Drosophila split ends (spen) gene encodes a large nuclear protein containing three RNP-type RNA binding motifs, and a conserved transcriptional co-repressor-interacting domain at the C-terminus. Genetic analyses indicate that spen interacts with pathways that regulate the function of Hox proteins, the response to various signaling cascades and cell cycle control. Although spen mutants affect only a small subset of morphological structures in embryos, it has been difficult to find a common theme in spen mutant structural alterations, or in the interactions of spen with known signaling pathways.The spen mutant cells in wing imaginal discs, we show that spen function is required for the correct formation and positioning of veins and mechanosensory bristles both on the anterior wing margin and on the notum, and for the maintenance of planar polarity. Wing vein phenotypic alterations are enhanced by mutations in the crinkled (ck) gene, encoding a non-conventional myosin, and correlate with an abnormal spatial expression of Delta, an early marker of vein formation in third instar wing imaginal discs. Positioning defects were also evident in the organization of the embryonic peripheral nervous system, accompanied by abnormal E-Cadherin expression in the epidermis.By generating clones of spen is necessary to maintain the correct positioning of cells within a pre-specified domain throughout development. Its requirement for epithelial planar polarity, its interaction with ck, and the abnormal E-Cadherin expression associated with spen mutations suggest that spen exerts its function by interacting with basic cellular mechanisms required to maintain multicellular organization in metazoans. This role for spen may explain why mutations in this gene interact with the outcome of multiple signaling pathways.The data presented indicate that the role of  Dfd encodes a Hox transcription factor that specifies maxillary segment identity during development [spen was subsequently found to enhance embryonic thoracic defects resulting from loss of function mutations in the Hox gene Antennapedia [Drosophila spen as modifiers of mutations in components of Ras/MAP kinase pathways, including Raf kinase [spen gene have also been identified as enhancers of gain of function phenotypes caused by overexpression of E2F or Cyclin E in eye cells [Dacapo, a cyclin dependent kinase inhibitor [spen function is required for the maternal expression of the Notch pathway transcription factor encoded by Suppressor of Hairless (Su(H)) [spen may participate in the transduction of the Wingless (Wg) signal within a subset of cells in the wing imaginal disc [The morphological complexity of metazoans is achieved through the regulation of multiple genes in an orchestrated spatial and temporal manner. One of these genes, osophila . Additioelopment . spen wannapedia . Other sf kinase , kinase f kinase , loss off kinase , and in f kinase ,8. Mutatye cells ,10, bothnhibitor . Overexpnhibitor , and spe (Su(H)) . Recent nal disc .spen results in isoforms encoding at least two proteins of ~5500 amino acids containing three tandem RNP-type RNA binding domains and a SPOC  domain at the carboxy terminus [osteocalcin promoter, via its RNP motifs, and repressing transcription in a binding complex with the homeodomain protein Msx-2 [-/- precursors reveals a defect in B cell development that could be attributed to defects in Notch signaling [The Spen protein is ubiquitously expressed throughout embryogenesis. Differential splicing of terminus . These din Msx-2 . The intin Msx-2 ,16. Bothin Msx-2 . Althougignaling .Drosophila remains unclear. Because wing development is a well characterized system for the study of primary pattern formation, diverse signaling pathways, and cell cycle control [spen mutant mosaics. An additional advantage is that, because wings are not essential for adult viability, the study of a large number of specimens is possible. In this study, we show that the function of spen is necessary for the maintenance of planar polarity, and for the correct formation and positioning of veins and mechanosensory bristles on the anterior wing margin and the notum. Alterations in vein formation in spen clones correlate with abnormal spatial expression of Delta, an early marker of vein formation in third instar wing imaginal discs. All wing phenotypes are enhanced in a crinkled (ck) mutant background, a gene encoding the non-canonical myosin VIIa. The abnormal position of sensory organs is also observed during embryonic PNS development. In contrast with previous reports, we show that spen is not essential for the determination of specific cell fates nor for cell survival, nor is it directly required for the outcome of the Ras, Notch, and Wingless signaling pathways. Based on our observations, we propose that spen is required for cells to maintain positional identity and tissue cohesiveness during the organized growth of wing and notum epithelia.Despite the sum of genetic and biochemical evidence, a selective role for Spen-like proteins in a particular pathway in mammals or  control ,20, we hspen has been shown to be involved in many processes during embryonic development. Mutant modifier and gain of function screens indicate that spen participates in a variety of developmental processes in Drosophila, including Hox gene function [spen functions through a common mechanism of action. To better understand a role for spen, we have generated genetic mosaics in adult tissues by using Flp1-mediated mitotic recombination [function ,3, cell function ,10 and tfunction -8, Notchfunction ,12, and function . Given tbination .spenpoc361 and spenpoc231 mutant alleles have been previously described [spenpoc361 is a null allele. First, in maternal and zygotic spenpoc361 mutant embryos, Spen protein cannot be detected with a polyclonal antibody raised against the region encoding amino acids 3203–3714 [[spenpoc361 allele displays nearly the same strength as the TE21A deficiency, which deletes the entire locus [The spen alleles were recombined onto chromosomes containing an FRT sequence at 40A [M(2)F 36FRTA 40chromosome. Heat shocks delivered at different times during larval and pupal development rendered few escapers (<1%), which presented with little or no mosaicism as revealed by the absence of the M dominant marker (not shown), indicating that the function of spen is essential during all stages of development.Both e at 40A , and werspen mRNA is expressed ubiquitously as revealed by RNA in situ hybridization (not shown). Using the MS1096-GAL4 driver [[UAS-Flp1 transgene [FRT chromosome bearing a spen mutant allele and an FRT chromosome containing a ubiquitously expressed GFP transgene on 2L. The homozygous viable 2piM FRTA 40chromosome [GFP transgene. In order to prevent the evaluation of non-specific abnormalities caused by the MS1096-GAL4 insertion on the X chromosome of hemizygous males, only heterozygous females were analyzed.Because the studies described above did not provide any information about the nature of this lethality, mosaics were generated through expression of the Flp1 recombinase in wing imaginal discs, where  driver [, Figure  driver [, the expransgene  induced romosome  was usedspen mutant cells was comparable in size to that obtained in the control crosses , as well as subtle mis-localization of both longitudinal and cross veins, frequently accompanied by thickening of the veins , or the UAS-Flp1 transgene, were independently present in a spen heterozygous background , a commonly used recessive marker on 2L. While the presence of ck homozygous clones had no effect on wing vein patterning and morphology . This process is followed by the initiation of vein formation, and finally, vein differentiation . The phed Figure . One gend Figure .2 piM, and the GFP FRT marker chromosomes, which confer nuclear GFP expression  ,25. The e margin ,27, and utations -13. As sn Figure , coincidspen mutant clones on the spacing of dorsal sensory bristles at the anterior wing margin was due to abnormal positioning or to a defect in the correct specification of sensory organ precursors. To determine if this effect could be generalized to other adult structures, expression of the Flp1 recombinase was directed to the anterior compartment of the wing disc with a dppDISC-GAL4 driver [spen mutant clones were generated throughout the anterior compartment of the wing disc, including most of the prospective notum (data not shown).It was unclear whether the effect of 4 driver . Althougspen mutant clones in the notum severely affected the final adult pattern of both the macro and microchaetae , whose loss of function results in holes in the epithelium. These holes, which later appear in the cuticle, presumably arise from a failure to re-establish a status quo at sites of high morphogenetic activity [Peripheral neurogenesis starts at the epidermis, where sensory organ precursors are specified, and give rise to sensory organ cells through carefully polarized cell divisions. Aside from these divisions, additional cell types might be recruited from the epidermis to take part in the formation of internal sensory organs [reviewed in ]. Increaactivity .spen mutant embryos die at the end of embryogenesis with sclerotic patches and holes in their epidermis in the ventrolateral thorax and lateral abdomen [[spen function display abnormal PNS neuron positioning and morphology compared to wild type as revealed by 22C10 staining . It is possible that, because the wing disc epithelium is not subject to the same level of stress as the embryonic epidermis at the sites of internal sensory organ formation, upregulation of E-Cadherin expression is specific to the embryonic phenotype, and does not occur in wing discs. Therefore, it remains unclear how spen could affect, if at all, cell adhesion in this tissue.Because E-cadherin is an important intercellular adhesion molecule, we wanted to test whether the defects seen in the developing adult wing were also associated with changes in its expression. Additionally, we assayed the expression of Crumbs, another component of the adherens junction, and β-tubulin, a marker of cell polarity. None of these proteins showed any detectable changes in expression or localization within spen mutant clones in adults, using heat-shock driven expression of FLP recombinase in a Minute background indicated that spen function was essential during all stages of development. This result is consistent with the pleiotropic effects previously described for spen. However, when clones were generated specifically in wing imaginal discs, we observed that mutations in the spen gene did not affect the viability or the size of mutant cells during growth of the disc, as evidenced by normal cell size and number within the adult clones. These observations are intriguing given previous reports linking the function of spen to cell cycle progression [spen mutations were shown to enhance morphological abnormalities upon overexpression of wild type E2F [spen has a negative regulatory role on these cell cycle regulators. If spen had a negative role in cell cycle