Acheta domesticus L. (Orthoptera: Gryllidae). Crickets reared on high phosphorus diets ate more food, gained more weight, were in better condition at maturity, and contained more phosphorus, nitrogen, and carbon in their bodies at death than crickets reared on low phosphorus diets. There was also a trend for crickets reared on high phosphorus diets to become larger adults (interaction with weight prior to the start of the experiment). These findings can be added to the small but growing number of studies that reveal the importance of phosphorus to insect life history traits. Future research should explore the importance of dietary phosphorus availability relative to protein, lipid, and carbohydrate availability.Phosphorus is extremely limited in the environment, often being 10–20 times lower in plants than what invertebrate herbivores require. This mismatch between resource availability and resource need can profoundly influence herbivore life history traits and fitness. This study investigated how dietary phosphorus availability influenced invertebrate growth, development time, consumption, condition, and lifespan using juvenile European house crickets,  Organisms require nitrogen and phosphorus to build their proteins, RNA, DNA, and ATP . ObtainiFor decades, a mismatch in nitrogen content between insects and their food plants has been recognized to be a critical factor influencing herbivore success . Aphids,the essential limiting element, phosphorus is also limited in a diverse number of ecosystems  reared on natural and artificial diets with reduced levels of phosphorus exhibited significantly decreased growth rate and increased time to final moult compared to caterpillars reared on phosphorus sufficient diets , were used as a model organism. Male cricket lifetime reproductive success is dependent upon their ability to attract mates. Cricket mate attraction abilities are, in turn, dependent upon acoustic mate attraction signal quality and quantity  in an insect rearing facility in the Nesbitt Biology Building at Carleton University. The rearing facility had a temperature range of 26 ± 4°C and 12 hour light :12 hour dark cycle. All crickets were provided with cardboard cartons for shelter, and unlimited access to food and water. Food consisted of powdered Harlan Teklad Rodent diet no. 8604  until the day they were included in the experiment.European house crickets, th instar to ensure crickets would have two full instars of growth on the phosphorus diets prior to undergoing final moult  with shelter and unlimited access to water.The experiment was initiated at the start of the 7Approximately 58 males (range = 57–60) and 58 females (range = 53–62) were placed on each of the five different phosphorus diets ranging from low to high phosphorus content . This phosphorus range was designed to mimic the range of food in the wild. Since crickets are omnivores they are capable of consuming a variety of foods, from plants to fungi to insects. These foods vary from an average of 0.2% phosphorus found in terrestrial plant matter to an average of 0.8% phosphorus found in insects . Experimwww.denverinstruments.com) on the day it reached its seventh instar. Change in body mass over time was quantified by weighing each cricket every week until they died naturally.Each cricket's initial body mass was obtained using a Denver Instruments Precision Analytical Balance model P-114 , or died. Developmental time was calculated as the difference between start date  and the date it moulted to adulthood (adult date). Lifespan was calculated as the difference between the start date and the date each individual died a natural death (death date). Adult lifespan was also quantified using the difference between the date each individual moulted to adulthood and their death date. The few individuals that escaped prior to dying or did not have their death date or maturity date recorded were excluded from analyses.www.zeiss.com) following their natural death. These size measurements were significantly positively correlated with each other . A principal component analysis was used to reduce the number of variables. The 1st principle component explained 98% of the variation in body size  and was used as an overall size measure.Each cricket's head width, thorax width, thorax height, and thorax area were measured to the nearest 0.1 mm using a Zeiss Discover 4. V 12 dissection microscope . A temperature of 130° C was used because this was the lowest temperature our drying oven could reach. Unfortunately, this high temperature might have resulted in some of the volatile organics evaporating. If so, the dry weights might be slightly lower than normal. Dry weight was quantified using a Denver Instruments Precision Analytical Balance (model P-114).Cricket condition at maturity was quantified using the residuals from an allometric regression of body weight (wet weight at maturity) on body size. Cricket condition at death was quantified using the residuals from an allometric regression of body weight at death (dry weight) on body size (PC1). To obtain dry weight measures each cricket was dried at 130°C for at least 24 hours in a Thermal Scientific (6520 series) drying oven  Vario Micro Cube CHN analyzer was utilized for the Dumas method. The Dumas method involves combusting the samples at high temperatures using oxygen. The gas that is produced is then passed through a special column where carbon dioxide and water is absorbed. The carbon and nitrogen content is then analyzed from this gas. The product that remains from thecombusted sample can then be analyzed for nitrogen content.Cricket total body phosphorus and nitrogen content were quantified on a subset of the individuals  to ascertain how phosphorus influenced body stoichiometry. Each dried cricket was pulverized to a uniform powder using a mortar and pestle. Approximately 1–2 mg of powder was then used to assess individual body stoichiometry. To quantify total body phosphorus content a persulfate oxidation technique followed by orthophosphate analysis using the acid molybdate technique was utilized . To quanwww.sas.com). Shapiro-Wilk goodness-of-fit tests were used to ensure the data do not differ significantly from normality. When data were not normally distributed (lifespan), they were transformed using a log transformation to approximate a normal distribution. A repeated measures analysis of variance was used to determine how body weight changed through time. A nominal logistic fit model was used to explore the factors influencing whether or not crickets survived through adulthood. Multiple regression models were used to quantify whether phosphorus availability influenced compensatory feeding behaviour, body size, condition, and body stoichiometry. Cox proportional hazard survival models were used to determine the factors influencing cricket lifespan , juvenile lifespan , and for the individuals that died as adults, juvenile development time and adult lifespan. Parameters and interactions included in each of the multivariate models are shown in Statistical analyses were conducted using JMP 8.0.1 statistical software  was not influenced by the availability of phosphorus in the diet. Development time was, however, influenced by the sex of the cricket, with females taking almost five days longer to develop to adulthood than males ,3. DevelThe model exploring survival to adulthood (successful maturation) was not statistically significant . SuccessCricket lifespan was not influenced by the availability of phosphorus in the diet. Lifespan was, however, influenced by cricket sex, condition at adulthood, and condition at death . FemalesFurther, crickets in good condition at adulthood lived longer than crickets in poor condition at adulthood. Crickets that survived a long time also tended to be in better condition at death than crickets that died earlier in the experiment .The factors that influenced adult body size were sex  and an interaction between start weight and dietary phosphorus availability . Together these factors explained 13% of the variation in body size and suggest phosphorus availability positively influences cricket body size, depending on start weight.Cricket condition at maturity was significantly influenced by the availability of phosphorus in the diet. Crickets with access to high phosphorus diets were in significantly better condition at maturity than crickets reared on low phosphorus diets. Condition at maturity was also influenced by cricket weight prior to the start of the experiment, and developmental time . CricketCricket condition at death was not influenced by the availability of phosphorus in the diet. It was, however, dependent on cricket weight at the start of the experiment and cricket lifespan . CricketCrickets did not compensate for poor phosphorus availability by consuming more food as would be expected if crickets were compensating. Instead, crickets tended to eat less when the diet contained little phosphorus . ForaginCricket body stoichiometry was highly variable across individuals , and theCricket total body phosphorus and nitrogen contents were positively correlated with each other . Further, nitrogen content was positively influenced the availability of phosphorus in the diet. Nitrogen content was also influenced by cricket sex , an interaction between start weight and phosphorus availability, and an interaction between condition at maturity and phosphorus availability . TogetheCrickets reared on high phosphorus diets gained weight faster, were in better condition at maturity, and had more phosphorus and nitrogen in their bodies than crickets reared on low phosphorus diets ,3. High The finding that dietary phosphorus availability impacts cricket weight gain  is consiIncreased dietary phosphorus availability did not influence the likelihood that crickets would successfully develop into adults, nor did it shorten the time it took them to undergo their final moult. These findings conflict with those of Perkins et al.  who reveDietary phosphorus availability did not directly influence cricket lifespan. This finding supports all other cricket studies to date that have investigated whether phosphorus availability influences length of life .Crickets reared on high phosphorus diets were in better condition at maturity than those reared on low phosphorus diets. Further, crickets reared on high phosphorus diets tended to be larger than crickets reared on low phosphorus diets (interaction with body weight). These findings suggest important fitness ramifications, as body size and condition can influence lifetime reproductive success. The quality and quantity of acoustic mate attraction signals are partially dependent on cricket body size and condition . This begs the question of where the phosphorus and nitrogen reside once the crickets consume it. Phosphorus could be incorporated into cricket RNA ; this is a likely scenario given growth requires extensive protein synthesis and crickets reared on the high phosphorus diets gained more weight over time than crickets on low phosphorus diets. Crickets could also store excess phosphorus in their hemolymph . NitrogeThe present study was a sequel to the earlier papers that investigated how phosphorus availability during adulthood influenced reproduction . The aduM. sexta caterpillars fed high phosphorus diets contained significantly more phosphorus it their bodies compared to caterpillars fed low phosphorus diets (The juvenile and adult experiments differed in how dietary phosphorus availability influenced body stoichiometry. There was no relationship between dietary phosphorus availability and body stoichiometry in the adult experiment . Howeverus diets . TogetheTogether the cricket experiments suggest that cricket growth, condition at maturity, lifetime reproductive efforts, and body stoichiometry are positively influenced by the availability of phosphorus during development and into adulthood (present study, The findings presented herein and in previous published studies support the small but growing number of studies that reveal phosphorus' importance in influencing insect life history traits. Given the plethora of research suggesting that proteins, lipids, and carbohydrates also influence insect life history traits e.g.,  the impo


