Myzus persicae, is recognized as one of the most important agricultural pests worldwide. Uses over 400 plant species and has evolved different insecticides resistance mechanisms. As M. persicae feeds upon a huge diversity of hosts, it has been exposed to a wide variety of plant allelochemicals, which probably have promoted a wide range of detoxification systems.Among herbivorous insects that have exploited agro-ecosystems, the peach-potato aphid, M. persicae can give an advantage in terms of reproductive fitness when aphids face two hosts, pepper (Capsicum annuum) a suitable host and radish (Raphanus sativus) the unfavorable host and (ii) examined the transcriptional expression of six genes that are known to be up-regulated in response to insecticides. Our results show a significant interaction between host and IRM on the intrinsic rate of increase (mr). Susceptible genotypes (not carrying insensitivity mutations) had a higher mr on pepper, and the transcriptional levels of five genes increased on radish. The mr relationship was reversed on the unfavorable host; genotypes with multiple IRM exhibited higher mr, without altering the transcriptional levels of the studied genes. Genotypes with one IRM kept a similar mr on both hosts, but they increased the transcriptional levels of two genes.In this work we (i) evaluated whether insecticide resistance mutations (IRM) in mr than susceptible genotypes on radish, the more unfavorable host. Susceptible genotypes should be able to tolerate the defended host by up-regulating some metabolic genes that are also responding to insecticides. Hence, our results suggest that the trade-off among resistance mechanisms might be quite complex, with a multiplicity of costs and benefits depending on the environment.Although we have studied only nine genotypes, overall our results are in agreement with the general idea that allelochemical detoxification systems could constitute a pre-adaptation for the development of insecticide resistance. Genotypes carrying IRM exhibited a higher  The evolution of insecticide resistance is one of the best-known examples of Darwinian microevolution on an ecological time-scale Myzus persicae (Sulzer), is recognized as one of the most important agricultural pests worldwide M. persicae has developed resistance to all of them through either metabolic or target site mutation mechanisms kdr) and super-kdr mutations in voltage-gated Na+ channels Rdl receptor M. persicae shows resistance through over-production of E4 or EF4 esterases Among the groups of herbivorous insects that have successfully exploited the agricultural environment, the green peach aphid, Regarding those metabolic and target site insensitivity mechanisms, several authors have proposed that plant allelochemical detoxification systems found in insects have served as a pre-adaptation for the acquisition of insecticide resistance M. persicae feeds on a wide diversity of hosts, therefore being exposed to a range of phytochemicals, thus favoring a great diversity of enzymatic detoxification systems M. persicaeMyzus persicae nicotianae) to feed on tobacco plants (Nicotiana tabacum) Two lines of evidence have contributed to this hypothesis. First, insecticides not only resemble plant chemical defenses in their structure, but some are also derived from them(e.g. pyrethroids and neonicotinoids) M. persicae clones carrying different insecticide resistance mechanisms under distinct environmental regimes imposed by the host plant. First, we evaluated the reproductive fitness of different aphid genotypes carrying or not MACE and kdr insensitivity mutations, which were reared on suitable and unfavorable host plants. Second, we compared the transcriptional levels for six specific genes on aphid genotypes reared on both hosts. In particular, we selected some genes coding for Cathepsin B, Heat Shock Protein 70, Glutathione S-Transferase, Carboxylesterase and Cytochrome p450 family CYP6 and CYP4. We selected those genes because they showed the highest up-regulation in expression (ranging 2–5 fold change) in a previous work where M. persicae individuals were subjected to insecticides The fecundity of adult aphids is one of the most frequently studied traits used to characterize the ability to feed on different hosts, because it is expected that specialization in herbivorous insects has evolved towards an optimal exploitation of the host in terms of maximizing individual fitness kdr mutation and five were heterozygote for both kdr and MACE mutations. These genotypes were labeled simple resistant (i.e. SR) and multiple resistant (i.e. MR), respectively. No genotypes was found to carry either MACE or kdr mutations in homozygote state, or carrying a super-kdr mutation. Of the 44 genotypes evaluated for constitutive carboxylesterase activity (EST activity), thirty-two genotypes were classified as susceptible (S), ten as moderately resistant (R1) and two were highly resistant (R2), following the nomenclature proposed by Devonshire et al. (1992) Of a total of 44 multilocus genotypes studied, thirty-three did not carry any resistance mutations and were labeled as sensitive (i.e. S). Six genotypes were heterozygote for mr) and body mass, for each tested genotype on both host plants (suitable and unfavorable). The results shown no variation for reproductive fitness . In addition, not all genotypes responded in the same fashion to both hosts; that is, there was an interaction between genotype and host . In particular, on pepper, the S genotypes had a higher reproductive fitness than SR and MR genotypes. However, the relationship was reversed on radish: S genotypes showed a lower reproductive fitness than SR and MR ones  when aphids were reared on radish (n radish . GenotypHeat Shock Protein 70 gene – A significant up-regulation was observed only in the genotype MR (16A) when reared on radish. The transcriptional levels for the remaining genotypes was not significantly different from 1  showed an up-regulation of the E4/FE4 gene when aphids were reared on radish (E4/FE4 gene were not significantly different from 1 in the other genotypes.n radish . The traCytochrome P450 genes – Two genes belonging to this family were assessed (CYP6CY3 and CYP4). The genotypes SR and MR evidenced a slight but not significant down-regulation for both genes  found in a pest aphid in Chilean agroecosystems can provide an advantage in terms of reproduction success when aphids are faced with well-defended host plants. This is particularly interesting when studying a highly polyphagous insect that is able to feed on more than 50 different plant families exhibiting a vast range of chemical defenses against herbivorous. The subject of this study was to determine whether the diversity of possible hosts and their defenses has promoted the evolution of different aphid counter-defense mechanisms that can be also involved in insecticide resistance. Therefore, we evaluated the impact of host plants with different levels of allelochemicals on: (i) the reproductive performance of aphid genotypes carrying or not MACE and kdr insensitivity mutations and, (ii) the transcriptional expression for six selected genes. Particularly, we selected genes coding for Cathepsin B, Heat Shock Protein 70, Glutathione S-Transferase, Carboxylesterase and Cytochrome p450 family CYP6 and CYP4, as they were observed to be highly up-regulated (ranging 2–5 fold change) in a previous work where M. persicae genotypes were subjected to insecticides In this work we examined the potential involvement of specific genes to determine whether point mutations at insecticide target sites . This significant inter-clonal variation in performance has also been reported by other authors kdr mutation on important behavioral traits have been reported for M. persicae, particularly in response to environmental signals such as presence of natural enemies (parasitoids) and low temperatures, which ultimately could lead to reduced fitness Although we have only studied nine genotypes, in overall terms our results are in agreement with the general idea that allelochemical detoxification systems can serve as a pre-adaptation for the development of insecticide resistance. In our case, the SR and MR genotypes exhibited a higher reproductive fitness than S genotypes on the defended host. However, costs and benefits associated with phenotypic expression depend on the locations of fitness optima in different environments On the other hand, the overall lower reproductive fitness showed by S genotypes on radish might be indicative of a cost. Two S genotypes (N36-1 and Sur25A) showed the greatest variation in fitness between hosts and thus were selected for transcriptional analyses. Those S genotypes were also the most plastic in terms of transcriptional variation, showing an important up-regulation in five of the six genes evaluated on the defended host. Therefore, the lower reproductive fitness shown by these two genotypes on the defended host might be indicative of an energetic trade-off, suggesting non-adaptive plasticity pre-adaptation that enables aphids to deal with insecticides, then it would be expected that genotypes carrying no resistance mechanism should be able to tolerate the defended host by up-regulating specific metabolic genes in the same way as they do when exposed to insecticides cathepsin B clado N; heat shock protein 70; glutathione S-transferase; Esterase E4/FE4, cytochrome p450 family CYP6CYP3 and CYP4).If resistance to plant chemical defenses is a M. persicae, PIs interfere with aminoacid assimilation, having a negative impact on fitness cathepsin B gene in both S genotypes. This could be a counter-defense mechanism against the PIs in radish plant or to supplying aminoacids for the biosynthesis of the enzymatic machinery needed for detoxification.The Cathepsins B are enzymes with cysteine protease activity that process exogenous polypeptides into aminoacids, which are used to synthesize their own proteins by sap-sucking insects Heat shock proteins-70 (Hsp70) are a family of well known proteins involved in cell protection and repair, reducing protein aggregation and unfolding no-native protein conformations caused by environmental stress glutathione S-transferases (GST), cytochrome P450  and carboxylesterases (ESTs).GST enzymes participate in the detoxification of xenobiotic substrates by conjugation of glutathione to electrophilic toxic molecules M. persicae have been reported as a response to glucosinolates and isothiocyanates GST gene was consistently higher in S and RS genotypes reared on radish.The other studied genes belong to the three gene families that typically participate in detoxification of plant chemical defenses and metabolic resistance to insecticides: M. persicae. However, this metabolic resistance is due to up to 80-fold duplication of the carboxylesterases genes (E4 and FE4) E4/FE4 gene when aphids were reared on the defended host. The other S genotype