Nucleosomes regulate DNA accessibility and therefore play a central role in transcription control. Computational methods have been developed to predict static nucleosome positions from DNA sequences, but nucleosomes are dynamic in vivo.Motivated by our observation that transcriptional interaction is discriminative information for nucleosome occupancy, we developed a novel computational approach to identify dynamic nucleosome positions at promoters by combining transcriptional interaction and genomic sequence information. Our approach successfully identified experimentally determined nucleosome positioning dynamics available in three cellular conditions, and significantly improved the prediction accuracy which is based on sequence information alone. We then applied our approach to various cellular conditions and established a comprehensive landscape of dynamic nucleosome positioning in yeast.Analysis of this landscape revealed that the majority of nucleosome positions are maintained during most conditions. However, nucleosome occupancy at most promoters fluctuates with the corresponding gene expression level and is reduced specifically at the phase of peak expression. Further investigation into properties of nucleosome occupancy identified two gene groups associated with distinct modes of nucleosome modulation. Our results suggest that both the intrinsic sequence and regulatory proteins modulate nucleosomes in an altered manner. Nucleosomes are the fundamental repeated units of eukaryotic genomes . They arThe coordination of nucleosome positions is a complex process involving combined interactions among multiple factors. Experimental evidence indicates that certain DNA sequences have strong ability to wrap around the histone octamer . ConsequIt has become clear that nucleosome positions are highly dynamic -23. RecePrevious computational methods have predicted static nucleosome positions using DNA sequences with nucleosome formation or inhibition signals -19. HoweP < 10-27, Mann-Whitney U-test). Nucleosome occupancy profiles could be well discriminated by the information of TF binding, an important type of transcriptional interactions. This result demonstrates that transcriptional interaction is discriminative information for nucleosome occupancy.A recent study has used nucleosome occupancy information to assist identification of transcription factor (TF) binding sites . ConversMotivated by the observation above, we asked whether it is possible to employ TF binding information to predict nucleosome positioning. Indeed, the positions of TF binding sites are strongly associated with nucleosome positions ,12. NuclIt is well accepted that TFs bind their targets in a dynamic manner, and their corresponding nucleosomal templates undergo dynamic changes . HoweverBased on dynamic transcriptional interaction and genomic sequence information, we developed a novel computational approach for identifying dynamic nucleosome positioning at promoters Figure ; MethodsWe first applied our method to cell cycle-regulated genes. Yeast cell cycle-regulated genes have been identified and grouped into five phases during which they display peak expression: M/G1, G1, S, S/G2, and G2/M . We direP < 0.003, Mann-Whitney U-test). We asked whether transcriptional interaction information contributes to the significant correspondence between our predictions and experimental measurements. To this end, we predicted nucleosome positions using only genomic sequence information (Methods). Predicted nucleosomes still had higher H3 occupancy than predicted nucleosome-free regions , but the statistical significance became less. This result validates that transcriptional interaction is important information for nucleosome positioning.To examine whether our approach is applicable to exogenous conditions, we identified nucleosome positioning in the response of cells to hydrogen peroxide . PokholoRecently, Shivaswamy et al. have measured genome-wide nucleosome positions after subjecting cells to heat shock for 15 minutes . We applTaken together, these results validate our method in three available datasets of dynamic nucleosome occupancy. It is noteworthy that both expression data and nucleosome occupancy were not measured in exactly the same experimental medium for either exogenous condition. As expression data is prior information for our method and experimentally measured nucleosome occupancy is used to evaluate our method, these discrepancies in experimental conditions inevitably limit assessment of our method. Nevertheless, our results show that TF binding information improves significantly the performance of prediction, which is based on the intrinsic DNA sequence alone.Having validated our approach in available datasets, we applied it to 22 cellular conditions to study global properties of the dynamic nucleosome organization ,29,32-34Second, we studied nucleosome organization by comparing nucleosome occupancy at the phases of peak expression to those at other phases Figure . Indeed,Third, we further investigated mechanisms of nucleosome positioning. Genes sharing the same phase of peak expression during each cellular condition were clustered into a group. In this way, we obtained a total of 132 groups. Region between -400 and -1 bp upstream of the gene is important for transcription. We focus on nucleosome positioning in this region. Visual comparison between nucleosome occupancy at the phase of peak expression and DNA sequence-directed nucleosome occupancy divided these gene groups into two clusters Figure . Genes iP < 10-4, Mann-Whitney U-test), whereas genes in cluster 1 did not show this property (mean transcription frequency = 7.39 for genes in cluster 1 and 7.42 for the rest of the genes). We hypothesize that the underlying DNA sequence plays less important roles in nucleosome positioning for genes in cluster 2, as their DNA sequence-directed nucleosome occupancy can suffice transcription, which is contradictive with their low transcription activity. To test this hypothesis, we compared experimentally measured nucleosome occupancy profiles to DNA sequence-directed nucleosome profiles in promoter regions [P < 10-5, Mann-Whitney U-test). This result demonstrates that the intrinsic DNA sequence explains less nucleosome positioning in promoter regions of gene cluster 2. In other words, regulatory proteins should account for more nucleosome positions in promoter regions of gene cluster 2 over the cell cycle. We further analyzed gene activity in various conditions for gene cluster 2. We compiled gene expression data from 1,082 published microarray experiments under various cellular conditions (Methods). For each gene, we calculated the proportion of experiments in which it displayed significantly up-regulated expression changes, and defined the normalized resulting value as open rate. The open rate reflected the general gene activity in various conditions. Genes in cluster 2 showed lower open rates than the rest of the genes . Overall, as DNA sequence-directed nucleosome occupancy at gene promoters of cluster 2 may permit accessibility to TFs, regulatory proteins should reposition nucleosomes to prevent improper transcription in repressed state. In contrast, as DNA sequence-directed nucleosome occupancy at gene promoters of cluster 1 can not suffice transcription for peak expression, nucleosome remodeling is required for transcription.We next sought to understand mechanisms of nucleosome positioning in other conditions for genes in cluster 2. As microarray experiments for all cellular conditions were carried out with the cell cycle as the start point, we analyzed gene transcription frequency over the cell cycle . Genes i regions . Genes iR > 0.5) between linker DNA lengths and gene expression levels during the corresponding condition , indicating that their nucleosome occupancy in promoter regions is relatively maintained during the corresponding condition. Furthermore, these 44 groups showed a moderate overlap with gene cluster 2 , implying that their maintenance of nucleosome occupancy is linked with the intrinsic DNA sequence.Finally, we sought to explore the relationship between nucleosome occupancy and gene expression level. As mentioned above, we used total length of linker DNA to represent the enrichment for nucleosome-free regions at the promoter. 88 of 132 gene groups showed high positive correlation . Although we cannot rule out the possibility that other nucleosome remodeling-related TFs have not yet been identified, one plausible explanation for this observation is that TF binding is not the main source of nucleosome remodeling. We speculate that the main manner of nucleosome remodeling is through nonspecific remodeling complexes to permit or impede site-specific access to TFs. Other study has indicated that nucleosome occupancy plays an instructive role in determining TF Leu3 targeting [Our identifications, based on TF binding and genomic sequence information, showed stronger correspondence with in vivo data than predictions on the base of genomic sequence information alone. This result suggests that TF binding is critical information for nucleosome positioning. However, whether the changes in nucleosome occupancy facilitate TF binding or occur as a consequence of TF binding is not known. TFs Abf1, Cbf1 and Rap1 are involved in nucleosome remodeling as indicated by Gene Ontology . The 132argeting . A recenargeting . The stuWe found that the global characteristics of nucleosome occupancy landscape persist throughout most conditions. Experimental evidence has supported our observation in two conditions (i.e. heat shock and cell cycle) ,24. The A key finding of this study is that genes exhibit distinct modes of nucleosome modulation. Nucleosome positions are determined by a combination of DNA sequence composition and regulatory proteins. However, it is unclear how these factors work in concert. For genes whose DNA sequence-directed nucleosome occupancy can suffice transcription, regulatory proteins are required to remodel nucleosomes in repressed state. On the other hand, regulatory proteins remodel nucleosomes to activate transcription for genes whose DNA sequence-directed nucleosome occupancy does not enable transcription. We found that these two gene clusters are not cellular condition-specific, and there is no significant difference in DNA sequence preferences for nucleosomes between these two clusters (data not shown). We speculate that these two modes of nucleosome modulation are linked with evolutionarily conservation in nuclear organization and sequence composition. Genes in the former cluster display lower transcription activity over the cell cycle, implying that regulatory proteins are involved in nucleosome positioning for these genes. The interaction between regulatory proteins and nucleosomes should be relatively stable. Regulatory proteins and their targets should be adjacent in nucleus. Otherwise the interaction between them may be transient. For genes in the latter cluster, DNA sequence-directed nucleosome occupancy does not enable transcription, TF binding sites thus should be enriched in regions with high DNA sequence preferences for nucleosomes. Nevertheless, the cause for these two modes of nucleosome modulation remains to be explored.We have only begun to explore the potential of the application of factors besides genomic sequence information to predict nucleosome positions. Despite the successes described above, our approach still has limitations. Nucleosomes govern the access of DNA to transcription apparatus. However, known TFs represent most, but not all, transcription apparatus. This discrepancy may limit the performance of our approach. As discussed above, TF binding seems to be a consequence of the changes in nucleosome occupancy. Although TF binding is critical information for nucleosome positioning, ATP-dependent