Corneal transplantation is among the most successful solid organ transplants. However, despite low rejection rates of grafts in the ‘low-risk’ setting, rejection can be as high as 70% when grafted into ‘high-risk’ recipient beds. Under normal homeostatic conditions, the avascular cornea provides a unique environment that facilitates immune and angiogenic privilege. An imbalance in pro-inflammatory, angiogenic and lymphangiogenic mediators leads to a breakdown in corneal immune privilege with a consequent host response against the donor graft. Recent developments in lamellar and endothelial keratoplasties have reduced the rates of graft rejection even more, while providing improved visual outcomes. The corneal layer against which an immune response is initiated, largely determines reversibility of the acute episode. While epithelial and stromal graft rejection may be treated with topical corticosteroids with higher success, acute endothelial rejection mandates a more aggressive approach to therapy due to the lack of regenerative capacity of this layer. However, current immunosuppressive regimens come with the caveat of ocular and systemic side effects, making prolonged aggressive treatment undesirable. With the advent of biologics, efficacious therapies with a superior side effect profile are on the horizon. In our review we discuss the mediators of ocular immune privilege, the roles of cellular and molecular immune players in graft rejection, with a focus on human leukocyte antigen and antigen presenting cells. Furthermore, we discuss the clinical risk factors for graft rejection and compare rates of rejection in lamellar and endothelial keratoplasties to traditional penetrating keratoplasty. Lastly, we present the current and upcoming measures of therapeutic strategies to manage and treat graft rejection, including an overview of biologics and small molecule therapy. Corneal transplantation is among the most prevalent and successful organ transplants performed worldwide  with morAll cells of individuals within a single species express surface polymorphic proteins known as major histocompatibility complex (MHC) antigens, which differentiate one individual from another. MHC genes are prolifically polymorphic with over 200 allelic variants, therefore further increasing the odds of mismatched antigens between donor and host corneas in allogeneic transplants, creating an optimal opportunity for graft rejection; these antigens are thus colloquially termed “major transplantation antigens” . ChromosCorneal DCs play a critical role in graft rejection through their ability to regulate T cell responses to both self and foreign antigens in the donor button, based on molecular cues received from the tissue microenvironment . AntigenThe evidence on the benefit of class I HLA tissue matching in reducing allogeneic corneal graft rejection is fairly conclusive with exclusion of the Collaborative Corneal Transplantation Studies (CCTS) data, demonstrating a clear benefit of prolonged graft survival in type I HLA-matched corneal transplant cases ,23,24. UMurine studies have provided a sound platform to advocate the utility of tissue matching in human corneal transplantation to prevent sensitization of the host to donor MHC antigens leading to reduced incidence of graft rejection . HLA matAnother class of cell-surface proteins are the minor histocompatibility antigens, which unlike the MHC, are coded for throughout the genome at various loci. In order to be recognized as non-self antigens, minor histocompatibility antigens must be processed by host APCs and presented with MHC-II molecules. ABO blood antigens are a relevant example of such antigens and found on corneal epithelial cells whose expression is increased in graft rejection. The CCTS proposed that ABO matching may reduce the risk of allogeneic graft rejection in high-risk corneal transplantation . It alsoThe concept of corneal immune privilege is entertained by observations of high survival rates of allogeneic corneal grafts despite HLA mismatching between donor and host tissues. It is the uniquely configured corneal anatomy and physiology of the anterior chamber that evade a host immune response through low immunogenicity and generation of alloantigen tolerance ,19,43. T+ APCs that orchestrate allotolerance by upregulating the expression of TGF-β with downregulation of the co-stimulatory molecule CD40/CD40L and interleukin-12 (IL-12) [in vitro [+ T cell responses, confirming the antigen-dependent effect of ACAID on CTL [+ regulatory T cells (Treg) in modulating CD8+ T cell function during ACAID have been explored [+ T and CD8+ T cell populations in the spleen proliferate upon MHC alloantigen-specific ACAID induction, however, once ACAID is expressed, the percentages of these T cells decrease substantially, suggesting ACAID-mediated inhibition of both CD4+ T and CD8+ T cell function. Therefore, we now know that solubility of the antigen is not a necessary determinant of ACAID-mediated CTL immune suppression. Therefore measures to promote ACAID-mediated inhibition of DTH and CTL could prove beneficial in prolonging graft survival. Interestingly, while FoxP3+ regulatory T cells (Treg) increase upon ACAID induction, they have not been shown to be directly involved in the modulation of ACAID-mediated MHC alloantigen-specific T cell function and response [The anterior chamber is rich in soluble immunosuppressive factors such as TGF-β, alpha-melanocyte stimulating hormone (α-MSH), calcitonin gene-related peptide (CGRP), CRP, somatostatin (SOM), indoleamine dioxygenase (IDO), vasointestinal peptide (VIP) and macrophage migration inhibitory factor (MIF), which inhibit T cell and complement activation ,51. The  (IL-12) ,56,57. S (IL-12) –60. Whilin vitro , making D on CTL –66. SincD on CTL –70, the explored . Both CDresponse .direct pathway constitutes presentation of donor antigens to naïve T cells directly by donor APCs through non-self MHC-II recognition on their surface, resulting in proliferation of direct alloreactive T effector cells [indirect pathway yields donor antigens to host APCs that travel the cornea, capture donor antigens, and transport them to draining lymph nodes where antigen presentation occurs through recognition of self MHC-II by naïve T cells [indirect pathway [direct and indirect pathways are implicated in the immune-mediated rejection of orthotopic corneal allografts, especially in high-risk corneal beds with higher immunogenicity and compromised immune privilege [Corneal graft rejection occurs when the host immune response is directed toward antigens in the donor corneal button, leading to tissue destruction brought about by cells and mediators of the innate and adaptive immune responses. An immune response may target any of the main layers of the cornea selectively, or, in combination. Compromise of the corneal epithelium and stroma may be reversible, but, rejection of the endothelium invariably results in irreversible endothelial cells loss and may result in permanent graft failure, if not treated judiciously . Sensitior cells . In cont T cells . While i pathway , accumulrivilege –78. The rivilege ,79, namerivilege . These rrivilege ,79,81,82rivilege –86. Oncerivilege .chemotactic cytokines), are small-molecule-weight cytokines that modulate recruitment of leukocytes and immune cells to the inflamed cornea [Lymph nodes serve as the priming hub for T cell allosensitization and activation, which then drives the subsequent “efferent” arm, or the expression phase, of immune-mediated graft rejection. It is this phase that results in the actual destruction of the graft, making lymph nodes profoundly critical to the process of rejection . In suppd cornea ,89. Immud cornea ,90–92.+ CTL, and CD4+ T-helper (Th) lymphocytes, otherwise known as DTH cells. Even though the role of CTL in corneal graft rejection remains somewhat controversial, they are believed to be sufficient but not necessary for corneal graft rejection [+ Th1 cells secrete IL-2, IFN-gamma and lymphotoxin, which lead to inflammation as an attack on the inciting antigen. IL-2 is critical to a sustained immune response by its positive feedback on T and B cell activation and proliferation. IFN-γ ensures that macrophages are activated at the site of inflammation, and facilitates further expression of MHC-II antigens in the donor button. Th17 cells on the other hand secrete IL-17, IL-21 and IL-22 [+CD25+Foxp3+ Tregs [+CD25+Foxp3+ Tregs is largely dictated by the cytokine milieu of the tissue microenvironment [Once the graft and infiltrating leukocytes release late chemokines, guidance of alloreactive T cells towards the graft begins ,93. Alloejection ,94. Baseejection ,96. Th1 ejection ,97. CD4+nd IL-22 . TGF-β ind IL-22 –103. IL-nd IL-22 . Interes3+ Tregs . Therefoironment ,106,107.ironment . Howeverironment , IL-17 kironment . This haironment ,110. Howironment .Both host and donor factors may contribute to the development of immune-mediated graft rejection. Among the most widely accepted and well-established risk factors for corneal allograft rejection are: presence of stromal blood vessels in one or more quadrants of the recipient cornea (high risk), with at least a 6 clock-hour distance between the two blood vessels, corneal neovascularization after surgery, prior graft rejection episodes, pre-operative glaucoma, young age, prior anterior segment surgery, active ocular inflammation, ocular surface disease, herpes simplex keratitis, neurotrophic keratopathy, large and eccentric grafts, and anterior synechiae –114.A high-risk cornea is defined by the CCTS as one with two or more quadrants of deep stromal vessels prior to surgery, or, one in which a prior graft has been rejected . PresencBased on the number of corneal quadrants with stromal vascularization, a low-risk transplant is defined as a graft placed in a recipient bed that is avascular. The 2-year survival rate associated with low-risk grafts is an impressive near 90% ,120,121,With the recent development of more sophisticated sutureless surgical techniques that dissect and replace the selected layer of diseased cornea, lamellar and endothelial keratoplasties have led to reduction in graft rejection rates and improved surgical and visual outcomes ,124.Descemet’s stripping automated endothelial keratoplasty (DSAEK) and Descemet’s membrane endothelial keratoplasty (DMEK) allow selective removal of the diseased Descemet’s membrane and endothelium, without disturbing the epithelium and much of the stroma ,15. ThisDMEK, a procedure that involves removal and replacement of the Descemet’s membrane and endothelium has generally a better visual acuity outcome than DSAEK . In addiA preferred procedure for stromal diseases with a healthy endothelium is a technique called deep anterior lamellar keratoplasty (DALK), where the stroma and epithelium are replaced leaving the host endothelium intact . DALK boTopical corticosteroids are the mainstay of treatment in the management of acute graft rejection and as post-operative prophylactic therapy for high-risk transplant patients. Topical 1% prednisolone acetate is typically prescribed in acute graft rejection. A commonly prescribed regimen involves 1% prednisolone acetate every 1–2 hours for