studied (South 25 A), had a constitutive E4/FE4 expression more than twenty-fold than genotype N36-1 , which could explain the absence of up-regulation of this gene in that genotype.ESTs participate in the sequestration and hydrolysis of esters and amides CYP genes have been characterized in several insect orders CYP6, CYP3 and CYP4 families being the most important in detoxifying plant defenses and insecticides P450 enzymes oxidize a broad range of endogenous and exogenous lipophilic compounds GST), it showed a constitutively high level of EST activity (E4/FE4 expression among genotypes reared on the suitable host showed that SR genotype presented fifty times more transcripts in average (data not show) than the other genotypes. In the case of the MR genotype, only the hsp-70 gene was up-regulated, but constitutively showed approximately three times more transcripts for GST than in the other genotypes (data not shown). This kind of canalization in response to defended plants has been recently reported for the grain aphid Sitobion avenae. Using the so-called ‘superclones’ (i.e. the most common and time-persistent genotypes) of the grain aphid reared on highly defended cereals, aphids demonstrated a rigid detoxifying capacity. That is, they did not modify detoxification enzyme activities with a similar cost across defended and non-defended plants M. persicae at the whole genome level showed that in general the S genotype avoided the lethal effects of insecticide by up-regulating 183 genes In general, for the six genes studied, the low transcriptional plasticity exhibited by aphid genotypes SR and MR suggest a greater constitutive expression of these or other related genes, including paralogs for those studied here. While the SR genotype up-regulated only one detoxifying gene  might be quite complex, with a multiplicity of costs and benefits between environments. All of the selective agents  play key roles in shaping population structures. Studies that consider the spatial and temporal dynamics of aphids are needed to understand the cost/benefit balance of the mechanisms herein studied. Finally, more research is certainly needed to confirm the generality of our findings and to determine and understand the wide arrange of phenotypic correlates of detoxification responses that M. persicae can exhibits.In conclusion, our results suggest that aphids under “insecticide” or “chemically defended plants” conditions have similar adaptive solutions to two different selective agents. Although we have only studied nine genotypes, their overall response in both reproductive performance and transcriptional expression was fairly consistent across genotypes carrying or not carrying MACE and Capsicum annuum var. grossum) in Blackman boxes under conditions that ensure parthenogenetic reproduction (20±1°C and L: D 16∶8). Colonies were maintained by transferring 5 wingless adults on new 7-day-old pepper seedlings every 10 days for at least 20 generations before the experiments.Ninety four clonal lineages (genotypes) previously sampled and established in the laboratory were used in this study and genotyped using six microsatellite loci  kdr (L1014F) and super-kdr (M918T) mutations, and by Anstead et al, (2008) for the MACE mutation The presence of insecticide resistance mutations (IRM) was screened in the 44 genotypes, using allelic discrimination based on quantitative-PCR assays developed by Anstead et al. (2004) for M. persicae was conducted by estimating the intrinsic rate of natural increase (mr) when aphids were reared on pepper (C. annuumvar. grossum), the most suitable host for M. persicaeRaphanus sativus var. sparkler), an unfavorable host species The host-plant effect on the fitness of One single adult wingless aphid  from each selected genotype  was tranmr) for each genotype on each host was estimated following Wyatt and White (1977). The days from birth to first reproduction (Td) and the number of offspring produced in that time (Md) were determined. Then, the intrinsic rate of natural increase was calculated as mr = 0,738 (logeMd)/Td, where 0,738 is a correction factor The intrinsic rate of natural increase  that are known to be regulated when S and SR aphid genotypes for IRM are exposed to carbamate insecticides mr, at least 3 nymphs from each line were maintained on its host plant until they were up to 12 days old. Aphids were collected from their host plants and immediately frozen in liquid N2 until RNA extraction.Transcriptional levels of six genes  and one endogenous control gene (DW011095), using the FastPCR (V 5.4.30) and AmplifX (V 1.3.7) packages, and checked in NCBI/Primer-BLAST  Total RNA was isolated using the RNeasy Plant Mini Kit  from three aphids per genotype and condition. From approximately 1.5 µg total RNA (previously treated with DNA-free™ kit Ambion), cDNA synthesis was conducted using AffinityScript QPCR cDNA Synthesis kit (Agilent). Then, the cDNA was diluted to 1∶10, taking 2 µl for PCR reactions . Each PCR reaction mix contained 10 pmol of each primer, 12.25 µl SYBR Green PCR Master Mix (Applied Biosystems) and 0.375 µl of Rox (dilution 1∶500), the latter used as passive reference dye. Negative controls were included for detecting foreign contamination, and all PCR reactions were performed in triplicate in a Mx3000P QPCR Systems (Stratagene) under the following cycling conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 57°C and 20 s at 72°C. A dissociation curve was included immediately after each PCR using a ramp of 65–95°C, to confirm the absence of nonspecific amplifications. Primers were designed from the sequences of mr, taking into account the presence of random factors , the nested structure of our design  and some minor unbalances. Body mass was included as a covariate. Hypothesis testing for fixed effects was based on marginal F tests and for random effects was based on likelihood ratio tests of nested models We used a linear mixed modeling approach to evaluate the effect of IRM and host on glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene as normalizing endogenous control. Several studies have validated the use of GAPDH as a reference gene for normalization M. persicae (fold change range 0.94 – 0.99) The relative expression ratio of a target gene was computed by relative quantification using the comparative Ct method


Background. We investigated the feasibility of helical tomotherapy (HT) for inoperable large breast tumors, after failing to achieve adequate treatment planning with conformal radiation techniques. Material and Methods. Five consecutive patients with locally advanced breast cancer (LABC) were treated by preoperative HT. All patients received up-front chemotherapy before HT. Irradiated volumes included breast and nodal areas (45–50 Gy) in 4 patients. One patient received a simultaneous integrated boost (55 Gy) to gross tumor volume (GTV) without lymph node irradiation. Acute toxicity was assessed with Common Toxicity Criteria for Adverse Events v.4. Patients were evaluated for surgery at the end of treatment. Results. Patients were staged IIB to IIIC (according to the AJCC staging system 2010). HT was associated in 4 patients with concomitant chemotherapy (5-fluorouracil and vinorelbine). Two patients were scored with grade 3 skin toxicity (had not completed HT) and one with grade 3 febrile neutropenia. One patient stopped HT with grade 2 skin toxicity. All patients were able to undergo mastectomy at a median interval of 43 days (31–52) from HT. Pathological partial response was seen in all patients. Conclusions. HT is feasible with acceptable toxicity profiles, potentially increased by chemotherapy. These preliminary results prompt us to consider a phase II study. Locally advanced breast cancer (LABC), defined mainly by stage III disease  and by aRecent studies have used preoperative radiotherapy (combined with chemotherapy) in an attempt to downsize the tumor –11 makinHelical Tomotherapy (HT) is a new form of intensity-modulated radiation therapy (IMRT) that delivers a modulated fan beam using a 6 MV linear accelerator mounted on a ring gantry that rotates around the patient as he/she advances slowly through the gantry bore . Its advWe sought to report our early experience with the use of HT (with or without CCT) for inoperable LABC not eligible to conformal radiation techniques due to disease extension.From November 2007 to February 2011 five consecutive women with stage IIB–IIIC LABC (according to AJCC staging system 2010) were seen at our multidisciplinary clinic. All patients had histological confirmation of malignancy by tumor biopsy with determination of tumor oestrogen and progesterone receptor (ER/PR) status and HER-2. The workup included history and physical examination with recording of size and location of the tumor on a diagram of the affected breast and a photo evaluation. Adequate biology lab tests were undertaken. Imaging studies included bilateral mammogram and breast ultrasound or breast magnetic resonance imaging (MRI), bone scan, thoracic-abdominal and pelvic computed tomography scan (CT), and fluorodeoxyglucose (FDG) positron-emission tomography scan (PET/CT) in one case. Genetic counseling was necessary in one patient. All patients had advanced voluminous breast tumors judged not amenable to any form of surgery . Inoperable breast cancer was defined as a combination of at least 2 of the following criteria (except for inflammatory breast carcinoma): fixation of the axillary nodes to overlying skin or deeper structures of the axilla, skin ulceration, inflammatory breast carcinoma, solid fixation of tumor to the chest wall, extensive edema of the skin (involving more than one-third of the skin over the breast), massive involvement of axillary lymph nodes (measuring 2.5 cm or more in transverse diameter), or clinically involved periclavicular lymph nodes and internal mammary metastases as evidenced by a parasternal tumor . ResectaAll patients received up-front NCT before radiation delivery due to size and extent of disease and thus for the risk of micrometastatic disease. Chemotherapy regiments used before HT are detailed in In all 5 cases the choice of HT was done after careful dosimetry planning in three-dimensional conformal radiotherapy (3D CRT). An “optimized” 3D field-in-field technique, associated with internal mammary (IMN) electron-beam planning, was used, which is the current standard in our department . Two tanThe treatment planning CT scan was performed 1-2 weeks after the last cycle of NCT. Patients were placed in the supine position, on a breast board, with both arms abducted alongside the head. The palpable breast tissue contour and the tumor were delineated with radioopaque wires. Radioopaque markers were also placed along the midsternum, as well as 1-2 cm below the palpable breast limits. Images were acquired from the upper neck to the midabdomen, using a 3 mm slice thickness and separation. The CT data were