chromatin remodeling and histone modification are the main ways of nucleosome remodeling. But they still lack comprehensive experimental data in multiple conditions. Until recently, models for dynamic nucleosome modification have been developed ,41, whicYeast genome sequences were downloaded from the Saccharomyces Genome Database . The TF-We compiled available gene expression data from the Saccharomyces Genome Database , a totalGiven gene expression data during one cellular condition, DNA sequences at gene promoters, and known PWMs that correspond to candidate TFs, we used following procedures to identify nucleosome positioning dynamics during the condition. First, we identified genes displaying significantly up-regulated changes in gene expression (hereinafter referred to as condition-regulated genes) and assigned them to phases (time points for exogenous conditions) at which they display peak expression (hereinafter referred to as phase-related genes), for example, G2/M-phase genes of cell cycle. To identify nucleosome positions for more genes, we set a less strict threshold (2-fold) for significantly up-regulated changes in gene expression. Genes displaying peak expression at the same phase during one condition should tend to be regulated by similar TFs, for example, TFs Mcm1 and Fkh2 regulate G2/M-phase genes . The genSecond, we used known PWMs and statistical test to derive functional and nonfunctional TFs for each condition-regulated gene. We collected 135 known PWMs that correspond to TFs from MYBS , a comprThird, we determined the presence or absence of each TF and the phases of presence for each present TF. We can identify TFs that can potentially bind their targets at one phase by determining their presence or absence in the cell from their concentration. However, there still lacks dynamic protein concentration data during multiple cellular conditions. Previous studies have shown that there is a strong correlation between protein and mRNA levels , and havFourth, we integrated transcriptional interaction information obtained above and DNA sequence-directed nucleosome formation potential to identify nucleosome positions at each phase. We divided the promoter into some segments, each of which began with the right end of one nucleosome-depleted locus and ended with the left end of the next nucleosome-depleted locus. The genomic DNA sequence itself is one dominant determinant of nucleosome positioning in vivo. The nucleosome positioning in each segment was identified by DNA sequence-directed nucleosome formation potential and the known nucleosome-covered loci. Lee et al. have integrated most sequence features related to nucleosome positioning to model nucleosome occupancy , and we We applied our approach to the following 22 cellular conditions: cell cycle (1 condition) , stress The approach for predicting nucleosome positions based on genomic sequence alone is as follows: we determined nucleosome positions by iterating over the sequence-directed nucleosome formation potential in decreasing order as long as the new nucleosome did not overlap with any previously determined nucleosome. This iteration proceeded until no more nucleosomes could be laid at the promoter.We used a Gaussian kernel to model predicted nucleosome occupancy. The mean of the Gaussian was taken as the centre of the identified nucleosome position under consideration, with the standard deviation set at 25 bp. This threshold is set according to the length (147 bp) of nucleosome so that the modelled nucleosome occupancy of predicted linker DNA approximates to 0.The authors declare that they have no competing interests.ZD and XD designed the study, analyzed the results and drafted the manuscript, and ZD also implemented the algorithms, carried out the experiments. QX, JF, YD, JW and CH participated in the analysis and discussion. All authors read and approved the final manuscript.


Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation.Drosophila forward genetic screen, we identified a mutation in capulet-encoding a conserved actin-binding protein-that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other.From a in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods  Actin is a major component of the cellular cytoskeleton and one of the most abundant cellular proteins. However, actin must be exquisitely regulated during cell migration, cell adhesion, cell division, and many other essential cellular functions. Actin also forms the core of many cellular structures including filopodia, lamellipodia, microvilli and stress fibers. Actin exists predominantly in one of two forms: monomeric actin (G-actin) and filamentous actin (F-actin). The inter-conversion between these two actin forms is tightly regulated by a diverse array of proteins that bind actin directly or indirectly. Actin depolymerizing factor (ADF), also known as Cofilin, represents one actin-binding protein that can disassemble actin by severing and depolymerizing actin filaments , which exists in all eukaryotes Drosophila CAP orthologue, has been shown to be involved in a number of developmental processes including oocyte polarity Drosophila as it works directly with Abl to control axon guidance Another actin-binding protein that works in conjunction with Cofilin is In neurons, extremely long neurites filled with G-actin must regulate the formation of F-actin in response to dynamic events such as synapse formation or axon guidance during sensation of chemo-attractive/chemo-repulsive cues. In addition, formation of ectopic F-actin must be suppressed to avoid physical blockages that could impede important transport functions within relatively thin neurites and produce deleterious cellular effects. For example, neurites contain a microtubule-rich cytoskeleton that provides a physical scaffold for delivery-both in anterograde and retrograde directions-for cargoes required to maintain proper neuronal function. Energy-dependent molecular motors, including dyneins and kinesins are ATPases that physically help deliver targeted cargoes by directional movement along these microtubules. In particular the kinesin superfamily protein KIF5 can transport diverse cargoes including membranous organelles, cytoskeletal proteins, and mRNAs , intriguingly only within neuronal dendrites but not axons capulet, are responsible for numerous actin-related phenotypes. Further, loss of function mutations in different genes synergize to produce severe phenotypes not observed by single loss of function mutations. In particular, we show that capulet genetically interacts with kinesin heavy chain and affects kinesin-mediated transport in the dendrites including mitochondrial distribution. Remarkably, the double mutant capulet and khc phenotype includes the formation of actin rods similar to those described in a Drosophila Alzheimer's model and in mammalian tissue culture neurodegeneration models. This study suggests a mechanism by which robust phenotypes may result from multiple seemingly mild impairments that interact with each other.Forward genetic screens in model organisms provide one potential method for identifying molecules that regulate actin dynamics punctate”, displayed actin::GFP-rich accumulations within dendrites but not within axonal shafts. However, neuron numbers and gross aspects of neural development including dendritic filopodia formation and dendritic branching appeared relatively normal within this mutant. These actin::GFP puncta, which are absent in wild type larvae, occur at an average of 38 puncta (standard deviation = 10) in each sensory neuron dorsal cluster of the punctate mutant. To determine the molecular lesion responsible for this phenotype, we used deficiency mapping and identified two molecularly defined deficiencies, Df(2L)Exel7006 and Df(2l)Exel6004, that failed to rescue the lethality of the punctate mutant. These two deficiencies overlapped by only 36 nucleotides within the Drosophila capulet (capt) gene. The Capulet protein encodes 424 amino acids with 3 recognizable motifs, namely a polyproline domain, a WH2 domain and two CARP domains. To confirm that capt was the affected gene, we sequenced the capt locus from the punctate mutant and found a nonsense mutation that changed amino acid lysine 304 to a premature stop codon. This mutant allele of capt, termed captK304, eliminated the last 121 amino acids of the Capulet protein, which includes both CARP domains. CARP domains are secondary structure predicted homology, not amino acid similarity, found in both Capulet and a gene mutated in Retinitis Pigmentosa We previously conducted a forward genetic screen to isolate mutations affecting neuronal dendrite development capt, we determined whether deficiencies that uncover the capt locus would also display the actin::GFP phenotype seen in captK304 (actin::GFP transgene onto Df(2L)ast5, a deficiency that lacks the capt locus, which also fails to complement the lethality of captK304. Indeed, we observed the actin::GFP phenotype when the deficiency was transheterozygous with captK304 (capt that encoded a nonsense mutation at tryptophan 145 (captW145), and introduced the actin::GFP transgene into this mutant background. The captW145 mutant also displayed the same actin::GFP phenotype when either homozygous or transheterozygous with captK304 (captk01217 and capt06955 (data not shown). In addition to demonstrating nearly identical phenotypes with different alleles, we tested whether expression of a capt transgene in a captK304 background would be sufficient to rescue the actin::GFP phenotype. To accomplish this, we made a fluorescently-tagged capt construct (UAS-mCherry::capulet) and recombined it onto the K304capt chromosome. The K304capt, UAS-mCherry::capulet recombinant did not display any actin phenotype . In both cases, Capulet localized within cytoplasmic compartments including axons, cell bodies and dendritic shafts but did not localize to the F-actin-rich dendritic filopodia Drosophila, whether in epithelial cells increased F-actin in loss of function capt mutant cells. The converse hypothesis suggests that elevated capt function might decrease F-actin formation. We therefore tested this putative role on an F-actin-rich cellular structure, dendritic filopodia, by overexpressing wild type Capulet. We found that overexpression did suppress formation of F-actin-rich dendritic filopodia -actin rods cofilin is overexpressed within a captK304 background  had similar phenotypes in establishing anterior-posterior asymmetry. The anterior localization of bicoid mRNA and posterior localization of oskar mRNA are essential in organizing the anterior-posterior asymmetry in Drosophila oocytes. One study documented that a mutation in capt alters the posterior localization of oskar mRNA kinesin heavy chain (khc) was also shown to perturb oskar mRNA distribution capt and khc mutations produced the same oskar mRNA mislocalization phenotypes in oocytes and Khc transports mitochondria, we investigated whether there was any genetic interaction between capt and khc. To determine whether genetic interactions exist between capt and khc, we placed an actin::GFP transgene in a khc mutant (khck13314) background. Homozygous khck13314 mutants exhibited swellings in dendrites , whereas the actin::GFP rods we observed in the capt, khc double mutant had an average length of 5.97 µm (standard deviation = 1.75 µm). The average number of actin rods (>2 µm in length) found in the dorsal cluster sensory da neurons was ∼7 per cluster . Importantly, actin rods were never seen in wild type larvae. We tested the genetic interactions between capt and khc using a different allele of capt (captW145) or a different allele of khc (khce02141) and we were able to replicate the same findings (data not shown).We wondered what genes, other than actin-binding proteins, might be relevant to endrites , similars