progression in the wing imaginal disc, we would expect that spen mutant clones would be larger than their twin spots after mitotic recombination. However, other authors have observed that spen mutant clones are indeed smaller than their wild type counterparts [spen mutant cells would increase their size, in order to offset a decrease in cell division rates, as it is the case for E2F mutants [spen mutant and wild type cells. This observation is in agreement with our observations in spen maternal and zygotic mutant embryos, which do not show differences in cell cycle progression versus wt embryos as assessed by 8-Bromo deoxy-Uridine incorporation, string transcript expression, or Proliferating Cell Nuclear Antigen (PCNA) protein expression (data not shown). Alternatively, there is no evidence of increased cell death in spen mutant cells as revealed by Acridine Orange staining, or reaper mRNA expression (data not shown). Therefore, we conclude that the function of the spen gene per se is not essential for cellular viability and normal progression through the cell cycle.The generation of large gression ,10. In ttype E2F  and Cyclterparts . If that mutants . Howeverspen gene might be an integral component of the Ras signaling cascade. Such an interaction has been found in the search for genes interacting with a viable allele of corkscrew [kinase supressor of ras (ksr) [Drosophila provides an amenable system to analyze mutations affecting the MAPK pathway, as it depends on the function of the EGF receptor (DER), as well as other genes encoding components of the pathway, such as Star (S), rhomboid (rho), and the DER ligand, vein (vn) [rho and S function result in non-autonomous loss of vein phenotypes, gain of function of DER and rho have the opposite effect [Recently, there has been increasing experimental evidence suggesting that the product of the orkscrew , and in orkscrew , kinase as (ksr) , and a cas (ksr) . Wing veein (vn) ,31. While effect ,31,32.spen gene were an integral component of the Ras pathway, we would expect that the loss of function of spen would generate similar phenotypes as those obtained with other genes acting in the DER pathway. However, this was not the case: spen mutant clones generated during wing development showed both indiscriminate loss and gain of vein material, resulting in a vein phenotype that could not be directly correlated with any mutant in the Ras pathway known to date. Similarly, the phenotypes observed in spen mutant embryos do not clearly correlate with mutations in components of the Ras signaling pathway. For instance, the expression of orthodenticle (otd) mRNA at the ventral midline, which is dependent on DER function, and is abnormal in mutants that are defective in this signaling pathway [spen maternal and zygotic mutant embryos and wild type embryos (data not shown). Thus, our data do not support a direct correlation between the loss of spen function and a specific defect in DER/Ras-dependent signaling during embryogenesis or during imaginal disc development.If the product of the  pathway , was indspen and Notch (N) function has been previously suggested, where the former appears to be necessary to maintain the embryonic expression of Suppressor of Hairless (Su(H)) [spen may also interact with N signaling [spen function. Additionally, there is supporting biochemical evidence indicating that both the human and murine orthologs of Spen, interact with RBP-Jκ/CBF-1, a mammalian ortholog of Su(H) [versus B cell fates in the lymphoid lineage, also dependent on N signaling, appears unaffected [A relationship between  (Su(H))  a downst (Su(H)) . The obsignaling  further of Su(H) ,18. The affected . Thus, ispen mutant clones, frequently accompanied by widening of the veins, is a phenotype consistent with defects in the N signaling pathway [spen mutant clones, and in cells adjacent to these clones. However, the role of the N signaling pathway has been well established as crucial for the determination of cell types during the development of external sensory organs. Defects in the N pathway alter sense organ cellular composition by affecting alternative cell fate decisions [spen mutant clones did not interfere with bristle formation per se, but with their spatial distribution. Both micro and macrochaetae were incorrectly positioned throughout the notum and, in some cases, chaetae were absent, but ectopic supernumerary bristles were also seen. Nevertheless, all external sensory organs appeared to have normal morphology, suggesting that there were no mis-specifications of cell fates, as it would be expected for N signaling defects. Additional evidence that supports this is that the expression of Cut at the wing margin, which is dependent upon both Su(H) and N function [spen mutant clones. Therefore, we conclude that the function of spen is not essential for N signaling in wing imaginal discs.The displacement of veins observed in adult wings in the presence of  pathway . Furtherecisions ,37. In ofunction ,27, was spen is required to transduce some aspects of the Wingless (Wg) signal in the wing imaginal disc, showing a requirement for spen for the expression of senseless, a downstream target of the Wg pathway [spen mutant clones would be predicted to lead to the absence of external sensory organs in adult wings [spen mutant clones to known defects in the Wg signaling pathway. Clonal analysis in third instar imaginal discs did not reveal specific defects in the Wg pathway either: Wg signaling is required for Dl expression at the wing margin [spen mutant clones. Taken together, it appears that spen function is required for one Wg signaling target (Senseless) [spen alleles used, do not seem to support a principal role for Spen in Wg signaling during wing imaginal disc development.Recent evidence suggests that  pathway . The loslt wings , which ig margin , and Dl nseless) , but notspen in any signaling pathway in particular. We would like to propose that the common theme that could best define spen function at the morphological level is that it appears necessary for the correct spatial organization of individual cells within a specific group during growth and development.Based on previous reports and the data presented herein, the available evidence does not support a specific role for spen mutant embryos, the expression of E-cadherin was up-regulated at sites of high epithelial morphogenetic activity, generating a phenotype similar to the E-cadherin mutant embryos, as it has been observed in other studies [spen mutations [K. Mace, J. Pearson, and W. McGinnis, submitted]. A defect in cell adhesion and/or cytoskeletal rearrangements could also explain specific aspects of the spen embryonic phenotype. The holes that result in abnormal cuticle deposition in the embryonic epidermis are due to a failure of epidermal epithelial integrity. These cells subsequently undergo a wound response at the end of embryogenesis. Additionally, some of the phenotypes resulting from the loss of spen are indeed similar to those seen in mutants for the gene encoding Daschous, a cadherin involved in cell adhesion [spen mutant clones.How could Spen instruct cells to maintain a specific position, without affecting their fate directly? A plausible explanation is that it could affect cell adhesion. In fact, in  studies . It is padhesion . Howevercrinkled (Myosin VIIA), and the planar cell polarity phenotype observed in mutant cells for spen in the wing blade. Myosin VIIA is associated with the cadherin-catenins complex and participates in the creation of a tension force between the actin cytoskeleton and adherens junctions, which is predicted to strengthen cell-cell adhesion [ck acts downstream of Drosophila Rho-associated kinase (Drok), which links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton [shaker-1 mutation in mice [mariner mutation in zebrafish [spen may regulate the expression or function of components affecting the outcome of pathways involved in cytoskeletal rearrangements and epithelial planar polarity and, hence, affect cell positioning. However, a direct requirement for spen function in the Ck or Drok pathways is unlikely, since mutations in these genes result in different phenotypes than those observed in spen mutants.A role for Spen in cell adhesion and/or cytoskeletal rearrangements could also be inferred through its genetic interaction with adhesion . Furtherskeleton . Myosin skeleton , the sha in mice , and theebrafish . Interesspen in mechanisms of intercellular adhesion and/or cytoskeletal rearrangements may also be relevant to understanding its suggestive role in human cancer. The search of public human sequence resources [spen ortholog (SHARP), and three putative Short Spen-like Protein (SSLP) orthologs in the human genome O, Cy}, {MS1096-GAL4; spenpoc361, ck, FRTA40/In(2LR)O, Cy, wg-lacZ}, {MS1096-GAL4; spenpoc231, FRT A40/In(2LR)O, Cy, wg-lacZ}, {MS1096-GAL4; ck, FRTA40/In(2LR)O, Cy, wg-lacZ}, {MS1096-GAL4; 2 piM FRTA40/ In(2LR)O, Cy, wg-lacZ}, {ck FRTA40/ In(2LR)O, Cy; dppDISC-GAL4/TM6B}, {spenpoc361, ck FRT A40/In(2LR)O, Cy; dppDISC-GAL4/TM6B}, {spenpoc361, FRTA40/In(2LR)O, Cy; dppDISC-GAL4/TM6B}, {spenpoc231, FRTA40/In(2LR)O, Cy; dppDISC-GAL4/TM6B}, and {y, w; GFP(2L), FRTA40; UAS-Flp1/TM6B} were generated with the following lines: {spenpoc231/In(2LR)O, Cy, wg-lacZ}, {spenpoc361/In(2LR)O, Cy, wg-lacZ} [ck, FRTA 40/In(2LR)O, Cy} (obtained through recombination from the line {SX155, ck, FRT A40/In(2LR)O, Cy} [w*; P{wmC += UAS-Flp1.D}JD2/TM3, Sb1}, {w1118; P{wmC += Ubi-GFP(S65T)nls}2L P{ryt7.2 += neoFRTA40}/In(2LR)O, Cy}, {w1118; P{wmC += piM}21C P{wmC += piM}36F P{ryt7.2 += neoFRTA40}, {w1118; +; dppDISC-GAL4/In(3LR)TM6B} [MS1096-GAL4X} [hsFlp1; M(2)36F, FRTA40} was generated with {hsFlp1; nocSco/In(2LR)O, Cy}, {y, w; FRTA40}, and {M(2)36F/SM5}. Other stocks used were {ck14/In(2LR)O, Cy}, {ck16/ In(2LR)O, Cy}, and y, w*; P{wmC += UAS-GFP::lacZ.nls}15:3.All flies were grown at 21°C in standard medium, and were obtained from the Bloomington Stock Center, unless indicated otherwise. The stocks {LR)TM6B} , and {MS6-GAL4X} . The linovoD technique [Df (2L)TE21A/In(2LR)O, Cy, wg-lacZ} males, that carry a deletion spanning the spen locus, to obtain maternal and zygotic mutant embryos that were collected at 25°C.The echnique  was usedechnique . These fDrosophila Delta 594-9B monoclonal antibody (MAb) [To preserve GFP fluorescence, third instar imaginal discs were collected in cold PBS and fixed for 10–20 minutes on ice with methanol free 10% formaldehyde (Polysciences, Inc, Warrington, PA). After washing thoroughly with PBT (PBS with 0.1% Tween 20), and preincubating in the same buffer containing 10% Bovine Serum Albumin (BSA), the fixed discs were incubated with antibodies in PBT with 1% BSA. The mouse anti dy (MAb)  was useddy (MAb) , and antdy (MAb)  MAbs werdy (MAb)  was usedThe embryonic expression of the neuron-specific marker Elav was immunodetected as described  with theAdult flies were collected in 70% ethanol, and stored in isopropanol. Wings were detached from the dehydrated adults and mounted with DPX (Fluka, Buchs, Switzerland). Notums were dissected, embedded in Lactic Acid: Hoyers (1:2) , and phoDrosophila cDNA sequences were retrieved from the Ensembl Genome Server [spen related cDNAs in the human genome were analyzed by using GENESCAN [Human genomic, and human and e Server , and froe Server . Sequence Server . Composie Server  and Cluse Server . GenomicGENESCAN .DAB: Di amino benzidineGFP: Green Fluorescent ProteinHRP: Horseradish peroxidaseLacZ: β-galactosidaseMAb: Monoclonal antibodySSLP: Short Spen-like proteinspen mutant embryos, and examined the expression of cell adhesion and polarity markers in spen mutant clones in wing discs, most of which are results not shown. She also actively participated in the writing and the elaboration of the conclusions of this work. AT did the rest.KM performed the experiments shown in Figure