the first few weeks with gradual tapering over several months. Surgeons may maintain indefinite treatment with low-dose steroids . For nonIn cases of severe endothelial graft rejection that present early, there is debate over the benefit of oral systemic steroids versus pulse therapy. In a randomized clinical trial that evaluated the outcomes of topical corticosteroid therapy with either 60 to 80 mg of oral methylprednisolone daily, or, a single intravenous pulse of 500 mg methylpredinosolone, it was observed that pulse therapy was superior oral methylpredinoslone. While the graft survival rates were comparable between the two groups, only 26.3% of the surviving grafts in the pulse therapy group went on to have a second rejection episode in comparison to 66.7% of the grafts in the oral steroid group, with added benefit to repeated pulse therapy ,137. MetAlternatively, intravenous dexamethasone, at a dose of 1 mg/kg in patients with severe endothelial rejection can be used for severe rejection. Reversibility of graft rejection is reported to be better with intravenous dexamethasone (72.3% vs. 45%) as observed in a retrospective study comparing combination therapy of topical steroids with either dexamethasone, or, methylprednisolone . ChronicCyclosporine A (CsA) and tacrolimus have proven efficacious to varying degrees in the treatment of graft rejection and management of high-risk patients post-transplantation. They provide viable options for sustained immunosuppression as djuvants to corticosteroids. Several groups have investigated the benefit of combination therapy with topical steroids and topical CsA in the management of acute graft rejection and as prophylactic therapy in high-risk patients following keratoplasty. The results of such studies are mixed and controversial. A randomized controlled trial failed to demonstrate any additional benefit of adding 0.05% topical CsA to 1% topical methylprednisolone acetate in the treatment of acute endothelial graft rejection . This stTacrolimus (FK-506), also a calineurin inhibitor and thus similar in its mechanisms of action to CsA, can be used topically or systemically in the management of low- and high-risk corneal transplantation. Randomized clinical trials have demonstrated that low-dose (0.06%) topical tacrolimus at a frequency of thrice daily for 6 months proved superior to topical steroids in preventing rejection episodes (100% vs. 84%) in low-risk patients following corneal transplantation . LikewisMycophenolate mofetil (MMF), an inhibitor of inosine monophosphate dehydrogenase (IMPDH), inhibits the de novo synthesis of DNA in T and B lymphocytes, making these immune cells cytostatic. Randomized clinical trials testing MMF have demonstrated that the efficacy of MMF in preventing graft rejection in high-risk patients is comparable to that of CsA ,156. MMF+ cell recruitment [Corneal neovascularization is the strongest determinant of graft rejection prior to transplantation and poses a challenge to reinstating ocular immune privilege in the wake of corneal transplantation . Patent ruitment . Antisesruitment .Adhesion molecules mediate trafficking of immune cells to from the lymph nodes towards sites of inflammation such as the graft. Animal studies of transplant rejection among various conditions of allodisparity and expression of intercellular adhesion molecule 1 (ICAM-1) in transgenic mouse models revealed that deficiency of ICAM-1 lead to significant improvement in graft survival in MHC-mismatched grafts, suggesting the role of ICAM-1 in allosensitization of T cells to MHC antigens . Other pRapamycin (sirolimus), an inhibitor of a serine-threonine protein kinase, mammalian target of rapamycin complex-1 (mTORC1), inhibitis effector T cell proliferation and activation without compromising the function of regulatory T cells. In an open-label study, rapamycin has been shown to have a 1-year rejection prevention rate of 78% and was comparable in its efficacy to MMF in the management of high-risk transplant patients . MMF andAnother evolving approach to regulating effector T cell response is by delivering monoclonal antibodies against T cell antigens. Monoclonal antibodies to CD3 and CD6 can successfully treat episodes of acute graft rejection when delivered intracamerally ,177. DacActivated T cells are critical to immune-mediated graft destruction. Complete and effective activation of T cells is contingent upon binding of T cell receptor/CD3 with MHC molecules on APCs, and interaction of T cell co-stimulatory molecule CD28 with B7 antigens on APCs (CD80 and CD86) . CytotoxThe pathophysiology of corneal allograft rejection is highly complex and still, in part, elusive. Nevertheless, significant advances have been made over the past several decades in our understanding of the mechanisms of corneal graft rejection. Investigations probing into the mechanisms and dissecting immune cell signaling and trafficking pathways are needed to further unravel the cellular and molecular mediators in corneal graft rejection. Development of targeted biological therapy in the future may provide new avenues in the treatment of corneal graft rejection with much safer side effect profiles and sustained therapeutic effects at lower doses. Further, the prospect of modulating immune cell activity through the use of morpholine oligonucleoitides, RNA interference and targeted microRNAs could be promising toward immunotherapy with minimal off-target effects. With the development of new cellular and molecular therapeutic approaches, meticulous clinical trials may then be designed to test their clinical utility. Currently, there is paucity of randomized controlled trials in corneal transplantation research, making conclusions about the safety and efficacy of treatment regimens limited.


We herein report our experience with pancreas transplantation in 26 patients at a single institution in Japan between August 2001 and December 2011.We reviewed the medical records of 26 pancreas transplantations performed in our institute.The early complications (within 2 weeks) included one graft venous thrombosis, one arterial thrombosis, and two reoperations for bleeding. Of the 26 pancreas transplant recipients, five lost pancreas graft function. Of 24 simultaneous pancreas–kidney recipients, three lost kidney graft function due to noncompliance. The patient, pancreas, and kidney survival rates were 100, 96 and 93 % at 1 year; 100, 80 and 93 % at 5 years; and 100, 67 and 68 % at 10 years, respectively. Of all these complications, venous thrombosis after pancreas transplantation was the most critical.As the largest series of pancreas transplantations in a single institution in Japan, our series yielded better results than the worldwide data recorded by the International Pancreas Transplant Registry. Routine postoperative anticoagulation therapy is not necessary for the prevention of graft thrombosis if sufficient fluid infusion is strictly controlled and the graft blood flow is frequently monitored. When graft thrombosis occurs, both early detection and appropriate intervention are extremely important if the pancreas graft is to survive. In patients with type 1 diabetes, pancreas transplantation normalizes the glucose metabolism, prevents the progression of secondary complications, and greatly improves the quality of life. About 35,000 pancreas transplantations had already been performed worldwide by 2010 . –12. 4–12n = 1), postoperative bleeding (n = 2), arterial thrombosis (n = 1), intestinal obstruction (n = 1), and bladder-to-enteric drainage conversion due to bleeding from the graft duodenum requiring duodenectomy (n = 1). Among the 26 pancreas transplant recipients, five pancreas grafts were lost due to venous thrombosis on postoperative day (POD) 9 in one patient, recurrent insulin-dependent diabetes mellitus caused by autoimmune isletitis on POD 2560 in one patient, noncompliance on PODs 519 and 1195 in two patients, and rejection on POD 730 in one patient. Of the 23 pancreas–kidney transplant recipients, three kidney grafts were lost, all of which were due to noncompliance .The patient, pancreas graft, and kidney graft survival rates were 100, 96 and 100 % at 1 year; 100, 80 and 93 % at 5 years; and 100, 67 and 68 % at 10 years, respectively. Complications requiring reoperations included venous thrombosis and graft loss  compared with the mean BMI of the others (20.1 kg/m2) were significant among the various risk factors for thrombosis (Table In terms of the recipient factors, the presence of preoperative dialysis and the higher BMI of the recipient 27.2 kg/m7.2 kg/m2The average hematocrit, urine volume, and fluid infusion volume in the 24 patients who underwent whole pancreas transplantation are shown in Fig. Pancreas transplantation improves not only quality of life, but also the survival, of patients with type 1 diabetes. Pancreas graft survival has been improving following the recent developments in immunosuppressive drugs and improvements in surgical techniques and postoperative management. In patients with type 1 diabetes on hemodialysis and on the waiting list, the 5-year survival is <50 %. Japanese recipient candidates on the waiting list tend to be older and have a longer diabetic and/or hemodialysis period. In addition, many donors are considered to be marginal according to Kapur’s criteria . In thisCertain postoperative complications, such as thrombosis, bleeding, infection, anastomotic leakage and pancreatitis, may occur after pancreas transplantation. Moreover, patients with type 1 diabetes and end-stage renal failure are reportedly in a hypercoagulable state, which is one of the causes of susceptibility to thrombosis after pancreas transplantation . It was A variety of risk factors for graft venous thrombosis in pancreas transplantation have been reported –9. We suWe were unable to perform a statistical analysis because there was only one case of venous thrombosis in our series. There were no differences in the donor- and operation-related factors between the patient with graft venous thrombosis and the others. However, the recipient BMI was higher than the average BMI of the other patients. Obesity may have led to a higher abdominal pressure and caused graft vein compression and congestion, leading to venous thrombosis.Virchow’s triad has been a major concept considered to lead to thrombus formation . In pancSufficient fluid infusion is a basic policy for the prevention of thrombosis used in our department, because the concentration of hematocrit secondary to dehydration increases the viscosity of the blood and becomes a risk factor for thrombosis . The posEarly detection is extremely important for adequate treatment of graft venous thrombosis. Recipients with complete occlusion of the portal vein reportedly develop hyperglycemia, epigastric pain, and hyperamylasemia . HoweverIn conclusion, as the largest series of pancreas transplantation performed so far at a single institution in Japan, our series yielded 10-year patient, pancreas and kidney survival rates of 100, 67 and 68 %, respectively, which are better than the worldwide data reported in the International Pancreas Transplant Registry. Among the 26 pancreas transplantations, there was one graft loss due to graft venous thrombosis in a recipient with a high BMI. Although pancreas transplantation may be complicated by graft thrombosis, routine postoperative anticoagulation therapy is not necessarily required for its prevention if sufficient fluid infusion is strictly controlled and the graft blood flow is frequently monitored. When graft thrombosis occurs, early detection and medical intervention are extremely important before complete occlusion of the portal vein in order to improve the survival of the pancreas graft.


