Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide.The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate.In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite spatiotemporal expression profile of peripheral nerve regeneration. Peripheral nerve injury has been estimated to occur in about 3% of trauma patients and can lead to life-long disability . Transecin vitro models of migration [Regeneration in the peripheral nervous system is a complex process which requires the careful orchestration of multiple factors and cues to create the optimal microenvironment for regeneration to occur [reviewed in  and 3]]]3]]. Pasigration -15.The underlying events that drive the regenerative process have yet to be fully elucidated; although there has been extensive research looking at both inhibitory and promotional factors. Peripheral nerve regeneration research efforts can be generally divided into two categories, those that evaluate materials used to create a physical scaffold or nerve guide to direct the regenerating nerve   and thosResearch focused on altering the microenvironment of the regenerating nerve has also shown promise. A number of molecules have been shown to enhance nerve regeneration including nerve growth factor (NGF) -32, fibrTo further enhance our understanding of the regenerative process it would be beneficial to gain a more global view of the cellular and biochemical events that underlie peripheral nerve regeneration from both a spatial and temporal perspective by evaluating protein expression in different segments of the regenerating nerve over time. Proteomic techniques such as Western blots and immunohistochemistry haven been used successfully for spatiotemporal profiling, but these techniques are typically low-throughput and most reports focus on just a few proteins. Due to differences in experimental models, it is difficult to combine observations between studies. Proteomics techniques such as two-dimensional gel electrophoresis and mass spectrometry have been instrumental in identifying candidate proteins involved in peripheral nerve regeneration and have demonstrated the complexity of protein expression in the regenerating nerve -53. The In this study we evaluate the use of RPPAs in peripheral nerve regeneration using an established rat sciatic nerve transection injury model combined with a polyethylene nerve guide. The non-permeable, non-degradable polyethylene guide was selected for this study over a resorbable guide to simplify the harvesting of regenerated tissue. Previous studies by our group using this model have reproducibly resulted in regenerated nerve cables within 4-8 weeks  and the All antibodies were tested for their suitability for use in RPPA by western blot analysis  was compared with day 28 . The change in N-cadherin expression between day 0 and day 28 was significant in the distal portion only . Interestingly, the levels of N-cadherin in the proximal segment appeared to peak at day 14 and the expression level at 14 days was significantly higher than baseline . Both E-cadherin and N-cadherin displayed continued elevated expression up to 21 days in the distal segment followed by a plateau in expression up to 28 days. There was no significant change in E- or N-cadherin levels in the guide contents when comparing the day 0 to the day 28 time point. The guide segment results compare intact nerve (day 0 expression levels) to the regenerate which, at least for the early time points, contains no nerve. Since the guide segment contains only regenerated tissue, the lack of significant difference from day 0 represents a return to the baseline protein levels seen in intact nerve. For N-cadherin this return to baseline occurs by day 14 while fluctuations in E-cadherin expression were recorded throughout the 28 day regenerative period with above baseline peaks at ≤ 7 and 21 days and troughs that approached baseline levels at 14 and 28 days. In contrast, β-catenin, an integral member of the cadherin complex, showed a significant change from baseline only at day ≤ 7 in the proximal  and distal  segments, but in the guide tissue displayed peaks and troughs that mirrored N-cadherin, but were opposite to that recorded for E-cadherin in the same site.FAK has been shown to promote neurite outgrowth and in this model displayed an increase in expression throughout the regenerative process in the proximal and distal segments with an initial, significant drop at day ≤ 7  and recovery to higher than baseline levels by day 14 in the guide portion. The overall increase in FAK levels between day 0 and day 28 was significant in the distal segment only .In the distal nerve stump GAP-43 levels increased rapidly up to 14 days followed by a relative plateau for the remainder of the experimental course. A sharp increase in GAP-43 was also seen in the proximal nerve up until 7 days with a slower, but marked increase through 28 days. In contrast, GAP-43 protein level showed no significant change from baseline throughout the course of the experiment in the guide contents indicating an early up-regulation of GAP-43 in the regenerating tissue. The overall increase in GAP-43 between day 0 and day 28 was significant in the proximal and distal segments only .Amongst the molecules included in the growth factor group, a significant increase in the expression level of FGF-1, FGF-2 and EGF was recorded between 0-14 days in the regenerating distal nerve followed by a plateau in expression between 14 and 28 days . In the proximal nerve segment, expression levels of each of the aforementioned growth factors continued to rise past 14 days although none of the changes after day 14 reached significance. Comparing day 0 to day 28, both FGF-1 and FGF-2 demonstrated a significant increase in protein levels in the proximal nerve segment, but EGF did not . Interestingly, significantly reduced EGF expression was recorded in the guide tissue over the 28 day regeneration period . In contrast, a comparison of day 0 to day 28 protein levels for FGF-1 and FGF-2 did not reveal a significant change in the guide contents indicating a return to the baseline levels of intact nerve and both proteins displayed a temporary increase in expression that peaked at 14 days and reached significance for FGF-2 .CNTF displayed only a limited, insignificant increase in expression in the proximal and distal segments relative to the other growth factors in the study and, like EGF, significantly reduced expression in the guide tissue between day 0 and day 28 . However, unlike EGF, CNTF did not decrease throughout the entire regenerative period, instead displaying an increase in expression at 21 days followed by a further decline at 28 days.NGF displayed a biphasic expression profile in the regenerating distal nerve with significant peaks occurring at 14 and 28 days post-axotomy . No significant change in NGF expression was recorded in the proximal segment or guide compartment of the regenerating nerve between baseline and day 28. In the guide compartment the level of NGF was not significantly different than baseline by day ≤ 7 indicating an early recovery of NGF protein in this segment.The ECM proteins have been shown to play a role in growth promotion and cell migration involving directional guidance, both important processes in nerve regeneration. Fibronectin displayed a significant increase in protein levels over baseline at 21 and 28 days in the proximal segment , 21 days in the distal segment , and even at the earliest time point of ≤ 7 days no significant change from intact nerve in the guide segment. A significant increase from baseline levels was observed in the guide segment at day 14 and day 21 . Laminin displayed a significant increase in both the proximal and distal segments between 0 and 28 days , whereas the guide contents did not show a significant change when comparing the same time points indicating a return to the levels of intact nerve. Like fibronectin, day ≤ 7 laminin expression was also not significantly different than baseline in the guide segment. Collagen I/III had a distinct temporal sequence of expression which peaked at days ≤ 7 and 21 in the proximal and guide segments, respectively, and was significantly reduced compared to baseline in the distal segment  at 14 days followed by restoration to expression levels recorded in the intact nerve for the remainder of the experimental course. At day ≤ 7 the guide segment had recovered to baseline collagen I/III expression levels. Nidogen and versican protein levels were elevated overall although the change was only significant in the distal segment . Nidogen, a potentiator of Schwann cell proliferation, showed biphasic expression with peaks at ≤ 7 and 21 days post-axotomy most notably in the guide segment indicating an early increase even higher than baseline expression. Versican protein levels were elevated early in the proximal and guide segments with a significant increase at 14 days  followed by a slow decline with levels increased, but not significantly different than baseline at 28 days. In the distal segment protein levels were significantly lower than baseline at day ≤7 and day 28 .Significant advances in promoting peripheral nerve regeneration have been made on multiple fronts including the use of bioresorbable polymer guides to serve as conduits and delivery devices for different molecules that have been implicated as promotional in the regenerative process. Knowledge of the expression pattern of proteins essential to regeneration have helped predict the best candidates for exogenous administration, however this information must be pooled from individual studies with variable models in order to generate a global expression profile. In this study we used RPPA to facilitate the simultaneous temporal and spatial expression mapping of key proteins in the nerve regeneration process using a rat sciatic nerve transection injury model. The benefit of RPPA analysis is that many samples can be analyzed simultaneously. Since this technique requires very little starting material, it also enables the analysis of a large number of proteins. The limited number of proteins reported in this study was not due to a limitation in source material, but instead the limited availability of antibodies that cross-react with rat proteins and were suitable for use in RPPA. In this study every antibody was first validated by western blot and any antibody that did not produce a single predominant band by western was excluded. Although an analysis of 15 proteins does not represent the full capability of the RPPA technique, we believe that this paper provides significant insight into the regulation of these proteins and sets the stage for further proteomic evaluation using this model.The phases of regeneration in a non-permeable, non-resorbable nerve guide chamber have been described and include a fluid, matrix, cellular, and axonal phase . Within Protein expression analysis in the guide content compares intact nerve (day 0 expression levels) to the regenerate which initially contains no nerve. Based on the described regenerative process, an early increase (manifested as a return to baseline levels) in the proteins included in the ECM category would be expected in the guide portion of the regenerate. Indeed, all of the ECM proteins studied demonstrate not only a return to baseline, but an increase in expression at ≤ 7 days over intact nerve. In contrast, several of the cellular proteins such as β-catenin, FAK, and N-cadherin show an initial decline from the protein level of intact nerve in the guide segment and peak later at day 14 corresponding with the cellular phase of regeneration. This initial decline from baseline is to be expected since the guide segment does not contain intact nerve at