The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H A new method combines automated imaging with serial sectioning inside the chamber of a scanning electron microscope Many cellular structures are so small that they can only be resolved in the electron microscope. Furthermore, it is often crucial to visualize and reconstruct the three-dimensional (3D) structure of biological tissue. One prime example of where 3D information is indispensable is in exploring the connectivity of local networks of neurons. While axonal and dendritic processes have been traced using the light microscope from the very beginning of cellular neuroscience , light microscopic tracing is only possible if staining is restricted to a small subset of cells, as results, for example, from the Golgi method  or from Truly 3D information in the TEM can be obtained using either tilt-series-based tomography  or seriaCaenorhabditis elegans has been reconstructed in this way  can be used at higher accelerating voltages and is, therefore, compatible with BSE contrast. Since scattering of electrons is strongly dependent on the charge of the atomic nucleus, the BSE signal provides a clear distinction between heavy-metal-stained and unstained structures  and S1 tThese results show that we can obtain sufficient contrast from uncoated block faces. On this basis we decided to construct an ultra-microtome appropriate for sequential sectioning inside the sample chamber of an SEM. There are a number of special requirements for such a microtome. (1) The position of the block has to remain stable or be returned to the same location after each image is taken in order to ensure alignment of subsequent images, which is one of the primary objectives of serial block-face imaging. (2) The distance between the block face and the SEM objective lens has to be sufficiently small to allow high-resolution imaging and the efficient collection of backscattered electrons under low-vacuum conditions. (3) The block face has to be perpendicular to the optical axis of the electron optical column to keep the whole block face in focus; for all commercial SEM instruments this means horizontal orientation. (4) The operation of the microtome must be remotely controllable to allow automation. (5) The microtome has to be compatible with low vacuum. Finally, (6) the range of the fine advance mechanism has to be at least several hundred microns to permit continuous sectioning of volumes large enough to be of interest in the study of local neuronal circuits. Conditions 1, 2, and 3 rule out the modification of a conventional ultra-microtome, in which the sample block, its face oriented vertically, is moved against a stationary knife. Condition 2, in addition, rules out the use of a conventionally mounted diamond knife, since the boat would protrude upwards, colliding with the BSE detector and even with the objective lens.We, therefore, decided to construct a microtome de novo , which dPreparation of samples for the serial block-face SEM (SBFSEM) was done using procedures common for preparing samples for viewing in the TEM . When trying to cut thinner sections the actual thickness became uneven or the knife would skip every other cut, as recognized by the lack of change in structural detail. Another indication of uneven cutting is alternating brightness of the whole images or image regions. The reason for this is that the backscattering efficiency generally increases after beam exposure see C, which Usually the cut-off slices pile up on the top surface of the knife see B. Only rWe found, somewhat surprisingly, that cutting quality was often better in the area scanned by the electron beam than outside see C, presumSince lateral registration between successive slices is crucial for the applicability of the SBFSEM technique for automated 3D reconstruction, we quantified the registration using cross correlation. The depth resolution depends, of course, on the section thickness but also on the depth below the sample surface from which the BSE signal originates, which, in turn, scales with the electron penetration (see chapter 3 of E1.67). This means, for example, that at 4 keV the depth is reduced to about one-third of that at 7.5 keV. The actual value for the depth penetration is difficult to estimate for a composite material such as plastic-embedded heavy-metal-stained tissue. For a detailed discussion of the issues surrounding depth penetration see, for example, chapter 3 of Electron penetration depends strongly  on the eWe have shown that fully automated acquisition of truly 3D datasets at nanoscopic resolution is possible with serial block-face imaging in the SEM. The lateral resolution is sufficient to recognize most cellular organelles and synaptic specializations and does not appear to be limited by the beam spot diameter but by the spread of the beam in the sample . This spIt would be highly desirable to reliably cut sections thinner than 50 nm, which appears to be the current limit in our hands. The exact reason for this thickness limit is not clear as yet. Much thinner sections have been cut from plastic blocks , albeit In spite of the similarity in appearance, it is important to keep in mind a number of differences between transmission imaging of sections and backscattered SBFSEM. First, in the SEM, a large fraction of the entire beam energy is deposited inside the sample, leading to much increased radiation damage per primary electron. This is partly counteracted by the fact that BSE imaging is a “dark field” technique and, therefore, each detected electron carries more information. Furthermore, because the primary-electron energies can be lower by a large factor in the SEM (4 keV) than in the TEM , scattering cross sections are much larger in the SEM. This may, however, not always translate into improved contrast, since in the SEM only electrons scattered by angles larger than 90° can contribute to the image, while rather small deflections are enough to remove an electron from the beam in the TEM. A major practical advantage of SEM block-face imaging is that there is no need to manually handle sections, and no occlusion occurs by EM-grid bars. On the other hand, it is, unlike in TEM, impossible to reimage a section at higher resolution. Finally, it is unlikely that SEM surface imaging will ever reach the lateral resolution that can be achieved by imaging thin sections in the TEM.The total volume that can be reconstructed by SBFSEM is currently limited in the lateral dimension by the digital resolution available on commercial SEM instruments. It should, however, be straightforward to increase the digital resolution by modifying the scan and data-collection cir-cuitry. Eventually, off-axis electron-optical aberrations will become significant, but the total field of view can then be increased by mechanical translation of the microtome and tiling of multiple images. In the z-direction the stack height that can be cut continuously is, first, limited by the travel of the fine advance. Second, and more fundamentally, the cutting pyramid becomes unstable and too compliant if it is too high. However, a single step of image realignment after retrimming and repositioning will allow the continuation in the z-direction for almost unlimited distances.SN).(τ using the backscattering coefficient (η) as follows:A major practical limitation is, of course, the acquisition speed of the SEM, which is ultimately dependent on the required signal-to-noise ratio BI is the beam current and e is the elementary charge. The available beam current depends on the electron-gun type, the desired resolution, and the electron energy, but a value of BI = 1 nA is realistic for our purposes. We set η to 0.1, which is the value for carbon and thus a lower limit since backscattering is, of course, higher for the heavy-metal-stained areas  electron guns may be sufficient, we feel that the long-term stability of field-emission emitters is essential for imaging large volumes.where ed areas . To get The approach described here will speed up the collection of medium-to-high resolution 3D electron microscopic datasets by several orders of magnitude. The acquired data will, in addition, not require time-consuming and error-prone alignment and distortion correction. This should have wide-ranging applications not only in biomedical research but also in materials characterization . We are It is quite likely that scanning-probe methods such as atomic-force or near-field microscopy could be used instead of SEM to image the block face. It would be particularly interesting if multiple fluorescent dyes could be detected and discriminated in this way since they still provide unmatched specificity in labeling biological samples.All data shown were taken on a environmental SEM with a field-emission electron gun (QuantaFEG 200, FEI, Eindhoven, the Netherlands) mostly at an electron energy of 7.5 keV (exceptions as noted, see Muscle tissue was prepared as described in Reslicing of image stacks see  was doneThe microtome was constructed using mostly custom-machined parts made from nonmagnetic stainless steel. The leave springs were made out of bronze. The specimen advance contained a lever mechanism that reduced the motion of the motorized micrometer (M227.10 with controller C862, Physik Instrumente, Karlsruhe, Germany) by a factor of 0.11. This scaled the position uncertainty of the motorized micrometer (50 nm) down to a value 5.5 nm. Retraction of the sample during reverse motion of the diamond knife was driven by a closed-loop piezo element (P841.10, with controller E-610.S0, Physik Instrumente). The cutting motion was driven by a large-displacement piezo element (PX-1500, Piezojena, Jena, Germany). Suspension of the microtome on steel balls sliding on sapphire plates allowed lateral positioning driven by piezo-actuated slip-stick motors . The microtome was controlled using a computer interface designed originally for electrophysiology applications (1401 power, CED, Cambridge, United Kingdom) using scripts written in the Spike2 programming environment (CED). Analog voltages that were generated by the computer interface drove the cutting and the retraction piezos. The specimen-advance motor was controlled via serial interface. During automated stack acquisition the QuantaFEG 200 microscope was