney, but their levels were relatively low and only detectable predominantly during the first few time points, and rapidly resolved back to normal before 14 days, as compared with ATN mice injected with PBS. In addition, in these mice, there was an increase of levels in IL-10 and IL-1Ra and IL-6 following hAFSC injection (similar to that noted in the mice injected with hAFSC following ATN), there was no increase in SDF-1 and also they expressed lower levels of NK attractors as well as macrophage attractors. Thus, hAFSC do stimulate a modest tissue reaction by nu/nu mice, but this did not cause any measurable perturbation of kidney function. In addition, the human cytokines produced by the injected cells in mice that did not undergo ATN disappear quickly and with no apparent adverse consequences.To rule out whether hAFSC alone can cause damage to the kidney and/or produce cytokines once injected In conclusion, we have demonstrated that early direct injection of hAFSC into the kidney strongly ameliorates ATN injury, as reflected by more rapid resolution of tubular structural damage, by tubular cell proliferation and by normalized creatinine and BUN levels. In addition, our data show evidence of potent immunomodulatory effects of hAFSC that appear to control the local immune response in favor of a tissue cytokine and cellular milieu that promotes prevention or resolution of tissue damage. Taken together these findings suggest that hAFSC may have therapeutic potential in ATN and, by extrapolation, perhaps in other kidney diseases.et al. et al. in vivo injection, were rekaryotyped at passage 38 using standard protocols by the Core Laboratory of Clinical Cytogenetics directed by Dr Wu at Childrens Hospital Los Angeles.Samples of human amniotic fluid from male fetuses (12–18 week of gestation) were provided to our laboratory by Genzyme Genetics Corporation  after karyotyping analysis. No written or verbal consent was required since samples were not identified and information obtained about the samples was limited to karyotype and fetal health status. The stem cell population was separated from the general human amniotic cellular milieu using standard Magnetic Sorting (MACS) techniques [Miltenyi Biotech] against the cell surface marker, c-kit, as described by Atala Before injection, a clonal hAFSC population at passage 40 was trypsinized in 0.05M trypsin/EDTA [Gibco/BRL] solution and centrifuged at 1500 rpm for 5 min, and then labeled with a cell surface marker CM-DiI [Molecular Probe] following the manufacturer's instructions, in order to track the cells after injection. Briefly, the cells were incubated with a working solution of 1 mg/ml of CM-Dil for 5 minutes at 37°C followed by an incubation of 15 minutes at 4°C and then three washes with phosphate-buffered saline (PBS) [Gibco/BRL].nu/nu mice [Jackson Laboratories] by intramuscular injection with 50% hypertonic glycerol solution in water (10 ml/kg body/wt) [Sigma-Aldrich] following deprivation of water for 22 hours. Controlled intramuscular injection of glycerol was performed under anesthesia by surgically exposing the caudal thigh muscle and slowly injecting the glycerol solution prior to delivery of cells. In nu/nu mice the amount of glycerol required for induction of ATN was 50% higher than the dose needed in wild type mice (data not shown). This suggests that the T-deficient mice are relatively protected as compared to wild type mice against glycerol-rhabdomyolysis-induced ATN Rhabdomyolysis-related ATN was induced in female 6 resuspended in 50 µl of saline vehicle solution, PBS) at passage 40 were carefully injected into the renal cortex of both kidneys with a 30–33 gauge needle, using a microinjector Eppendorf TransferMan NK2 Injector [Eppendorf], 2 hours after intramuscular glycerol injection. The kidneys were then replaced into the retroperitoneum, the incision closed with polypropylene suture and the mice were allowed to recover from anesthesia. The animals were maintained on a heating pad throughout the period of anesthesia. 0.1 mg/kg of buprenorphine was administered subcutaneously and 1 mg/kg bupivicaine  along the incision margins just prior to wound closure to provide post-operative pain relief. The animals were sterilely draped to prevent contact of the kidneys with the skin of the animal to reduce risk of peritonitis. As control, mice were injected with saline vehicle solution (PBS) 2 hours after glycerol damage. In addition, another animal group was injected only with hAFSC without previous glycerol damage using the same technique described above for the cell suspension and surgery.Animal experiments were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with institutional Animal Care and Use Committee approval. The mice were carefully anesthetized using isofluorane inhalation. Once satisfactory anesthesia was achieved, the mice were prepared for surgery using chlorhexidine. A 1 cm dorsal incision was made, both kidneys were carefully delivered via the incision, and the labeled hAFSC (1×10At different times points (from 24 hours to 3 weeks), the injected and the control mice were sacrificed. The kidneys were extracted, washed in PBS, and processed in one of the following ways depending on the analysis performed.The kidneys were minced in small pieces and the RNA extracted using Qiagen RNeasy kit [Qiagen] according to the manufacturer's instructions. Total RNA was isolated using the RNeasy Mini Kit [Invitrogen] as described on the data sheet. Briefly, with the use of silica-gel columns RNA is separated from DNA through centrifugation after lysis and homogenization of the samples. Ethanol addition allows RNA to bind the silica-gel before the centrifugation step. The RNA solution obtained was then processed with DNAse treatment  to avoid any possible genomic contamination. 1 µg of total RNA was reverse transcribed using SuperScript II reverse transcriptase [Invitrogen]. Amplification of the resulting cDNA was carried out using only specific human primers not coding for mouse sequences. A PCR thermal cycler [Eppendorf] was employed after an initial denaturation step at 95°C for 10 minutes. We used a denaturation step at 95°C for 30 seconds, an annealing step at the temperature specific for each primer (ranging from 54°C to 60°C) for 45 seconds, and an extension step at 72°C for 45 seconds for a total of 35 cycles. Detection of the PCR amplification products was performed by size fractionation on 1% agarose gel electrophoresis. As a housekeeping gene, amplification of fragments of the human β-actin RNA was performed. Specific human primer sequences, predicted sizes of amplicons and specific annealing temperatures are shown in In order to perform PCR on the genomic DNA to evaluate the presence of the luciferase gene, DNA extraction was performed following standard protocols of the Qiagen DNeasy kit . Briefly, the kidney was cut in small pieces and incubated at 55°C with proteinase K until completely lysed. To the mixture was then added buffer AL and ethanol 70% furnished by the kit to obtain ideal DNA binding conditions to the DNeasy Mini Spin column. After centrifugation and washing steps DNA was collected and PCR was performed as previously described.Kidneys were fixed in 4% paraformaldehyde[Sigma-Aldrich] in PBS for 8 hours at 4°C, dehydrated through a gradual series of alcohol, embedded in paraffin, and sectioned at 4–5 µm. The sections were deparaffinized for 15 minutes in histochoice [Sigma-Aldrich], 5 minutes in 100% alcohol, 5 minutes in 95% alcohol, 5 minutes in 70% alcohol; 5 minutes in 50% alcohol, 5 minutes in 30% alcohol and then water. The sections were then stained with Periodic Acid Schiff (PAS) [Sigma-Aldrich] to evaluate kidney morphology. The slides were immersed in Periodic Acid Solution [Sigma-Aldrich] for 5 minutes at room temperature; rinsed in several changes of distilled water, then stained with Schiffs reagent [Sigma-Aldrich] for 15 minutes at room temperature, washed in running tap water for 5 minutes and finally counterstained with hematoxylin solution Gill [Sigma-Aldrich] for 90 seconds. In the end the slides were rinsed in running tap water, dehydrated, cleared and mounted in mounting medium PROTOCOL XYLENE BASED [Fisher Scientific].In addition, some kidneys were frozen in liquid nitrogen using Tissue-Tek O.C.T. compound [Finetek] and stored at −20°C. When required, kidneys were cryosectioned at 5 µm and then used for immuno-histochemistry.et alin vitro analysis. The percentage of the transfected cells was 40%; positive clones were selected and used for the experiments. Before in vivo injections, a simple in vitro test was employed to determine the minimum amount of hAFSC detectable by bioluminescence. Different concentrations of the cells ranging from 5×104 to 2×106 were evaluated. In addition, the expression of the luciferase gene was confirmed by PCR after 20 passages in culture. A number of 25 animals, 10-week old nu/nu mice, obtained from Jackson Laboratories were injected directly into the kidney with luciferase-transduced hAFSC (1×106 cells/mouse diluted in 50 µl ofPBS) after glycerol damage. In vivo optical imaging was performed with a prototype IVIS 3-dimensional bioluminescence/fluorescence optical imaging system  at different time points.Clones of hAFSC were transduced with a lentiviral vector (SMPU-R-MNCU3-LUC based on HIV-1 that transduces the firefly luciferase gene) made by the Vector Core Facility at Childrens Hospital Los Angeles following standard protocols as reported in the paper by Crooks Prior to imaging, each mouse was given an intraperitoneal injection of luciferin [Promega] at a dose of 125 mg/kg, as previously described Frozen and paraffin slides were stained for immunofluorescence. Paraffin slides were deparaffinized as previously described while the frozen slides were fixed for 5 minutes in 80% methanol. The slides were placed in 1% Triton x-100 [VWR] in PBS for 5 minutes (if the antigen was nuclear) and briefly washed in PBS. The slides were then placed in working solution of Vector Antigen Retrieval as described in the data sheet. [Vector Laboratories] for three cycles at high power for 4 minutes each afterwards cooled down at room temperature. After Avidin/Biotin blocking using the Blocking Avidin/Biotin Vector kit [Vector Laboratories] for 30 minutes at room temperature, a second block was carried out for 30 minutes using 5% of Bovine Serum Albumin  diluted in PBS at room temperature. The slides were then incubated in primary antibody at different concentrations in solution a of 5% of BSA in PBS  solution for one hour at room temperature (Dolicholus Biflorus and Peanut Agglutinin) or overnight at 4°C (Luciferase and GDNF). Afterwards, the slides were washed in PBS for 5 minutes for 3 times. Secondary antibodies [Vector Laboratories] were diluted 1∶200 in 5% BSA in PBS – slides were incubated in this solution for 1.5 hours at room temperature, followed by washes in PBS for 5 minutes for 3 times. The appropriate fluorescent marker [Texas Red or Fluorescein Avidin DCS from Vector Laboratories] was then applied in a concentration of 1∶500 in PBS buffer for 5–10 minutes, followed by a final wash in PBS for 5 minutes for 3 times. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).A Leica DM RA fluorescent microscope was used in conjunction with Open Lab 3.1.5 software to image the staining.1. 10 control animals, no damage and no treatment; 2. 10 animals that underwent only ATN; 3. 10 animals that underwent ATN and intrarenal injection of hAFSC; 4. 10 animals that underwent ATN and intrarenal injection of saline vehicle solution (PBS). 5. 10 animals that underwent intrarenal injection of hAFSC but not ATN) as suggested by the kit data sheet. All the reagents were furnished by the kit. Briefly, after deparaffination and rehydration, the slides were incubated with Proteinase K solution for 30 minutes at 37°C, washed in PBS for 2 minutes, and after blocking incubated with a TUNEL Reaction Mixture for 1 hour at 37°C, followed by the Streptavidin –HRP solution for 30 minutes at 37°C and finally revealed with the DAB Substrate. If the staining was performed in frozen section the fluorescent TUNEL  was used. Briefly, the cells were incubated at 37°C for one hour with the TUNEL reagent and then washed in PBS. Slides were mounted with Vector DAPI mounting medium [Vector Laboratories].The number apoptotic cells were determined using the TUNEL Apoptosis Detection Kit for paraffin-embedded tissue sections  at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks after injections in every experimental group  at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks after injections in every experimental group above described as suggested by the kit data sheet. Briefly, after deparaffination and rehydration and blocking, the slides were incubated with a biotinylated mouse anti-PCNA primary antibody for 1 hour at room temperature followed with incubation with Streptevidin-HRP and revealed with DAB substrate. In all the experimental groups the proliferative nuclei were counted as a fraction of the total number of nuclei present in the section using consecutive, non-overlapping fields of PCNA-stained specimens. The percentage of proliferative cells was estimated without knowledge of the experimental group.1. 10 animals for measuring baseline creatinine and BUN levels; 2. 10 animals that underwent only ATN; 3. 10 animals that underwent ATN and intrarenal injection of hAFSC; 4. 10 animals that underwent ATN and intrarenal injection of saline vehicle solution (PBS). 5. 10 animals that underwent intrarenal injection of hAFSC but not ATN.The facial vein was lanced with a 5 mm animal lancet and blood collected using standard protocols approved by the Animal Core Facility at Childrens Hospital of Los Angeles and Saban Research Institute. Animals were divided into different groups per each time point as follow : The blood samples (30 µL) were collected into plasma separation tubes with lithium heparin. They were centrifuged at 13,000-RPM for 3 minutes and the plasma (upper layer) was removed and stored at −80°C until analysis. A maximum of 15% of circulating blood was sampled in a given 14-day period . Post-damage measurements were obtained every 24 hours. The blood samples were used to monitor renal function, by analyzing creatinine and BUN levels. Expression levels were determined by a quantitative colorimetric assay [BioAssay Systems Cat # DUCT-500 for creatinine Cat # DIUR-500 for BUN], using an improved Jaffe method for creatinine, and an improved Jung method for BUN without any pretreatment of the samples.1. Sections obtained from mice that underwent ATN with no injection of hAFSC sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 2. Section obtained from mice that underwent ATN and injection of hAFSC sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 3. Sections obtained from mice that underwent ATN with and injection of PBS sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 4. Sections obtained from mice that underwent only intrarenal injection of hAFSC and sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks. Tubular injury was evaluated based on different parameters using PAS staining: including disruption of tubular membranes and brush borders, but mainly relaying on cast formation since it is the most evident sign of damage in stained sections.Paraffin embedded kidney sections were prepared at 4 µm thickness by a routine procedure and stained with PAS reagents as described above. The kidney sections were divided as follow: In the experimental groups, the tubular injury was counted as a fraction of the total number of tubules present in the section using consecutive, non-overlapping fields of PAS-stained specimens. The percentage of damaged tubules was estimated without knowledge of the experimental group.1. control animals, no damage and no treatment; 2. animals that underwent only ATN; 3. animals that underwent ATN and intrarenal injection of hAFSC; 4. animals that underwent ATN and intrarenal injection of saline vehicle solution (PBS). 5. animals that underwent intrarenal injection of hAFSC but not ATN) using a multiple cytokine array technique Proteome Profiler Array Kit, for human c# ARY005 and for mouse c# ARY006, as suggested from the protocol [R&D Systems].To examine if the injection of hAFSC would interfere with the modulation of inflammation after acute kidney injury human and mouse cytokines levels were measured in digested mouse kidneys at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks in every experimental groups  was incubated for one hour at room temperature with a cytokine cocktail and each sample was then added to the membrane and incubated overnight at 4°C. The following day after several washing steps in Washing Buffer the membranes were incubated with a streptavidin-HRP secondary as suggested by the manufacturer (concentration 1∶2000) for 30 minutes at room temperature. After washing in Washing buffer, it was added the Super Signal West Pico Chemiluminescent Substrate was added [ThermoScientific] to detect the signal. After 1 minute incubation the membranes were exposed for 20 seconds to x-ray film. The signal was measured by counting the pixel density and the data were analyzed using the Array Vision Program [R&D Systems].Kidneys are briefly washed in PBS, cut in pieces and added to 1 ml Phosphatase Inhibitor solution [ActiveMotif] and then mechanically homogenized on ice for 1 minute, then 10 µl of Triton X-100 [Sigma-Aldrich] is added before vortexing for 1 minute. The lysate is frozen at -80°C and thawed at room temperature. The sample is centrifuged at 10.000 rpm for 20 minutes at 4°C. The supernatant is then transferred to a clean tube and the protein concentration measured by UV-vis spectroscopy. An equal amount of protein (300 µg) for the samples derived form the different experimental groups  with Bonferroni post-test correction was used, and for all other data Mann-Whitney U test was applied to compare two ore more (ANOVA) independent sets of data. A P value less than 0.05 was considered statistically significant.Table S1In the table are reported the P values for the cytokine analysis at 1,2, 3, 7 and 14 days. Column 1: Cytokines are grouped by target and/or effects. Column 2: Mice with ATN and injection of hAFSC versus normal nu/nu mouse . Column 3: Mice with ATN and injection of PBS versus normal nu/nu mouse . Column 4: Mice with ATN only versus normal nu/nu mouse . Column 5: Mice with no ATN and injection of hAFSC versus normal nu/nu mouse . P values are expressed as follows: * P<0.05; ** P < 0.01; *** P< 0.001 and they represent the deviation from the control ; ⇑: increase of cytokine levels in the experimental groups compared to the control; ⇓: decrease of cytokine levels in the experimental groups compared to the control. Blank cells in the table indicate no statistically significant change in cytokine expression.(0.08 MB DOC)Click here for additional data file.Table S2In the table are reported the P values for the mouse cytokine analysis at 1, 2, 3, 7 and 14 days. Column 1: Cytokines are grouped by target and/or effects. Column 2: Mice with ATN and injection of hAFSC versus mice with ATN and injection of PBS . Column 3: Mice with ATN and injection of hAFSC versus mice with ATN only . Column 4: Mice with ATN and injection of PBS versus mice with ATN only . P values are expressed as follows: * P < 0.05, ** P < 0.01, *** P< 0.001. Blank cells in the table indicate no statistically significant change in cytokine expression.(0.08 MB DOC)Click here for additional data file.Table S3In the table are reported the P values for the human cytokine analysis at 1, 2, 3, 7 and 14 days. Column 1: Cytokines are grouped by target and/or effects. Column 2: Mice with ATN and injection of hAFSC versus hAFSC in vitro  . Column 3: Mice without ATN and injection of hAFSC versus hAFSC in vitro  . P values are expressed as follows: * P < 0.05, ** P < 0.01, *** P< 0.001. Blank cells in the table indicate no statistically significant change in cytokine expression.(0.06 MB DOC)Click here for additional data file.