the earlier time points. Interestingly, the growth factors represent a mixed group in the guide portion with an early return to baseline or increase over intact nerve for NGF, FGF-1 and FGF-2, and overall decline in the levels of CNTF and EGF in the solid guide contents. Overall, we saw a decrease in the expression of all growth factors in the guide contents from day 21 to day 28, the time at which axonal migration and myelination is underway. This is consistent with a previous study that demonstrated that the neuronotrophic activity of the fluid that fills the guide chamber is highest in the first few days of regeneration suggesting that the role of at least some of these molecules peaks early in the regenerative process . The genGAP-43 is present in the presynaptic terminal of novel neuromuscular junctions , and hasMembers of the cadherin family and the associated proteins that form the functional cadherin/catenin complex have been shown to play important roles in cell migration. E-cadherin is associated with the adherens junctions of the Schmidt-Lanterman incisures and is unique because it forms an autotypic adherens-type junction confined to the plasma membrane of a single Schwann cell . ExpressAs cadherin complexes assemble, there is a distinct hierarchy of catenin association led by E-cadherin/β-catenin binding followed by additional catenin members. Interestingly, β-catenin that binds to E-cadherin within the cadherin complex, revealed no significant change in expression profile throughout the regenerative process in the proximal and distal segments in our study. β-catenin levels in the guide decreased significantly at ≤ 7 days followed by a rebound to baseline levels and a significant but lower decline from baseline at 21 and 28 days. Our observation is in contrast to a report by Hatoko et al. that showed increased β -catenin expression in the graft portion of a nerve graft model of regeneration, however this difference might be explained by the fact that the previous study was evaluating expression in intact nerve grafts where our study included just the regenerated contents within a hollow guide .in vitro 85]]. Sevin vitro [While EGFR mRNA and protein levels increase following nerve injury , and an in vitro , Dubuissin vitro . We founNGF is a neurotrophin that has been shown to play a role in nerve regeneration in a large number of studies. Rich et al. demonstrated improved peripheral nerve regeneration with added NGF, as have others -32,98. NCNTF increases neuronal survival, neurite outgrowth, and axonal regeneration with exogenous administration ,100,101,in vitro and in vivo [ECM components play an essential role in nervous system development and repair. Schwann cells assemble a fibrillar network that consists of fibronectin, laminin, and collagen type IV which is thought to play a role in proliferation . The dif in vivo ,105,106  in vivo -109. We  in vivo .in vivo by regulating axonal growth [Laminins are key components of the basal lamina and laminin has been shown to play a prominent promotional role in nerve regeneration l growth ,111. Thel growth . There il growth ,114. In l growth . In bothl growth ,114. ThiCollagen is the major component of the ECM and several groups have shown an increase in collagen I and III mRNA after nerve injury as well as functional improvement with exogenous administration ,115-118.Nidogen is a component of the basement membrane. Nidogen protein and mRNA levels have been shown to be up-regulated post transection injury  and nidoThe chondroitin sulphate proteoglycan, versican, isoform V2 has been shown to be an inhibitor of axonal growth while V1 has been shown to promote neurite outgrowth -122. We Not surprisingly, cluster analysis of protein expression over time did not always separate the proteins into the functional groups we defined, but other interesting patterns were revealed Figure . In the Analysis of expression levels in the guide content is particularly interesting as this compares intact nerve (day 0 expression levels) to the regenerate which, at least for the early time points, contains no nerve. It is not surprising that a greater number of proteins show an initial decline in this segment, but interestingly many are up-regulated even at the earliest time point. In the guide segment groupings more closely follow functional roles. For example, all of the ECM proteins are represented in a single branch (early up-regulated expression) and the majority of the adhesion and motility proteins in the other (early down-regulated expression). The pattern of expression correlates to the temporal role of these proteins groups in the regenerative process where ECM proteins play an early role in the matrix phase and adhesion and motility proteins a later role in the cellular phase of regeneration. GAP-43 is clustered in the up-regulated branch and grouped closely with laminin. A functional relationship between GAP-43 and laminin has been implicated in several other studies and this expression pattern may reflect the complex role of GAP-43 in the regenerative process ,125.The expression of multiple cellular and extracellular molecules is finely orchestrated and integrated leading to the regeneration of a new nerve. Past work using this or similar models of peripheral nerve regeneration have taken a reductionist approach evaluating the contribution of individual or small numbers of proteins in the regenerative process. The RPPA approach taken in this study allows expression profiling of multiple proteins from a large number of samples simultaneously. In this study we focused on proteins known to be involved in the regenerative process and with known expression profiles. The results both validated previous observations and provided new detail and complexity, but the application can be easily expanded to include any protein with a suitable, validated antibody including posttranslational modifications such as phosphorylation. In this study we have demonstrated that RPPA is a reliable, high-throughput approach for the analysis of overall protein expression profiles with the potential to identify the temporal sequence of events underlying complex, multifaceted, biological processes.-1 sodium pentobarbital. All underwent an initial surgery during which the right sciatic nerve was exposed via a three centimeter incision made just posterior to the femoral head. The dissection was carried down through the gluteal muscle at which point the sciatic nerve was identified. The remainder of the dissection was performed under an operating microscope at 30 × magnification. This same approach was carried out during a second harvesting procedure.All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at Lahey Clinic. Forty male Sprague Dawley rats weighing between 225-250 g served as the model for nerve guide repair. All animals were anesthetized via an intraperitoneal injection of 45 mg kgA length of at least 20 mm of nerve was exposed. A 10 mm section was removed via sharp transection with straight micro-scissors leaving at least a 5 mm proximal and distal sciatic nerve stump. A 14 mm long polyethylene nerve guide  was sutured in place with 10-0 nylon suture, using a horizontal mattress stitch. The suture was placed 2 mm from the end of the guide and through the epineurium on each respective nerve stump. The full circumference of each stump was therefore drawn entirely inside the guide with 2 mm of length lying inside each end of the guide, resulting in the proximal and distal stumps positioned 10 mm apart. The 10 mm resected section of nerve was divided into two 5 mm sections  and the two sections were placed in individual tubes, and frozen immediately in liquid nitrogen. The gluteal muscle was then closed with three buried interrupted 3-0 chromic sutures and the skin was closed with a running 4-0 braided polyglycolic acid suture.At each time point , 10 rats underwent a second procedure to harvest the proximal and distal nerve ends and the contents of the guide. All adhesions on the guide were carefully taken down with sharp dissection under the operating microscope. Sharp transection of the proximal and distal stump was carried out, 5 mm from each end of the guide, with straight micro-scissors. The entire specimen, proximal and distal stumps within the guide, was taken out as one piece. The 10-0 nylon sutures were cut and the remaining tissue was separated into proximal and distal nerve stumps and solid guide contents. Proximal and distal segments included a total of 7 mm of nerve . At each time point of ≤ 7, 14, 21, and 28 days there were 5, 2, 6, and 4 rats with solid material spanning the guide, respectively (see Table 2) and sheared through a 26-gauge needle. β -mercaptoethanol (1%) was added to each sample which was boiled for 5 min. The resulting lysate was loaded across the top of a 7.5% polyacrylamide resolving gel, with one lane containing a marker. Proteins were transferred overnight onto nitrocellulose. Membranes were blocked in 10% milk in TBS with 0.05% Tween-20, cut into strips and placed on primary antibody overnight at 4°C. Blots were washed in TBS with 0.05% Tween-20, three times for 15 min each, and secondary antibody linked to horseradish peroxidase was incubated with the blots for 60 min at room temperature. Blots were then washed as described above and developed with an ECL kit . Only antibodies that produced a single band by western blot analysis were used for RPPA.Nerve lysate for antibody validation was prepared by crushing a pooled sample of the excised normal 5 mm nerve segments under liquid nitrogen, using a mortar and pestle. The finely ground tissue was lysed in hot sample buffer ; Nidogen and FGF1 ; CNTF and FAK ; FGF2, Fibronectin, GAP-43, E-cadherin and β-catenin  EGF and laminin-1 gamma-1 ; Collagen Type I/III ; Versican ; and N-cadherin . The manufacturer of the fifteen antibodies as well as the concentrations used in RPPA and western blot are shown in Table Each nerve sample was crushed under liquid nitrogen with a clean mortar and pestle and suspended in 1.0 ml ice-cold PBS and centrifuged at 4000 rpm for 2 min. After removing the PBS, a volume of lysis buffer, composed of 9 M Urea, 4% Chaps, 2% Pharmalyte pH 8.0-10.5 and 65 mM Dithiothreitol, equal to the size of the pellet, was added and aspirated up and down 3 times. Samples were centrifuged at 14,000 rpm for 30 min. The supernatant was collected and stored at -80°C and the pellet was discarded. All steps following the initial PBS wash were performed at 4°C. Ten, 2-fold serial dilutions were made from each lysate with a buffer containing 6 M urea, 2.7% CHAPS, 1.3% Pharmalyte (pH 8.0-10.5), and 43.6 mM Dithiothreitol ,127.Samples were arrayed using the Aushon 2470 solid pin, contact microarrayer equipped with 185 μm pins . The print matrix was a nitrocellulose-cast glass slide . Each slide contained 10, 2-fold serial dilutions of each sample and included a technical replicate totaling 3,360 lysate spots per slide. All printing was carried out at 80% relative humidity.Printed nitrocellulose-coated slides were washed for two 15-min periods in deionized water. Slides were blocked for 1 h in I-Block  with continuous shaking at room temperature. Immunostaining of microarrays was performed on an automated stainer  using a tyramide-based catalyzed signal amplification system according to the manufacturer's protocol . A negative control was included using a non-specific antibody solution  substituting for the primary antibody.25 [25 value of each nerve sample was normalized for total protein concentration determined by colloidal gold staining.The slides were scanned at 2400 dpi resolution in an uncompressed, 16-bit TIFF image format using an optical flatbed scanner. Images were processed by the P-SCAN