Dendritic cells (DC) are known to present exogenous protein Ag effectively to T cells. In this study we sought to identify the proteases that DC employ during antigen processing. The murine epidermal-derived DC line Xs52, when pulsed with PPD, optimally activated the PPD-reactive Th1 clone LNC.2F1 as well as the Th2 clone LNC.4k1, and this activation was completely blocked by chloroquine pretreatment. These results validate the capacity of XS52 DC to digest PPD into immunogenic peptides inducing antigen specific T cell immune responses. XS52 DC, as well as splenic DC and DCs derived from bone marrow degraded standard substrates for cathepsins B, C, D/E, H, J, and L, tryptase, and chymases, indicating that DC express a variety of protease activities. Treatment of XS52 DC with pepstatin A, an inhibitor of aspartic acid proteases, completely abrogated their capacity to present native PPD, but not trypsin-digested PPD fragments to Th1 and Th2 cell clones. Pepstatin A also inhibited cathepsin D/E activity selectively among the XS52 DC-associated protease activities. On the other hand, inhibitors of serine proteases  or of cystein proteases (E-64) did not impair XS52 DC presentation of PPD, nor did they inhibit cathepsin D/E activity. Finally, all tested DC populations  constitutively expressed cathepsin D mRNA. These results suggest that DC primarily employ cathepsin D (and perhaps E) to digest PPD into antigenic peptides. Dendritic cells (DC) are professional antigen presenting cells that induce primary antigen specific T cell responses  and exhiIn the present study, we sought to characterize the Ag processing capacity of DC, as well as the enzymes previously involved in this process. In this regard, several groups have previously reported that epidermal LC and splenic DC, both of which contain small numbers of non-DC contaminants, exhibit significant Ag processing capacities -12. LC fStaphylococcus aureus nuclease [With respect to the mechanisms by which DC process complex protein Ags, chloroquine has been shown to inhibit this process; this suggests that Ag processing primarily occurs within acidic compartments -8, 10-1-110-12. nuclease -35. Thesnuclease . Little The XS52 DC cell line , a long-term DC line established from the epidermis of newborn BALB/c mice , were ex+ cells , and T cells (with GK1.5 and 3.155 mAbs). The remaining I-A- cells were then cultured in the presence of GM-CSF (10 ng/ml). The purity of bone marrow derived DC was more than 95% as determined by flow cytometry using anti-CD11c and anti-I-A antibody (not shown).Splenic DC were purified from BALB/c mice  by a series of magnetic bead separations as before ,25. BrieCells were lysed in 0.1% Triton X-100 in 0.9% NaCl; extracts were then examined for protease activities using the following substrates: a) Z-Arg-Arg-βNA , b) denatured hemoglobin , c) Arg-βNA , d) Z-Phe-Arg-MCA , e) Z-Phe-Arg-MCA , f) Gly-Phe-βNA , g) BLT ester , and h) Suc-Ala-Ala-Pro-Phe-SBz and Suc-Phe-Leu-Phe-SBz . Samples were incubated at the indicated pH and enzymatic activities were assessed by colorimetric or fluorogenic changes . Enzymat4CL (15 mM). Subsequently, the XS52 cells were washed 3 times with PBS to remove unbound PPD and then cultured in 96 round-bottom well-plates (104 cells/well) with either the PPD-reactive Th1 or Th2 clone (105 cells/well) in the presence of the same inhibitor at the above concentration. In some experiments, XS52 DC were pulsed overnight with PPD in the presence of an inhibitor and then fixed with 0.05% glutaraldehyde in PBS for 30 seconds at 4°C; the fixation reaction was stopped by adding 0.1 M L-lysine. These XS52 cells were then washed with PBS and examined for their ability to activate Th1 or Th2 clones in the absence of protease inhibitors. In order to determine the mechanism of action for pepstatin A, XS52 cells were pulsed in its presence with PPD either in a native form or following digestion with trypsin-conjugated sepharose beads  for 15 minutes at 37°C. We also examined the effect of added pepstatin A on the capacity of XS52 cells to activated allogeneic T cells isolated from CBA mice (Jackson Laboratories). Samples were pulsed for 18 hr with 1 μCi of 3H-thymidine and then harvested using an automated cell harvestor.XS52 DC were γ-irradiated (2000 rad) and then pulsed for 8 hr with 100 μg/ml of PPD  in the presence of each of the following inhibitors (or vehicle controls): a) pepstatin A , b) DCI 100 μM, Sigma), c) E-64 , d) DMSO (1%), and e) NHmRNA expression for cathepsin D was examined by RT-PCR. RNA isolation, reverse-transcription, and cDNA amplification were carried out as previously described . The fol+ splenic DC, and GM-CSF-propagated bone marrow DC. As noted in Table In the first set of experiments we sought to identify which protease activities were expressed by DC. A panel of synthetic peptide and protein substrates was incubated with extracts prepared from three DC populations: the XS52 DC line, 4F7We next asked whether DC are capable of digesting a complex protein Ag into antigenic products. In this regard, it has been reported previously that the original XS52 DC, as well as clones derived from this line, are capable of presenting KLH to the KLH-specific Th1 clone HDK-1 . These rTo identify the proteases responsible for processing PPD, we employed three inhibitors: pepstatin A (aspartic acid protease inhibitor), DCI (serine protease inhibitor), and E-64 (cysteine protease inhibitor). XS52 DC were pulsed for 8 hr with native PPD in the presence of each inhibitor and then examined for the capacity to activate PPD-reactive Th1 and Th2 clones. To ensure a maximal effect, inhibitors were also added to cocultures of XS52 DC and T cells. As noted in Figure To identify the protease(s) inhibited by pepstatin A, XS52 DC were pretreated for 1 hr with pepstatin A (100 μg/ml), and extracts prepared from these cells were then examined for enzymatic activities. As noted in Figure + splenic DC and a bone marrow derived DC line, indicating that DC do possess the capacity to produce cathepsin D  during Ag processing by DC. Taken together, these results reinforce the concept that DC are fully capable of processing complex protein Ag into antigenic peptides.The experiments reported in this study provide new information with respect to complex Ag processing by DC. First, the long-term DC line, XS52 DC, was capable of processing PPD into immunogenic peptides, in the complete absence of other cell types. Although previous studies using several different DC preparations have documented similar results (3–12), this is the first report validating the Ag processing capacity of DC, in the absence of contaminating cells. Second, we have characterized the protease profiles expressed by DC. XS52 DC, 4F7Staphylococcus aureus nuclease (17–22). Here we report that DC also employ cathepsin D and/or E to digest PPD into an immunogenic Ag-product. This conclusion is supported by several lines of evidence: a) pepstatin A, but not other protease inhibitors, completely blocked the presentation of intact PPD by XS52 DC to PPD-reactive Th1 and Th2 clones, whereas it did not affect the presentation of PPD fragments; b) pepstatin A pretreatment inhibited cathepsin D/E activity selectively among the DC-associated protease activities; and c) all tested DC preparations expressed cathepsin D mRNA constitutively. In this regard, DC isolated from the mouse thoracic duct have been reported to produce neglible, if any, cathespin D immunoreactivity (assessed by immunofluorescence staining), whereas peritoneal macrophages produced relatively large amounts [As described before, macrophages and B cells have been reported to employ cathepsins B, D, and E primarily to digest complex protein Ag, such as ovalbumin (OVA), hen egg white lysozyme (HEL), myoglobin, exogenous IgG, and  amounts . Also co amounts  may be uIt is important to emphasize that different protein Ag may be degraded by different proteases in DC. Moreover, DC isolated from different tissues or in different maturational states may employ different proteases. For example, murine DC isolated from the thoracic are unable to digest human serum albumin effectively , and murNone declared.Dr. Mohamadzadeh is the major contributor (15%) of the experimental data and a rough draft of the paper. The next three intermediate authors' contributed remaining data and advice. Dr. Luftig was the overall individual who directed the several drafts and contributed to providing a new set of references to the manuscript.


