Pilot studies are often used to transition therapies developed using animal models to a clinical setting. Frequently, the focus of such trials is on estimating the safety in terms of the occurrence of certain adverse events. With relatively small sample sizes, the probability of observing even relatively common events is low; however, inference on the true underlying event rate is still necessary even when no events of interest are observed. The exact upper limit to the event rate is derived and illustrated graphically. In addition, the simple algebraic expression for the confidence bound is seen to be useful in the context of planning studies. While pilot studies may vary in the fundamental objectives, many are designed to explore the safety profile of a drug or a procedure ,2. Oftenα (the Type I error rate) and β (the Type II error rate) may be inappropriate since the objective of the research is not to provide definitive support for one treatment over another [In the context of pilot studies, traditional levels of  another . For exaβ) is of less practical importance in a single arm, non-comparative  pilot study since the results would almost always require confirmation in a controlled trial setting. Shih et al [Similarly, power % confidence interval is to be generated for π and an estimate of the sample size, n, is desired. Denote X as the number of patients sampled who experience the adverse event of interest. Then, the probability of observing x events in n subjects follows the usual binomial distribution. Namely,For ease of presentation, assume the pilot study will involve πu as the upper limit of the exact one-sided 100 × (1 - α)% confidence interval for the unknown proportion, π [πu is the value such thatDenote rtion, π . Then πux = 0), equation (1) reduces toA special case of the binomial distribution occurs when zero events of interest are observed. In pilot studies with relatively few patients, this is of practical concern and warrants particular attention. When zero events are realized n = α.(1 - α)% confidence interval for π isAccordingly, the upper limit of a one-sided 100 × (1 - πu = 1 - αn1/.     (2)α)% one-sided confidence interval is .The resulting 100 × % confidence that the true rate did not exceed a pre-specified π, say π0, given that zero adverse events were observed. Using (2), it follows that:Furthermore, one can consider using (2) in other clinically important manners. For instance, an investigator may be planning a pilot study and want to know how large it would need to be to infer with 100 ×  declare that they have no competing interests.RC and RW contributed to the conceptualization, writing and editing of this manuscript.


Incidence and mortality of human malignant melanoma has risen rapidly over recent decades. Although the notorious resistance to treatment is characteristic for metastatic malignant melanoma, only a few experimental models have been established to study the metastatic cascade or to test new alternative treatment modalities. Thus, new human models are wanted. Here, we describe the metastatic behaviour of seven human melanoma cell lines derived from two primary cutaneous melanomas  and five metastases established from liver (UKRV-Mel-4), skin , pleural effusion (UKRV-Mel-2) and lymph node (MV3). All cell lines were analysed for their capacity to grow in nude mice after s.c. and i.v. administration. M13 cells developed liver metastases spontaneously after s.c. injection, and subsequent passages of M13 and M7 melanoma cells caused liver metastases after i.v. injection, whereas MV3 and WM98-1 gave rise to lung metastases, using the same inoculation route. In contrast, WM 1341, UKRV-Mel-2 and UKRV-Mel-4 grew only very slowly in nude mice after s.c. injection and did not cause any metastases after i.v. or s.c. administration. The pattern of metastases or growth kinetics did not correlate with the interleukin 8 or tumour necrosis factor secretion of cell lines. Adhesion molecules and growth factor receptor expression on the cell lines differed widely, as determined by flow cytometry, with the low metastatic cell lines  demonstrating a marked reduction in VLA-1 and VLA-5 expression compared with the metastatic lines . Expression of pigment-related proteins such as tyrosinase, TRP-1, TRP-2, Melan-A/MART-1, gp100, MAGE1 or MAGE-3 was not associated with growth and metastatic characteristics of the melanoma cell lines analysed. In conclusion, the established human melanoma cell lines exhibited diverse growth behaviour in nude mice in congruence with some early established prognostic markers such as VLA-1 and VLA-5. The xenografts provide good models for further study of metastatic processes as well as for evaluation of alternative treatment modalities including new pharmaceutical drugs and gene therapeutic targeting using tissue-specific gene regulatory elements for gene targeting.


PTEN-controlled PI3K-AKT-mTOR pathway represents one of the most deregulated signaling pathways in human cancers. With many small molecule inhibitors that target PI3K-AKT-mTOR pathway being exploited clinically, sensitive and reliable ways of stratifying patients according to their PTEN functional status and determining treatment outcomes are urgently needed. Heterogeneous loss of PTEN is commonly associated with human cancers and yet PTEN can also be regulated on epigenetic, transcriptional or post-translational levels, which makes the use of simple protein or gene expression-based analyses in determining PTEN status less accurate. In this study, we used network component analysis to identify 20 transcription factors (TFs) whose activities deduced from their target gene expressions were immediately altered upon the re-expression of PTEN in a PTEN-inducible system. Interestingly, PTEN controls the activities (TFA) rather than the expression levels of majority of these TFs and these PTEN-controlled TFAs are substantially altered in prostate cancer mouse models. Importantly, the activities of these TFs can be used to predict PTEN status in human prostate, breast and brain tumor samples with enhanced reliability when compared to straightforward IHC-based or expression-based analysis. Furthermore, our analysis indicates that unique sets of PTEN-controlled TFAs significantly contribute to specific tumor types. Together, our findings reveal that TFAs may be used as “signatures” for predicting PTEN functional status and elucidate the transcriptional architectures underlying human cancers caused by PTEN loss. PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor gene is mutated frequently in human cancers and cancer predisposition disorders PTEN genomic deletion, gene mutations, epigenetic silencing (e.g. silencing by DNA methylation or miRNAs), impaired membrane recruitment (e.g. loss of interaction with MAGI2) or decreased protein stability/activity mediated by various post-translational modification  The Determination of PTEN functional status can be further complicated by the intricate signaling pathways that are regulated by PTEN. Through its lipid phosphatase activity, PTEN regulates PI3K-AKT-mTOR signaling that are involved in downstream transcription machineries, such as NF-κB, FOXO, and p53 PTEN expression using global gene expression profiling in a PTEN inducible system. We further hypothesized that vast target gene expression changes may be controlled by a few key transcription factors (TFs), which can be a more sensitive and accurate “signature” for PTEN status. However, expression levels of these TFs will not always be sufficient to reflect their activity since the activity of a transcription factor (TFA) is controlled by various post-translational modifications as well as co-activator and co-repressor activities. Previous works by us and others have shown that TFA can be best inferred from the transcript levels of its direct target genes, rather than its mRNA level using Network Component Analysis (NCA) To decipher functional status of PTEN and PTEN-controlled signaling network, we first analyzed the transcriptional targets which are immediately regulated by PTEN (see Pten null mouse embryonic fibroblasts (MEFs). We found that the activities of these PTEN-controlled TFs are significantly altered in prostate cancer mouse models. Furthermore, the TFAs of these TFs show enhanced sensitivity and specificity when used to predict PTEN status in human prostate, breast and brain tumors, as compared to the gene expression-based analysis.In this study, we identified 20 TFs whose activities are immediately altered upon the reexpression of PTEN re-expression, an inducible system was generated in which PTEN expression can be controlled in a doxycycline-dependent manner in the Pten null Δloxp/ΔloxpPten cells L/LPten is a Pten WT line and isogenic to Δloxp/ΔloxpPten). Consistent to our previous study, re-expression of PTEN was able to suppress the gene expression of p90MDM2 isoform without significant change in p76MDM2 isoform as previously reported (To identify transcription factors whose expressions or activities are directly regulated by reported  [21].PTEN re-expression by comparing gene expression levels 1 and 2 days after doxycycline (20 µg/mL) treatment with those before the treatment  are up regulated and 216 (61.4%) are down regulated by PTEN re-expression. Next we analyzed global gene expression alterations immediate following We reasoned that the PTEN-inducible genes must be regulated by the key transcription factors whose activities are controlled by PTEN. Therefore, we investigated PTEN immediately controlled TFs via network component analysis (NCA) in which the activities of TFs (TFAs) are deduced based on expression of their target genes. Different from conventional gene expression analysis, which focuses on statistically significant changes of individual genes, NCA deduces TFAs based on the concordant variations of all, rather than individual target genes, and that does not require the analyzed gene expressions to be statistically significant PTEN re-expression  while LEF1 expression level remained constant  analysis. c-MYC target genes BCAT1, CDK4, EIF4E, and SHMNT1 were selected because (1) their proximal promoter regions contain c-MYC consensus binding sequence (CACGTG), which is conserved between human and mouse; and (2) the control strengths by c-MYC were highly significant based on NCA analysis . Interestingly, hi-c-Myc shares 13 