Many fundamental cell processes, such as angiogenesis, neurogenesis and cancer metastasis, are thought to be modulated by extracellular matrix stiffness. Thus, the availability of matrix substrates having well-defined stiffness profiles can be of great importance in biophysical studies of cell-substrate interaction. Here, we present a method to fabricate biocompatible hydrogels with a well defined and linear stiffness gradient. This method, involving the photopolymerization of films by progressively uncovering an acrylamide/bis-acrylamide solution initially covered with an opaque mask, can be easily implemented with common lab equipment. It produces linear stiffness gradients of at least 115 kPa/mm, extending from ∼1 kPa to 240 kPa (in units of Young's modulus). Hydrogels with less steep gradients and narrower stiffness ranges can easily be produced. The hydrogels can be covalently functionalized with uniform coatings of proteins that promote cell adhesion. Cell spreading on these hydrogels linearly correlates with hydrogel stiffness, indicating that this technique effectively modifies the mechanical environment of living cells. This technique provides a simple approach that produces steeper gradients, wider rigidity ranges, and more accurate profiles than current methods. The mechanical properties of the extracellular matrix (ECM) contribute to the regulation of many important cell processes that determine cell fate and function In order to study these processes, it is necessary to develop methods to fabricate defined-stiffness gradient profiles on hydrogel substrates to which ECM components are covalently bound. One commonly used scheme is to vary the crosslink density of a polyacrylamide (PAA) hydrogel which underlies the surface upon which cells are deposited. The crosslink density has often been modulated by varying the ratio between acrylamide and the crosslinker bis-acrylamide, and initiating the reaction with the soluble initiator TEMED Here, we introduce a new method which produces inexpensive, high gradient matrices whose stiffness profile at the micrometer scale can be tightly controlled. This method allows fabrication of well defined stiffness profiles in PAA hydrogel matrices. Subsequent addition of a layer of ECM protein is unaffected by the varying stiffness of the underlying hydrogel. This method can be easily implemented with common lab equipment and produces stiffness gradients and rigidity ranges higher than the ones prepared with microfluidic devices, allowing for precise control of the gradient profile at the micrometer scale. Although the scheme described here has more general applicability, we focus on PAA hydrogels because they have been widely used as a matrix support for cells Stiffness gradient hydrogels were obtained by irradiating an acrylamide/bis-acrylamide solution (containing the photoactivatable initiator Irgacure) with a varying dose of light, achieved by covering the solution with an opaque mask and then moving the mask at controlled speed to progressively uncover the gel solution . In thisδ<0, the cantilever was not in contact with the hydrogel. As the cantilever pyramidal tip contacted the hydrogel at δ = 0 (arrow), the force recorded by the cantilever started to increase, exhibiting a non-linear relationship with the indentation. This non-linear relationship is caused by an increase of the contact area as the pyramidal tip indents the sample. By fitting the contact part of the force-indentation curve to the 4-sided pyramidal indenter model E) as a function of position on the gel surface.Thin PAA hydrogels polymerized in this manner were characterized with Atomic Force Microscopy (AFM). An example of a force-indentation curve recorded from the hydrogel is shown in E, of a hydrogel produced by a linear UV irradiation profile. At the beginning of the fabrication process, the opaque mask was placed at gel position x = 0 mm . Depending on the mask speed, we obtained different gradient slopes while maintaining the same rigidity range  display a similar correlation between spreading and stiffness with a slightly higher stiffness sensitivity . Notably, cells located at the stiffer part of the hydrogel had a spreading comparable to cells seeded on glass when also coated with fibronectin.We seeded early passage fibroblasts (NIH3T3 cell line) on our stiffness gradient hydrogels. To maximize the stiffness range accessible per field of view, we used a hydrogel whose stiffness varied linearly from ∼1 kPa to 240 kPa across 2 mm and then non-linearly to 360 kPa across an additional 2 mm. Phase contrast images reveal that cell spreading strongly correlates with hydrogel stiffness , panel aChanges in cell spreading correlate with changes in cytoskeleton organization . Cells lWe have developed a new method to fabricate hydrogels having well defined, linear stiffness gradients. AFM reveals that our technique produces stiffness gradients up to 115 kPa/mm, with a rigidity range that varies linearly from ∼1 kPa to 240 kPa, followed by a region of increasing but non-linear stiffness change in the 240–360 kPa range. Fabrication of gradients is difficult in this upper range which is near the limit of stiffness possible with this material. Hydrogels with less steep gradients and narrower stiffness ranges can easily be produced as well. The hydrogels can be covalently functionalized with proteins that promote cell adhesion. Immunofluorescense against fibronectin reveals that the ligand density is independent of the stiffness gradient. The spreading of cells attached to stiffness gradient hydrogels correlates linearly with hydrogel rigidity. Cell spreading strongly differs in regions with different rigidity, indicating that hydrogels produced with this technique effectively mimic the mechanical environment of living cells.The method presented here utilizes the photoinitiator Irgacure 2959 to fabricate elasticity gradients in polyacrylamide hydrogels. When illuminated with UV light, Irgacure molecules decompose into free radicals that initiate polyacrylamide polymerization Other methods have been developed to obtain PAA hydrogels with a stiffness gradient that covers part of the physiological range. As mentioned earlier, the simplest technique is to place two droplets having differing concentrations of acrylamide/bis-acrylamide next to each other on a common coverslip By contrast, our method, allows us to create accurate linear profiles with a 3-fold steeper gradient and a 4.5-fold larger rigidity range than those obtained with microfluidic gradient generators. In contrast to hydrogels produced with microfluidic techniques, our method does not need access to clean room facilities and costly equipment. In addition, the fabrication process is faster, simpler, and might be extended to 3D cell culture. To our knowledge, our fabrication method is the easiest and most precise way to obtain hydrogels with a high stiffness gradient and a broad rigidity range.et al. utilized this approach to crosslink a methacrylated hyaluronic acid system with both DTT and UV polymerization. The sliding opaque mask was used to control the radical polymerization in further stiffening the gels, achieving an average gradient of 6.5 kPa/mm and a stiffness range from ∼3 to ∼100 kPa over a 15 mm gel et al. used a similar opaque mask to degrade PEG gels with photodegradable crosslinkers linear gradient hydrogels requires varying mask speed, a constant mask displacement produces hydrogels with a steadily decreasing rigidity  on the PAA hydrogels are hard to obtain. This difficulty possibly occurs because the photopolymerization of acrylamide depends not only on the amount of light received, but also on nucleation mechanisms of polymerization and perhaps also on the diffusion of the photoinitiator . This limitation may be overcomed by the use of another photocrosslinkable polymer, such as styrenated gelatin, which has been shown to polymerize in tight correlation with irradiation profile in vivo, as the resulting hydrogels provide linear rigidity gradient profiles that cover most of the stiffness range physiologically relevant to cells. When we seed cells on our hydrogels, we observe that cell spreading correlates with substrate rigidity. This positive correlation has been extensively documented in various cell types The method presented here allows us to reproduce most of the conditions observed 2, as measured with a calibrated photodiode . In general, the time needed to crosslink the acrylamide/bis-acrylamide solution depends on the amount of Irgacure used and the intensity of the UV irradiation, as well as its wavelength. The irradiation of the sample is minimally dimmed by the coverslip as we measured a 50% cut-off at 310 nm by the coverslip alone . The optimal Irgacure 2959 absorbance peak is 280 nm and, at 310 nm, the absorbance is 2/3 of the peak. So our use of a longer wavelength lamp slightly increases the exposure time needed to crosslink the