animal . In a carequency , suggestrequency , not seeAlteration of kinesin function is associated with swellings in the plasma membrane in axons capt background, we expressed a Khc::EGFP transgene in the PNS neurons. We could not easily visualize Khc::GFP along the dendrites of PNS neurons in both WT and capt backgrounds with a single overexpressing transgene. However, Khc::EGFP appears to accumulate in the cell bodies in a capt heterozygote which is not observed in a WT background 80; UAS-Khc::GFP transheterozygote is synthetic lethal and dies at the pupal stage. This observation further supports a capt and khc genetic interaction. Possibly, the increased amount of Khc::GFP exceeds the level of Capt function in a heterozygote required for unimpeded Kinesin function.To compare the neuronal distribution of Khc in a WT and ckground , suggestRegulation of actin dynamics has important consequences in all cell types but special significance in neurons as dendritic spines – the sites of synapse formation in the brain – and axon growth cones are highly enriched for F-actin. In addition, neurites, either axons or dendrites, span very long distances from the cell body and therefore must allow continued transport of proteins to and from the cell body to either axon termini or dendritic spines. Blockage of relatively small diameter dendrites could hinder this transport and presumably have deleterious affects on neuronal function.Drosophila have identified capulet loss of function mutations that lead to ectopic increased F-actin accumulation capulet function lead to increased actin in small aggregates, while over-expressed Cofilin in a capulet mutant background leads to larger accumulation in actin rod-like structures.Capulet plays a vital role in regulating actin dynamics and can reduce filament formation by binding/sequestering actin monomers and preventing new polymerization or by recycling Cofilin activity by removing actin monomers from it, thus freeing Cofilin for another round of actin severing and depolymerization Drosophila oocyte Perturbations in Capulet function not only affect actin dynamics, but also microtubule-based processes. A Recent study demonstrated that the actin nucleators, Spire and Cappuccino, have major affects on the regulation of the microtubule network and cytoplasmic flows in the capulet and khc as well as the robust double mutant phenotype that produces actin rods in dendrites suggests a strong cellular interdependence between these two molecules  direction along microtubules capulet only functions in dendrites, as roles for capulet in actin regulation have already been demonstrated in diverse cell types from follicle epithelial cells to the adult compound eye capulet mutants, however, relatively smaller Drosophila sensory dendrites would be more susceptible to impaired transport and decreased ATP.A fundamental question relates to why we only observe dendrite and not axon phenotypes. It is highly unlikely that The role of altered actin polymerization and aggregation in neurodegenerative disease is relatively unexplored, although some studies have begun to examine actin aggregates in neurodegenerative disease. In Alzheimer's disease brain, aggregates of ADF/Cofilin have been described in association with both amyloid deposits and neurofibrillary tangles Drosophila brain with altered actin dynamics. In this study, the authors documented that ectopic expression of a mutant human Tau protein, associated with fronto-temporal dementia, increases the levels of F-actin in the brain Drosophila model and in a mouse transgenic tauopathy model. Some of the rods stained for actin, Cofilin and Tau, which are all characteristic of Hirano bodies. It is interesting to note that in their study, co-expression of Cofilin with the mutant Tau protein significantly reduced the effects of expressing mutant Tau alone, suggesting that they genetically antagonize each other. Conversely, over-expression of human wild type Tau enhances the toxicity of β-amyloid (Aβ). These authors suggest that interactions between the microtubule binding protein Tau and actin may be a key mediator of Tau-related neurotoxicity capulet background when expressing the human mutant Tau protein capulet function interacts with impaired Kinesin function to produce similar actin rods. Interestingly, both studies suggest that proteins normally associated with microtubules can genetically interact with the actin cytoskeleton under pathological conditions.Recently, investigators have described an Alzheimer's disease model developed in the Drosophila βAPP results in axonal transport defects, which are enhanced by genetic reductions in the amount of functional Kinesin In many neurodegenerative models impaired Kinesin function and the failure of motor-driven transport in long axonal compartments is hypothesized to play a major role in neurodegeneration. This transport failure leads to formation of axonal swellings, which are the physical manifestation of accumulation of axonal cargoes. For example, overexpression of capt/khc double mutant larvae do not survive long enough under time lapse to observe the disappearance of actin-rods (data not shown).Although actin rods are induced under conditions that diminish neuronal function or with expression of neurodegeneration-associated proteins, at least one study postulates that actin rod formation may be a neuroprotective response In addition to forming actin rods by overexpressing Cofilin or microinjecting Cofilin, rod formation can also be induced simply by depleting ATP. Decreasing ATP would alter numerous cellular pathways. However, Cofilin has a higher affinity for ADP/actin than ATP/actin and by self-assembling ADP/actin/Cofilin into rods, the exchangeable ADP/actin could be removed from the dynamic actin pool decreasing ATP turnover. Interestingly, actin rods are not unique to neurons, as non-neurons under stress can also be induced to form Cofilin positive, phalloidin negative actin rods khc phenotypes-paralytic tail flipping and axonal swellings – in a sensitized Abl background capulet and abl to also have a common function in promoting kinesin movement in microtubules, as both of these mutants form swellings in neuritic processes when transheterozygous with khc. Furthermore, both Abl and Capulet prevent formation of F-actin, either by Capulet depolymerizing F-actin via Cofilin, or by Abl negatively regulating Ena.A second, line of thought suggests that other actin regulatory proteins may directly interact with kinesin and could provide another route for Capulet ultimately affecting Kinesin-mediated transport. Enabled (Ena) is an actin-polymerizing factor that genetically antagonizes the Abl tyrosine kinase  or by over-expressing proteins such as Cofilin, Tau and β-Amyloid peptide in transgenic models.www.flybase.org) for nomenclature. Gene names are italicized, whereas proteins are not. To visualize PNS neurons, Gal4 109(2)80  was recombined with UAS-actin::GFP  to produce the Gal4 109(2)80, UAS-actin::GFP recombinant. The following fly lines were obtained from the Bloomington Stock Center : DrosDel and Exelexis deficiency stocks on chromosome 2, deletion mutants Df(2L)Exel7006 (stock # 7776), Df(2L)Exel6004 (stock # 7491) and Df(2L)ast5 (stock # 6344); captE636 allele which we refer to as captW145 (stock # 5944) and UAS-capt line (stock # 5943); khck13314 (stock # 11084) and khce02141 (stock # 18018); UAS-tsr (stock # 9235); UAS-myr-mRFP (Stock # 7119), UAS-mitoGFP (stock # 8443) and UAS-Khc::EGFP (stock # 9647). Second instar larvae were used for all experiments unless otherwise indicated. All flies were maintained at 25°C in yeast-cornmeal (Bloomington recipe) vials and bottles.We follow guidelines from Flybase (punctate (punc/CyO) were crossed with males from each stock of the Chromosome 2 Deficiency Kit from Bloomington. We noted which deficiency stocks did not generate F1 progeny with straight wing flies . We identified the common chromosomal locus among all the deletions in these stocks, which is within the capulet gene. To identify the nature of the EMS mutation, we amplified and sequenced overlapping segments of capt from genomic DNA of punctate and compared it to the WT capt sequence.Virgin females of mCherry::capt (monomeric Cherry::Capulet) fusion construct, mCherry::Tubulin was taken out of pcDNA3.1 (gift from the R. Tsien lab) by cutting with EcoRI. Both ends of the excised mCherry sequence were made blunt by Klenow fragment  treatment and the 3′ end was made sticky by digestion with XhoI. The pUAST vector was linearized by digestion with HindIII and the ends were made blunt by Klenow fragment treatment. The 5′ end of the pUAST vector was cut with XhoI to create a sticky end. The mCherry::Tubulin fragment was then inserted into the pUAST vector by blunt-sticky ligation. The capulet gene sequence was amplified from the cDNA clone LD24380 by using a forward primer containing a BglII restriction site and a reverse primer containing an XhoI restriction site. The capulet PCR product was digested with BglII and XhoI. The tubulin sequence was excised out of the newly constructed pUAST-mCherry::Tubulin by digestion with BglII and XhoI and was replaced with the capulet sequence. The pUAS-mCherry::capulet construct was transformed into electrocompetent DH10B bacteria, amplified and injected into w1118Drosophila embryos by P element mediated transformation to make transgenic fly lines (Duke Drosophila Transgenic Core).To make the pET28a vector with BamH1 and XhoI digestion and ligating the amplified capulet PCR product (see above) that had been digested with BglII and XhoI. The capulet in the pET28a vector was transformed into competent cells, amplified, sequence verified, and transformed into BL21 cells. The His-Capulet protein was extracted and purified using the TALON system and sent to QED Bioscience Inc.  for antibody production.Polyclonal antibodies against His-Capulet were generated by linearizing the The dissection of larvae, immunostaining procedures and confocal microscopic visualizations were done essentially as described . Briefly, the larva was washed with water and placed in a droplet of 4% para-formaldehyde. The head and the tail sections of the larva were excised using a pair of fine scissors and the guts and other visceral organs were pulled out with forceps. An anterior-posterior cut was made along the mid-dorsal area of the larva and the resulting larval fillet was cleaned out of muscle tissue with forceps. The larval fillet was fixed in 4% paraformaldehyde for 20 minutes and then washed with PBT 3 times. This was followed by incubation with a blocking solution of 5% normal goat serum and PBST for one hour at room temperature. The larval fillet was incubated with the primary antibody overnight at 4°C, washed 3 times with PBT, incubated with the secondary antibody for 4 hours at room temperature and then washed again 3 times with PBT. The larval fillet was mounted on a slide with glycerol to prepare for viewing. The primary antibodies used were anti-twinstar/cofilin , anti-actin , and anti-GFP . Staining actin with phalloidin  was performed according to manufacturer's instructions, and when double labeling, was done after washing off the secondary antibodies.For live and fixed larva visualization of the PNS, images were obtained using a Zeiss LSM (Laser Scanning Microscope) 510 (Germany) confocal microscope with a 40× and a 63× oil immersion lens. For live specimens, larvae were picked from food bottles and vials, cleaned with water, mounted on a slide with halocarbon and covered with a cover slip. Fixed specimens were washed with 1× PBT and mounted on a slide with glycerol and covered with a cover slip. Database microscopic images taken using the LSM510 software exported as TIFF files, cropped and sized using Adobe Photoshop  and arranged and labeled using Adobe Illustrator .