Several sawfly larvae of the Tenthredinidae (Hymenoptera) are called easy bleeders because their whole body integument, except the head capsule, disrupts very easily at a given spot, under a slight mechanical stress at this spot. The exuding haemolymph droplet acts as a feeding deterrent towards invertebrate predators. The present study aimed to describe the cuticle surface, to consider it from a mechanistic point of view, and to discuss potential consequences of the integument surface in the predator-prey relationships.®), which was related to the sawfly larvae's ability to bleed easily.The integument surface of sawfly larvae was investigated by light microscopy (LM) and scanning electron microscopy (SEM) which revealed that the cuticle of easy bleeders was densely covered by what we call "spider-like" microstructures. Such microstructures were not detected in non-easy bleeders. A model by finite elements of the cuticle layer was developed to get an insight into the potential function of the microstructures during easy bleeding. Cuticle parameters  were measured on integument sections and used in the model. A shear force applied on the modelled cuticle surface led to higher stress values when microstructures were present, as compared to a plan surface. Furthermore, by measuring the diameter of a water droplet deposited on sawfly larvae, the integument of several sawfly species was determined as hydrophobic (e.g., more than TeflonEasy bleeders show spider-like microstructures on their cuticle surface. It is suggested that these microstructures may facilitate integument disruption as well as render the integument hydrophobic. This latter property would allow the exuding haemolymph to be maintained as a droplet at the integument surface. Aneugmenus, Athalia, Monophadnus, Phymatocera and Rhadinoceraea. Recently, the mechanical strength of dissected pieces of larval integument was measured in a calibrated manner. The force needed to damage the integument can vary in more than one order of magnitude from one species to another [The integument of insects is very often involved in defence strategies towards predators and pathogenic agents ,2. Gener another . Easy bl another . As soonAn ecological implication of easy bleeding is that the emission of a haemolymph droplet will deter an attacking predator from killing and feeding on an easy bleeder. Indeed, the haemolymph is feeding deterrent towards foraging ants and wasps -8. BirdsHowever, integument disruption remains puzzling from a morphological and mechanistic point of view. The present study is based on a comparative analysis of the larval integument surface in several sawfly species, which comprise easy bleeders as well as non-easy bleeders. We wanted to describe the geometry and to approach the mechanical properties of the integument surface, and to consider proximate, ecological implications.P = 0.0001, Fisher exact probability test, N = 24 species; Table The larvae of sawfly species observed by SEM showed above surface microstructures of their cuticle and which are described below. These microstructures were strikingly more complexly structured in easy bleeders than in non-easy bleeders Fig. , 2 and t2. They possess fine ridges (carinulate) in a radiated way , but can be elongated (length: 35 μm in A. rosae). The ridges can be reduced . The height of microstructures was measured on LM views and reaches 23 μm (in P. aterrima). For further measurements by LM, see Table In easy bleeders, the cuticle is covered with irregularly shaped wart-like microstructures (verrucose). Their density is approximately of 15 units per 0.01 mmway Fig. , 2b,2d, T. nigritus, H. australis, Nematus, Fig. S. multifasciata, Fig. H. testudinea). In some genera such as Nematus and Craesus, each swelling shows a very small prickle (echinulate). Several swellings are sometimes aligned and then can be joined, several together, to form a low ridge of approximately 35 μm long .Compared to easy bleeders, the cuticle surface of non-easy bleeders was much smoother. It only shows blister-like swellings (pustulate) which have a diameter of 3–4 μm  of these two species as well as T. scrophulariae are covered with a layer of waxy powder. Setae were observed instead of microstructures in the outgroup species G. hercyniae  versus easy bleeders (M2), when a same loading is applied Fig. . The maxIn Table M. monticola and P. aterrima, the apical part of the microstructure was extremely minute. All stresses were concentrated in the tip of the microstructure, which lead to non-physical deflections. The obtained values for these two species are probably irrelevant in a comparison with other species.With a normal force, M1 and M2 resulted in stress values which were in the same range of magnitude Table  to 3f. TIt was difficult or even impossible to deposit a water droplet on the larval body of some sawflies Table . When thP = 0.045, Fisher exact probability test, N = 12 species, Table In species where the integument was less hydrophobic, the diameter of the droplet on it ranged from 1.5 to 1.9 mm (2 μl droplet) and from 2.0 to 2.6 mm (4 μl). Considering this latter droplet size, a small diameter (2.0 mm) or an immeasurable diameter (see above) was associated with sawfly species which are easy bleeders, whereas a larger droplet diameter (> 2.0 mm) was associated with non-easy bleeders  integument of non-easy bleeders. The 2 μl droplet was apparently light enough to impede its adhesion on the Teflon® surface, but not the larger droplet size tested.On inert surfaces the diameter of 2 and 4 μl droplets was constantly as follows: immeasurable (see above) and 2.2 mm on TeflonAneugmenus padi and absent in the non-easy bleeders Strongylogaster spp. Both genera are closely related since they belong to the same subfamily and have the same host plant [Dolerus  species presents "meshes of microsculpture not sharply defined", with a dimension ranging from 20 to 40 μm, and these microstructures are often fused [Several sawfly larvae showed a characteristic cuticle surface with spider-like microstructures and this was associated with a low mechanical strength of their integument. For instance, these microstructures were present in the easy bleeder st plant . It is len fused . They maen fused  and in aen fused ,15. Theien fused .P. aterrima). This biological observation is in agreement with our model results. The regions subject to high stresses are not restricted to the zone of the microstructure, but extend deeper into the cuticle mass  of the procuticle with a reinforcing effect of chitin microfibriles. Differing chitin and protein patterns are observed in the cuticle when easy bleeders are compared to non-easy bleeders . Ongoing research aims to investigate these physiological aspects as well as the healing process, and to link them with the phenomenon of easy bleeding.The question arises to know whether in sawfly larvae able to bleed easily the microstructures are directly involved in integument disruption. We compared cuticle models of non-easy bleeders versus easy bleeders and applied a unit force on it. Compared to the real-life, the model was simplified by considering a linear elastic behaviour of the cuticle , because we do not know the exact physical properties of the cuticle. Nevertheless, a comparison of geometrical parameters from easy bleeders versus non-easy bleeders revealed that by applying a shear force the cuticle stresses both in compression and tension were higher in the presence of microstructures Table . This suass Fig. . From th®. There is a trend for the integument of easy bleeders  to appear as mat, in contrast to the brilliant aspect in non-easy bleeders  . We believe that the hydrophobic property is ecologically relevant during predator-prey interactions. When a predator, typically an insect with biting-chewing mandibles [The cuticle surface of easy bleeders was highly hydrophobic Table  as compaandibles , bites iandibles ,22. But andibles .® [Eriocampa ovata [A parallel can be drawn between the integument surface of easy bleeders and the one of several plant leaves. The lotus leaf led recently to the so-called Lotus-effect® . Particu® ,24. The ® . This co® . There apa ovata , a non-eWe suppose at least two types of functions in the occurrence of spider-like microstructures, which we observed specifically on the body surface of easy bleeders. Firstly the damage provoked by a biting predator could be facilitated. Secondly the integument of easy bleeders could be rendered hydrophobic, which helps stop the emitted haemolymph droplet from spreading out.A. rosae and G. hercyniae that came from indoor populations. The larvae were identified according to Lorenz & Kraus [All sawfly larvae . The geometry was discretized with 3D solid linear finite elements (prisms or bricks). The patch used to model non-easy bleeders was composed of two layers, procuticle and epicuticle, of different properties in height and Young's modulus. It is supposed that generally the epicuticle of insects is pliant but not extensible, stronger in compression than tension, and that the epicuticle is less elastic than the procuticle [A. padi  and parallel to it (shear force).Myrmica rubra and reached 20 μm as the smallest value. In the model, the shape of the contact point made by the mandible was a disc (radius = 10 μm) on which the force was applied. This force was applied either on the upper centre of the epicuticle for non-easy bleeders  was considered to express the hydrophobic or hydrophilic property of the surface.This property of the integument was estimated by a simple method that allowed the use of insects alive. The first step was to notice whether a 2 and 4 μl droplet of charcoal filtered water could adhere, gently depositing it with a 1–10 μl pipette on the thoracic or abdominal integument of a sawfly larva that was resting on a leaf of its host plant. If an adherence was possible, the diameter of the deposited droplet was measured under a stereomicroscope with micrometer. Six full-grown larvae were tested per species. As control the following substrates were tested in the same manner: TeflonJLB collected and identified the insects, performed the tests on the hydrophobic property, measured the cuticle parameters for the model on LM views, and wrote the manuscript, except the parts about this model in Results and Methods. VD obtained most SEM views. TM and PB performed the model by finite elements and wrote the two related parts in the manuscript. SA maintained indoor populations of two sawfly species and carried out and photographed the integument sections used in LM and, thereby, in the model.