TFAs changes with Pten null or Pten null and mAKT1 models, of which seven are concordantly regulated, including Ar, Creb1, Hoxc8, Elk1, Smad1, Hif1α, Stat6 and c-Myc  PTEN deficiency frequently occurs in these cancers As shown in PTEN-controlled TFA patterns can also be used as signatures to separate PTEN negative from PTEN positive breast cancers  altered in the PTEN negative group in all three cancers. STAT6 TFA is decreased while the others TFA are increased in the PTEN negative group. The degree of overlap of the tumor type-specific PTEN-controlled TFAs is summarized by a Venn diagram in Given that the PTEN-controlled TFA signatures are associated with PTEN status in prostate, breast and brain tumors, we next asked if a particular subset of the transcription factors play more important role in each individual tumor type. To this end, we first compared each TFA between PTEN positive and negative samples, identified by both IHC/CN and TFA-based analysis. We further investigated the possible interactions among the TFAs by examining pair wise correlation coefficients of inferred TFAs across patient tumors in each tissue type. The absolute correlation coefficients between the pairs are illustrated in In this study we used NCA and its complementary trimming algorithm to reveal 20 TFs that immediately respond to the expression of PTEN in a PTEN inducible system. We found that the PTEN immediate responsive gene-based TFA signatures are more accurate and sensitive than either cancer-based TFA or gene expression-based analyses in predicting PTEN functional status in human cancers. These TFA-based signatures, therefore, provide readout of transcription factor activity even if their mRNA levels do not change, and help to overcome the complexity of multifactorial post-transcriptional PTEN signaling pathway regulations. Since mRNA profiles are currently measureable in clinical settings, our TFA-based signatures provide new rationales for stratifying patients according to their PTEN functional status and for monitoring treatment outcome in PI3K-targeted therapies.Saccaramyces cerevisiaeOur study testifies to the power of combining traditional genetic and biochemical approaches with mathematic algorithms in deciphering complicated transcription regulatory networks. NCA complements other classical bioinformatics methods, such as Principal Component Analysis (PCA) and Independent Component Analysis (ICA). In contrast to PCA and ICA, NCA utilizes biochemical constraints, i.e., the relationship between transcription factors and their regulated genes, rather than statistical or mathematical constraints in data deconvolution. This means if target genes of a certain TF concordantly altered their expressions, even though at the subtle levels upon stimulation, its NCA-derived TFA will show significantly changes. Besides, NCA also detangles effects of multiple TFs regulating a same gene. TFA profiles are more robust and reliable in representing the real activities than the expression of a TF specific target gene which may not be expressed in all tissues. This explains why TFA-based signatures are more reliable than gene-based signatures in predicting PTEN status in different tumor types. NCA has been used to reveal biological relevant network structure and regulatory dynamics in bacteria A shortcoming of NCA is that it depends on the information of TF and target gene relationship. For instance, although FOXO activity is known to be regulated by PTEN controlled PI3K/AKT pathway, FOXO TFA cannot be derived in PTEN inducible system because its target gene expression values are not available in the database we used. Nevertheless, the results obtained from our analyses are quite robust.Pten null mouse exhibit concordant alterations in their activities when the PTEN downstream AKT/mTOR pathway is manipulated genetically or pharmaceutically by the mTOR inhibitor rapamycin. This result implies that PTEN controls TFAs through its phosphatase activity by regulating the PI3K/AKT/mTOR pathway, which is known for regulating activities of several TFs including NF-κB hi-c-Myc and Pten null or mAKT1 prostate cancer models in their associated prostate cancer-associated TFAs. It is worthy to note that c-MYC target genes that are perturbed by PTEN expression involve the regulation of cell growth, cell metabolism, and protein synthesis Although PTEN is not a TF, it can regulate TFAs through either phosphatase-dependent or -independent mechanisms. In the prostate cancer mouse models, the majority of TFs perturbed in the Our study reveals common and cancer tissue type-specific regulation of TFAs by the PTEN tumor suppressor. The six common PTEN-controlled TFAs including c-MYC most likely play an essential role in tumor development caused by PTEN loss and their activity may serve as surrogate markers for determining PTEN functional status and measuring response to targeted therapy. Other tissue-type-specific regulated TFAs may help us understand PTEN's tissue-specific function. The PTEN-controlled TFAs deduced by NCA, therefore, will aid in stratifying cancer patients according to PTEN functional status and in deciphering the complicated transcription regulatory networks controlled by the PTEN tumor suppressor. Although targeting transcription factors with small molecules remains challenging, recent works by Bradner and colleagues on selective inhibitions of BET bromodomains PTEN-inducible mouse PtenΔloxp/Δloxp MEF cells Total RNAs were extracted using RNeasy Mini kit (Qiagen). RNAs were reverse-transcribed by oligo(dT) primer using Superscript RT-PCR kit (Invitrogen), according to the manufacturer instructions. PCR reaction was performed under the following conditions: 94°C for 3 min; 94°C 30 Sec; 58°C for 30Sec; 72°C for 30 Sec for 40 cycles; and 72°C for 10 min, using iQ SYBER Green Supermix Kit from Bio-Rad. Chromatin immunoprecipitations were modified from the EZ- ChIP (Upstate) protocol using antibodies: anti-c-Myc  anti-LEF1 . The percentage of the bound DNA was quantified against the original DNA input using real time PCR analysis (Biorad). Primer sequences used for ChIP are as follows: AATCCGCTAGGTCGCGAGT; BCAT-3′: AGCAAGACCTGGGGCAGTBCAT-5′: TTACACTCTTCGCCCTCCTC; CDK4-3′: ATGTGACCAGCTGCCAAAGCDK4-5′: CAGGGCCAAACGGACATA; EIF4E-3′: CAATACTCACCGGTTCGACAEIF4E-5′: GCAGAGTGCACCTTCCTGA;SHMNT1-5′: GTGCCACCAGTCCCAGACSHMNT1-3′:GGGATAGCAAGCATCCAGAG;WISP1-5′:  CCTTCATGACACGTGAAAGCWISP1-3′:All the array datasets were downloaded from public domains, and were MIAME compliant. Mouse and MEF expression data was available through NCBI's Gene Expression Omnibus (GEO) with accession IDs GSE29010 and GSE1413, and normalized by RMA method. Human expression data sets were downloaded from GEO and other public domains see . If a geNCA is built based on log-linear model in which gene expression ratios are log-linearly proportional to activity ratios of their regulators. In the NCA pre-processing steps, expression data sets from single channel Affymetrix arrays were set in log ratios comparing the conditions of interest  to the references . Biological repeat arrays were averaged first before calculating the log ratios to filter out extreme value of log-ratio.We used the network structure information provided by Transcriptional Regulatory Element Database (TRED) from Cold Spring Harbor Laboratory Cluster 3.0 was used for the unsupervised hierarchical clustering analysis using TFAs/gene expression. The similarity between samples was represented by the cosine (or un-centered correlation) metric. Complete linkage was used to clusters samples. In the heatmap of prostate data the TFAs are colored code based on their relative values to the respective averages of normal samples to illustrate the direction of TFA variation to normal prostate tissue.In each human cancer dataset, Pearson correlation coefficient between each pair of TFAs was calculated. The unsupervised hierarchical clustering analysis was then used to rearrange the order of TFAs in the matrix of the absolute correlation coefficients.Whole-cell extraction was described detail in Figure S1Validation of PTEN-inducible systems. (A) Restoration of PTEN expression suppressing the expression of the p90 isoform, but not p76 isoform of MDM2 in PTEN inducible PC3 cells. (B) PTEN re-expression does not change the c-MYC, STAT6 and c-JUN total protein levels but does alter the ratio of phosphor-c-JUN to total c-JUN. Numbers indicate the relative ratio of phosphorylated to total protein, or the levels of c-Myc protein, with the PTEN null state defined as unity.(TIFF)Click here for additional data file.Figure S2Heatmap of TFAs changes deduced from gene expression profiles in PTEN inducible MEFs and prostate cancer mouse models. Heatmap showing PTEN-controlled TFAs that are significantly altered in PTEN-inducible MEF tissue culture cells; and a set of prostate cancer-related TFAs that are significantly altered during tumorigenesis in murine prostate cancer models, but not by re-expression of PTEN in the PTEN-inducible MEF system (Rapa: Rapamycin treatment).(TIF)Click here for additional data file.Figure S3The PTEN-control TFA-based unsupervised clustering analysis. Unsupervised clustering analysis, based on PTEN-controlled TFAs, was used to classify human tumor samples. (A) the first and (B) the second (NKI) breast cancer data sets and in (C) brain cancer dataset. In the first breast tumor data set (A), PTEN-controlled TFA-based unsupervised clustering yields a clustering pattern of tumor PTEN negative status (Group 1). As for the second breast cancer data set the dendrogram also illustrates the association of PTEN-negative Group 1 with poorly differentiated, ER-negative and basal-like phenotype.(TIF)Click here for additional data file.Figure S4NCA-inferred TFAs significantly altered in human breast cancer based on PTEN IHC. Log10-transformed t-test p values for each TFA between samples of different IHC-based PTEN status. The graph shows the 45 TFs with the highest log10-transformed p-values. The p-values >0.1 of the other 25 TFs are not shown. The dashed line (p = 1e-4) indicates the threshold value for selecting the TFA-based PTEN-IHC-derived signatures used in the analysis in (TIF)Click here for additional data file.Table S1List of PTEN immediately controlled genes in MEFs.(XLS)Click here for additional data file.Text S1Supporting Information.(DOC)Click here for additional data file.