Listeria monocytogenes is a model organism for cellular microbiology and host–pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes  in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype , with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence. Listeria monocytogenes is a pathogen transmitted through contaminated food and is responsible for severe infections, including meningitis and abortion in animals and humans. It is known that many distinct strains of this pathogen exist, and that they differ in their virulence and epidemic potential. Unfortunately, there is currently no standard definition of strains and no comprehensive overview of their evolution. To tackle these serious limitations to the control of listeriosis and to improve knowledge of how virulence evolves, we characterized a large collection of isolates with sequence-based genotyping methods. We were thus able to identify precisely the most prevalent clones of L. monocytogenes, i.e., groups of isolates that descend from a single ancestral bacterium, which can now be characterized further for diagnostic purposes and determination of their precise ecology and virulence potential. We also determined how these clones evolved from their common ancestor and the evolutionary history by which they acquired their phenotypic characteristics, such as antigenic structures. Finally, we show that some particular strains tend to lose a virulence factor that plays a crucial role in infection in humans. This is a rare example of evolution towards reduced virulence of pathogens, and the discovery of the selective forces behind this phenomenon may have important epidemiological and biological implications. Listeria monocytogenes causes life-threatening infections in animal and in human populations at risk. This facultative intracellular bacterium is widespread in the environment and infections occur through ingestion of contaminated food L. monocytogenes has long been known to be genetically diverse The opportunistic pathogen L. monocytogenes strains Listeria serotyping scheme L. monocytogenes isolate typing and investigations into the ecological distribution, epidemiology and virulence of strains. Unfortunately, serotyping discriminates only 13 serotypes, many of which are known to represent genetically diverse groups of strains, and only four serotypes  cause almost all cases of listeriosis in humans L. monocytogenesL. monocytogenes isolates L. monocytogenes is currently not available. The molecular factors that determine ecological differences among strains are also poorly understood.Several methods have been used to differentiate L. monocytogenes is the distinction of three phylogenetic lineages. Initially, two major lineages were distinguished, mainly based on multilocus enzyme electrophoresis and PFGE L. monocytogenes clones is needed to determine which ones mostly contribute to human or animal infection One salient feature of the population structure of L. monocytogenes strains L. monocytogenes into various non-phagocytic human eukaryotic cells expressing its receptor E-cadherin inlA, onto the phylogenetic framework of the genomes in which they are presently distributed.Several virulence genes play an important role in the virulence of L. monocytogenes; (ii) the evolutionary origin and stability of serotypes; and (iii) the patterns of variation of the virulence gene inlA with respect to the evolution of the core genome.The aims of this study were to provide a robust phylogenetic framework based on MLST analysis of a highly diverse isolate collection and determine (i) the population structure of Listeria monocytogenes and four L. innocua isolates were selected from the collections of the French National Reference Centre for Listeria and the WHO Collaborative Centre for foodborne listeriosis  a diversity subset of 171 isolates, which included representative isolates of the distinct L. monocytogenes serotypes, atypical strains from lineage III, isolates that caused major epidemics throughout the world, strains for which the complete genome sequence is available, 75 historical strains collected from 1924 to 1966 and belonging to H.P.R. Seeliger Listeria Culture Collection , isolates from the environment, food or animals, and research strains from several countries used in previous studies involving the Institut Pasteur Listeria laboratory . Identification was confirmed and subdivided into serotypes by classical serotyping L. monocytogenes isolates into four major groups  corresponding to groups of serotypes , bglA (beta-glucosidase), cat , dapE (Succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (lactate deshydrogenase), and lhkA (histidine kinase). This MLST scheme was adapted from the MLST system proposed by Salcedo and colleagues ldh was extended from 354 to 453 nucleotides, thus improving strain discrimination. Second, gene templates were shortened because the extremities of the previous templates correspond to parts of the PCR primer sequences, thus possibly not corresponding totally to the genomic sequence of the isolates analyzed. Third, we incorporated universal sequencing tails to the PCR primers , and 72°C for 2 min; and a final incubation at 72°C for 10 min. The PCR products were purified by ultrafiltration  and were sequenced on both strands with Big Dye v.1.1 chemistry on an ABI3730XL sequencer (Applied BioSystems).The MLST scheme used to characterize  primers Table 1,inlA gene was sequenced from 157 isolates (L. monocytogenes (see below). DNA extraction was performed with the Wizard® kit . The PCR amplification conditions were as follows: an initial cycle at 94°C for 5 min; 35 amplification cycles, each consisting of 94°C for 30 s, 55.2°C for 30 s, and 72°C for 1 min 30; and a final incubation at 72°C for 10 min. We used external primers for amplification and internal primers for sequencing  number was attributed to each distinct combination of alleles at the seven genes. Numbers were initially based on highest frequency for the frequent alleles and STs, and were subsequently incremented arbitrarily. In order to define the relationships among strains at the microevolutionary level, we performed allelic profile-based comparisons using a minimum spanning tree (MST) analysis with the BioNumerics v5.10 software . MST analysis links profiles so that the sum of the distances  is minimized Neighbor-joining tree analysis was performed using MEGA v4 To test for phylogenetic congruence among genes, one strain of all 39 STs with allelic mismatch distance >0.65 was used in order to exclude the expected congruence among genes at small evolutionary scale due to common clonal descent, as proposed previously The relative contribution of recombination and mutation on the short term was calculated using software MultiLocus Analyzer  and the simplest implementation of the clonal diversification method tructureK, and also estimates q, the combined probability of ancestry from each of the K groups for each individual isolate. We chose three groups for this report because repeated analyses  with K between 1 and 10 showed that the model probability increased dramatically between K = 2 and K = 3 and only slowly thereafter.We used the linkage model in Shathat employs a parametric approach, based on the neutral coalescent, to estimate the scaled parameter 2Ner where eN is the effective population size, and r is the rate at which recombination events separate adjacent nucleotides. The crossing-over model L was used for the analysis of biallelic sites.The population recombination rate was estimated by a composite-likelihood method with LDomegaMapindel: improper inverse, ω: inverse with range 0.0001–10, ρ: inverse with range 0.01–10. The variable block model was chosen for both ω and ρ, with block sizes of 10 and 30, respectively. We created 10 subsets of 50 randomly drawn inlA sequences each, and analyzed each subset with 50,000 iterations and 10 reorderings. The first 20,000 sequences were discarded as burn-in period.We also tested for presence of positively selected sites using the software www.pasteur.fr/mlst for the seven MLST genes. inlA sequences have been deposited in GenBank/EMBL/DDBJ databases under the accession numbers FM178779 to FM178796 and FM179771 to FM179785. Alleles of the seven MLST genes were deposited under the accession numbers FM180227 to FM180445.Sequences generated in this study are available at L. monocytogenes isolates, harbored a total of 438 polymorphisms  consisting in bi-allelic (404 sites), tri-allelic (32 sites) or four-allelic (2 sites) single nucleotide polymorphisms (SNPs). The average nucleotide diversity π was 2.91%, ranging from 1.18% to 5.98% per gene , while it differs from all other 1/2a strains by several genes. CC9 also comprised the only included isolate of serotype 3c.Remarkably, most isolates within a given clone had the same serotype, or a restricted set of serotypes. CC1 and CC2 were dominated by isolates of serotype 4b, and included all isolates of serotypes 4d and 4e. CC3 comprised a large proportion  of isolates of serotype 1/2b, and included all isolates of serotypes 3b, and all but one (ST75) isolates of serotype 7. These results suggest that serotypes 4d and 4e each derived at least twice from 