controlled using a modified keyboard that allowed simulated key pressings via a serial interface. Both serial interface connections were driven by commands in Spike2 software scripts.The diamond knife used was custom made by Diatome, derived from their ultra 35° type. The cutting edge was 1.5 mm long. Unlike in the standard knife, in which the diamond is soldered to a piece of hard metal—increasing the clearance height necessary above the cutting edge and thereby increasing the working distance—our diamond was directly clamped in a custom-made stainless-steel holder. The clearance angle was fixed at 6°. The section thickness values quoted were calculated using the nominal micrometer position change and the mechanical reduction ratio of the advance mechanism. For some of the data  the knifDataset S1(248.1 MB ZIP).Click here for additional data file.Dataset S2(252.6 MB ZIP).Click here for additional data file.Dataset S3(252.7 MB ZIP).Click here for additional data file.Dataset S4(252.6 MB ZIP).Click here for additional data file.Dataset S5(251.9 MB ZIP).Click here for additional data file.Dataset S6(252.2 MB ZIP).Click here for additional data file.Dataset S7(253.7 MB ZIP).Click here for additional data file.Dataset S8(255.9 MB ZIP).Click here for additional data file.Dataset S9(256.1 MB ZIP).Click here for additional data file.Dataset S10(253.8 MB ZIP).Click here for additional data file.Dataset S11(252.6 MB ZIP).Click here for additional data file.Dataset S12(252.6 MB ZIP).Click here for additional data file.Dataset S13(251.9 MB ZIP).Click here for additional data file.Dataset S14(251.8 MB ZIP).Click here for additional data file.Dataset S15(250.7 MB ZIP).Click here for additional data file.Dataset S16(251.4 MB ZIP).Click here for additional data file.Dataset S17(252.7 MB ZIP).Click here for additional data file.Dataset S18(250.5 MB ZIP).Click here for additional data file.Dataset S19(253.4 MB ZIP).Click here for additional data file.Dataset S20(254.9 MB ZIP).Click here for additional data file.Figure S1Complete field of view for dataset underlying (10.8 MB TIF).Click here for additional data file.Figure S2Bottom slice of a stack of 2,000 images taken at 4 keV. Slice thickness was 55 nm. Spotsize was 3.4. Pixel size is 26.7 nm. The pixels correspond to the original data. The area shown corresponds to 54.8 × 47.3 μm. The raw data can be found as numbered TIF images in (10.6 MB TIF).Click here for additional data file.Figure S3Same volume data as used for (8.2 MB TIF).Click here for additional data file.Figure S4Same as (7.1 MB TIF).Click here for additional data file.Figure S5Manual reconstruction of selected processes in cortical tissue (data from (17.5 MB TIF).Click here for additional data file.


PLoS Medicine suggests that oats are not safe in all cases.Most patients with celiac disease can eliminate their symptoms—at a price: life-long adherence to a gluten-free diet. This means no wheat, rye, barley, and, until recently, no oats. Then some recent studies suggested that oats did not cause the intestinal inflammation characteristic of the disease, and thus oats are now often included in the celiac disease diet. This is good news for patients coping with severe restrictions on what they can and must not eat, but a study by Ludvig Sollid and colleagues in this issue of Like other chronic inflammatory diseases, celiac disease is caused by a complex interplay between genetic and environmental factors, but it is better understood than most. Long believed to be a relatively rare disorder, it is now thought to affect about one in 250 people worldwide. Clinical symptoms are present in less than half of patients and vary considerably. Genetically, almost all patients have one of two predisposing HLA molecules, which determine the context in which their immune system encounters foreign antigens, including gluten proteins found in wheat and other cereals. In individuals with celiac disease, the immune system mounts an abnormal response to gluten, which is characterized by gluten-reactive intestinal T cells and by inflammation and compromised function of the small intestine.Ludvig Sollid and colleagues applied the current understanding of celiac disease and a range of molecular pathology tools to studying the response to oats of nine patients with celiac disease. The nine patients were not a random sample: all of them had been eating oats, and four of them had shown clinical symptoms after oats ingestion. The goal of the study was to characterize the intestinal T cell response to oats in these patients, and to relate it to clinical symptoms and intestinal biopsy results. All patients were on a gluten-free diet and ate oats that were free of contamination by other cereals.Three of the four patients who had reported problems after eating oats showed intestinal inflammation typical of celiac disease, and Sollid and colleagues studied intestinal T cells from these three patients. Two of the five patients who seemed to tolerate oats also had oats-reactive intestinal T cells. Functional study of these T cells showed that they were restricted to celiac-disease-associated HLA molecules and that they recognized two peptides derived from oat avenin that are very similar to peptides of gluten.Taken together, the findings show that intolerance to oats exists at least in some patients with celiac disease, and that those patients have the same molecular reaction to oats that other patients have to wheat, barley, or rye. However, identical reactions were also seen in two of the patients who were clinically tolerant to oats. The authors suggest that these reactions could develop into symptomatic disease after some time delay, but there is no proof that the presence of oats-reactive T cells is an indicator of future symptoms or even of enhanced susceptibility to clinical oats intolerance.Oats are not safe for all patients with celiac disease, but future studies are needed to determine the frequency of oats intolerance.


It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole – time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects.This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined. The protein constituents (proteome) of cerebrospinal fluid (CSF) are altered in disease states such as meningitis, but may also be more subtly altered in many other neural conditions. CSF has been difficult to investigate because of the need for invasive lumbar punctures and the small volumes of CSF available for analysis. This situation is now rapidly changing as methods requiring microliter volumes and sophisticated analysis tools such as proteomics become available ,2. ProteAphrodite aculeate intracellular globin (30% identify) [One such protein is neuroglobin. Neuroglobin is a recently identified member of the globin family. It binds oxygen with an affinity between that of myoglobin and hemoglobin ,4. Neurodentify)  than to r) for each peptide. The numbers of matching peptides were 5 in sample #1, 2 in sample #2, and 3 in sample #3. The peptides mapped 31.1%, 13.9% and 17.2%, respectively, of the total neuroglobin protein sequence. They matched amino acids 1–10 and 15–30 of the N-terminal and 131–151 of the C-terminal. Trypsin digestion missed cleavage sites at amino acids 18 and 146. Reproducibility was demonstrated by the consistent retention times for the same peptides from different subjects. No low abundance neuroglobin peptides were found in other samples. BLAST sequence analysis of all six peptides identified only one protein: hypothetical 16.9 kDa protein .Five peptides derived from neuroglobin (NCBInr ID accession 10864065) were identified using both the MASCOT software with NCBInr database and ProteinLynx Global Server with SwissProt database  in a new globin subfamily that forms hexacoordinated heme iron complexes ,13. ThesThe source of neuroglobin in the CSF is likely to be brain regions such as the subthalamic nuclei , frontal lobe, thalamus, occipital pole, pituitary gland, and medulla oblongata ,14. ImmuNeuroglobin mRNA was also present in adrenal cells and the β cells of the pancreatic islets of Langerhans . Roles iNeuroglobin is likely to serve as an intracellular oxygen depot to facilitate oxygen diffusion to the mitochondria. A role in oxygen supply was supported by the very high expression of neuroglobin mRNA in retinal neurons but not the supporting ocular epithelium and other structures . RetinalNeuroglobin might act in certain circumstances to limit neural cellular damage during hypoxia. Neuroglobin expression was inversely correlated to the sensitivity of the brain regions to ischemia . For exa2+-E7His) and displace the protein from the globin [Plasmodium and Trypanosoma from the antiparasitic effects of nitric oxide [Neuroglobin was also colocalized with nitric oxide synthase in the lateral tegmental nuclei, stria terminalis, habencule, nucleus of the tractus solitarius, periaqueductal grey matter, amygdala and subfornic organ . The proe globin . This afic oxide . Neuroglic oxide .In this study, neuroglobin was qualitatively identified in CSF from 3 female subjects with chronic pain conditions. Females have greater pain sensitivity to pressure and other stimuli (lower pain thresholds) , but paiIt is tempting to speculate that the source of neuroglobin in our samples was from nuclei involved in pain transmission or regulation such as the thalamus, prefrontal cortex, amygdala, or spinal cord dorsal horn somatic pain synaptic regions (e.g. layers 1 and 2 of Rexed). The fact that neuroglobin was not detected in any of the control females in our study makes it unlikely that the expression was related to gender. Examination of additional normal and chronic pain subjects is underway to determine the factors that may be responsible for neuroglobin expression. It is also possible that the proteomic detection of neuroglobin varies depending upon sample preparation, signal-to-noise ratio for relatively low abundance proteins compared to albumin and immunoglobulins that are present in high abundance, duration of storage, factors related to trypsin digestion, capillary liquid chromatography, mass spectrometry or bioinformatic neuroglobin peptide detection. These technical factors are unlikely to be significant since our samples were treated identically and were stored for approximately equal amounts of time.In contrast to neuroglobin's localization, cytoglobin-immunoreactive material was localized to the cellular nucleus in all tissues examined . MammaliThis is the first description of neuroglobin in cerebrospinal fluid and in humans. Neuroglobin was identified in 3 of 9 female pain subjects. The role(s) for this ancient oxygen and nitric oxide binding protein in humans, and potential links to pain, remain to be fully determined.After obtaining informed consent, lumbar punctures were performed on 17 subjects as part of an evaluation of pain mechanisms. Twelve patients had musculoskeletal pain and five were healthy control subjects. Cerebrospinal fluid samples were aliquoted and frozen at -70°C. Lipids and peptides were extracted from 200 μl of thawed CSF by adding an equal volume of 50% ethanol, 50% 0.2 N acetic acid 0.02% sodium bisulfite ("acid-ethanol") . Centrif and NCBInr protein database [) [The column eluate was pumped through a nanoelectrospray interface into a quadrapole – time of flight (Q-TOF-2, Waters, Milford, MA) mass spectrometer. MASSLYNX version 3.5 software was used to control the CapLC and Q-ToF-2, data acquisition, processing, and determination of peptide sequences. The protein identification was performed with the MASCOT MS/MS ion search software database ,8, and wabase [) ,10, protSamples were assessed by CapLC-Q-ToF-2 in triplicate. At least 2 separate peptides from neuroglobin had to identify in each individual sample to ensure that this protein, and not a related protein, was present. In an attempt to detect low abundance expression of neuroglobin peptide ions that were not selected by MS-MS , all MS data from the appropriate CapLC retention times were reassessed at high resolution. Positive results (MS data) were checked to see if ions in MS were present but not in MS-MS for other pieces. All putative neuroglobin peptide spectra were sequenced using PepSeq (Waters) and confirmed by visual inspection.The author(s) declare that they have no competing interests.1,4, Sample preparation, chromatography, mass spectrometry and manuscript preparationCasado B 2, Supervision and assistance with chromatography and mass spectrometry and manuscript preparationPannell LK 1, Sample preparationWhalen G 3, Clinical investigation of subjectsClauw DJ 1,*, Organization of study and selection of samples, preparation of manuscriptBaraniuk JN