Furthermore, the decision as to which journal a given study should be submitted, may well be based on the trend in the journal's overall quality. This study sought to assess time trends in journal IF in the field of public, environmental and occupational health.Journal impact factor (IF) is linked to the probability of a paper being cited and is progressively becoming incorporated into researchers' We used the IFs of 80 public health journals that were registered by the Science Citation Index from 1992 through 2003 and had been listed for a minimum period of the previous 3 years. Impact factor time trends were assessed using a linear regression model, in which the dependent variable was IF and the independent variable, the year. The slope of the model and its statistical significance were taken as the indicator of annual change.The IF range for the journals covered went from 0.18 to 5.2 in 2003. Although there was no statistical association between annual change and mean IF, most of the fastest growing journals registered mean IFs in excess of 1.5, and some represented emerging areas of public health research. Graphs displaying IF trends are shown.In view of the delay between the publication of IFs and that of any given paper, knowing the trend in IF is essential in order to make a correct choice of journal. In Spain, publication in top-IF journals has occupational implications in terms of academic careers and obtaining research grants [Scientific journal impact factor (IF) is directly linked to the probability of a paper being cited. It is currently accepted that a higher IF indicates a better journal quality, existing a correlation among IF and quality indicators at least in some health disciplines . As a reh grants . The mosh grants .This pressure means that when it comes to having to select a journal in which to publish their studies, researchers turn to journals with IF, and assess the possibility of publishing in those that have the highest IF possible. Journals having the top IF within each medical specialty tend to be those with the greatest international prestige and highest profile, i.e., the most widely read by researchers and most in demand to publish their studies. However, the publication of extraordinary relevant scientific findings are concentrated in a small list of core journals, most of them not directly related with the specialty of the author ,4Positive evaluation of IF which failed to take account of its trend over time would tend to favor publications of recent, but not necessarily lasting, interest. In contrast, publications of steadily growing interest and stable impact would be undervalued, though there may be differences of opinion about this .Hence, in addition to journal quality, the decision as to which journal a manuscript should be submitted may be based on the trend in its IF over time. The aim of this study was to analyze IF time trends of journals in the "Public, Environmental and Occupational Health" category, thereby furnishing a new criterion on which to base the choice of journal for publication.For study purposes, we selected 80 journals that: were included in the "Public, Environmental and Occupational Health" category of the hard copy version of the Journal Citation Reports (JCR) from 1992 through 2003 ; were liThe impact factor is one of the quantitative tools provided by JCR for ranking, evaluating, categorizing, and comparing journals. The annual impact factor of a journal is calculated by dividing the number of current year citations to the source items published in that journal during the previous two years .Impact factor time trends were assessed using a linear regression model, in which the dependent variable was IF and the independent variable, the year. The slope of the model  and its statistical significance were taken as the indicator of year-to-year variation.Shown in Table The IF range for the journals covered went from 0.18 to 5.2 in 2003. Although there was no statistical association between annual change and mean IF, most of the fastest growing journals registered mean IFs in excess of 1.5 , and some represented emerging areas of public health research  declare that they have no competing interests.CMT was responsible for the development of intellectual content and the study design, collected the data, data coding and entry, interpretation of the results, manuscript drafting and the critical revisions of manuscript. GLA was responsible for the development of intellectual content, statistical analyses, interpretation of the results and manuscript drafting. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


X-linked Emery-Dreifuss muscular dystrophy is caused by loss of emerin, a LEM-domain protein of the nuclear inner membrane. To better understand emerin function, we used affinity chromatography to purify emerin-binding proteins from nuclear extracts of HeLa cells. Complexes that included actin, αII-spectrin and additional proteins, bound specifically to emerin. Actin polymerization assays in the presence or absence of gelsolin or capping protein showed that emerin binds and stabilizes the pointed end of actin filaments, increasing the actin polymerization rate 4- to 12-fold. We propose that emerin contributes to the formation of an actin-based cortical network at the nuclear inner membrane, conceptually analogous to the actin cortical network at the plasma membrane. Thus, in addition to disrupting transcription factors that bind emerin, loss of emerin may destabilize nuclear envelope architecture by weakening a nuclear actin network. Loss of emerin leads to Emery-Dreifuss muscular dystrophy (EDMD). Biochemical studies presented here suggest that emerin drives the formation of an actin-based cortical network at the nuclear membrane, and that network destabilization may contribute to EDMD LMNA, encoding A-type lamins, and STA, which encodes a nuclear membrane protein named emerin  is inherited through mutations in either of two different genes: d emerin . Lamin fd emerin . Togethed emerin , suggestd emerin . Both emd emerin . There id emerin . Althougd emerin  and in aCaenorhabditis elegans emerin is expressed ubiquitously . Mass spectrometry (data not shown) and Western blotting identified β-actin as a major emerin-binding protein A. Six otg. Approximately 75% of input emerin pelleted in the presence of F-actin, compared to 15% in the absence of F-actin  and then pelleted at 100,000 F-actin C, demonsn = 32; one experiment is shown in The above results suggested that in vivo emerin might (a) use actin filaments as anchors, (b) stabilize F-actin networks, or (c) actively influence actin dynamics. To test these models, we first used reactions containing 5% pyrene-labeled actin  to determine if emerin influenced actin polymerization in vitro. Results were graphed as the rate of emerin-induced polymerization (R) divided by the control rate . At concentrations ranging from 0.1 to 4.4 μM, emerin increased the rate of pyrene–actin polymerization 4-fold  in the pyrene–actin polymerization assay B. Five md) of emerin for F-actin was 430 nM , and assayed immediately. Preformed actin filaments depolymerized rapidly in the absence of emerin B, arrow,n = 30). However, in the presence of either emerin or tropomodulin, a pointed-end binding protein, most red filaments lacked green segments (n = 60) and 0.09 ± 0.13 μm (n = 50), respectively. Single filament assays were also done using small red filaments formed from gelsolin–actin seeds (n = 40). However, when emerin was present, the preformed filaments remained predominantly short and red, demonstrating that emerin blocks pointed-end growth (n = 40) and 0.1 ± 0.11 μm (n = 40), respectively. These experiments also show that emerin does not stimulate branching (n = 306). Interestingly, 10% of actin-associated emerin proteins localized to “aster-like” structures  were preformed from 2 μM actin, then capped on the barbed end with capping protein, and diluted 2-fold into a final concentration of 1 μM emerin. Pointed-end growth was then initiated by increasing actin to 3.2 μM in the presence of 3.2 μM Alexa-488 phalloidin (green) for 2 min. In the absence of emerin C, demonssegments C, consisin seeds D. Here, in seeds D. The avd growth D. Similad growth D, Tmod. ranching D. To dirranching E–3H. Undructures G and 3H,d = 110 nM) and stimulates actin polymerization by stabilizing the actin filament  and lamin A  includes integral membrane proteins , anchoring proteins (ankyrin), spectrin filaments, and “junctional complexes”  was used at 1:1,000 for immunoblotting. An antibody specific for β-actin  was used at 1:10,000 for immunoblotting and 1:1,000 for immunoprecipitation. Our rabbit polyclonal emerin antibody (serum 2999), described previously , was use10 HeLa-S3 cells, obtained as frozen cell pellets from the National Cell Culture Center. For each affinity purification, we incubated 50 mg of nuclear lysate proteins with 2 ml of either emerin beads (0.5 mg/ml) or BSA beads in binding buffer  for 4 h at 4 °C. Beads were collected by centrifugation at 500g, washed five times with binding buffer, and eluted with SDS-PAGE sample buffer. Dr. Robert Cole at the Johns Hopkins Mass Spectrometry Facility performed MALDI-TOF. Actin and αII-spectrin were two of seven emerin-associated proteins identified unambiguously in this work, which will be reported separately .Wild-type emerin residues 1–222 (comprising the entire nucleoplasmic domain of emerin and lacking the transmembrane domain) or BSA (as a negative control) were coupled to Affigel-15 beads  per manufacturer's instructions. Nuclear extracts were prepared by hypotonic lysis  from 101F-actin columns were assembled as described . Equal aCoimmunoprecipitation assays were performed as described . Brieflyg for 60 min. For experiments with unlabeled emerin, corresponding load, supernatant, and pellet fractions were resolved by SDS-PAGE and stained with Coomassie blue. For experiments with phalloidin-546-labeled F-actin, filaments were viewed using a Zeiss Axiovert 200 fluorescent microscope  and images captured using a Quantix CCD camera  attached to an Apple G4 computer using IPLab  software.To measure emerin binding to single actin filaments, actin was polymerized by the addition of KMEI buffer . After 30 min, the indicated form of emerin  was added to the filaments and incubated 30 min; we lastly added Alexa-546 phalloidin . These red F-actin polymers were then pelleted at 100,000Actin polymerization assays were performed per manufacturer's instructions (Cytoskeleton). Rabbit actin  plus pyrene–actin  were used in all assays, unless otherwise stated. Actin polymerization was measured in a fluorimeter , with excitation wavelength 365 nm and emission wavelength 407 nm, and plotted using DataMax-Std . Graphs were refined using Cricketgraph III  and Kaleidagraph . Pyrene–actin was always present at 5% of total actin. Increasing concentrations of recombinant emerin were added just prior to initiating actin polymerization. The actin depolymerization assays were performed exactly as described  with incActin polymerization in the presence or absence of emerin was monitored by fluorescent microscopy, as described . Samples