4b ancestors, consistent with previous data L. monocytogenes isolates from a single year (year 2000) and the three major clinical presentations in humans: bacteremia (n = 25), CNS infections (n = 20), and maternal-fetal infections (n = 18). These isolates corresponded to 28 STs, distributed into 7 CCs and 13 singletons , we compared ngletons Table S1Possible trends in the relative prevalence of CCs over time were investigated based on 126 isolates from maternal-fetal cases of listeriosis, collected from 1987 to 2005 Table S1L. monocytogenes alleles are five times more likely to change by mutation than by recombination (r/m = 0.197). In addition, the r/m rate for nucleotides was 0.59, indicating that nucleotides are approximately twice more likely to change by mutation than by recombination. As an independent approach, the composite likelihood of r/mr/m values of some of the observed housekeeping genes exceeded 1, but lacked statistical significance  method . Close inspection of the sequence alignment showed that a large proportion of nucleotide changes that distinguished CLIP98 from lineage II strains were clustered in a small number of short segments and corresponded to nucleotide bases also observed in L. innocua strains.One exceptional isolate, CLIP98 (serotype 1/2a) isolated from a human blood infection in Canada, was placed at the tip of a long branch, thus representing an apparent fourth lineage. Individual gene genealogies based on the neighbor-joining method also placed CLIP98 outside the three lineages, except for genes The phylogenetic relationships within lineage I  as MLST genes. In contrast, over the long term, inlA sequence divergence was restricted, as housekeeping genes were on average more divergent between the three major lineages than are inlA sequences . This constraint on inlA sequence divergence resulted in the lack of phylogenetic demarcation of lineages I and III  polymorphic sites and 33 alleles undergoing a distinctive pattern of evolution Figure 4inlA was considered, purifying selection against amino-acid changes appeared more relaxed than for housekeeping genes, with a Ka/Ks ratio for inlA (0.094) higher than for the seven concatenated MLST genes (0.039 for the same 157 isolates). However, the distribution of ω (the Bayesian estimate of the rate of synonymous vs. non-synonymous substitutions) along the sequence was heterogeneous, with a highly constrained LRR-region (ω≈0.04) and moderately constrained Ig-like and B-repeats regions, with peak values of 0.5. It is worth mentioning that no single stretch of the molecule displayed a significant signature of positive selection, a result that contradicts previous analyses in which recombination was not incorporated Second, when the entire length of inlA alleles that had premature stop codons (PMSC) at positions 492, 539, 577 and 685  to the inlA sequences that are specific of clone CC9, strongly suggesting that they first occurred in isolates of clone CC9 as well. The remaining PMSC (at codon 492) was observed in ST121 (1/2a), and three other PMSCs from previous reports were also observed in inlA alleles unrelated to those in CC9 , with the exception of the three STs  inferred to derive by a recombination event that changed more than two SNPs. Hence, no significant loss of information was incurred by collapsing nucleotide sequence information into allelic profiles, as expected given limited amounts of recombination. Interestingly, the major clones were almost always separated from other strains by at least three allelic mismatches, indicating ancient divergence. Hence, a relaxed criterion  would have little impact on the assignment of L. monocytogenes isolates to particular clones. Given the neat delineation of the major clones and the fact that they account for a large proportion of clinical L. monocytogenes isolates, we propose that these genetic entities represent reference units for future studies on strain virulence, ecology or epidemiology.A majority of L. monocytogenes and are freely available for comparison at www.pasteur.fr/mlst. Other sequence-based strain characterization methods have been developed L. monocytogenes strains, as well as establishment of the correspondence among the sequence types they define, is required for optimal communication and will provide Listeria specialists with a choice of methods that may be suited for distinct purposes .For global population biology and international surveillance purposes, a definitive strain typing scheme is greatly needed L. monocytogenes clones, we found different distributions of serotypes and clones among animal, environmental and clinical isolates that are consistent with previous reports L. monocytogenes is an environmental saprophyte, which does not need to infect animals for survival and propagation. The diversity of clinical isolates may thus only represent a particular subset of the entire diversity of this species.Although our strain collection was not designed to address the important question of ecological or epidemiological differences among The evolutionary relationships of serotypes within the major lineages have not been previously defined, although this knowledge is particularly important for correct interpretation of serotyping data. We found that all serotype 4b strains belonged to a unique branch, consistent with early MLEE data  and Groups of distinct serotypes observed in isolates with the same ST or CC reflect relatively recent evolution of antigenic structures. Under our evolutionary scenario  and 2, seroL. monocytogenes strains, in agreement with a wide set of previous studies based on alternative markers L. monocytogenes lineage. Instead, the fact that two genes of CLIP98 were typical of lineage II, whereas numerous polymorphisms in the other five genes were shared with L. innocua, suggests that CLIP98 could have a ‘hybrid’ genome derived from lineage II, but which has received from L. innocua donors enough recombined fragments to now appear poorly affiliated with its ancestral lineage. Because putative recombined segments are very small and often consist of only two or three SNPs, these imports were overlooked by ClonalFrame. As three nucleotides of CLIP98 (between positions cat 364–368) were also uniquely shared with lineage III strains, CLIP98 may correspond to a recombinant strain with multiple ancestries, possibly due to an increased capacity for incorporation of foreign DNA. Further genomic characterization of this isolate is needed to clarify these aspects.The distinct phylogenetic methods used herein consistently identified three major lineages of L. monocytogenes lineages differed from L. innocua, their closest relative, by 11% of nucleotide positions on average. This is consistent with the monophyly of L. monocytogenes and does not support the hypothesis of a descent of L. innocua as a whole from L. monocytogenesL. innocua, whereas strains within a particular lineage generally shared at least one allele. Hence, consistent with DNA hybridization data L. monocytogenes needs careful evaluation. In particular, improved sampling  would be necessary to challenge the neat demarcation among lineages and characterize their ecology.L. monocytogenes undergoes low levels of recombination among housekeeping genes L. monocytogenes is one of the bacterial species with the lowest rate of recombination L. monocytogenes EDGe (CC9), but the regulatory genes for their expression have not been identified, and a noncoding RNA (RliE) may regulate negatively the L. monocytogenes orthologs required for competence in Bacillus subtilisL. monocytogenes retained the ability for localized recombination is clearly provided by the inlA gene encoding InlA, in agreement with previous reports L. monocytogenes strains may coexist. Hence, rapid diversification of inlA sequences contrasts with the inferred high stability of clonal backgrounds defined by housekeeping genes. This illustrates how the phylogenetic structure based on MLST genes provides a scaffold, which sheds light onto the evolution of individual genes exposed to selective pressures, such as virulence genes.The rate of homologous recombination within bacterial species can differ widely from one species to another L. monocytogenes clones, but a realistic scenario is that different clonal families might be adapted to different niches, and their occurrence as mammalian pathogens may be of limited significance for their evolutionary success in the long term. Among the four alleles of gene inlA identified among ST9 isolates, the one corresponding to the non-truncated form (inlA-8) can be inferred to be ancestral, as the three inlA alleles corresponding to truncated forms  differed by only one mutation from inlA-8, whereas they differed by two mutations from each other , which was inferred to branch off before diversification of other CC9 members . Alleles resulting in truncated forms of the protein InlA are indicated by a delta letter followed by the position of the stop codon. Isolates in which the alleles were observed are indicated in (0.25 MB EPS)Click here for additional data file.Table S1Strains(0.09 MB XLS)Click here for additional data file.Table S2RDP analysis(0.03 MB XLS)Click here for additional data file.