solution. In our experimental setup, we found that 4 min was sufficient to crosslink the acrylamide/bis-acrylamide solution and obtain a hydrogel with stiffness of 240 kPa.Preparation of matrices is carried out by photopolymerization of an aqueous solution of acrylamide and bis acrylamide . Acrylamide concentrations varied between 8–15% and bis-acrylamide between 0.48–1%. To polymerize the solution, we add 0.5 mg/ml of Irgacure 2959 , place the solution between two glass coverslips, one of them activated with 3-Aminopropyltriethoxysilane and glutaraldehyde. We irradiate the solution with a conventional ultraviolet (UV) lamp  held above the solution at a distance of 6 cm. The light flux delivered to the sample was 3.9 mW/cmE) gradient that changes from 240 kPa in the most irradiated region to ∼1 kPa in the least irradiated one. The use of the sliding mask simplifies the fabrication setup, as there is no need for UV light source collimation. We use a conventional microscope stage  to reach micrometer scale resolution in mask displacements. The irradiation is performed on an inverted microscope  which enables us to image the irradiation profile while the mask is uncovering the acrylamide-bisacrylamide solution. Custom LabView™ software  allows us to modulate the mask speed. Hydrogels are stored in water for two days prior to protein coating, to remove unreacted photoinitiator molecules.To obtain hydrogels having a stiffness gradient, we irradiate the acrylamide/bis-acrylamide solution with a spatially-varying light source. This light gradient is created by a programmable linear motion stage that progressively uncovers the acrylamide/bis-acrylamide solution by moving an opaque mask at a controlled speed Following irradiation of an acrylamide/bis-acrylamide solution, we found that the stiffness of the resulting hydrogel depends non-linearly on the total amount of light received see . As a reE). Values of the spatially-dependent moduli of the stiffness-gradient hydrogels were measured by AFM, using a Bioscope Catalyst® NanoScope® V device  attached to an inverted optical microscope . The gels were probed with a V-shaped cantilever  whose spring constant was calibrated by the thermal fluctuations method F-z) curves  by monitoring F and z while the piezo translator was ramped forward and backward at constant speed . Each experimental F-z curve was fitted to the four-sided pyramidal indenter model E is the Young's modulus,ν is the Poisson's ratio, θ is de semiincluded angle of the pyramidal indenter, and δ is the indentation depth. The parameter ν is assumed to be 0.5 , and the indentation depth is calculated as δ = z−z0−d, where z0 is the tip-gel contact point. E and z0 were estimated by least-squares fit of this equation to the F-z curve recorded on each gel point E. However, in our hydrogels, this part corresponded to a small region of <50 µm away from the hydrogel border. We measured the Young's modulus of the gel every 200–300 µm  along the axis of maximum gel stiffness change.We characterize the hydrogel stiffness by the Young's modulus (2). Due to the short half-life of Sulfo-SANPAH in water, these steps were done in less than 2 minutes. The hydrogels were irradiated under the UV light for 3 min and washed thoroughly with distilled water. A 20 µl drop of 0.1 mg/ml fibronectin  was placed in parafilm sheet. The sulfo-SANPAH activated hydrogel was inverted on top of the fibronectin drop and incubated at room temperature for 2 h (∼2.5 µg fibronectin/cm2). Previous work has shown that incubation of 0.1 mg/ml protein solutions for more than 30 min is sufficient to produce saturating levels of adhesion proteins on hydrogels independent of their substrate stiffness Fibronectin was covalently grafted to the polymerized hydrogels through free amino groups by succinimide chemistry We used immunofluorescence to confirm that the density of fibronectin on the hydrogel surface did not depend on gel stiffness and does not display obvious heterogeneity. Three typical gels, prepared as discussed above, were rinsed once in TBS , and incubated with anti-fibronectin primary antibody  for 1 h at room temperature. The gels were then rinsed five times in TBS and incubated in secondary antibodies . The resulting fluorescently stained hydrogels were imaged using an Olympus IX70 inverted wide-field microscope equipped with a mercury vapor lamp and a Hamamatsu C9100 camera. In addition, confocal cross-sectional fluorescence images were obtained with a Zeiss LSM 510 META microscope.−1 penicillin, 100 mg ml−1 streptomycin  and 10% fetal bovine serum . Cells were incubated at 37°C and 5% CO2. Two days before the experiments, cells were harvested by means of a brief exposure to trypsin EDTA (Life Technologies) and plated sparsely on 22 mm diameter glass cover slips (30 cells mm−2) upon which a stiffness gradient gel coated with fibronectin had been formed. After 20 h of incubation, cells were observed with phase contrast under the microscope. The public domain software ImageJ , along with the plug-in MosaicJ, was used to quantify cell spreading area. A minimum of 400 cells were used to quantify area variation as a function of substrate stiffness.Cell behavior on fabricated stiffness gradient hydrogels was tested principally with NIH3T3 fibroblasts, though some experiments used SY5Y neuroblastoma cells. The NIH3T3 cell line was a gift from Dr. Suresh Ambudkar, NCI/NIH Text S1Controlling the irradiation profile by varying mask speed.(PDF)Click here for additional data file.Figure S1Mask speed can modulate the hydrogel elasticity slope while maintaining a comparable stiffness range. Spatial map of elasticity of hydrogels obtained with the same acrylamide/bisacrylamide/Irgacure solution but different mask speeds. Hydrogels obtained with a mask speed of 7.5 µm/s (green line) resulted in a gradient slope of 170 kPa/mm. Hydrogels obtained with a mask speed of 30 µm/s (red line) displayed a gradient slope of 90 kPa/mm. Finally, hydrogels obtained with 100 µm/s (blue line) showed a slope that changed from 50 to 17 kPa/mm. Error bars in each hydrogel represent SE of 3 replicates.(TIF)Click here for additional data file.Figure S2Examples of irradiation profiles obtained by moving the mask using different speed protocols. The method presented here is suitable for obtaining linear (A), exponential (B) and general monotonically decreasing (C) irradiation profiles. Dashed line in panels A–C indicates the initial position of the mask. These data have been obtained by imaging the real movement of the mask and then summing all recorded frames. Bottom panels display the mask speed protocol used to obtain the linear (D), the exponential (E) and the general monotonically decreasing (F) irradiation profiles shown in A–C panels.(TIF)Click here for additional data file.Figure S3Correction for non-linear relation between irradiation and stiffness. (A) From the data in the calibration curve (t. The edge of the mask is positioned at x(t) = x and is moved at a speed v(t) = v. L is the hydrogel length and T is the maximum irradiation time (i.e. T is the time that needs the edge of the mask to travel from x = 0 to x = L). The exposure time of the hydrogel at position x is given by T – xt, where xt is the time at which the mask arrives at gel position x.on curve , we asce(TIF)Click here for additional data file.Figure S4Hydrogel stiffness profiles are reproducible. Spatial map of elasticity of hydrogels produced on different days with different solutions of acrylamide/bis-acrylamide/Irgacure.(TIF)Click here for additional data file.Figure S5Hydrogels treated with Sulfo-SANPAH incorporate substantially more fibronectin (FN) than untreated ones. Fluorescence intensity profile along a gradient hydrogel using the Sulfo-SANPAH protocol described in the (TIF)Click here for additional data file.Figure S6Stiffness gradient fabrication schematic. (A) The acrylamide/bis-acrylamide/Irgacure solution is placed between 2 coverslips and supported on the top of a 4× objective by means of a holder. The mask is attached to a microscope stage that allows precise control of the mask speed. The sample is illuminated by a UV bench lamp placed on the top of the setup. (B) The holder is an empty cylinder that allows one to image the solution as the mask progressively uncovers the polymerizing solution.(TIF)Click here for additional data file.


I had the privilege and opportunity of meeting Dr. Albert Kligman  in OctobAt the banquet dinner that evening I had the good fortune of exchanging a few words with him. Though there were other international dermatological celebrities like Jablonska, Cunliffe, falabella, Leyden and Goh, Dr. Albert Kligman, fondly addressed by everyone as AL, was the cynosure of all eyes.That was the first and the last time I saw AL Kligman but I shall forever remember him. Though he has departed from this world, as the hymn goes, “he will be remembered by what he has done.”May his soul rest in peace!