In particular, a new method for direct and real-time visualization of the Cd uptake by the roots in the culture was first realized in this work.Rice is a major source of dietary intake of cadmium (Cd) for populations that consume rice as a staple food. Understanding how Cd is transported into grains through the whole plant body is necessary for reducing rice Cd concentrations to the lowest levels possible, to reduce the associated health risks. In this study, we have visualized and quantitatively analysed the real-time Cd dynamics from roots to grains in typical rice cultivars that differed in grain Cd concentrations. We used positron-emittingjaponica type) showed rapid saturation curves, whereas three high-Cd accumulating cultivars (indica type) were characterized by curves with a peak within 30 min after107Cd supplementation, and a subsequent steep decrease resulting in maintenance of lower Cd concentrations in their roots. This difference in Cd dynamics may be attributable to OsHMA3 transporter protein, which was recently shown to be involved in Cd storage in root vacuoles and not functional in the high-Cd accumulating cultivars. Moreover, the PETIS analyses revealed that the high-Cd accumulating cultivars were characterized by rapid and abundant Cd transfer to the shoots from the roots, a faster transport velocity of Cd to the panicles, and Cd accumulation at high levels in their panicles, passing through the nodal portions of the stems where the highest Cd intensities were observed.Imaging and quantitative analyses revealed the different patterns in time-varying curves of Cd amounts in the roots of rice cultivars tested. Three low-Cd accumulating cultivars (This is the first successful visualization and quantification of the differences in whole-body Cd transport from the roots to the grains of intact plants within rice cultivars that differ in grain Cd concentrations, by using PETIS, a real-time imaging method. Since then, the contamination of rice by Cd has been monitored to prevent it from being distributed to consumers in Japan, in accordance with the Food Sanitation Act established in 1969 in Japan. Nevertheless, the Cd contamination of rice is still a serious threat to Japanese people and other populations in the world that consume rice as a staple food, because rice is a major source of dietary intake of Cd. Understanding how Cd is taken up by rice roots and subsequently transported to rice grains is necessary for reducing Cd concentrations in rice as much as possible, thus diminishing the risk that Cd poses to human health.Cadmium (Cd) has an important impact on agriculture, as the excessive consumption of Cd from contaminated food crops can lead to toxicity in humans. High-dose Cd exposure is particularly toxic to the kidney and leads to renal proximal tubular dysfunction . In Japa1B-ATPase transporter OsHMA3, which is involved in Cd sequestration in root vacuoles [Arabidopsis thaliana [OsHMA2 in yeast have suggested that this gene is a good candidate for the control of Cd xylem loading in rice [Plant roots are the first entry point for Cd uptake from soil solutions, and the transport processes of Cd into the roots have been well reviewed from the viewpoints of physiological and genetic studies . A dose-vacuoles ,10. Xylethaliana ,12. In r in rice . The pro in rice ,14. Tana in rice  estimate in rice  collecte109Cd has been widely used to visualize Cd distribution within plant tissues [109Cd and115mCd) supplementation at the early ripening stage, and lesser amounts of Cd were distributed to grains, whereas the lowest levels of Cd were present in the leaves. However, only the static distribution of Cd at a given moment can be obtained by autoradiography. In recent years, the positron-emitting tracer imaging system (PETIS) has been employed to study various physiological functions in intact, living plants [52Fe [52Mn [62Zn [107Cd tracer and PETIS. The movement of Cd in the aerial part of rice (cultivar Nipponbare) in the vegetative and reproductive stages was captured as serial images, and various parameters (e.g. transport velocity in the shoot) were analysed quantitatively. However, a method for direct imaging of the underground parts, which should provide valuable information about the root uptake, remained to be developed because of interference by the highly radioactive culture solution.In general, radioisotope tracers are useful tools for analysing the spatial distribution or temporal change in the amount of a substance in the plant body. tissues ,18. For  tissues  observedg plants ,20. Thists [52Fe ,52Mn [22Mn [62Zn . RecentlMn [62Zn  establisIn this study, we employed PETIS in our two objectives: to realize direct observation of Cd uptake by the roots in the culture solution, and to characterize clearly the differences in Cd dynamics from the culture to the grains between the high- and low-Cd accumulating cultivars.107Cd dynamics , which were classified as having markedly high Cd concentrations in their grains and shoots (herein collectively referred to as "high-Cd indica cultivars"), the amounts of Cd in the roots peaked within 30 min of exposure to107Cd, and the subsequent decreases in Cd were monitored until the 5 h point  with lower Cd concentrations in their grains and shoots (herein collectively referred to as "low-Cd japonica cultivars"), the amounts of Cd in the Nipponbare and Sasanishiki roots plateaued or increased slightly after peaking at approximately 1 h. A delayed Cd peak was observed in the Koshihikari roots. In this study,107Cd was supplied only at the beginning of the imaging, and almost all of the107Cd in the culture solution was absorbed by the roots within approximately 5 h in all cultivars  and ROI-2 (leaf sheaths and leaf blades) are shown in Figure 107Cd supplementation and increased dramatically up to 10 h, particularly for the high-Cd indica cultivars. The amounts of Cd in ROI-1 were significantly higher in the high-Cd indica cultivars than in the low-Cd japonica cultivars up to 36 h. After 10 h, the amounts of Cd reached plateaus for all cultivars, but slight decreases were found in the high-Cd indica cultivars. Unlike the accumulation patterns of Cd in ROI-1, the amounts of Cd in ROI-2 (leaf sheaths and leaf blades) continued to increase linearly until the end of the experiment. There was an approximately 3-fold difference in the amount of Cd between the high-Cd indica cultivars and the low-Cd japonica cultivars.Figure japonica rice cultivars accumulated in their roots, whereas only 60-70% of the Cd in the indica rice cultivars was distributed in their roots. In the shoot parts, Cd accumulated at the shoot base in the highest proportions; this accounted for approximately 15-20% of the total Cd in the plant body for the high-Cd indica cultivars, whereas it was less than 10% for the low-Cd japonica cultivars. On the other hand, the proportions of Cd in the shoot base were approximately 50% of those in the total shoot and did not differ greatly between cultivars. In the leaves (leaf sheaths and leaf blades), Cd was mostly distributed in the younger leaves, that is, the 4th and 5th leaves, suggesting that Cd moves preferentially to new leaves after moving from the roots to the shoot bases.After the PETIS experiment, autoradiography was performed to obtain static distributions of Cd for each plant part at the vegetative stage  use of a root box with flat, shallow compartments, allowing detectors to focus on the roots; 2) use of a simple nutrient solution to avoid competition between Cd and other minerals at adsorptive sites in roots; and 3) ensuring application of adequate radioisotope activity for the quantitative measurements by taking into consideration the dynamic range of the PETIS. These technical improvements first enabled direct visualization of real-time Cd dynamics in the whole plant body, that is, from roots to grains.We applied the improved system to analyse the time-varying distribution of Cd to characterize the differences in Cd dynamics in rice cultivars varying in grain Cd concentrations.japonica cultivars showed gentle saturation curves, whereas three high-Cd indica cultivars showed a drastic drop  supply in this study, and this could be considered as a kind of pulse feed experiment. The curves obtained would naturally be different from those of roots with continuous Cd supply. The point is that the pulse feed experiments provide snapshots  of dynamics and the result with continuous feed could be described as their integration. In fact, the results from this study agreed well with our previous results obtained from the rice genotypes grown continuously in the Cd-polluted soil [japonica cultivars than in the high-Cd indica cultivars.This difference most probably depends on whether the rice plant inherently conserves the functional OsHMA3, which is a membrane transporter protein involved in Cd storage in root vacuoles. All high-Cd vacuoles ,10,28. Ovacuoles . This trs Figure  suggeststed soil ; root Cd107Cd had a strong presence in the non-elongated stems at the shoot bases  was found to be slightly faster than that for Koshihikari. However, the differences in the Cd transport velocity between genotypes were likely to be small. Instead, a remarkable difference (approximately 5-fold) was observed in the slopes of Cd accumulation to panicles. Therefore, this result indicates that the differences in root Cd dynamics also influence the Cd concentration of the long-distance Cd transport to panicles in rice cultivars.The Cd accumulation pattern of the neck node for the high-Cd accumulator BIL48 plants corresponded well to that of the node at the shoot base, showing the characteristic steep and linear increase, and subsequent plateau pattern of Cd accumulation Figures  and 4c. s Figure . Fujimak107Cd was supplied to the genotypes with emerged ears  supply might be a description of the Cd dynamics in rice at the vegetative and heading stages after water drainage in the paddy fields.In paddy fields, rice is mostly grown under submerged conditions in which bioavailable Cd is limited because of the rise in soil pH and decrease in the redox potential. Midseason drainage in Japanese paddy fields is widely recommended at the vegetative stage to avoid the root rot induced by continuous soil reduction. In addition, early drainage after panicle emergence is often practised in paddy fields to facilitate machine harvesting. Thus, rice is not continuously exposed to high bioavailable Cd in the soil, and the PETIS data obtained by a limited Cd  consisting of three indica rice cultivars  with markedly high Cd concentrations in their grains and shoots, and another three major japonica cultivars from Japan  with lower Cd concentrations in their grains and shoots [4)2SO4, 0.36 mM Ca (NO3)2·4H2O, 0.54 mM MgSO4·7H2O, 0.18 mM KNO3, 0.18 mM KH2PO4, 40 μM Fe(III)-EDTA, 18.8 μM H3BO3, 13.4 μM MnCl2·4H2O, 0.32 μM CuSO4·5H2O, 0.3 μM