Macaca mulatta) are the primate most used for biomedical research, but phenotypic differences between Indian-origin and Chinese rhesus macaques have encouraged genetic methods for identifying genetic differences between these two populations. The completion of the rhesus genome has led to the identification of many single nucleotide polymorphisms (SNPs) in this species. These single nucleotide polymorphisms have many advantages over the short tandem repeat (STR) loci currently used to assay genetic variation. However, the number of currently identified polymorphisms is too small for whole genome analysis or studies of quantitative trait loci. To that end, we tested a combination of methods to identify large numbers of high-confidence SNPs, and screen those with high minor allele frequencies (MAF).Rhesus macaques (By testing our previously reported single nucleotide polymorphisms, we identified a subset of high-confidence, high-MAF polymorphisms. Resequencing revealed a large number of regionally specific SNPs not identified through a single pyrosequencing run. By resequencing a pooled sample of four individuals, we reliably identified loci with a MAF of at least 12.5%. Finally, we found that when applied to a larger, geographically variable sample of rhesus, a large proportion of our loci were variable in both populations, and very few loci were ancestry informative. Despite this fact, the SNP loci were more effective at discriminating Indian and Chinese rhesus than STR loci.Pyrosequencing and pooled resequencing are viable methods for the identification of high-MAF SNP loci in rhesus macaques. These SNP loci are appropriate for screening both the inter- and intra-population genetic variation. Macaca mulatta) are used more extensively as animal models for the study of human disease than any other primate species. They provide the primary model for research in infectious diseases, reproductive biology, behavior, neuroscience and immunology. More recently, they have been employed in research and vaccine development for the human immunodeficiency virus (HIV)  and itsEight of the approximately 23,000 candidate SNPs  were selMacaca mulatta individuals were selected for SNP confirmation. DNA was extracted from EDTA-preserved blood samples using the QIAmp Blood Mini Kit . All of these individuals  had previously been sequenced for 835 bp of mtDNA and classified into one of six regional haplotypes: IND1 (Indian); CHIE (Eastern China); CHIW1, CHIW2, and CHIW3 ; and CHIS (Southern China) . Each STR primer pair was optimized for annealing temperature and extension time: 94°C for 3 min., then 60 cycles of 94°C for 20 sec., 54–62°C (-0.1°C/cycle), 72°C for 45–90 sec., and a final extension at 72°C for 5–60 sec. All samples were analyzed on an ABI 310 sequencer using the Liz 500 size standard, and the ABI GeneScan software to assign genotypes. Some of the STR genotypes included in this study were also included in 4)2SO4, 0uded in [. Principuded in [ package Experiments conceived and designed by RSM, SK, and DGS. JAS and RYT performed the experiments. RSM, JAS, RYT and VSM analyzed the data. RSM and DGS contributed reagents, materials and analysis tools. JAS, RSM, SK and DGS wrote the paper.The file "Additional_File1.doc" has been uploaded. This file is a table in Microsoft Word format. The data is titled The file is titled: "Primer Information for Rhesus SNP Resequencing" and includes the name of the pyrofragment in which the SNP was located, the chromosome and nucleotide position, the direction of the change, the number of overlapping pyrofragments and the Phred score of the SNP.Click here for fileThe file "Additional_File2.doc" has been uploaded. This file is a table in Microsoft Word format. The data is titled: "All validated SNPs", and includes the chromosome, nucleotide position, the name of the 454 fragment in which the SNP was originally discovered, the polymorphism, the observed heterozygosity in the sample of Chinese animals, observed heterozygosity in the sample of Indian animals, the gene or feature in which the SNP is located (if any), the nearest genes or features at both the 5' and 3' sides (with a maximum distance of 1.5 Mb).Click here for file


Economic assessments and modeling studies suggest that these programs yield cost savings and public health benefits. 2  and ≈$612/km2 [$500/km2 in 1999 Can$], respectively), but these tactics have proven crucial to elimination of raccoon rabies in Canada and to maintenance of ORV zones for preventing the spread of raccoon rabies in the United States. Economic assessments have shown that during rabies epizootics, costs of human postexposure prophylaxis, pet vaccination, public health, and animal control spike. Modeling studies, involving diverse assumptions, have shown that ORV programs can be cost-efficient and yield benefit:cost ratios >1.0.Progressive elimination of rabies in wildlife has been a general strategy in Canada and the United States; common campaign tactics are trap–vaccinate–release (TVR), point infection control (PIC), and oral rabies vaccination (ORV). TVR and PIC are labor intensive and the most expensive tactics per unit area (≈$616/km Canis familiaris) in both countries, but rabies persists in wildlife reservoirs. In 2007, a total of 6,776 cases in wildlife were reported for the contiguous United States  is an evolving rabies control technology for use in wildlife (Vulpes vulpes) in Switzerland; subsequent programs were reported throughout much of western Europe (We reviewed the literature on ORV programs and economics in Canada and the United States. The first use of ORV sought to control rabies in red foxes (Alopex lagopus)–variant rabies that had spilled into  red foxes and spread southward . The strategy was termed progressive elimination and resembled an expanding ORV wedge, which started near the center of the outbreak and expanded during successive years  and livestock dropped from preepizootic (30-year) means of >36 and >42, respectively, to 0 by 1997  and skunks, trap–vaccinate–release  was integrated into ORV campaigns in the Toronto area (2) in a 60-km2 portion of the city, and 66,168 ORV baits were distributed by hand in natural areas (20–40 baits/km2). Of sampled foxes, 46%–80% had biomarkers from baits, and only 1 rabid fox was found during 1987–1992  were distributed in an arc-shaped band over a 24-county area  ≈200 km north of Laredo  and 241 other domestic and wild animals in west-central Texas were confirmed positive for a unique rabies variant typically found in gray foxes (2) was conducted annually by encircling the epizootic area using ≈32-km–wide ORV strips; an added 16- to 24-km vaccination buffer of ORV baits was created along the northern and eastern edges of the rabies-variant area; this tactic has been referred to as a purse string–like tactic   . An areaIn 2007, new cases of gray fox rabies occurred northwestward along the Pecos River and in west-central Texas. To prevent further spread of this variant, ORV was used . The rabies-control goal has not changed from one of containment and elimination of the gray fox variant from Texas. However, in light of recent surveillance, the anticipated strategy of establishing and maintaining an ORV zone along the Rio Grande River to prevent potential reemergence from Mexico has been delayed and is being refined to include prolonged enhanced surveillance as a key factor in allocating resources and gauging success .2) to create a 40–50 km zone of vaccinated raccoons to help prevent the spread of the virus  have characterized this effort as of 2007 . On the basis of rates of spread of 30–60 km/year in the Mid-Atlantic states before 1997 (2). Estimated raccoon vaccination rates, based solely on the index of rabies virus neutralizing antibody response, range from 10% to 55%  along the Appalachian Ridge. A total of 58 bait distributions  totaling ≈41,018,800 baits and covering ≈530,825 kmTo maintain the integrity of the Appalachian Ridge ORV zone, contingency actions have been needed. In 2004, emergency ORV baiting and TVR were used in northeast Ohio between the established ORV zone and the eastern suburbs of Cleveland . Currently, ORV is used twice a year  in the eastern half of the Cape, and baiting is moved gradually westward until an ORV zone can be reestablished along the Cape Cod Canal . Additionally, to prevent raccoon rabies from reemerging in southern Ontario, ORV baiting for raccoon-variant rabies continues in northern New York. Confirmed positive raccoon-variant cases in southern Quebec have led to extensive PIC and ORV campaigns to prevent the disease from reaching Montreal. Together, these events and contingency actions illustrate the challenges posed by raccoon rabies, the importance of enhanced surveillance, plus the need to anticipate unexpected contingency actions and their related costs as a component of ORV campaigns.–2  to $16.13/county resident at $1,503/km2   was $3,356/patient ; 69% of the cost was for biologics  (Recently (1998–2002), rabies exposure costs were estimated at $4,066/patient  in southern California ; costs reported for 3 PIC operations for raccoons totaled $469,247  and $198/km2 . Cumulative cost of the Appalachian Ridge ORV program has totaled ≈$57 million since its inception in 1997; baits accounted for 72% of the funds expended . Because of improved production efficiency, bait prices have decreased slightly during the past 5 years.One ex post study  provided detailed estimates of PEP administrations in Ontario during 1956–2000 , $10.78/km2  aerial distribution, and $18.75/km2  post-ORV evaluation. The effect of an epizootic was calculated in terms of unit human population within bands. Results showed that all 8 scenarios, except the 40 km/year spread rate with 20-year fixed baiting costs, yielded BCRs >1.1 and that total estimated net present values of the program were $48–496 million with >$96 million in discounted program costs  ranged from $35.4 million to $99.3 million  . BCRs ranged from 0.49 to 1.36, and outputs implied a lag effect for savings; BCRs were <1.0 during 1990–1992 and Recently, an ex post modeling analysis was performed for the 1995–2006 ORV program that eliminated canine-variant rabies from southern Texas  has been spent on ORV programs in North America during the past 10 years. Programs have proved lengthy , have required enhanced surveillance, and have often required contingency actions to ensure rabies elimination without reintroduction.Rabies campaigns have been relatively expensive. We estimate that Most economic assessments and modeling studies indicate that ORV programs can yield cost savings Correction for Inflation of Selected Costs in Original PublicationsPrinciples of an Economic Analysis of Oral Rabies Vaccination Programs