transferred to a commercial treatment planning system . The breast clinical target volume (CTV) was defined as the tissue delineated by the aforementioned radioopaque wire. In practice, on each transverse slice, the breast volume extended from the pectoralis major muscle to the skin, excluding the pectoralis muscle, ribs, or the first 3 mm of skin except in inflammatory tumors. Breast planning target volume (PTV) was generated by adding a tridimensional margin of 5 mm around the breast CTV. The gross tumor volume (GTV) was defined on the planning CT as the tissue delineated by the radioopaque wire. Margins were then added to GTV based on the information from initial clinical and radiological reports (boost CTV). Boost PTV was defined adding an additional margin of 5 mm beyond boost CTV. However, a simultaneous integrated boost (SIB) was delivered in only one patient. The regional lymph nodes (axillary (ALN), internal mammary (IMN), supraclavicular (SCV)/infraclavicular (IFC)) were delineated (whenever indicated) using our atlases , 18. The2/d, administered by continuous intravenous infusion over five consecutive days (d1–d5), and vinorelbine, 25 mg/m2, short intravenous infusion on days 1 and 6. Courses were repeated every 3 weeks for a total of four courses. Radiotherapy started on day one of the second course of chemotherapy. Two cycles were prescribed during radiotherapy. This CCT protocol was tested in our institution in a phase II trial and was previously published [Concomitant chemotherapy (CCT) consisted of 4 cycles of 5-fluorouracil (5-FU), 500 mg/mublished , 23. Patients were seen on a weekly basis during HT. All toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v.4 . Pathological response assessment on surgical specimen took into account the proportion of residual tumor cells, the location of this malignant component , the mitotic index in malignant cells, and the status of the metastatic axillary nodes. The response was considered as pathologically complete (pCR) when there was no residual invasive malignant epithelial cells in both the breast and the axillary lymph nodes. Tumors with an epithelial malignant residual component strictly in situ or representing less than 5% of the breast and/or axillary tumor mass and without any mitosis were also classified in the group of pCR. The response was considered as absent (pSD) when no histological modification of the tumor tissue could be related to therapy and as partial (pPR) in the remaining cases. This is according to the interpretation at the Institut Curie of the definition proposed by Sataloff and colleagues of a “total or near total therapeutic effect” , 26. Patient and initial tumor characteristics are described in Dmean) and maximum dose (Dmax) as well as V20 constraints for ipsilateral lung were not achieved with 3D CRT in 4 patients. Equally, Dmax and V25 for the heart had not been achieved with 3D field-in-field technique in one patient with a left-sided large tumor.Planning with 3D CRT revealed that the doses to PTV did not attain the 95% constraint in 3 cases (<85%). Furthermore, mean  with axillary lymph node dissection (ALND) of the first two levels was performed in all cases. Median time to surgery from last day of radiotherapy was 43 days (range: 31 to 52). Pathological response assessment on surgical specimen revealed pPR in all patients, according to the modified Sataloff criteria . No patiMargins were negative in all cases . No fibrosis was described in the surgical reports. One patient had wound infection and needed surgical drainage. Two patients had aspirations of lymphoceles. Adjuvant treatments were decided according to pathological criteria and consisted of either chemotherapy  and/or endocrine therapy (presence of positive expression of ER/PR).Median follow up was 15.4 months (range: 2 to 25.1). At last follow up, 2 patients were still alive and free of disease, presently undergoing endocrine therapy. One patient was lost to follow up, and 2 patients had died from metastatic disease. In the present study we have tested a relatively new form of radiation combined with sequential and/or concomitant chemotherapy. To the best of our knowledge this is the only exploratory study of HT in inoperable LABC. As it can be seen in HT appears to improve target coverage while sparing OAR because of its ability to achieve a higher degree of conformity to the PTV. The well-known ability of HT to treat breast cancer with complex treatment volumes  and regiDmax < 40 Gy) and reduce the lung V20 and V5 below tolerance levels. Wang et al. [In the current study, we have seen that HT can significantly spare the ipsilateral lung  versus 5.5 Gy ± 1.4 Gy (P = 0.2). Similar results were published by Goddu et al. [Dmean to the heart compared to 3D CRT (12.2 ± 1.8 Gy versus 7.5 ± 3.4 Gy). HT was also used in our series with the intention to avoid eventual cardiovascular toxicity, knowing that patients had previously received anthracycline (with or without bevacizumab) or taxane-based NCT. The reported rates of cardiac dysfunction vary from 4 to 7% in patients receiving Trastuzumab alone and up to 27% with concomitant trastuzumab, antracycline, and cyclophosphamid . Epirubir et al.  also repThe same protective cardiac feature of HT on the heart (from high doses) was also described by Coon and colleagues  in patieRegarding skin toxicity, our findings indicate that the rate of severe acute events grade ≥ 3 CTCAE) is potentially increased by CCT, high radiation dose (>45 Gy/25 fx to lymph node volumes), and outspread of target volumes (breast only versus breast and lymph nodes). Doses of 50 Gy/25 fx to whole breast seem tolerable (without CCT or lymph node irradiation) with possibility of simultaneous boost to gross tumor volume . Thus, small community centers may not have sufficient financial resources for HT or human personnel for the HT workload.Preoperative HT with or without CCT appears to be a feasible and promising alternative to highly conformal techniques in the treatment of large inoperable breast cancers. Particular attention should be given to evaluate acute skin toxicity especially in patients receiving CCT. Larger studies are warranted to better define HT doses and to evaluate long-term toxicities.


Posttraumatic stress disorder (PTSD) is common in survivors of acute life-threatening illness, but little is known about the burden of PTSD in survivors of stroke and transient ischemic attack (TIA). This study estimated the prevalence of stroke or TIA-induced posttraumatic stress disorder (PTSD) using systematic review and meta-analysis.Potentially relevant peer-reviewed journal articles were identified by searching the Ovid MEDLINE, PsycINFO, PILOTS Database, The Cochrane Library and Scopus from inception to January 2013; all searches were conducted on January 31, 2013. Observational cohort studies that assessed PTSD with specific reference to a stroke or TIA that occurred at least 1 month prior to the PTSD assessment were included. PTSD rates and characteristics of the study and sample were abstracted from all included studies. The coding of all articles included demographics, sample size, study country, and method and timing of PTSD assessment.Q1 = 10.30; P = .001). Using a random effects model, the estimated rate of PTSD following stroke or TIA was 23%  within 1 year of the stroke or TIA and 11%  after 1 year.Nine studies  met our inclusion criteria. PTSD rates varied significantly across studies by timing of PTSD assessment (i.e., within 1 year of stroke/TIA versus greater than 1 year post-stroke/TIA; 55% of heterogeneity explained; Although PTSD is commonly thought to be triggered by external events such as combat or sexual assault, these results suggest that 1 in 4 stroke or TIA survivors develop significant PTSD symptoms due to the stroke or TIA. Screening for PTSD in a large population-based prospective cohort study with cardiovascular outcome assessments is needed to yield definitive prevalence, and determine whether stroke or TIA-induced PTSD is a risk factor for subsequent cardiovascular events or mortality. Observational evidence suggests that PTSD is related to increased risk of incident cardiovascular disease Until recently, only a few studies assessed PTSD due to stroke, another potentially traumatic acute cardiovascular event. Prevalence estimates in these studies has varied from 3–37% stroke or TIA-induced PTSD. For the one study that reported multiple PTSD assessments, we abstracted only the earliest estimate (at 1 month post-stroke) We sought to identify all studies that reported a valid prevalence estimate of PTSD due to stroke or TIA. Included studies must have been observational cohorts and must have assessed PTSD with specific reference to a stroke or TIA that occurred at least 1 month prior to the PTSD assessment. Further, studies must have used a self-report PTSD screening instrument or clinical interview designed or specifically altered to query about only Potentially relevant peer-reviewed journal articles published in English were identified by a search of the biomedical electronic databases Ovid MEDLINE, PsycINFO, PILOTS Database, The Cochrane Library and Scopus conducted by an information specialist (L.F.) with expertise in behavioral medicine. Databases were searched from inception to January 2013; all searches were conducted on January 31, 2013. All relevant subject headings and free-text terms were used to represent PTSD and stroke or TIA and the sets of terms were combined with AND. Terms for MEDLINE included: exp Stress Disorders, Traumatic/OR ptsd.tw. OR (post-traumatic OR (post adj traumatic)).tw or posttraumatic.tw. AND exp stroke/OR Stroke$.tw OR cerebrovascular.tw. OR  adj2 (accident$ or infarct$ or event$ or attack$)).tw. Search terms were adapted for the other databases. Additional records were identified by scanning the reference lists of relevant studies and reviews and by employing the Related Articles feature in PubMed/Medline and the Cited Reference Search in Scopus. To determine the studies to be assessed further, two authors  independently read the title, or title and abstract of every record retrieved. Opinion differences were resolved by consensus. All potentially relevant articles were investigated as full text.7 and another’s demographic characteristics were uncertain specifically due to stroke or TIA.We abstracted PTSD rates and characteristics of the study and sample from all included studies Table 1.Comprehensive Meta-analysis  served as the statistical platform for completing all statistical tests and associated graphic results. To summarize the prevalence findings, we computed prevalence point estimates using these formulas:Logit Event Rate = Log [Event Rate/(1 − Event Rate)].Event Rate Standard Error = √(1/(Event Rate ·Sample Size)/(1/[(1−Event Rate) · Sample Size]),We computed 95% confidence intervals (CIs) using this formula:Lower Limit = Logit Event Rate − (1.96 · Logit Event Rate Standard Error).Upper Limit = Logit Event Rate+(1.96 · Logit Event Rate Standard Error).Results are presented after back-transformation.There was statistically significant heterogeneity in prevalence estimates, so random-effects models were used. In addition, we used sensitivity analyses to assess evidence of moderator effects for the prevalence results across two methodological factors: (1) method and (2) timing of PTSD assessment. Finally, we produced a forest plot and calculated both the classic and Orwin’s fail-safe N to address the issue of publication bias.Our search identified 1238 articles, and 2 coders agreed that 10 articles required full reading. Of those, 9 met our criteria for inclusion yielding a total of 1,138 participants Figure 1The timing of PTSD assessments ranged from 1 month to 60 months after the stroke or TIA. PTSD was assessed by clinical interview in 2 studies and using a screening questionnaire in the remaining 7 studies. All included studies met our minimum criteria for quality. Study quality across the included studies was highly similar, with very good internal validity  but only moderate external validity .9Q = 85.64; P<.001; I2 = 89.49).The prevalence estimate of stroke or TIA-induced PTSD across all studies included in the review was 13% . However, there was significant heterogeneity in prevalence rates, with reported rates ranging from 3% to 37% . The aggregate prevalence estimate in the 7 studies in which PTSD was assessed with diagnostic self-report questionnaires was 28% , compared with 6%  in the two studies in which PTSD was assessed with clinical interviews.The PTSD prevalence estimate varied significantly by type of PTSD assessment . Of the 9 included studies, 7 assessed PTSD within 1 month to 1 year after the index stroke The PTSD prevalence estimate varied significantly by timing of assessment .As substantial heterogeneity explained by timing of PTSD assessment suggested that two underlying prevalence estimates existed, we conducted another meta-analysis with the estimates from Kronish et al The forest plot given in To our knowledge, this is the first meta-analytic review of stroke or TIA-induced PTSD. We found an overall prevalence of 13% among stroke/TIA survivors, with 23% prevalence in the first year post-stroke and 11% after the first year. Given that about 85% of the 795,000 patients who experience a stroke each year in the US specifically due to the stroke event, so these estimates may represent an underestimate of the total burden of PTSD symptoms in these participants in so much as PTSD due to other types of events may have been present.Important caveats to this review include the fact that only studies with relatively small sample sizes have addressed stroke-induced PTSD, and one of the studies represented half of all of the participants included in this review. Also, none of the included studies used population-based sampling, and all but one study The estimated prevalence of stroke-induced PTSD was lower when PTSD was assessed by clinical interview. This may be due to the fact that the prevalence of psychiatric disorders is often higher when measured using diagnostic instruments as compared to clinical psychiatric interviews Nevertheless, research on the association between ACS-induced PTSD and ACS recurrence and mortality risk suggests that even elevated symptoms of PTSD, not clinical diagnosis, is associated with increased risk. Hence, the estimate of the prevalence of PTSD symptoms based on diagnostic instruments may still be clinically relevant Prevalence estimates were limited to patients who could participate in PTSD assessments and hence should be extrapolated to patients with severe cognitive impairment or aphasia with caution. Similarly, these results should be interpreted with awareness that the mean age of the participants was slightly low relative to the entire population of stroke survivors and PTSD is associated with younger age in ACS-induced PTSD Only one of the studies included in this review evaluated for stroke lesion localization It is also important to note the difference in prevalence estimates reported in studies that assessed PTSD by screening questionnaire versus clinical interview. Although screening questionnaires are often used to identify patients who should be further assessed for PTSD diagnosis–and therefore may overestimate prevalence–reliance on screening questionnaires for estimates of stroke-induced PTSD prevalence may further inflate those estimates due to overlap in symptoms of stroke and PTSD. Very little is known about the validity of PTSD questionnaire items in stroke survivors, but the evidence that exists suggests that PTSD questionnaires are valid in this population. In one study, confirmatory factor analysis of responses to the PTSD Checklist-specific for stroke conformed to the established factor structure for PTSD due to other types of events Across published studies estimating the prevalence of stroke-induced PTSD, two general conclusions can be drawn: (1) stroke-induced PTSD is relatively common, with approximately one in four experiencing PTSD in the first year after stroke and one in nine experiencing chronic PTSD over a year later, and (2) based on a single study, stroke-induced PTSD symptoms appear to influence important secondary prevention behaviors such as medication adherence Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


Marine cyanobacteria scavenge PO4 using the high-affinity periplasmic phosphate binding protein PstS. The pstS gene has recently been identified in genomes of cyanobacterial viruses as well. Here, we analyse genes encoding transporters in genomes from viruses that infect eukaryotic phytoplankton. We identified inorganic PO4 transporter-encoding genes from the PHO4 superfamily in several virus genomes, along with other transporter-encoding genes. Homologues of the viral pho4 genes were also identified in genome sequences from the genera that these viruses infect. Genome sequences were available from host genera of all the phytoplankton viruses analysed except the host genus Bathycoccus. Pho4 was recovered from Bathycoccus by sequencing a targeted metagenome from an uncultured Atlantic Ocean population. Phylogenetic reconstruction showed that pho4 genes from pelagophytes, haptophytes and infecting viruses were more closely related to homologues in prasinophytes than to those in what, at the species level, are considered to be closer relatives (e.g. diatoms). We also identified PHO4 superfamily members in ocean metagenomes, including new metagenomes from the Pacific Ocean. The environmental sequences grouped with pelagophytes, haptophytes, prasinophytes and viruses as well as bacteria. The analyses suggest that multiple independent pho4 gene transfer events have occurred between marine viruses and both eukaryotic and bacterial hosts. Additionally, pho4 genes were identified in available genomes from viruses that infect marine eukaryotes but not those that infect terrestrial hosts. Commonalities in marine host-virus gene exchanges indicate that manipulation of host-PO4 uptake is an important adaptation for viral proliferation in marine systems. Our findings suggest that PO4-availability may not serve as a simple bottom-up control of marine phytoplankton.Phosphate (PO PO4 depletion in particular is thought to cause intense competition between different taxa in the North Atlantic Ocean and to have influenced gene content in different strains of the picocyanobacteria Prochlorococcus and Synechococcus through selective adaptations  and Pst genes. Many eukaryotes and bacteria encode PHO4 superfamily members. This superfamily includes high- and low-affinity inorganic phosphate transporters. In eukaryotic phytoplankton a pho4 gene has been identified in expressed sequence tags from the haptophyte Emiliania huxleyi  known to inhabit warm, oligotrophic waters where PO4 is often depleted , but the source waters the viruses came from were collected at different times of the year . Hence, different nutrient conditions associated with seasonal changes may explain the fact that only BpV-1 encodes the pho4 gene. Different Micromonas clades also vary along environmental gradients  and one Major Facilitator Superfamily  member was identified (Helicoverpa zea. A putative ion channel transporter gene (belonging to the VIC Superfamily) was detected in several of the Mamiellales viruses  and mimivirus . A putative ATPase-encoding gene, phoH, was also identified in most of the viral genomes  and 46 herpesvirus genomes sequences available when that Pfam alignment was generated. The majority of these viruses infect terrestrial hosts including mammals, non-mammalian vertebrates and invertebrates. Furthermore, pho4 genes were not detected in newly available genomes from viruses that infect other hosts, including non-marine unicellular eukaryotes. In environments like the mammalian cellular milieu or many freshwater systems, PO4 is not considered limiting and therefore selection pressure favouring viruses that encode a PO4 transporter (or retention of this gene) is presumably weak. In contrast, manipulation of PO4-uptake in marine hosts that often encounter PO4-deplete conditions could ensure enough PO4 is available for viral replication.The only viral sequence included in the full PHO4 Pfam alignment at the time of our analysis was from EhV-86. pho4 gene, including hosts for the analysed viruses. In addition to the previously described E. huxleyi pho4 gene (Aureococcus anophagefferens (a pelagophyte) and all genome sequenced Ostreococcus strains . They were also found in Micromonas RCC299 and CCMP1545 but with low similarity to the Ostreococcus pho4 genes (see discussion below).Many previous studies have suggested that viruses acquire genes from their hosts . Results for 16S rDNA clone sequences were less clear and an unknown endosymbiont or other material may have been present (Micromonas and Ostreococcus), that fit other criteria as well , were considered derived from the Bathycoccus nuclear genome and used in subsequent analyses. Two distinct G + C signatures were observed in the Bathycoccus nuclear metagenome similar to what has been seen in whole genome sequences from cultured members of the Mamiellales. The ‘anomalously low-G + C’ region of 38% G + C was about 10% lower than the overall metagenome average of 48%. These regions of considerably lower G + C content were first observed in O. tauri (58% for the whole genome and 52% for the low G + C region) and Micromonas sp. RCC299 (64% and 50% respectively) and have been hypothesized to serve as a sex chromosome that encodes convergent, overlapping genes . Similarities between pho4 genes from these Mamiellales and those in Micromonas were low (Bathycoccus had 29% similarity to Micromonas RCC299 ACO69637 and 27% to M. CCMP1545 XP_003062965). Surprisingly, the similarities between pho4 from Ostreococcus or Bathycoccus were