and ProteinScan (version 0.21) programs written using Matlab software  by the National Institutes of Health . Outliers traceable to defects in the arrays were eliminated and the data were analyzed. The numerical output for each sample was generated by a modified Dose Interpolation algorithm, DI25 . ProteinThe protein intensity data was log-transformed to ensure data normality. To investigate the spatial and temporal differences in the protein expression levels, a linear mixed effects model was applied to each protein separately. The models contained fixed effects for different times and sample types and are adjusted for baseline protein expression levels. To account for correlated data structure random rat effect was used in the models. To determine which particular times and samples were significantly different, Scheffe's test  was usedECM: Extracellular matrix; NGF: Nerve growth factor; FGF: Fibroblast growth factor; CNTF: Ciliary neurotrophic factor; RPPA: Reverse phase protein array; GAP-43: Growth associated protein 43; FAK: Focal adhesion kinase; EGF: Epidermal growth factorAHH and JA were employees of Aushon BioSystems (manufacturer of the 2470 microarrayer) at the time that this study took place. AHH's affiliation at the time of publication is with Lahey Clinic.CRL and JVM performed animal surgeries. CRL performed antibody validation experiments, reverse phase microarray processing and assisted with manuscript preparation. DJB contributed to study design, data interpretation, and critical manuscript review. TL performed statistical analysis. AHH contributed to study design and data interpretation, performed reverse phase microarray printing and drafted the manuscript. JA-performed reverse phase microarray printing and critical review of the manuscript. ICS contributed to study design and data interpretation. KMR-C contributed to study design, data interpretation, and critical review of the manuscript. All authors read and approved the final manuscript.q-values; The statistical significance of expression data was determined and results are presented as q values, false discovery rate adjusted p-values.Click here for file


NOD2 polymorphisms, identified as disease-associated variants in Crohn's disease, have recently been suggested as important candidate gene markers in the outcome of HSCT. It was originally postulated that as the clinical manifestation of inflammatory responses characteristic of several post-transplant complications was of notable similarity to those seen in Crohn's disease, it was possible that they shared a common cause. Since the publication of this first paper, numerous studies have attempted to replicate the results in different transplant settings. The data has varied considerably between studies, and as yet no consensus on the impact of NOD2 SNPs on HSCT outcome has been achieved. Here, we will review the existing literature, summarise current theories as to why the data differs, and suggest possible mechanisms by which the SNPs affect HSCT outcome.Haematopoietic stem cell transplantation (HSCT) is a valuable tool in the treatment of many haematological disorders. Advances in understanding HLA matching have improved prognoses. However, many recipients of well-matched HSCT develop posttransplant complications, and survival is far from absolute. The pursuit of novel genetic factors that may impact on HSCT outcome has resulted in the publication of many articles on a multitude of genes. Three  The improvement in transplant techniques and practice has resulted in similar survival prospects for recipients of a well-matched UD as that using a sibling [Allogeneic haematopoietic stem cell transplantation (HSCT) is an important treatment option in the management of many diseases including malignant and non-malignant haematological disorders, immune deficiencies and inborn errors . The inc sibling , 3. Howe sibling .The vital role of HLA matching in transplant outcome is accepted, but there is still controversy as to which of the six major HLA genes are most important. The current perspective on what constitutes a well-matched donor is a 10/10 HLA allele match that is matched at an allele level for HLA-A, -B, -C, -DRB1, and -DQB1 , 5–7. CoWhile the current donor selection criteria for matching donors and recipients usually refer to five of the classical HLA genes , the impact of a sixth gene, HLA-DPB1, on the outcome of UD-HSCT is emerging. Current data suggests that nonpermissive HLA-DPB1 mismatches increase the risk of GvHD and transplant-related mortality –15.NOD2) gene  gene). The data from these studies is conflicting. Here, we will review the current data, on the impact of NOD2 polymorphisms on the outcome of HSCT, potential causes of differences in the data and possible mechanisms by which the variants affect outcome.Despite the benefit resulting from having a 10/10-matched donor, the survival of such a group of individuals is far from being absolute. Recipients receiving a graft from a well-matched sibling donor can be susceptible to getting GvHD. Conversely, some recipients of ≤9/10 HLA-matched grafts do survive and can achieve full remission of their disease . While cNOD2 gene is located in humans on chromosome 16 (16q21) [NOD2 encodes the NOD2 protein, a member of the NLR  containing) protein family [The  (16q21) . It is an family –22. Othen family . These pn family –26.Early functional studies identified NOD2 expression in antigen-presenting cells, specifically intestinal epithelial cells , Paneth κB and mitogen-activated protein kinase pathways [in vitro to viral infection by the recognition of a single-stranded (ss) RNA ligand [κB pathway. The NOD2 protein functions as a regulator of infection by the recognition of pathogenic ligands and the induction inflammatory responses via a number of pathways. The most studied interaction is the response to the bacterial ligand muramyl dipeptide (MDP), a derivative of peptidoglycan, which is a component of both Gram-positive and -negative bacterial cell walls , 38. Recpathways –41. In apathways , 43. NODA ligand . Here, sNOD2 gene is proving to be highly polymorphic with over 660 single nucleotide polymorphisms (SNPs) reported to date both in the literature [http://www.genecards.org/, http://www.ensembl.org/ and http://fmf.igh.cnrs.fr/ISSAID/infevers/) [The terature –47 and ifevers/) –50. The NOD2 as a susceptibility locus [NOD2 gene.Early studies to identify possible genetic factors that were affecting the incidence of Crohn's disease, a chronic inflammatory disorder of the gastrointestinal tract that can be complicated by anaemia, stenosis, and fistulae, mapped ty locus . Furtherty locus , 51. It ty locus . Other dty locus . SubsequNOD2 exons 4, 8 and 11 respectively. SNPs 8 and 12 are nonsynonymous nucleotide substitutions that result in amino acid changes, SNP 8 (coding (c.) 2104C>T, protein (p.) R702W) and SNP 12 . SNP 13 differs in that it involves the insertion of a nucleotide that results in a frameshift within the coding sequence causing the introduction of an early termination codon and thus a truncated protein . SNP 8 is located within the central NBD region of the molecule, while SNPs 12 and 13 are found within LRRs 7 and 10, respectively, of the NOD2 LRD [SNPs 8, 12, and 13 are located within NOD2 LRD , 46. NOD2 gene have been implicated in numerous diseases. SNPs 8, 12, and 13 have been correlated with increased risk of ankylosing spondylitis [NOD2 SNPs 8, 12, and 13 have also been correlated with an increased risk of malignant diseases such as colorectal [NOD2 genotype were limited to the presence of SNP 13. More recently, NOD2 SNPs have been shown to affect graft survival and mortality post renal transplantation [Following the early studies in Crohn's disease, polymorphisms throughout the ndylitis , psoriatndylitis , and morndylitis . Three andylitis . In addilorectal , gastriclorectal , breast,lorectal  as well lorectal , althougantation  and coroantation .κB, resulting in reduction in the production of cytokines and antimicrobial peptides [NOD2 variant similar to SNP 13 had increased sensitivity to MDP and elevated levels of NF-κB activation when compared to WT mice suggested a gain-of-function effect of NOD2 SNPs [NOD2 variants contributed to the onset of Crohn's disease, these findings have not been replicated in human studies, and further data has been published that confirm the loss-of-function mechanism [NOD2 variants appear to reduce the ability of NOD2 to recognise MDP and consequently to stimulate NF-κB responses. It has been suggested that the inflammatory response seen in Crohn's disease results from the inability of toll-like receptor-2 (TLR-2) to become tolerant to its ligand in the absence of appropriately functioning NOD2, resulting in upregulation of proinflammatory cytokines [SNPs 8, 12, and 13 are thought to reduce the ability of MDP to activate NOD2 and consequently the activation of NF-peptides –66. ThesOD2 SNPs , 68. Whiechanism –72. Thusytokines , 74. In ytokines . NOD2 variants other than the three aforementioned SNPs has not been investigated to the same extent. NOD2 polymorphisms outside of the LRD do not appear to alter the ability of MDP to stimulate NOD2. In the case of the variants associated with Blau syndrome, all of which are located within the central NOD region of the protein, an increase in NF-κB activity has been reported [The impact of reported , 65. ThiNOD2 variants that are purported to increase the risk and severity of Crohn's disease might also contribute to the risk of GvHD, particularly gastrointestinal GvHD, due to their notable similarity in clinical symptoms [It was originally postulated that the symptoms . In the NOD2 genotyping for SNPs 8, 12 and 13. The cohort consisted of a mix of HLA-matched related donor, unrelated donor and a small number of one HLA antigen-mismatched related donor, transplants. Transplants were performed as a therapy for acute leukaemia, myeloproliferative disorder, lymphoma, or myeloma. Approximately 44% of the cohort underwent T-cell depletion, predominantly with antithymocyte globulin (ATG), while a small number of individuals were treated with alemtuzumab or CD34+ cell selection. The results of this study showed that 29.5% of HSCT pairs in this cohort had at least one of the NOD2 variants. The authors correlated the presence of any of the three SNPs in the genotype of the pair  with increased severe aGvHD, (grades III-IV), severe gastrointestinal aGvHD and nonrelapse mortality [In the first published study by Holler et al.  169 HSCTortality . When thNOD2 genotyping of recipients and donors showed similar frequencies of SNPs 8, 12, and 13 to their earlier study and, importantly, between the different cohorts that were included in the study. The data showed that the effect of NOD2 variants on clinically significant aGvHD (grades III-IV) and gastrointestinal GvHD persisted in this new cohort, while a trend for increased cGvHD was also noted. A dosage effect of the SNPs was seen in this study where individuals with increasing numbers of SNPs correspondingly had an increasing risk of aGvHD. The SNP dosage effect was also seen on the incidence of nonrelapse mortality. Survival was affected, but only when variants were present in the recipient genotype or in both the recipient and donor genotypes. The authors also described how the use of particular gastrointestinal decontamination agents could reduce the risk of aGvHD and nonrelapse mortality seen with NOD2 SNPs. Specifically, the effects of NOD2 variants were only seen in individuals who received either no decontamination or those whose protocol included the antibiotic Ciprofloxacin. In their subsequent analysis, the authors extended the cohort to 303 HLA-matched sibling HSCT pairs, transplanted at