Fisheries catches represent a net export of mass and energy that can no longer be used by trophic levels higher than those fished. Thus, exploitation implies a depletion of secondary production of higher trophic levels (here the production of mass and energy by herbivores and carnivores in the ecosystem) due to the removal of prey. The depletion of secondary production due to the export of biomass and energy through catches was recently formulated as a proxy for evaluating the ecosystem impacts of fishing–i.e., the level of ecosystem overfishing. Here we evaluate the historical and current risk of ecosystem overfishing at a global scale by quantifying the depletion of secondary production using the best available fisheries and ecological data . Our results highlight an increasing trend in the number of unsustainable fisheries  from the 1950s to the 2000s, and illustrate the worldwide geographic expansion of overfishing. These results enable to assess when and where fishing became unsustainable at the ecosystem level. At present, total catch per capita from Large Marine Ecosystems is at least twice the value estimated to ensure fishing at moderate sustainable levels. Fisheries can cause depletion of target and non-target species c, 5), which allows for the evaluation of the trophic position of marine organisms removed from exploited ecosystems, and showed a global trend of fishing down marine food webs; and (iii) the Fishing in Balance index , which integrates the TL of caught species and the Transfer Efficiency (TE) of energy flows in the food web, and it allows to evaluate if exploitations at different trophic levels are ecologically balanced over time. In order to obtain a comprehensive measure to quantify the consequences of marine fishing activities at an ecosystem level, the total losses in secondary production, due to fishing was recently formulated integrating previous analyses Several efforts to quantify the pressure exerted by fishing activities on marine ecosystems worldwide have led to the development of ecological indices. Three important ones are (i) the Primary Production Required to sustain marine catches , normalized to the primary production (%PPR) and representing catches of different species in uniform energetic units; (ii) the mean Trophic Level of catches (TLsensu Murawski sust). These estimates allowed deriving a relationship between L and psust that can be used to evaluate the risk of ecosystem overfishing sust at for e.g., 75% (EMSC75) and 95% (EMSC95) and assuming constant fishing strategies, i.e. by maintaining current TLcFisheries catches represent a net export of mass and energy that can no longer be used by trophic levels higher than those fished. Thus, exploitation implies a depletion of secondary production of higher trophic levels due to the removal of prey. Based on this assumption, a new method was developed to quantify the loss in secondary production (L index) due to the removal of marine organisms through catches  compared to a theoretical unfished situation c, and contains P1 estimates. These, together with frequency distribution of TE for ecosystem types sust for 66 Large Marine Ecosystems (LMEs), and 17 FAO areas (open sea oceanic areas outside LMEs). The L index and psust were computed for each area and for each year of the period 1950 to 2004 using both official catches, and catches corrected to include discards In order to quantify L at a global scale and evaluate the global sustainability of marine fisheries we use here the best available geographically referenced database of world catches sust≪75%) (sust = 40.9%), West Greenland Shelf (33.2%), Norwegian Shelf (27.4%), North Sea (24.2%) and North-eastern US Continental Shelf (22.9%), Faroe Plateau (15.1%), Iceland Shelf (14.3%), and Yellow Sea (12.9%). Within tropical LMEs, the Sulu-Celebes Sea (37.9%) and the Gulf of Mexico (35.4%) had the lowest psust values. Seas around China showed particularly intense exploitation , despite the SAUP database has been corrected for over-reporting of catches in the area sust≥95%), with the Western-Central Pacific ocean (psust = 63.7%) scoring the lowest. When aggregating data on the basis of ecosystem type (sust = 53.8%). This is chiefly due to the high catch rates of lower trophic organisms, mainly small pelagic fish . Temperate and high latitude, and tropical areas, ranked intermediate with mean psust values of 63.7% and 71.60%, respectively. All FAO areas combined showed the highest mean probability of being sustainably fished (psust = 95.7%). However, higher risk of overfishing was assessed when higher percentages of IUU were considered  and top-down (fishing pressure) effects. However, this analysis could be certainly improved by including higher spatial resolution of catch statistics and new environmental data, such as time series on primary production when such information is available with global coverage. Given the importance of discards and unreported catches on the quantification of ecosystem impacts of fishing, the current assessment using official catch data and conservative IUU% estimates may be too optimistic for several areas known to have high levels of unreported catches and limited monitoring of fisheries such as in open seas and coastal areas under the control of countries with low monitoring capacity L index results might be affected, other than by uncertainty on input data, by limitations in capturing some dynamic changes occurring in the ecosystem Despite some limitations, L index allows for a robust evaluation of the risks of marine ecosystems to overfishing and illustrates when and where fishing became unsustainable at the ecosystem level since the 1950s. It highlights notable risk of ecosystem overfishing for several LME areas, which supply the bulk of marine production to human populations. These results confirm previous concerns about the sustainability of fishing activities at a global scale i, 8) and the ecological role  of caught organisms and incorporates elements of ecosystem function . As secondary production we intend the production of mass and energy by herbivores and carnivores in the ecosystem.The Loss in secondary production index (L) due to fishing takes into account the amount , the Trophic Level (TLi) of these species, P1, and the TE in the ecosystem's trophic flows:c, 5). Similarly to FiB index and PPR, L weights the catches by using the TL of fished species. However, whereas other indices measure what is taken from the system Thus, L for a given ecosystem can be expressed as a function of the PPRc), primary production required to sustain the catch (PPR), primary production (P1) and transfer efficiency (TE) were calculated from 51 ecosystems using mass-balance model results Reference levels for the L index were quantified by using a set of well documented mass balance models representative of exploited ecosystems distributed worldwide, considering model-independent information on ecosystem status sensu Murawski Each model was then classified as representing an overfished or sustainably fished ecosystem based on the ecosystem overfishing definition sust). For any given value of the L index, say L*, the number of models of overexploited ecosystems with L<L*, i.e. P(L1<L*), and the number of models representing sustainably fished ecosystem with L>L*, i.e. P(L2>L*) allowed the estimation of psust for L* as in the following:Using the frequency distribution of L values it was possible to calculate, for each ecosystem, the probability for the ecosystem to be classified as sustainably fished . Limitations of the L index have been discussed in detail elsewhere A jackknife resampling method p, based on a reference psust = p, can be achieved with different strategies combining appropriate values of target TLc, %PPR, TE and P1. Assuming a constant fishing strategy fishery  and a constant ecosystem function , target %PPRp can be estimated by inverting Eq. (1). On the basis of target and actual PPR  along with actual catches (Y0), the corresponding Ecosystem-based Maximum Sustainable Catches (EMSCp) The target value Lp