The assessment of a tumour's angiogenic potential, by measuring the microvessel density in histological sections, assumes that a 4-microm section is representative of whole tumour vascularity. This study has examined this assumption by comparing the vessel density found radiologically, after injecting specimens with contrast, with that found immunohistochemically. Twenty-one breast angiograms were performed following mastectomy for carcinoma and graded 1-3 for vessel density. Sections (4 microm) from these carcinomas were labelled for endothelial cells using anti-CD34, and the vessel counts were compared with the radiological grades. A significant correlation was found between the densities . We therefore conclude that the microvessel density measured in histological sections is representative of whole tumour vascularity.


Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density. DNA damage is a constant problem that cells must deal with to maintain viability. Proper duplication and segregation of undamaged genetic material to daughter cells is essential for survival. DNA damage may come from endogenous sources including reactive oxygen species produced by cellular metabolism, spontaneous depurination of DNA and replication fork collapse at various replication fork barriers, or from exogenous sources including ionizing and ultraviolet radiation. Failure to alter DNA metabolism to properly respond to damaged DNA can lead to genetic instability, resulting in cell death and, in multicellular organisms, oncogenesis .To ensure each daughter cell receives a full complement of undamaged DNA, cells have evolved checkpoints. These checkpoints are surveillance mechanisms employed by the cell to detect and respond to DNA damage. They halt the cell cycle, allowing time to repair DNA damage before the crucial processes of DNA replication and chromosomal segregation ,2. CheckSeveral checkpoints regulate the cell cycle. The G1/S and G2/M DNA damage checkpoints prevent cell-cycle progression into S-phase and M-phase, respectively. Additionally, the S-M checkpoint, also known as the replication checkpoint, prevents mitosis in the presence of arrested replication forks. The cell cycle targets of the G1/S, G2/M, and S-M checkpoints have been characterized . In eachThe S-phase DNA damage checkpoint is a bit different from the other DNA damage cell-cycle checkpoints. Instead of preventing a cell-cycle transition, this checkpoint reduces but does not absolutely halt DNA synthesis in the presence of damaged DNA during S-phase. In addition, there is not a strong correlation between checkpoint activity and DNA damage resistance. For these reasons, it has been suggested that the checkpoint may be more involved in accommodating and tolerating damage during replication than actually repairing the damage .in trans to DNA lesions that may be far from the origins being regulated [in cis to DNA damage, only forks encountering DNA damage would be slowed. The contribution of fork slowing to total reduction in DNA synthesis would thus depend on lesion density. The idea that the checkpoint may act locally to slow replication at sites of DNA damage is consistent with checkpoint helping to coordinate replication and repair and to allow cells to tolerate damage during S-phase.The hallmark of the S-phase DNA damage checkpoint is the slowing of replication in response to DNA damage. Bulk replication can be slowed by inhibiting origin firing or reducing the rate of replication fork progression and both mechanisms appear to be used Figure . Origin egulated . CheckpoChanges in the rate of DNA synthesis have been measured using a variety of bulk methods. Before the establishment of DNA as the critical target of radiation or even the genetic material, it was shown that replicating cells are more sensitive to ionizing radiation (IR) than stationary cells . FurtherThe first efforts to deconvolve origin and fork regulation used DNA fiber autoradiography and alkaline sucrose gradient centrifugation. Both techniques allow detection of new DNA synthesis by pulse-labeling replicating DNA. Fiber autoradiography permits direct visualization of DNA synthesis originating from multiple origins of replication on individual DNA fibers. Alkaline sucrose gradient centrifugation separates newly synthesized DNA of various sizes from the bulk of unreplicated DNA. The quantity and sizes of small species are used to measure the rates origin firing and fork progression. Experiments using these techniques suggested DNA synthesis was reduced due to inhibition of origin firing at low doses of UV or IR -18. ConcMeasurement of bulk DNA synthesis by radioactive label incorporation shows that cells respond in a biphasic manner to increasing doses of IR: a steep initial decline at low doses followed by a much more shallow reduction at higher doses  and Figu. EstimatThe study of checkpoints began with the realization that cell-cycle effects were not a passive consequence of DNA damage itself but an active regulatory response . The firThe AT RDS phenotype appeared to be primarily origin based. AT cells lacked the steep initial decline in DNA synthesis in response to low doses of IR. Analysis by both alkaline sucrose gradient centrifugation and autoradiography indicated that ATM mutants lacked control of origin firing in response to IR ,28,31,32The role of ATM in regulating fork progression is less clear. First of all, fork slowing was observed only in response to high doses of radiation and most studies concentrated on the low dose results ,33-35. ICheckpoint inhibition of origin firing is by necessity a global response to DNA damage. To prevent origins from firing, checkpoint components must act at origins distant from sites of DNA damage. Consistent with this idea, low doses of IR inhibit origin clusters not necessarily directly impacted by DNA damage and episomal DNA synthesis is prevented even when only nuclear DNA damaged by IR ,18,36,37in vitro in Xenopus extracts [Significant progress has been made in elucidating the mechanism underlying checkpoint-regulation of origins in higher eukaryotes. Activation of either ATM or the ATM and Rad3-related (ATR) kinase prevents replication extracts ,39. In vextracts ,41. Likeextracts ,43. DegrAlthough checkpoint inhibition of origin firing is conserved between vertebrates and yeast ,45, the Inhibition of origin firing is a general checkpoint response to replication fork stalling. Hydroxyurea (HU) treatment leads to nucleotide depletion, fork arrest, and replication checkpoint activation early in S-phase. Like the S-phase DNA damage checkpoint, the replication checkpoint prevents origin firing. Although the replication and S-phase DNA damage checkpoints are activated by different substrates, arrested replication forks and DNA damage, respectively, both checkpoints prevent origin firing via activation of the same checkpoint kinases. Gel-electrophoretic detection of replication intermediates at several origins showed that prevention of late origin firing upon HU arrest occurred in a checkpoint dependent manner ,48. MicrCheckpoint kinases regulate origin firing even in the absence of insult. AT cells were originally observed to replicate more quickly than wild-type . Likewisin cis to DNA damage for the following three reasons. First, there is no evidence that checkpoint activation can slow fork progression in trans. Second, there is no reason to believe that slowing in the absence of encountering damage would be beneficial to a replication fork. And third, as described below, there is evidence that the degree of slowing is correlated with the density of DNA damage. Therefore, we imagine a scenario in which forks transiently pause at sites of damage in a checkpoint-dependent manner. Each individual pause would not significantly delay replication, but the cumulative effect of forks pausing at many sites would lead to an overall reduction in fork rate. Therefore, even though the overall effect is referred to as 'fork slowing', we propose that forks are never slowed, per se; either they are replicating normally or they are transiently paused at sites of damage. This pausing would be checkpoint-dependent, since in checkpoint mutants replication is not slowed. It should be noted that although in our model fork slowing is a local, in cis effect, the checkpoint signaling involved need not be. For instance, forks encountering damage may activate checkpoint kinases that can act globally to inhibit replication origins or to regulate other forks, but such global signaling would slow only those forks which are also encountering DNA damage.In contrast to origin firing, fork slowing may represent a local checkpoint response to DNA damage. It is possible that checkpoint regulation of fork progression is a global mechanism in which, upon checkpoint activation, the rate of progression of every fork is slowed Figure . HoweverThe checkpoint-dependent pausing could allow a number of responses to DNA damage: the replicative polymerases could be exchanged for translesion DNA polymerases allowing error-prone replication through the damage, the DNA lesion could be repaired after replication fork regression or the paused fork may undergo replication-coupled recombination allowing bypass of DNA damage through sister chromatid exchange. In the absence of the checkpoint, the fork would not pause at damage, possibly bypassing it by downstream repriming.Chk2 mutants still display intermediate slowing. MRN mutants also display residual slowing even in response to IR fully capable of activating Chk2 and preventing origin firing [Recent genetic evidence suggests that replication fork slowing contributes to checkpoint-dependent replication slowing. Two pathways downstream of ATM are required for robust slowing in response to IR Figure . Mutation firing . These on firing .Direct physical evidence for checkpoint-dependent slowing of fork progression has come from fluorescent DNA fiber analysis, an updated version