lower to Micromonas than to Tetraselmis chui (50% similarity with Bathycoccus), a more distant prasinophyte. Bathycoccus and Ostreococcus also appeared more closely related to each other than to Micromonas based on 18S rDNA analyses  to one another than to homologues from Ostreococcus itself. A pho4 sequence is not available from Ostreococcus RCC393, the host strain against which OtV-2 was isolated, although a sequence from another Ostreococcus Clade OII member (Ostreococcus RCC809) was included. The fact that OlV-1 and OtV-2 sequences branched together could indicate they originated from a common ancestor rather than from their respective hosts. Without an OtV-2 host-encoded pho4 sequence in the tree it is not possible to determine if placement of the OtV-2 version would differ if the host sequence was included. Finally, host cross-infectivity levels of Phycodnaviridae family members are not well known and could influence acquisition and retention patterns.Recovery of  huxleyi . The anapho4 gene in marine systems have been reported for E. huxleyi and EhV-86 sphingolipid biosynthesis genes. Sphingolipid biosynthesis genes were hypothesized to have been acquired by the virus from its host , suggesting this gene may have been acquired through HGT and is sufficiently advantageous to be retained by the recipient genome. Homology has been reported between 11 predicted genes in OtV-1 and host-encoded (O. tauri) genes  and the haptophyte E. huxleyi. High representation of green algal or plant-like genes in haptophytes has been reported previously . There are several possible explanations for the observed phylogenetic distribution of pho4 genes in eukaryotic algae. The fact that the stramenopile and haptophyte pho4 homologues grouped with green algae may have been influenced by taxon undersampling, particularly the lack of available homologues from red algae apart from C. merolae. However, it may also reflect ancestral characteristics with differential loss or divergence in some taxa (e.g. diatoms) or gain from a proposed cryptic green algal endosymbiont in the chromalveolates  acts as a high-affinity PO4 permease and the gene is specifically upregulated in P-deplete conditions  regulon of E. coli and other bacteria, although its precise role is unclear  but not EhV-86 (phoH genes in cyanobacteria and cyanophages (present in both hosts and viruses) or pstS and pho4 genes, which were found in all sequenced hosts, phoH genes appeared to be present only in the O. tauri and E. huxleyi genomes, based on BLASTp and tBLASTn similarity searches against available proteomes and genomes. In addition, the O. tauri-encoded version did not appear to be closely related to versions in viruses infecting Ostreococcus.Most of the eukaryotic viral genomes analysed herein also encoded the gene phoH . Althougt EhV-86 . Unlike 4), mesotrophic (moderate PO4) and oligotrophic (moderate PO4 and low nitrogen) for members of the PHO4 superfamily  branched in the same region of the tree. WH5701 is a halotolerant strain and some environmental sequences assigned to this group might be from the nitrogen fixing cyanobacterium Crocosphaera. The Crocosphaera gene model containing the PHO4 domain (ZP_00517140) appeared to be missing a section of the conserved C-terminal domain that is part of the PHO4 Pfam model and was therefore not included in the tree, but had high similarity to the WH5701 version . Prochlorococcus and Synechococcus do not appear to encode genes containing the PHO4 Pfam domain. Environmental sequences were assigned to other bacterial groups as well  clade appeared to be from viral DNA templates (i.e. those assigned to OlV-1/OtV-2 or BpV-1) rather than host-derived templates. For the haptophyte lineage, environmental sequences were not placed with E. huxleyi or its virus EhV-86, but rather branched in a position basal to the E. huxleyi-EhV-86 node, suggesting they may have originated from other prymnesiophyte taxa, which include widespread uncultured marine groups (Pelagomonas (or Pelagomonas-infecting viruses); therefore, a pho4 gene from this taxon (assuming it contains the pho4 gene) was not included in our analysis. Several Pelagomonas, but not Aureococcus, SSU rDNA sequences were present in clone libraries (data not shown) from the Pacific sites  as opposed to sampling protocols that target the viral size fraction , indicates that the virally derived environmental sequences were present in infected hosts, especially for the two largest size fractions we sequenced. The proportion of viruses in nature that encode the pho4 gene is still not known.Three hundred and nineteen environmental sequences were assigned to branches within the region of the PHO4 superfamily tree containing cultured eukaryotic algae, the ed above . These eho4 tree , which iic sites . Hence,  replete  and deplter gene . Given t4-uptake via different molecular mechanisms mitigates P-starvation and is important for survival in oligotrophic waters  and the other on R/V Western Flyer (October 2007). The first cruise was in the tropical Atlantic Ocean and the sample sequenced here was collected on 12 July 2006 at Station 13 (SJ0609) from the deep chlorophyll maximum (http://oceancolor.gsfc.nasa.gov).Samples were collected using a rosette equipped with Niskin bottles, a CTD and fluorometer on two research expeditions, one aboard the  maximum , Table 2 maximum ; Fig. S3Bathycoccus. The analysed cells were first sorted by flow cytometry based on distinctive scatter and chlorophyll signals (2O and 750 ml were run through the instrument after cleaning (immediately before sorting). Contamination control materials were from left and right test deflections run for 1 min (sample flow rate ∼25 µl min−1). All sort droplets were immediately frozen cryogenically. Upon return to land the population sort was resorted to enhance purity levels  and the traditional metagenomes (size fractionated filters) was different. For the former, a 454-FLX and Sanger blended metagenomic assembly was generated for  signals  using any levels . The samBathycoccus sort we constructed 18S rDNA clone libraries using universal 18S rDNA primers  were retrieved using EMBOSS GetORF  was retrieved using the NCBI taxonomy database and assigned to the corresponding ORF. A scaffold was classified as Bathycoccus-like if more than half of its predicted ORFs had their best BLASTp hits  against a protein sequence originating from any of the five available prasinophyte genomes , and among these 5.1 Mb were scaffolds of 7 kb or more (183 scaffolds).In order to further insure that only scaffolds originating from For each Pacific Ocean traditional metagenome, three different size fractions were extracted and sequenced independently. The nominal size fractions were 0.1 to < 0.8 µm, 0.8 to < 3 µm and 3 to < 20 µm. DNA was extracted from a fragment of each 293 mm filter using a sucrose protocol as described in E. huxleyi, A. anophagefferens, Ostreococcus sp. RCC809) and NR-DB, allowing the detection of E. siliculosus  was retrieved and used to build a hidden Markov (HMM) model based on amino-acid sequences using hmmbuild [part of HMMer 3 ]. This HE-value threshold of 1e−10 and gathering cutoff. The percentage of pho4 genes detected in our 454-FLX reads was lower than from GOS samples (Sanger sequences) or some of our Sanger libraries  were considered further.PHO4 superfamily members detected in traditional metagenomes from the Pacific (as well as in GOS data) using our HMM search were then assigned to branches or nodes on the PHO4 protein family tree using pplacer  were exthttps://portal.camera.calit2.net/gridsphere/gridsphere). The targeted Bathycoccus metagenome was deposited in NCBI genomes under accession AFUW00000000.Traditional metagenome Sanger sequences from this study have been deposited in GenBank under Accession Numbers JF837193-JF837211 and 454-FLX sequences are in the CAMERA database (


Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility  Nanomedicine has a leading role in pharmaceutical research and development of clinical protocols, mainly in the form of nanoparticle-based delivery systems for drugs and imaging agents, especially in the field of stem cell (SC) therapies http://www.epa.gov/nanoscience/index.htm).Ferumoxides (a suspension of Super Paramagnetic Iron Oxide nanoparticles (SPIOn)), are (FDA)-approved agents which may be accurately, sensitively and easily detectable by non-invasive Magnetic Resonance Imaging (MRI) to monitor grafted cell distribution over time SPIOn molecular interactions may exert metabolic or mutagenic effects on the surroundings, especially in the long term, limiting their diagnostic and therapeutic potential Alterations in migration capability are primarily involved in pathological conditions  and amniotic fluid-  derived cells. hCVCs and hAFCs, conversely to embryonic SCs, do not raise special ethical concerns. If compared to adult SCs, they display higher multipotentiality and proliferative capabilities, a low immunogenicity as well as an easy accessibility. Moreover, they can be expanded in the long term without tumorigenic risk Cell movements are finely regulated by Reactive Oxygen Species (ROS) Herein, we report that SPIOn loading, dose-dependently, affect migration capability, but not ROS production in two fetal SC populations, thus enlightening novel side effects of SPIOn labeling and potential caveats for SPIOn application to (pre)clinical therapy.hAFCs and hCVCs were collected from amniotic fluid or chorionic villi of pregnant women respectively, after specific written informed consent for this research  (19 luglio 2006) n. 4).2 in a fully humidified atmosphere for one week before fetal karyotypization by standard procedures (QFQ banding technique) th gestational week for hAFCs and 4  from women between 11–13th gestational weeks for hCVCs.Cells were isolated and grown in Amniomax II  at 37°C and 5% COCells obtained from every sample were harvested after gentle trypsinization, centrifuged at 1,000×g for 10 minutes, pooled together and counted. Dividing cells in suspension were collected as well. Pools were then frozen  and stored in liquid nitrogen. A periodic monitoring of karyotype was conducted to check if subculturing has altered chromosome constitution. hAFCs and hCVCs were loaded with the commercially available MRI agent, dextran coated SPIOn  coupled to Poly-L.Lysine  in Amniomax II, to maximize cell internalization, as already described To specifically verify SPIOn influence on the main biological processes  which affect the therapeutic potential of SCs we sequentially analyzed them.Cell proliferation was performed on 75,000 cells/well in untreated 12-wells plate (NUNC) and each sample was tested in quadruplicate. Cells after SPIOn loading for 72 hrs, were cultured for around 3 weeks, with weekly medium change. At different time points, as detailed in the corresponding graphic, cells were detached and counted . BrieflyCell metabolism