ZnSO4·4H2O, and 0.03 μM (NH4)6Mo7O24·4H2O. Kimura B solution has been widely used for growing rice plants [For the experiments conducted at the vegetative seedling stage, we used six rice cultivars  for 24 h before the start of the107Cd supplementation experiment. The solution was continuously aerated, and the surface levels were set a few centimetres below the boundaries between the shoot bases and roots by automatically supplying fresh solution from the reservoir tank as the plants took up the water. Purified107Cd and nonradioactive Cd at concentrations of 0.1 μM were simultaneously supplied as carriers to the 0.5 mM CaCl2 solution in which the plants were grown. Plants were placed in the mid-plane between the two opposing detector pairs of the PETIS apparatus . A pair of annihilation γ-rays emitted from the decaying positrons was detected simultaneously, and the emission point was then determined as the middle point of the two incident points. Repeated determinations of the emission points reconstructed a static image of the tracer distribution. One frame, which is the unit of time required to obtain one static image with sufficient quality, was set to 4 min, and 540 (36 h) frames were collected to yield serial time-course imaging. The detectors were set at the roots, non-elongated stem bases (shoot bases), and panicles to monitor the dynamics of Cd in each part. The typical size of the FOV in the detector head was 12 cm in width and 19 cm in height, and the spatial resolution was approximately 2 mm. All PETIS experiments were conducted in a growth chamber at 30°C and 70% humidity, with continuous light at a density of 400 μmol m-2 s-1.The PETIS imaging experiments were conducted following the method of Fujimaki et al.  with modi et al. . Finallyhttp://rsb.info.nih.gov/ij). Because the ROI can be selected freely from the image data using this software, the radioactivity of107Cd over time within each ROI was extracted from the data. A time-course curve of Cd accumulation within the ROI indicated the amounts of total Cd, consisting of the sums of radioactive and nonradioactive Cd. All PETIS experiments were conducted two or three times, and the representative data are shown in this paper.To determine Cd dynamics in the plant body qualitatively and quantitatively, the dataset obtained from the PETIS apparatus was reconstructed using the NIH Image J 1.42 software . This isotope has a longer half-life (461 days) than107Cd (6.5 h), and it was absorbed by the plants during the PETIS experiments but not detected by the PETIS apparatus because it is not a positron emitter. After sufficient decay of107Cd within the test plants, they were separated into several parts and set on imaging plates  in cassettes. After a few days of exposure, the imaging plates were scanned using a bio-imaging analyser  to obtain the autoradiographic images for examining109Cd distribution in the plant bodies. The Cd concentrations in each plant part were determined with a well-type gamma counter .In the production process of1, NS, and SF initiated and coordinated the study. SI1, MI, TA, and MK prepared the experimental plants and participated in the PETIS imaging. NS, SIT, SI2, NK, and SF produced the107Cd tracers and carried out the PETIS imaging. NS and SIT processed the imaging dataset obtained by the PETIS. SI1 drafted the manuscript with the assistance of NS and SF. All authors discussed the results and commented on the draft manuscript, and read and approved the final manuscript.SI107Cd dynamics in the roots of six rice cultivars at the vegetative stageAnimation film of.Click here for file107Cd transport into shoots of six rice cultivars at the vegetative stageAnimation film of.Click here for fileAutoradiography of detached parts of plants at the vegetative stage 36 h after Cd supplementation. A, Nipponbare. B, Koshihikari. C, Sasanishiki. D, Choko-koku. E, Jarjan. F, Anjana Dhan.Click here for file107Cd accumulation in the panicles of Koshihikari and BIL48 at the grain-filling stageAnimation film of.Click here for file


A large amount of data supports the view that PTEN is a bona fide tumor suppressor gene. However, recent evidence suggests that derailment of cellular localization and expression levels of functional nonmutated PTEN is a determining force in inducing abnormal cellular and tissue outcomes. As the cellular mechanisms that regulate normal PTEN enzymatic activity resolve, it is evident that deregulation of these mechanisms can alter cellular processes and tissue architecture and ultimately lead to oncogenic transformation. Here we discuss PTEN ubiquitination, PTEN complex formation with components of the adherens junction, PTEN nuclear localization, and microRNA regulation of PTEN as essential regulatory mechanisms that determine PTEN function independent of gene mutations and epigenetic events. This inhibits PI3K downstream targets, mainly PKB-Akt [PTEN (phosphatase and tensin homolog deleted on chromosome ten)/MMAC (mutated in multiple advanced cancers) has been identified simultaneously by two research groups as a candidate tumor suppressor gene located at 10q23 and encoding 403 amino acids [ PKB-Akt –10. It s PKB-Akt –15. So f PKB-Akt . By virt PKB-Akt . PTEN co PKB-Akt , and dow PKB-Akt . Exogeno PKB-Akt , but the PKB-Akt  also ind PKB-Akt –23.In tumor tissue, proper function of PTEN acts as a tumor suppressor primarily through the ability to suppress proliferation and decrease cell survival. The frequent loss of PTEN function, through deletion, mutations, and/or decreased expression, is observed in hereditary cancers as well as sporadic cancers . In manyIn the absence of germline and monoallelic mutations, PTEN protein levels have been found to be progressively lost during cancer progression . A numbeThe relevance of downregulated PTEN protein levels, as opposed to a complete loss of PTEN, is best observed in mouse models where PTEN is genetically manipulated –37. In mIn some cancers, like nonsmall cell lung carcinoma (NSCLC), where PTEN expression is reduced or lost in 55%–74% of patients, genetic alteration such as loss of heterozygosity and epigenetic silencing were not good predictors of PTEN protein levels , 30. Witβ, have been reported to phosphorylate PTEN [NEDD4 ubiquitin-ligase activity, which downregulates PTEN protein levels, is enhanced by the small endosomal PY-motif containing membrane proteins Ndfip1 and Ndfip2 . This suate PTEN . In lighCalcium-dependent homophilic binding of the adhesion protein E-cadherin is vital for the cell-cell interaction found in epithelial tissue, while loss of E-cadherin expression is associated with transformation and metastatic cancers –55. It hα- and β-catenin, PTEN, PI3K, and E-cadherin [−/− cells by the overexpression of MAGI-2 or by expressing an E-cadherin-α-catenin fusion protein. [β-catenin in a nonmalignant mammary acini model in laminin-rich extracellular matrix. In this model, either inhibition of E-cadherin function or reduction of PTEN protein levels abrogated acini organization and proliferation control suggesting that PTEN roles in cell adhesion and proliferation may be interconnected [MAGI (membrane-associated guanylate-kinase inverted) family members are multidomain scaffolding proteins with multiple sites for protein interaction. There are multiple splice variants of MAGI; MAGI-1a, MAGI-1b, MAGI-2, and MAGI-3, where each is expressed in a tissue-specific manner. MAGI proteins are part of a PDZ subfamily of proteins called MAGUK (membrane-associated guanylate kinases). Members of this family, including MAGI-2 and -3, were shown to directly bind PTEN in yeast two-hybrid screens , 69, 70.cadherin . In thiscadherin , 59. Indcadherin , 72 and cadherin . PTEN leprotein. . Our preonnected . The surβ-catenin is part of the cadherin-catenin complex and indirectly connects E-cadherin to the cytoskeleton. Studies have suggested that β-catenin negatively regulates PTEN transcription by blocking early growth response gene 1 (Egr1) [β-catenin at the plasma membrane. Thus, β-catenin cannot translocate to the nucleus and inhibit Egr1 transcription, the effect of which is continued PTEN transcription. Additionally, PTEN is protected in the signalosome from proteosomal degradation, resulting in sustained PTEN protein levels in the cytosol and subsequently negative regulation of the PI3K/AKT pathway. Loss of E-cadherin expression in cancer causes disruption of the signalosome, thus freeing β-catenin and PTEN from the MAGI scaffolding protein. The net effect of this is the proteosomal degradation of PTEN and the inhibition of PTEN transcription by β-catenin that has translocated to the nucleus.1 (Egr1) . Egr1 ha1 (Egr1) . Taken tThe existence of nuclear PTEN was reported in a number of cell models including primary neurons and endothelial cells , myoepit+2-regulated interaction and the binding was specific between the C2 PTEN domain and the calcium binding motif (EF-hand pair) of MVP [+2/calmodulin-dependant protein kinase) mediated activation of AMPK α1/2 can bypass the inhibition of PTEN nuclear entry achieved with mTOR/S6K downregulation [The absence of a “true” nuclear localization signal within PTEN has led to a number of alternative mechanisms for nuclear entry of PTEN . One mec) of MVP . Other m) of MVP . Phospho) of MVP , 93–98. ) of MVP . Finallygulation  and thusIn seminal work, the functional significance of nuclear PTEN with regard to proliferation and cell cycle control has been revealed . In thisMicroRNAs (miRNAs) are short 18–25 nucleotide long noncoding RNAs involved in posttranscriptional regulation of gene expression . They nein vivo mouse model in which miR-21 was knocked out. This increased the expression of several miR-21 target genes including PTEN [In 2007, Meng et al. reported overexpression of miR-21 in human hepatocellular cancer using miRNA microarray assay . Inhibiting PTEN . In conting PTEN . Recent ing PTEN .MicroRNAs other than miR-21 have been shown to be involved in various biological events and disease through PTEN regulation. For instance, MiR-214 is involved in cell survival  and cellThere is ample evidence that the full functionality of PTEN is modulated by alternative mechanisms beyond gene mutations and epigenetic processes. For instance a number of tissue-specific cancers strongly associate with PTEN deregulation at the gene expression level, changes in PTEN posttranslational modifications, miss-guided PTEN subcellular localization, and PTEN-specific microRNA upregulation. Determining whether these alternative mechanisms are causal or a consequence of tumor initiation and progression or whether they produce tissue-specific effects is still an ongoing area of important research. Clinically, as PTEN-regulating pathways become fully resolved, essential factors will arise, hopefully providing new targets for the development of novel and effective anticancer therapies and diagnostic tools.


PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PIP3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein–protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease. Phosphatase and tensin homolog located on chromosome 10 (PTEN) was originally characterized as a tumor suppressor that can inhibit proliferation, migration, cell growth, and apoptosis in a number of different cells. Subsequently it became apparent that, in addition to its role as a tumor suppressor, PTEN has many roles in the central nervous system (CNS) during the different stages of brain development and in adulthood. PTEN is highly expressed in neurons  domains, to recognize and bind this lipid on the plasma membrane. As suited for a second messenger, PIP3 is also subject to tight regulation by specific phosphoinositide phosphatases. PTEN is one of the most important phosphatases because it directly antagonizes the PI3K reaction by its 3-phosphatase activity and dephosphorylates PIP3 back to PIP2. In this sense, PTEN represents a brake built into PI3K signaling and its absence or dysfunction results in the constitutive and unregulated elevation of the signaling output of PI3Ks. Alternatively, PIP3 can be dephosphorylated by type II phosphoinositide 5-phosphatases like SH2-domain containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) /H(+) exchanger regulatory factors) or β -arrestins, which recruit PTEN to growth factor receptors  sensors to study the role of PIP3 at the synapse compartment, Ueda and Hayashi showed that PIP3 is primarily enriched in spines and not in dendritic shafts .A recent study demonstrated the presence of PTEN at the endoplasmic reticulum (ER) with apparent specific enrichment in mitochondria-associated membranes (MAMs) , the GTPase Ran, or the protein PNUFTS (a nuclear targeting subunit of PP1) may have a function in transporting or sequestering PTEN to the nucleus  , refers to a collection of clinically distinct syndromes molecularly defined by germline PTEN mutations. These include Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), Proteus syndrome (PS), and Proteus-like syndrome (PSL) Eng, . CS is aThe majority of PTEN missense mutations found in PHTS occur in the phosphatase domain of PTEN Eng,  and mostThe PTEN-associated neurological deficits frequently observed in this group of syndromes are macrocephaly, developmental delay, and mental retardation. Ataxia, tremor, and epilepsy have been also reported   results in increased neural stem cells proliferation, enlarged cell size, and brain enlargement . This PTEN cKO model recapitulates many of the late-onset morphological and behavioral abnormalities associated with autistic symptoms as these mice exhibit macrocephaly, neuronal hypertrophy, and deficits in hippocampus-based social and cognitive behavior, hypersensitivity to sensory stimuli, anxiety, and epileptic seizures  and long-term depression (LTD)  under basal conditions , the knock down of PTEN rescued disease associated defects in axon length, increased survival and restored growth cone sizes  is the second most prevalent neurodegenerative disorder after Alzheimer's. It is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Earlier studies provided links for PTEN's involvement in the pathogenesis of PD by virtue of its indirect and direct interactions, respectively, with two prominent PD-associated genes, PINK1 and DJ-1. However, more recent studies utilizing primarily neuron subtype-specific PTEN cKO mice have suggested diverse roles for PTEN and the Akt/mTOR pathway in dopaminergic neurons. It has been shown, for instance, that inhibition of PTEN in dopaminergic cell lines significantly inhibits the neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an established in vivo. Interestingly, although PTEN-deficient dopaminergic neurons show no alteration at the axon terminal, concomitant deletion of PTEN and Atg7 results in a exacerbation of the axon terminal size seen in Atg7 deficient neurons in the absence of any degeneration phenotype (Inoue et al., A specific requirement for PTEN in the regulation of axon terminal morphology of dopaminergic neurons has been uncovered in neurons deficient in an essential macroautophagy component, Atg7 (Inoue et al., In conclusion, an overall protective effect against neuronal death has been demonstrated when PTEN is deleted or inactivated in dopaminergic neurons, which is likely to reflect a generalized response due to activation of the PI3K/Akt/mTOR signaling pathway.Numerous specific modes of PTEN functions have been identified that are restricted to diverse subcellular compartments and involved in mediating a plethora of cellular responses. It has become increasingly clear that PTEN functions are not exclusively restricted to targeting phosphoinositides in membranous compartments; instead, PTEN specific cellular responses also involve its protein phosphatase activity or, no phosphatase activity at all. On one hand, PTEN inhibition has become a potentially attractive therapeutic intervention in certain settings. Yet, on the other hand, PTEN activation could be beneficial in other conditions. So far, chemical compounds that can act as PTEN inhibitors have been shown to substantially corroborate findings from studies utilizing deletion and silencing approaches. In principal, rational design of chemical compounds or peptides to shift, or prevent PTEN's localization to a specific subcellular compartment, to alter binding to a prominent protein partner, or to impose specific structural conformations on PTEN are plausible. Furthermore, the implications of using PTEN itself as an exogenous factor are only now beginning to be appreciated. In this review, we have attempted to detail some of the roles of PTEN that are, or may turn out to be involved, in mediating cellular responses in the developing, the mature, as well as the diseased neuron. No doubt, the interplay between the mechanisms that coordinate subcellular targeting in the context of controlling enzymatic activity will require detailed attention and have to be further explored.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results.We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed.Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%).Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer. Survival for patients with metastatic colorectal cancer (mCRC) has improved significantly over the past 15 years, largely due to improved systemic treatment options-6. In adPTEN is an important negative regulator of PI3K/AKT pathway and controls cell proliferation, survival and angiogenesis. Loss of PTEN function leads to persistent activation of the PI3K pathway and has been observed in breast, prostate, glioblastoma, endometrial and colon cancers,13. LossSeveral crucial factors make testing and interpretation of PTEN difficult. Loss of PTEN function results from several genetic mechanisms including small scale PTEN gene mutations , allelic loss at chromosome 10 and epigenetic silencing via hypermethylation of the PTEN promoter region. PTEN geFurther complicating the situation, the frequency of loss of PTEN expression increases from progression from normal colonic mucosa to adenoma, primary CRC and ultimately metastasis. The resClearly the role of PTEN is more complex than KRAS gene mutation where a single identifiable mechanism (activating mutation), largely concordant between primary and secondary tumours, confers near complete resistance to anti-EGFR MoAbs. Understanding this complexity is central to interpreting the current literature relating to PTEN and its potential role as a predictive biomarker. Recently reported cohorts of mCRC patients receiving anti-EGFR MoAbs have used PTEN loss of IHC expression to report loss of PTEN function. While this represents the functional outcome of several genetic mechanisms of PTEN loss, IHC relies on subjective interpretation and has the potential for inter reporter variation. Furthermore there is variability over the definition of 'loss of PTEN’ based on IHC scoring. In the largest cohort of mCRC patients, PTEN loss was defined as no staining in any cells at any intensity, while oOur group undertook an analysis of PTEN status in the AGITG MAX study of mCRC patients to identify the rate of inter-observer variability in IHC assessment and the rate of discordance between IHC and PCR assessment of PTEN status.The MAX study design and eligibility criteria have been reported previously.The primary objective of this Phase III randomized trial was to evaluate the effect of adding bevacizumab to capecitabine (with or without mitomycin C) on progression free survival (PFS) among patients receiving first line chemotherapy for mCRC. Four hundred and seventy-one patients were enrolled between July 2005 and June 2007. We have used the TaqMan® Copy Number Assay  to assess for PTEN allelic loss and have previously reported that loss of PTEN copy number was not prognostic nor predictive of outcome in the MAX trial cohort. In thisFormalin-fixed, paraffin-embedded (FFPE) samples of tumor tissue from archival specimens collected at the time of diagnosis were retrieved from storage at hospital pathology departments. For Copy Number PCR, genomic DNA was extracted from FFPE tissue sections with the use of the QIAamp DNA FFPE tissue kit . Manual micro-dissection was performed on samples with less than 80% malignant cells when visualized by microscopy. The same tissue blocks were used to make tissue microarrays (TMAs) and were assessed for PTEN expression by IHC. Researchers who assessed PTEN IHC expression were blinded to the PCR results.Immunohistochemical staining was carried out on TMAs using the PTEN monoclonal antibody 6H2.1  that has been used previously-36. EssePTEN, at cytoband 10q23.31a, location Chr.10:89727445 on NCBI build 37 (Life Technologies). The assay is a duplex PCR for the PTEN gene and the reference gene, RNaseP , set up according to the supplier’s protocol and run on the Rotorgene 6000 real time PCR instrument . The results are calculated as a ratio relative to a 2-copy control using the 2-∆∆Ct method (Rotorgene software), and multiplied by 2 to give the copy number. We tested DNA from colon cancer cell lines to determine the reproducibility of the assay and to select cell lines to use as copy number controls. HT29 (ATCC) is known to have 3 copies of chromosome 10 as determined by spectral karyotyping and comparative genomic hybridization[The PTEN TaqMan® copy number assay  was performed using 10 ng DNA in quadruplicate PCR. The primers provided in the assay were entirely within exon 9 of dization. For theFifty-nine tumor specimens were analysed for loss of copy number by Taqman® and for loss of protein expression by IHC. Eight samples were found to contain no tumor tissue and were excluded from further analysis..Two blinded pathologists assessed 51 specimens independently for PTEN protein expression with IHC. Pathologist JC assessed 29/51 (57%) as having PTEN expression loss, while pathologist AR assessed 17/51 (33%) as having loss of PTEN expression. Concordance between pathologists on final IHC assessment (PTEN loss/no loss) was 37/51 (73%), indicating in 14/51 (27%) of specimens there was discordance in the final assessment of IHC PTEN loss  had ≤1.5 copy number and were thus classified as PTEN loss.® PCR concordance analysis. Fifteen specimens had PTEN loss on IHC of which 10 (67%) also had PTEN allelic loss on Taqman® PCR. Seventeen specimens had PTEN allelic loss on Taqman® PCR of which 10 (58%) had PTEN loss on IHC. Fifteen specimens had preserved PTEN on both IHC and Taqman® PCR analysis. Overall concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%)  whether protein expression is reduced and whether such reduction confers a growth advantage is unknown. Sood et al. also demonstrated monoallelic PTEN dysfunction (by mutation or promoter methylation) resulted in loss of protein expression in only 38% of samples, while biallelic inactivation resulted in loss of PTEN expression in 80% of cases. Ali et In our cohort 25% of cases without PTEN allelic loss demonstrated complete absence of PTEN expression on IHC. These findings confirm alternative genetic mechanisms, beyond allelic loss, are responsible for loss of PTEN protein expression. Several authors have undertaken more comprehensive analysis of PTEN status on CRC specimens and provide an important insight into the often coexisting genetic mechanisms of PTEN dysfunction. Goal et al. demonstrated hypermethylation of the PTEN promoter region occurred in 10/132 (7.6%) sporadic CRC specimens, with a higher rate (19.1%) in microsatellite unstable CRCs. PTEN mutations coexisted in 4/10 (40%) of hypermethylated PTEN specimens. Eighty percent of patients with promoter hypermethylation had reduced (+1) or loss of PTEN protein expression and in the 3 cases of complete loss of PTEN staining, promoter hypermethylation coexisted with PTEN mutation or allelic loss. PerroneAlternatively, focusing on loss of protein expression at least represents the functional outcome of any such genetic insult. We have demonstrated the current limitations of IHC for this purpose. In our cohort, IHC assessment of PTEN loss by two pathologists was 33% and 57%, with overall concordance of 73%. As this was designed as a validation subset we did not ask the two pathologists to discuss the results that were not concordant, nor seek a further opinion, methods commonly described in papers reporting PTEN IHC aimed at reducing the apparent discordance rate. In all Overall the current literature highlight the difficulty in accurately measuring PTEN function to date; measurement of a single genetic insult, while minimizing inter-observer variability, does not capture the often coexisting mechanisms required for biallelic inactivation. The use of IHC, while potentially a better measure of PTEN function, is observer dependent and there remains a lack of consensus on optimal methodology and scoring.Given the limitations of PTEN assessment discussed here, it is not surprising reports of the predictive value of PTEN as a biomarker in CRC remain conflicting,17,27,31The lack of standardization in assessing loss of PTEN function appears to have contributed significantly to the conflicting results from retrospective cohort studies. Further elucidation of PTEN as a potential biomarker for colorectal cancer relies on defining PTEN loss of function and standardizing analytical methods and scoring systems. Future studies assessing PTEN function may be better served by obtaining a more comprehensive analysis of PTEN function by assessing PTEN mutation, hypermethylation of PTEN promoter, PTEN allelic loss and protein expression on each specimen. An alternative approach may be to explore improved methods of measuring reduced protein expression beyond IHC, given reduced or absent protein expression should reflect the functional outcome of PTEN loss irrespective of the genetic mechanism. Immuno-PCR may provide an option of combining the protein-specific capability of antibodies with the objective quantification of real-time PCR. This wiCNV: Copy number variation; CV: Coefficient of variability; EGFR: Epidermal growth factor receptor; FFPE: Formalin-fixed, paraffin-embedded; FISH: Fluorescent in situ hybridization; IHC: Immunohistochemistry; mCRC: Metastatic colorectal cancer; MoAbs: Monocloncal antibodies; PTEN: Phosphatase and tensin homologue; TMAs: Tissue microarrays; VEGF: Vascular endothelial growth factor.The authors declare that they have no competing interest.CH drafted the manuscript. JH conceived the study and coordinated all laboratory aspect of the study. VB participated in the design of the study and assisted with writing of manuscript. JW was involved in performing the DNA isolation and copy number PCR. NT contributed to trial conception and design as well as assisting with manuscript preparation. JC undertook the immunohistochemistry of the specimens and contributed to study design. AR undertook the immunohistochemistry of the specimens and contributed to study design. CL undertook data analysis and statistical analysis. TP conceived the study and participated in its design and coordination of all aspects of the study including drafting of the manuscript. ART designed study and assisted with manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/128/prepub


Patients with severe burn injury experience a rapid elevation in multiple circulating proinflammatory cytokines, with the levels correlating with both injury severity and outcome. In animal models, accumulations of these cytokines have been observed in remote organs, including the heart, brain and lungs. However, data are lacking regarding the long-term levels of cytokines in the heart following severe burn injury and also how infusion of parenteral estrogen, a powerful anti-inflammatory agent, would affect these levels. Using a rat model, we studied the effects of a full-thickness third-degree burn on cardiac levels of IL-6 and TNFα over 45 days with and without 17β-estradiol infusion.n = 8); (2) Placebo (no treatment) burn group (n = 80); and (3) E2 (estrogen treatment) burn group (n = 80). Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fluid resuscitation and, 15 minutes post burn, 0.5 mg/kg intraperitoneal estrogen (Group 3) or placebo (Group 2). From each group of 80, eight animals were sequentially sacrificed  at one of 10 respective time points: 0, 0.5, 1, 2, 4, 6, 8, 18, 24 hours and day 7 post burn (at day 7 only for the eight shams). The markers were measured by ELISA method.A total of 168 male rats were assigned randomly to one of three groups: (1) Sham burn  group  more comparable with the estradiol group (70 pg/mg), and significantly less than the placebo animals (332 pg/mg). Similarly, IL-6 levels in the sham animals (70 pg/mg) were comparable with the estradiol group (86.5 pg/mg), and significantly less than the placebo group (730 pg/mg), even at 45 days post burn.Following severe burn injury in an animal model, an early single dose of estrogen can decrease the prolonged let alone the early onset of cardiac inflammation. Based on these data, clinical studies of estrogen infusions should be seriously entertained as estrogen may not only be an inexpensive, simple adjunctive therapy in burn management, it may also obviate the need for many subsequent interventions altogether and even diminish mortality.