of the fiber autoradiography. Using nucleotide analog labeling and fiber analysis, replicated regions on individual DNA molecules may be visualized. From this data, replication fork progression and origin firing frequency can be analyzed. This data explicitly shows DNA damage causes fork slowing, a possibility only indirectly supported previously by bulk assays. Both ATR and Chk1 are required to slow replication forks in response to UV and camptothecin (CPT) ,60. BesiLike regulation of origin firing, checkpoint kinases regulate fork progression even during unperturbed S-phase. Depletion of the ATR-Chk1 mediator Claspin or target Tim reduces fork progression in the absence of damage ,59. PresIn addition to checkpoint-dependent slowing, bulky DNA lesions can slow replication forks independently of checkpoint activity. Density shift experiments conducted in budding yeast followed replication of a long region initiated from a single origin. Results suggested replication fork progression was reduced in the presence of MMS in a checkpoint-independent manner . MoreoveThe idea of the checkpoint as a fork modulator is consistent with the known vital functions of S-phase checkpoints in stabilizing stalled replication forks. First, fork stabilization allows for the completion of replication in the presence of damage. Two-dimensional gel electrophoresis and density shift analysis show checkpoint kinases prevent fork stalling and accumulation of unreplicated DNA in the presence of MMS -69. SimiFork slowing in the presence of damage appears to be a delicate balance between stalled fork stability and fork restart. Dynamic regulation of checkpoint kinase activity is required for efficient fork progression even when forks are slowed by DNA damage. Deactivation of Rad53, the budding yeast checkpoint effector kinase, is required for efficient replication fork progression in the presence of MMS . PresumaAlthough genetic and physical evidence suggests checkpoint activation pauses replication forks, the exact mechanism by which the checkpoint accomplishes this feat is unknown. One possible mechanism for fork pausing involves recombination. Vertebrates require Rad51 for fork slowing in response to DNA damage . AlthougAlthough involvement of recombination in replication fork slowing differs amongst eukaryotes, recombination still appears to be important for replication fork metabolism. A possible mechanism involving recombination and fork pausing is polymerase template switching. Recombination intermediates or joint molecules are observed during replication ,81-83. Erqh1Δ and sgs1Δ display hyper-recombinant phenotypes and S-phase slowing defects [sgs1Δ mutants accumulate X-shaped intermediates during S-phase suggesting a correlation between failure to slow and unregulated template switching [Some data implicates that the restraint of recombination is important for slowing replication forks. Both budding and fission yeast helicase mutants  defects ,82. sgs1witching . A numbewitching  and Willwitching . This epThe role of recombination in replication fork slowing is further supported by the conserved requirement of the MRN recombinational-repair complex for replication slowing in budding yeast, fission yeast and vertebrates ,86-88. IXenopus, the Tim homologue is required for fork restart [Like the regulation of origin firing, the phenomenon of replication fork slowing is conserved, although the mechanism seems to vary among eukaryotes. For example, components of the Replication Fork Protection Complex, namely Timeless/Swi1/Tof1, Tipin/Swi3/Csm3 and Claspin/Mrc1, serve different roles in stalled fork metabolism but are all required for some aspect of replication fork progression. In fission yeast, Swi1 is not required for slowing but is responsible for slowing defects displayed by other mutants. In contrast, both components of the vertebrate Tim-Tipin complex are required for slowing replication forks and fork progression in the absence of damage and, in  restart ,80,93,94 restart . HoweverAt first glance, yeast, frogs and mammals seem to differ in S-phase DNA damage checkpoint response to different damaging agents. However, differences in slowing may simply be related to the frequency of DNA lesions produced in these different systems. For example, vertebrates slow replication in response to low doses of IR while fission yeast do not ,95. In tet al. observed in vertebrate cells that low doses of IR (1-5Gy), which cause 100s of lesions, prevented origin firing and did not slow forks but that MMS, which causes 10,000s of lesions, prevented origin firing and robustly slowed forks [DNA lesion density may contribute to whether checkpoint-dependent fork slowing is manifest as slowing of bulk replication. Unlike global checkpoint inhibition of origin firing, which inhibits all origins once a threshold of checkpoint signaling is crossed, if fork slowing is local in nature, its contribution to bulk slowing would depend explicitly on the number of forks directly encountering DNA lesions. The greater the lesion density, the more often forks would pause and the greater the contribution of fork slowing to overall reduced DNA synthesis. Data from titration experiments using MMS shows replication slowing in fission yeast is directly related to the concentration of MMS used . DNA fibed forks . These oed forks . Lack ofDespite some important differences between model systems, we propose checkpoint regulation of both origin firing and fork progression play important roles in checkpoint-dependent replication slowing. Direct evidence for both these mechanisms has recently been shown using fluorescent DNA fiber analysis. We propose that checkpoint activation prevents origin firing in a global manner but slows forks only when they encounter DNA damage. Thus, the contribution of fork slowing to the overall reduction in DNA synthesis is dependent on the density of DNA damage. The importance of proper checkpoint regulation of origin firing and fork progression in response to damage is emphasized by the fact that defects in the S-phase DNA damage checkpoint response are often associated with genomic instability, inability to tolerate DNA damage during replication and a predisposition to cancer development. Thus continued analysis of the mechanisms of the S-phase DNA damage checkpoint will undoubtedly produce interesting and important insights into how cells deal with DNA damage during S phase.The authors declare that they have no competing interests.NW and NR collaborated on the writing of this review.


Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level.Phenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells.The investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike LexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogenous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes. Bacillus subtilis, lysogenic versus the lytic cycle of bacteriophage lambda, biofilm formation, toxin production and antibiotic persistence [Genetically identical bacterial cells can exhibit heterogeneity as the population bifurcates into distinct subpopulations. Such heterogeneity within clonal populations is a bet hedging strategy as a small fraction of a population is either prepared to survive adverse environmental conditions or sacrifice itself to enhance the likelihood of survival of clonal siblings. Examples of phenotypic heterogeneity include: development of competence and sporulation in sistence -4.Escherichia coli DNA damage induces the expression of more than 40 genes leading to arrest of cell division and the induction of DNA repair, prophages, toxin production and mutagenesis [10-CAG designated as SOS boxes [In agenesis . The keyagenesis , is the OS boxes . Upon DNOS boxes .E. coli strains, designated as colicins, are plasmid encoded and are found with high frequency among natural isolates [E. coli populations in the mammalian colon [SOS induction often results in the production of antimicrobial toxins such as bacteriocins. The bacteriocins of isolates . These tan colon ,10. Colian colon -13. Prodan colon . Some coan colon . Colicinan colon ,17. Expran colon ,18.cka is expressed in only a small fraction of a bacterial population while the immunity gene encoding the immunity protein is expressed in the large majority of the cells [recA, lexA and umuDC, and finally we examined the simultaneous expression of the colicin encoding cka gene and the lexA gene.In previous studies we showed that the pore forming colicin K activity gene he cells ,19. In t-1 and 30 μg l-1, respectively.The bacterial strains and plasmids used in this study are presented in Table Plasmid DNA isolation was performed with the GeneJET™ plasmid miniprep kit . Standard procedures were used for gel electrophoresis, ligations and transformation experiments . RestricBamHI and XhoI restriction sites. The PCR generated fragments were cut with BamHI and XhoI (Fermentas), and ligated into the low copy number pSC101 [(GFPmut3) gfp also cut with the same two enzymes.PCR was carried out to amplify the promoter regions with an additional 73 - 93 bp of the flanking colicin encoding gene for colicins A (486 bp), E1 (508 bp), E7 (501 bp), N (499 bp) and M 298 bp) with the primers listed in Table 98 bp witr pSC101  based plDsRed-Express2 gene, which is part of a gene cassette on plasmid pDsRed-Express2-N1, was cloned into the natural colicin K encoding plasmid pColK-K235 manipulated to carry the Apr gene as a selectable marker, and a KpnI restriction site in the cka gene. A cassette carrying the promoterless gfp was inserted at the KpnI restriction site [cka-DsRed-Express2 transcriptional fusion was constructed by replacing the KpnI fragment harboring the promoterless gfp with the in frame promoterless DsRed-Express2 [KpnI. Only cells with an active cka promoter can express DsRed-Express2.The promoterless ion site . The ckaExpress2  amplifieHM449002 (caa promoter region), HM449003 (cna promoter region), HM449004 (ce1a promoter