In our last Editorial  we highlighted that the second edition of the International Congress “Health, Nutrition, Fitness, and Wellbeing – SANABUNAINT 2012”, took place in Falticeni, between 19-21 of October 2012, within the context of the 9th edition of the Apple Festival – “The Apples of Rădășeni”, and as an expression of the responsible preoccupation of the Falticeni Town Hall and Suceava Town Council in promoting the Public-Civic-Private Partnership, while considering the entire series of potential benefits . A first image in this Editorial showed the Opening Ceremony of the SANABUNA International Congress, which took place at the prestigious “Ion Irimescu” Art Museum, Falticeni.Since 110 years from the birth of Master Ion Irimescu are celebrated, our welcoming “host” of the recent event “Health, Nutrition, Fitness and Welfare” – SANABUNA, which took place in Falticeni, in October 2012 , our thoughts turn, with a natural emotion, to this important moment of recognizing some exemplary achievements which confirm the tough work, the commitment and the talent of the Master. The anniversary program organized by Falticeni City Council, Falticeni City Hall, “Ion Irimescu” Art Museum and “Ion Irimescu” Cultural Foundation confirm the respect of the community regarding the real values of our nation. It does not seem a coincidence that Titu Maiorescu, Founding Member of the Romanian Academy, who organized a long time ago, the well-known „Junimea” Circle meetings at Falticeni, used to say: „The art of life? Care, discreetness, moderation, basically negation and briefly abnegation”. With all his unrest in the loneliness of his workshop, Master Ion Irimescu had expressed his feelings according to the job of the local and national community, giving sense and life to the art.I am pleased to share a significant „fresh” connection between Falticeni and Brussels, the european capital from which I have recently returned home. The words „Falticeni” and „SANABUNA” have repeatedly been pronounced at „Sfanta Ana” Castle, the office of the Belgian Diplomatic Club, on Friday the 15th of February 2013, our greeting becoming famous due to the International Congress which took place in Falticeni. This happened in the context in which: “Carol Davila” Academic Publishing House, of “Carol Davila” University of Medicine and Pharmacy in Bucharest, has been awarded The Belgian Prize of Innovation for its contribution in the organization and the successful development of SANABUNA International Congress, which took place in Falticeni ; the rigorous Laudatio, presented by the prestigious National School of Politic and Administrative Studies (SNSPA) Bucharest, on the occasion of granting the Doctor Honoris Causa title to Léon F. Wegnez, A.I.D.A General Secretary in Brussels, General Manager of the Belgian Diplomatic Club and co-founder of Brussels Diplomatic Gazette (http://www.crd-aida.ro/2013/02/leon-f-wegnez-doctor-honoris-causa-prestigious-snspa-bucharest-romania/), finally referred to the concept of cooperation, thanks to SANABUNA International Congress which took place in Falticeni. I am proud to say that we managed to promote the country brand in Brussels, with the help of the same professional and dedicated team in Falticeni, namely three Members of the Scientific Committee of SANABUNA International Congress, Prof. Remus Pricopie, Minister of National Education, Prof. Petru Filip, President of the Commission of Foreign Policy of the Romanian Senate, Prof. Theodor Valentin Purcărea, President of the Romanian Distribution Committee and Member of the Board of A.I.D.A. Brussels, and last, but not least, the President of the National Organizing Committee of SANABUNA International Congress, Assoc. Prof. Victor Lorin Purcărea, Director of “Carol Davila” Academic Publishing House, President of the Commission for Internal and International Policy of the Senate of “Carol Davila” University of Medicine and Pharmacy Bucharest, and Advisor to the Vice-President of the Commission of Public Health, Senate, Romanian Parliament.Being inspired by SANABUNA International Congress, our distinguished colleague, Prof. Eliot Sorel, talked about the “Magic of Falticeni” and the uniqueness of “Ion Irimescu Museum” both in Romania and in Europe. Imbued with this state of mind from Falticeni, which we also managed to impart to Brussels, I transmitted  to the Mayor of Falticeni collegial greetings, wishing a full success for the significant anniversary event of Master Ion Irimescu. Highlighting the connection between SANABUNA International Congress and Brussels, I cannot stop remembering the moment in which the idea of launching SANABUNA brand shaped, on the grounds of our constant preoccupations for “Medicine and Life”. In fact, I believe that the attached images, beginning with the Brussels Diplomatic Gazette, December 2004, speak for themselves. Moreover, our discussions in the context were centered towards the legitimacy of “SANABUNA” brand. This legitimacy depends both on the vitality and the status of the brand, and on its consistency and continuity; on truly understanding the experience of the patient, while the benefits and costs for the creation of an adequate patient experience were being professionally and responsibly taken into account, while assembling tools, processes, abilities and proper measures necessary to meeting and overcoming the needs of the patients, rethinking the system of improving the healthcare system, while having the patient as the core element. All these imply a greater commitment in accepting the challenge of a creative thinking, managing the problem with regard to the quality of life, which is connected to health and the health perceived by the patient, making a progress by knowing and understanding the reciprocal confidence between the healthcare provider and the patient, while approaching the relationship between medicine (as a science and art) and life, finally giving life to the years. Prof. Dr. Florian Popa


Introduction. Systemic heparinisation is advocated during laparoscopic live donor nephrectomy (LDN) as a preventative measure against renal vascular thrombosis during the warm ischaemic interval. This study compares the outcome with and without the administration of systemic heparinisation. Methods. A retrospective analysis was performed on 186 consecutive LDN patients between April 2008 and November 2012. Systemic heparin (2000–3000 IU) was administered intravenously to donors (hep n = 109). From January 2010, heparin was not used systemically in this group of LDN (no hep n = 77). Outcome measures included donor and recipient complications, initial graft function, and 12 month graft survival. Results. The demographics of both heparinised and non-heparinised donors were similar. The warm ischaemic time (WIT) was comparable in both groups . There was no difference in complication rates, no episodes of graft thrombosis, and no incidences of primary nonfunction in either group. Delayed graft function occurred in 4/109 and 1/77  and there was no significant difference in graft survival (P = 0.650). Conclusion. Omitting systemic heparinisation during laparoscopic donor nephrectomy is a feasible and safe approach that does not compromise donor or recipient outcome. Minimally invasive techniques of surgery for live donor nephrectomy have been rapidly adopted across the UK. Unquestionably this has helped to increase the number of live donor kidney transplants . KidneysDuring laparoscopic live donor nephrectomy (LDN) the kidney endures a period of warm ischaemic injury before it is retrieved and flushed with cold preservation solution . SystemiA retrospective analysis was performed on 219 consecutive patients undergoing LLDN from April 2008 to November 2012. Three donors were converted from laparoscopic surgery to an open procedure due to a complication during surgery; however all 3 conversions were carried out before heparin was administered and these cases were consequently excluded from the study. Thirty patients were also excluded due to lack of completed documentation. Therefore, 186 LDN were analysed in this study. All LDN were performed by the same consult transplant surgeon (MLN).n = 109). From December 2010 the remaining donors in the series did not receive intraoperative systemic heparin (n = 77).Patient's notes and computerised records were manually assessed for donor and recipient complications, including complications throughout the operative procedure and graft function of the recipient. Graft outcome measures were collected up until 12 months after transplant. All donors who underwent LDN between April 2008 and December 2010 received systemic heparin . The laparoscopic surgical procedure was consistent throughout this cohort of 186 patients. A pure laparoscopic, nonhand assisted procedure was used throughout.A 4-port transperitoneal access was used. Kidneys were extracted via a pfannenstiel incision (6–8 cm), using a fully transperitoneal approach. Two 10 mm ports were used; one placed close to the umbilicus and the other in the ipsilateral iliac fossa. Five mm ports were placed in the epigastrium and the lumbar region. The renal artery was secured with a linear cutting stapler or lockable silastic clips . The renal vein was divided after controlling with Hem-o-lok clips. Systemic heparin (2000–3000 IU) was administered intravenously to donors 5 minutes prior to arterial clamping.Donor and recipient demographics and the incidences of intra- and postoperative complications in the donor and recipient were assessed. In the recipient, the incidences of graft thrombosis, graft function, and graft survival were recorded. The total ischaemic time was defined from the start of arterial clamping of the donor vessels to reperfusion of the kidney.Recipient graft function was measured daily using levels of serum creatinine, and eGFR on day 7, 1 month, and 12 months after transplant.Delayed graft function (DGF) was defined as any form of renal replacement therapy (RRT) needed in the first 7 days after transplant. Acute rejection was diagnosed by histopathological examination of a renal biopsy and treated with 3 × 0.5 grams methylprednisolone for 3 consecutive days. Resistant rejection was treated with antithymocyte globulin (ATG). Graft and patient survival were monitored up to 12 months after transplant.t-test or contingency test (Fisher's exact). P ≤ 0.05 was considered to be statistically significant.Statistical analysis was performed using an integrated measurement using Excel  and Graph Pad Prism 5 . Results were displayed as mean ± standard deviation. Mean