is deeply influenced by intra-cellular and extra-cellular conditions with potential implications on cell migration, therefore we tested if our experimental conditions (presence of increasing SPIOn doses or 6-hydroxydopamine (6-OHDA)) may affect this biological parameter.Cell viability was evaluated by -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) MTS assay  following manufacturer’s instructions. To test the potential toxicity of SPIOn, first hAFCs and hCVCs were loaded with increasing concentrations of SPIOn , 5, 10, 25 and 35 µg/ml) for 72 hours, as detailed in the corresponding figure. Cells were then collected and plated  in 96-well plates (NUNC). One hour after a resting time at 37°C, MTS solution (20 µl/well) was added and cells were incubated for another hour at 37°C. The supernatant of cell cultures was collected, centrifuged at 2,000×g for 5 minutes and read at 490 nm with a microplate reader . Results were expressed as a percentage of viable cells versus CTR (unlabeled cells) using the following function: “labeled cells value”×100÷“unlabeled cells value”.Toxicity of 6-OHDA was also assessed on both unlabeled and SPIOn labeled  hAFCs and hCVCs . SH-SY5Y was used as positive control: this is a cell line subcloned from a bone marrow biopsy of a patient affected by a neuroblastoma. SH-SY5Y cells are susceptible to 6-OHDA and therefore represent a good positive control to test the effect of this neurotoxin. Cells were plated and placed at 37°C for 1 hour before being treated with 6-OHDA , as detailed before.This assay employs a simplified Boyden chamber-like design that consists of two chambers separated by a filter coated with different extra-cellular matrix components. The cell suspension is placed in the top chamber and incubated in the presence of test media eventually containing specific chemoattractants in the bottom chamber. Cells migrate from the top chamber through the coated filter pores to the bottom of the filter. Detection of cell invasion is quantified using calcein acetoxymethyl ester . Cell dissociation/Calcein-AM solution is placed in the bottom chamber to dissociate the migrating cells from the filter. Calcein-AM is internalized by the cells, and intra-cellular esterases cleave the acetomethylester moiety. Free Calcein fluoresces brightly and is used to quantify the number of cells that have invaded or migrated by comparison with a standard curve.Migration capability of hAFCs and hCVCs was evaluated by a Cultrex 96 Well Cell Migration Assay kit  following manufacturer’s instructions. Briefly, hAFCs and hCVCs were loaded with 0 , 5, 10, 25, 35 µg/ml of SPIOn at time T0 ), the bottom chamber was loaded with serum free media or with 10, 20 or 30% FBS respectively.We also mimicked a pathophysiological condition with a well standardized cell model of neurodegeneration to observe any eventual influences of SPIOn presence on cell migration in this experimental setting. Presently, cell models for neurodegenerative diseases, (i.e. Parkinson’s disease (PD)) primarily include neuronal tumor cell lines, such as the human neuroblastoma cells (SH-SY5Y). These cells mimic many aspects of the dopaminergic neuron death observed in PD in a well standardized way, when treated by neurotoxins such as 1-methyl-4-phenyl-pyridinium (MPP+), 6-OHDA, or rotenone without the high variable response of primary dopaminergic cultures derived from different brain regions, after the addition of 6-OHDA 2 until adhesion. SCs were then fixed with 4% paraformaldehyde  for 20 minutes. Following manufacturer’s instructions, after several washings with distilled water, SCs were incubated 20 minutes with a mixture of reagent A™ and reagent B™  then washed with distilled water. All the passages were done at RT. Coverslips were mounted on microscope slides, observed and images were acquired with a CCD camera, directly connected with a Leica DMRS/HCS  microscope.Evaluation of the internalization of SPIOn in hAFCs and hCVCs was performed by Prussian Blue staining (which labels iron deposits) using a diagnostic kit specific for the detection of ferric iron overload in cells and tissue . Prussian blue reaction involves the treatment of sections with acid solutions of ferrocyanides. Any ferric ion (+3) present in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called Prussian blue, or ferric ferrocyanide. This is one of the most sensitive histochemical tests and will demonstrate even single granules of iron in cells. Labeled cells were rinsed with Phosphate Buffered Saline , collected after trypsinization, plated in 24-well plate, at a density of 30,000 cells/well on glass coverslips and cultured at 37°C with 5% CO2·6 H2O, 1 mM MgCl2·6 H2O), washed and incubated for 72 hours with SPIOn (35 and 100 µg/ml in buffer). Fluorescence was measured both during incubation with SPIOn  and after SPIOn removal  by a microplate reader . Thereafter, values were correlated to cell viability measured by digitonin  and propidium iodide  exposure for 20 minutes at 37°C and subsequent reading with the same instrument  For analysis of intra-cellular ROS, hAFCs and hCVCs were labeled with the oxidation-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate . Briefly, the day before the experiment, cells were plated in a Fluorometer/Luminometer white 96-well plates  and left overnight at 37°C to allow adhesion. Cells were then incubated with DAF-FM (5 µM) for 15 minutes in the specific buffer  as previously reported Cells  were mixed immediately before flow cytometric analysis, but they remained easily distinguishable by their excitation and emission spectra (respectively phycoerythrin and fluorescein). Samples were acquired after lysis by a flow cytometer (FACSCanto II Becton Dickinson) and analyzed using the dedicated Diva software. All possible combinations of the fluorescent dyes were used in order to validate our results in triplicate.Labeled cells were also observed by fluorescent microscopy to confirm flow cytometry data on a confocal microscope .Elemental analysis was carried out by inductively coupled plasma optical emission spectrometry  using an Optima model 8300 instrument . Samples were analyzed after digestion using concentrated nitric acid (Sigma Aldrich).Each experiment was run in triplicate on 5–6 different pools with at least three replicates for each condition and representative values were expressed as mean ± SD.post hoc test (as specified in the respective figure or table), using a dedicated statistical software .Data were analyzed using one or two way analysis of variance (ANOVA) followed by Bonferroni in vitro  did not significantly alter proliferation, metabolism and morphology of hCVCs in vitro  a, b. Inin vitro  a. Similin vitro  b.In our experimental conditions, we found that hAFCs and hCVCs (labeled with SPIOn 35 µg/ml for 72 hours) did not shown morphological differences, but a significant reduction of migration capability, when compared with unlabeled cells (CTR). Migration rate of loaded cells was approximately halved in respect to CTR .Excess ROS are generated during a variety of cell stresses and may contribute to inflammation and cell/tissue damage. Therefore, to check if SPIOn internalization in cells in these conditions could interfere with physiological cell processes, we measured the ROS formation at different time points during incubation and after SPIOn removal  the level of intra-cellular ROS was unaffected and comparable between CTR and SPIOn labeled cells for the first 24 hours . FluoresIn a second set of migration experiments, we wanted to verify if impairment of cellular motility in our system was due to the concentration of SPIOn used. The same experiments were performed with decreasing concentration of the nanoparticles (from 35 to 5 µg/ml). We observed a progressive significant recovery of migration, inversely proportional to SPIOn concentration. Graph reported in MTS assay was performed to check cell survival under every condition and ascertain that reduced cell migration was not due to hAFC and hCVC death. We observed no significant differences in hAFC and hCVC viability, even when the higher concentration of SPIOn was used  in the same experimental conditions . This diEach naïve cell type was loaded with the same amount of SPIOn (35 µg/ml) for 72 hours and separately labeled with a specific fluorescent membrane linker . The two cell types were then mixed for FACS analysis , medium with 10% FBS was added in the bottom chamber, as detailed in the experimental design, We also investigated if increasing percentages of FBS  could dose-dependently restore migration in SPIOn-labeled cells (35 µg/ml for 72 hours), as detailed in the experimental design, SC rate of migration is strongly influenced by the surroundings: their movements can be stimulated by soluble factors  released by damaged cells or tissues. To observe if a lesioned environment may influence migration of SPIOn loaded hAFCs and hCVCs we exploited SH-SY5Y cell line exposed to 6-OHDA, as a well standardized model of cell neurodegeneration.First we checked the sensitivity to 6-OHDA for all our cell types. SH-SY5Y, hAFCs and hCVCs were plated and treated with 6-OHDA (100 µM). By MTS assay, viability was then tested after 1, 4 or 6 hours of exposition to the toxin. We found that SH-SY5Y survival was significantly reduced starting from 4 hours after 6-OHDA exposure . On the Thereafter, we performed a migration assay plating SH-SY5Y on the bottom chamber while unlabeled (CTR) and SPIOn-labeled hAFCs and hCVCs were seeded in the upper chamber, as illustrated in in vivo characterization of cell biological processes over time, especially in SC therapy Molecular medicine imaging is a recent biomedical approach that allows the non invasive in vivo over time Recently, it has been reported that implanted SPIOn patches (with an iron concentration of 552 mg/ml) in animals can undergo slow progressive degradation in vitro confirming a previous report on altered migration rate of neural progenitor cells loaded with SPIOn in vivoin vitro effects of SPIOn presence in human fetal SCs may therefore negatively influence the outcomes of SPIOn loaded cells in clinical regenerative protocols.Our data indicate that SPIOn labeling dose-dependently affect human fetal SCs in vitro (as presented here) and hAFCs both in vitro and in vivoin vitro, in vivo and ex vivoin vivo survival of SPIOn loaded hAFCs, till 56 days after transplantation, in the lateral ventricles of wobbler mice  jointed to the long-term permanence of grafted cells even in damaged areas demonstrating the usability of these SPIOn labeled cells in animal models of neurodegeneration In this study we used a limited dose of SPIOn (35 µg/ml for 72 hours) that has no collateral effect and is safe on the most important biological properties of hCVCs Nevertheless, we observed an enhanced ROS production, after 48 hours of SPIOn loading at low dose, without reaching statistical significance for