This is followed by a systemic surge in these markers and correlated with subsequent multiorgan failure (MOF). In animal models, this response can be somewhat blunted by early debridement, but such early intervention is not usually feasible in most clinical settings. As estrogen is a powerful anti-inflammatory/anti-apoptotic agent, we tested parenteral 17β-estradiol (En = 168) were assigned randomly to one of three groups: (1) sham (no) burn (n = 8); (2) burn given placebo (n = 80); and (3) burn given E2 (estrogen). Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fluid resuscitation and 0.5 mg/kg i.p. estrogen (or placebo) 15 minutes post burn. From each group of 80, eight animals were sequentially sacrificed  at one of 10 time points as follows: 0.5, 1, 2, 4, 6, 8, 18 and 24 hours and 7 days (7 days only for the eight shams).Male rats (In placebos, very high levels of cytokines appeared almost immediately in the echars and circulation, persisting 7 days post burn. In the estrogen group, cytokines, including tissue and circulating IL-6, the greatest predictor of MOF, remained suppressed at all time points, even day 7 Figure .Early single-dose parenteral estrogen can dramatically suppress both the local and systemic massive proinflammatory responses in severe burns. Based on these data, estrogen may not only be an inexpensive, simple, adjunctive therapy in burn management, it may obviate the need for many subsequent interventions altogether.


Severe burn patients are often noted to have subsequent neurocognitive problems. Experimentally, we have found striking, prolonged elevations of inflammatory markers in the brain  even when the injury occurs in a remote anatomic location. This neuroinflammatory response can also be significantly blunted by a single post-burn dose of estrogen. Sonic hedgehog (SHH), an important signaling protein found in the brain, controls and directs differentiation of neural stem cells, influencing brain regeneration and repair by generating new neurons throughout life. As estrogens not only blunt inflammation but also exert an influence on a variety of stem cells, we hypothesized that 17β-estradiol (E2) might affect levels of SHH in the post-burn rat brain.n = 44) were assigned randomly into three groups: controls/no burn (n = 4); burn/placebo (n = 20); and burn/E2 (n = 20). Burned rats received a 40% 3° TBSA dorsal burn, fluid resuscitation and one dose of E2 or placebo  15 minutes post burn. Eight animals from each of the two burn groups (burn/placebo and burn/E2) were sacrificed at 24 hours and at 7 days, respectively (sham group at 7 days only), with four each of the two burn groups sacrificed at 45 days. Brain tissue samples were analyzed by ELISA for SHH.Male rats  as compared with the placebo-treated burned animals (<700 pg/mg) and controls (<300 pcg/mg). See Figure Early, single-dose estrogen administration following severe burn injury significantly elevated levels of SHH in brain tissue. This finding may represent an extremely novel and important pathway for both neuroprotection and neuroregeneration in burn patients.


Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT).In  S. aureus. Integrated prophages encode S. aureus-specific toxins such as Panton-Valentine leukotoxin (PVL) and staphylococcal enterotoxin A Escherichia, Bacillus, Halomonas, Chlamydia, and Borrelia species S. aureus.Chromosomally integrated prophages are known to play key roles in the pathogenicity and virulence of Borrelia burgdorferi, B31, contained 12 different plasmidial prophages, , while Chlamydia pneumoniae contained only one B. burgdorferi and C. pneumoniae is attributed to evolutionary pressure on their smaller genomes to remove all non-essential DNA B. burgdorferi because of its higher copy number prophages combined with its small chromosome, allowing for the identification and sequencing of these additional, smaller, genetic elements. Thus, plasmidial prophages might not be as rare as this study indicated, but rather difficult to identify. Additional evidence also suggests that lysogeny in the environment favors the plasmidial form. B. anthracis strains isolated worldwide have nearly identical chromosomal prophage sequences, yet up to 20% of isolates encode a diverse array of additional inducible phages B. anthracis phages  with significant roles in the adaptive behavior of the pathogen A report by Casjens in 2003 showed the existence of only two plasmidial prophages in over 80 published bacterial DNA sequences among diverse species S. aureusS. aureus phages Φs80b and Φs84b could integrate into the chromosome of S. aureus strain s64c but remained plasmidial or solely extra-chromosomal in S. aureus strain 8325–4. Thus, the S. aureus host cell determined the extra-chromosomal state of these prophages: episomal in S. aureus s64c, and plasmidial in 8325–4. In strain s64c, this extra-chromosomal state was reported as ‘transient’, where the integrated and extra-chromosomal states could switch during serial passages σH, was found to directly affect integration and excision of S. aureus phages; sigH deletion from the S. aureus genome caused an increase in the episomal state of the phage −5) in the bacterial population that they cannot be detected using current sequencing techniques.In this study we term transient phages as ‘episomal’, and those existing solely in the cytoplasm as ‘plasmidial’. Two previous reports have examined extra-chromosomal prophages in Streptococcus pyogenes chromosomal island in an M1 serotype (SpyCIM1), can integrate at a specific site in the chromosome during stationary phase of growth, blocking transcription of a mismatch repair gene, mutL, thereby increasing the mutation rate. During exponential growth however, SpyCIM1 excises and replicates in the extra-chromosomal compartment of the cell, enabling mutL transcription E. coliBacillus subtilisClostridium difficileEnterococcus faecalis V583 S. aureus ExPΦ is unclear, these studies suggest it will likely have an important role in cellular processes.MGEs in both the integrated and circularized states can have major effects on the bacterial cell. For example, a phage-like MGE in S. aureus has been biased toward sequencing the bacterial chromosome; not surprisingly, smaller, low-copy, extra-chromosomal elements (like ExPΦ) would have been overlooked unless specifically isolated S. aureus may be a transient event and therefore potentially difficult to detect due to extremely low-copy numbers. In this study, we developed a method to effectively enrich extra-chromosomal DNA that includes low-copy number episomal and/or plasmidial ExPΦs and other extra-chromosomal elements from S. aureus. In addition, a NGS approach was used to achieve extremely high coverage and delineate genetic diversity of MGEs. Our results revealed naturally present and potentially widespread ExPΦs in virulent and antibiotic-resistant strains of S. aureus. ExPΦs were shown to be either episomal or plasmidial. The complete genomic sequence of one extra-chromosomal S. aureus phage ФBU01 from a VISA strain, was assembled and annotated, and found to contain scn, chp, sak, and sep IEC virulence genes associated with β-hemolysin containing phages S. aureus pathogenesis.Next-generation sequencing (NGS) in Staphylococcus aureus strains were cultured in Bacto Brain Heart Infusion  and incubated at 37°C. S. aureus from freezer stocks were revived on BHI agar plates and incubated for 16 to 24 hrs. One to two-day-old single colonies were inoculated into BHI broth and grown overnight (12–15 hrs). Cultures were then diluted 1∶100 in 150 mL BHI and grown to an OD600 of 0.6 to 0.9. Cultures were then centrifuged at 4,000 RPM for 20 minutes at 4°C. Cell pellets were used immediately or stored overnight at −20°C.Bacterial strains used in this study are described in Table S1 in S. aureus cell pellets were resuspended in 12 mL of the QIAGEN  Resuspension Buffer  supplemented with 200 µg lysostaphin from Staphylococcus simulans , 800 ng of PlySs2 lysin 2O at 65°C.g for 30 min or as needed at 4°C. The centrifugation force was kept below 5000×g to prevent shearing of large plasmids . When needed, samples under a total volume of 100 µl were concentrated further by Speedvac (Thermo Scientific Savant) set at low drying rate. Samples were stored at −20°C and shipped overnight on ice to the sequencing facility. Samples treated with Plasmid-Safe linear DNase were incubated for 2–4 hrs at 37°C with 1 µl 25 mM ATP, 2.5 µl 10× Reaction Buffer and 1 µl (10 U) Plasmid-Safe Linear DNase (25 µl reaction volume). Heating at 70°C for 30 min then inactivated the linear DNase.DNA was separated by electrophoresis at 50 V for 60 min on a 0.7 % Agarose, 1X, TAE gel. DNA was stained with ethidium bromide, and visualized under ultraviolet transillumination on an Alpha Imager HP . Total DNA was estimated by measuring final concentrations of extra-chromosomal DNA preparations using Thermo Scientific  NanoDrop 1000, software version 3.1.0. If needed, samples were concentrated using Amicon Ultra 0.5 mL 30,000 MWCO centrifugal units ; centrifuged at 2000×et al. 2005 De novo assembly of sequences was performed using Roche gsAssembler Newbler at default settings. Final assemblies were submitted to Genbank with the following accession numbers: ΦBU01, KF831354; pBU108a, KF831355; pBU108b, KF831356; and pBU108c, KF831357.