data was compared using the appropriate P = 0.386). More kidneys in the heparin group had multiple arteries compared to the nonheparinised group (P = 0.027). Several kidneys in each of the groups had dual renal veins (P = 0.473).Donor and recipient demographics are outlined in P = 1.000) (range 1 to 13 min versus 2–8 min) or in the total ischaemic time  between the groups. The warm ischaemic time was significantly longer in kidneys with multiple arteries compared to those with single vessels .There was no significant difference in the duration of warm ischaemia .Patients in the heparin group stayed in hospital significantly longer compared to those in the nonheparinised group . There was no significant difference in the anastomosis time in kidneys with single or multiple arteries .The anastomosis time was 26 ± 6 minutes in the heparin group and 30 ± 8 minutes in the nonheparinised group (P = 0.740).There were no episodes of graft thrombosis or primary nonfunction in either group. In the heparinised group six recipients received blood transfusions, compared to three in the nonheparin group . Day 7, 1 month, and 12 months serum creatinine and eGFR levels were not significantly different between the groups  of heparinised patients and 1/77 (1.2%) of the nonheparinised group (P = 0.326).There was a similar incidence of acute rejection in the two groups . Patient survival at 12 months was 98.2% (hep) versus 96.1% . Three grafts failed in the heparin group within the first 12 months. One due to recurrence of primary end-stage renal disease, and two due to rejection that did not respond to treatment. The single graft loss in the nonheparinised group was due to rejection within the first 3 months after transplant. There were 2 patient deaths in the heparin group. One due to a cardiac arrest 3 days after transplant and the other due to ischaemic bowel leading to sepsis 7 days after transplant. In the nonheparinised group one patient died due to adenocarcinoma of the lung, another due to a cerebral head trauma secondary to an epileptic episode, and the third due to a hypoxic brain injury at 11 months after transplant.Graft survival at 12 months was similar, 97.2% (hep) versus 98.7% in the nonheparinised group;  or cause graft loss . In renaThe duration of warm ischaemia varies significantly between centres in live kidney donation (range 2 to 17 minutes), although this is reported to have no adverse effect on short term graft outcome . The aveIn addition to the increased warm ischaemic interval other factors during LDN can also contribute to complications. These may also advocate the use of heparinisation. The left kidney is the preferred choice for LDN due to the longer renal vein. The shorter renal vein in the right kidney has been associated with increased incidence of renal vein thrombosis . HoweverIn this present series, the patients were well matched with a comparable ratio of right and left kidneys donated. There was a higher incidence of multiple arteries in the patients receiving heparin. Nonetheless, the complication rate was extremely low with no incidences of thrombosis, only 3 recipients in each group requiring reexploration and no significant difference in graft function or survival. Delayed graft function (DGF) occurred in 3.6% versus 1.2% in the nonheparinised group. These rates of DGF fall within ranges reported in literature sources from other worldwide live donor transplant centres , 17. FurProtamine Sulfate can be used to reverse the anticoagulation effects of heparin before completion of the operation. Nonetheless, it has been linked with severe complications and is not commonly used during LDN and was not used in this study . There wThree donors were converted to open surgery due to complications during surgery. They were excluded from the analysis because the complications arose before heparin was administered. Conversion to open surgery was a necessary step to prevent any further complications. Hospital stay was increased by one day in donors receiving heparin compared to those without. Systemic heparinisation was used early in the series and since this time there has been a change in practice and a more rapid recovery protocol in place to enhance a faster recovery and reduce hospital stay.In conclusion omitting systemic donor heparin during LDN is a feasible and safe approach that does not adversely compromise donor or recipient outcomes.


Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes  in barley (This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure. Starch, a polysaccharide composed of glucose molecules, is a common constituent of higher plants and can be found in all the organs being the major forms in which carbohydrates are stored ,2. BiosyGlucose moieties in starch form two structural arrangements called amylose and amylopectin. Amylose is a linear, or slightly branched molecule in which the glucose units are joined end-to-end by α-1,4 linkages and typically represents about 25% of the starch granule . AmylopeStarch is economically important. It is the major source of calories in food and feed worldwide. It is also a functional polymer with potential to provide environmental friendly biomaterials . In rece+, K+, Ca2+ and Mg2+[A rapid hydrolytic degradation of the bulk of dietary starch takes place in the lumen of the small intestine, making it a highly glycemic carbohydrate . However and Mg2+-17. RS c and Mg2+, stimula and Mg2+,19, gene and Mg2+.retrogradation[The proportion of amylose in starch has a direct positive correlation with RS content, reduced digestibility and lower glycaemic responses -25. The gradation,26.In planta production of starches with high proportion of RS has caught considerable attention since such an approach can generate valuable enhanced health promoting qualities directly in the crop. Bioengineering has successfully provided increased amylose starches in wheat [in wheat ,27, ricein wheat ,29, potain wheat -33 and bin wheat . Howeverin wheat  and a puin wheat .Agrobacterium-mediated transformation.We intended to silence all three genes of the family of starch branching enzymes in barley by RNAi. Earlier studies suggests that silencing of an entire family of genes may be done either by targeting a highly conserved region, if one exists in all members of the gene family, or alternatively by designing a chimeric single construct with a sequence of multiple specific targets . We did 0) independent transgenic plants were generated. Insertion of the selection marker gene (Hpt) was confirmed by PCR (gDNA from leaves). Expression of hairpin construct was detected in 6 of the lines (cDNA from developing endosperm 20 DAP) by RT qPCR (data not shown). The two lines SBE RNAi3 and SBE RNAi4, which showed the highest transcription levels of hairpin construct were selected and ten grains per line were propagated to generate T1 lines.11  called A, B and C for each of the plants there will be some that are not transgenic. In agreement with this we observed both grains with SBE silencing and grains without gene silencing from some of the plants. Two plants, SBE RNAi4.1 and SBE RNAi 4.9, showing the highest level of gene expresssion suppression, were selected. The transcript levels of the three SBE isoforms detected in SBE RNAi4.1 endosperms as compared to control were 13% for SBEI, 27% for SBEIIa and 26% for SBEIIb. For the SBE RNAi4.9 line the values were 15% for SBEI, 48% for SBEIIa and 36% for SBEIIb . SBE RNAi4.1 and control plants were further propagated (T2) in greenhouse and in semifield trials.Transcription levels of the 3) and control plants grown in the greenhouse. With similarity to the reduction in gene expression  from SBE RNAi4.1  and iodine complexation staining approach Figure . The chrThermal properties were analysed with DSC for the control starch and for starch extracted from the SBE RNAi 4.1 barley line Figure . ControlSwelling power trials demonstrated that the control starch started to swell at 68°C correlating with the DSC point of gelatinization at 66°C Figure a. The swTransgenic grains had a characteristic wrinkled shape Figure . To chec1 plant were collected and the ratios of wrinkled/wild type (wt) grains were assessed for each plant (Table 1 grains (SBE RNAi 4.9) and tested for insertion of transgene by PCR using the primers Hairpin Fw and Rev  SBE RNAi4.1, SBE RNAi4.9 and control mature grains was used for β-glucan analysis. β-glucan contents in SBE RNAi4.1 and SBE RNAi4.9 wrinkled grains were 22% and 33% higher than the content of control grains  crystalline polymorphs with totally 25% crystallinity of the starch and no trace of the original A-type polymorph was found.The diffraction peaks detected with XRD at 2θ for the control starch were typical for A-type  crystalline polymorphs with approximately 20% crystallinity. For the SBE RNAi4.1 starch a combination of B-type  and VAverage number of spikes per plant was calculated from all semifield plants Figure a. A samp2 plants from control and SBE RNAi4.1 lines growing in greenhouse were measured at 20, 40, 60, 80, 100 and 120 days after sowing  GBSSIa, GBSSIb, glucan water dikinase (GWD) GWDI in T2. RT-qPCR was performed in 20 DAP developing endosperms of SBE RNAi4.1 and control lines. Genes with significant up-regulation in the SBE RNAi 4.1 line as compared to the control line were SSI (1.7-fold), SSIIa (1.7-fold) and GWDI (2.2-fold). The most evident up-regulations were found for GBSSIb (5.7-fold) and SSIV (3.6-fold). All of these were mean values of 3 biological and 9 technical replicates  s Figure .In vitro degradation by pancreatic α-amylase and glucoamylase was employed to simulate the effects of small intestine hydrolysis and subsequent glycemic response of the engineered starch [d starch . The assBased on the degradation data, the content of rapidly digestable starch (RDS), slowly digestable starch (SDS) and resistant starch (RS) was calculated according to Englyst  definitiStarch branching enzymes (SBEs) play a pivotal role in amylopectin biosynthesis by catalysing chain transfer by cleavage of an α-1,4 linkage following a condensation of an α-1,6 linkage . In cereUsing size exclusion chromatography (SEC), we found that the amylose fraction in control starch was 29.9% of the total starch. In contrast, the amylopectin constituted less than 1% of total starch in SBE RNAi4.1, where instead a major double peak characteristic for amylose was identified Figure . The λ-mSBEIIa and SBEIIb in barley increases amylose content to a certain degree (70%) [Arabidopsis leaves [Amylopectin is a semi-crystalline