one of five European centres . The undNOD2 variant genotype was analysed separately in the related and unrelated donor cohorts. The presence of any NOD2 variant in the genotype of the pair was correlated with significantly increased severe aGvHD (grades III-IV), non-relapse mortality and reduced overall survival in recipients of a related donor HSCT. In the UD-HSCT cohort, aGvHD was the only outcome affected by the presence of any of the three SNPs, while detrimental effects on nonrelapse mortality and survival were associated with the presence of SNP 13 within the donor's genotype. The association of specific gastrointestinal decontamination protocols (either none or Ciprofloxacin-based therapies) with increased effects of NOD2 variants was confirmed in these cohorts.In their third and most recent study, Holler and colleagues have extended their cohorts further to include 358 HLA matched related donor and 342 unrelated donor HSCT pairs . ApproxiNOD2 variant genotype on HSCT outcome described by Holler et al. A recent study by a group in The Netherlands described the effects of NOD2 SNPs 8, 12, and 13 on the outcome of 85 HLA-identical sibling transplants [NOD2 variant frequencies were similar to those reported in the earlier studies and in the general Dutch population. The authors confirm the detrimental effect of any NOD2 variant on the risk of clinically significant aGvHD and nonrelapse mortality. As described in the earlier studies, the effect was most profound when both the recipient and donor were positive for any one of the SNPs.Other groups have confirmed the effects of nsplants . The cohNOD2 polymorphisms with GvHD. Elmaagacli and colleagues published data on the effect of the variants in a cohort of 403 related and unrelated donor transplants [NOD2 variants in this cohort was similar to those described in other studies. Although an increased risk of aGvHD (grade III-IV) was seen when recipients and donors were both positive for one of the NOD2 variants, a protective effect was associated with an SNP in the donor genotype. The protective effect was also seen on disease relapse in pairs where both the recipient and donor had at least one NOD2 SNP. Unlike previous studies, no effects of NOD2 genotype on nonrelapse mortality or survival were seen. The authors suggested that the possible reason for the lack of association here was due to their routine use of gastrointestinal decontamination with agents to target both Gram-positive and negative bacteria.Not all studies have been able to demonstrate an association of nsplants . The recNOD2 variants in a more homogeneous cohort [NOD2 genotyping was performed on a cohort of 142 AML recipients and their HLA-matched sibling donors. As in previous studies, the reported frequency of SNP-positive recipients and donors was similar to those found elsewhere. The cohort only included recipients who received myeloablative conditioning regimens and T-cell replete grafts. Unlike in their previous study, no protective effects of NOD2 SNPs were associated with GvHD. A significant association was seen between SNP-positive recipients and an increased risk of any aGvHD (grade I–IV) and severe aGvHD (grades III-IV). Interestingly, after multivariate analysis, only a correlation with grade II–IV remained significant (relative risk (RR) 3.7652, P < 0.002). No impact on overall survival or nonrelapse mortality was reported.In a recent update by this group, the authors have investigated the affects of s cohort . NOD2 geNOD2 genotype with increased aGvHD [NOD2 genotyping was performed on 85 HLA-matched sibling HSCT pairs. The underlying diseases of the recipients were acute leukaemia, myeloproliferative disorder, lymphoma, myeloma, myelodysplasia, aplasia, and chronic lymphocytic leukaemia. All recipients had T cell depletion included in their conditioning regimens, although the method was not reported. The authors report an association of recipient NOD2 variant genotype with significantly reduced event-free survival. No other variable was significantly affected [Granell et al. also failed to correlate ed aGvHD . Here, Naffected .NOD2 genotype on HSCT outcome [NOD2 genotype was investigated in a cohort of 196 recipients of an unrelated donor HSCT for an acute leukaemia. T-cell depletion was included in the conditioning regimens of 83% of recipients, with in vivo alemtuzumab being the preferred method. We reported a significant correlation between SNP-positive pairs  and increased risks of disease relapse and death. In accordance with the data published by Granell et al. [NOD2 SNPs on aGvHD was noted although it remained nonsignificant. Despite failing to achieve statistical significance, this data was in accordance to that reported by Elmaagacli and colleagues [Our group has also reported the effects of  outcome . Here, tl et al. , we werelleagues .NOD2 genotype and disease relapse [in vivo ATG included in their conditioning regimens. The authors performed extensive analyses to determine if an association between NOD2 genotype and aGvHD could be identified. A trend towards reduced gastrointestinal aGvHD was reported in recipients positive for any NOD2 variant, but this affect was limited to univariate analyses. There were no significant differences in GvHD in any of the other models tested. Recipients positive for any of the three SNPs did have a significantly increased risk of disease relapse, although this was only a trend after multivariate analysis (P = 0.056). A study published in 2010 from a group in Dresden, Germany also reported a correlation between  relapse . This siNOD2 SNPs in a large, multicentre, paediatric cohort [P = 0.049). This study also confirmed the effects of NOD2 genotype on overall survival. A trend for lower survival was reported in pairs where the recipient had at least one of the three variants. Additionally, survival was also lower in recipients only positive for NOD2 SNP 13. A brief communication published last year highlighted the impact of c cohort . A totalNOD2 variants on bronchiolitis obliterans (BO) and bronchiolitis obliterans organising Pneumonia (BOOP), two serious late-onset, non-infectious pulmonary complications that can occur after HSCT. Hildebrandt et al. [NOD2 SNPs, effects that persisted after multivariate analysis. It is important to point out, however, that the overall number of recipients who developed BO was very low in this cohort . In contrast to this data, Ditschkowski et al. did not find an association between NOD2 genotype and the incidence of BO/BOOP in their cohort of 281 sibling donor HSCT pairs [in vivo T cell depletion. As in the previously described study, the overall incidence of BO/BOOP was low .Two studies have specifically reported data on the impact of t et al.  analysedCT pairs . TransplNOD2 variants, several studies have suggested that there are no significant effects on HSCT outcome. Groups from Sweden [Despite the plethora of data available showing an effect of m Sweden , Germanym Sweden , the Unim Sweden  and The m Sweden  have perNOD2 variants on the outcome of HSCT. It would be reasonable to assume that the potential mechanisms of how the SNPs cause functional irregularities may be common but that the manifestation of the effects differs between groups. Here, we will discuss possible mechanisms by which NOD2 genotype may affect HSCT outcome. There does not yet appear to be a consensus on the impact of κB pathway [NOD2 variants can affect the outcome of HSCT. NOD2 is known to function as a regulator of cytokine production and a mediator of proinflammatory responses upon recognition of the bacterial ligand muramyl dipeptide , 93. Fun pathway , 94. ThiNOD2 genotype causes an effect after HSCT is that the inability of the NOD2 variant proteins to initiate cytokine production could, in theory, lead to a massive disruption of the cytokine storm, resulting in a lack of GvL or GvHD responses. An early event posttransplant is the onset of the “cytokine storm” , an extrNOD2 genotype-related dysregulation of cytokine production may not be the only contributing pathway to the cytokine storm, the role of NOD2 and other sensors of bacterial infection has long been proposed as major factors in GvHD responses. Studies that have shown that gastrointestinal mucosa damaged by aggressive treatments such as the conditioning regimens used in HSCT allow bacterial ligands, specifically the MDP homologue Lipopolysaccharide (LPS), to seep into systemic circulation. Once there, T cells specific for these ligands are capable of stimulating cytokine production and eliciting GvHD responses [NOD2 SNPs can increase the permeability of the gastrointestinal mucosa and potentially increase the ability of bacterial ligands to enter systemic circulation [NOD2 variant genotype result in an increased level of circulating LPS, which are able to prime T cells and thus initiate strong GvHD responses. These effects are in concordance with the data published by numerous groups correlating NOD2 variant genotype and increased aGvHD. While the effect of esponses –103. It culation . It is pNOD2 are known to cause a reduction in IL-12 production [NOD2 is also known to have a synergistic relationship with TLRs and is thought to provide some regulatory control over their ability to stimulate cytokine production , 104–106oduction . Interesoduction  without oduction , 109. NOD2 variant genotypes results in their evasion of immunesurveillance activity. This escape mechanism would lead to the proliferation of leukaemic cells and thus disease relapse after transplant. This theory is consistent with the observations that NOD2 polymorphisms cause disruption of GvL responses. NOD2 is expressed both intracellularly and on the cell surface of epithelial cells. It has been suggested that this membrane recruitment of the protein is necessary to initiate a functional response , 35. TheNOD2 SNPs 8 and 12 on the membrane recruitment of NOD2 has been reported to date, it is possible that they have an alternative mechanism by which they cause cells to evade immune responses. SNP 8 is located within exon 4 of the NOD2 gene and is found between the NBD and the LRD of the protein [NOD2 molecule to recognise MDP, leading to the failure of NOD2 to initiate NF-κB signalling and its related downstream events.Although no effect of  protein , 110. Se protein , 111. ItA logical explanation for the divergent results could be the heterogeneity in the characteristics and treatment of the recipients, not only between studies but also within each of the cohorts themselves. An obvious difference between the studies is donor source. The advances in transplant techniques and practice have resulted in similar survival prospects for recipients of a well-matched UD and related donor HSCT , suggestNOD2 SNP association studies reported the use of T-cell depletion in their treatment protocols, several methods  were included, and thus it is important to consider the effectiveness of these different methods. For example, the anti-CD52 antibody alemtuzumab targets all human cells of lymphoid lineage, although NK cells appear to be relatively spared [A second and strikingly different factor between the cohorts is the use of T-cell depletion within the conditioning regimens. T-cell depletion is used as a mechanism of reducing the risk of GvHD, although a consequence of this may be an increase in disease relapse , 112. Why spared –115. CD3y spared . The effNOD2 genotype and GvHD were either T-cell replete regimens or included ATG or partial CD34+ cell-selected grafts [NOD2 variants with impaired Graft-versus-leukaemia (GvL) effects included consistently higher numbers of recipients treated with T-cell-depleted protocols (85–100%) and in some cases included alemtuzumab [In addition to the method of