Gefitinib and erlotinib are two new treatments for advanced lung cancer. Gene mutations in the cancer may help predict which patients will respond to these treatments Lung cancer is the leading cause of death in many countries. To date, cause of cancer death in chemotherapy with cytotoxic agents has been the mainstay of treatment for advanced lung cancer. However, the activity of these agents is quite limited, and they have severe adverse effects. Recent, rapid advances in molecular biology have led to the development of many new agents that inhibit the activities of specific molecules related to tumor growth, invasion, or metastasis Gefitinib is the first “molecular-target agent” for lung cancer that inhibits the tyrosine kinase of the epidermal growth factor receptor . EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC), especially in squamous cell carcinoma, and its expression is related to the cancer's proliferation. Initial clinical trials of gefitinib showed its modest clinical activity in patients who had failed previous standard chemotherapy. But subsequent randomized trials in patients with previously untreated, advanced NSCLC have not shown a clinical advantage of gefitinib combined with standard chemotherapy over chemotherapy alone ,3,4,5.Erlotinib, another EGFR tyrosine kinase inhibitor for treating NSCLC, which was approved by the United States Food and Drug Administration in November 2004, has a therapeutic profile similar to that of gefitinib. Erlotinib has shown a survival benefit in a phase III trial for chemo-refractory NSCLC that compared erlotinib to best supportive care Science and the New England Journal of Medicine helped to explain at the molecular level the low clinical activity of EGFR inhibitors [In April and May 2004, two new studies published in hibitors ,8. TheseThese findings should hopefully lead to the identification of subgroups of patients who are likely to benefit substantially from such EGFR inhibitors. But another question was raised by these studies: is it only EGFR gene mutations that determine the response to EGFR inhibitors?PLoS Medicine suggests that the K-ras mutation is an important predictive factor in defining which patients will benefit from receiving EGFR inhibitors The RAS proteins are low-molecular-weight GTPases that are bound to the inner side of the cell membrane. They are involved in signal transduction pathways: they regulate downstream effector proteins such as Raf/MAP kinase and PI3 kinase, under the influence of various cell surface receptors including EGFR . MutatioAlthough the number of tumors examined in this study may be too small to lead to a definite conclusion—and, furthermore, about half of tumors were retrospectively collected—this study is the first to show that mutations of EGFR and K-ras are not related and that K-ras mutations are associated with a lack of sensitivity to EGFR inhibitors. However, since the sensitivity of the method for finding each mutation influences how often it is detected, standardization of detection methods is important. Hence, the true incidence of K-ras mutations in NSCLC (including non-adenocarcinoma) and their refractoriness to EGFR inhibitors need to be established in further studies. In fact, the reported incidence of EGFR mutations differs between institutions. In addition, although Pao and colleagues' study examined such mutations only by DNA sequence, mRNA or protein expression would show mutation status more accurately.In the clinical setting, prolonged disease stabilization with no measurable reduction in tumor size is seen in about half of patients treated with EGFR inhibitors, and a significant survival benefit in this group was shown in a phase III trial It will also be important to establish whether, in tumors with an EGFR mutation that eventually acquire resistance to EGFR inhibitors, the resistance is associated with a change of EGFR status (mutation or change in expression) or is associated with a change of K-ras status. Such changes can be revealed by re-examination of tumors at the time of relapse. If resistance is K-ras dependent, the new “K-ras inhibitors” (unfortunately not yet available) may be of help for patients who have developed a K-ras mutation, as well as for patients whose tumors harbor K-ras mutations from the beginning.Although many issues still need to be resolved step by step through prospective trials to show the benefit of this new strategy, these mutations are promising predictive factors for the success of EGFR inhibitors. Even if the population who may benefit from EGFR inhibitors (such as patients who are positive for an EGFR mutation and negative for a K-ras mutation) is very small, the response rate of over 80% is encouraging, and has never before been achieved in advanced NSCLC. Finally, it is important to detect which patients derive no benefit from EGFR inhibitors because severe adverse effects such as acute lung injury can occur in any patient treated with these drugs


Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy. In the present study, we used a first trimester human extravillous CTB cell line, termed SGHPL-4, to investigate HCMV replication. SGHPL-4 cells were derived from first trimester chorionic villous tissue and have been described previously. Importantly, these cells share many characteristics with isolated primary cells, including the expression of cytokeratin-7, HLA class I antigen, HLA-G, BC-1, CD9, human chorionic gonadotrophin, and human placental lactogen[Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that is the leading cause of congenital viral infection in the United States and Europe. Intrauterine transmission of the virus occurs in approximately 40% of pregnant women with primary HCMV infection, and the incidence of congenital HCMV infection is an estimated 1% of newborns . Althougin vitro ,9. Additothelium . Despite lactogen-12.The lytic replication cycle of HCMV is a temporally regulated cascade of events that is initiated when the virus binds to host cell receptors. Upon entry into the cell, the viral DNA translocates to the nucleus where viral gene expression occurs in a stepwise fashion beginning with the expression of immediate early (IE) genes (reviewed in ). To iniAlthough several studies have shown that first trimester primary trophoblasts can be permissively infected with HCMV, some reports have demonstrated that progression through the replicative cycle was slow and progeny virus remained predominantly cell associated ,15,16. Tin vitro studies of placental HCMV infection. We propose that the permissiveness for HCMV replication in SGHPL-4 cells may allow for future studies in elucidating the molecular mechanism(s) of HCMV infection and pathogenesis at the maternal-fetal interface during early pregnancy.In the present study, we demonstrate that the first trimester extravillous CTB cell line SGHPL-4 is fully permissive for HCMV replication. The utilization of a CTB cell line, rather than primary CTBs and explant cultures that are short-lived cultures, may provide an experimental advantage for human cytomegalovirus (HCMV), cytotrophoblast (CTB), human foreskin fibroblasts (HFFs), immediate early (IE), hours (h), post-infection (p.i.), multiplicity of infection (MOI), plaque forming unit (PFU), 4', 6-diamidino-2-phenylindole, dihydrochloride(DAPI)The authors declare that they have no competing interests.HL participated in the experimental design, performed all experiments and drafted the manuscript. BS participated in the experimental design and assisted with viral propagation and viral replication assays. CM conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