material with distinct thermal characteristics . The theee (70%) . Our dats leaves  and wheas leaves .Swelling and solubilisation of starch in aqueous systems, e.g. during cooking, are crucial for efficient enzymatic starch digestion leading to glycemic response. Heating of granular starch in excess water disrupts the crystalline structure as an effect of breakage of the extensive hydrogen bonding network between water molecules and the hydroxyl groups of the starch. This causes granule swelling and gelatinization  and the Major suppression of enzymatic degradation rates and a major increase in the RS content fraction were found for the SBE RNAi4.1 starch as compared to control starch as evaluated by the Englyst method for determination of RS. The amount of RS in the amylose-only starch was 90%, 65% and 68% respectively for native, gelatinized and retrograded starches  compared with control grains (49%). This demonstrates the physiological importance of amylopectin in the structural ordering of carbohydrate to allow a more rapid energy remobilization.No difference was found in the germination frequency of SBE RNAi4.1 and control grains  and Isoamylase 1 (ISAI) has been detected [The endosperm of developing SBE RNAi grains had increased expression levels of a number of key starch biosynthetic enzymes. The most prominent increases were found for ndosperm . In durudetected . This gedetected .An amylose-only starch was produced with high yield in the barley endosperm by implementing a new method of simultaneous suppression of the entire complement of genes encoding SBEs.h crystallites and that amylopectin is not essential for granule crystallinity and integrity. Such polysaccharides can have significant applications such as food additives to ensure improved health via large bowel fermentation of resistant starch.The severe yield penalty most often observed for these kind of enzyme suppressions is supposedly counteracted by pleiotropic stimulation of a number of starch biosynthetic enzymes and, generally, high yield storage of pure amylose in cereal seeds is of interest for industrial-scale production of this polysaccharide. The high amylose grains had a characteristic wrinkled phenotype, which segregated in a 3:1 ratio. The fact that amylose-only barley can be obtained only when suppressing the expression of all the SBE genes reveals the functional importance for all SBE genes. Finally, we demonstrated that amylose-only starch granules can be synthesized and deposited with very high proportion of VThe tri-antiSBE sequence was synthesized artificially by Eurofins Medigenomix GmbH (Germany). For each SBE gene, 300 base pairs of coding sequences from the cDNA sequence were used as targets as shown in Additional file The 300 base-pair fragments were designed to be flanked by the restriction-enzyme target sites for SalI and XhoI. This resulted in a DNA fragment with the following syntax: SalI-SBEIIa-SBEIIb-SBEI-XhoI, which was cloned into the RNAi silencing vector pSTARGATE forming a chimeric triple RNAi hairpin construct.E. coli DH5α as described by the manufacturer (Invitrogen). Plasmid DNA was purified by a ‘Fastplasmid Mini Kit’ as described by the manufacturer  and analyzed by restriction enzyme digestion (BamHI or SmaI). Valid vectors were transfected to Agrobacterium tumefaciens (AGL0) and used for transformation of Hordeum vulgare var. Golden Promise. Transgenic barley transformation was performed as described [To secure high expression, the construct was expressed under control of the maize Ubiquitin-2 constitutive promoter (2 kb) . This frescribed .GAPDH as an internal control as described [Genomic DNA and RNA analysis using quantitative real-time PCR (RT qPCR) was performed as described . Relativescribed . All the−1 mg protein−1.Barley endosperms at 15–20 DAP were collected from 3 different plants (1 spike from each plant). Endosperms (5–7) were collected from each spike and pooled. The endosperms were homogenized in ice cold buffer . The homogenate was centrifuged at 15,000g for 15 min at 4°C. The supernatant was re-centrifuged at 15,000g for 10 min at 4°C to remove any traces of debris. Protein concentration was determined using a standard Bradford reagent  and the samples were diluted to 1mg/mL protein and stored at −20°C until analysis. SBE activity was measured as the decrease in absorbance of the amylose-iodine complex after SBE catalyzed branching as described by . AcarbosStarch was extracted and purified using a modified version of the protocol described . To avoiIodine colorimetric analysis was carried out as described .SEC was performed as described .Samples were analyzed using a Perkin Elmer Diamond DSC instrument operated from 30 to 100°C at a scanning rate of 10°C per minute. The starch granules were analyzed in aqueous slurries consisting of of 3 mg starch granules and 12 μL 10 mM NaCl in technical duplicates. Perkin Elmer Pyris 7.0 software was used to determine the parameters peak temperature (TP) and enthalpy change (ΔH).2O was placed in a pre-weighed centrifuge tube and vortexed. After heating for 20 min in a shaking thermomixer at 56, 68, 78, 90 or 100°C, the tube was cooled to 15°C and centrifuged at 15.000 ×g for 10 min. The supernatant was removed by siphoning, and the swollen, precipitated starch was weighed. The total carbohydrate content of the supernatant was determined in triplicate using a modified method of Dubois et al. (1956) [2SO4 (200 μL) was added and the absorbance read at 490 nm using glucose as standard.Swelling power was determined using a modification of the method of Schoch (1964) . For sol. (1956) . ProperlMedian cross sections were prepared using a scalpel and examined with a Wild MZ8 Leica stereo microscope. For light microscopy analysis 50 μm thin sections were cut from fresh frozen 40 dap grains using a HM 550 OM Cryostat microtome, stained with I2/KI and mounted on a Zeiss Axioplan 2 Imaging microscope.β -glucan content was determined using the β-Glucan (Mixed Linkage) kit by Megazyme International Ltd.  following the manufacturers protocol.Starch content was determined using the ‘Total starch AOAC Method 996.11/AACC Method 76.13’ kit from Megazyme International Ltd.  using the protocol recommended by the manufacturer for samples containing resistant starch.Phosphate content was measured as described .Polarization light microscopy was performed as described .Scanning electron microscopy was carried out as described .Powder X-ray diffraction was performed following appropriate hydration as described . XRD diaSemifield experiments were conducted in a locked out-door cage raising 50 plants in 5 100 L soil pots, 10 plants each pot. SBEs suppression and amylose-only starch composition in this consecutive generation was confirmed by RT qPCR, iodine complexation and DSC respectively (data not shown).t-test (PROC ANOVA) with k-1 and n-k degrees of freedom, where k is 2 (control and SBE RNAi4.1) and n is the number of observations. A value of P < 0.05 was considered to indicate statistical significance.Average number of spikes per plant was calculated from all plants. Average mass of the grains, average number of grains per spike and average yield per plant were calculated on a sampling. Statistical comparisons between the two lines (control and SBE RNAi4.1) were evaluated using a 2 plants growing in greenhouse  were measured at 20, 40, 60, 80, 100 and 120 days after sowing and the average height was calculated.Heights of T2) from lines SBE RNAi 4.1 and control each were sown in semifield trial and the germination ratios were calculated by inspection of germinated seedlings after 2 weeks.60 grains  in 20 mm sodium phosphate buffer with 6.7 mM sodium chloride (pH 6.0) at 37°C for 0, 0.5, 1, 2, 6, 12, 24 hours. Enzyme treatment was terminated by adding 30 μl 0.1 M HCl and 250 μl of 50% ethanol on ice and centrifuged  and the supernatant was collected. The amount of soluble reducing sugars was measured [t method , using nmeasured  and the The authors declare that they have no competing interests.in vitro starch degradation analysis and prepared text and figures relative to these experiments. AH and AB carried out X-ray diffraction analysis. PBH assisted in the writing of the manuscript. KHH designed the transgenic construct, carried out the transgenic transformation, assisted with binocular stereo microscopy of grains and size exclusion chromatography, prepared text and figure relative to construct design and transgenic transformation, assisted in the writing of the manuscript and planned, supervised and coordinated the project. All authors read and approved the final manuscript.MC carried out transgenic plants regeneration, quantitative real-time PCR, starch extraction and purification, iodine complexation analysis, size exclusion chromatography, binocular stereo and light microscopy of grains, β-glucan content, starch content, phosphate content, scanning electron microscopy, yield assessment, plant height analysis, germination frequency test, starch remobilization analysis and wrote the manuscript. AB assisted in the writing of the manuscript, supervised, planned and coordinated the work on starch analysis and assisted in the size exclusion chromatography. SLJ carried out differential scanning calorimetry, swelling power and solubility analysis, prepared text and figures relative to these experiments and collaborated in the size exclusion chromatography and X-ray diffraction analysis. SSS carried out starch extraction and purification, SBE activity assay, polarization light microscopy, SBE target sequences. Target sequence of SBEI, SBEIIa and SBEIIb for the chimeric SBE RNAi construct.Click here for fileTransgene expression analysis. Relative expression levels of selection marker gene (Hpt) and transgenic hairpin construct analysed by RT qPCR in leaves of control and transgenic T1 lines . SE is indicated.Click here for fileDifferential scanning calorimetry table. Differential scanning calorimetry (DSC) in aqueous suspension of starch extracted from control and from SBE RNAi4.1 lines. ΔH: Change in enthalpy due to starch thermal dissolution in water.Click here for filePCR of SBE RNAi 4.9 T1genomic DNA. A PCR was performed using the primers Hairpin Fw and Rev .Click here for filePolarization microscopy. Bright field microscopy image (left) and polarized microscopy image (right) of control starch granules  and amylose-only granules from line SBE RNAi4.1 .Click here for fileYield. (a) Average number of spikes per plant calculated in all plants from a semi-field trial (sample population: 50 plants each line). (b) Average number of grains per spike in 25 plants per line from a semi-field trial. (c) Average single grain mass calculated for 25 plants per line from a semi-field trial. (d) Average yield per plant in milligram of grain produced calculated in a sample of 25 plants per line from semi-field trial. (e) Correlation coefficients (r) and significance level as P values for the three yield components: average yield per plant, average number of spikes per plant and average grain mass.Click here for fileGermination frequency. Germination frequency in soil of barley grains from SBE RNAi 4.1 and control lines.Click here for filePrimers. Primers for RT qPCR.Click here for file