T-cell depletion used, notable differences in the number of recipients treated varied between the studies (approximately 30–100%). It is interesting to note that a high number of studies that reported a correlation between d grafts , 79, 85.mtuzumab , 82, 84.NOD2 SNPs may be more evident in recipients who received either no decontamination or those who were treated with Ciprofloxacin-based therapy [NOD2 variants in cohorts stratified by protocol. It would be prudent for future studies to include this data in their analyses where possible in order for the exact relevance of this information to be obtained.Gastrointestinal decontamination, a method of using drugs to control levels of bacteria within the gastrointestinal tracts, may also be used all around transplantation as a method of controlling GvHD , 117. Ho therapy , 78. Elm therapy . In addi therapy . UnfortuNOD2 genotype in recipients diagnosed with an acute leukaemia [NOD2 genotype with any posttransplant complication had a notably low number of recipients with acute leukaemia in their analyses [NOD2 SNPs alter the responsiveness of recipients with an acute leukaemia to their treatment. This may occur by modulation of the pathways of disease progression, rendering recipients resistant to treatment. While no direct evidence of the involvement of NOD2 variants in leukaemia progression exists, there is much data to show how it can affect the other diseases that are associated with the polymorphisms. In Crohn's disease, NOD2 SNPs 8, 12, and 13 have been correlated with distinct disease phenotypes, in particular with the site of Crohn's disease within the gastrointestinal tract and with the age of onset [NOD2 genotype may also alter the recipient's response to drugs or conditioning therapies. Studies have shown that NOD2 polymorphisms can affect the response to antibiotic treatment of perianal fistulas in Crohn's disease patients. The data showed that patients with an NOD2 WT genotype had a 33% rate of complete response to treatment as compared to none of the patients with NOD2 variant genotypes [Several studies, including ours, have demonstrated the effects of eukaemia , 83, 85.eukaemia . Other sanalyses , 92. A pof onset –122. NODenotypes . NOD2 genotype on transplant outcome, data has been published that contradicts these findings [NOD2 SNPs reported. The overall SNP frequencies were between 10–15% lower than reported elsewhere. The difference in the frequency of NOD2 SNPs between different ethnic and geographic populations has been widely discussed [While the majority of studies have shown an effect of findings –92. As discussed –127. ThuNOD2 genotype and detrimental posttransplant outcomes. This may imply that cells which express NOD2 and remain in the recipient after their conditioning regimens, such as tissue macrophages, dendritic cells, and Paneth cells, may facilitate GvHD or GvL responses, and that these responses are limited in recipients with NOD2 variant genotypes. The ability of recipient cells, specifically dendritic cells, to initiate GvHD effects has been reported [NOD2 genotype in murine models of GvHD [NOD2 knock-out mice showed no significant differences in the ability of the repopulating cells to proliferate, to be activated, or on their expression of gut-homing molecules. The risk of developing GvHD was similar in the two groups. Conversely, NOD2 knock-out recipient mice showed significantly higher levels of GvHD than their WT counterparts, and importantly, the organs targeted were the liver and the small and large bowels. Further tests showed that recipient NOD2 genotype was also able to effect donor T-cell functional capabilities. While the translation of murine studies into human models does not always result in the same findings, these data in combination provide some evidence to substantiate the observation that recipient genotype appears to significantly correlate with HSCT outcome in humans.A common feature of many of the studies is the correlation between recipient reported . Additio of GvHD . Here, mNOD2 genotype results in impaired GvL responses do not fit this model. A possible explanation for this is that recipient cells that are more resistant to the effects of pretransplant conditioning regimens  are responsible for the lack of GvL effects. NK cells have been shown to be more resistant to the T-cell depletion agent alemtuzumab than other targeted subgroups [NOD2 variant genotypes, this NK cell activation is limited, resulting in a reduced ability to initiate GvL responses. Interestingly, in our study, where predominant T-cell depletion with alemtuzumab was used, an increase in disease relapse was seen in recipients with NOD2 polymorphisms. The studies that have suggested the ubgroups . The impubgroups , and theubgroups , 130. Imubgroups . NK cellubgroups . It is pNOD2 polymorphisms other than SNPs 8, 12, and 13 may have on HSCT outcome. It is possible that these SNPs are only markers for detrimental outcomes and that the true association is with one or more untested polymorphisms that may be in linkage disequilibrium with these known variants. As stated previously, NOD2 is highly polymorphic with some minor allele frequencies reaching 40% in certain populations. It would be prudent for future studies to consider the effects of the previously unstudied variants in any future analyses. It is possible that reanalysis of the published data including novel variants may result in concordance between different groups and potentially elicit an effect of NOD2 genotype in cohorts where no association has been demonstrated previously. Finally, it is important to consider what impact NOD2 genotype on HSCT outcome, it must be concluded that the gene and its variants currently indicate an important role in transplant biology. The published data also reaffirms the belief that personalised medicine based on a combination of recipient and donor characteristics, HLA matching, and non-HLA genetics could provide the key to superior outcomes after HSCT.Despite the many questions that remain even after eight years of investigation into the importance of


We investigated if the changes in pancreatic islet sst expression in diabetic NOD mice compared to normoglycemic mice are a consequence of hyperglycemia or the ongoing immune reaction in the pancreas. Pancreatic islets were isolated from NOD mice precultured for 5 days and further cultured for 3 days at high or low glucose before examined. Islets were also isolated from NOD mice and transplanted to normal or diabetic mice in a number not sufficient to cure hyperglycemia. After three days, the transplants were removed and stained for sst1–5 and islet hormones. Overall, changes in sst islet cell expression were more common in islets cultured in high glucose concentration in vitro as compared to the islet transplantation in vivo to diabetic mice. The beta and PP cells exhibited more frequent changes in sst expression, while the alpha and delta cells were relatively unaffected by the high glucose condition. Our findings suggest that the glucose level may alter sst expressed in islets cells; however, immune mechanisms may counteract such changes in islet sst expression.Somatostatin acts via five receptors (sst In rodents, somatostatin inhibits secretion of several hormones such as insulin, glucagon, GH, TSH, and prolactin. It can also regulate gastrointestinal motility, gastric acid, and exocrine pancreatic secretion [Somatostatin is a peptide hormone, known to have several biological functions, and its effects are mediated via five specific membrane-bound receptors sst-5. In roecretion –8.in vivo syngeneic islet transplantations to diabetic as well as normoglycemic NOD mice and cultured NOD mouse islets in vitro at different glucose concentrations and subsequently evaluated islet sst expression. The rationale for this design is that the in vitro system would provide an environment with only an elevated glucose concentration, whilst in vivo after islet transplantation both an influence from autoimmunity and a diabetic milieu can be envisaged.Type 1 diabetes mellitus is considered to be caused by autoimmune destruction of the pancreatic beta cells, rendering the pancreas unable to synthesize and secrete sufficient amounts of insulin. The nonobese diabetic (NOD) mouse is an animal model with many features resembling human type 1 diabetes, such as infiltrating immune cells (insulitis) accompanied by spontaneous hyperglycaemia . We haveInbred NOD mice , originally obtained from the Clea Company, Aobadi, Japan, were used for this study. The animals had free access to water and pelleted food and were housed in a room with a 12-hour light/dark cycle. All experiments were approved by the local animal ethics committee at Uppsala University  and were in accordance with international guidelines (NIH publications 85–23). Blood glucose determinations  were performed on blood samples taken from the tail tip, and the weight of the animal was measured. 2 before experiments. Medium was changed every second day.For islet isolation, male NOD mice (4–7 weeks of age) were killed by cervical dislocation followed by resection of the pancreas. Pancreatic islets were isolated by collagenase digestion procedure , and islin vitro, groups of 100 pancreatic islets (n = 4) were cultured in RPMI 1640 + 10% FCS supplemented with 5.6 mM or 28.8 mM glucose for three days . The men = 6) and diabetic female (n = 6) NOD mice were anaesthetised with an intraperitoneal injection of 0.02 mL/g body weight of Avertin  in 10 mL 2-methyl-2-butanol ). Cultured pancreatic islets, 200 islets per graft, were collected in a braking pipette and implanted beneath the left kidney capsule. It should be noted that the number of implanted islets was on purpose chosen to be too low to normalize the hyperglycemia of the diabetic mice. The blood glucose levels were determined daily, and the animals were killed after three days, and the graft-bearing kidney was dissected free. The part of the kidney containing the islet graft was excised leaving a margin of 1 mm of each side of the graft.A female NOD mouse was considered diabetic when the nonfasting blood glucose concentration exceeded 10 mM and served as recipient of grafted islets to diabetic mice, whereas females with blood glucose below 10 mM were designated as normoglycemic. Normoglycemic .The production and specificity of subtype-specific somatostatin receptor antibodies has been described earlier , 12. TheThe specimens were single stained for the five sst receptors . The imm1-5 on the transplanted islet cells as well as the cultured islets, we used the immunofluorescence method described previously [1-5 in a cocktail with chicken anti-insulin , chicken antiglucagon ), sheep antisomatostatin , or sheep anti-PP  as previously described [To investigate the coexpression of ssteviously . The traIn the double fluorescence study, the kidney grafts or cultured islets were examined in a Leica Leitz DMR fluorescence microscope  equipped with filters of 492 nm to 510 nm for Cy2 (green) and 550 nm to 570 nm for Cy3 (red). Pictures from a Zeiss Axiocam camera  of each transplants, using both filters, were merged together with Adobe Photoshop 7.0 software , in which a yellow colour indicated coexpression of the sst subtype with any of the four islet hormones tested in this study. The results are expressed as a percentage of ssts-positive cells in relation to the total number of the respective islet cell type in a specific pancreatic islet.t-test, where P < 0.05 was considered as statistically significant. The computer program used was SigmaStat 2.0 .Data are presented as means ± SEM, and groups of data were compared using Student's in vivo at least during the last two days. NOD mouse islets grafted into normoglycemic