Although Western models of education are being used to establish health professional programs in non-Western countries, little is known about how students in these countries perceive their learning experiences. The purpose of this qualitative study was to describe the reflections of physiotherapy students from a Middle East culture during their clinical placements and to compare them to reflections of physiotherapy students from a Western culture.Subjects were six senior students  and 15 junior, female students (mean age 20.1 years) in the baccalaureate physiotherapy program at a university in the United Arab Emirates (UAE). They wrote weekly entries in a journal while in their clinical placements. They described an event, their reaction to it, and how it might affect their future behavior. Two evaluators independently read and coded the content of all the journals, and then worked together to categorize the data and develop themes. A third evaluator, an UAE national, independently read the journals to validate the content analysis. A feedback session with students was used to further validate the data interpretation. The themes were compared to those derived from a similar study of Canadian physiotherapy students.The content of the students' reflections were grouped into 4 themes: professional behavior, awareness of learning, self-development and shift to a patient orientation, and identification and analysis of ethical issues. Although the events were different, students from the UAE considered many of the same issues reflected on by Canadian students.Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others. Reflection is considered by many -3 in theJournal writing is a method that has been used to promote reflective thinking in health science students and professionals ,6, to faTwo recent Canadian studies ,13. haveIn considering the student reflections, the investigators wondered about the potential impact of culture and working environment on the content of the reflective journals. Would students from a non-Western culture and a different health care system be concerned about the same issues? Would they feel comfortable with the reflective process, particularly documenting their thoughts in a journal? How might the hospital/institutional infrastructure and norms affect their learning?We had a unique opportunity to answer some of these questions by comparing the themes from reflective journals of physiotherapy students in the United Arab Emirates (UAE) with those taken from the above mentioned studies ,13. condAlthough both the UAE and Canada have many of the conveniences of modern society, there are major societal and cultural differences. The students in both countries have similar standards of living and have ready access to education and health services. Many of the health problems of modern society – diabetes, heart disease, arthritis, cancer, and trauma from traffic accidents – are common in Canada and the UAE. However, Canada is a democracy with a predominantly Christian and European population, whereas the UAE is a system of emirates with a population comprising mainly Arabic and Eastern cultures and where the Muslim religion predominates. The students in the two countries might be expected to have different values and beliefs, and different societal norms that could affect their reflections.The purpose of this study was to describe the reflections of physiotherapy students from the University of Sharjah during their clinical placements. A second objective was to compare the themes generated from the Sharjah data to the themes from the previous two studies on reflections of McMaster students during their clinical placements. Because the students in the two programs have similar socioeconomic status and similar curricula, it was anticipated that differences in reflections would be due to differences in culture and life experiences related to the cultures.This qualitative study was conducted during the students'clinical placements in a summer semester of the Physiotherapy Program at the University of Sharjah, United Arab Emirates, a program developed in affiliation with McMaster University, Hamilton, Ontario, Canada.The baccalaureate physiotherapy program in the UAE comprises one pre-professional year (year 1) and three professional years (years 2–4) of study. To be admitted to the program, students must have completed the UAE Secondary School Scientific Certificate , or equivalent program accredited by the Ministry of Education. They must have also scored at least 500 on the Test of English as a Foreign Language (TOEFL).While in the physiotherapy program, students need to complete university required and elective courses. Basic science courses are offered in years 1 and 2. Physiotherapy courses in years 2–4 include small-group, problem-based learning tutorials and clinical skills laboratories based on problems/ case scenarios enhanced with resource sessions. The male and female students are taught separately in discrete areas of the campus.Six senior physiotherapy students  with a mean age of 22.6 years (range 20.5 to 23.1) and 15 junior, female students with a mean age of 20.1 years (range 19.9 to 22.8) formed the subject group of this study. These students were respectively from the first and second cohorts of students admitted to the new physiotherapy program at the University of Sharjah. All students spoke Arabic and English. The senior students had completed the academic component of year 3 and were in their third clinical placement, which provided them with six weeks experience primarily with clients with cardio-respiratory conditions. The junior students had just completed two academic semesters of year 2, and were in their first clinical placement – four weeks with clients with peripheral musculoskeletal conditions. Students practiced in five different clinical sites in the UAE, under the supervision of clinical preceptors and university instructors/coordinators. The study was approved by the Ethics Committee of the College of Health Sciences, and all participating students signed an informed consent.Two instructors were involved in reading, coding and identifying the major themes that emerged from the students' reflective journals. One instructor (HL) from the University of Sharjah, Department of Physiotherapy, had taught one course to the junior group and two courses to the senior group. The second instructor (JW) from McMaster University Physiotherapy Program, had taught one course to the senior group as a consultant to the program. The two instructors were not involved with the students during their summer clinical placements. A third evaluator (AAS) independently read all the journals. She was an instructor in the Sharjah program, a UAE national, and fluent in both Arabic and English. She had taught two courses to the junior students. Her analysis was used to confirm the interpretation of the entries made by the other two evaluators who were both from Western cultures.The students were asked to write a reflective journal, a new exercise for all students. This assignment was one of the requirements for a Pass mark in their summer clinical placement. The students were required to make one entry per week for the duration of their clinical placement (four weeks for junior students and six weeks for senior students), and to submit the journal weekly to the university instructor involved in their clinical supervision. However, the instructor did not provide any feedback to the student at that time. The students were instructed to include the following in their reflective journals:Reflective journals should include observations, impressions, and reactions to what you have learned in the academic portion of the semester and how you are applying it to clinical practice. How does the clinical experience change what you thought, felt, or did in the past, and how you may respond in the future? You are expected to write at least one journal entry per week during your clinical placement.1. Describe the learning event, issue or situation. Describe prior knowledge, feelings or attitudes with new knowledge, feelings or attitudes.What happened?2. Analyse the learning event, issue or situation in relation to prior knowledge, feelings or attitudes.What was your reaction to the learning event, issue or situation? Your response may include cognitive and emotional reactions. Why did it happen?3. Verify the learning event, issue or situation in relation to prior knowledge, feelings or attitudes.What is the value of the learning event, issue or situation that has occurred? Is the new knowledge, feeling or attitude about the learning event, issue or situation correct?4. Gain a new understanding of the learning event, issue or situation.What is your new understanding of the learning event, issue or situation?5. Indicate how the new learning event, issue or situation will affect future behavior. Determine the clarification of an issue, the development of a skill or the resolution of a problem.How will you approach the same or similar event, issue or situation in the future?The method described by Coffey and Atkinson  was usedThe following two methods were used to further validate the analysis and interpretation of the data. 1) The third evaluator independently read and labelled all journal entries to confirm the interpretation of the content. 2) The themes established by the evaluators were presented to two students who had completed the journals, and who volunteered to provide feedback on the authors' interpretation of their reflections.Themes for the present study were developed without direct reference to the themes found in the Canadian studies ,13. The The two initial evaluators  came to a consensus on four themes concerning the clinical learning experience of the students. These themes did not change as a result of the coding of the third reader (AAS) or the feedback from the students. However, these additional data occasionally resulted in a change in emphasis of some of the content within a theme. Whenever this happened, the differences are described.All students outlined the positive impact and value of their learning from the clinical placement. They discussed events and issues that fit into the following four themes:1. Professional behaviors: skills and attitudes, scope of practice, time management, professional boundaries, and respect for clients and colleagues.2. Awareness of learning: clinical versus academic learning, gaining of important new knowledge, self-directed and life-long learning, and client as a source of learning.3. Self-development and shift to a more client-oriented focus over the time of the clinical placement.4. Identification and analysis of ethical issues.Each of these themes is discussed below with quotes provided for illustrative purposes.The students appeared very aware of their own behavior and that of other health professionals and students. They reflected on how they should behave as professionals, but this also included how they should behave in general. The events that precipitated discussion on this theme included actions of themselves and other health professionals and included what they regarded as both positive and negative behaviors. They referred to physiotherapists and other health professionals who helped the students and who modelled good professional behavior. They also described some situations where health professionals did not show sufficient respect for their clients or their colleagues, or did not provide 'best practice'. They commented on professional boundaries, being concerned about the client-therapist relationship becoming too