Objective: Facial allotransplantation is fast becoming a reconstructive option for severely disfigured individuals, but it is still in experimental stage. Facial allotransplantation will be considered fully ethical only when it addresses the recipient's identity concerns. There exist no such studies that quantify and predict identity changes following change in geometry of underlying facial skeleton and overlying soft tissue morphology. I objectively address these identity concerns in the present study. Methods: Using software , I developed 3-dimensional facial models from the computed tomographic images of 4 identical and 1 nonidentical twin pairs. On the basis of cranial base anatomy, 3-dimensional coordinate values of selected landmark locations were measured  and a morphometric method was applied to quantify identity differences between them. Identity parameters were drawn on a diagrammatic chart that depicted the biophysical identity range. Using software (Mimics version 14.0), facial allotransplant simulation was done involving most of the nasal bone, maxilla, and zygoma. Morphological parameters of resulting new identity were drawn on diagrammatic chart and if they fell within the chart, facial allotransplantation resulted in identity transfer. Moreover, a scale was used to quantify identity transfer and identity restoration resulting from facial allotransplantation. Results: Identity changes of facial allotransplantation are objectively quantified that can be used as a communication tool in consent taking process. Conclusion: My findings suggest that donor facial allograft may be identified on the recipient's face depending upon underlying craniofacial morphology and accordingly, identity changes can be suitably predicted. The first radical facial surgery that restored facial appearance was done in India in 1994, when Dr Thomas, a microsurgeon, replanted the entire face of a 9-year-old girl, who had lost her face and scalp in a threshing machine accident.3Few authors have investigated the identity issues of facial allotransplantation. Siemionow and AgaogluHowever, no study quantifies and predicts identity changes accompanying altered morphology of overlying soft tissues and geometry of underlying bony framework. Only 9 face transplants have been done around the world,predict and quantify identity changes of facial allotransplantation andpredict identity changes relative to recipient's family facial phenotype.The aim of the study was toFive twins—aged between 20 and 33 years —were selected on the basis of lack of any craniofacial trauma, surgery, congenital deformity, or unknown lesion and the fact that they were not being treated for any medical deformity, underwent computed tomographic (CT) scan. All twins were of north Indian descent that had symmetrical appearances and had been living together for most of their lifetime. Four twin pairs were monozygotic and 1 twin pair was dizygotic as was determined at the time of their birth on the basis of anthropometric and serologic methods. They were explained in details about the purpose and nature of study in their native language before informed consent was taken.Computed tomographic (multislice) images, with a high-resolution bone algorithm , were taken using CT scanner , and image covered the area from vertex to submandibular region of craniofacial complex. Acquired CT slice data were imported to workstation  and reconstructed in 3 dimensions. A life size facial model was developed using segmentation and volume rendering techniques . Hard and soft tissues were separated by threshold-based segmentation, based on differences in their permeability to x-rays.Software  allowed creating a template of landmark locations, Table  and measX-axis was determined roughly parallel to right-left direction of subject's maxillofacial skeleton. Y-axis and z-axis corresponded to anterior-posterior and inferior-superior directions, respectively. Three-dimensional coordinate values  were calculated for landmarks Bc, Pr, and Mx with origin  set at Ba. For Pr, only y- and z-axis values while for bilateral landmarks, x-axis , y- and z-axis coordinate values  were measured Fig . DisplacMean and standard deviation values of identity indices (mm) and chin protuberance difference (mm) of identical twins were drawn on a diagrammatic chart that indicated the biophysical identity range Fig .Four subjects  were randomly chosen as single family unit excluding recipient, and mean values of 3D coordinates  at set landmark locations defined shape of family facial phenotype (F).Displacement vector was calculated as the difference between 3D coordinate values at set landmark location of individual family member and corresponding mean family values and was called family identity index for that landmark.Mean and standard deviation values of identity indices (mm) of a particular landmark and nose length (mm) of all family members were indicated on a diagrammatic chart that depicted family identity pool Fig .It has 3 parts:Using point registration (Mimics version 14.0) at infraorbital margin, nasion, and prosthion on facial skeleton, recipient and donor's transparent facial images were superimposed; donor graft in the mid face area was cut out using a cutting plane that included lower half of nasal bone, whole maxilla including most of the hard palate and zygoma; and was transplanted onto recipient's face Fig . Four suIn addition, a donor compatible with family phenotype was chosen, and recipient's family identity indices were plotted on chart. If they fell within identity pool, recipient's new identity became part of family phenotype Fig . where “d” is the difference between double measurements and “n” is the number of paired double measurements.All processes were performed by the author (A.M.). Errors in landmark localization were evaluated by comparing differences between 3D coordinates and linear measurements of original and repeated examinations of 10 subjects during a 2-week time interval. Method error was calculated as Table To study variations among human faces, it is necessary to study the structures that compose it—skull characteristics, musculature, and associated soft tissue. The mimetic musculature is stretched across facial skeleton like a mask and to a great extent; any change in form of facial bones causes variations in facial appearance.It is important to know the fundamentals of shape perception in 3 dimensions to understand identity variations of human face. Perception of shape depends upon viewing direction, distance, and illumination. Shapes look different in 3 dimensions depending upon how much light is reflected on their surface and illumination of adjacent environment.I hypothesize that craniofacial complex has 3 different optical environments that are contained by different size and contours of its skeletal components and, overlying soft tissues mask them. These three environments are inner, middle, and outer. Maxilla forms inner optical environment and is central to human identity. It not only supports the nose and lip but also there exist varied eminences, and angles within its small surface, along with overlying soft tissues, contribute to unique facial appearance. Its immediate outer boundaries form middle optical environment that is marked by landmarks buccal, prosthion, and maxillare. Boundaries of outermost optical environment are ill-defined and depend upon face index and chin characteristics Table . In addiContours of a bony segment are a true expression of its total morphological configuration,To study a shape, one needs a constant stimulus that illuminates it.Donor's facial graft will retain its native identity only when recipient's parameters of outer optical environments match with the donor's. Face index indicates the overall shape of craniofacial complex, and average face index ranges from 85% to 89.99 %.Aesthetic outcome of the facial allotransplantation will depend upon number of craniofacial parameters that are identity-compatible between recipient and donor. Scoring them will make easy to communicate identity issues with donor's family and recipient as well. “AM” scale, denoting letter “M,” is proposed in this study to quantify identity changes of both types of facial allotransplantation Fig . Lee andUsing this morphometric method and values of biophysical variations, identity transfer to recipient can be objectively predicted and quantified from donor's face CT scan. A drawback of this study is the small number of subjects. There needs collaboration among institutions to establish a twin research study with large sample size that measures and standardize the numerical values of biophysical variations in morphology of craniofacial components.Humans use facial comparisons to recognize their relatives and regulate their behavior.This study can also be helpful (1) for organ banks to record the identity databases of facial grafts that can be used in future to choose appropriate graft in accordance with recipient's identity expectations from facial allotransplantation, (2) to measure and restore the facial symmetry of subjects that require treatment with facial implants for facial asymmetry, trauma, and congenital deformities, and (3) in twin studies, to accurately measure the inheritance patterns of craniofacial components keeping in mind the limitations of 2D methods.23Severe facial disfigurement disrupts ability to recognize oneself and may constitute a major life crisis with a potential change in personality traits.The author thanks his family, especially his beloved Mom, Mrs Urmila Devi , for her indispensable spiritual inspiration and support that was a guiding force to finish this study. He also thanks his uncle, Mr Vijay Garg , who was instrumental in arranging the twins. Lastly, he thanks to Materialise team at Michigan, United States, particularly Eric Renteria, for the technical assistance regarding software.