represents the maximum catch that allows to achieve a given reference level of psust = p. Therefore EMSC75 and EMSC95 for the two reference values chosen  represent a practical, though approximate, guide for fishery management.EMSC−2·y−1, P1* = 200 gC·m−2·y−1, TE* = 10% and TLc* = 2.5).In order to explore the propagation of uncertainty of each of the input variables to the value of the L index, local analytical and global numerical sensitivity analyses were performed from representative nominal values  confirmed that a) L index errors are highly dependent on uncertainty connected with TLc, b) TE and other parameters have secondary effects, c) overall, L estimates are robust to realistic levels of uncertainty in input values. These results imply the need for great accuracy when estimating TLc, by using the best available estimates for species TLs and catch statistics disaggregated at the lowest possible level.Global sensitivity was also explored by means of MonteCarlo simulations simultaneously accounting for errors in PPR, P1, TL−1) from 1950 to 2004, by Large Marine Ecosystems (LMEs) and open sea areas , were obtained from the Sea Around Us project (SAUP) Global Fisheries Mapping data . This database contains the available data from FAO (www.fao.org), complemented with regional and national catch statistics, which are re-expressed on a spatial basis. The SAUP database also provides primary production (mgC·m−2·day−1) from 1998 and surface (km2) estimates for each LME and open sea (FAO area). The primary production data are based on a model whose parameterization varies between biomes and biogeochemical provinces, and that estimates depth-integrated primary production based on chlorophyll pigment concentration as derived from SeaWiFS data and photosynthetically active radiation calculated with a spatial resolution of 9 km www.seaaroundus.org.Data concerning landings  catches for LMEs and open sea (FAO areas). Discard data were available for the period 1992–2002 Following the climatic distribution i, was taken from Fishbase (www.fishbase.org), the Catalogue of fishes  and Cephbase (www.cephbase.utmb.edu) to estimate the mean trophic level (TLc) of the catch (Y) Trophic level of each caught species or group of species (i), i.e. TLc has the major influence on index values. Therefore, this parameter was estimated as accurately as possible for each ecosystem by using disaggregated data. The %PPR was calculated applying the following equation 1) for each area.According to sensitivity analyses, TLst quartile = 3.9, 3rd quartile = 6.1), temperate and high latitude areas , tropical areas  and global marine ecosystems  . Instead of using a single representative value of TE for each group of models, the original distribution of TE values for each ecosystem type was used for estimating L index, psust and EMSC. This was done by implementing repeated calculations using TE values extracted from the distribution of TE typical for each ecosystem type. This procedure generated distributions for L, psust and EMSC for which statistical indices were calculated . Since obtained distributions are seldom symmetrical, the median and interquartile range are reported and used for summarizing results.Characteristic frequency distribution of TE values by ecosystem type were derived from a set of ecosystem models http://www.census.gov/ipc/www/idb/).Global population estimates were taken from the U.S. Census Bureau, International Data Base  as resulting from changes in input parameters  around their nominal value. Each curve results from sensitivity analyses on one single parameter indicated between parentheses, L(X).(0.04 MB DOC)Click here for additional data file.Figure S2Results of global sensitivity on L index obtained by randomly choosing the four input parameters from normal distributions . White and red circles represent median and mean values, respectively.(1.12 MB DOC)Click here for additional data file.Table S1Global assessment of ecosystem overfishing for Large Marine Ecosystems (LME) and Open Sea (FAO areas) for the period 2000–2004.(0.07 MB DOC)Click here for additional data file.Table S2Assessment of ecosystem overfishing for Open Sea (FAO areas) for the period 2000–2004 including higher estimates of IUU catches (results follow the ones presented in (0.03 MB DOC)Click here for additional data file.Table S3Global assessment of ecosystem overfishing for Large Marine Ecosystems (LME) and Open Sea (FAO areas) for the period 1990–1999.(0.05 MB DOC)Click here for additional data file.Table S4Global assessment of ecosystem overfishing for Large Marine Ecosystems (LME) and Open Sea (FAO areas) for the period 1970–1979.(0.06 MB DOC)Click here for additional data file.Table S5Global assessment of ecosystem overfishing for Large Marine Ecosystems (LME) and Open Sea (FAO areas) for the period 1950–1959.(0.05 MB DOC)Click here for additional data file.


We describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS.The assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing.The simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories. Genetic differences contributed to phenotypic diversity of humans or pathogens, including variation in disease susceptibility and drug response. The genotypic analysis to identify the polymorphisms that differentiate one individual or strain from another has become increasingly important as a prognostic measure of disease courses and to enable choice of more efficacious therapeutic or preventive options based on individual genetic makeup. Due to the complexity of many common, chronic diseases and quantitative traits and the confounding effects of disease heterogeneity, gene-gene interaction, and gene-environment interaction, a large number of the polymorphisms must be surveyed in numerous individuals. These progresses highlight the need for rapid, accurate, and efficient methods that permit high throughput genotyping.The most commonly used methods for genotype readout are based either on fluorescence or mass spectrometry (MS). Fluorescence readout is quite sensitive but often relies on secondary reporter systems for detection ,2. In coMTHFR) gene, known to be associated with hyperhomocysteinemia and cardiovascular diseases, using RFMP assay and also assessed the potential for application to determination of allele frequencies in DNA pools.Here, we describe the development of a novel MALDI-TOF MS-based SNP scoring strategy, termed RFMP, that is suitable for genotyping SNPs in a simple, efficient, and high-throughput manner. The assay is based on PCR amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. We demonstrated fast, reliable genotyping of five SNP markers in methylenetetrahydrofolate reductase (FokI site (an isoschizomer of BstF5I) in the amplified product by substituting the restriction recognition sequence GGATG for the ninth base upstream of the polymorphic nucleotide. The backward primer was designed to make the resulting amplicon as short as possible while both primers' Tm values matched with each other for better PCR yield. This short inserted sequence in general is not expected to base pair to the template strand, but rather loops out when the primer is bound to the template. When the complementary strands are copied the inserted sequences are incorporated into the product. Both FokI and BstF5I are typeIIS restriction enzymes that cleave DNA outside the recognition sequence. The FokI enzyme cleaves DNA 9 bases 3' to the recognition site on one strand and 13 bases from the recognition site on the other strand, leaving a four base overhang protruding 5' end. BstF5I cleaves DNA 2 bases 3' to the recognition site on one strand and immediately 3' to the recognition site on the opposite strand, leaving a two base overhang. Thus digestion of the resulting amplification product with FokI followed by BstF5I should result in excision of a 7-mer fragment and a 13-mer fragment. As summarized in Table The RFMP assay is based on mass spectrometric analysis of small DNA fragments containing site of polymorphism, as illustrated in Fig. RFMP assay established in this study exploits differences in the molecular masses of oligonucleotides comprising the nucleotide variations. The assay is based on amplification and mass detection of oligonucleotides excised from typeIIS enzyme digestion using MALDI-TOF MS. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose mass was determined by MALDI-TOF MS. This genotyping assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a fluorescent or radioactive report tag is interpreted. Further, both DNA strands can be analyzed in parallel, and the specific target amplification can be validated simultaneously with mass analysis, providing a level of internal confirmation not achievable by other methods. The use of a typeIIS restriction enzyme makes this assay independent of the fortuitous occurrence of restriction sites, because these enzymes have cleavage sites distal to their recognition sites. Recognition sites are incorporated into the amplification primers, and short fragments that contain the polymorphisms can be generated for mass spectrometric analysis. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel.Mass spectra were acquired on a linear MALDI-TOF MS  workstation equipped with a 337 nm nitrogen laser and a nominal ion flight path length of 1.25 m as previously described with slight modification . The samMTHFR gene by RFMP method . We used C18 reverse phase micro-column chromatography as an effective and inexpensive means for desalting oligonucleotides with since it was recyclable by repeated washing with isopropanol up to ten times without remarkable loss of efficiency. We have used fructose as matrix additive for less dependency of the quality of mass spectra on laser power for better ion abundance without compromising mass resolving power as previously described ,22. Furt2 = 0.984, slope = 1.033, p = 0.755; rs2066462: R2 = 0.965, slope = 1.011, p = 0.650). Using the pooling strategy a minor allele with a frequency as low as 0.05 in the pool could be accurately detected for both markers. We have not observed that the significant difference obtained in dynamic range depends on which spectral band is chosen for quantitation between 2 restriction fragments, 7 mer and 13 mer. The 7 mers showed a lower limit of minor allele detection as low as 5%, and a dynamic range of 0.05 to 0.95 with R2 equal to a mean of 0.912 for both markers. Considering that 13 mer fragment has 4 additional bases adjacent to the polymorphism originated from target sequences while the 7 mer has only the polymorphic base, the result suggest better reflection of 13 mer fragment on real abundance due to the advanced target specificity.To test for a relationship between estimated frequencies in pools and direct counts from individuals, ratios were calculated using the mean peak areas generated from multiple mass spectra and compared to the defined pooling ratios. For the markers rs1801133 and rs2066462, we tested if there was a linear relationship between estimated and real allele frequencies. By analysis of artificial pools representing one allele in the frequency range from 0.05 to 0.95 a linear relation of estimated allele frequencies to the expected ratios was observed with the 13 mer fragment (rs1801133: RAllele frequency estimation of five SNPs selected from public databases was made in pools generated from the Korean population (192 subjects), and compared with results from individual genotyping. The minor allele frequency by the annotation standards of dbSNP database for the particular marker ranged from 0.093 to 0.872  have been suggested as a cause of cardiovascular disease, colorectal neoplasias, neural tube defects, and pregnancy complications, especially in homozygotes for C677T, but also in compound heterozygotes for C677T/A1298C [Hyperhomocysteinemia is caused by low intake of folate and other B vitamins and by genetic factors, including polymorphisms of genes encoding enzymes involved in homocysteine remethylation, such as  pathway . SNPs wie status . The polT/A1298C ,28. The MTHFR 677T alteration has occurred once on the 31T-1298A haplotype (93.7%) as previously described by Rosenberg et al [MTHFR 677T alteration on a founder haplotype that may have had a selective advantage. The presence of a high prevalence of the specific haplotypes within MTHFR gene suggests compounding effects of the SNPs in occurrence of complex diseases and might be worth further investigation for better predicting power in genotype-phenotype association studies.For analysis of a possible linkage among C677T (rs1801133), A1298C (rs1801131) and other polymorphisms , haplotypes of the five variations in Korean population (N = 192) were built by employing a computational method . The results showed that the population are composed of 8 haplotypes, and haplotype M1 and M2 were the most prevalent, constituting 71.9% of the samples, which are 677T  or 677C  with residual SNPs all common alleles Table . Of interg et al . It is aMTHFR gene, known to be associated with hyperhomocysteinemia and cardiovascular diseases, and also provided the potential for application to determination of allele frequencies in DNA pools as a means of efficiently screening SNPs and prioritizing them for further study. Therefore, we believe that the simplicity, accuracy and amenability to high-throughput screening analysis make the RFMP assay suitable for large-scale genotype association study and a routine genotyping platform in clinical laboratories.In conclusion, the RFMP assay for SNP scoring utilizing mass difference of oligonucleotides requires the simple steps of single PCR amplification and restriction enzyme digestion, and is amenable to high-throughput system. The assay represents an improvement over previous methods in reliance on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted, both DNA strands being analyzed in parallel, and the ensured specific target amplification simultaneously with mass analysis, providing an additional level of assay precision. Using RFMP assay, we demonstrated highly reliable genotyping of five SNP markers in SNPs rs1801133, rs2066462, rs1994798, rs2066470, and rs1801131 . The amplification conditions included initial denaturation at 94°C for 2 min, 10 cycles of denaturation at 94°C for 15 sec, annealing at 50°C for 15 sec and extension at 72°C for 30 sec, followed by 35 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 30 sec. The respective sequences of forward and reverse primers used in the PCR for each SNP site are summarized in Table FokI site in amplicon as shown in Figure Informed consent was obtained form all subjects and experimental protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. 50 ng of the genomic DNA was used for the PCR reaction. PCR was performed in 18 μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.2 mM of each dNTP, 10 pmol of each primer, and 0.4 units of PlatinumFokI and BstF5I at 37°C for 15 min. The resulting digest was purified by vacuum filtration through a 384-well sample preparation plate containing 5 mg of polymeric sorbent  per well using Microlab 4200 robotic liquid handler . Each well was equilibrated with 90 μl of 1 M triethylammoninumacetate (pH 7.6). Each cleavage reaction mixture was added to 70 μl of 1 M TEAA, pH 7.6 and loaded into a well. After rinsing 5 times with 85 μl of 0.1 M TEAA pH 7.0, the plate was reassembled on a vacuum manifold and eluted with 60 μl of 60% aqueous isopropanol into a collection plate, which was placed on a heating block at 115°C for 90 min. The desalted reaction mixtures were resuspended with matrix solution containing 50 mg/ml 3-hydroxy picolinic acid, 0.05 M ammonium citrate, 5 mg/ml of fructose, and 30% acetonitrile, and were spotted in 3 μl volumes on a polished anchorchip plate  using Microlab 4200 robotics or resuspended with distilled water and dotted in 2 μl to pre-spotted anchorchip plate which had only matrix crystallized in advance. Mass spectra were acquired on a linear MALDI-TOF MS  workstation. Spectra were acquired in a positive ion, delayed extraction mode. Typically, time-of-flight data from 20 – 50 individual laser pulses were recorded and averaged on a transient digitizer, after which the averaged spectra were automatically converted to mass by data processing software .Restriction enzyme digestion of PCR products was performed by mixing the PCR reaction mixture with 10 μl of buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, and 1 unit of each The RFMP results were compared with the results from either direct sequencing or the clonal sequencing assay. When direct sequencing results were not decisive, we cloned the PCR products into the pCR-Script Amp cloning vector , for sequence analysis of each clone. Sequence analysis was performed by ABI PRISM 310 Genetic Analyzer . The primers used for Snapshot assay were designed and the primer extension reactions were carried out with SnaPshot™ multiplex mix (Applied Biosystems) according to manufacturer's recommendations. The reaction mixtures were run on ABI3700 (Applied Biosystems) using POP6 polymer and analyzed by Genescan program.et al [The concentration of the DNAs used to construct pools was measured using the Picogreen reagents and kits . The DNAs were diluted to a final concentration of 8 ng/μl and equal amounts of DNA were mixed to form the pools. Range pools were constructed by mixing appropriate volumes of homozygote DNA. The concentrations ranged from 50–50% to 95-5%, with 5% increments. Allele frequencies were calculated using peak areas generated from mass spectra. All spectra were smoothed by applying a 21-point Savitzky-Golay filter function  to minimize noise errors. Peak areas were estimated using TOF 1.6 m taking into account baseline correction and the noise level of the spectrum. To evaluate the reproducibility of the frequency estimates, assays were performed in 5 replicates for each pool and marker. In order to take unequal representation of both alleles in the mass spectrum into account, at least five heterozygotes were genotyped individually as recommended by Le Hellard et al . The meaet al .MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight; RFMP: restriction fragment mass polymorphism; PCR: polymerase chain reaction; MS: mass spectrometry; RFLP: restriction fragment length polymorphism; SNPs: single nucleotide polymorphisms; MTHFR: methylenetetrahydrofolate reductase.Experiments were designed by SPH, WY and S–OK. SPH, SIJ, SKS and SYH performed the experiments. SPH, WY, H–BO, SHL, SDL and S–OK analyzed the data. HR contributed software and analysis tools. SPH and S–OK wrote the paper. All authors read and approved the final manuscript.PickCamp ver9. Software for designing engineered primers and calculating predicted mass values for SNP scoring by RFMP assay.Click here for file