Cryptosporidium parvum genes of likely endosymbiont or prokaryotic origin supports the hypothesis that C. arvum evolved from a plastid-containing lineage.An analysis of  Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium.The apicomplexan parasite Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum.We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite. Cryptosporidium is a member of the Apicomplexa, a eukaryotic phylum that includes several important parasitic pathogens such as Plasmodium, Toxoplasma, Eimeria and Theileria. As an emerging pathogen in humans and other animals, Cryptosporidium often causes fever, diarrhea, anorexia and other complications. Although cryptosporidial infection is often self-limiting, it can be persistent and fatal for immunocompromised individuals. So far, no effective treatment is available [Cryptosporidium continues to be a security concern as a potential water-borne bioterrorism agent [vailable . Furthersm agent .Cryptosporidium is phylogenetically quite distant from the hemosporidian and coccidian apicomplexans [Cryptosporidium contained a plastid, even though many of its descendants (for example, the ciliates) appear to lack a plastid. Although indirect evidence has been noted for the past existence of an apicoplast in C. parvum [mplexans  and, depmplexans ,5. It ismplexans ,7. The amplexans . All thamplexans . The apimplexans ,11. In amplexans . Plastidmplexans -17. Amonmplexans ,18. If t. parvum ,20, no rCryptosporidium is thus of evolutionary and practical importance.Gene transfers, either intracellular (IGT) from an endosymbiont or organelle to the host nucleus or horizontal (HGT) between species, can dramatically alter the biochemical repertoire of host organisms and potentially create structural or functional novelties -23. In pC. parvum ) [C. parvum. The fate of several of these transferred genes in C. parvum is explored by expression analyses. The significance of our findings and their impact on the genetic makeup of the parasite are discussed.In this study, we use a phylogenomic approach to mine the recently sequenced genome of es (Mb))  for evidCryptosporidium, like that of Plasmodium falciparum [Arabidopsis and Oryza than to any other non-apicomplexan organism currently represented in GenBank. The program Glimmer predicted 5,519 protein-coding sequences in the C. parvum genome, 4,320 of which had similarity to other sequences deposited in the GenBank nonredundant protein database. A significant number of these sequences, 936  or 783 , had their most significant, non-apicomplexan, similarity to a sequence isolated from plants, algae, eubacteria (including cyanobacteria) or archaea  were used as input for phylogenetic analyses using the PyPhy program [The Glimmer-predicted protein-coding regions of the  program . In thisC. parvum predicted gene set with the 551 P. falciparum predicted nuclear-encoded apicoplast-targeted proteins (NEAPs) yielded 40 significant hits , 23 of which were also identified in the phylogenomic analyses. A combination of these two approaches identified 410 candidates requiring further detailed analyses. Of these candidates, the majority were eliminated after stringent criteria were applied because of ambiguous tree topologies, insufficient taxonomic sampling, lack of bootstrap support or the presence of clear vertical eukaryotic ancestry . Thirty-one genes survived the screen and were deemed to be either strong or likely candidates for gene transfer . Two experiments were performed. In the first, nearly complete genomic sequences (generated in a different laboratory) from the closely related species C. hominis were screened using BLASTN for the existence of the predicted genes. Twenty out of 21 C. parvum sequences were identified in C. hominis. The remaining sequence was represented by two independently isolated expressed sequence tag (EST) sequences in the GenBank and CryptoDB databases (data not shown). In the second experiment, genomic Southern analyses of the IOWA isolate were carried out , as confirmed by similarity searches against ApiDB . In P. falciparum, leucine aminopeptidase is a predicted NEAP and possesses an amino-terminal extension with a putative transit peptide. Consistent with the lack of an apicoplast, this gene in Cryptosporidium contains no evidence of a signal peptide and the amino-terminal extension is reduced. Similarity searches of the GenBank nonredundant protein database revealed top hits to Plasmodium, followed by Arabidopsis thaliana, and several cyanobacteria including Prochlorococcus, Nostoc and Trichodesmium, and plant chloroplast precursors in Lycopersicon esculentum and Solanum tuberosum (data not shown). A multiple sequence alignment of the predicted protein sequences of leucine aminopeptidase reveals overall similarity and a shared indel among apicomplexan, plant and cyanobacterial sequences  , plants (Arabidopsis, Oryza), cyanobacteria (Trichodesmium, Nostoc and Synechocystis), a ciliate (Tetrahymena) and the kinetoplastids (Leishmania and Trypanosoma). Arabidopsis thaliana apparently contains at least two copies of this gene; the protein of one (accession number NP_565734) is predicted by ChloroP [Another 1) Table . Similar ChloroP  to be chP. falciparum, T. gondii [Giardia intestinalis and Spironucleus and the parabasalid Trichomonas vaginalis [Giardia, Spironucleus and Trichomonas group with the cyanobacterium Nostoc, as previously published. In the midsection of the tree, the G6PI of apicomplexans and ciliates forms a well-supported monophyletic group with the plants and the heterokont Phytophthora. The multiple protein sequence alignment of G6PI identifies several conserved positions shared exclusively by apicomplexans, Tetrahymena, plants and Phytophthora. This gene does not contain a signal or transit peptide and is not predicted to be targeted to the apicoplast in P. falciparum. The remainder of the tree shows a weakly supported branch including eubacteria, fungi and several eukaryotes. The eukaryotes are interrupted by the inclusion of G6PI from the eubacterial organisms Escherichia coli and Cytophaga. This relationship of E. coli G6PI and eukaryotic G6PI has been observed before and may represent yet another gene transfer [Only one gene of algal nuclear origin, glucose-6-phosphate isomerase (G6PI), was identified by the screen described here. Several other algal-like genes are probable, but their support was weaker Table . A 'plan. gondii ) and a 'aginalis . Figure transfer .Our study identified HGTs from several distinct sources, involving a variety of biochemical activities and metabolic pathways Table . NotablyC. parvum is tryptophan synthetase β subunit (trpB). This gene has been identified in both C. parvum and C. hominis, but not in other apicomplexans. The relationship of C. parvum trpB to proteobacterial sequences is well-supported as a monophyletic group by two of the three methods used in our analyses  monophyletic grouping with eubacterial homologs (data not shown). The eubacterial origin of 1,4-α-glucan branching enzyme is shown in Figure T. gondii. It is possible that these genes are of plastidic origin and were transferred to the nuclear genome before the divergence of C. parvum and T. gondii; the phylogenetic analysis provides little direct support for this interpretation, however.Other HGTs of eubacterial origin include the genes encoding α-amylase and glutamine synthetase and two copies of 1,4-α-glucan branching enzyme, all of which are overwhelmingly similar to eubacterial sequences. α-amylase shows no significant hit to any other apicomplexan or eukaryotic sequence, suggesting a unique HGT from eubacteria to trpB gene and the gene for aspartate ammonia ligase are located 4,881 base-pairs (bp) apart on the same strand of a contig for chromosome V; there is no annotated gene between these two genes. Both genes are of eubacterial origin and are not found in other apicomplexan organisms. While it is possible that they have been acquired independently with this positioning, or later came to have this positioning via genome rearrangements, it is interesting to speculate that these genes were acquired together. The origin of trpB is proteobacterial. The origin of aspartate ammonia ligase is eubacterial, but not definitively of any particular lineage. In the absence of genome sequences for all organisms, throughout all of time, exact donors are extremely difficult to assess and inferences must be drawn from sequences that appear to be closely related to the actual donor.We examined the transferred genes for evidence of non-independent acquisition, for example, blocks of transferred genes or evidence that genes were acquired together from the same source. Examination of the chromosomal location of the genes listed in Table C. parvum encodes two genes for 1,4-α-glucan branching enzymes. Both are eubacterial in origin and both are located on chromosome VI, although not close together. They are approximately 110 kb apart and many intervening genes are present. The evidence that these genes were acquired together comes from the phylogenetic analysis presented in Figure C. parvum and T. gondii represent one copy each of this ancient duplication. This suggests that the ancestor of C. parvum and T. gondii acquired the genes after they had duplicated and diverged in eubacteria.In the second case, C. parvum-infected HCT-8 cells was shown to be free of contaminating C. parvum genomic DNA by the lack of amplification product from a reverse transcriptase reaction sham