‘Whole-genome sequencing’ (WGS) on extra-chromosomal DNA samples was performed using the Roche-  454 Sequencing Genome Sequencer FLX as described in Margulies http://www.nibi.nih.gov/BLAST). Bacteriophage gene identification was performed using BLASTP analysis. Homologies found in the GenBank database were based on greater than 50 percent identity Contigs generated by Roche gsAssembler Newbler resulting from the Roche-454 sequencing runs were identified as plasmid, bacteriophage, chromosome, or miscellaneous MGE by BLASTN analysis comparing to GenBank database 5′-CCTGTTGCTTGGGTAACTGTATC-3′ and 5′-AATGGCAGAAAGTGGCTGG-3′ for identified bacteriophage in the NRS19 strain and primers 5′TGCCATTGTGATGAGGAGGG-3′ and 5′-GCAACGCAGATTGTTTGAGTG-3′ for the identified bacteriophage of the NRS26 strain. These PCR products were purified with QIAquick PCR Purification Kit # 28106 and labeled for use as a Southern blot probe using Biotin DecaLabel DNA Labeling Kit . DNA transferred to nylon membrane was pre-hybridized and hybridized based on standard Southern blot procedures Aliquots of 200–400 ng extra-chromosomal DNA were separated (50 volts for 60 min) on a 0.7 % agarose, 1X TAE gel. Invitrogen  SYBR Safe DNA gel stain (# S33102) was added to the gel at a 1∶10,000 dilution. DNA was treated and transferred from gel to Positively Charged Nylon Transfer Membrane  using standard Southern blot procedures et al. 2000 S. aureus cultures were grown overnight, then diluted 1∶100 in BHI, and 10 mL samples were taken at 6, 8, and 24hrs. Samples of φBU01 were also taken at an OD600 of 0.12, 0.65, 1.3, 2.0 and 2.5 (24 hrs). Samples were concentrated to an adjusted OD600 of 5.0–10.0. Cultures were added to Lonza  SeaPlaque GTG low melting agarose (# 50111) and allowed to solidify. Samples were treated in 0.75 % agarose disks with 100 µg/mL RNase A, 50 µg/mL lysostaphin, 1 mg/mL lysozyme, and 25 µg/mL PlySs2 for 5 hrs. Agarose disks were then treated with 1 mg/mL proteinase K for 17 hours and overnight with SmaI. DNA was separated by pulse-field electrophoresis for 23 hours at 6.0 V/cm. Gel was stained with ethidium bromide for 30 min and destained in dH20 for 30 min.Pulsed field gel electrophoresis was performed with a modified procedure from Chung De novo assembly of the sequence contigs was performed using MacVector assembly (without template) with the PHRAP assembly algorithm, default settings Primers were designed based on preliminary mapping of the contigs to the reference genome  was spotted on BHI soft-agar overlays  of S. aureus reporter strains NRS77, RN4220, and Newman , 0.002% (w/v) gelatin). Prior to verification of bacteriophage DNA by PCR, bacteriophage samples were treated with 2 µl DNase  at 37°C for 30 min. DNase in the bacteriophage samples was inactivated by incubation at 75°C for 10 min.Phage induction with mitomycin C was performed following a modified protocol described by Cao 2O, and stained with 2% 0.2 µm filtered uranyl acetate. Excess solution was wicked away and grids were allowed to dry at least 30 min before visualization. Grids were examined on the JEOL 100CX transmission electron microscope using AMT V600 software (Advanced Microscopy Techniques) and images captured at 33,000x and 50,000x magnification.PEG-precipitated phage samples as described above were visualized at The Rockefeller University Electron Microscopy Resource Center. Briefly, 10 µL of sample was spotted on glow discharged 200-mesh copper grids with carbon film  and incubated for 2.5 min. Grids were then washed 2x in ddHPCR amplicons for Southern analysis, primer walking for sequence gap closure, and for verification of the presence of free phage particles in culture supernatants were all generated using NRS19 extra-chromosomal DNA preparation as template. Two DNA polymerases were used: (1) Taq PCR Master Mix  and (2) Phusion DNA Polymerase . The reaction condition for the Taq polymerase were as follows: (1) 95°C for two min; (2) 95°C for 30 sec; (3) appropriate annealing temperature for 30 sec; (4) 72°C for one min per kb; (5) repeat steps two through five 29 times; (6) 72°C for 10 min; and (7) 4°C indefinitely. The reaction conditions used for the Phusion polymerase were as follows: 1) 98°C for 30 sec; (2) 98°C for 10 sec; (3) appropriate annealing temperature for 30 sec; (4) 72°C for 30 sec per kb; (5) repeat steps two through five 29 times; (6) 72°C for 10 min; and (7) 4°C indefinitely. Following PCR, DNA was separated by electrophoresis at 100 V for 20 min on a 1.5 % agarose gel, run in 0.5 X TAE buffer. DNA was stained with EtBr, and visualized under ultraviolet transillumination on a Cell Biosciences  Alpha Image HP.Staphylococcus NARSA strains (Table S1 in S. aureus phage genomes S. aureus (VISA), three of seven methicillin-resistant S. aureus (MRSA), and three of five methicillin-sensitive S. aureus (MSSA) extra-chromosomal DNA samples possessed phage genes (S. aureus (VRSA) strains (VRS2 and VRS3a) and the one vancomycin-intermediate S. epidermidis (VISE) strain (NRS53). Thus, Roche-454 based sequencing of S. aureus extra-chromosomal DNA revealed evidence of the possible widespread existence of ExPΦs for the first time in natural isolates of these human pathogens.Extra-chromosomal DNA from 24 ge genes . Phage DS. aureus strains in more detail to categorize the MGEs found in our sequencing data. From BLASTN sequence analysis, all S. aureus extra-chromosomal contigs could be categorized as either plasmid, phage, or in some cases chromosomal DNA , in that the fewer the number of contigs, the lower the score. Assemblies with large numbers of contigs, such as that of NRS26 (1993 contigs) and NRS145 (500 contigs), generally had higher Q39 scores , possibly indicating that either the depth of coverage was relatively low, or the presence of more than one genomic species. This could occur with either low-level chromosomal contamination or the presence of multiple phages.Each extra-chromosomal DNA fraction was prepared as described in S. aureus ФNM3 phage genome (Siphoviridae family of phages. The assembled circular sequence of ФBU01 and Southern blot identification of a Plasmid-Safe (a DNase specific for linear DNA) treated sample of NRS19 , was determined by primer walking on PCR-amplified extra-chromosomal phage DNA . Using ae genome . The pree genome  were usee genome ; PCR#1. of NRS19  providedBacillus anthracis) S. aureus.  or linear  and NRS26 . These eThe efficacy of the linear DNase is shown in a control study that removed sheared, contaminating chromosomal DNA from an extra-chromosomal DNA sample , and NRS26 by pulsed-field gel electrophoresis (PFGE)  . Primerss (PFGE) . After 1s (PFGE) . Howevers (PFGE) , suggestS. aureus strains NRS19 and NRS26 were treated with mitomycin C S. aureus reporter strains; controls without mitomycin C treatment were negative. Polyethylene glycol-precipitated phage preparations were first treated with DNase to remove any host chromosomal DNA not contained within the phage particles. Phage particles were then disrupted, and the released DNA was used as template in PCR. DNA extracted from mitomycin C-induced culture supernatants of NRS19 (which includes ΦBU01) and NRS26 was subjected to PCR analysis. Primers bdu211, bdu212 and bdu215, bdu216 described above were used to determine if ExPΦ DNA could be detected in the induced phage fraction. The primers specific for the ExPΦs of NRS19 and NRS26 detected the presence of phages from both strains , then ФBU01 DNA should represent approximately 0.0078% of the extra-chromosomal DNA sample. Thus, ФBU01 is represented in our extra-chromosomal sample at a nearly 100-fold higher percentage  and NRS109 (MSSA) represent two such samples. Since these strains did not appear to contain ExPΦs, we were curious if they contained integrated prophage. Upon treatment with mitomycin C, we could induce phage from NRS24, which plaqued on S. aureus strain RN4220 . The lack of any extra-chromosomal phage contigs in the prophage-positive strains NRS24 and NRS109 indicates that the extra-chromosomal phage DNA of strains NRS19 and NRS26 are not the result of spontaneous excision but rather more stable, potentially plasmidial forms.In addition, some of the extra-chromosomal S. aureus strains identified several putative, phage-encoded virulence factors with the potential to be horizontally transferred to other bacteria , and Bacillus cereus plamidial-prophage pE33L54 contains multiple integrases Vibrio parahaemolyticus is 92% identical to integrated VHML phage. The replication of linear plasmidial-phages requires a replication protein (RepA), an origin of replication (ori), a protelomerase (Tel), and a telomere resolution site. Both Vp58.5 and VHML phage contain these elements. Therefore the presence of single genes such as integrases, is not a reliable indicator of whether a bacteriophage can integrate or is solely excised S. aureus.However, an integrase does not always determine the fate of plasmidial and integrated bacteriophages. g to reduce potential shearing of large DNA elements. Furthermore, Roche-454 sequencing was selected as it generated DNA sequencing read lengths longer than other NGS platforms de novo assembly of contig sequences of low-copy DNA elements.Extra-chromosomal DNA from nine VISA, seven MRSA, two VRSA, one VISE and five virulent MSSA strains were isolated and screened for ExPΦs using Roche-454 sequencing  containing a region of 98% percent identity with a query coverage of 83%. ФNM3 is a defective β-haemolysin (hlb) converting phage essential to the virulence of S. aureus Newman strain sea, sak, chp, and scn), the CHIPS gene responsible for reduction in neutrophil recruitment, phage structural genes, and an integrase gene (tyrosine recombinase family XerC). ФNM3 is unique in that it does not encode an excisionase, and primarily remains integrated within the chromosome ФBU01 shares sequence homology to several phages, including S. aureus MGEs that may be identified in this analysis include phages, plasmids, transposons, S. aureus pathogenicity islands (SaPIs), and staphylococcal cassette chromosomes (SCCs), all of which may be transferred horizontally between staphylococci Extra-chromosomal isolations separated by electrophoresis contained multiple bands  and 4 in−4, while φ11 in the same host has an excision rate closer to 10−2. In the same study, ΦSA2mw had excision rates over 10−2 in NCTC8325, yet under 10−3 in host strain MW2 −4 are within the label of “spontaneous excision”, with a range of 10−4 to 10−5. However, rates of 10−2 represent a two-log increase and potentially occur in 1% of the population. Based on our calculations, we predict that at a minimum, the episomal phages identified by our technique would excise at a rate of 10−2 or greater  into S. aureus recipient strains mec elements in the extra-chromosomal sequencing results. Therefore, it is plausible that MGEs detected in this study, including ExPΦs, could be transferred by phage transduction.Integrated phages can be vertically transferred from mother to daughter cells, passing on the phage's genetic information, associated virulence factors, and transcriptional regulators that can have important effects on the bacterial cell. The extra-chromosomal