both SC types with respect to controls. ROS production was lower in hCVCs than in hAFCs at any time point considered, although comparable cell size and iron uptake were demonstrated in both fetal SCs over time. Moreover, in our experimental conditions, SPIOn loaded hCVCs displayed lower ROS production, at least until 48 hours after SPIOn removal. Similar ROS levels were observed after 72 hours in serum free medium for both SC types, while only 9–12 hours are required in human embryo kidney (HEK 293) and HeLa cervix carcinoma cells to observe major ROS-induced apoptotic cell death triggered by withdrawal of cell survival factors Our data also show that the higher dose of SPIOn loading , although a time dependent reduction of iron content by exocytosis partially restored the physiological migration rate in vitro a reduced migration rate of human SCs. We also speculate that only a careful choice of SPIOn dose and the SC source could guarantee best imaging, physiological cell migration rate and the correct assessment of the eventual therapeutic effects, both in animal models and clinical applications.Migration is an essential process during cell life in organism from development to ageing The dose-dependent impairment of cell motility we describe here, may therefore affect the outcomes of (pre)-clinical trials of SPIOn loaded SCs, especially in a neurodegenerative/pathological environment where misregulation of metal homeostasis in vivoMoreover, diffuse neurotoxicity/multiple side effects of iron nanoparticle have been described in several cell systems from neuronal cell models to Central Nervous System (CNS) microglia and primary neuronal cells. Intra-cellular delivery of even moderate levels of iron oxide nanoparticles (iron concentration from 0.15 to 15 mM) adversely affects cell functions of the rat pheochromocytoma cell line (PC 12), an ideal model system for the study of neural stem cell development and differentiation. Multiple side-effects have been described: i) statistically significant reductions in PC12 cell viability, coupled to significant cell detachment, within the first 48 hours after nanoparticle loading; ii) changes in morphology and cytoskeletal structure of cells after differentiation into neuronal type cells post Nerve Growth Factor exposure (100 ng/ml) with minimal axonal/microtubule sprouting deprived of mature neurites and neuronal branching, dose dependently correlated to the iron content; iii) significantly diminished expression of the neuronal protein growth-associated protein-43 associated with axonal growth, neuronal plasticity, learning and development as well as physiological function of neuronal system Our data may be also useful to efficiently calibrate the best SPIOn content and incubation time to maximize beneficial effects in cells preserving best imaging resolution, especially for novel therapeutic approaches, such as the application of magnetic iron nanoparticles to reduce the amyloid aggregates typical of Parkinson’s, Alzheimer’s Disease and type II diabetes in vitro which may interfere with the outcomes of SC grafts: 1) long-term increase of ROS (over 24 hours incubation time) independently from SPIOn presence; 2) dose-dependent impairment of migration in SPIOn loaded cells which appears correlated to the SC type used in the study. However we also detected a partial recovery of cell motility, dose-dependently, by a chemoattractant (here FBS) or, time dependently, by a degenerative surrounding (here SH-SY5Y exposed to 6-OHDA).Our data enlighten novel collateral effects of SPIOn presence in human fetal SCs in vivo for its safe application to nanomedicine and nanodiagnostic, especially for the treatment of neurodegenerative diseases.Our results also suggest that SC SPIOn labeling is a promising strategy, but requires future experiments and optimization Figure S1Representative confocal image of PKH26-labeled hCVCs (red) and PKH67-labeled hAFCs (green) showing comparable cell size between the two populations, thus supporting the flow cytometry data. (ry data. .(TIF)Click here for additional data file.File S1This file contains Tables S1 and S2. Table S1: Scheme of experiment for SPIOn labeling. Table S2: Experimental protocol for migration assay.(DOC)Click here for additional data file.


TYRP1, an important melanosomal enzyme associated with human pigmentary differences. We identify alternate alleles of TYRP1 that induce or disrupt miR-155 regulation and demonstrate that these alleles are selected with different modes among human populations, causing a strong negative correlation between the frequency of miR-155 regulation of TYRP1 in human populations and their latitude of residence. We propose that local adaptation of microRNA regulation acts as a rheostat to optimize TYRP1 expression in response to differential UV radiation. Our findings illustrate the evolutionary plasticity of the microRNA regulatory network in recent human evolution.MicroRNA (miRNA)–mediated gene regulation is of critical functional importance in animals and is thought to be largely constrained during evolution. However, little is known regarding evolutionary changes of the miRNA network and their role in human evolution. Here we show that a number of miRNA binding sites display high levels of population differentiation in humans and thus are likely targets of local adaptation. In a subset we demonstrate that allelic differences modulate miRNA regulation in mammalian cells, including an interaction between miR-155 and  TYRP1, a melanosomal enzyme associated with human pigmentation. We found that the two alternate alleles on the 3′ UTR of TYRP1, either inducing or disrupting repression by miR-155, are under opposite selections among human populations. This results in a strong negative correlation between the degree of fixation of miR-155–mediated repression of TYRP1 in a population and the population's latitude of residence. These observations collectively suggest miR-155 acts a rheostat to optimize TYRP1 expression for local adaptation to differential UV radiation along the latitudes. Our findings demonstrate the plasticity of miRNA regulation in recent human evolution.MicroRNAs (miRNAs) are endogenous small RNAs that bind to their target mRNAs to post-transcriptionally repress protein production. miRNA–mediated gene regulation is usually considered to be strongly conserved among and within species, and thus alteration of such regulations is usually considered as detrimental. However, it is likely that evolutionary divergence of miRNA regulation may actually be selectively advantageous and could even serve as a genetic reservoir for innovation and adaptation. Towards this goal, we identified a number of polymorphic miRNA binding sites that display extreme population differentiation and show evidence of positive selection. We experimentally validated 3 regulations, including a regulation by miR-155 on  They recognize their target mRNAs primarily through sequence complementarity between the seed region of a miRNA  STF. For example, Coop et al. showed that SNPs with high STF values in humans are enriched for genic SNPs in comparison to non-genic SNPs, and interpreted this as evidence of local selection targeting sites in genic regions STF values into different bins  were significantly enriched for miRNA binding sites . Positive selection by definition acts on functional loci so these findings clearly indicate that miRNA binding sites with extreme population differentiation (STF≥0.5) are likely targets of positive selection.Extreme population differentiation can be attributed to several possible factors, such as outliers from neutral drift, population structure, or positive selection for local adapation. Thus we sought to determine the potential sources accounting for the observed differentiation events. Recent population genetic analyses of human variation have shown that much of the recent local adaptation in humans may be caused by subtle changes in allele frequencies in many genes rather than strong changes in a few genes ent bins , and comSTF≥0.5); however we also attempted to control, at least partially, for hitchhiking effects by excluding target-site SNPs linked to any annotated functional variant that lies 500 Kb upstream or downstream of the SNPs , SLC25A19 , and TYRP1 (tyrosinase-related protein 1) showed significant miRNA-dependent inhibition of luciferase expression in an allele-specific manner , the signal of positive selection can be localized directly to the miRNA binding sites using the CLR method  TYRP1 is important for adjusting skin reflectance to protect against excessive UV exposure TYRP1 harbors 2 common SNP variants, rs683 and rs910 that reside in two putative miR-155 binding sites  is thus a target of miR-155. Analysis of the ancestral alleles revealed substantial suppression when compared to a mutant in which all three miR-155 targets were deleted , we next tracked the local selection within individual populations. As shown in de novo binding sites on TYRP1 in YRI, CHB and JPT, likely reflects a requirement in these populations to induce miR-155 suppression on TYRP1.Previous studies suggested that TYRP1. Taken together, these results suggest that in YRI, and particularly in CHB and JPT, the derived alleles of rs683 and rs910 have been a target of positive selection . We investigated this hypothesis further by extending our analysis from the 4 HapMap populations to 37 representative indigenous populations genotyped in the Human Genome Diversity Project (HGDP), spanning 650,000 common SNPs R = −0.63). As the values for different populations might be correlated due to miration history, we cannot apply standard statistical methods to test whether the correlation is significant. However, we note that the correlation between latitude and allele frequency in rs683 is among the 1% most extreme of such correlations in the genome. This is also true if we restrict ourselves to analyses of SNPs with STF≥0.5  incoherent control or incoherent feed-forward loop in describing generic regulatory networks). Such a network architecture is important in maintaining target protein homeostasis and in fine-tuning and buffering target protein expression. Indeed, the amount of skin pigmentation is thought to be balanced between two conflicting and UV-dependent physiological needs, the production of vitamin D and folate TYRP1 in Africans (in low-latitude regions) might ensure proper expression of this gene by dampening potential fluctuations that may shift its expression away from the optimal level, conferring unfavorable pigmentation phenotypes  in this study since we aimed to find the most extreme cases of population differentiation. Future comparative studies aimed at miRNA regulatory sites may reveal many more examples of rewired miRNA interactions. Second, our target prediction was based on TargetScanS, which uses seed matches as the principal mechanism for target recognition by