control. RT-PCR detected no signals in cDNA samples from mock-infected HCT-8 cells. On the other hand, RT-PCR product signals were detected in the C. parvum-infected cells of six independent time-course experiments for each of the genes examined ). The expression profiles of the acquired genes show that they are regulated and differentially expressed throughout the life cycle of C. parvum in patterns characteristic of other non-transferred genes , gene trees obtained from automated PyPhy analyses indicate a strong monophyletic grouping of the C. parvum gene with plant or eubacterial homologs, but this topology disappears when sequences from other unicellular eukaryotes, such as Dictyostelium, Entamoeba and Trichomonas are included in the analysis (data not shown).The limitations of similarity searches and incomplete taxonomic sampling are well evidenced in our phylogenomic analyses. From similarity searches, lciparum , is moreia Table . In manyC. parvum instead of a definitive list. As genomic data are obtained from a greater diversity of unicellular eukaryotes and eubacteria, phylogenetic analyses of nearest neighbors are likely to change.The list of genes in Table C. parvum sequences of cyanobacterial and algal origin reported here had to enter the genome at some point during its evolution. Formal possibilities include vertical inheritance from a plastid-containing chromalveolate ancestor, HGT from the cyanobacterial and algal sources (or from a secondary source such as a plastid-containing apicomplexan), or IGT from an endosymbiont/plastid organelle during evolution, followed by loss of the source. Cryptosporidium does not harbor an apicoplast organelle or any trace of a plastid genome [Cryptosporidium or the lineage giving rise to it. The exact position of C. parvum on the tree of life has been debated, with developmental and morphological considerations placing it within the Apicomplexa, and molecular analyses locating it in various positions, both within and outside the Apicomplexa [C. parvum is an apicomplexan, and if the secondary endosymbiosis which is believed to have given rise to the apicoplast occurred before the formation of the Apicomplexa, as has been suggested [C. parvum would have evolved from a plastid-containing lineage and would be expected to harbor traces of this relationship in its nuclear genome. Genes of likely cyanobacterial and algal/plant origin are detected in the nuclear genome of C. parvum  provides the best evidence against this scenario as multiple independent transfers would be required and so far there is no evidence for intra-alveolate gene transfer. Vertical inheritance is more difficult to address as it involves distinguishing between genes acquired via IGT from a primary endosymbiotic event versus a secondary endosymbioic event. Our data, especially the analysis of G6PI and BT-1 are consistent with both primary and secondary endosymbioses, provided that the secondary endosymbiosis is pre-alveolate in origin. As more genome data become available and flanking genes can be examined for each gene in a larger context, positional information will be informative in distinguishing among the alternatives.What about other interpretations? While it is formally possible that these genes were acquired independently via HGT in C. parvum leucine aminopeptidase does contain an amino-terminal extension of approximately 85-65 amino acids  relative to bacterial homologs, but this extension does not contain a signal sequence. The extension in P. falciparum is 85 amino acids and the protein is believed to be targeted to the apicoplast [C. parvum and P. falciparum amino-terminal extensions (data not shown).The plastidic nature of some genes is particularly apparent. There is a shared indel among leucine aminopeptidase protein sequences in apicomplexans, cyanobacteria and plant chloroplast precursors Figure . The C. icoplast ,37. No sP. falciparum [Trypanosoma [C. parvum and P. falciparum, with an identity of 60%. In our phylogenetic analyses, C. parvum clearly forms a monophyletic group with Plasmodium, Toxoplasma and Eimeria. This branch groups with Dictyostelium, Kinetoplastida and cyanobacterial lineages, but bootstrap support is not significant. The sister group to the above organisms are the plants and additional cyanobacteria, but again with no bootstrap support . Another gene, enolase, contains two indels shared between land plants and apicomplexans (including C. parvum) and was suggested to be a plant-like gene [Other genes were less informative in this analysis. Among these, aldolase was reported in both lciparum  and the panosoma  as a plaike gene , but altike gene .C. parvum [Cryptosporidium genome sequences deposited in CryptoDB using plant (Cucumis sativa, GenBank accession number AAP36992), cyanobacterial  and other bacterial (Yersinia pestis, NP-404547) homologs.The biochemical activity of the polyamine biosynthetic enzyme arginine decarboxylase (ADC), which is typically found in plants and bacteria, was previously reported in . parvum . HoweverC. parvum [C. parvum genome sequence , trpB and the gene for aspartate ammonia ligase).Several HGTs from bacteria have been reported previously in . parvum ,32,40. WC. parvum which does not contain a typical mitochondrion [The endosymbiotic event that gave rise to the mitochondrion occurred very early in eukaryotic evolution and is associated with significant IGT. However, most of these transfer events happened long before the evolutionary time window we explored in this study . Many IGhondrion ,42-44) aC. parvum are known to be expressed. IMPDH has been shown to be essential in C. parvum purine metabolism [C. parvum no longer possesses the ability to synthesize nucleotides; instead it relies entirely on salvage.Gene transfer is an important evolutionary force ,22,45,46tabolism  and TK htabolism . It is nC. parvum, acquired plastidic proteins are theoretically destined to go elsewhere. In the absence of an apicoplast, it is tempting to suspect that plastid-targeted proteins would have been lost, or would be detected as pseudogenes. No identifiable pseudogenes were detected and at least one gene is still viable. The C. parvum leucine aminopeptidase, which still contains an amino-terminal extension , is intact and is expressed, as shown in Figure C. parvum genome. One copy has a long amino-terminal extension (97 amino acids) beginning with a signal peptide. The extension does not contain characteristics of a transit peptide. Expression of a fluorescent reporter construct containing this extension in a related parasite, T. gondii, did not reveal apicoplast targeting but instead secretion via dense granules . Exactly how and where intracellularly transferred genes  have become incorporated into other metabolic processes remains a fertile area for exploration.Many apicoplast and algal nuclear genes have been transferred to the host nuclear genome, where they were subsequently translated in the cytosol and their proteins targeted to the apicoplast organelle. However, as there is no apicoplast in Cryptosporidium is the recipient of a large number (31) of transferred genes, many of which are not shared by other apicomplexan parasites. The genes have been acquired from several different sources including α-, β-, and ε-proteobacteria, cyanobacteria, algae/plants and possibly the Archaea. We have described two cases of two genes that appear to have been acquired together from a eubacterial source: trpB and the aspartate ammonia ligase gene are located within 5 kb of each other, while the two copies of 1,4-α-glucan branching enzyme represent copies of an ancient gene duplication also observed in cyanobacteria.Arabidopsis genome [Giardia [Trypanosoma [Entamoeba [Euglena [Cryptosporidium [Once thought to be a relatively rare event, reports of gene transfers in eukaryotes are increasingly common. The abundance of available eukaryotic genome sequence is providing the material for analyses that were not possible only a few years ago. Analysis of the s genome  has reve[Giardia ,49, Tryppanosoma , Entamoentamoeba ,49, Eugl[Euglena , Cryptosporidium ,32,40 anporidium .C. parvum genome, and in two cases (IMPDH and TK), the transferred genes have been shown to be functional [Cryptosporidium genome has changed the genetic and metabolic repertoire of the parasite.As discussed earlier, genes transferred from distant phylogenetic sources such as eubacteria could be potential therapeutic targets. In apicomplexans, transferred genes are already some of the most promising targets of anti-parasitic drugs and vaccines ,25,52. Wnctional . The sucC. parvum and C. hominis were downloaded from CryptoDB [C. parvum (IOWA) sequence as previously described using the Glimmer program [Cryptosporidium coding sequences [C. parvum annotation did not consider ORFs of less than 100 amino acids that did not have significant BLAST hits and thus may be a slight underestimate [Genomic sequences for CryptoDB . Genes w program  trained equences . A few pequences ,20; thusestimate .C. parvum, ORFs of more than 120 amino acids from Theileria annulata, and more than 75 amino acids from consensus ESTs for several apicomplexan organisms. Genomic sequences for T. gondii (8x coverage) and clustered ESTs were downloaded from ToxoDB [Entamoeba histolytica [D. discoideum [Leishmania major [T. brucei [T. cruzi [Tetrahymena thermophila [T. annulata, E. histolytica, D. discoideum, L. major and T. brucei were produced by the Pathogen Sequencing Unit of the Sanger Institute and can be obtained from [T. thermophila was obtained from TIGR and can be accessed at [An internal database (ApiDB) containing all available apicomplexan sequence data was created . A seconm ToxoDB ,56. Genom ToxoDB  and extetolytica , D. discscoideum , the kinia major , T. bruc. brucei , T. cruzT. cruzi , and a crmophila . Sequencned from . Prelimiessed at .The source code of the phylogenomic software PyPhy  was kindC. parvum gene does not form a monophyletic group with prokaryotic or plant-related taxa regardless of rooting, the subject gene was eliminated from further consideration. If the topology of the gene tree is consistent with a phylogenetic anomaly caused by gene transfer, but may also be interpreted differently if the tree is rooted otherwise, it was removed from consideration at this time. If the top hits of both nr and dbEST database searches are predominantly non-plant eukaryotes, and the topology of the tree was poor, the subject gene was considered an unlikely candidate. Finally, all 551 protein sequences predicted to be NEAPs in the malarial parasite P. falciparum [C. parvum genome and the results were screened using a BLAST cutoff E-value of 10-5 and a length coverage of 50%. Sequences identified by these searches were added to the candidate list  for manual phylogenetic analyses to verify their likely origins. It should be noted that all trees were screened for the existence of a particular phylogenetic relationship. In some cases the proteins utilized to generate a particular tree are capable of resolving relationships among many branches of the tree of life, and in others they are not. Despite these differences in resolving power, the proteins which survive our phylogenetic screen and subsequent detailed analyses described below exhibit significant support for the branches of the tree in which we are interested. Similar procedures were used to characterize the complement of nuclear-encoded genes of plastid origin in the Arabidopsis genome [Output gene trees with phylogenetic connections (that is, the nearest non-self neighbors at a distinct taxonomic rank)  to prokalciparum  were uses genome . BLAST ss genome .Detailed phylogenetic analyses of candidate genes identified by phylogenomic screening: candidate genes surviving the PyPhy phylogenomic screen were reanalyzed with careful attention to taxonomic completeness, including representative species from major prokaryotic and eukaryotic lineages when necessary and possible. New multiple sequence alignments were created with ClustalX , followeC. parvum (IOWA) oocysts (108) were obtained from the Sterling Parasitology Laboratory at the University of Arizona and were lysed using a freeze/thaw method. Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen). Genomic DNA (5 μg) was restricted with BamH1 and EcoR1 respectively and electrophoresed on a 0.8% gel in 1x TAE buffer, transferred to a positively charged nylon membrane (Bio-Rad), and fixed using a UVP crosslinker set at 125 mJ as described in [C. parvum genomic DNA for the probes (700-1,500 bp) was amplified by PCR .ribed in . C. parvC. parvum (IOWA isolate) oocysts were used to infect confluent human adenocarcinoma cell monolayers at a concentration of one oocyst per cell as previously described [C. parvum-infected HCT-8 cultures at 2, 6, 12, 24, 36, 48 and 72 h post-inoculation by directly lysing the cells with 4 ml TRIzol reagent (GIBCO-BRL/Life Technologies). Purified RNA was resuspended in RNAse-free water and the integrity of the samples was confirmed by gel electrophoresis.Sterilized escribed . Total RC. parvum 18S rRNA were used to normalize the amount of cDNA product of the candidate gene to that of C. parvum rRNA in the same sample. PCR products were separated on a 4% non-denaturing polyacrylamide gel and signals from specific products were captured and quantified using a phosphorimaging system (Molecular Dynamics). The expression level of each gene at each time point was calculated as the ratio of its RT-PCR product signal to that of the C. parvum 18S rRNA. Six independent time-course experiments were used in the analysis.Primers specific for several transferred genes identified in the study were designed  and a semi-quantitative RT-PCR analysis was carried out as previously described . PrimersC. parvum phosphoglucomutase in T. gondii; table of primers used for RT-PCR experiments; phylogenetic tree of aldolase; alignment files for phylogenetic analyses in Figure Additional data is provided with the online version of this paper, consisting of a PDF file (Additional data file C. parvum phosphoglucomutase in T. gondii; table of primers used for RT-PCR experiments; phylogenetic tree of aldolase; alignment files for phylogenetic analyses in Figure 4; and the alignment of 1,4-α-glucan branching enzyme sequences used in Figure 5This file contains the materials and methods for genomic Southern analysis; the amino-acid sequences of genes listed in Table 2; accession numbers for sequences used in Figure 4; accession numbers for sequences used in Figure 5; expression of Click here for additional data file


Quantitative studies are becoming more recognized as important to understanding health care with all of its richness and complexities. The purpose of this descriptive survey was to provide a quantitative evaluation of the qualitative studies published in 170 core clinical journals for 2000.All identified studies that used qualitative methods were reviewed to ascertain which clinical journals publish qualitative studies and to extract research methods, content , and whether mixed methods  were used.60 330 articles were reviewed. 355 reports of original qualitative studies and 12 systematic review articles were identified in 48 journals. Most of the journals were in the discipline of nursing. Only 4 of the most highly cited health care journals, based on ISI Science Citation Index (SCI) Impact Factors, published qualitative studies. 37 of the 355 original reports used both qualitative and quantitative (mixed) methods. Patients and non-health care settings were the most common groups of people studied. Diseases and conditions were cancer, mental health, pregnancy and childbirth, and cerebrovascular disease with many other diseases and conditions represented. Phenomenology and grounded theory were commonly used; substantial ethnography was also present. No substantial differences were noted for content or methods when articles published in all disciplines were compared with articles published in nursing titles or when studies with mixed methods were compared with studies that included only qualitative methods.The clinical literature includes many qualitative studies although they are often published in nursing journals or journals with low SCI Impact Factor journals. Many qualitative studies incorporate both qualitative and quantitative methods. Quantitative studies provide answers or insights for many important questions or issues in health care and clinical research. Other important questions dealing with why, how, contexts, and experiences of individuals or groups, can be best addressed using qualitative methods. Other issues benefit from interleaving or integration of both research traditions. Miller and Crabtree , describMost studies in the major clinical journals have been quantitative studies. Very few qualitative studies and even fewer that combine both qualitative and quantitative approaches are published. An example of the breadth of qualitative studies and how findings and results can be combined across paradigms is a study by Jolly and Wiles ,4 who usAnother example of the use of mixed methods was research done by Willms and Wilson and their colleagues -7 on smoAnother effective example of integrated qualitative (ethnography) and quantitative (epidemiology) methods was a study done by Borkan and colleagues  to deterDonovan and colleagues  used mixWe postulate that qualitative studies, either stand-alone reports or studies with mixed methods, are occurring more frequently in health care. This paper was done to describe the publishing of qualitative studies in 1 year of clinical literature, document and present the range of content and techniques in these studies, and establish a baseline for subsequent studies. We defined our sample to include all articles published in a set of major general medical, mental health, or nursing journals during 2000. We determined how many qualitative studies were published and in which journals, and extracted design methods and healthcare content, and how often studies used mixed methods and analyses. Because the nursing literature published a higher proportion of qualitative studies in our sample we also compared studies published in nursing journals with other journals to ascertain if quantitative differences exist across disciplines in the use of qualitative methods. Our analysis is a quantitative review of qualitative studies in health care in 2000.ACP Journal Club , Evidence-Based Medicine , Evidence-Based Nursing , and Evidence-Based Mental Health . Their purpose is to provide enhanced abstracts and commentaries on important high-quality original studies and review articles for their respective clinical audiences. To identify these studies and review articles, 6 research staff read major clinical journals to ascertain if articles were in 1 or more categories of therapy, diagnosis, prognosis, etiology, economics, clinical prediction guides, differential diagnosis, and qualitative studies and if so, did each meet predefined methodology criteria for study quality[The Health Information Research Unit of the Department of Clinical Epidemiology and Biostatistics, Faculty of Health Sciences at McMaster University in Hamilton, Ontario, Canada was the editorial office for four evidence-based summary journals in 2000: y quality. For 200y quality. One hunThe staff of the Health Information Research Unit has established quality criteria for the 8 categories of clinical literature that must be met before articles are judged appropriate for clinical application and publication in an abstract journal. Qualitative studies have 3 criteria:• content relates to how people feel or experience certain situations, specifically those that relate to health care• data collection methods and analyses are appropriate • units of collection and analysis are ideas, thoughts, concepts, phrases, incidents, or stories that become categories or themes.The reading methods have been developed during the past 13 years and inter-rater reliability kappa (chance adjusted agreement) for identifying categories and applying criteria is consistently > 80%.For this paper, KAM, one of the readers, analyzed the qualitative studies. Qualitative systematic reviews were excluded leaving only reports of original studies. These were assessed to extract journal title, qualitative study type, data collection methods, research question, persons studied, setting, and disease or health condition considered. In addition, studies with mixed methods were further analyzed although we did not use stringent criteria for assessing the quality  of the cIn addition, Giacomini and Cook ,15, as pnd edition by Denzin and Lincoln [Data were taken from article abstracts and if needed, the full text was reviewed. Methodologies assessed were phenomenology, grounded theory, ethnography, case studies, narrative analysis, participant action, critical incident techniques, and discourse analysis. Author descriptions were used and if an additional methodology was found it was added to the list of types using definitions and descriptions from the Handbook of Qualitative Analysis, 2 Lincoln . Data coThe 170 journals included 60 330 articles of which 31 496 (52%) contained original data or were review articles. 3830 of these (6%) passed criteria for being high-quality and clinically relevant in 1 of the 8 categories. 