female NOD mice were used as control, and these mice remained normoglycemic throughout.The blood glucose concentrations for the transplanted mice during three days are shown in 1-5 antibodies together with the islet hormones insulin, glucagon, somatostatin, and PP. To investigate the influence of elevated glucose concentrations on the islet sst expression pattern, grafts or cultured islets were double stained with sst1-5 with insulin, that is, beta cells in in vitro cultured islets  in vitro was higher in comparison to the glucose concentration of the transplanted diabetic mice (15–20 mM). It is possible that this difference in islet exposure to glucose had an impact on changes in islet cell sst expression. Moreover, the changes observed in vitro did not correspond to changes obtained in vivo, which could argue in favour of the assumption that the level of glucose is of importance. The changes in sst expression observed after either culture at high glucose or after islet transplantation to diabetic recipients are schematically summarized in in vivo and in vitro data suggests that the alpha and delta cells were relatively unaffected by the high-glucose conditions, whilst beta and PP cells exhibited more dynamic changes. Concerning the specific sst, it was found that in vitro exposure to high glucose affected sst5 expression on alpha, beta, and delta cells. Sst5 activation is suggested to inhibit the insulin secretion in rodents [5 expression seen in this study is a downregulation of receptor expression to avoid signalling which may inhibit the insulin secretion from beta cells. Interestingly a similar decrease in beta cell sst5 expression was found in the spontaneously diabetic NOD mice [5 expression on delta cells is unclear, but it might contribute to an autocrine-mediated dampered secretion of somatostatin which would promote insulin secretion. The combined analysis of the  rodents , 15–17. 3 was increased on beta cells in isolated islets cultured at higher glucose concentrations. The insulin-producing cell line MIN6 has previously been reported to express sst3, and this was suggested to be related to MAP kinase and c-fos signalling in inhibition of cell growth by somatostatin [3 by a high glucose concentration could result in a restriction on beta-cell proliferation promoted in a milieu of a high-functional demand on the beta cells. Moreover, sst3 may also affect signal transduction associated with adenyl cyclase activity [The expression of ssttostatin . An upreactivity , which cactivity .in vitro, the sst1 expression was increased and sst3 expression decreased on PP cells. It is possible that these combined changes in expression of sst is part of the regulation of the PP-cell response to high glucose. Further studies, however, are needed to address this question.PP secretion is inhibited by somatostatin and by elevated blood glucose levels . At highin vivo. Either immune-related mechanisms or other in vivo regulatory mechanisms may counteract effects of elevated glucose on islet cell sst expression. Future studies should address the mechanisms of the immune response for the changes in sst expression in pancreatic islets observed in NOD mice with a spontaneously developed diabetes.In our earlier study in NOD mice, we reported a higher sst expression in the pancreatic islets of diabetic mice as compared to normoglycemic NOD mice . The mec


The folding pathway, three-dimensional structure and intrinsic dynamics of proteins are governed by their amino acid sequences. Internal protein surfaces with physicochemical properties appropriate to modulate conformational fluctuations could play important roles in folding and dynamics. We show here that proteins contain buried interfaces of high polarity and low packing density, coined as LIPs: Light Interfaces of high Polarity, whose physicochemical properties make them unstable. The structures of well-characterized equilibrium and kinetic folding intermediates indicate that the LIPs of the corresponding native proteins fold late and are involved in local unfolding events. Importantly, LIPs can be identified using very fast and uncomplicated computational analysis of protein three-dimensional structures, which provides an easy way to delineate the protein segments involved in dynamics. Since LIPs can be retained while the sequences of the interacting segments diverge significantly, proteins could in principle evolve new functional features reusing pre-existing encoded dynamics. Large-scale identification of LIPS may contribute to understanding evolutionary constraints of proteins and the way protein intrinsic dynamics are encoded. Protein dynamics range from local fluctuations of specific regions loci. Thus, there is a need for simple and reliable methods of computational analysis that could help to identify and delineate the boundaries of such regions.At present, the intrinsic flexibility and dynamic behavior of individual proteins can be investigated at atomic or residue level, in a one-to-one basis, by using well established techniques, such as hydrogen exchange NMR Proteins are generally organized into folding domains, some proteins consisting of just one. The interior of protein domains is well packed on average but significantly heterogeneous, such that tightly-packed regions, usually hydrogen bond-rich To test our hypothesis, we define protein interfaces as surface patches buried by the interaction of a contiguous protein segment of an arbitrarily defined length with the rest of the protein. Stable protein cores are characterized by a high content of hydrophobic residues, a fine matching of buried polar groups through hydrogen bonding, and tight packing. Most protein interfaces should, therefore, be highly apolar and the protein segments at the interface should be tightly packed. In contrast, protein interfaces involved in dynamics need to be intrinsically unstable and should display physicochemical features indicative of a low stability, such as a higher polarity and a lower packing density than stable interfaces.i.e. 7-to-9-long probes give rise to almost identical profiles. However, short probes tend to make the profiles noisier while longer probes tend to average the properties of distant regions that may include both unstable and stable interfaces. We have thus set probe length to eight residues in all the cases reported. We have additionally tested whether the resolution of the structures could affect the outcomes of our method. Basically, the polarity profiles do not change significantly in the 1.2–2.8 Å resolution range (not shown), while the packing density profiles retain their shape –i.e. the position of maxima and minima– and exhibit slightly lower packing densities, which is in agreement with previous reports relating lower structural resolution with lower computed packing To analyze the properties of protein interfaces, we have probed protein structures using a sliding-window approach. To that end, the three-dimensional structure of a given monomeric protein, as defined by a PDB file, is scanned from end-to-end using a contiguous peptide probe. For each peptide probe, two relevant properties of the probed interface are computed: the ratio of polar/apolar buried area, and the packing density (see methods). The corresponding computed values are assigned to the central residue of the probe. When the scanning is completed, property sequence profiles are built. The profiles so obtained are not very sensitive to probe length –Anabaena PCC 7119. The polarity profile represents the ratio of polar over apolar surface area buried in the interface. A baseline with a polarity ratio of around 0.5 can be observed by visual inspection of the profile. Such a baseline is present in all polarity profiles we have built , α/β (folding Lysozyme-like), all α (folding Cytochrome c) and all β (folding Immunoglobulin-like β-sandwich) can be visually assessed in Tight packing, high hydrophobic burial and good pairing of buried polar groups are key ingredients of protein stability The correlation between LIPs and unstable foldons identified by their fast proton exchange rates can be illustrated by cytochrome c. The native basin of this protein has been characterized in detail by equilibrium proton exchange Due to their low stability, LIPs are expected to become unfolded in solution conditions that are nevertheless compatible with the rest of the protein retaining the native conformation. LIPs should therefore correlate with the unfolded regions of equilibrium intermediates. These partly unfolded conformations tend to accumulate at moderately high temperatures or denaturant concentrations, or at extreme pH values, usually low pH. The apoflavodoxin intermediate discussed above is a fine example of the autonomous unfolding of LIPs in a thermal intermediate. The free energy difference between this intermediate and the native state is of just 1.5–2.0 kcal/mol A second common type of equilibrium intermediate is the molten globule A third common type of equilibrium intermediates is that found in chemical unfolding. The role of LIPs in chemical intermediates can be exemplified by the equilibrium intermediate accumulating in the urea unfolding of indole-3-glycerol phosphate synthase (IGPS) The free energy difference between the IGPS equilibrium intermediate and the native conformation is of 8.5 kcal/mol In addition to kinetic intermediates, a key species in protein folding reactions are transition states of folding. These ephemeral conformations are of high energy and can only be characterized by a combination of protein engineering and fast kinetics -values for the comparison of the distributions returned by the test, the alternative hypothesis, determining statistical significant differences between the two distributions, should be accepted in all cases.An important issue to take into account is trying to estimate the statistical significance of the observations reported in this work regarding the special characteristics of buried interfaces related to unstable protein regions. Although the polarity ratio profiles included in this study , 3, 4, 5-values obtained prove that the residue stability values of unstable regions are lower than those of stable regions. However for the three other proteins there are no statistical differences between the distributions. In all cases, the ℘-values obtained are higher than those obtained using our methodology. Not surprisingly, in only one of the five proteins tested (indole-3-glycerol phosphate synthase) there is a statistical correlation between COREX predicted unstable regions and LIPs.We also tried to compare our results with those obtained using a well-established software such as COREX On the hypothesis that the intrinsic dynamics of protein domains could be related to the presence of buried interfaces of low stability, we have devised a tool that allows to scan protein interfaces and to compute relevant physicochemical properties of the interfaces. Two key properties have been selected as indicative of low stability: the ratio of polar over apolar surface buried in the interface and the packing density at the interface. For a 200-residue protein it takes less than 2 minutes of CPU time in an average personal computer to calculate the polarity ratio and packing density profiles. Therefore, calculation of protein interface properties in a proteome scale is feasible. Our analysis indicates that protein interfaces display significant heterogeneity in polarity ratio and packing density. The protein examples discussed in this work contain interfaces, established by contiguous segments of 8 residues, whose polarity ratios vary from 0.3 to 1.2. In all proteins analyzed, a polarity baseline can be observed around 0.5, which appears to