region), HM449005 (ce7a promoter region), HM449006 (cma promoter region).Nucleotide sequencing was performed to confirm that no base changes had occurred during amplification. The sequences have been deposited in the GenBank Nucleotide sequence database under accession numbers, gfp transcriptional fusions, and control strains without plasmid carrying gfp fusions, were grown with aeration at 37°C. Samples were removed at early stationary phase and chloramphenicol (500 μg ml-1) (Sigma) was added to block protein synthesis. Prior to microscopy, cells were attached to glass slides coated with 0.1% (wt vol-1) poly-L-lysine (Sigma). Fluorescence microscopy to detect expression in single cells was performed using an inverted microscope (Nikon Eclipse TE300), equipped with a Nikon digital camera DXM 1200, and a 488 nm Argon-Ion laser as well as bright field microscopy.Strains RW118 and RW464 carrying different colicin promoter region-http://www.cs.tut.fi/sgn/csb/cellc/. The fluorescence intensity of individual cells was estimated using image analysis software Scion Image http://www.scioncorp.com as previously described [The examined cells were counted with software for quantification of bacteria by automated image analysis cellC escribed . The flucka-DsRed-Express2 and the lexA-gfp fusions was investigated employing a laser scanning Confocal Microscope .Simultaneous expression of the cka gene, encoding the pore forming colicin K; in the absence of exogenous DNA damaging agents cka is expressed in only a small fraction of the population [gfp transcriptional fusions. Our results revealed that, as was previously shown for the cka gene [caa, Figure gfp fusion with the colicin M activity gene promoter, cma-gfp, revealed low level expression in the large majority of the investigated cells. Colicin M was shown to be tightly connected with the upstream colicin B encoding genes and it is presumed that expression of both colicins B and M is regulated from common SOS boxes situated upstream of the colicin B activity gene [E. coli strains [cma and found neither colicin regulatory motifs nor any consensus promoter sequence (data not presented). Nonetheless, we detected uniform low-level fluorescence mediated by the colicin M promoter  and no detectable basal level of expression was observed among the large majority of the population. Our results show that in the absence of exogenous DNA damaging agents very low levels of umuDC promoter activity is detected. As translesion synthesis must be employed only when necessary, synthesis of the specialized polymerases is under physiological conditions controlled by complex regulation at the level of transcription and posttranslation.The SOS genes, ized DNA . The opeized DNA . It was ized DNA . AccordiStaphylococcus aureus [E. coli [recA, lexA and umuDC genes is dependent upon an SOS response under physiological conditions, expression was investigated in an isogenic recA defective strain RW464  and colicin E7 (approximately three fold) was observed, while the percentage of cells expressing colicin E1 and N activity genes remained essentially unaltered  of the cells harboring gfp fusions with the colicin activity genes were intensely fluorescent, indicating high level expression (data not shown).To resolve the effect of LexA regulation at the single cell level, expression of the investigated gene fusions was also studied in strain RW542 encoding a LexA protein defective in binding to LexA boxes. Fluorescence microscopy revealed that in the cka and the lexA genes was followed at the single cell level in strain RW118 harboring two plasmids: pKCT10 with a cka-DsRed-Express2 fusion and the pSC101 derivative vector harboring the lexA-gfp fusion. As is evident from Figure lexA gene also expressed the cka gene. Nonetheless, individual cells (approximately 0.1%) highly expressing only the cka gene could be detected suggesting, that in a very small fraction of the population the colicin K activity gene is expressed in the absence of the SOS response most probably stochastically, due to perhaps intracellular fluctuations of the LexA protein. Filamentation while a hallmark of SOS induction due to binding of SulA to the FtsZ proteins is also evident in cells not expressing lexA-gfp , horizontal gene transfer (induction of prophage) and virulence among pathogenic E. coli strains. The same might apply to other gram negative  and gram positive  bacterial species that possess a system similar to the E. coli SOS system.Expression of colicin genes under physiological conditions by a small subpopulation may promote strain and genetic diversity and due to lysis of producing cells could provide resources to facilitate growth of non-expressing cells. On the other hand, a subpopulation of cells with higher levels of the RecA protein may be more proficient in recombination, e.g. for the stable incorporation of horizontally acquired DNA or a rapid response to DNA damage. We can speculate that heterogeneity of expression of recA and lexA, the colicin activity genes and umuDC exhibit no basal level expression. Heterogenous expression is established primarily by stochastic factors as well as the binding affinity of LexA to SOS boxes.LexA regulated SOS genes exhibit heterogeneity as they are highly expressed in only a small subpopulation of cells. Unlike SK performed all experiments. ZP contributed to analysis of the results. OG and DŽB participated in the design of the experiments and SK, OG and DŽB in preparation of the manuscript. All authors read and approved the final manuscript.


The Swine Genome Sequencing Consortium (SGSC) was formed in September2003 by academic, government and industry representatives to provide internationalcoordination for sequencing the pig genome. The SGSC’s mission is to advancebiomedical research for animal production and health by the development of DNAbasedtools and products resulting from the sequencing of the swine genome. Duringthe past 2 years, the SGSC has met bi-annually to develop a strategic roadmap forcreating the required scientific resources, to integrate existing physical maps, and tocreate a sequencing strategy that captured international participation and a broadfunding base. During the past year, SGSC members have integrated their respectivephysical mapping data with the goal of creating a minimal tiling path (MTP)that will be used as the sequencing template. During the recent Plant and AnimalGenome meeting , presentations demonstrated thata human–pig comparative map has been completed, BAC fingerprint contigs (FPC)for each of the autosomes and X chromosome have been constructed and that BACend-sequencing has permitted, through BLAST analysis and RH-mapping, anchoringof the contigs. Thus, significant progress has been made towards the creation of aMTP. In addition, whole-genome (WG) shotgun libraries have been constructed andare currently being sequenced in various laboratories around the globe. Thus, ahybrid sequencing approach in which 3x coverage of BACs comprising the MTPand 3x of the WG-shotgun libraries will be used to develop a draft 6x coverage ofthe pig genome.


Exposure to organophosphate insecticides is of particular concern because these widely used compounds have been shown in rodents to induce persistent synaptic abnormalities in neural acetylcholine (ACh) systems at doses too low to cause symptoms of systemic exposure. Pilot studies have reported some evidence of improvement when a “ketogenic” diet—high in fat and low in carbohydrates—was used to treat certain neurologic disorders. Drawing from this preliminary clinical research, researchers have now demonstrated that many of the abnormalities in ACh systems produced by neonatal organophosphate exposure were not evident in adult rats fed a high-fat diet Rats were injected with the organophosphate parathion on each of postnatal days 1–4, at doses of 0.1 or 0.2 mg/kg/day—these dosages straddle the threshold at which cholinesterase inhibition is first detectable. In adulthood, half the animals were switched to a high-fat diet for 8 weeks. The investigators then examined brain regions of the rats to assess specific aspects of ACh synaptic function, including nicotinic ACh receptor binding, choline acetyltransferase activity, and hemicholinium-3 binding to the presynaptic choline transporter.Adult rats on a standard lab chow diet showed multiple abnormalities in regional ACh synaptic markers following parathion exposure. All seven abnormalities observed in parathion-exposed females on the standard diet were absent in exposed females on the high-fat diet, and eight of ten abnormalities observed in parathion-exposed males on the standard diet were absent in exposed males on the high-fat diet. The results suggest that diet may offer a way to ameliorate the effects of developmental neurotoxicant exposure.However, the authors offer several caveats. Their earlier work showed that neonatal exposure to organophosphates produced long-term changes in metabolic function that have been linked with obesity, prediabetes, and cardiovascular risk factors such as elevated serum lipids. Because these metabolic abnormalities could be exacerbated by a high-fat diet, future studies should seek to uncover whether and how specific aspects of the diet influence abnormalities in ACh systems. Moreover, although this study showed that dietary modifications may offset synaptic changes, future studies will need to determine whether such modifications can actually lead to improved neurobehavioral outcomes.The authors also highlight a potential connection between early-life toxicant exposure and subsequent diet-related disease. If a high-fat diet can indeed ameliorate the impact of developmental neurotoxicants, then this might serve as an underlying, subconscious reinforcement to consume a high-fat diet as a way of self-remediating underlying neurobehavioral deficits—potentially expanding the public health implications of the developmental effects of neurotoxicant exposure.