367 articles met quality criteria for original studies or reviews of qualitative studies. Table The reproducibility of the categorization was measured by kappa (chance adjusted agreement): 0.92 for disease/condition, 0.83 for groups studied, 0.81 for setting, 0.73 for data collection, and 0.63 for data analysis type. The agreement for data analysis type was disappointing but not surprising in that 20% of the studies did not label their analyses necessitating assignment of analysis type by the data extractors. Agreement was low for participant selection methods (kappa 0.5) and therefore data on participant selection methods are not reported.The 355 qualitative studies appeared in 48 journals . These 48 journals were only 28% of the 170 clinical journals being read. Most of the qualitative studies were published in nursing journals: The 17 nursing titles included 214 qualitative studies .BMJ publishing 12. The highest-ranking journal with qualitative studies was Annals of Internal Medicine, ranked number 6. JAMA, ranked number 2, published articles about qualitative studies in 2000 [Few qualitative studies were published in the high-circulation, general healthcare journals. Using SCI Impact Factor ranking for 2000, only 4 of the top 20 journals  included qualitative and quantitative methods and analyses. These were published in 17 journals with only 1 article in 1% includContent of the studies is shown in Table Disease/condition breakdowns represented common health care situations: cancer (11%), mental health (10%), pregnancy and childbirth (9%), cerebrovascular disease (10%), general issues such as vaccinations or Internet use, and nonspecific spectrum of diseases  (12%). Many uncommon issues were also assessed. For example, Tongprateep  reports Analysis of the 37 articles with mixed methods showed similar patterns for settings, persons studied, and disease/condition evaluated except that more physicians were studied (P < 0.025) and more situations dealing with injury (P < 0.001) were evaluated. For the 211 articles in Nursing journals, very little difference was also seen except that fewer physicians were studied (P < 0.001) and more studies were done outside clinical settings (P < 0.001).Phenomenology (37%), grounded theory (35%), and ethnography (18%) were used most often with some case studies (7%), narrative analysis (6%), participant action (3%) research, critical incident techniques (1%), and discourse analysis (1%) Table . More thSemi-structured interviews were used (77%) with some focus groups (18%) and observation (14%). These methods are major data gathering techniques in qualitative studies. Questionnaires (7%), document analysis (6%), and structured (4%) and unstructured interviews (1%) were used less often. For mixed methods studies, patterns were similar although questionnaires were used more frequently (24% vs. 7%, P < 0.01). Nursing studies did not differ for data gathering techniques.Sampling is important in all studies–often no single right way exists for a study question. Purposive, snowball, and theoretical sampling are often used in qualitative studies and random and consecutive sampling for quantitative studies. All methods were represented in this analysis but the breakdowns are not reported because of low inter-rater agreements for categorization and missing author information.BMJ and Annals of Internal Medicine also published several. Three of the high circulation medical journals  and 16 of the top 20 clinical journals, based on SCI Impact Factors, did not publish any qualitative studies. This is likely a reflection on the emphasis on a positivist, numerical approach that many of these journals embrace. The difference in proportion of qualitative studiers published in nursing journals is probably because of two historical, but linked factors. Qualitative studies have roots in women's studies and the nursing profession has always dealt with the patient as much more of a whole person rather than basic sciences facts and numbers. Both of these factors lead to more emphasis on understanding and embracing qualitative methods for research and practice. This view is substantiated by the fact that MEDLINE indexes most of the qualitative studies under the term Nursing Methodology Research until 2003.In 2000 the major clinical journals published many qualitative studies–approximately 9% of all high-quality, clinically relevant articles. Most of the qualitative studies were reports of original research although 12 (3%) were systematic reviews. Most of the qualitative studies were in nursing journals although some medical journals such as A substantial proportion of the qualitative studies 11%) included both qualitative and quantitative (mixed) data. In general, these mixed methods studies were similar to the single methodology studies except they did more assessments of physicians and relied more on questionnaires to gather data for analysis. The presence of these mixed methods or multipardigmatic studies as described by Miller and Crabtree 1% includ is encouIt is also interesting comparing qualitative studies in Nursing and non-Nursing journals. Regardless of the differences in proportion of qualitative studies published, from a content point of view few differences exist between the Nursing and non-Nursing journals except that more physicians were studied in the non-Nursing journals and fewer studies were done in clinical settings–not unsurprising findings. This indicates that the content and methods of qualitative studies seem to be similar across disciplines or if the methods are combined with quantitative methods.This review of the publication of qualitative studies is limited in several ways. The proportion of journals studied was very low in relation to the total number of journals published. MEDLINE indexes over 4000 journals and this number is still a relatively small proportion of all journals that deal with health care. In addition, all of the journals searched were published in English so we do not know about qualitative studies in other languages. Although our criteria were relatively strict for including qualitative studies, our criteria for mixed methods studies could certainly have been stronger. We did not count the number of high-quality quantitative studies that could have included some qualitative analyses. We studied only 1 year of publishing; much could have changed since 2002.Qualitative studies provide insight into social, emotional, and experiential aspects of health and health care and as such, they have an important place in understanding health and health care. Hopefully more studies will be published and more will be published in the high impact (high circulation) journals. This paper provides a basis for measuring increases.Qualitative studies are being done and are published in a wide range of healthcare journals. These journals however are not the highest impact journals. It is encouraging to see that the number of qualitative studies that were published in 2000 and also the number of studies that combined qualitative and quantitative methods. More can be done however to complete and publish qualitative studies, and where appropriate, integrate the best of both methodologies. Both qualitative and quantitative researchers and clinicians need to work together to make this happen. Journal editors can also encourage submission of qualitative and mixed methods studies and facilitate publication of those they do receive.SCI ISI Science Citation IndexNone declared.This work was done in partial fulfillment of PhD requirements for KAM. Both authors have supplied intellectual input in designing and implanting the survey. KAM has collected and analyzed the data and both authors have contributed to writing the paper and agree on its content.The pre-publication history for this paper can be accessed here:


Late-stage cancers are notoriously unresponsive to treatment, making certain hard-to-detect cancers particularly insidious. Ovarian cancer, for example, most often escapes diagnosis until the tumor has already metastasized. At this stage, ovarian cancer is classified as peritoneal carcinomatosis, a terminal condition characterized by widespread tumor growth throughout the peritoneum, the large serous membrane that lines the abdominal cavity, pelvis, and associated organs. Advanced cases of peritoneal carcinomatosis are largely resistant to chemotherapy and account for the bleak 15%–20% survival rate of ovarian cancer.Biologists often view cancer as an evolutionary process in which cells that would normally cooperate with their neighbors begin to compete with them. Selective advantage for cancer cells often begins with mutations that inhibit tumor suppressor pathways. p53, like other tumor suppressor genes, arrests cell growth and induces apoptosis (programmed cell death) in response to cellular stress, such as chromosomal damage. Cells with p53 mutations often escape these constraints, leading to the uncontrolled growth characteristic of “immortal” cancer cells. Nearly all types of tumors have mutations in the p53 pathway. Treatments focused on restoring p53 function—which is likely to be defective only in cancer cells—should prove more effective than chemotherapies, which indiscriminately kill all dividing cells, healthy or cancerous. With the goal of developing targeted therapeutic strategies, Steven Dowdy and colleagues show that restoring p53 protein function in tumor cells not only dramatically increases lifespan in mice but also eliminates disease.While past efforts to restore tumor suppressor function in cancer cells have focused on gene therapy, Dowdy and colleagues introduced modified p53 peptides, or protein fragments, into cancer cells. p53 works as a “transcriptional” activator that binds to specific sequences of DNA and triggers apoptosis in response to DNA damage. Its biological function flows from this binding ability. One region of this large protein, called the C-terminal domain, facilitates effective binding. In cancer cells, synthesized peptides  derived from this region can induce apoptosis by activating p53—which is normally present in low levels in a biologically inactive form—and restoring function to p53 proteins with DNA-binding mutations.To get p53C′ peptides into cancer cells, Eric Snyder et al. used a technique pioneered by Dowdy that delivers large proteins into the cell interior. Since the cell membrane normally limits passage to only small molecules , this is no small feat. The technique exploits the ability of a small peptide region from the HIV TAT protein to smuggle macromolecules through cell membranes that normally prohibit entry to such large molecules. After synthesizing a structurally modified form of p53C′ less prone to degradation, the researchers first confirmed that the peptide was functional and then that it activates p53-specific genes in tumor cells, but not in normal cells. Testing the effectiveness of the peptide therapy on mouse strains used to model human metastatic disease, they found that mice treated with the TATp53C′ peptide showed a significant reduction in tumor growth and lived six times longer than both mice treated with a control peptide and untreated mice, with some mice remaining disease-free more than 200 days after treatment.This macromolecular delivery approach, Snyder et al. argue, works with greater specificity and avoids the tumor-generated neutralizing effects observed in small molecule strategies. Because a mutation in the p53 gene is one of the most common events in the development of cancer, these results could have implications for a wide variety of cancers. And by working with mouse models that approximate the physiological burdens metastatic cancer imposes on humans, Dowdy's team demonstrates the promise of developing targeted “intracellular biologic” therapeutics that treat the systemic pathology of cancer—inhibiting tumor growth as well as alleviating the lethal complications associated with the disease.