be the typical average polarity ratio of protein cores . Above tOn the other hand, the packing densities vary from 0.65 to 0.9, with local minima along the profiles, but no obvious baseline value shared by the different proteins. Nevertheless, it is clear that most interfaces of high polarity ratio appear at packing density minima below the cutoff established. This can be quantitatively assessed by calculating for each particular protein its average packing density and then determining whether the peaks of high polarity display packing densities below that average minus two standard deviations. Such is the case of 13 out of 14 polar interfaces discussed in this work, the only exception being the red foldon of cytochrome c, which, as explained in the results section, appears at the wall of a deep minimum.Proteins thus contain interfaces of high polarity and low packing density. We have termed them LIPs , they are expected to exhibit low local stability and they can be easily identified. To test the hypothesis that LIPs are related to the structure of protein folding intermediates we have defined LIPs in a quantitative manner as interfaces including at least one residue with polarity ratio greater than 0.8 and extending to those flanking residues with polarity ratios greater than 0.5. In addition, the potential LIP should contain a clear minimum, defined as above, in the packing density profile.The correspondence between LIPs, so defined, and the unfolded regions in protein equilibrium intermediates of different kinds is excellent. Altogether, our analysis reveals that protein domain cores contain interfaces of high polarity and low packing density that appear to be involved in protein dynamics, as they correlate with late folding events and with local instability in the native state that can lead to alternative partly unfolded conformations. Some of these conformations will be energetically distant form the native state, while others will be close in energy. The latter are expected to populate under native conditions and to get involved in function more easily.e.g those exhibiting the characteristic low polarity of protein cores.As can be seen in loci is simple and fast. A brief comparison with the COREX algorithm http://best.bio.jhu.edu/BEST/index.php). The stability plots calculated for apoflavodoxin, cytochrome c, α-lactalbumine, indole-3-glycerol phosphate synthase and barnase are shown in -values returned by the assay, the performance of COREX in distinguishing stable from unstable regions is lower than that of our method.Importantly, the computational methodology developed here to identify these proteins' dynamic i.e. recognition of new partners– because they could be mutationally tailored without seriously compromising the intrinsic dynamic nature of the interface. Actually, for the experimentally determined unstable regions and LIPs in Although an analysis of the evolutionary significance of protein LIPs is clearly beyond the scope of this work, we would like to note some features of those buried interfaces that might turn out to be relevant. As we prove in this work, the methodology presented here is useful to identify unstable regions in proteins by means of our definition of LIPs, which in some cases match fairly well the location of unstable regions in proteins. Because those interfaces are simply characterized by displaying outlying values for averaged properties, their evolutionary conservation may not require a high conservation of the sequences involved. To illustrate this fact, structural multiple alignments of flavodoxin, cytochrome c and α-lactalbumine protein families are shown in ad hoc Perl and Tcl scripts to estimate, from the 3D structure of a given protein, the ratio of polar/apolar surface area of its buried interfaces. The input coordinates are used to extract a fragment of variable length –eight residues was the window size used–, then the cropped protein and the extracted fragment are processed using NACCESS PRinterface profile of all interfaces along the structure of a protein. In these profiles, the PR of each interface appears assigned to the fourth residue of the 8-residue probe. 7-residue or 9-residue probes give rise to close to identical profiles (not shown).We have developed a set of interfaceρ) using the following expression:We have computed the packing density of buried interfaces  to estimate the local stability of protein regions. For each protein, a structure ensemble was first generated, which is used by the program in subsequent calculations. Based in the ensemble of structures generated, entropy-weighting factors were determined and stability constants calculated. The results obtained are represented in The structures of the five proteins analyzed in this work  corresponding to different representative folds were processed with the software COREX In order to evaluate the significance of our results we performed a non-parametric test to compare the polarity ratio of buried interfaces adjacent to structurally unstable regions and of our predicted LIPs with conformationally stable protein segments or with non LIP regions, respectively. For each polarity profile we performed a one-sided Mann-Withney-Wilcoxon rank-sum test with a confidence interval of ℘<0.05 to test the significance of obtaining higher buried interface polarities in unstable segments confirmed experimentally or in LIPs, when compared to the polarities of stable regions, see upper half of Figure S1Conservation of the polarity and packing density profiles in the flavodoxin family. The polarity and packing density profiles of some members of the Flavodoxin family with known structure are shown. The different members of this family were structurally aligned and the polarity and packing profiles obtained were superposed taking the structural alignment as template. The proteins analyzed were (PDBID: 1AHN), (PDBID: 1FUE), (PDBID: 1OFV) and (PDBID: 1FTG), the rmsd of the alignment is 3.20 Å.(EPS)Click here for additional data file.Figure S2Conservation of polarity profiles in representative folds in SCOP  database.A) SCOP class α/β (fold TIM α/βbarrel), B) SCOP class α+β (fold Lysozyme-like), C) SCOP class all α (fold Cytochrome c) and D) SCOP class all β (fold Immunoglobulin-like β-sandwich). The PDB IDs of the proteins analyzed are indicated and a cartoon representation of each protein fold is shown. The apoflavodoxin profiles shown in (EPS)Click here for additional data file.Figure S3Anti-correlation of the polarity ratio and packing density of buried interfaces. The packing density of buried interfaces calculated for apoFlavodoxin is plotted against their polarity . The resulting dispersion plot shows a propensity of high polar interfaces to be less packed than other interfaces of the protein, resulting in an anti-correlation between these properties. The correlation coefficient of the adjust line is R = 0.52.(EPS)Click here for additional data file.Figure S4Conservation of packing density profiles in representative folds in SCOP  database.A) SCOP class α/β (fold TIM α/β barrel), B) SCOP class α+β (fold Lysozyme-like), C) SCOP class all α fold Cytochrome c) and D) SCOP class all β (fold Immunoglobulin-like β-sandwich). The PDB ids of the proteins analyzed are indicated and a cartoon representation of each protein fold is shown. The apoflavodoxin packing density profile shown in (EPS)Click here for additional data file.Figure S5Residue stability estimations using COREX. For each protein studied in this report using our methodology we also calculate the local stability using this alternative procedure. As described in methods, for each protein we first generated the structure ensemble used in the calculations, then determined the entropy-weighting factor before obtaining the corresponding stability constants. The residue stability obtained are plotted in this figure in the following order: A) apoflavodoxin, B) cytochrome c, C) α-lactalbumine, D) indole-3-glycerol phosphate synthase and E) barnase. In each case the unstable regions determined experimentally are colored in light grey.(EPS)Click here for additional data file.Figure S6Sequence conservation of LIPs in three protein families. Each protein family was aligned as described in methods and the corresponding structural alignments were processed using JOY to obtain a detailed representation of the alignment including structural information. In the alignments the solvent accessible residues are in lower case and buried residues in uppercase. We also show in the top line of each alignment fifty-column block the qualitative representation of conservation and in the bottom line we include the histogram with the quantitative estimates of conservation scores in a given position as calculated by CLUSTAL. With these data, the average conservation scores for alignment columns  have been calculated for the complete alignments, for the experimentally determined unstable regions, and for the LIPs. We report the PDB codes of the proteins aligned, color in light grey the experimentally unstable regions, and remark the LIPs with a light blue line. A) Structural alignment of Flavodoxin family (the identity percentage (PID) is 34%). The unstable regions 87–108 and 118–152 are highlighted. The average column conservation score is 24%. However, the average column conservation score for buried and exposed residues in LIPs are 29 and 9%, respectively, and for buried and exposed residues in the experimentally unstable regions are 30 and 10%, respectively. B) Structural alignment of the Cytochrome c family (the PID is 47%) showing the 40–57 flexible region. The global average column conservation score is 33%, while the average column conservation scores for buried and exposed residues in LIPs are 49 and 30%, respectively, and for buried and exposed residues in the experimentally unstable regions are 42 and 25%, respectively. C) Structural alignment of α-Lactalbumin family (PID 50.4%) with the unstable region 40–80 in grey. The global average column conservation score is 43%, while the average column conservation scores for buried and exposed residues in LIPs are 73 and 41%, respectively, and for buried and exposed residues in the experimentally unstable regions are 70 and 44%, respectively.(EPS)Click here for additional data file.Table S1Estimations of solvent accessibilities for a group of proteins. From a set of 19 proteins from different folding families, different sizes and sharing less than 20% of sequence similarity we obtained the polar and apolar solvent exposed areas in the folded state (columns 3–5) and in the unfolded ensemble (columns 9–11) as described in (28). We also estimated the polar and apolar buried surface in the core of the folded state (columns 6–8). The averages ± SD of the ratio of polar and apolar areas for the three states of each protein are indicated in bold characters in the bottom line of the table.(EPS)Click here for additional data file.Table S2Statistical significance of interfacial polarity and of COREX stability estimates. We show the results of a one-sided Mann-Withney-Wilcoxon test performed on the interfacial polarity and the residue specific stability profiles obtained with our methodology and COREX, respectively. In the first case the alternative hypothesis H1 test the significance of obtaining higher polarities in unstable segments determined experimentally (column: Unstable regions) and in the segments corresponding to our definition of LIPs (column: LIPs). For the stability estimates obtained with COREX the alternative hypothesis H1 test the significance of obtaining lower stability values in the same protein segments described above. The confidence interval was set to ℘<0.05 in all cases.(EPS)Click here for additional data file.


Farnesyltransferase enzyme (FTase) is considered an essential enzyme in the Ras signaling pathway associated with cancer. Thus, designing inhibitors for this enzyme might lead to the discovery of compounds with effective anticancer activity. In an attempt to obtain effective FTase inhibitors, pharmacophore hypotheses were generated using structure-based and ligand-based approaches built in Discovery Studio v3.1. Knowing the presence of the zinc feature is essential for inhibitor’s binding to the active site of FTase enzyme; further customization was applied to include this feature in the generated pharmacophore hypotheses. These pharmacophore hypotheses were thoroughly validated using various procedures such as ROC analysis and ligand pharmacophore mapping. The validated pharmacophore hypotheses were used to screen 3D databases to identify possible hits. Those which were both high ranked and showed sufficient ability to bind the zinc feature in active site, were further refined by applying drug-like criteria such as Lipiniski’s “rule of five” and ADMET filters. Finally, the two candidate compounds (ZINC39323901 and ZINC01034774) were allowed to dock using CDOCKER and GOLD in the active site of FTase enzyme to optimize hit selection. The established link between human cancers and mutant Ras Proteins has prompted considerable concern to target Ras pathway for cancer remedy. Based on such efforts a group of rationally designed drugs targeting the farnesyltransferase (FTase) enzyme which post-translationally modifies Ras oncoproteins has been discovered ,2.Ras proteins require farnesylation step  which is essential for their binding to plasma membrane and performing their function in signal transduction. This farnesyl moiety is transferred by FTase enzyme to Ras protein bearing C-terminal amino acid sequence known as CAAX motif  in the carboxyl terminus of a group of membrane-bound G-proteins ,4,5,6.FTase is heterodimeric zinc containing enzyme of two subunits α and β with molecular weights of 49 and 46 kDa, respectively . The α sFTase is classified as a metalloprotein, in which the metal-binding capabilities are encoded in the primary sequence that further plays a crucial role in determining the three dimensional structure ,10. The Various research groups have published structure and ligand-based pharmacophores in order to reveal the paramount features in FTase’s active site for inhibitor binding. However, they treated zinc atom as hydrogen bond acceptor and ignored the fact that the main determinant of inhibitors' binding to this enzyme is the presence of zinc cation ,15,16. TFilters ligands using Lipinski and Veber Rule protocol. As the extracted ligands are crystallographically determined biological conformations, there is no need to perform Diverse conformation generation protocol in order to cover the possible biological space. The fourteen selected compounds were divided into two sets, a training set and a test set by exploiting a protocol in DS 3.1 called Generate training and test data where the splitting method is based on structural diversity of the ligands and the splitting percentage for the training set is 70% , and then the ligands were extracted from the active sites. Fourteenth co-crystallized inhibitors were chosen as drug-like compounds based on the Lipinski rule of five filter using the Accelrys DS 3.1 t is 70% . The Comshown in , using tshown in . The cheshown in .Common feature pharmacophore generation protocol no conformation generation was performed, Principal and MaxOmitFeat values of 2 and 0 were set for all compounds in the training set as the chosen compounds were the most active and they also were co-crystallized in the active site of the enzyme. Since the active site of the FTase enzyme contains a zinc cation and of all the ten chosen compounds interact with that zinc cation by performing a coordination bond, the features selected to generate the hypotheses were hydrogen bond acceptor 05, hydrogen bond donor 05, hydrophobic 05, ring aromatics, zinc binder 05, maximum pharmacophores 10, minimum interfeature distance 2.0Å. All other control parameters were kept at their default values. The zinc binder feature was modified to be able to identify the zinc binding groups in the active inhibitors that are not included originally in DS 3.1. The best common feature of pharmacophore hypothesis (Pharm-3A) was selected based on the inclusion of the zinc binder after customization in its features , hydrogen bond acceptors (HA) and hydrophobic pockets (HY) by referring to the active site residues. As a final step to optimize the structure-based pharmacophore, Edit and Cluster tool was utilized to cluster and remove any redundant features or features with no catalytic importance.Structure-based pharmacophore modeling has been extensively implemented by researchers world-wide to provide successful novel drugs with potent activity. Mainly, it is used whenever there is a shortage of information of ligands that bind to the receptor or to get more insight into the geometry of the active site. In this study, a crystal structure of FTase enzyme with a bound ligand (PDB code: 3E33) crystallized at 1.9 Å resolution was utilized to generate the structure-based hypothesis. A sphere of 10 Å radius which covers the most important residues that bind with the twenty three crystallized ligands was generated using Ligand pharmacophore mapping Protocol available in DS 3.1 with Best flexible fitting method was used to measure the extent to which the active molecules could match the pharmacophore and the results were represented by percentages. Finally in the third level, within the common feature pharmacophore , an internal validation performed by providing a set of active and inactive molecules where the results presented as roc curve files.Validation was conducted on three separate levels; the first level was performed using the ligand pharmacophore mapping protocol, where the four test compounds mapped to the generated pharmacophores. In the second level a set of 286 ligands extracted from literature and divided into active and inactive molecules based using activity of 500 nM as threshold. Running validation through the mapping method will test the ability of the generated pharmacophores to distinguish the active molecules from inactive molecules. The last step in this level, Ligand Pharmacophore Mapping protocol with Best Flexible Conformation Search method. The candidate molecules were further subjected to numerous filters in DS 3.1 (Lipiniski’s and Veber rules) to select the drug-like compounds. The designated molecules were considered in molecular docking stage.The generated pharmacophore hypotheses based on the aforementioned two approaches were used as 3D queries to extract chemical compounds from commercially available databases. This process will help in finding potential leads for this target that can be optimized further by investigating drug likeness and synthetic feasibility. A selected compound from the screening method should map all the pharmacophoric features in order to be considered a hit for the common feature pharmacophore hypothesis. On the other hand, a one feature miss was tolerated for considering a compound to pass the screening for structure based pharmacophore. The screening processes were performed using CDOCKER docking protocol in DS 3.1 and GOLD (Genetic Optimization for Ligand Docking) program version 5.1 from the Cambridge Crystallographic Data Centre (CDCC). CDOCKER uses a CHARMm-based molecular dynamics (MD) scheme to dock ligands within the active site of enzymes and receptors. First, random ligand conformations are generated using high-temperature MD, and then the produced conformations are translated into the binding site. Candidate poses are further created using random rigid-body rotations followed by simulated annealing. A final minimization is used to refine the ligand poses [Prepare Protein protocol that removes water, corrects any defects in the protein structure and adds hydrogen. The Input site sphere was 10 Å in radius using the ligand in the active site as a reference point for both CDOCKER and GOLD.Molecular docking was conducted using nd poses ,18. GOLDnd poses . GOLD hand poses  of FTaseFor CDOCKER, the candidate molecules from the screening process were docked in the active site using CHARMm forcefield with top 10 poses to be presented and scored and keeping the other options in their default values. On the other hand, for GOLD, early termination step was activated if the first three poses have an rmsd value of less than 1.5 Å, other parameters were set as default. The final hit molecules were selected based on a combination of docking score, mode of binding and molecular interaction within the active site.Generate training and test data protocol at DS 3.1. Splitting of these molecules is based on diversity and it is assumed that 70% of them (10 compounds) are the training set. The crystallized biologically active conformations were mapped without any conformation generation as they were considered to possess the required and optimum conformation for pharmacophore hypothesis generation. In this study, the selected features are HBA, HBD, hydrophobic, ring aromatic, and zinc binder. Ten pharmacophore hypotheses based on common features were generated and they are displayed in  containing six features that are complementary to Tyr361, Tyr166, Phe360, Leu96, Tyr93 and the Zn2+ atom. The third stage involved customization of the pharmacophore by selectively replacing HBA feature pointed to the Zn2+ atom by zinc feature available on DS 3.1 , ZINC-MayBridge_(75443), and ZINC-Key Organics_(54869) [Ligand Pharmacophore Mapping protocol was used with MaxOmitFeat value of zero as the four features of the Pharm-3A should be fulfilled. A compound will be determined as a hit if it maps all the four features that are included in the pharmacophore hypothesis.On the other hand, a relaxed criteria was used for Pharm-B where the hit is accepted if it can map five features out of the six and hence the Ligand Pharmacophore Mapping protocol which allows some of the features to be missed was used. The extracted compounds were then subjected to further screening processes relying on their fitness value, drug likeness according to Lipinski's rule of five and Vebers’ rule. A compound is determined positive according to Lipiniski and Veber if (i) less than five hydrogen bond donor groups; (ii) less than 10 hydrogen bond acceptor groups; (iii) a molecular weight less than 500; (iv) A LogP value less than 5; (v) number of rotatable bonds less than 10; polar surface area less than 140 Å2; and hydrogen bond donors and acceptors less than 12. A subsequent level of filtration to achieve drug like compounds is to perform ADMET screening techniques in DS 3.1 in which the databases are filtered according to their solubility, intestinal absorption and Blood Brain Barrier penetration. A total of 1,296 compounds (885 from the structure-based design and 411 from the common feature pharmacophore) were found to satisfy the assigned filters for the molecular docking step (Both the common feature pharmacophore and the structure-based pharmacophore hypotheses that were generated previously were subjected to database screening process utilizing _(54869)  which weing step .The 1,296 compounds emerged from both structure and ligand-based pharmacophores conjointly with the training set were submitted to docking programs CDOCKER and GOLD. Consensus scores of the candidate compounds from both CDOCKER and GOLD were performed, eventually compounds enumerated the highest value from score summation of the two docking programs were chosen to be candidate molecules. These scores of the candidate compounds were contrasted by scores obtained for the training set compounds (the highest consensus score for the training set compound is 147.4) and only compounds that scored equally or higher than the training set compounds were fully investigated by studying their binding mode. Of the 1,296 compounds, twenty one compounds had shown scores higher than training set scores. Based on molecular fitting mode inside the active site and their proper interaction with zinc atom, two compounds were chosen as potential virtual leads namely ZINC39323901, and ZINC01034774. Compound ZINC01034774 which was identified from structure-based pharmacophore has a fit value, CDOCKER, and GOLD fitness scores of 3.28, 64.65, and 98.11 respectively. The imidazole ring is supposed to form a coordinate bond with zinc cation which has a perfect pose when the pyridine-like nitrogen points directly at zinc cation of the active site with optimum distance of 1.76 Å. In addition, internal π-π interaction is shown between the imidazole ring and one of the benzene rings of the compound. The other benzene ring is positioned in a hydrophobic pocket within the active site formed from the hydrophobic tail of farnesyl pyrophosphate (FPP), Tyr166, and Lys164. The sulphonamide moiety interacts via hydrogen bond with Tyr361 phenol side chain . CompounTwo distinctive techniques; ligand-based and structure-based drug design techniques were used independently to create two pharmacophore hypotheses, Pharm-3Aand Pharm-B, respectively. The two hypotheses were exploited to find novel inhibitors of FTase enzyme as anticancer agents. Both hypotheses were customized by adding a zinc-binder feature to emphasize the importance of the zinc cation in the active site of this enzyme which is a crucial structural and catalytic feature. Pharm-3A was comprised of four features including a zinc binder, one hydrophobic, one ring aromatic and one hydrogen bond acceptor whereas Pharm-B was composed of six features including zinc binder, one hydrophobic, one ring aromatic and three hydrogen bond acceptors. Both of these pharmacophores were validated for their ability to discover reliable chemical drug candidates. The validation process included internal pharmacophore validation using test set compounds, ROC curve and fit value calculations. All the training and test set compounds have mapped all the features of Pharm-3A and mapped five features of the six in Pharm-B and in both cases; the zinc binder feature was mapped. Three chemical databases were screened and molecules were selected as candidates if they mapped all the Pharm-3A features and at least five features of Pharm-B then using filtering based on Lipinski and Verber drug-like properties and ADMET to acquire favorable drug like properties. Docking of compounds resulted from screening using CDOCKER and GOLD followed by choosing compounds that have higher consensus score compared to the training and test compounds afforded possible candidates which upon studying their binding modes, two potential lead compounds as FTase inhibitors were identified. The structures of all hit compounds will not be revealed at this stage of the research as extensive biological evaluation of these compounds will be under investigation and further steps needed to discover the lead compound.