Catheter ablation is a first line treatment for many cardiac arrhythmias and is generally performed under x-ray fluoroscopy guidance. However, current techniques for ablating complex arrhythmias such as atrial fibrillation and ventricular tachycardia are associated with suboptimal success rates and prolonged radiation exposure. Pre-procedure 3D CMR has improved understanding of the anatomic basis of complex arrhythmias and is being used for planning and guidance of ablation procedures. A particular strength of CMR compared to other imaging modalities is the ability to visualize ablation lesions. Post-procedure CMR is now being applied to assess ablation lesion location and permanence with the goal of indentifying factors leading to procedure success and failure. In the future, intra-procedure real-time CMR, together with the ability to image complex 3-D arrhythmogenic anatomy and target additional ablation to regions of incomplete lesion formation, may allow for more successful treatment of even complex arrhythmias without exposure to ionizing radiation. Development of clinical grade CMR compatible electrophysiology devices is required to transition intra-procedure CMR from pre-clinical studies to more routine use in patients. Radiofrequency (RF) catheter ablation has advanced over the last 25 years from an experimental procedure to the first line treatment for a number of cardiac arrhythmias including atrio-ventricular reentrant tachycardia, accessory pathway associated tachycardias, and typical atrial flutter . These pThe clinical indications for anatomy based catheter ablation have since expanded to more complex arrhythmias such as atrial fibrillation and scar based ventricular tachycardia ,5. The bModern imaging techniques such as Cardiovascular Magnetic Resonance (CMR), intra-cardiac ultrasound, and x-ray computed tomography (CT) are increasingly used to approach the shortcomings of current mapping and ablation systems. CMR is a particularly flexible imaging modality that offers excellent soft tissue contrast, well characterized gadolinium enhancement techniques for myocardial scar visualization, 3-D imaging of complex cardiovascular anatomy, real-time 2-D imaging along arbitrary imaging planes, and the ability to quantify cardiac motion and blood flow. This article will review the application of CMR to current clinical procedures and ongoing advances toward full CMR guidance of electrophysiology procedures.CMR has been used most extensively to assist planning and guidance of atrial fibrillation (AF) ablation procedures. AF is the most common clinically relevant arrhythmia affecting 0.4% of the general population . The priIn an effort to improve procedural success and reduce complications, 3-D CMR angiography (MRA) has been used to assist planning of AF ablation. Kato and colleagues used MRA to study left atrial anatomy in normal subjects and patients with paroxysmal atrial fibrillation and found that 38% of people had pulmonary vein anatomic variants . IdentifCMR also has the potential to guide the treatment of scar based monomorphic ventricular tachycardia (MVT), a potentially lethal arrhythmia that is difficult to treat medically or with current ablation techniques. Ventricular tachycardia that results in uniform repetitive electrical activation of the heart arises from anatomically fixed arrhythmia substrate that can be targeted for ablation. Myocardial scarring due to infarction, cardiomyopathy, sarcoidosis, ARVD, or cardiac surgery is a common cause of MVT . Scar reThe use of CMR for assisting MVT ablation is still in the investigational stages but shows promise. Late gadolinium enhancement CMR (LGE-CMR) has been used extensively to characterize regions of scar in ischemic and non-ischemic cardiomyopathy Figure . A numbeThe detailed anatomic information available from CMR is now being used as a roadmap for guiding placement of ablation lesions -30. A nuThese sub-optimal results call attention to limitations of this current state-of-the-art in 3D image guided cardiac ablation. First, pre-acquired roadmap images may not correspond to the anatomy during the procedure. Changes in cardiac chamber size associated with variations in heart rate, rhythm, and volume status are not accounted for by pre-acquired imaging and could lead to catheter position registration errors . AdditioIntracardiac echocardiography (ICE) addresses some of these shortcomings and is increasingly used in clinical practice . ICE hasThere are a number of reasons intra-procedure CMR is an attractive option for guiding future electrophysiology procedures. First, CMR offers a number of ablation lesion imaging techniques. In addition, the ability to obtain images in arbitrary orientations opens the potential for high quality visualization of catheters, anatomy, and electrode tissue contact. Further, the position errors introduced by registering catheter position to pre-acquired 3D images can be largely avoided because both real-time CMR images and 3D CMR images are acquired in the same coordinate system and 3-D images can be reacquired during the procedure if needed.Over the last 15 years the basic techniques to enable fully MR guided EP procedures have been developed. Lardo and colleagues introduced the potential of CMR for guiding EP procedures in 2000 . ContinuSubsequent work in our lab has demonstrated the ability to use real-time CMR to perform basic diagnostic EP studies . ImagingOther techniques relevant to EP procedures have also been performed using real-time CMR guidance. Trans-septal catheterization is required for left atrial catheter ablation. While generally safe, the procedure can be difficult in the setting of distorted atrial anatomy and carries the risk of serious complications such as aortic puncture. The ability of real-time CMR to guide trans-septal punctures under direct visualization of the needle, atria, fossa ovalis, and surrounding vasculature has been nicely demonstrated ,41. RetrRecent advances promise to take MR guided EP from these initial feasibility studies to safe, efficient practice. These advances include 1) intra-procedure ablation lesion monitoring techniques, 2) faster 2-D and 3-D imaging, 3) improved device visualization, 4) intuitive 3-D anatomy and intracardiac electrogram visualization, and 5) CMR safe device construction techniques.Perhaps the most significant advantage of CMR-guided ablation therapy is the potential to visualize ablation lesions with high spatial and temporal resolution. The typical endpoint of current ablation procedures is absence of electrical conduction across the ablated region and/or an inability to re-induce the clinical arrhythmia with cardiac pacing and medications. However, propagation of electrical signals through the heart is affected by a number of factors including the tissue temperature change induced by ablation ,44. Some500 kHz radiofrequency (RF) current is the most commonly used ablation source used for electrophysiology procedures. Cryothermy, ultrasound, laser, and microwave ablation are also being investigated. Ablation lesions can be visualized because CMR is able to detect specific changes in proton precession and relaxation properties resulting from heating and heat induced biophysical changes in cardiac tissue including interstitial edema, hyperemia, protein conformational changes, cellular shrinkage, and tissue coagulation . Acute iT1 weighted non-contrast enhanced MR imaging of RF ablation lesions has also been investigated . T1 chanLGE-CMR can provide better visualization of RF ablation lesions compared with non-contrast imaging techniques Figure . The timOther methods for monitoring ablation lesion formation during RF energy application are also being investigated. Proton resonance shift thermography is an CMR technique that takes advantage of the decrease in the proton resonance frequency with increasing temperature . This teWhile most cardiac ablation lesion CMR studies have been performed in roughly 10 mm thick ventricle, imaging the less than 3 mm thick human atria is of particular clinical interest given the difficulty of achieving long term pulmonary vein isolation following atrial fibrillation ablation. Peters et.al. demonstrated 3D LGE-CMR of left atrial ablation lesions 10 to 15 minutes after contrast injection using image based respiratory gating . This ga2. The target temporal resolution is 7 frames per second (fps), the usual x-ray fluoroscopy frame rate for clinical EP procedures.While cardiac gating can be used to generate MR images with excellent spatial resolution by splitting data collection over multiple heartbeats, real-time CMR requires a more deliberate trade off between temporal and spatial resolution. To adequately visualize catheters, CMR guided EP procedures require an in-plane spatial resolution of around 2 mmSince the initial 1 fps imaging used to guide the first MR guided EP procedure  faster, While the current imaging rates are adequate for a single 2-D image plane, ideal visualization of the device, target anatomy, and surrounding reference anatomy may require multiple 2-D image planes or even 3-D imaging. Other techniques that can improve imaging speed while balancing imaging quality include non-Cartesian k-space sampling, temporal data sharing between images, and adjusting the tradeoff between temporal and spatial resolution . These tWhile fluoroscopy provides projection images where the entire catheter body and tip are easily visualized, 2-D MR images typically depict a slice through the body that is around 5 to 10 mm thick. Curved devices such as catheters may pass in and out of the MR imaging plane leading to misinterpretation of the device tip position. We have noted in preclinical studies that poor delineation of the tip position can result in tissue contact trauma, such as local hemorrhage. In addition, for electrophysiology ablation procedures the device tip contains the energy source. Misestimating the tip/tissue contact region can lead to inaccurate placement of ablation lesions.During our feasibility studies, tip location has mostly been performed using interactive real-time sequences with a user interface that permits adjustment of the scan plan during image acquisition. Part of the catheter is first identified on some imaging plane and the plane is manually adjusted until the tip is located. For vascular procedures where the device is constrained to a co-planar segment of blood vessel, manual plane manipulation is acceptable since only minor image plane translations are needed to visualize the device tip and relevant anatomy. For navigation in cardiac chambers where the device tip location is less constrained, the frequent need for manual plane manipulation necessitates a skilled operator for image plane manipulation and can distract from efficient procedure workflow.One approach to this problem is to automatically direct imaging to the device location using position sensors located in the catheter. 15 years ago Dumolin et.al. described using 1-D projection MR imaging along the x, y, and z directions to identify the 3-D position of small receiver coils located in a catheter tip . This poAn alternative technique for device visualization uses non-slice selective imaging to produce an effect similar to projection x-ray fluoroscopy ,75. The Another factor that affects device navigation in the CMR environment is the physical constraint of performing procedures near and within the narrow CMR bore. The availability of shorter, wider bore, high-field CMR scanners are making this less of an issue. Remote catheter steering is also gaining interest in the EP field to facilitate point-by-point electrical mapping of the cardiac chambers and assist stable device placement during ablation ,80. SomeThe ability to generate real-time images with arbitrary orientations in addition to anatomically detailed 3-D images with flexible tissue contrast makes CMR well suited for navigating complex arrhythmia anatomy and delineating complex ablation patterns. This flexibility also introduces the potential for disorientation and information overload. Appropriate displays and user-interfaces tailored to the workflow of an EP procedure are needed to manage this flexibility. Because thin-slice real-time imaging can intersect anatomy in unfamiliar ways, 3-D visualizations that plot real-time images oriented relative to reference images can be helpful Figure . A basicThe most important consideration for any new diagnostic or therapeutic approach is safety. While a number of studies have been performed to determine the safety of conventional CMR with regard to electromagnetic energy exposure and tissue heating ,85, inteAn additional safety concern particular to CMR guided cardiovascular procedures is the significant heating that can result from RF transmission induced current in extended metallic objects such as guidewires, wired electrodes, and metal braided catheters ,88. ThisTransitioning proof of concept studies to clinical electrophysiology procedures requires collaboration between academic centers, imaging and device companies, and regulatory agencies. The first CMR guided electrophysiology procedure in a patient was performed using custom catheters made to clinical specifications by a clinical catheter manufacturer . Prior tThe compatibility of CMR with implanted devices such as pacemakers and implantable cardiac defibrillators (ICDs) is also an important consideration in performing interventional CMR studies in the electrophysiology patient population. Particular concerns include static magnetic field induced movement of the device and scanning induced programming changes, device inhibition, activation of tachyarrhythmia therapies, and lead currents leading to heating or cardiac stimulation -106. ModIncreasing knowledge of the anatomic basis for cardiac arrhythmias has extended the role of catheter ablation to curing even complex rhythms such as atrial fibrillation and scar based ventricular tachycardia. CMR has demonstrated a number of uses for procedural planning, particularly for treatment of atrial fibrillation. The use of LGE-CMR for planning VT ablation procedures also shows promise. Real-time CMR combined with intra-procedural lesion imaging could allow physicians to accurate guide devices and establish completeness of ablation lines without concern for radiation exposure. This could significantly improve the way current ablation procedures are performed and open the way to ablative cure of arrhythmias such as permanent atrial fibrillation that currently respond poorly to minimally invasive approaches .Kolandaivelu:Imricor: OptionsLardo:Zoll Medical: ConsultantImricor: EquitySurgivision: EquityHalperin:Zoll Medical: Consultant, GrantImricor: RoyaltiesSurgivision: EquityAK was responsible for manuscript integration and co-author of all sections. ACL was responsible for lesion visualization and fast imaging. HRH was responsible for real time guidance and lesion visualization.