personal and limits to the scope of physiotherapy practice. In addition, the students noted that self-directed learning and life-long learning were professional responsibilities. This topic is discussed under the next theme, Awareness of Learning.The students providing feedback emphasized that professional behavior (both negative and positive) was also learned from patients who might show their appreciation, be patient or uncooperative, or provide specific feedback regarding the student's communication or skills. The students also provided examples of "crossing boundaries" in terms of professional competencies and relationships with their patients and supervising therapists.One patient asked me angrily where [were the physiotherapists]? I responded politely to wait until they finish their work with the patients... I calmed her down and asked her to be patient...I really felt that the [physiotherapist] and I should maintain and manage our time...It is really a big responsibility we have. The child came crying and refusing to look at her forearm. We should respect the emotional status of the child and try to understand it because the child has been through a lot. The accident..., surgery,...and functional limitations...The stress should be controlled before giving physiotherapy...I should be patient and try to give the most enjoyable set of exercises to gain the child's trust. Sometimes patients are shy to tell that there is no benefit from the treatment programme. So I asked the patient if he feels any improvements and if not to inform me in order to change the plan of treatment. The patient responded very well to that. Although the entire reflective journal assignment was designed to have students reflect on their learning, this theme was included to capture the many components of learning discussed by the students. They gained knowledge, but also commented on the unique aspects of clinical learning. Clinical experiences provided an opportunity to apply and enhance their academic learning. Junior students particularly noted how observing surgery helped them understand their anatomy, or how working with "real patients' was not as straight forward as their academic learning. The senior students realized the need to be life-long and self-directed learners because they were always encountering new conditions/situations and they wanted to provide their clients with 'best practice'. Students acknowledged the clients as a source of knowledge, and noted that clinical situations provided a stimulus to learn.In the feedback session, students particularly emphasized how the clinical experience had enhanced their learning "a lot!" They also indicated that clinicians reinforced the need for life-long learning because of the constant changes in science. Some therapists welcomed new knowledge from students, for example, learning about outcome measures, while others indicated they did not have the time.It was really good to have a mix of people [clinicians] and to learn from their experience in the work field...Each one of them has given us a new way to deal with patients and... also a new experience to add to our future knowledge... The learning is not finished...We have to keep our hands on the best information available and the best experience. [I] try to apply the theoretical bases learned at the university. [I] interact with patients and take their histories, try to know their goals and what they need from physiotherapy. I try to invent new materials for the exercises. In my opinion, when we apply what we learned in the theory, the information sticks in our minds and is hardly forgotten. The theme of self-development was derived from the content of individual student entries, but was also seen in the change in the reflections as students proceeded through their placements. Often at the beginning of the clinical encounter, the students were unsure of themselves, but they gained confidence as they worked with the client and/or learned from their colleagues. The change across the journal entries was even more noticeable. In many of the early entries, students described their own emotional response to a client or a situation. They were concerned about their own performance, and about what they were learning from the placement. They indicated how an event had an impact on them. The later entries were more client-centred. The focus was more on how the client responded to an event, or how helpful a physiotherapist was. Students also referred to an increased confidence, the more experiences they had.It became evident from the third reader's summary that students were aware of how their professional behavior, outside direct patient care, could ultimately have an impact on the patient. They referred specifically to the maintenance of knowledge and skills and to relationships with other health professionals.Students in the feedback session referred to their initial lack of confidence which improved during the clinical placement. They added, however, that it was difficult to demonstrate the "correct confidence level" to their instructor/evaluator, particularly when students were occasionally told they were "over-confident". They felt that self-development was affected by behavior of other physiotherapy students, method of feedback from instructors and whether their clients improved or not.The quote immediately below illustrates how a student confronted his/her uncertainties during one client encounter. The next quote concerns a student's acknowledgement of the change in his/her confidence with increasing experience.First patient with tetraplegia...What if I do something wrong?...First, worried... and unable to do anything, I observed other colleagues... I could do it too! A one-month old infant was in the ICU. I thought I would not be able to touch her as usual, but things are now different. I did all the treatment... with my hands with no hesitation and I was not scared... My reaction was totally changed and it is becoming more regular... I am happy... I trust myself better and it is increasing with time...and will increase the more cases I face. In the quotes below, the student initially talks about his/her own emotional response to a patient, but later in the placement discusses the need to adapt to patients' situations and provide them with the necessary treatment. In both journal entries, the student discusses a client with a novel and somewhat "unpleasant" condition. However, the student reacts differently in week 5 than week 1.At that moment I was shocked because I was not familiar with the neurology cases. When the physiotherapist asked me to help...I hesitated in the beginning. ...This situation had a bad effect on me ... I was depressed and I couldn't do any single movement I have seen a patient who was always under sedation due to his pain and his anger which was there almost all the time. ... I was always confused ...not knowing what to do for him and what is the best for him. ... Such a case made me think [very] much before any and each simple movement. ... So this made me prevent [other] problems such as ulcers, and invent any simple movement just to change his lying position As the quotes illustrate, the student was concerned about what to do in both cases, but by week 5 was able to overcome the emotional response, problem solve about the management of the client, and take action.Students commented on what they believed to be ethical or unethical behavior of the health professionals they were in contact with. They noted when informed consent was not obtained from clients and they discussed ways to get consent from uncooperative clients. They talked about having respect for their clients, and maintaining confidentiality. They noted "ethical" and "unethical" behavior in the same professionals under different circumstances.The staff is cooperative and helpful, respect each other, but there is no confidentiality or respect of time with patients. They were concerned when individuals  were being treated unfairly or differently because of their health condition or socio-economic status. In some cases they thought that the client might be harmed by the practice. One student was concerned about how to 'tell the truth' to a client who had a poor surgical result possibly due to the questionable practice of the surgeon.One student described an event where a therapist refused to see a patient when she changed her appointment time, but treated another patient who did not have an appointment. The student felt that the latter patient was treated because she was a "VIP (very important person)".I felt sad for the [person who did not receive treatment], and I said to myself, where are the ethics? I learned before ... that all patients should be at the same level without any bias. ...all persons who work in health care fields specially the professional therapists must [follow] the code of ethics because they have humans in their hands. ... I will delete the word VIP from my life work. In the feedback session, the students also indicated how clients could be discriminated against because of their health condition and the therapist's level of confidence. Therapists might provide less treatment to a child if they felt uncomfortable treating children. They might leave a student to treat an older person if they felt the management of older persons was less important or less effective.The students agreed with the four themes and did not think that any major concept was omitted in this analysis. However, they added some insight to the content and interpretation of each theme. The students emphasized that many people affect their learning – university instructors, therapists, other health professionals, students and patients/clients. They felt that the information derived from the analysis of the reflective journals was very important. They strongly recommended that the results be disseminated to clinicians, instructors and future students to increase their awareness of the learning process and help the students to improve.The content of the journals indicated that students from both universities were comfortable with the reflective process. They frequently shared quite personal information, including their positive and negative emotions, concerns about their own behavior, and a discussion of their own beliefs. Some students in both countries, simply described an event and provided minimal personal reflection, but these students were in the minority.Table In the area of professional behavior, students in both countries commented on role models (both positive and negative) in the clinical environment, as well as on their own behavior or response to clients. Both groups demonstrated a respect for their clients and saw their role as going beyond physical treatment. Canadian students mentioned advocacy in the community while students in the UAE discussed supporting their clients within the hospital milieu.Both groups talked about the uniqueness of learning in clinical placements. The clinical experience enhanced and confirmed their academic learning, and stimulated them to learn more. They enjoyed learning new information and using their knowledge to help others. They were anxious when they "didn't know what to do", but expressed increased confidence with each experience. They appreciated constructive feedback on their performance from clinical supervisors, health professionals and clients. Both groups acquired new skills related to direct client care (therapeutic techniques and communication) and administrative activities . Only Canadian students referred to writing reports and billing, tasks that students in the UAE were not expected to do in their clinical placements. Both groups, however, broadened their view of the scope of practice of physiotherapy.Students from both countries noted ethical issues pertaining to inequalities in their respective health care systems. Canadian students were concerned about the shortfall of resources , and about differences in health care provided to private clients or those funded through provincial health insurance. Emirati students noted that the health or societal status of a client could affect their treatment within the health system.Physiotherapy students from Sharjah demonstrated the ability to reflect on their clinical practice at the highest level (discussion of how experience will impact on future behavior). They described not only the learning event, but also their emotional and cognitive reactions to it. They analysed the situations to arrive at new understandings and considerations of how they would behave differently in the future.Like the students in the Canadian studies, the subjects in the present study described three types of learning that are considered essential for clinical decision-making. These are propositional , professional craft (learning from experience), and personal knowledge (knowledge of self and unique frame of reference) . The stuThe reflection on one's own development is a common theme in the reflections of health professionals. Jensen and Denton referredJensen and Paschal suggest One other study  examinedLanguage might have been a factor in the reflections of the students from the UAE, however. Although the students worked in Arabic in the clinical environment, they conducted their physiotherapy education and wrote their journals in English. It is possible that some students had difficulty accurately expressing their reflections in English, and that there was some misinterpretation by the evaluators. However, two of the evaluators  knew the students well and were used to their writing. One of them (AAS) was a UAE national and fluent in both English and Arabic. When the two initial evaluators  worked together in Phase 1 to determine coding methods, part of the discussion was on how to interpret the "second-language writing" of the journals. In spite of these precautions, it is still possible that the themes may have varied somewhat if the journals had been written in Arabic and interpreted by Arabic-speaking evaluators.Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others.The author(s) declare that they have no competing interests.HL and JW were involved in the conception and design of the study, in the qualitative analysis, and in the writing of the manuscript. HL coordinated the data collection. AAS assisted with data analysis. All authors were involved in the preparation of the manuscript, and read and approved the final version.The pre-publication history for this paper can be accessed here:


By default, all published works are copyrighted. Creative Commons provides means for authors to share their work more freely When did calling a lawyer become part of the scientific process? It hasn't officially, of course. But as a generation of researchers has grudgingly come to know, navigating the legal red tape of universities, corporations, and publishers is an inevitable part of the practice. Whether seeking access to information or sharing one's own findings with others, scientists increasingly find themselves having to ask an intermediary's permission.This “ask first” culture has developed at just the moment when technology has opened vast new possibilities for collaboration and information-sharing. The timing is not coincidental. Policymakers, under the influence of lobbies defending pre-digital business models, have reacted to new technology with ever more extreme intellectual property laws. The result is a legal regime tailored to a powerful minority but ill-suited to a number of other constituencies—scientists and scholars chief among them—that thrive on openness. Worries over broad notions of “piracy” and “asset management” have insinuated themselves into fields where those terms, until recently, held no meaning.The Public Library of Science (PLoS) is at the vanguard of a growing cross-disciplinary movement to counteract this trend by demonstrating that voluntary models of open publishing are not only viable, but crucial to scientific innovation. Yet PLoS’ goal of “immediate, unrestricted access to scientific ideas, methods, results, and conclusions” is not immediately compatible with the stringent rules of copyright, which apply fully and automatically to all published works, by default. 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With a portion of a new US$1 million grant from the Hewlett Foundation , we hope to build the Science Commons, a branch of the organization dedicated to bringing a measure of reason, and restraint, to the legal thicket that has grown around scientific research.Like PLoS, Creative Commons’ goals and methods are designed to make the most of the opportunities created by new communications technology. But, also like PLoS, our inspiration reflects the wisdom and optimism of the Enlightenment as much as that of the Digital Age. We are trying to restore the sense of legal moderation that policymakers of a bygone era, heavily influenced by the philosophy of the first scientific revolution, understood would “promote the progress of science and the useful arts,” as the U.S. Constitution puts it.“Knowledge [is] not the personal property of its discoverer, but the common property of all,” wrote Benjamin Franklin, the great cosmopolitan, polymath, and patron saint of innovation. As we enjoy great advantages from the inventions of others, we should be glad of an opportunity to serve others by any invention of ours, and this we should do freely and generously.”Franklin, who knew a thing or two about both publishing and science, never practiced law.


While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRbf loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRbf function; Rbf inactivation by T121, a fragment of SV40 T antigen that binds to and inactivates pRbf proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb Inactivation of the three retinoblastoma genes in the mouse mammary gland provides a new animal model for human breast cancer INK4a), cyclin D1 (CCND1), or cyclin-dependent kinase 4 (CDK4), is observed in the majority of human sporadic cancers  pathway activity, resulting from defects in pRb itself, cyclin-dependent kinase inhibitor 2A  inactivation in multiple cell types of the mouse by dominant expression of T121, a truncation mutant of simian virus 40 (SV40) T antigen that inactivates all three pRb-related proteins  regulates the apoptosis, and reduction in its function accelerates tumor growth  transcriptional signals  and a line 3 F1 mouse shows that the impact of Rb perturbation is severalfold. Compared to an age- and parity-matched control tissue, the normal architecture of the lactating mammary tissue is disturbed. In contrast to normal tissue where acini consist of a single layer of secretory epithelia with milk-filled lumen  from single-pregnancy females of the line 2 founder . Transgene expression was induced by a single pregnancy, and mammary glands were examined on lactation day 1. As expected, nontransgenic mammary glands showed no appreciable apoptosis regardless of p53 status  (wingless-related murine mammary tumor virus (MMTV) integration site 1 (Wnt-1)  mice were assessed for the expression of nuclear PCNA. Unlike the tumors initiated by activated Ras or Wnt-1, p53 heterozygosity or nullizygosity had no significant impact on the level of cell proliferation   or wingl (Wnt-1) , inactivferation C. This e121 in nonmammary tissues, further characterization focused on line 3. For this line, the median time following initial transgene induction until a palpable tumor appeared was 10 mo, and within 16 mo, all mice developed palpable tumors  failed to nurse pups because of lactation defects and developed mammary tumors after multiple pregnancies. Because line 4 mice expressed Te tumors A. Interee tumors  was redun = 3; data not shown), which indicates that the transgene and not its insertion caused tumorigenesis. With two exceptions, line 3 121 WAP-Tmice, regardless of p53 status, developed a single palpable tumor . A single mouse with either two or three palpable tumors was also observed in both p53 +/+ and +/− backgrounds. At least one additional nonpalpable tumor was visible during necropsy in approximately one-third of all tumor-bearing mice. While the two lower-expressing lines, lines 1 and 2, were able to nurse pups and appeared grossly normal, both had hyperplastic lobular alveoli associated with increased levels of proliferation and apoptosis. However, females from low-expressing lines did not develop adenocarcinomas after at least four pregnancies and 20 mo of age  (data not shown).The median onset for mammary tumors in line 4 was 14 mo , consistent with published reports (−/−p53 mice (n = 4) succumbed to these tumors by 4 mo of age, prior to developing palpable mammary tumors, so acceleration of this phenotype could not be assessed. In +/−p53 animals, mammary tumors were detected significantly earlier (see p = 0.0003) compared with +/+p53 mice. Furthermore, once palpable, 121;p53+/−WAP-T tumors grew significantly faster than the p53 wild-type counterparts  and suggests that the reduction of p53 activity drives tumors to the adenocarcinoma phenotype. Taken together, these studies indicate that p53 heterozygosity leads to increased tumor growth rates and/or progression and may alter the spectrum of tumor morphologies.Since 75% of the apoptosis induced by Rb reports . All p53arts see B. The ob121;p53+/−WAP-T mammary tissue compared with that of 121;p53+/+WAP-T mice, it was possible that p53 heterozygosity was sufficient for tumor acceleration. To assess whether this was the case or whether there was selective pressure for p53 inactivation during tumor progression, real-time PCR analysis was employed to determine the status of the wild-type p53 allele in 121;p53+/−WAP-T tumors. Of ten tumors, eight showed loss of the wild-type p53 allele : chromosome-based CGH (cCGH) . Among samples tested by both methods, there is strong concordance among large chromosomal changes, encompassing multiple cytological bands to whole chromosome lengths. For example, there is apparent whole chromosomal duplication of Chromosomes 6 and 15 in tumor C and of Chromosomes 8 and 18 in tumor H, monosomy of Chromosome 10 in tumor J, and loss of X Chromosome in tumors E and H . Making comparisons among imbalances spanning shorter chromosome lengths was more difficult, mainly due to the challenge of reconciling cytological and physical maps. Furthermore, technical limitations may account for real differences between the two assays: small imbalances detected by one to several bacterial artificial chromosome (BAC) clones are irresolvable by cCGH; on the other hand, the relatively low density of BAC clones may not adequately sample smaller regions detected by cCGH. Nevertheless, on average, about five imbalances per tumor were detected by cCGH. This number is comparable to the number of changes observed in myelocytomatosis oncogene (c-myc)-induced mouse mammary tumors (Brca1)-deficient mouse tumors (8.0) and more than v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER2/neu)-induced tumors (2.8)  .Here we report that loss of pRb family function in mammary epithelium predisposes to malignant adenocarcinoma. Using a single transgenic allele, we have thus far inactivated the pRb pathway in several cell types in the mouse: brain choroid plexus epithelium, astrocytes, and mammary epithelium. In each case, despite the marked differences among these divergent cell types, pRb inactivation causes a similar response, initially evoking increased proliferation and apoptosis and, ultimately, predisposing to tumorigenesis .f inactivation. Previous models using wild-type large T antigen . Importantly, the Rbf deficiency-induced apoptotic response and inhibition of tumor progression are not universally dependent on p53. In astrocytes, we recently showed that PTEN, and not p53, modulates these same responses to Rbf inactivation. In contrast to the p53-dependent apoptosis of mammary epithelial cells in response to pRbf deficiency, apoptosis associated with normal mammary involution subsequent to lactation does not require p53  and brain (85%) epithelia is p53-dependent as determined by comparing +/+p53 and −/−p53 tissue. However, while p53 heterozygosity had no impact on the level of apoptosis in the brain epithelium, in the mammary gland the level was reduced by half in +/−p53 tissue. Given that apoptosis is the basis for selective inactivation of p53 in the brain tumor model , the mechanism of loss may be distinct. Chromosome loss clearly explains p53 LOH in the brain carcinoma model . One explanation for this observation is that, because the brain is a vital organ, animals succumb to their illness when the brain tumor is at a relatively earlier stage at which fewer changes have accumulated. However, chromosome content of choroid plexus tumors passaged further in xenografts remained stable . The converse experiment, analyses of early mammary tumors subsequent to p53 loss, will be required to determine the kinetics of chromosomal changes in this tissue.Genomic instability is a hallmark of most human solid tumors, and a widely held view is that p53 represses instability to suppress tumorigenesis, although evidence for this activity has been mostly correlative. Contrary to this model, we demonstrated previously that in the absence of p53 activity in brain epithelia, tumors progress without chromosomal instability; except for Chromosome 11 loss, in a  diploid . Here wef, leads to mammary adenocarcinoma , along with the further characterization of 121WAP-T tumors, should help provide additional insights into human breast cancer biology.The y cancer . In addiy cancer , and as dl1137′t plasmid  and therefore are designated as B6;D2-Tg(WAP-T121) Tvd. To study the effect of background differences, 121WAP-T males were backcrossed to BALB/cJ (Jackson Laboratory) female mice. To increase sample size, tumor onset analysis for BALB/c background mice combined data for N6 (n = 6), N7 (n = 1), and N9 (n = 4) generation mice. For tumor induction, female mice, unless noted as virgin, were housed with male mice to maximize the number of pregnancies, because WAP promoter activity is lactation-dependent  and was plasmid . We targactation  . p53 genotypes were determined by PCR using two reactions  and a second that amplifies the endogenous p53 allele . Cycling parameters were the same as the above 121WAP-T reaction. We performed the cross −121;p53+/−WAP-T × +121;p53+/−,WAP-T and transgenic female mice that were +/+p53, +/−p53, or −/−p53 were used for analyses while nontransgenic littermates served as controls.To study the effect of eactions , one tha® homogenizer . Total protein (10 μg) was electrophoresed through a 15% polyacrylamide denaturing gel and then transferred to nitrocellulose membrane . Alternatively, for low-expressing lines, immunoprecipitation was performed prior to electrophoresis as previously described  for histopathological examination. Apoptosis levels were evaluated by TUNEL assay  containing AmpliTaq Gold polymerase, deoxynucleoside triphosphates, and PCR buffer. The cycling conditions were 50°C for 2 min and 95°C for 10 min for 1 cycle, and 95°C for 15 s and 60°C for 1 min for 40 cycles. The reactions were performed using an ABI 7700 Sequence Detection system (Applied Biosystems), and the data analyzed using Sequence Detector 1.7 (Applied Biosystems) and standard protocols (http://www.appliedbiosystems.com). The copy number of each sample was determined by calculating ΔΔCt based on the formula ΔΔCt = [sample Ct(p53) − sample Ct(β-actin)] − [p53+/+ control Ct(p53) − p53+/+ control Ct(β-actin)], where Ct is the number of cycles required to reach a threshold based on linear amplification. Analyses of standard samples  indicate copy numbers of 2, 1, and 0 are indicated by 2-ΔCtn values of greater than 0.6, 0.15–0.6, and less than 0.15, respectively. Standard samples analyzed along with experimental samples confirmed the accuracy of these assignments.Quantitative real-time PCR analysis was performed using a TaqMan approach on DNA derived from terminal tumors to determine the status of the wild-type escribed . The priKaplan–Meier survival analysis was used to determine median tumor latencies , and the Log-Rank  test was performed to evaluate significance. The equivalence of tumor morphology distributions was tested using the Fisher–Freeman–Halton's exact test.2O. cCGH was performed as described in Cot-1 DNA to either normal metaphase chromosomes from cultured cells (cCGH) or microarrayed BAC clones containing mouse genomic DNA (aCGH). Nonequivalent fluorescence intensities indicate relative imbalances of genomic DNA. Aneuploidy and partial chromosome gains and losses are detectable by cCGH with approximately 10 Mb resolution. Graphical output of cCGH data was generated using the National Cancer Institute and National Center for Biotechnology Information Spectral Karyotyping SKY and Comparative Genomic Hybridization CGH Database (http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi). For aCGH, approximately 1,500 BAC clones span the entire mouse genome with 2–20 Mb spacing. Tumor DNA and normal DNA were sex-mismatched; thus, the X Chromosome served as an internal control, while normal tail DNA was used as a negative control. Gains or losses were scored based on tumor:normal fluorescence ratios that were greater than 1.25 or less than 0.75, respectively.Genomic DNA was extracted from end-stage tumors (1 cm in diameter) or tails using a DNeasy genomic tip  and further purified by proteinase K digestion followed by phenol/chloroform extraction, ethanol precipitation, and resuspension in sterile HBrca1 (LocusLink ID 12189), CDK2 (LocusLink ID 1017), CDK4 (LocusLink ID 1019), CDK6 (LocusLink ID 1021), c-myc (LocusLink ID 17869), cyclin D1 (LocusLink ID 595), cyclin E (LocusLink ID 898), E2F (InterPro ID IPR003316), HER2/neu (LocusLink ID 13866), histone deacetylase (LocusLink ID 3065), p16INK4a (LocusLink ID 1029), p53 (LocusLink ID 7157), p107 (LocusLink ID 5933), p130 (LocusLink ID 5934), p300 (LocusLink ID 2033), PCNA (LocusLink ID 18538), pRb (LocusLink ID 5925), PTEN (LocusLink ID 5728), v-Ha-ras (LocusLink ID 3265), WAP (LocusLink ID 22373), and Wnt-1 (LocusLink ID 22408).The accession numbers for the genes and gene products discussed in this paper are www.ncbi.nlm.nih.gov/LocusLink/ (LocusLink), and www.ebi.ac.uk/InterPro/ (InterPro).These databases may be found at


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