The discovery of small molecule and biologic drugs offers treatments applicable to a wide variety of infectious and chronic human diseases. Antibiotics have, since World War II, been the weapons of choice in humanity's on-going battle with pathogenic bacteria, and more recently the discovery of antiviral drugs has been an area of active endeavor. Yet the emergence of multiresistant bacteria, as well as the continuing prevalence of antigenic drift in viruses, has shown the fundamental limitations of postinfection drug therapy. Fortunately, the scientific exploitation of host immunity provides an even more powerful approach to forestalling infection and counteracting chronic disease states. The supreme embodiment of this is the vaccine. Indeed, prophylactic, and to a lesser extent therapeutic, vaccines continue to be the most cost-effective and efficient alternative to other medicines for the treatment and prevention of infectious diseases, cancer, and allergy conditions. Importantly, unlike drug-based medicines, vaccines are primarily curative not palliative remedies.Vaccines work by inducing profound changes in the cellular components of adaptive immunity, comprising T- and B-cells. The first vaccines targeted infections pathogens and were composed of killed or attenuated pathogens. However, our emerging understanding of antigen presentation and subsequent recognition has enabled the development of several new types of vaccine, including those based on single proteins or on synthetic peptides encompassing many B- and T-cell epitopes . PeptideThe design of peptide-based vaccines takes advantage of an emergent computational paradigm that couples immunoinformatic prediction with rigorous experimental validation, thus facilitating the identification of epitopes within protein antigens . AmongstHowever, development of effective peptide-based vaccines requires science to overcome a number of significant difficulties, including identifying optimal delivery routes, overcoming the low intrinsic immunogenicity of individual peptides, and the task of combining different types of epitopes to engage the humoral and cellular arms of the adaptive immune response properly, as well as compensating for the poor population coverage of individual T-cell epitopes due to MHC restriction. On a more technical level, certain antigenic epitopes, specifically B-cell epitopes or antibody determinants, are conformational and need to be mimicked by linear sequences or placed onto suitable spatial frameworks. Moreover, methods for predicting B-cell epitopes need to be improved as they are notoriously unreliable in predicting both linear  and confIn this special issue on peptide-based vaccines, we have gathered together a varied array of cutting-edge reports and reviews detailing several important and innovative approaches to the design and discovery of epitope-based vaccines. Articles range from reports on new computational methods for epitope prediction and selection for vaccine design to clinical trials using peptide-based vaccines.in silico with in vitro and in vivo techniques. Indeed, the combination of computational and experimental epitope mapping offers many tools and techniques appropriate to the identification and optimization of vaccine candidates. As this special issue demonstrates so well, such methodology is fast becoming one of the most pivotal tools currently available to vaccine developers; and it foreshadows the day when this variety of approaches will fully synergize to speed up the development of powerfully efficacious vaccines against both infective and chronic disease states.We hope that readers will find this special issue not only interesting but also enlightening, exciting, and inspiring. As more and more pathogen genomes are revealed and our understanding of fundamental immunological mechanisms deepens, it becomes ever clearer that the only tractable approach to vaccine discovery and design is through the interplay of Pedro A. RechePedro A. RecheEnrique Fernandez-CaldasEnrique Fernandez-CaldasDarren R. FlowerDarren R. FlowerMasha Fridkis-HareliMasha Fridkis-HareliYoshihiko HoshinoYoshihiko Hoshino


Current Neuropharmacology. The impact of Current Neuropharmacology is steadily increasing and this volume marks another important milestone as the journal increases to six issues per year. The standard and range of reviews and special issues consistently attains high standards. For Current Neuropharmacology to continue to thrive it is important that diversity and high standards are maintained. Of course, this depends on the dedicated efforts throughout the year of authors, Editorial Board members, Special Guest Editors, anonymous reviewers, and of course the efforts of Bentham Science Publishers. I express my thanks to all of you.This Editorial marks the beginning of the eleventh year of publication of Current Neuropharmacology is reflected in the content of the present issue of the journal. A series of ‘Hot Topic’ papers on Advances in Dystonia is guest-edited by Christopher Bragg and Nutan Sharma. This is followed by a group of papers that reflects the diversity and wide scope of neuropharmacology. With its impact, visibility and availability on PubMed Central, the journal offers an excellent vehicle for the publication of novel reviews and hypotheses.The diversity and high standard of work published in


PLOS Computational Biology, I have decided to step to the side. It's time to bring in new leadership and a new vision. As scientists we generally do not learn a lot of management skills (a mistake in my opinion), but if I have learnt two management skills it is the value of enablement and to start planning for your successor on day one. Well, I did not start on day one, but Ruth Nussinov has been the Deputy Editor-in-Chief since October 2008, and she is an outstanding scientist and editor ideally suited to take over as editor-in-chief. So please welcome Ruth to this leadership role. The journal is in very safe hands. The journal staff, editors, and Ruth have not seen the last of me, however—this is just too much fun. As Founding Editor-in-Chief, I will continue to be involved with the journal, with a focus on special projects, helping Ruth and the team where I can, and, of course, continue as an author of both research articles and front matter, such as the Ten Simple Rules series.After seven years as the Editor-in-Chief of In changing roles, I would like to make a few personal comments about the evolution of the journal and where it might go next. When Steven Brenner, Michael Eisen, and I founded the journal, we had a vision for how it would fill a gap between journals supporting purely computational methods and the array of experimental journals with the odd, token computational paper I must confess that when we started planning for the journal in 2004, support for open access seemed like the right thing to do, but it was not a major driver for me. That changed when, early on in my tenure, I realized open access is critical to maximizing the rate of scientific discovery. Supporting open access, and the new forms of scholarly communication it fosters, enriched my career and brought me into contact with many amazing people I would not otherwise have met. Emphasizing that enrichment, of the 22 invited lectures I gave in 2011, 18 were evangelizing about the importance of open access and open science, and four were directly related to my science. In my opinion, we have yet to see open access reach its full potential, but we will It only remains for me to thank and acknowledge a variety of people who have all been so critically important in the past seven years. First and foremost are the approximately 150 editors who have worked tirelessly to shape and ensure a high-quality product over the years. There is no journal without community-driven efforts, which offer limited reward beyond a job well done. I can't mention everyone, but I must call out Karl Friston, who between 2005 and 2010 worked tirelessly to make computational neuroscience such a rich part of the journal, and Steven Brenner, Simon Levin, and Sebastian Bonhoeffer, who, since the journal's inception, have always responded with good advice.Thanks are also due to Mark Patterson, who, until late 2011, was Director of Publishing at PLOS. Mark was critical to the success of the journal and to PLOS as a whole. We first began talking about the journal in May 2004, and he listened to, refined, and contributed to a lot of crazy ideas that define what the journal is today. To Catherine Nancarrow, who managed the journal from 2005 to 2010. A nicer and more dedicated person will not be found. In the current era of 140 character snapshots, her emails conferred a quality, beauty, and caring that you just don't see anymore. To Evie Browne, who contributed so much as Publications Assistant and Publications Manager between 2006 and 2009. Her spreadsheet analyses of how the journal was doing were amazing. To Andy Collings, who in various roles from Publications Assistant to Editorial Manager from 2005 to 2012 just made all ideas work, however outside the box they were. To Fran Lewitter, who, as Education Editor since the beginning, has created an important community jewel. To Scott Markel, who has been a voice of reason and an interface to the International Society of Computational Biology (ISCB) over the years. Finally, to all the other staff who have contributed over the past seven years: Emily Stevenson, Johanna Dehlinger, Helen Budd, Sheran Basra, and Cecy Marden, and the amazing current staff of Laura Taylor, Clare Weaver, and Chris Hall, all so well led by Rosemary Dickin and Theo Bloom.PLOS Computational Biology.PLOS is a family of journals and a family of people; in short, a family organization whose goal is to disseminate science in the most open and useful way possible. It is a successful organization able to recruit the best and most dedicated staff, and to adjust to its growing success and the success of open access itself. The world of scientific publishing will never be the same again because of PLOS and the people who drive it. I am proud to continue to be a member of this amazing family. There is no other publisher like PLOS and no other journal like


One of the recognised complications of resurfacing a wound with a split skin graft is collection of haematoma or seroma under the graft. This can result in graft loss and delayed wound healing. The risk can be reduced by meshing or fenestrating the graft, which permits fluid escape, but both techniques may compromise the final cosmetic appearance compared with a sheet graft.We promote a technique that combines the excellent cosmesis of a sheet graft but with the safety of meshed or fenestrated grafts. First described for the management of burn wounds,Though the technique of micromeshing was first described in 1999,