miRNAs. However, other mechanisms might also exist, such as sites with central pairing There are likely many more changes in miRNA interactions that have contributed to human adaptation than reported here. First, we only report signals relating to increased levels of population differentiation, which most likely only reveal a small fraction of the selection that has acted during human evolution since the divergence with chimpanzees. We also used a very stringent cutoff for Fst values for ∼3 million HapMap Phase II SNPs. When controlling for potential hitchhiking effects, we followed the protocol used in HapMap database and computed the pairwise linkage disequilibrium  between the SNPs in question and its flanking SNPs within 500 Kb downstream and upstream. Any SNPs having R2≥0.5 with annotated functional sites within this distance were excluded from the analysis. The function annotation of SNPs was retrieved from dbSNP 130 queried from UCSC Table Browser. Following previous protocol SNP annotations were based on a previous study Allele frequencies of rs683 in world populations were collected from the Human Genome Diversity Project http://haplotter.uchicago.edu/). CLR test was performed using SweepFinder The iHS values and Fay and Wu's H for HapMap populations were extracted from Haplotter  R2 with rs683 using PGEToolbox TYRP1 and none of the SNPs mediates interaction with known miRNAs. Similarly in CHB and JPT, we did not find any known functional variants on TYRP1 in strong LD with rs683. We also expanded the analysis from HapMap SNPs to dbSNP132 by querying the UCSC Table Browser, which includes SNPs genotyped in the 1000 Genomes Project To explore the possibility that rs683 and rs910 changed frequency due to hitchhiking with some other functional variants on this gene, we analyzed all the known HapMap SNPs on We first extracted all SNPs annotated to be in 3′ UTR of human genes, which were annotated by HapMap , and all the SNPs are polarized to the plus strand. These 3′UTR SNPs were then mapped onto the 3′UTR sequences of RefSeq transcripts , and we retained the longest transcripts when multiple sequences are annotated under the same transcript. Sequences with inconsistent annotations were discarded from our analysis. With this mapping procedure, we then generated a set of polymorphic 3′UTR segments, which is a 15 nucleotide window centered at the SNP position. Therefore for each SNP, sequences within the window will present twice each carrying the alternate alleles. TargetScanS All the constructs for this study were derived from the pMIR-REPORTER (Ambion). Human 3′UTR sequences in this study were amplified from genomic DNA of HEK293T cells using PCR. The PCR products were subcloned into pMIR-REPORTER vector. Overlapping PCR and QUICKCHANGE II XL Site-Directed Mutagenesis Kits (Agilent) were used to generate the mutants of 3′UTRs containing different SNPs and deletions of miRNA target sites. pMIR-REPORTER β-galactosidase vector was used as the transfection control. G3R is a gift from Dr. Burton Yang' lab at the University of Toronto, which has the coding sequence of the chicken versican G3 domain in the pMIR-REPORTER vector, and was used as a negative control. All the synthesized miRNA mimics and the mimic negative control were purchased from Dharmacon. miR-CTL in this study is a negative control with sequence from a C.elegans miRNA cel-miR-67, which has minimal sequence identity with miRNAs in human, mouse and rat (by Dharmacon).HEK293T cells were grown in high glucose DMEM containing 10% FBS (Thermo). They were transiently transfected with DNA constructs and miRNA mimics at 40% confluency in 24-well plates using the calcium-phosphate precipitation method. Cells were lysed 48 hours after transfection, and the activities of firefly luciferase and β-galactosidase of total cell lysates were determined using the Firefly Luciferase Assay System (Promega) and the β-gal assay previously described, respectively. To obtain the relative activity, the activity of the firefly luciferase was first normalized to the activity of β-galactosidase to obtain normalized firefly luciferase activity (nFFLuc), and the data were then determined using the following formula: Relative Activity = log2(nFFLucmiR-155/nFFLucmiR-CTL), where nFFLucmiR-155 or nFFLucmiR-CTL is the nFFLuc in the presence of mir-155 mimics or mimic negative control. The concentration of miR-155 was increased as indicated in the figures , with thSK-MEL-19 cells were grown in RPMI 1640 (GIBCO) containing 10% FBS and antibiotics. For genotyping, the lysates of SK-MEL-19 cells were subjected to PCR. The forward primer starts from 116 bps upstream of rs683 whereas the reverse primer starts from 118 bps downstream of rs683. The PCR products were cleaned and sent to DNA sequencing using the forward primer. The sequencing spectra were processed and analyzed using MacVector. SK-MEL-19 cells were transiently transfected with miRNA mimics following the instructions of the RNAi MAX transfection kit (Invitrogen). For immunoblotting analysis, cells lysates were collected 40 hours after transfection. The antibodies used for immunoblotting analysis are: TYRP1  and GAPDH . The ΔCT values were obtained by comparing the amplification of the target cDNA to that of HPRT. The TapMan SNP qPCR kit for rs683  was used to analyze the expression of the TYRP1 transcripts carrying its alternate alleles. This kit included two probes, one conjugated with VIC fluorescence dye to monitor transcripts carrying the ancestral allele and the other conjugated with FAM fluorescence dye to detect the derived allele. To evaluate the cross-hybridization between the probes, a pilot qPCR analysis was performed for the constructs A and B in Figure S1TYRP1 are indicated in block III.Identifying the predicted miRNA binding sites showing population differentiation. (A) Distribution of the context score among the predicted sites affected by SNPs. Prediction confidence was quantified by context score where more negative score indicates more confident prediction. Based on the distribution, a site considered confident if its context score no more than −0.2. (B) Hierarchical clustering of the ancestral allele frequencies in the 4 populations, YRI, CEU, CHB and JPT. The SNPs were clustered into 4 blocks as indicated from block I to block IV, with highly differentiated patterns among the 4 populations. The two SNPs (rs683 and rs910) on (TIF)Click here for additional data file.Figure S2(A–D) The CLR test localizes selection signal on the polymorphic miRNA binding sites (indicated in blue below the X-axis in each panel). The dotted lines indicate the loci for each SNP analyzed.(TIF)Click here for additional data file.Figure S3(A–D) The luciferase reporter assay in HEK293T revealed four genes did not respond to their predicted miRNA regulators. The ancestral alleles are shaded in blue whereas the derived alleles are shaded in red. The reporter constructs were individually transfected into HEK293T cells with miR-CTL or with the predicted miRNAs as indicated. The luciferase relative activities were obtained and analyzed as described in (TIF)Click here for additional data file.Figure S4Lack of selection signal on the derived allele of rs7198 in YRI (A–B) and CEU (C–D) revealed by CLR test  and Fay-Wu's H test . The derived allele in CEU is 0.67, whose H is marginally significant as shown in (D), but is not supported by CLR test (C). The threshold of statistical significance was not shown in (B) as all the values were insignificant, far beyond the threshold.(TIF)Click here for additional data file.Figure S5Statistical tests for positive selection on the derived allele of rs683 in YRI (A–B) and CEU (C–D). CLR and Fay-Wu's H tests consistently localized the selection signal around the rs683 locus in YRI (A–B), but not in CEU (C–D). The dotted line for H statistic indicates the 5% extreme value among the genome-wide SNPs.(TIF)Click here for additional data file.Figure S6TYRP1 and its 5 kb upstream in YRI (the first row) and CEU (the second row). Each column represents one SNP, and different genomic regions are separated by red vertical bars, including a 5 Kb upstream region. The brighter color indicates higher association (2R) of a given SNP with rs683. The 6th SNP rs12001162 is absent in HapMap CEU population. In CEU and YRI, the strongly linked loci with rs683 are intronic and do not overlap with known splice sites. The only missense SNP is not associated with rs683. The two other SNPs in close proximity to rs683 and rs910 on the 3′ UTR do not interact with any known human miRNAs.Linkage disequilibrium between rs683 and all other known variants on (TIF)Click here for additional data file.Figure S7Plots of the extended haplotype homozygosity (EHH) in the 0.5 Mb region centering at rs683 in CEU (A), East Asia (B) and YRI (C). Haplotypes carrying the ancestral or the derived allele of rs683 are in blue or red, respectively.(TIF)Click here for additional data file.Figure S8STF (≥0.5) (B). In either case the correlation derived by rs683 is among the extreme 1% of all the SNPs analyzed (the red bar).Histograms of the Pearson's correlation coefficients between the absoulte latitides of HGDP populations and the derived frequencies of SNPs across the genome (A) or SNPs with high-(TIF)Click here for additional data file.Figure S9Evaluation of the cross-hybridization between probes for the TaqMan SNP qPCR assay for rs683. In this assay one probe conjugated with VIC fluorescence dye monitors the ancestral allele (the blue circles) and the other probe conjugated with FAM fluorescence dye detects the derived allele (the red squares). Constructs A and B used in (TIF)Click here for additional data file.Table S1The 30 SNPs and 26 human genes identified in this study. The confidence of miRNA target prediction and the associated selection statistics are also shown. Alternative transcripts of these genes as annotated by RefSeq are shown individually. Note that more than one miRNAs can share one target site and thus a single SNP can impact the regulation by multiple microRNAs, for example the SNP rs9893667 on gene NM_006380 (APPBP2) can influence the target site of hsa-miR-362-3p, hsa-miR-329, and hsa-miR-603 (highlighted in bold).(XLS)Click here for additional data file.Table S2http://david.abcc.ncifcrf.gov). In this table, each gene is annotated with its OMIM disease association, gene ontology and KEGG pathways. Note C4orf46 is a hypothetical gene with no function annotation.Summary of molecular functions of the identified genes. Annotations were queried from DAVID ((XLS)Click here for additional data file.Table S3Functional annotation of the 7 genes selected for experimental validation.(XLS)Click here for additional data file.Table S4miRNA families considered in this study. miRNAs were grouped into families based on their seed identity. The miRNA family information was deposited in TargetScanS.(XLS)Click here for additional data file.