Expanded polytetrafluoroethylene (ePTFE) vascular grafts frequently develop occlusive neointimal hyperplasia as a result of myofibroblast over-growth, leading to graft failure. ePTFE exhibits higher ultrasound attenuation than native soft tissues. We modelled the selective absorption of ultrasound by ePTFE, and explored the feasibility of preventing hyperplasia in ePTFE grafts by ultrasound heating. Specifically, we simulated the temperature profiles of implanted grafts and nearby soft tissues and blood under ultrasound exposure. The goal was to determine whether ultrasound exposure of an ePTFE graft can generate temperatures sufficient to prevent cell growth on the graft without damaging nearby soft tissues and blood.® Multiphysics heat transfer model. 50°C is known to cause cell death and therefore the transducer powers were adjusted to produce a 13°C temperature rise from 37°C in the ePTFE.Ultrasound beams from two transducers (1.5 and 3.2 MHz) were simulated in two graft/tissue models, with and without an intra-graft cellular layer mimicking hyperplasia, using the finite-difference time-domain (FDTD) method. The resulting power deposition patterns were used as a heat source for the Pennes bioheat equation in a COMSOLSimulations showed that both the frequency of the transducers and the presence of hyperplasia significantly affect the power deposition patterns and subsequent temperature profiles on the grafts and nearby tissues. While neither transducer significantly raised the temperature of the blood, the 1.5-MHz transducer was less focused and heated larger volumes of the graft and nearby soft tissues than the 3.2-MHz transducer. The presence of hyperplasia had little effect on the blood's temperature, but further increased the temperature of the graft and nearby soft tissues in response to either transducer. Skin cooling and blood flow play a significant role in preventing overheating of the native tissues.Modelling shows that ultrasound can selectively heat ePTFE grafts and produce temperatures that cause cell death on the graft. The temperature increase in blood is negligible and that in the adjacent soft tissues may be minimized by skin cooling and using appropriate transducers. Therefore, ultrasound heating may have the potential to reduce neointimal hyperplasia and failure of ePTFE vascular grafts. There are two primary types of permanent hemodialysis accesses, native arteriovenous (AV) fistulae and expanded-polytetrafluoroethylene (ePTFE) AV grafts. When compared to the fistula, AV grafts provide vascular access with higher blood flow rates, do not require a maturation period, and can be used sooner after surgical placement. Grafts are a particularly useful form of vascular access in patients whose vasculature is insufficient to support a functional native AV fistula. The latter is important because up to 60% of AV fistulae in the United States fail to mature and become functional. Despite these advantages, only 23.3% of the permanent hemodialysis vascular accesses in the United States are AV grafts , largelyThe low primary patency rates of ePTFE grafts are attributed to their propensity to develop excessive cell growth in the graft lumen, preferentially near the graft-venous anastomosis site . This grStrategies to prevent or reduce NH in synthetic AV grafts include the use of drug-eluting stents ,6, endotin vitro  ,32 31,32x, y and z grid space. The absorbed power density Q, in W/m3, is the time average (within each cycle) of power deposited per unit volume throughout the 3D model. It depends on the acoustic pressure and the material through which the wave is travelling as follows , k is thermal conductivity [W/m·K], T is temperature [K], A is a metabolic heat generation term [W/m3] , Cp is specific heat capacity [J/kg·K], and perf Qrepresents heat transfer loss due to perfusion [W/m3]. Because the general heat transfer application in COMSOL does not include the thermal effects of perfusion included in the Pennes bioheat equation, the effects of perfusion were simulated in the muscle and fat tissues usingwhere ω is the blood perfusion rate of the tissue [s-1] and κ is a term to account for the incomplete thermal transfer to perfused blood. In our simulations κ was assumed to be zero  to modeThermal damage to healthy native tissues, a major concern during hyperthermia, can be reduced with active skin surface cooling ,36. The 5 N/m2 and 1.2 × 105 N/m2, respectively.Our simulated pressure results showed that, while the focus was positioned at the rear graft wall for both transducers, the focal zone of the 1.5-MHz transducer spanned the width of the graft Figure  while thQ for the 1.5-MHz transducer was 2.75 × 107 W/m3 in the front graft wall . The attenuation of ePTFE is almost 10 times greater than the tissues surrounding the ePTFE graft Table . ConsequQ approximately three times more than the 1.5-MHz transducer  of the 3.2-MHz transducer caused a higher, more concentrated power deposition pattern. Although the 3.2-MHz transducer operated at 63% of the power of the 1.5-MHz transducer it deposited a maximum  Figures  and 3b. Understanding cellular response to specific temperatures provides relevance for the thermal modelling. While cellular response to thermal exposure varies with cell species and origin , both apDifferences in thermal exposure between the 1.5- and 3.2-MHz transducer simulations may influence the extent of cell death on implanted ePTFE grafts. For example, the 3.2-MHz transducer heated the graft to a higher temperature after 5 seconds Figures  and 5d. Our modelling results suggest that the ultrasound should be delivered in an intermittent manner in our target clinical application. Non-continuous ultrasound therapy helps to reduce unwanted heating of healthy tissues by allowing cooling between the ultrasound pulses. Pulsed ultrasound exposure could also help ensure that hyperplasia receives only mild heat, thereby avoiding tissue necrosis. To avoid residual temperature buildup during pulsed ultrasound exposure, a 30-second exposure should be followed by a 30-second rest, for a duty cycle of 0.5. Shorter rest times would not allow sufficient time for the tissue to return to 37°C before the next ultrasound pulse exposed the ePTFE graft. Consequently, a treatment equivalent to 30 minutes at 45°C would take twice as long, lasting up to an hour.During the ultrasound exposure the low thermal conductivity of the tissue slowed thermal diffusion into the tissue. Thus, after 30 seconds of exposure, the rise in temperature was confined to and in close proximity of the ePTFE material Figure . FurtherDue to convection, the blood-ePTFE boundary Figures  and 5c aResults of our modelling showed selective ePTFE graft heating could be achieved by either the 1.5-MHz or the 3.2-MHz transducer. Both transducers can lead to temperatures capable of inducing apoptosis near the NH-ePTFE boundary. The temperature increase in blood was negligible using either transducer, while the temperature increase in the adjacent soft tissue was smaller when the 3.2-MHz transducer was used. Tissue temperature variations due to the presence or absence of NH suggest the need of patient-specific ultrasound exposure protocols. Overall, intermittent ultrasound heating may have the potential for clinical application to reduce neointimal hyperplasia and failure of ePTFE vascular grafts.The authors declare that they have no competing interests.MRB carried out the modelling and drafted the manuscript. RJS, DAC and YES conceived of the study. AKC, DAC and YES participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Acoustic waves can be described  using Newhereū = x ẋ + uy ẏ + uz ż uis the particle velocity,xu, yu, and z uare the respective particle velocities in the ẋ, ẏ, and ż directions,p is the pressure,η is the dynamic coefficient of shear viscosity of the material,η' is the dynamic coefficient of bulk viscosity of the material,o ρis the average mass density of the material, andΚ is the compressibility of the material.p in finite-difference form, (A2) yieldsConsidering compression waves only (shear waves attenuate rapidly with distance ), these x, Δy, and Δz are the lengths of the voxels in the computational grid space, the letters i, j, and k denote the x, y and z indices, respectively  is given in finite-difference form in (A4). The y uand z usolutions (not shown) are similarly defined.Here Δondition . The supu is defined on the boundaries of each voxel, the material density ρ' also must be defined on the edges. As a result, ρ' is approximated as the average density of two adjacent cells:Because the velocity y uand z uvelocity solutions not shown here, to calculate the pressure and velocities at each time step throughout the entire model grid space.The FDTD algorithm works iteratively between (A3) and (A4), as well as between the respective equations for the


The beginning of 2011 marks the 4th year of publishing of ‘Journal of Medicine and Life’, under the aegis of the prestigious ‘Carol Davila’ University Press, ‘Carol Davila’ University of Medicine and Pharmacy, Bucharest, becoming, presently, an important tool for publishing studies, research and reviews from different branches of medicine, both for the professors and doctors and for the students and PhD.For ‘Journal of Medicine and Life’, the new year presupposes a change, a change which will improve the quality of the published material and the co–optation of new prestigious authors. The final purpose is the indexing of the journal in as many international databases as possible, an action which will bring a high degree of visibility to the medical research in Romania, as well as to the authors who publish articles in it. Starting with the first issue of this year , Journal of Medicine and Life aligns to the peer–review criteria and methodology, used by the international publications.I wish to thank the authors who have contributed and who are still bringing their contribution to the improvement of the journal, but also the board and the editorial team who have made the image of the journal known at a national and international level, by participating in different events and by indexing it in some prestigious international databases such as PubMed, Index Copernicus, EBSCO Publishing and ProQuest.In order to be helpful for the ones who constantly bring their contribution to the process of raising the notoriety of the journal, we intend that each issue of the journal to have a section dedicated to the young researchers and PhDs who want to make their researches or studies known in the pages of Journal of Medicine and Life. Accordingly, we have created, starting with the first issue of 2011, a new category called ‘Young Researchers Area’. Moreover, we wish to offer the possibility to some of the world's medical and academic personalities to publish prestigious articles in the journal, by creating another section named ‘Guest editor’.The journal remains a tool of promoting the visibility of the researchers engaged in doctoral and postdoctoral studies, as well as a tool for information dissemination through the financed research projects. So, once again, we open the pages of the journal with pleasure, to all the friends, professors in the universities of medicine and doctors in the hospitals in our country but also from abroad, to the researchers, students and PhDs who wish to facilitate the professional dialogue with the whole world, for the good of all those who are suffering.


Although there is no data to prove the role of renal re-transplantation, but it is presumed to be a favorable option for many patients after graft loss. Re-transplantation often provides a better survival and good health in comparison with dialysis, that is more obvious for younger recipients and whom receives transplantation either preemptively or shortly after the need for renal replacement therapy has arisen . AlthougThe etiology of their initial graft loss varies in candidates of kidney re-transplantation depends on the time of graft loss. If graft loss occurred within the first year after transplantation, the most common etiologies are acute rejection and graft thrombosis and if this occurred after 1 year of transplant, chronic rejection accounts for nearly two thirds of the graft loss .In recent decades, kidney re-graft survival has been improved and this improvement is more obvious for grafts from cadaveric source  in comparison with living donors .Many variables have an effect on re-transplantation outcome, such as recipient comorbidities, outcome of primary transplant, risk of recurrent disease and the wait time on dialysis before transplantation . CKD wilEarly graft loss after previous transplantation is a predictor of the occurrence of the same process after re-transplantation and so the recipient and potential donors should be evaluated very precisely. This early graft rejection can be due to many factors such as acute rejection, FSGS, IgA nephropathy, MPGN, thrombotic microangiopathy or vascular events . PolyomaThe proper time for evaluating recipient and donors for re-transplantation is when the GFR falls to less than 20 mL/min , 10. RouPresence of antibodies, that may be created previous blood transfusion, pregnancies or previous transplantation, is a great concern before transplantation . AntibodIn re-transplantation immunosuppressive agents are used to prevent acute graft loss but risk of infection and malignancies increase. It should be kept in mind that re-transplant patients are at higher risk for acute rejection in comparison with first graft patients .Reasons for previous transplant nephrectomy include: symptoms of graft loss, infections, proteinuria, severe hematuria, the need for more space in the pelvic fossa but in about 80% of these patients, there is no need for previous graft nephrectomy before re-transplantation .In theory, there isn’t any limitation for the number of renal re-transplantation in a patient with ESRD but vascular anatomy can restrict it, so evaluation of the vascular anatomy of recipients before re-transplantation is very important , 17.


As the new Editor in Chief, I will use this editorial to give a brief outline of my vision for the future of the journal. As the english-language successor to the dutch-language journal of the Dutch Association for Medical Education (NVMO), ‘Perspectives on Medical Education’ has become a fully-fledged international scientific journal. I commend the editorial board on this achievement. It provides me, together with the editorial board, with an excellent starting point for further growth. We opt to position the journal on the cutting edge of educational research and development, offering quality papers that are of interest to teachers, educational developers, policy makers and researchers: a position between more theory-oriented journals, such as Advances in Health Sciences Education and Medical Education on one hand and on the other hand journals with a more practical focus, such as clinical teacher. We invite authors to submit papers that focus on the transition of theory to practice and if possible on the impact on patient care. Clinicians are an important target group for Perspectives. In the coming period we will communicate this course through the editorials, commentaries, some new rubrics and manuscript categories. Firstly, we will update and globalize the Practical Guidelines to Clinical Education that were developed for the dutch-language version of the journal. Two associate editors—Bob McKinley and Fedde Scheele—will invite international experts to update the existing guidelines for practice on topics such as multi-source feedback, entrustable professional activities, and the interpretation of assessment data. Second, a new section for papers in which experienced experts give a state of the art overview of their field has been added. The first author to contribute to this section is one of our new editors—Cees van der Vleuten. In one of the coming issues he will give his perspective on how evidence can be translated to medical education. Third, we would like to focus the journal more broadly than medicine and also give space to the other health professions. We will make this more explicit in the definition of the aim of the journal and in the membership of the editorial board. In line with this is our plan for a section for papers on inter-professional education. Finally, we will start a section with papers that give a policy perspective to medical education. We will appoint associate editors with expertise in these domains.In my opinion, it is essential that Perspectives achieves an impact factor, as this is likely to entice significantly more researchers to opt for Perspectives as the platform for their publications. Important requirements for eligibility for an impact factor are: papers which are cited by other researchers in the field, reliable and timely publication of the journal, short lead time of submitting an article to decision and possible publication, and an international team of editors. We will focus our attention on good quality papers and a timely response to authors. For good quality papers, constructive feedback from editors and reviewers to the authors is essential. To achieve this, the review process has been reorganized. The Editor in Chief reads and makes an initial assessment of all incoming papers. At this stage, unsuitable papers are rejected and others are sent for assessment to one of the associate editors—who are committed to the journal and have a network of potential reviewers. The associate editor rejects or sends the paper for review to a reviewer who is knowledgeable on the topic in question. These first two screenings by the Editor in Chief and associate editor will promote economical use of reviewer time without concessions as regards the quality of the review process.I would like to thank my predecessor Jan Borleffs for his contributions to the development of perspectives. Under his 12 years of leadership, the journal has developed into a truly international journal both in terms of authors and readers. Perspectives’ articles are downloaded by readers from all over the world, and each issue includes authors from different countries. For example, authors from Belgium, Canada, Ireland, New Zeeland, Saudi Arabia, the United Arab Emirates, UK, and USA contributed to this issue. In addition to Jan Borleffs, Onno Terpstra has also left the editorial board. I would like to thank Onno for all his work for the journal.Bob McKinley of Keele University Medical School, Keele, Staffordshire, UKBrian Hodges of Toronto General Hospital, Toronto, ON, CanadaCees van der Vleuten of Maastricht University, Maastricht, the NetherlandsCharlotte Ringsted of Toronto General Hospital, Toronto, ON, CanadaChristopher Watling of Schulich School of Medicine and Dentistry, London, CanadaLorelei Lingard of Schulich School of Medicine and Dentistry, London, CanadaMahan Kulasegaram of Toronto General Hospital, Toronto, Toronto, ON, CanadaMartin Grønnebæk Tolsgaard of Copenhagen University Hospital Rigshospitalet, Capital Region, Denmark.Fortunately, we have been able to expand the editorial board with new members. We are pleased to welcome the following persons:We are very excited about the new members of the editorial board; they bring a new scope, energy and expertise to our team.Our team is completed by Lieda Meester, editorial coordinator, Bohn Stafleu van Loghum.With these new team members, we strive to make perspectives an exciting and thought-provoking journal that caters to the interests of all those involved in medical education.


We start a new series with this issue.The Looking Glass will depict how others look at medical practice, its practitioners, mental health workers and philosophers. It will also reflect on happenings in the world of Medicine and Science.CMAJ…We start with Medical Journals. The CMAJ , John Hoey, and a Senior Deputy Editor Anne Mary Todkill. The Editor appointed in the interim, Stephen Choe and another Deputy Editor Sally Murray, also resigned in a week's time, on 28 Feb to be precise. Along with them also went Jerome Kassirer, a former CMAJ editorial board member and a former editor of the New England Journal of Medicine (NEJM), appointed to frame regulations or governance plans about editorial independence.The date: Feb 20, 2006. Medical publishing was rocked by the sudden dismissal of the Editor In Chief of the All these are well-respected professionals in the field of biomedical publication and there has been a huge outcry, both in the medical as well as popular press, at their abrupt sacking.CAMJ also resigned, precipitating a grave crisis. In the meanwhile, the CMA, in an attempt at damage control, appointed an interim Editor, Noni Macdonald, along with an Editor Emeritus, Bruce Squires, who has been an earlier editor of CMAJ, and a founder member of WAME. A retired Chief Justice of the Canadian Supreme Court has been appointed to chalk out a Governance Plan for Editorial independence. Changes are also envisaged in the JOC . The CMA President is exhorting everyone concerned to move on, and has assured a policy of editorial independence. The atmosphere, at the time of writing this, is combative and smoldering, and a distinct unease prevails.If this were not enough, fifteen out of the nineteen members of the editorial board of the CMAJ, and the Editor of CMAJ over editorial independence. John Hoey has been asking for greater editorial independence since the journal, although belonging to the CMA, according to him, actually belongs to the whole world of medicine and science, and is, really speaking, accountable mainly to them.There has been a long-standing feud between the CMA, the organisation that owns and tries to control CMAJ from a modest journal to one whose impact factor is today fifth in the world of general medical journals, only less than that of the NEJM (38.6), JAMA (24.8), Lancet (21.7) and BMJ (7.0). He brought it from an impact factor of 1.6 in 1997 to an impressive 5.9 in 2004. (It has since gone to 7.4 in 2005. See ‘About CMAJ’ at http://www.cmaj.ca/misc/about.shtml).: John Hoey, in nearly a decade at the helm (he was hired in August 1996), has brought CMAJ closely approximates what happened with JAMA in 1999 when George Lundberg, the editor for 17 years at JAMA (Jan 1982 onwards), and who brought it to scientific respectability, was summarily sacked by the executive vice-president of the American Medical Association (AMA), E Ratcliffe Anderson, Jr., during the Bill Clinton oral sex episode. He was sacked because he fast tracked an article on college students′ perception whether oral sex constituted sex .The CMAJ. . The pharmacists had a big stake in the product. It was changed from a prescription to a non-prescription emergency contraceptive drug, but with an important rider. The pill was costly enough, but the pharmacist was supposed to charge almost an equal amount for counselling about the appropriateness of the drug. Moreover, they were supposed to collect personal details, including sexual history, during this counselling. The CMAJ probed this by asking a few ladies whether they approved of this, which obviously they did not , a pill for contraception to be used by females, was investigated in the  did not . The CanWe have a transgression to report. While the Dec. 6, 2005, issue was in preparation, the editorial independence of the journal was compromised when a CMA executive objected strenuously to a news article we were preparing on behind-the-counter access to emergency levonorgestrel (Plan B). The objection was made in response to a complaint from the Canadian Pharmacists Association, who had learned about the article when they were interviewed by our reporters. The CMA's objection was conveyed to CMAJ's editors, and to our publisher, who subsequently instructed us to withhold the article.The stated objection was to our reporting method; as one component of the story, we had asked 13 women from across Canada to attempt to purchase Plan B from a pharmacy in their community and then tell us what the experience was like. The CMA questioned the propriety of our investigation and the boundary between news reporting and scientific research. Our story was not scientific research, however, but legitimate journalism .The editorial further said:We felt that we had a choice between pulling the entire story, or getting most of it out by publishing a negotiated revision. We opted for the latter: what our readers saw omitted the results of our informal survey. This transpired without the story having been read by those who were raising the objection.Our objective in making this incident public is to set in motion a process to ensure the future editorial independence of the journal. Readers expect CMAJ editors to select content without interference, and authors expect their work to be judged without regard to the interests of any third party. Readers and news media who rely on our reporting need to know that our journalists are not subject to censure .CMAJ and the CMA. On Sept 17 2002, the journal had carried an editorial criticising Bill 114, a proposed Quebec legislation that was meant to force family physicians to work on emergency duty during shortage of staff . The article was a report on the appointment of the federal Minister of Health by the new Conservative government. It pointed out the health minister's favourable stance toward privatization of health care delivery during his tenure as the Minister of Health for Ontario. On Feb. 22, 2006, a different report on the federal Minister of Health appeared in the original's place . Though the revised article contains some of the same phraseology as the original, it is more supportive and less critical of the health minister and seems more beneficial to the CMA. We pose the question as to whether the extensive revision of this article is another instance in which the political interests of the CMA exerted an influence on CMAJ publishing decisions. Some days before the firing of the editor in chief and senior deputy editor, the JOC was informed about a disagreement concerning the original Tony Clement article, but the JOC turned down a request for an emergency meeting. The editors are not willing to comment on how the changes came about; the publisher has also declined comment.As this report on the Plan B commentary was being finalized, we became aware through a communication of the Canadian Health Coalition that another news story published electronically on Feb. 7, 2006, was subsequently removed from the CMAJ Web site  used the services of a newly appointed President, Graham Morris, to do the act. The salvo was fired in a three-paragraph termination by the last named gentleman. :Under such circumstances, to continue to have an upright editor at the helm of an important opinion maker like the The Canadian Medical Association Journal (CMAJ) announced that Dr. John Hoey would be leaving his post as Editor-in-Chief today.During the ten years that Dr. Hoey has been Editor-in-Chief of CMAJ, he has broadened the scope of the publication and raised its international reputation as a respected peer-reviewed scientific journal.The CMAJ's mission in serving its 69,000 readers is to provide accurate and up-to-date scientific and clinical information on the promotion of health and the treatment of disease .So, if the first reason was economic, the second was political. And the two scourges of modern medicine, economics and politics, managed to do in the credibility of a respectable Journal. While we know the economic-political compulsions of Associations and its office bearers, that they could not rise above their petty considerations in the larger interest of science and research is a sorry tale to recount.BMJ , some things become clear.As the dust settles on this episode, which it inevitably will,  were set up in the wake of the Associations, which own journals, will have to decide to give up ownership rights over journals. This must come sooner or later, and the sooner the better. The financial and other logistics have to be worked out, of course. But most prestigious journals manage to earn revenues for themselves too, so it is not as formidable a task as may first appear.BMJ where the Editor is appointed by the Council of the BMA and, if at all, would have to be fired by it. He/she is on par with the Secretary of the Association (here the BMA), something equivalent to the Executive Vice-President of the AMA for example, and reports directly to a Council which is the servant of the Annual Representative Meeting, and also if needed to the Annual Meeting of the BMA. Not to any individual who can hire or fire him. The major difference between BMA and many other medical associations, including those like the AMA, as also the Australian and the Canadian, is that the Editor does not report to the Secretary. In fact both are chief joint executives. This has ensured the reduced coming and going of editors in the BMJ for example, with hardly such problems as the CMAJ faces today, or the NEJM and JAMA faced not so long back. It proudly claims it has had only six editors in a century, certainly an enviable record, and worthy of emulation (JAMA is another worthy claimant (Those Journals that still depend on Associations may work out a system like the mulation . The Ediclaimant .Other solutions are when the Journal publishes the Presidential Address or Orations/Award Papers of a Society/Association, and has a regular column for Events/Announcements of the Society/Association, but with regard to everything else, it is absolutely independent. Such a step is essential if a Journal has to earn respectability as a biomedical publication and not become only an Association mouthpiece or Bulletin.A Society/Association should know that a Journal's reputation, beyond a certain limit, is independent of its originator. It must reach the broader constituency of the world of biomedical science and researchers spread all over. It must allow for, even encourage such flowering, for in so doing the Association truly appreciates the potential of its worthy progeny. It is very much like a father and a son. The son needs the father in the initial stages. But when he outgrows that need, it is suicidal for the father to continue to control, or try and milk the son. That can only lead to bitterness and rancour. What western society has forsaken in its family structure by giving up on the joint family, unfortunately, it is still clinging to here, creating untold hardships in the bargain.Editors should not, reading the writing on the wall, become handmaidens of the establishment. They may survive, even prosper, that way, but will never leave their impress ‘on the sands of time’. Their role, fundamentally, is to further scientific advancement, irrespective of the compromises circumstances force on them. That does not sanction gladiator roles, nor does it mean every editor must perennially fight against an establishment. It just means the Editor, and the Journal he heads, by their very nature, have to be in pursuit of scientific evidence and patient welfare. That may occasionally bring them in conflict with powerful economic, political and bureaucratic forces, including Association office bearers. But that they must accept as a professional hazard, and plod on nevertheless.scientific evidence and patient welfare, the twin pillars of biomedicine, howsoever high and mighty, can become a predominant concern for an Editor, or Journal. Ever. That is the bottom line, and the very raison d′ etre for their existence.No consideration or power that tries to over ride If ever a lesson has to be reiterated, this episode and its unfolding aftermath should italicise it.Hopefully.Ajai R. SinghShakuntala A. SinghMens Sana MonographsEditors,


In the Results section the last two sentences are incorrect. The corrected sentences can be seen below.Incorrect sentences:Diagnostic scores were moderately correlated with CON1 copy number, exhibiting a Pearson's r of 0.49 and 0.67 in social and communicative domains, respectively. Repetitive behavior score demonstrated a more modest correlation with CON1 copy number, with a Pearson's r of 0.26.Corrected sentences:As might be expected diagnostic scores were moderately correlated with each other, ranging from a Pearson′s r of 0.49 to 0.67 in social and communicative domains on the measures utilized. However repetitive behavior score was not as correlated with social and communicative domains ranging from Pearson′s r of 0.26 to 0.55.


Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics.The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four , out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully .Eleven peptides derived from T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents.We have precisely located the epitopes of  Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds, causing toxoplasmosis [T. gondii is an important food-borne parasite, and the primary route of transmission from animals to humans is through the consumption of infected meat [T. gondii[T. gondii have focused on SAG1 and shown encouraging results [T. gondii. Therefore, B cell epitopes of SAG1 were analyzed using a synthetic peptide technique in combination with software-based prediction.lasmosis ,2. T. goted meat ,4. In soT. gondii. In addiT. gondii. Therefo results . Further results -10. HoweT. gondii-positive sera, which had been previously collected from experimentally infected pigs by our lab, were investigated. Twelve pig serum samples were collected at the time of presentation of clinical symptoms (G1); 18 follow-up specimens were taken on days 14 to 35 after the onset of symptoms (G2), and 21 further serum samples were taken on days 60 to 120 after the onset of symptoms (G3). Toxoplasma IgM and IgG antibodies were confirmed by T. gondii lysate antigen-ELISA. The serum samples in G1 and G2 were positive for IgM and IgG against T. gondii. The serum samples in G3 were only positive for IgG against T. gondii. Ten sera that were negative for T. gondii IgM and IgG were used as controls. The experimental protocol was approved by the Ethical Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.A total of 51 T. gondii SAG1 (Genbank Accession No. FJ455529), 20 non-overlapping or overlapping 12–36 mer peptides were synthesized by GL Biochem Ltd . Peptide sequences are shown in Table Based on the sequence of et al. [Toxoplasma infection in humans [ELISA for each peptide were performed as described by Cardona et al. , except et al. . A robusn humans . HoweverTo further determine the epitopes of SAG1 and decrease the number of laboratory experiments, bioinformatics was used to predict the epitopes. The secondary structure and the surface properties of the SAG1 were analyzed as described by Zhang . The resT. gondii SAG1 to a shorter sequence than had been identified previously. The identified epitopes will be useful in vaccine and diagnostic reagent design.Conformational epitope selection needs to determine the tertiary structure of antigen in order to identify antibody interacting residues in antigen. Experimental techniques such as crystallography are expensive and time consuming. Thus, our work focuses on linear epitope selection. In this study, we have located the epitopes of The authors declare that they have no competing interests.YHW, HY and DLZ designed the experiment. YHW, GXW and MW performed lab work and drafted the manuscript. All the authors read and approved the final manuscript.


Twenty years ago, and still in some textbooks, allergy was regarded as a "disease of the rich," with a rising prevalence only in industrialized or western countries. This has changed: allergy has become a major health problem in almost all countries of the world, both in terms of individual suffering, medical management, and quality of life, as well as a socio-economic burden. People in Papua New Guinea or Tanzania are experiencing very similar allergic diseases and symptoms as patients from São Paulo, New York, Moscow, or Tokyo. Yet there are considerable differences in management, diagnostic and therapeutic modalities, and especially with regard to eliciting allergens and modifying environmental factors. Globalization is not only an economic phenomenon. In addition, the world has grown together in science and in the medical field.There is a need for more information, exchange of experience, and for a discussion forum at an international level. Allergy with its interdisciplinary character involves the expertise of various fields both in clinical medicine and in research and experimental science. Allergy manifests itself as various conditions with a colorful spectrum affecting different organs and following quite variable pathomechanisms.The World Allergy Organization (WAO), as a global federation of 74 national and regional societies for allergology and immunology, is launching the new journal "WAO Journal," addressing this need for information, exchange of experience, and international discussion.The journal will be practice-oriented and offer the highest level of information in a strictly peer-reviewed system. It offers space for reviews both at the postgraduate education as at the advanced research level, originals, and letters, where exceptional case studies, concise reports on study findings, or expression of opinions and discussion of recent topics of controversy will be published.The journal has established an excellent group of experts in the editorial board who represent at the same time the various features of clinical and experimental allergology and relevant subdisciplines and the various geographic areas of the world.The WAO Journal will reach over 30,000 allergists all over the world directly online; it will be available for free for the first year for all individual members of national allergy societies as well as nonmember subscribers. The new journal is in direct tradition grown on the basis of Allergy and Clinical Immunology International--The Journal of the World Allergy Organization, which has been the official organ of WAO for 19 years and has been discontinued by December 2007. This decision never was caused by dissatisfaction; Allergy and Clinical Immunology International--The Journal of the World Allergy Organization was one of the most successful allergy journals, at one time reaching 17,000 allergists, with editions in several languages. At this place, special thanks should go to the founding editor, Alain De Weck, and to the long-time editor, Allen Kaplan, who have done an excellent job together with Hogrefe Huber publishers, Dr Christiane Hogrefe, Dr Rob Dimbleby, and Christina Sarembe. The decision for a new journal reflects a new philosophy of publication, concentrating on the electronic way, with many advantages starting from the speed with which articles can be accepted, reviewed, and published, decreased costs, the opportunity for digital content (including videos and animation), easy searching of contents, and independence from postal services, just to name a few.Coeditor in Chief Lanny Rosenwasser and myself are eager to push "WAO Journal" to become one of the premium international journals in allergy in a truly global endeavor!Johannes RingEditor-in-ChiefMunich, Germany, January 2008


Celiac disease is caused by an abnormal intestinal T-cell response to gluten proteins of wheat, barley and rye. Over the last few years, a number of gluten T-cell epitopes restricted by celiac disease associated HLA-DQ molecules have been characterized. In this work, we give an overview of these epitopes and suggest a comprehensive, new nomenclature. Celiac disease (CD) is caused by an abnormal intestinal immune response to proline and glutamine-rich wheat gluten proteins and to similar proteins in barley and rye (Green and Cellier cis configuration), whereas in individuals who are DR5DQ7/DR7DQ2 heterozygous it is encoded by alleles located on opposite chromosomes (trans configuration)  and DQ8.5  found in DR3DQ2/DR4DQ8 heterozygous individuals   . Yet, DQ2.2 positive CD patients do not mount a T-cell response to the same gluten epitopes, but rather have responses to gluten peptides that bind stably to DQ2.2  resistance of the polypeptide sequence to proteolytic degradation, (b) specificity of TG2 and (c) HLA binding specificity. The proline-rich nature of gluten makes the gluten proteins resistant to proteolytic degradation in the gastrointestinal lumen, and long gluten peptide fragments ranging from 15 to 50 residues therefore survive in the small intestine , P6 or P7 in HLA-DQ2.5 restricted epitopes and at position P1 and/or P9 in HLA-DQ8 restricted epitopes Table . This rehttp://www.isscd.org/EpitopeNomenclature) will update this list as more epitopes are identified.The list of gluten epitopes recognized by T cells of CD patients presented in Table


Information-theoretic secrecy is combined with cryptographic secrecy to create a secret-key exchange protocol for wirelessnetworks. A network of transmitters, which already have cryptographically secured channels between them, cooperate to exchange a secret key with a new receiver at a random location, in the presence of passive eavesdroppers at unknown locations. Two spatial point processes, homogeneous Poisson process and independent uniformly distributed points, are used for the spatial distributions of transmitters and eavesdroppers. We analyse the impact of the number of cooperating transmittersand the number of eavesdroppers on the area fraction where secure communication is possible. Upper bounds on the probability of existence of positive secrecy between the cooperating transmitters and the receiver are derived. The closeness of the upper bounds to the real value is then estimated by means of numerical simulations. Simulations also indicate that a deterministic spatial distribution for the transmitters, for example, hexagonal and square lattices, increases the probability of existence of positive secrecy capacity compared to the random spatial distributions. For the same number of friendly nodes, cooperative transmitting provides a dramatically larger secrecy region than cooperative jamming and cooperative relaying. Information-theoretic secrecy has attracted a significant interest in recent years due to its possible applications in wireless communications and the growing significance of wireless networks. Wyner  first incooperative jamming [cooperative relaying [Secrecy capacity can be improved using cooperation with friendly nodes . In the  jamming , friendlrelaying , 7, frieInformation theory achieves perfect  secrecy as opposed to the computational secrecy provided by cryptographic algorithms. Here we examine the possibility for mutual applications of cryptographic secrecy and information-theoretic secrecy. A set of transmitters have already cryptographically secured the communication channels between them. When a transmitter wants to communicate securely with a new receiver , a presecret key message is created by the transmitter and broken into several data blocks, and a separate block is encrypted and sent to each of the other transmitters. Then each transmitter sends its data block to the receiver. The transmitters ensure that all the data blocks are received correctly at the receiving node, which is required for the computation of the secret key at the receiver. The secret key is securely and cooperatively transmitted to the receiver (without being divulged to the eavesdroppers) using information-theoretic secrecy if the secrecy capacity is positive for the communication channel between at least one transmitter and the receiver. As the number of transmitters grows, the eavesdroppers are facing a more difficult task of being able to intercept a larger number of transmitters. Once the secret key is exchanged, the legitimate parties can start communicating at the maximum data rate since their communication channel is cryptographically protected, achieving computational secrecy . Our modOur results are presented using the following organisation of the paper. In LT cooperating transmitters, a single receiver, and a network of LE passive eavesdroppers. The passive eavesdroppers do not transmit any signal and try to intercept the information that is transmitted between the pairs of legitimate nodes, hence reducing the secrecy capability of the network. Their locations are unknown to the transmitters. Each transmitter is equipped with only a single omnidirectional antenna.We consider two-dimensional wireless networks with the following communication nodes: a network of  L(A) is the area of a region A ∈ R2; LT is a random variable which denotes the number of transmitters in a region A; LE is a random variable which denotes the number of eavesdroppers in a region A; b||c is a concatenation of two data blocks b and c;  V, Ve are the additive noise at receiver and eavesdropper, which are independent zero mean Gaussian random variables with variance σ2; Ct,r is the capacity of the communication channel between transmitter t and receiver r; Cs:t,r is the secrecy capacity between transmitter t and receiver r; Cs is the secrecy capacity between a set of cooperating transmitters and a receiver; dj,i is the distance between nodes i and j.In the sequel, we use the following notation.r from the transmitter t isX is the transmitted signal from the transmitter t and β is the path-loss coefficient [e from the transmitter t equalsWe use the additive white Gaussian noise model. Then, the received signal at the receiver fficient . The rect and receiver r, and between transmitter t and eavesdropper e are given by [Pt is the transmitter's power. If the point to point capacity between the two communicating nodes Ct,r is larger than the capacity of the channel between the transmitter and the eavesdropper Ct,e, then Cs:t,r = Ct,r − Ct,e > 0. Otherwise, Cs:t,r = 0 [Cs:t,r > 0 if the receiver r is closer to the transmitter than the eavesdropper; that is, dt,r < dt,e. The disk Ds ⊂ R2 with center at the transmitter and radius equal to the distance between the transmitter and the nearest eavesdropper is called secrecy disk of the transmitter. If a receiver is inside the secrecy disk, then the secrecy capacity between the transmitter and the receiver is positive.The point to point capacities between transmitter given by 3)Ct,r=Ct,r=t anReceivers which are outside the secrecy disk for a given transmitter cannot communicate securely with that transmitter. In the next section, we explain a type of cooperation for a set of friendly transmitters that combines their secrecy disks and thus allows them to communicate secretly with receivers positioned in a larger region.ti and a new communicating node/receiver r want to communicate. ti generates a presecret key message B with arbitrary length, which it then divides into LT blocks b1, b2,…, bLT. Each block is sent to a different transmitter via a cryptographically secured channel. Then each transmitter ti sends its block bi to the receiver. The intended receiver correctly receives all blocks b1, b2,…, bLT and restores the presecret key message B = b1||b2||⋯||bLT. Both ti and r use a cryptographic hash function H to calculate the mutual secret key K = H(B), which is then used to cryptographically protect their mutual communication. The eavesdroppers have to be able to intercept the transmission from all LT transmitters. If at least one data block out of LT data blocks is not intercepted, then the secret key K cannot be computed at the eavesdropper. We call this strategy for cooperation—cooperative transmitting. Using cooperative transmitting, a transmitter can exchange a secret key with a receiver if the receiver is inside any of the secrecy disks for all LT transmitters. The impact of cooperative transmitting is quantitatively measured through the fraction Fs(A) of a region A covered by the union of secrecy disks. Fraction Fs(A) is equal to the probability of securely exchanging a secretkey with a receiver that is randomly positioned inside the region A:L(A) → ∞.The set of transmitters have already established a cryptographic secret key, and they can cryptographically protect their mutual communication channels. Let us assume that transmitter  Haenggi , 13. Ther is inside the secrecy disk of transmitter t4, and therefore block b4 cannot be intercepted by any of the eavesdroppers.P{Cs > 0} on the spatial distributions of transmitters and eavesdroppers. We analyse both random and deterministic models for the spatial distribution of transmitters and eavesdroppers. Two simple models for random spatial processes for the transmitters and the eavesdroppers will be used. The first model is homogeneous Poisson process on the plane characterised by the mean number of points λ in a unit area, also called rate or density of the Poisson Process. The number of points l inside a region A follows the Poisson probability distribution law with parameter λL(A):Fs(A). For the spatial distribution of the transmitters, we will also investigate two deterministic models: hexagonal lattice and square lattice.In the remainder of this paper, we analyse the dependence of  secrecy –15, the  secrecy , 17, it  secrecy . Second,A ∈ R2 obeys a IUD process with parameter nT. A fixed number of eavesdroppers nE are positioned according to an IUD process in the same region A. If nT = 1, then Cs > 0 if the receiver is inside the secrecy disk of the transmitter, that is, the receiver is closer to the transmitter than any of the nE eavesdroppers:In the first case, the position of the transmitters in a region nT > 1, we establish an upper bound for P{Cs > 0} as follows. For nT = 2, the secrecy region of the two transmitters is a union of their secrecy disks:nT > 1, thus obtainingFor y disks:P{Cs>0}=1nT and nE grow infinitely, while their ratio remains constant k = (nT/nE). This is a good first-order approximation when the area of region A grows infinitely and the densities of transmitters and eavesdroppers remain constant. ThenNext we consider the case when both P{Cs > 0} for real networks. P{Cs > 0} on nT and nE as obtained from the numerical simulations. Each point on the curves is averaged over 100,000 simulations of real networks.In order to evaluate the accuracy of the upper bounds  and 10)10), we hP{Cs > 0}, which are estimated through numerical simulations. Relative gap between the upper bound and the real values grows for larger nT due to the increasing number of overlapping secrecy disks (see (sks (see ).P{Cs > 0}. The relative gap between the upper bounds and the real values gets smaller for smaller k (larger nE) since the secrecy disks as well as their overlaps become smaller in size. Consequently, the neglected terms in  = 1 and thus the average number of transmitters in the region A is λL(A) = λ. The eavesdroppers' positions obey an IUD process and the number of eavesdroppers in the region A is nE.Next we consider the case where the transmitters are positioned according to a Poisson spatial process with rate LT is 1, then   LT is 1P{Cs > 0}. Similar to λT as a consequence of the increasing number of intersecting secrecy disks. Numerical simulation of a Poisson spatial process was done according to [λ in a region A, we first randomly select a value l for a Poisson variable with mean λL(A), and then we randomly position l IUD points in A. Observed dependence of P{Cs > 0} on λT and nE was similar to the one depicted in rding to . In ordenT are positioned at IUD points in a region A ∈ R2. The positions of the eavesdroppers follow a Poisson spatial process with average rate λE. For the sake of simplicity we again assume that L(A) = 1. Then the number of eavesdroppers in A is a Poisson random variable LE with average value λE. Its probability distribution function is given by (λ = λE. If there is only one transmitter randomly positioned in A, that is, nT = 1, then the secrecy region fraction is given byA fixed number of transmitters given by  where λ nT > 1, we ran numerical simulations, and the results are given in For A ∈ R2 is assumed for both the transmitters and the eavesdroppers. Again we have obtained very similar results to the previously analysed three combinations of IUD and Poisson spatial processes for transmitters and eavesdroppers. Following slight differences were observed. IUD spatial process for the transmitters gives slightly higher values for P{Cs > 0} than the Poisson spatial processes. On the contrary, the Poisson spatial process for the eavesdroppers gives slightly higher values for P{Cs > 0} than the IUD spatial processes.For the sake of completeness, we have also numerically analysed the case when a homogeneous Poisson process in a region P{Cs > 0} is higher for a deterministic lattice compared to stochastic spatial processes for the transmitters (see Next we analysed the case when the transmitters are positioned on a deterministic lattice, and the eavesdroppers obey a UID process. By means of numerical simulations we examined a square lattice and a hexagonal lattice. We observed similar shapes to the curves shown in Figures ters see  due to tIn this section, we compare cooperative transmitting with two other strategies for cooperation in wireless networks. Cooperative relaying and cooperative jamming increase the secrecy capacity by means of widening the gap between the SNR at the legitimate receiver and the SNR at the eavesdroppers. In the single hop cooperation with the best relay , 20, onlP{Cs > 0} as a quantitative measure of the positive impact of the different strategies for cooperation. We use the value for In this work, we propose to combine information-theoretic secrecy with cryptographic secrecy to increase the secrecy region and provide a novel solution to the key-exchange problem. Cooperative transmitting can significantly improve information-theoretic secrecy in wireless networks. The type of cooperation is quite important for the resulting secrecy region. For the same number of friendly nodes, cooperative transmitting provides a larger coverage area than cooperative jamming and cooperative relaying.


The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana. Musa spp.), one of the most important fruit crops in the world, is seriously threatened by cold stress and pests such as Fusarium oxysporum var. cubense. Development of new banana cultivars with resistance to diseases or cold stress is one of the best ways to overcome this problem. However, it is extremely difficult to create such cultivars through conventional breeding because most commercial cultivars are sterile triploids bearing parthenocarpic fruits. Plant regeneration via somatic embryogenesis is the base of banana germplasm improvement using biotechnological techniques. Unfortunately, some important banana cultivars are recalcitrant in regard to the embryogenic response The production of banana (Zea mays L.), chicory (Cichorium), coconut (Cocos nucifera L.), barley (Hordeum vulgare) and olive (Olea europaea L.) Cell wall plays a very important role in the plant development. The chemical components of the cell walls are modulated during plant growth and development. Several previous studies have reported about developmental changes in cell wall components such as arabinogalactan proteins and pectins in some plant species such as maize , pectins, and a small amount of structural proteins. Pectins, one major class of chemical components, make up to 35% of the primary cell walls in dicotyledonous plants and non-graminaceous (non-grass) monocots Somatic embryogenesis is characterized by well-defined embryogenic stages, which are generally similar to those in zygotic embryogenesis. This process requires strict spatial and temporal control over cell division and elongation in situ within complex tissues and organs. These techniques enable monitoring of structural changes, organization and partial changes of function in the plant cell wall Actinidia deliciosa) Triticum aestivum L.) Citrus, cork oak (Quercus suber L.), olive and Capsicum annuum L. Daucus carota L.) suspension cells and sugar beet (Beta vulgaris L.) calli. Another immunohistochemical study described the changes of JIM5 and JIM7 epitopes during somatic embryogenesis of coconut Currently, immunohistochemical techniques using well characterized antibodies have been applied to better define plant cell wall components and to localize cell wall polymers Ruthenium red is a cationic stain with six positive charges, which forms electrostatic bonds to the acidic groups of sugars, for example carboxyl groups and sulfate groups In this study, ruthenium red along with an arsenal of pectin antibodies, including six recently developed antibodies specifically recognizing HG epitopes with varying degrees of methyl-esterification Previously, PAS has been used to detect glycogen in animal tissues and carbohydrates, proteoglycans and glycoproteins in plant tissues. It is based on the reaction of periodic acid which selectively oxidizes the glucose residues and creates aldehydes reacting with the Schiff reagent (product of fuchsine or pararosaniline and sodium bisulfite) and producing purple-magenta color. In the present study, PAS staining was used to study the histology of NECs of cultivar ‘Baxijiao’ used as a control as well as in ECs and somatic embryos of cultivar ‘Yueyoukang 1’. This histological analysis revealed different cellular organization in NECs versus ECs as well as all basic developmental embryogenic stages including ECs, pre-globular, globular, pear- shaped and cotyledonary somatic embryos of cultivar ‘Yueyoukang 1’ . In moreA surface treatment of the whole mounted fresh material (not fixed and not embedded and sectioned) with ruthenium red was used to label extracellularly secreted pectins in the mucilage covering both non-embryogenic and embryogenic cell clumps as well as somatic embryos. Overall, the intensity of pink/red stain on NECs of cv. ‘Baxijiao’  was muchA representative set of six commercially available monoclonal antibodies  has been reported to bind to HG epitopes showing different degrees of esterification. The fully de-esterified and low methyl-esterified HG were recognized by CCRC-M38, JIM5, LM18 and partially to highly methylesterified HG were recognized by JIM7, LM20 and CCRC-M34. These antibodies were tested and selected to study developmental distribution and methyl-esterification of these pectic epitopes in the cell walls of NECs, ECs and somatic embryos of banana by immunodot technique. Furthermore, the 2F4 antibody binding to de-esterified/calcium cross-linked HG and by LM5 antibody mainly binding to (1→4)-β-D-galactan of rhamnogalacturonan I were also used (RG-I) , Fig. 3.in situ immunolabeling due to the possible molecular crowding and/or masking/blocking in intact cell walls. Therefore, both qualitative and quantitative statements about relative abundance of pectic epitopes  were avoided. Nevertheless, in situ immunolabeling revealed that low and/or fully de-esterified HG epitopes such as LM18, CCRC-M38 and JIM5 as well as in the LM5 epitope representing (1→4)-β-D-galactan  content in different developmental stages during banana somatic embryogenesis revealed that it was the highest in the late-stage embryos . The GalMusa, family Musaceae and order Zingiberales of monocotyledonous flowering plants. It has been reported that banana pericarp tissue contained high levels of both uronic acid and glacturonan Pectins are major plant cell wall polysaccharides representing up to 35% of the primary cell wall in dicotyledonous plants and non-graminaceous (non-grass) monocots Here we report on the developmental histochemical (ruthenium red) and immunofluorescence (using antibodies against diverse HG epitopes and the (1→4)-β-D-galactan epitope of RG-I) localizations of pectins along with developmental study on pectin DM during somatic embryogenesis of banana. Generally, all immunofluorescence data on intact cell walls should be interpreted with caution because immunolabeling of some pectic epitopes in certain cell walls/cell types might be blocked due to the sterical hindrance of these epitopes in these walls during immunolabeling procedure. We tried to partially avoid this problem by using several antibodies recognizing diverse epitopes at the same molecule, namely at pectic HG.Histochemical staining with ruthenium red revealed pink/red colored surface-localized pectins, especially at the surface of banana embryos at pre-globular, globular and pear-shaped developmental stages. This was in good correlation with immunolocalization patterns of surface-localized pectin epitopes  at the same embryogenic developmental stages. Roughly, it was also consistent with immunodot labeling of these pectin epitopes during embryo development. Further, immunodot analysis correlated to immunolabeling with pectin antibodies suggested that banana embryogenic cultures were relatively rich in the JIM5 and LM5 epitopes, but poor in the 2F4 and CCRC-M34 epitopes. Among pectic epitopes, especially the highly methyl-esterified HG ones, recognized by LM20 and JIM7 antibodies, appeared to be developmentally regulated. Thus, both immunodot and immunofluorescence labelings of these two epitopes were weaker in embryogenic cells but stronger in pre-globular/globular and late embryos during embryo development. The immunolabeling intensities of other pectic epitopes on immunodots and immunofluorescence-labeled tissue sections also varied during embryo development, though the differences between diverse developmental stages were not as obvious as for LM20 and JIM7 epitopes.This study revealed also some differences in pectins between nonembryogenic and embryogenic cultures of banana. For example, the LM5 epitope seemed to be more abundant in NECs as in ECs when immunodot and immunofluorescence labelings were compared to each other. This might be related to the fact that this epitope was mainly immunolocalized to the small groups of starch-rich cells in EC aggregates whereas it was detected in all cells in NEC aggregates. Moreover, clusters of NECs were covered by extracellular layer containing both LM18 and JIM5 epitopes. This might suggest extracellular secretion and local accumulation of these pectic epitopes in this ECM surface layer. On the other hand, the same epitopes were localized solely to the cell walls of individual ECs, suggesting that they were not secreted towards ECM surrounding ECs. Some of these results are in good agreement with previously published reports. For instance, larger intercellular spaces and the cell wall junctions contain JIM5 epitope in kiwifruit endosperm-derived callus Previously, it was reported for monocot maize that highly methyl-esterified pectins recognized by JIM7 antibody were localized to the ECM covering embryogenic cell clumps of embryogenic callus 2+ linkages, thus promoting the formation of so-called “egg-box” model structure Sinapis alba which is composed of tightly adhered cells Formerly, acidic (low methyl-esterified) pectins were thought to play a crucial role in cell-cell adhesion Citrus proembryos Modifications of HG such as methyl-esterification are important for cell fate determination and plant development This study provides new information about the developmental localization of cell wall pectins and about methyl-esterification patterns of pectin HG epitopes during banana somatic embryogenesis. The main conclusions are:1. De-esterified and low methyl-esterified HG epitopes were detected in the surface localized ECM surrounding banana ECs and embryos.2. Partially and highly methyl-esterified HG epitopes were more abundant in the cell walls of pre-globular and globular embryos.3. De-esterification of pectins with base treatment caused imunolabeling depletion of highly methyl-esterified HG epitopes but increased immunolabeling of low methyl-esterified ones.4. The data from the present study support the hypothesis that the mechanism of intercellular attachment and cementing of cells is likely determined not only by calcium pectate gels but also by certain acidic de-esterified pectins.These results could be potentially valuable for development of future strategies aiming to improve the regeneration capacity of ECs in banana. This can be done, for example, by manipulation of pectin modifying enzymes such as pectin esterases and pectate lyases, as it was recently proposed for cyclamen somatic embryogenesis Musa spp. AAA) as well as nonembryogenic cells (NECs) of cultivar Baxijiao (Musa spp. AAA) were cultured in ZZl medium Embryogenic cells (ECs) of banana cultivar Yueyoukang 1 - N,N,N',N'-tetraacetic acid (EGTA), pH 6.9] at room temperature for 1 h. After washing in MTSB and phosphate-buffered saline (PBS) (pH 6.9), samples were dehydrated in graded ethanol series diluted in PBS and infiltrated with Steedman's wax according to previous studies NECs, ECs (both at seven days after the last subculture), pre-globular/globular embryos  and late embryos  were collected and fixed with 3.7% (v/v) formaldehyde in stabilizing buffer MTSB [50 mM piperazine--N,N'-bis(2-ethanesulfonic acid (PIPES), 5 mM MgSOTo test surface-localized pectins, suggesting an extracellular transport and accumulation of these pectins in mucilage-like ECMSN around NECs, ECs and somatic embryos at different developmental stages, the whole mount samples  were collected at various developmental stages (see above) and incubated in 0.01% (w/v) ruthenium red by shaking (120 rpm) at 30°C for 2 h. Histochemical staining of surface pectins appeared as pink/red color and it was evaluated and documented under Leica binocular microscope .All antibodies used in this study and their corresponding epitopes/antigens are presented in Immunolabeling was carried out exactly as described by Xu et al. AIR was prepared according to the method described by Louvet et al. Pectin was extracted from AIR with 0.5% (w/v) ammonium oxalate buffer at 100°C and the concentration was adjusted to 1 mg/ml. Samples were spotted (as 5 µl drops) onto nitrocellulose membrane by a micropipette. The membrane with dots was air dried at room temperature for 1 h. Assays with JIM5, JIM7, LM5, LM18, JIM20, CCRC-M34 and CCRC-M38 antibodies were carried out as described by Willats et al. The DM was calculated as moles of methanol per mol of GalA. The methanol assay was adopted from the methods described by Klavons and Bennett Methanol in potassium phosphate buffer (pH 7.5) (0–20 µg/ml range) was used as a standard. GalA content of the sample was determined colorimetrically by the metahydroxydiphenyl assay adopted from Blumenkrantz and Asboe-Hansen The extensive degradation of pectic polymers was performed by base catalyzed de-esterification. Samples were prepared as described above for immunofluorescence labeling. After dewaxing, sample sections were treated with 0.05 M NaOH at 4°C for 30 min followed by modified immunolabeling protocol .