form provides multiple DNA copies of the phage, yet its relevance in vertical and HGT is currently overlooked. While the rate at which ExPΦs are transferred is unclear, it is possible that they could mediate their own transfer or be transferred by helper phages or other elements. Transduction by phage plays a pivotal role in S. aureus are needed before categorizing these genetic elements as true plasmidial prophages. The mechanism for the replication of ΦBU01 is presently unclear, however, a putative DnaD replication protein is encoded on ΦBU01. DnaD replication proteins have been found in S. aureus phages B. subtilisS. aureus phage DNA replication modules Further studies on the replication of ExPΦs in S. aureus. These elements may play a major role in bacterial virulence and human disease, as they encode multiple S. aureus virulence determinants. Perhaps their presence as multiple ExPΦ is beneficial by allowing for increases in gene dosage as a result of accumulative higher expression levels. Importantly, ExPΦs could potentially facilitate the dissemination of these virulence factors through transduction or in tandem with another MGE. Understanding the biology of these elements and their contribution to pathogenesis will pave the way for better epidemiology, diagnostics, and therapeutics for S. aureus. Furthermore, the approach described here can also easily be extended to reveal the cytoplasmic DNA elements of other bacterial pathogens.In conclusion, it is clear that advances in NGS have allowed for cost effective whole genomic sequencing of a number of organisms, leading to the sequencing of more 10,000 bacterial genomes and several thousand phage genomes to date File S1Supplementary Figures and Tables.(DOCX)Click here for additional data file.File S2Preliminary alignments of 454 sequencing contigs identified as bacteriophage DNA.(DOCX)Click here for additional data file.


Functional magnetic resonance imaging (fMRI) studies have revealed group differences in the frontal area between the subcortical vascular cognition impairment (SVCI) patients and the controls. However, most of the existing research focused on average differences between the two groups, and therefore had limited clinical applicability. The aim of our study was to investigate whether inter-regions functional connectivity of the dorsal frontal cortex (DFC) can be used to discriminate the SVCI from the controls at the level of the individual. Thirty-two SVCI patients and 32 demographically similar healthy individuals underwent resting-state functional magnetic resonance imaging. The DFC, derived from a prior atlas, was divided into 10 clusters. Features based on DFC were obtained through functional connectivity analysis between pairs of DFC. A nonlinear kernel support vector machine was used for classification and validated using 8-fold cross validation. An excellent classification accuracy was obtained from both the left and the right DFC functional connectivity . These findings shed further light on the pathogenesis of SVCI and showed promising classification performance using machine learning analysis based on DFC fMRI data, which may be useful for the differentiation of SVCI. Subcortical vascular cognition impairment (SVCI) is characterized by executive dysfunction, which was consistently thought to be associated with the dysregulation in frontal-subcortical loop . In thesFunctional MRI, especially the resting-state fMRI provides us a promising viewpoint to explore the function alteration of SVCI. However, in our daily life, the SVCI patients are prone to be ignored, especially during the early stages, due to the subtle clinic symptom or obscure onset. Therefore, it is necessary to find an objective biomarker, which could be used to assist the diagnosis of SVCI and to improve the accuracy. Multivariate pattern analysis (MVPA) is a promising and potentially powerful data-driven tool in clinical research that permits the differentiation of patients from healthy controls at individual subject level . The mosThus, in the present study, we aim to examine DFC functional connectivity maps in differentiating SVCI patients from healthy controls and to find out which side of the DFC brain functional connectivity contribute more to the discrimination. We also sought to examine the relationship between identified group differences in DFC region and behavioral measures of cognition.Demographic and clinical characteristics for all of participants are shown in Table The classification results indicated that the final correct classification rate of the training data set was 45.38% using the right DFC discriminating functional connections, while the classification rate in the left DFC was up to 75.07% Table . Receivet test.Voxel-based morphometry (VBM) results are shown in Table There was a positive correlation between CAMCOG-C and the strength of functional connectivity (area 46-pre-SMA) in the left DFC . We also found a trend for a positive relationship between praxis function and the connectivity between area 8d and area 9/46d   in the study, the obtained classifier was only specific to the current data and not general enough. Future investigations with large database and the integration of functional connectivity with other neuroimaging methods will be needed to confirm these preliminary findings.in vivo. Twenty-three healthy controls were recruited from either the spouses of patients or recruited via advertisement. All subjects performed a neuropsychological battery assessment, including CAMCOG-C, MMSE, ADL and CDR Clinical dementia rating to evaluate the function of episodic memory, attention, psychomotor speed, executive function, visuo-spatial skills and emotion respectively.Thirty-two right-handed SVCI patients and 32 age-matched controls were enrolled in our study. Each subject provided written informed consent, and this study was approved by the Institutional Review Board of the first affiliated hospital of Anhui medical university Subcommittee on Human Studies. Patients with SVCI met the previous criteria . ExclusiFunctional imaging data were acquired using a 3.0 T GE Signa HDxt MRI scanner . At the beginning of the resting scan, the participants were asked to keep their eyes closed without falling asleep and relax. Resting state images were obtained using echo-planar imaging (EPI) sequence . A 3D high-resolution T1-weighted anatomical images were also acquired using an inversion recovery fast spoiled gradient-recalled echo pulse sequence .t test, p>0.05). Moreover, to remove possible effects, six head motion parameters and the mean time series of gray matter, white matter and cerebrospinal fluid signals were introduced as covariates into the random effects model. A component-based noise correction method (Comp Cor) was also employed to reduce physiological and other noise artifacts [Functional connectivity analysis was performed on the MATLAB platform using the correlated and anticorrelated brain networks (CONN) Toolbox . The mairtifacts . A tempohttp://www.rbmars.dds.nl/CBPatlases.htm). It was noted that the DFC anatomical scheme appeared similar in both hemispheres according to a previous study [Ten subregions of the DFC in each hemisphere were defined in standard MNI space using the previous criteria, and then registered into each individual subject's MRI using FSL. We selected the DFC template as the ROI, which consists of 10 clusters Figure . The resus study .Regional mean time series were obtained for each individual by averaging the fMRI time series over all voxels in each ROI. Pearson's correlation coefficients were calculated between the time courses of ROI and ROI, and a 10×10 symmetric correlation matrix were yielded, which contained normalized z-scores for each subjects. Results of exploratory analyses were considered significant if clusters survived family wise error (FWE) correction p<0.05.To assess the influence of gray matter on functional connectivity, we analyzed our data for structural differences in ROIs between the groups using statistical parametric mapping (SPM) software and VBM toolbox. Structural MRI data processing was performed using VBM and the diffeomorphic anatomical registration using exponentiated Lie algebra (DARTEL) registration method, which has been reported in a previous study . In addiConnectivity matrices for each individual were converted to a feature vector containing 45 unique ROI-to-ROI connections (10×9/2).SVM is one of the most powerful classification algorithms in terms of predictive accuracy. In this study, SVM-based classification method adopted radius bases function (RBF) as a kernel function because of its suitability for nonlinear mapping, few parameters and low numerical difficulty. A grid search algorithm was used to optimize the two parameters of SVM: γ, width of the RBF, and C, an input parameter for the SVM algorithm. The detailed description of the application of RBF kernel SVM in MRI data has been introduced in previous studies –22. DuriTo estimate the performance of the classifiers, 8-fold cross-validations were used. The performance of each classifier can be quantified using sensitivity (SS), specificity (SC) and generalization rate (GR) ROC curves were used to quantify the sensitivity and specificity of the classifier . The AUCTo assess the statistical significance of the observed classification accuracy, permutation tests were then employed to estimate the probability of obtaining GR higher than those obtained using the correct labels by chance . We randTo identify the relationship between the strength of functional connectivity in DFC and the clinical scores in SVCI, the average strength of functional connectivity in the left and right DFC was extracted separately and correlated with the cognitive scores for all patients using Pearson's correlation analysis. Only the functional connectivity that differed significantly between groups was included in this analysis.This study used a MVPA method, which is based on functional connectivity pattern, to distinguish individuals with SVCI from the controls. The final model gave promising classification results with prediction accuracies from 45.38% to 75.07%. We proposed that functional connectivity within the DFC provided great potential for SVCI patient discrimination.