The face of prostate cancer has been dramatically changed since the late 1980s when PSA was introduced as a clinical screening tool. More men are diagnosed with small foci of cancers instead of the advanced disease evident prior to PSA screening. Treatment options for these smaller tumors consist of expectant management, radiation therapy  and surgery . In the highly select patient, cancer specific survival employing any of these treatment options is excellent, however morbidity from these interventions are significant. Thus, the idea of treating only the cancer within the prostate and sparing the non-cancerous tissue in the prostate is quite appealing, yet controversial. Moving forward if we are to embrace the focal treatment of prostate cancer we must: be able to accurately identify index lesions within the prostate, image cancers within the prostate and methodically study the litany of focal therapeutic options available. As defined by the International Task Force on Prostate Cancer and the Focal Lesion Paradigm, the goal of focal therapy for prostate cancer would be to "selectively ablate(s) known disease and preserve(s) existing functions, with the overall objective of minimizing lifetime morbidity without compromising life expectancy" . SeveralFocal therapy for the treatment of prostate cancer has been called the 'male lumpectomy', an analogue to women's breast lumpectomy for the treatment of breast cancer . In a luSuccessful adoption of focal therapy for the treatment of prostate cancer will hinge on several critical issues: 1) can we accurately identify index lesions within the prostate, 2) can we reliably image cancers within the prostate, 3) long-term efficacy of the technology to eradicate cancer, 4) appropriate follow-up of patients treated with focal therapy, and 5) limitations of PSA following therapy and how to detect recurrent/persistent disease.Extensive, bilateral disease precludes focal therapy. Multifocal prostate cancer has been reported in 67% to 87% of all cases of contemporary radical retropubic prostatectomy [In order to implement focal therapies, cancer must be accurately and reliably mapped within the gland. Initially, transperineal 3D mapping biopsies were reported to effectively exclude patients with clinically significant unsuspected cancer outside the area destined to be ablated more so than repeat transrectal ultrasound (TRUS) guided biopsies . Though st International Workshop on Focal Therapy and Imaging of Prostate Cancer.Perhaps the 3D nature in which the biopsies are obtained not the route the biopsies are obtained  is not the critical factor. Researchers using the TargetScan 3D transrectal ultrasound and prostatic biopsy system correctly characterize 88% of ex vivo prostatic octants in regards to whether or not they contained cancer . These 3Though standard imaging with ultrasound have limited sensitivity of identifying pathologic proven tumor within the prostate gland, novel techniques are being investigated with prostate biopsying, including the use of ultrasonographic contrast agents, color Doppler, power Doppler, and MR spectroscopy image-directed TRUS-guided biopsy in hopes of better identifying cancer at the time of prostate biopsy ,12.Equally important to the success of focal therapy of localized prostate cancer is imaging and its ability to more accurately provide information regarding tumor presence and localization before and after treatment. Tumor localization using imaging modalities allows for treatment of the area(s) involved with cancer and sparing of the normal, non-cancerous tissue. Our current radiologic staging modalities  lack sensitivity in depicting the presence and location of localized disease .Previous studies reported a variable sensitivity of magnetic resonance imaging (MRI) in localizing disease within the prostate ,15. ReceMRI, MRSI and DCE-MRI with a 3-Tesla whole-body scanner were performed in 30 patients with biopsy-proven prostate cancer before radical prostatectomy. The high signal-to-noise ratio (SNR) at 3 Tesla provided T2-weighted turbo spin echo (TSE) images with excellent anatomical detail (in-plane voxel size of 0.22 × 0.22 mm) and good T2 contrast, whereas dynamic contrast-enhanced images showed good temporal resolution . The comThough fascinating, delving into the history and biology of cryotherapy is not part of this review. Recently, the AUA released its best practice statement on cryosurgery for the treatment of localized prostate cancer . A corneFocal cryoablation was planned to encompass the area of known tumor based on staging biopsies. Forty-eight patients with at least 2-year follow-up had focal cryoablation. Mean follow-up was 4.5 years. Ninety-four percent of the patients treated had stable PSAs according to American Society of Therapeutic Radiology and Oncology (ASTRO) criteria. Of the 24 patients with stable PSAs who were routinely biopsied (n = 24) all were negative. No local recurrences were noted in areas treated. Potency was maintained to the satisfaction of the patient in of 36 of 40 patients who were potent preoperatively. Of the 48, all were continent . It is tThe use of hormone therapy must be accounted for when interpreting the results from the above studies. In the study by Onik, patients on combined hormone therapy had therapy stopped immediately after treatment in all cases. Nonetheless, the 3-month data should be cautiously interpreted due to potential residual effects of neoadjuvant hormone therapy . A 2005 First introduced in the 1940s, high-intensity focused ultrasound (HiFU) uses energy focused to a specific point within the prostate, thus enabling targeted destruction of prostatic tissue. Currently, two HiFU devices are attempting to get FDA approval for the treatment of prostate cancer in the US. However, HiFU therapy for localized prostate cancer has been widely accepted in Europe and Asia. In a large Phase II/III prospective multicenter study, Thuroff reported the results of 402 patients with stage T1-2, Nx-0, M0 prostate cancer treated with HiFU. Follow-up was extremely short at approximately 1 year, but the negative biopsy rate observed in the T1-2 primary-care population was 87.2% . The HiFSimilar to focal cryoablation, focal HiFU is planned to encompass the area of known tumor based on staging biopsies. In 2008, Muto and others published a study on 70 patients treated with HiFU. Twenty-nine of these patients had unilateral disease as determined by multi-regional biopsies. These patients received focal therapy while the remaining patients received whole organ ablation. Scheduled biopsies were performed at 6 and 12 months following treatment. After 12 months 84% of patients who received whole therapy were biopsy negative versus 77% in the focal therapy group. Using the ASTRO definition, the 2-year biochemical recurrence free survival rates for patients at low and intermediate risk were 91% and 50% for whole therapy, respectively. For the focal therapy group, biochemical recurrence free survival rates were 83% and 54%, respectively at 2 years. The authors also noted in this study that whole, but not focal therapy, resulted in a continuous decline in serum testosterone levels. Of the 52 patients who were continent prior to HiFU, 49 (94%) subjects were continent after HiFU therapy. Morbidity of whole gland HiFU compared to focal gland HiFU was not significantly different . These rSimilar with cryosurgery, in patients who received hormone therapy, caution must be taken when interpreting these results. The study by Thuroff excluded patients who had previous orchiectomy or hormone therapy . Muto do® (WST09) has been studied in the VTP of prostate cancer. Huang and colleagues demonstrated that Tookad-VTP could destroy a clinically-significant volume of normal canine prostate tissue [Vascular targeted photodynamic therapy (VTP) involves the generation of cytotoxic agents in situ that results in cell death and tissue ablation. This is achieved by systemic or local administration of a photosensitizing drug that is activated with light of a specific wavelength. The photosensitizer Tookade tissue . In a phe tissue . RegardlRecently, Emberton presented results of a phase I trial using VTP to treat low volume, primary prostate cancer. Preliminary results are encouraging, however the authors readily admit refinements in the technique are needed prior to embarking on a larger clinical trial . The encThe face of prostate cancer has been dramatically changed since the late 1980s when PSA was introduced as a clinical screening tool. More men are diagnosed with localized prostate cancer  and smalThough focal therapy of the prostate only treats a portion of the prostate it has been referred to as the 'male lumpectomy', but one must remember women who undergo lumpectomy usually will have other local or systemic therapies to ensure eradication of disease. Since men who are undergoing 'male lumpectomy' rarely undergo these addition therapies, comparison with women undergoing a lumpectomy should be limited. However, through rigorous clinical scrutiny the ideal treatment combinations were identified for breast cancer that successfully treated the cancer and limited the morbidity associated with more radical therapies. A strong push must be made in the Urologic community to rigorously scrutinize focal therapy in men with prostate cancer.As previously stated, successfully adoption of focal therapy for the treatment of prostate cancer will hinge on two critical issues: 1) accurately identifying index lesions within the prostate, 2) reliably imaging cancers within the prostate, 3) long-term efficacy of the technology to eradicate cancer, 4) appropriate follow-up of patients treated with focal therapy, 5) limitations of PSA following therapy and how to detect recurrent/persistent disease. Solving these critical issues not only will assist with pretreatment counseling of these patients, but could be used as effective monitors for ideal end points in cancer therapy. In addition, optimal patient selection using pretreatment risk stratification must be developed to ensure the treatment of subjects who would gain the most out of focal therapy. Despite our limitations in identifying disease that can be treated by focal therapy, several clinical trials utilizing some of the promising therapeutic modalities mentioned above are on the horizon. Hopefully with careful monitoring and critical review of these trials we can better determine if focal therapy is an appropriate treatment option to offer patients with localized, low-grade prostate cancer.PSA: prostate specific antigen; 3D: 3 dimension; TRUS: transrectal ultrasound; MRI: magnetic resonance imaging; DCE: dynamic contrast-enhanced; MRSI: magnetic resonance spectroscopic imaging; DWI: diffusion-weighted imaging; SNR: signal-to-noise ratio; AUA: American Urological Association; ASTRO: American Society of Therapeutic Radiology and Oncology; HiFU: high-intensity focused ultrasound; FDA: Food and Drug Administration; DFS: disease free survival; VTP: Vascular targeted photodynamic therapy; TSE: turbo spin echo.The authors declare that they have no competing interests.RT gathered all data and wrote the manuscript. CJR developed and was responsible for overseeing the project.The pre-publication history for this paper can be accessed here: