A 29-year-old man developed chronic allograft nephropathy 63 months after renal transplantation. He became symptomatic with advanced chronic graft failure; his immunosuppressive medications were reduced and he was commenced on haemodialysis. Two months following the withdrawal of immunosuppression, he presented with abdominal pain, haematuria, and a marked drop in haemoglobin. The patient was taken to the operating room, where the renal allograft was found to be ruptured, and graft nephrectomy was subsequently performed. Histological examination of the graft specimen showed severe haemorrhagic acute vascular cellular rejection in a background of marked chronic allograft vasculopathy. Immunostaining for C4d showed diffuse, strong, linear circumferential staining of the peritubular capillaries, indicating a concurrent antibody-mediated rejection. We report herein an unusual case of spontaneous renal allograft rupture that occurred long time after transplantation due to severe acute rejection following cessation of immunosuppressive medications for advanced chronic allograft failure. To the best of our knowledge, the time interval between transplantation and the rupture of this allograft is the longest of those reported in the literature. Renal allograft rupture is a well-recognised but rare complication of renal transplantation, which normally occurs in the first few weeks after transplantation. It is associated with severe graft pain, hypotension, and a drop in haemoglobin. The most common cause of allograft rupture is acute rejection . The incμmol/L. In addition, he was kept on intermittent self-catherization four times per day. Forty-eight months after transplantation, the patient was diagnosed in another institution to have chronic allograft failure based on slow rising serum creatinine. No renal allograft biopsy was obtained as the patient refused. His immunosuppression was modified, whereby tacrolimus was substituted for cyclosporine A, when his serum creatinine had increased to 400 μmol/L.A 29-year-old man received a living-unrelated kidney transplant abroad for end-stage kidney disease secondary to neurogenic bladder. The patient had an uneventful postoperative clinical course with good early graft function. The data of HLA matching and induction protocol are lacking. He was maintained on prednisone, cyclosporine, and myocophenolate mofetil. The patient was discharged on postoperative day 5, with an adequate urine output and serum creatinine 110 μmol/L. He was admitted to the intensive care unit and started on ventilatory support. Appropriate antimicrobial coverage was started for pneumonia and haemodialysis was initiated. During the hospital admission for about a month, the patient was diagnosed to have advanced graft failure based on his clinical progression. The patient refused the renal allograft biopsy. He was maintained on haemodialysis and discharged on prednisone 5 mg daily. Tacrolimus and mycophenolate mofetil were discontinued.Sixty-three months after renal transplantation, the patient was first seen in our hospital, when he presented with hypoxic respiratory failure secondary to pneumonia, pulmonary oedema, and deteriorating kidney allograft function. His blood urea and nitrogen (BUN) was 44 mmol/L and serum creatinine 700 μL, haemoglobin of 5.5 g/dL (dropped from 12.7 g/dL one month earlier), haematocrit of 16.6%, platelets count of 344 × 103/Ul, PT of 9.1, PTT of 33.9, BUN of 13.0 mmol/L, and serum creatinine of 809 μmol/L. The patient was resuscitated with crystalloid fluids and packed red blood cells. An enhanced abdominal computed tomography (CT) scan revealed few scattered hyperdense foci in the renal allograft, in keeping with microhaemorrhage and microperforations , the patient was admitted because of abdominal pain, graft tenderness, and gross haematuria. There was no history of abdominal trauma or recent renal allograft biopsy. Upon admission, the patient was pale, tachycardic, and hypotensive. His heart rate was 111 beat per minute, and the blood pressure 110/56 mmHg. Laboratory tests showed WBC of 2 × 103/orations . The renThe patient was emergently taken to the operating room, where urinary bladder irrigation and washout revealed 3 litres of partially clotted blood. Exploration of the renal graft through the old transplant scar and intracapsular approach showed ruptured graft. An oblique laceration that measured 4 cm in length and involved the upper pole and the mid portion of the graft was evident . The renlymphocytic tubulitis, acute tubular epithelial cell degenerative and regenerative changes in the viable renal tubules, and severe endarteritis with fibrinoid necrosis  rejection, in a background of marked chronic allograft arteriopathy. The interstitium was markedly oedematous and showed areas of extensive haemorrhage . Severe necrosis . The vasnecrosis . Marked necrosis . Peritubnecrosis . Immunos Immunologically, anti-HLA (PRA) antibody profile revealed the following specificities: Class I-A1, 24, 80, 34, 32, 68, 36, 33, 11, and 23 (A1 and A10 CREGs) and B51. Profiling for Class II PRA was negative. The donor HLA typing was not performed, since the transplantation was performed abroad.Spontaneous renal allograft rupture is one of the most serious complications of kidney transplantation , 9–11, wclinically labelled with advanced graft failure.One possible mechanism put forward for RAR is that cortical and capsular ischemia results from interstitial oedema and cellular inflammatory cell infiltration causes capsular tension and rupture. The main clinical manifestations of RAR are sudden onset of abdominal pain, graft bulging and tenderness, and haemorrhagic shock . RecipieOur case has the longest time interval between the time of transplant and the graft rupture in the literature. Nevertheless, the underlying cause of rupture was still severe combined acute cellular and antibody-mediated rejection, resulted from cessation of immunosuppression. Case with the second longest interval (21 months) was also a case of RAR secondary to acute rejection that followed discontinuation of immunosuppression following chronic allograft failure .In our case, the patient refused the renal allograft biopsy twice, leading to the clinical assumption of allograft failure without tissue diagnosis. Although severe chronic interstitial fibrosis and tubular atrophy (IF/TA) and chronic allograft arteriopathy were evident in the graft, severe acute cellular as well as antibody-mediated allograft rejection was severely affecting the graft. Our case indicates that RAR remains a possibility five years after transplantation, particularly when immunosuppressive agents are discontinued. This raises a question of what is the best approach for immunosuppressive drug withdrawal following graft failure; should it be abrupt or gradual? The answer for this is not clear. Abrupt cessation of immunosuppression might lead to graft rejection and subsequent RAR, as in our case. On the other hand, higher morbidity and mortality have been reported in patients continued on immunosuppressive therapy after graft failure and commencement of dialysis . In our Another important point to be emphasized is the role of renal allograft biopsy before the presumption of graft failure and withdrawal of the immunosuppressive agents. Our patient might started rejecting the graft long time before his initial clinical presentation 48 months after transplantation. A graft biopsy at that time would help identifying the immunological insult to the graft, dictating appropriate management, and preventing a subsequent graft rupture. Refusing graft biopsy is not uncommon in some regions and cultures of the world, and the patients should be educated about the reasons and benefits of the graft biopsy, in attempt to deliver best clinical practice.Although the donor HLA typing was not performed in our patient, the presence of PRA Class I specificities for CREGs A1 and A10 and B51 suggests that the antibody-mediated component in the rejection process is mediated by donor-specific antibodies. Most intriguing is what may initiate an antibody-mediated immune rejection in a sensitized patient after such a long time since transplantation. Likely possibility is that the pneumonia (as an infection) may have been the triggering stimulus in our patient.Renal allograft rupture is a rare but a serious complication of kidney transplant, which usually occurs few weeks after transplantation. The most common cause is acute antibody-mediated allograft rejection. Discontinuation of the immunosuppressive therapy in patients with graft failure may be a significant risk factor for late RAR and renal allograft biopsy should be performed before withdrawal of immunosuppressive agents. Following cessation of immunosuppressive therapy, careful clinical follow-up should be practiced to detect the symptoms and signs of RAR, even long time after transplantation.


In regard to the following paper published in the October 2010 issue of the International Journal of Inflammation: Aleksandra Malgorzata Urbanska, Arghya Paul, Jasmine Bhahena, and Satya Prakash. Suppression of Tumorigenesis: Modulation of Inflammatory Cytokines by Oral Administration of Microencapsulated Probiotic Yogurt Formulation, one of the authors' names was misspelled. We would like to correct the author's name as it appears in the paper from “Jasmine Bhahena” to “Jasmine Bhathena.”


Dear Colleagues:The official 2011 impact factor for the Journal of Epidemiology is 1.858. Our journal placed 66th among the 157 journals in the category of Public, Environmental & Occupational Health and top in the Asia-Pacific region. The proportion of self-citation was only 6%, which indicates that ours is a sound and promising journal in epidemiology. The 10 most frequently cited papers in the Journal of Epidemiology in 2010 and 2011 are shown below.The average time between submission and first response is 27 days. For this achievement, I sincerely thank the editorial committee members and reviewers for their outstanding efforts.Epidemiology—Scientific Wisdom and Perspectives from East and West, which are written by leading epidemiologists from Asian and Western countries. Such review series will continue this year.In addition to our regular review articles, we published 7 joint or single review articles in 2012 for various research fields, Our editorial team looks forward to receiving new high-quality submissions from around the world in a broad range of topics in epidemiology. In this way, our journal can continue to advance research in basic and clinical science, public health science, and health policy.Cordially,Hiroyasu Iso, MD, PhD, MPHEditor-in-ChiefJournal of EpidemiologyProfessor of Public HealthOsaka University Graduate School of Medicine


Journal of Community Hospital Internal Medicine Perspectives (JCHIMP) was conceived in 2010. This was done in the context of the rapid expansion of Internet-based journals. The number of Internet journals had increased worldwide, including in the United States. There were a significant number of Internet medical journals published in the United States, but what was lacking was a journal aimed at scholarly activity and community hospital departments of medicine. Community hospitals constitute more than half of the training programs in the United States  and donations from interested parties. The MedStar Union Memorial Department of Medicine Research Fund provided the balance of funding support.Journal of Medical Education Online. Dr. Solomon served not only as a trusted advisor but also as an inspiration in JCHIMP's conception and development. He has served as a guide in the continued evolution of the journal. Upon his recommendation, we have a contract with a publisher, Co-Action Publishing, based in Stockholm, Sweden. This has been an excellent relationship.An additional objective of our journal was to include trainees and faculty at community hospitals in the entire process, including peer review. This was a challenge considering a lack of training for students and residents in the journal publication process. The journal relied upon David Solomon as our advisor . Dr. SolOur peer reviewers are largely from the membership of the Association of Program Directors in Internal Medicine (APDIM). Many reviewers are also members of the Community Hospital Education and Research Network (CHERN), an informal association within APDIM. Many of the reviewers were novices at the review process until contacted by JCHIMP. We also encourage resident and medical student peer review involvement when appropriate.Our general content has evolved over the last 2 years. It includes: perspectives, original research, medical education, case reports, history of medicine, research of EKG cases, and radiologic imaging. We were particularly interested in community hospital editorial perspectives. We hoped the journal would serve as a platform to voice community hospital opinions. Case reports are also a major component of the journal (see below).One year ago, we summarized the accomplishments of the first year of JCHIMP. During 2011, 50 manuscripts were published on the topics related above. At the end of the first year, our readership included 2,500 ‘unique’ individuals from 88 countries . A totalIn this second year of the journal, we can list many accomplishments. The total number of manuscripts published in year 2 is 41. The review total in the first 2 years is 136. Active reviewers for the 2012 academic year are listed in Publishing case reports is one of the missions of the journal. The publishing of a case report and the research required to do it well, including the historical context, is crucial to the development of scholarly attributes of internal medicine trainees. The exercise is a goal unto itself. In this issue, there is an article on Pott's disease secondary to an unusual mycobacterium. The discussion mentions Dr. Percivall Pott and his cohort, Dr. James Paget. Also, looking at imaging of remains from a previous millennium shows that tuberculosis was endemic in the Americas long before it was in Europe .It is very encouraging to follow the web data of our journal. The journal has allowed us to introduce faculty, residents, and students to the concept of peer review. We have encouraged peer reviewing by those who are novices in this area. We seek to provide guidance when they need help .Going forward, we would like to stretch our publication schedule pending resources, financial and personal. A semi-monthly publication would be ideal. We believe that the importance of scholarly work will only increase as future residency program accreditations standards demand more scholarly work. We also wanted to maintain a trace of wit as the accompanying photos indicate. We intend to apply for indexing when it is advisable based on publication numbers.www.CHERNINFO.com) Survey Group on methods of teaching residents the nuances of patient health literacy (The editor will also continue to highlight the various papers that follow in each issue. Also in this issue are seven manuscripts. There are three research papers: a timely study on the variable effects of medical readmissions by a resident house staff team ; a literliteracy . A paperliteracy . In addiliteracy ; asymptoliteracy ; and an literacy .Robert P. Ferguson, MDChief of MedicineChristopher D. Kearney, MDChief of Geriatrics Medstar Union Memorial Hospital


Other-regarding preferences are a critical feature of human cooperation but to what extent non-human animals exhibit these preferences is a matter of intense discussion. We tested whether jackdaws show prosocial behaviour (providing benefits to others at no cost to themselves) and altruism (providing benefits to others while incurring costs) with both sibling and non-sibling recipients. In the prosocial condition, a box was baited on both the actor's and the recipient's side (1/1 option), whereas another box provided food only for the actor (1/0 option). In the altruistic condition, the boxes contained food for either the actor (1/0 option) or the recipient (0/1 option). The proportion of selfish (1/0 option) and cooperative  actors' choices was significantly affected by the recipients' behaviour. If recipients approached the boxes first and positioned themselves next to the box baited on their side, trying to access the food reward , actors were significantly more cooperative than when the actors approached the boxes first and made their choice prior to the recipients' arrival . Further, in recipient-first trials actors were more cooperative towards recipients of the opposite sex, an effect that was even more pronounced in the altruistic condition. Hence, at no cost to the actors, all recipients could significantly influence the actors' behaviour, whereas at high costs this could be achieved even more so by recipients of different sex. Local/stimulus enhancement is discussed as the most likely cognitive mechanism to account for these effects. Pan troglodytesHuman cooperation is characterized by a high degree of prosocial and altruistic behaviour Callithrix jacchus, fitted the hypothesis and altruistically provided food to kin and non-kin partners Saguinus oedipus, did not Cebus apella, showed prosocial food choices towards familiar kin and non-kin but not towards unfamiliar individuals Macaca fascicularis, directed prosocial behaviour mainly towards kin The second hypothesis assumes that cooperative breeders are motivated and psychologically predisposed to act prosocially What has been fairly neglected in these studies on other-regard is the role of the recipients. Melis and colleagues (2011) recently proposed a third hypothesis : recipients should signal their goals or requests for help to their conspecific partner because the acting subjects might have limitations in their ability to infer the recipients' goals and needs in the absence of overt actions or requests. In chimpanzees they found a significant positive effect of the recipient's behaviour on eliciting altruistic behaviour in a conspecific actor in a food and a non-food context Corvus corax, recruit conspecifics to foraging sites Corvus corone, are cooperative breeders Corvus monedula, food sharing includes a high percentage of actor-initiated behaviours Corvids are large-brained birds We subjected seven birds  to a prosocial and an altruistic test condition in which the respective actor individual could choose to open one of two boxes that were baited with preferred and identical food items (dry cat food). Boxes were divided into two compartments, one of which could be accessed by the actor, the other by the recipient . In the Boxes were placed onto a wooden platform that extended into both experimental compartments, each of which accommodated one bird. Boxes were therefore accessible by both birds and allowed either the actor or the recipient to approach them first. In actor-first trials the actor approached the boxes first and made its choice prior to the recipient landing on the platform. In recipient-first trials the recipient approached the boxes first and, although the boxes could only be opened from the actor's side, positioned itself next to the box baited on its side and/or tried to get access to the food reward by pecking/stepping onto the box. Therefore, when the actor later arrived and made its choice, the recipient was already present at its baited box.Before proceeding to the test conditions actor individuals received two training phases  and cooperative  choices was most strongly influenced by first approach . On the contrary, in recipient-first trials actors showed a nearly significant tendency to choose the cooperative option more often than the selfish one . In the control condition that resembled the prosocial condition without a recipient present, actors did not show a significant preference for either the 1/0 or the 1/1 option .Analyzing all actor-first trials together showed that actors chose the selfish option significantly more often than the cooperative one  significantly affected actors' choices  or in the altruistic condition  also yielded a significant effect on actors' choices .That jackdaws did not exhibit any other-regarding preferences per se is in line with results from some studies on chimpanzees Focusing on the recipient-first trials, our results are in accordance with a similarly significant effect of recipients' behaviour on actors' choices in chimpanzees object, whereas with local enhancement, the attention is drawn to a particular location In our study, jackdaw recipients showed their interest in the food by positioning themselves at the baited box and/or trying to get access to the reward by pecking or stepping onto the box. However, they did not emit any begging calls, nor did they show any begging-related behavioural displays Interestingly, females actors were more affected by the recipients' cues in the prosocial condition than in the altruistic condition  but onlyIn sum, this first study on other-regard in birds corroborates results from several experiments on non-human primates insofar that jackdaws did not show other-regarding preferences per se. However, they did respond to enhancement cues provided by recipients and increased their prosocial choices with either type of recipient and altruistic choices even more with recipients of different sex. The set-up used in this study aimed to control for reciprocity. Further studies are needed to investigate whether the same mechanisms would work in a more naturalistic setting, which allows individuals to interact freely and repeatedly.This study complied with Austrian and local government guidelines and permission was received from the Konrad Lorenz Forschungstelle and the ethics committee of BH Gmunden (Permit Number Sich71-1309) specifically approved testing the jackdaws for this study.Corvus monedula, 5 females, 7 males) that were housed together with six further conspecifics in one social group in an outdoor aviary at the Konrad Lorenz Research Station (KLF), Gruenau, Austria. All individuals were handraised in spring 2007 at the KLF under standardized conditions with sibling groups being raised together but in separate nests, i.e. cardboard boxes, to allow for familiarization with and later recognition of kin. The aviary consisted of one outdoor compartment  which was equipped with wooden perches, breeding boxes, rocks and natural vegetation and an outdoor experimental area  which was roofed and equipped with wooden perches and could be divided into smaller compartments via wooden doors. When not being tested in behavioural experiments, birds could move around freely in all areas. They had ad libitum access to water and were fed three times a day. Their diet consisted of dry insects, minced beef, various kinds of fruits, vegetables, grain, eggs, bread, and milk products.Subjects were 12 jackdaws  was composed into a dyad with a conspecific (recipient) which was chosen out of a set of 5 birds with regards to meeting certain requirements due to the experimental set up. Actors and recipients were of same age and recipients were either the actor's sibling (kin) or non-sibling (non-kin) and of the same or different sex. Each subject was tested in two dyads (with a sibling and with a non-sibling). The specific actor-recipient dyads were maintained over the entire experiment and actor-recipient roles were not reversed to exclude the possibility of short-term reciprocation within the context of the experiment. Each set of sibling and non-sibling dyads consisted of 3 male-male, 2 female-female, and 2 male-female dyads.Individuals from a dyad were placed in two adjacent compartments  of the experimental area that were divided by wire mesh. Hence, actors and recipients were physically separated from each other but in visual contact with each other. Test dyads were physically and visually separated from the rest of the group. The testing apparatus consisted of two boxes  made of acrylic glass with a likewise transparent lid that could be slid open to only one side hereby giving access to the contents of the box. Sliding the lid open was achieved by pulling the end of the lid which was only possible from the actor's compartment. Birds in the recipient compartment never had the possibility to open the box. A transparent partition separated each box into two equally sized fractions which were either baited with a food item or empty, depending on the test condition. Both boxes were placed onto a wooden platform  that extended into both experimental compartments and on which birds readily landed. The boxes were positioned at a distance of 40 cm to each other and centered in a size-matched gap in the separating wire mesh. Food rewards were single pieces of dry cat food which is a highly preferred food for jackdaws and was not part of the birds' maintenance diet.Prior to testing, the animals were subjected to a training phase to ensure that they were capable of manipulating the apparatus and that they understood the consequences of their choice. Birds were singly trained in the actor's compartment and training consisted of two steps.In step 1 one box was baited with cat food in each fraction (1/1 option) whereas the other box was left empty  and the other box was baited on the recipient's side  whereas the other box was baited on both the actor's and the recipient's side  and the other box was baited on the recipient's side  and three subjects were tested first with a non-sibling (one subject started with condition 1 and two subjects started with condition 2). A trial was finished when the actor had opened one box and the bird on the respective side had gained and eaten its reward, which was followed by immediate re-baiting by the experimenter and the start of the next trial. If a subject refused to participate in a trial, i.e. did not approach the platform for 3 minutes after baiting, the session was terminated and continued on another day until each subject had finished 20 trials, i.e. had opened one of the boxes 20 times. Left-right positions of the differently baited boxes were counterbalanced throughout the test phase in such a way that the same baiting pattern never occurred more than two times in a row.In the control condition subjects were singly tested in the actor's compartment in one session of 20 trials. It resembled the prosocial condition insofar that one box was baited only on the actor's side (1/0 option) whereas the other box was baited on both sides  and all trials were videotaped .We recorded the number of actor's choices for each of the boxes, i.e. how often the actor pulled which box open. Furthermore, we recorded how often the actor was on the platform first and opened one of the boxes prior to the recipient landing on the platform in its compartment (actor-first). We also recorded how often the recipient landed on the platform first which was always followed by immediately positioning itself next to the box baited on its side and/or trying to get access to the food reward by pecking or stepping onto the box and occurred prior to the actor opening one of the boxes (recipient-first).P<0.05 or P<0.1 (to account for tendencies) and, if applicable, corresponding lower order interactions and main terms were kept in the model. Excluded terms were reentered one by one into the final model to confirm that these terms did not explain a significant part of the variation.We analyzed the data by conducting a generalized linear mixed model (GLMM), applying the restricted maximum likelihood procedure (REML). We constructed the GLMM with actor choice  as the binomial response variable. Selfish choices were coded as 0, cooperative choices were coded as 1. Into the model we entered subject identity as random term to account for repeated measures. We included “first approach”, “condition”, “kinship”, “sex relation of dyad”, and “sex of actor” as fixed terms and combinations thereof as first order interactions. We further included “first approach*condition*kinship”, “first approach*condition*sex relation of dyads” and “first approach*condition*sex of actor” as second order interactions to explicitly test for our hypotheses. We sequentially deleted fixed terms in order of decreasing significance, starting with second order interaction terms in the model. Only terms with To compare selfish versus cooperative choices in actor- and recipient-first trials and in the control condition we used Wilcoxon signed-ranks tests with α<0.05. All analyses were performed using SPSS 19 (IBM SPSS Statistics 19.0.0).


Six class I- and seven class II- restricted epitope sequences within the core, NS3, NS4B and NS5B regions were compared across the six HCV genotypes using local genotype 5a sequence data together with global data. Common HLA alleles in the South African population are A30:01, A02:01, B58:02, B07:02; DRB1*13:01 and DRB1*03:01. Epitope binding to 13 class I- and 8 class –II alleles were described using web-based prediction servers, Immune Epitope Database, (IEDB) and Propred. Online population coverage tools were used to assess vaccine efficacy.Host genetics influence the outcome of HCV disease. HCV is also highly mutable and escapes host immunity. HCV genotypes are geographically distributed and HCV subtypes have been shown to have distinct repertoires of HLA-restricted viral epitopes which explains the lack of cross protection across genotypes observed in some studies. Despite this, immune databases and putative epitope vaccines concentrate almost exclusively on HCV genotype 1 class I-epitopes restricted by the HLA-A*02 allele. While both genotype and allele predominate in developed countries, we hypothesise that HCV variation and population genetics will affect the efficacy of proposed epitope vaccines in South Africa. This Despite the homogeneity of genotype 1 and genotype 5 over the epitopes, there was limited promiscuity to local HLA-alleles.Host differences will make a putative vaccine less effective in South Africa. Of the 6 well-characterized class I- epitopes, only 2 class I- epitopes were promiscuous and 3 of the 7 class-II epitopes were better conserved and promiscuous. By fine tuning the putative vaccine using an optimal cocktail of genotype 1 and 5a epitopes and local HLA data, the coverage was raised from 65.85% to 91.87% in South African Blacks.n vivo and in vitro studies are needed to confirm immunogenic epitopes, in silico HCV epitope vaccine design which takes into account HCV variation and host allele frequency will maximize population coverage in different ethnic groups.While i As a relatively “new” virus, only identified in 1989 , there i1) The virus is highly mutable and exists as a quasispecies within the host and genotypes cluster geographically.2) Host cell responses to HCV infection are poorly defined and inconsistent among infected individuals.CD4+ and CD8+ T-cell responses are also not cross-protective to heterologous genotypes  and, to 3) Humans are the only natural host of HCV, and suitable laboratory models have only been developed recently. The chimpanzee has been infected in the laboratory , but stuEpitope-based vaccines promote an immune response by presenting immunogenic peptides  bound to major histocompatibility (MHC) molecules (host specific) to the T cell receptor. Class II- proteins are presented to T helper cells by antigen presenting cells (APCs) with the aid of the CD4 co-receptor whereas class I- proteins are presented by the infected target cell to cytotoxic T cells with the aid of the CD8 co-receptor. The T helper response is important in directing and activating the immune response, including the effectiveness of CD8+ T cells -x (5–8)-[LFYRHK (MI)]. However, one would expect lower predictions for NS5B2422-2433 epitopes MSYSWTGAL and MSYTWTGAL (38%ProPredI 12%SYFPEITHI) since only the carboxyl anchor is present but this was not the case.The second exception observed was the failure of IEDB ANN to predict binding between any of the epitopes (or their variants) and HLA-B*27:05 which SYFPEITHI and/or ProPred I scored. There was no data supporting restriction of these particular peptides by B*27:05 in the IEDB epitopes database. Both SYFPEITHI and ProPred I use peptide motifs and amino acid matrix based prediction. The following scores using x-[R (K)]-x 1073-1081, NS4B1851-1859 and NS5B2727-2735 bound with high affinity to A*02:01 allele, regardless of genotypic variation  and TVGGVMWTV (genotype 3a) had changes from the wild type N (Asparagine) in position 3 but none of the variants had changes in positions 4, 5 and 7. Interestingly, when all possible alanine exchange peptides were placed into IEDB ANN, the output scores reflected the experimental binding changes for all of the alanine exchange peptides with the exception of the total abrogation of signal for substitutions in positions 3, 4 and 5 (data not shown).Of note, while consistent binding was observed across the supertype A02 for all of the variants of the A*02 restricted epitope NS31073-1081, epitopes of genotypes 1, 3a and 5a (variant) were found to be intermediate binders , while the genotype 1b variant had low affinity with this allele (2071 IC50nM) and the original genotype 1a peptide was not predicted to bind at all. The original peptide and one of the two of three variants of the published B*15-restricted NS5B2422-2433 epitope displayed intermediate binding IC50 nM values of 80 and 144 (Table The genotype 4a and 5a variants of the HLA-A*02 restricted epitope NS5B50nm) and intermediate (<500 IC50nm) affinities to two or more supertypes: LTGPTPLLY , FLSTATQTF , ITYSTYGKF , KVLVLNPSV , RAKAPPPSW . Of the five epitopes above, three were conserved among genotypes 1, 2, 4 and 5  and KLRDCTLLV .In a preliminary attempt to identify conserved epitopes showing greater promiscuity across supertypes, strings of epitopes (other than the ones selected from publications for this study) of the NS3 protein were placed into the IEDB server. Table 17-42, RRPQDVKFPGGGQIVGGVYLLPRRGP, returned two 9-mer peptides, VYLLPRRGP and VGGVYLLPR, which scored similarly for alleles HLA-DRB1*03:01 and HLA-DRB1*15:01  with the chosen allele Table . For exa1248-1261 and results showed coverage of 94.28% . The only other common amino acid in this anchor position was Valine (V). Valine was conserved at position 9 in all but the genotype 5a sequences where approximately one third of the sequences had a leucine (L) in this position. Despite the fact that substitutions at P2 were conservative (an I or V for the more favourable L), affinity of this epitope was lowered. When alanine exchange peptides were used in in vitro assays , NS4B [GenBank: JX571032-JX571039] and NS5B [GenBank: DQ482799-DQ482824] regions of genotype 5a.Care was taken to ensure that all our own data, as well as data used from public databases, corresponded to one sequence per subject. The study was retrospective and approved by the ethics committee of the University of the Witwatersrand, Johannesburg, South Africa (WITS HREC M051114), and was therefore performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. PCR and sequencing was performed as previously described ,31.http://weblogo.berkeley.edu/logo.cg; 2008-09-08). Sequence numbering is according to [BioEdit , was usrding to . WebLogording to . The WebThe relative conservation of each epitope was calculated as a percentage of the number of polymorphic sites over the epitope length when compared to the overall HCV consensus sequence. The HCV consensus was determined by taking the most common amino acid at each amino acid site of the 7 respective genotype consensus sequences , irrespective of representation in the database. A minimal class I-restricted epitope length of 9 nucleotides was used for all class I-restricted epitopes. Since class II-restricted epitopes are longer and are made up of numerous overlapping regions, the number of amino acids per epitope varied. The statistical analysis was performed using the analysis of variance (ANOVA) tests of significance in the Statistica software, version 9.1.http://www.allelefrequencies.net 2010-11-30). However, much of this data was low resolution with 2 digits. Hence, high resolution data [Initially, a literature search was conducted in order to collate available South Africa population HLA-A –B and –DR allele frequency data which included relevant data stored online in the New allele Frequency Database  and Proex.html, ) were chhttp://tools.immuneepitope.org/main/jsp/menu.jsp; version 2.0, accessed 2009-09-09 to 2011-03-14, [The IEDB is a manually curated database of experimentally characterized immune epitopes. Its companion site, the IEDB resource, is a collection of tools for prediction and analysis of immune epitopes . The “P1-03-14,  on the I1-03-14, .http://www.imtech.res.in/raghava/propredI/index.html, 2010-11-30, [50nM . IC50 values <50 nM indicate high affinity while values >500 but <5000 nM indicate low affinity and values in between the two extremes (>50 nM but <500 nM) indicate intermediate affinity (http://tools.immuneepitope.org/main/jsp/menu.jsp).In addition, however, the matrix-based methods, ProPred 1  and SYF0-11-30,  were use50nM to more than one HLA class I- allele was considered promiscuous.Sequence data in the NS3 region that was available on the database was used for the genotype 5 conservation score and binding to predominant HLA-alleles in the South African context were predicted.The promiscuity of “newly predicted” (i.e. other than published epitopes) class I-epitopes of the NS3 gene were analysed using the IEDB server. An epitope sequence that bound with <500 IChttp://www.imtech.res.in/raghava/propred/index.html, 2010-10-20, [A structure-based method with a quantitative matrix (QM) algorithm on the Propred II server  was useThe program is useful in locating promiscuous, versus allele specific, binding regions in a query peptide sequence. Note that, by comparison to IEDB ANN, a high score is indicative of good binding between the relevant peptide and the specific HLA allele and vice versa. The score represents the percentage binding of the query peptide when compared to the highest possible binding score for the optimal peptide with the given allele and thus reflects the binding characteristics of the query peptide. However, there is no clear cut off as with IEDB ANN scoring, and actual percentages should not be compared between alleles. The stringency threshold of the analysis can be set between 1% and 10% where the highest stringency guarantees no false positives and the lowest stringency guarantees no false negatives. The highest stringency was, therefore, used in all programme runs to minimize the number of false positives and ensure that all binding had significance.http://tools.immuneepitope.org/tools/population/iedb) for South African Whites and Blacks for both the published class I- and II- epitopes and an adapted “best mix” which took into account the most prevalent alleles and epitope variants in South Africa and their predicted binding. In order to assess the efficacy of a vaccine epitope, the IEDB resource Tool calculates the fraction of individuals predicted to respond to a given set of epitopes with known MHC restrictions . The calculation is based on input HLA genotypic frequencies.Population coverage was calculated by the Population coverage tool on the IEDB server , also shown in Table Click here for fileFigure S2. Epitope and population coverage in South African Blacks with original published epitopes, using IEDB.Click here for fileFigure S3. Epitope and population coverage in South African Whites with original published epitopes, using IEDB.Click here for fileFigure S4. Epitope and population coverage in South African Blacks with “best mix”, using IEDB.Click here for fileFigure S5. Epitope and population coverage in South African Whites with “best mix”, using IEDB.Click here for fileFigure S6. Epitope and population coverage in Caucasians (North American and Europe), using OptiTope.Click here for fileFigure S7. Epitope and population coverage in Zulus (South Africa), using OptiTope.Click here for fileFigure S8. A summary of the steps and results of the population coverage analyses, using the IEDB and OptiTope.Click here for file


In 2011, researchers around the world interested in extracellular vesicles (EV) joined forces and founded the International Society for Extracellular Vesicles (ISEV). Membership has grown to approximately 750 in eight months, and the Society's first meeting will take place in Gothenburg, Sweden, on 18–21 April 2012. Already approximately 500 participants have been attracted to this event. These are signs of rapid expansion in global research in the field of EV.Journal of Extracellular Vesicles (JEV). Scientific publications in the field of extracellular research have been dispersed across many journals, including in some publications lacking the necessary expertise to judge methodologies and nomenclature in the field. Furthermore, following a survey of the new membership and outreach to the community, the need for a journal in this field became evident. In this climate it was an easy decision for the interim board of ISEV to launch the journal, and also that it should be published according to the open access principles.It is with great pleasure that the interim board of ISEV launches the Society's official journal: Journal of Extracellular Vesicles aims to publish high-impact research relevant to scientists involved and interested in EV in the form of original research articles as well as review articles. Manuscripts selected for publication should contain data of high quality that advance the field and contribute to an in-depth understanding of the processes under study. Areas of interest include, but are not limited to:technological advances in isolation, quantification and characterization of EV, including exosomes, microvesicles, ectosomes, apoptotic bodies and any other type of EV;biological functions of EV in health and disease;intracellular molecular mechanisms of EV production and release, including membrane trafficking and protein sorting;EV as biomarkers;signal transduction, uptake and propagation of EV;stem cell biology and EV;informational databases as EV resources, including large sets of data on genomics, lipidomics, proteomics, high-throughput screening data and other techniques.Journal of Extracellular Vesicles’ is well reflected in the first scientific publications in the journal, which include work on both what are evidently exosomes as well as larger microvesicles. Interestingly, Bobrie and colleagues show that vesicles isolated via “a classical exosome isolation procedure” actually contain at least two separate types of extracellular nano-vesicles that appear to be of different intracellular origin (The scope of r origin . This isr origin , showingr origin  show thar origin  suggest r origin . The aimr origin , discussWe expect that JEV will become the key forum for exchanging research in the field. JEV is launching strongly, with six publications and a supplement consisting of 265 abstracts for the International Society for Extracellular Vesicles 2012 Meeting . In ligh


Ulva prolifera (Müller) J. Agardh has been identified as the causative macroalgal species. U. intestinalis, however, has been observed in the bloom areas, co-occurring with U. prolifera, but it has not been found to be causative. The Yellow Sea green tide has shown consistent phases of development that match corresponding environmental changes. U. prolifera, not U. intestinalis, is dominant. Our experimental design was based on these observed phenomena, and the results of our field investigation indicated a close relationship between changes in principal environmental factors  and the development of each phase of the bloom. These main environmental factors were simulated to allow estimation and comparison of the physiological responses of U. prolifera and U. intestinalis. Ecophysiological differences were found between these two species. (1) More photosynthetic activity and plasticity were detected in U. prolifera. (2) U. prolifera was found to be more sensitive to dynamic environments, especially harsh and changing environmental conditions. U. intestinalis was found to be more stable, probably due to the higher stress tolerance given by its antioxidant system. (3) Markedly higher nutrient absorption activity was observed in U. prolifera. Comparisons of the ecophysiological traits of these two species in this present study may foster understanding of their natural ecological processes. Specifically, U. prolifera seemed to be more engaged with the ephemeral blooms, while U. intestinalis seemed to be directed toward persistence. This also suggests that the ecological success of U. prolifera may be inextricably linked to its higher capacity for photosynthesis, nutrient absorption, and nutrient assimilation.Yellow Sea green tides have occurred in coastal China almost every year from 2007 to 2011.  Ulva, has been reported as cause of green tides in many parts of the world, including Europe, the Americas, Australia, and Asia Over the past decade, the excessive growth of some species of green algae, mainly within the genus Ulva species, the most relevant environmental factors were found to include irradiance, temperature and salinity Ulva species were sensitive to environmental stress conditions, so previous studies used them as indicators of physiological response states. However, they have only rarely been used to explain dominance among groups of species, especially co-occurring species. The environmental factors connected to the diminishment of green tides after the bloom period are not well understood. We therefore speculated that the dominant species gains a competitive advantage by exhibiting either pronounced adaptability to wide ranges of irradiance, temperature, and salinity or physiological adaptations to variable environmental conditions.Green tides are related to a series of complex coupled processes and various biotic and abiotic factors are thought to affect the spatial patterns of green tides Large-scale green tides in China's Yellow Sea, called the “Yellow Sea green tide,” have broken out continuously from 2007 to 2011. The first green tide occurred off the coast of Qingdao in late July, 2007 Ulva prolifera (Müller) J. Agardh U. linza-procera-prolifera (LPP) clade U. intestinalis, which has been noted worldwide as a green tide-forming species, especially in eutrophic estuaries in Europe and North America U. prolifera. The U. intestinalis population remained attached throughout a year , while the annual U. prolifera cycle involved a relative ephemeral bloom lasting from May to July followed by a drop in population in August. However, both species were always found to co-occur U. prolifera. Little attention has been paid to ecological and physiological differences between these co-occurring species.The dominant species of the Yellow Sea green tide was found to be U. prolifera in the green tides occurring off the coast of Qingdao.In the light of their co-occurrence and different natural ecological processes, we chose these two species of green macroalgae for the present study. We decided to assess (1) the relationship between the environmental factors and the bloom-forming period and (2) possible ecological and physiological mechanisms underlying the dominance of Ulva, free-floating U. prolifera is the dominant species. It often co-occurs with attached U. intestinalis during the green tide period. Our study field, located around Taiping Cape , is frequently studied by the Ocean University of China (OUC) and we had permission from OUC to conduct our experiments. The area is a seaside resort open to the public, and no endangered species live there.The field observations were carried out along the rocky intertidal shores around Taiping Cape , Qingdao, P.R. China. Green tides have broken out in this area during each of the past five years. Many natural macroalgal assemblages were found in these intertidal zones, both attached and free-floating. Within the genus U. prolifera and U. intestinalis were collected from coastal Qingdao in June 2010 during the bloom period. The thalli were rinsed gently in sterile seawater and cleaned thoroughly with a brush under a magnifier to remove the attached sediment, small grazers, and epiphytes. They were then cultured in sterile seawater enriched with f/2 medium at a constant temperature of 20°C and light intensity of 72 μmol photons.m−2s−1 in a 12:12 h light:dark cycle in a GXZ-280 C intelligent illumination incubator  for acclimation before of experiments 2) at a concentration of 0.5 mg l−1 was added to the cultures to suppress diatom growth The thalli of bloom.” The period during which the bloom diminished, from August to September, was designated “post-bloom.” All other months were designated “pre-bloom.” The environmental factors were analyzed simultaneously within the green tide. The aim of this part of the study was to determine whether these environmental factors varied along with the phases of the green tide.We divided the green tide bloom into three periods according to the field observations: The blooming period from May to July was designated “−2) from March 2010 to March 2011 (about three times per month). We transformed monthly means of total luminous flux to PAR  according to the method described by Thimijan The environmental factors measured in the present study mainly included photosynthetically active radiation (abbreviated “PAR” in the present study), surface seawater temperature (abbreviated “SST”), surface seawater salinity (abbreviated “SSS”), and dissolved inorganic nitrogen (abbreviated “DIN”). They were investigated from November 2009 to March 2011 at ebb tides. SST and SSS were measured with a T-S meter . Seawater samples for nutrient analysis were filtered through GF/C filters  and stored -80°C. DIN  concentrations were measured using standard procedures. Light intensity was recorded as total luminous flux (lm.mhttp://www.worldweather.org/).The number of typhoons  capable of affecting China's offshore regions and total rainfall was recorded from January 2010 to December 2010. Data were obtained from the World Weather Information Service Organization database ;bloom, at 20°C, 60 μmol photons.m−2.s−1, 30 PSU;post-bloom, at 26°C, 90μmol photons.m−2.s−1, 25 PSU.After acclimation, the thalli were randomly selected and cultured in separate 500 ml glass beakers with five duplicates. They were then transferred into beakers under the conditions given above. The culture experiments were repeated five times. Each lasted 7 days, covering two phases: short-term (24 hours) and long-term (7 days). Salinity was adjusted using NaCl and deionized water. The culture medium was renewed every 2 days to prevent increases in salinity and depletion of nutrients.The treated thalli were collected at 24 h and 7 d. Various factors related to photosynthesis, including photosynthetic pigments, optimal photochemical efficiency , and rapid light respond curves (RLCs) were analyzed.Samples  were ground in liquid nitrogen and extracted using 90% (V/V) acetone buffer (5 ml) and some grit to extract liposoluble pigments. The acquired mixture was then treated with 10,000 rpm at room temperature for 10 min and the supernatant was used for further analyses. The chlorophyll contents were determined spectrophotometrically with a Hitachi F-4500 Fluorescence Spectrophotometer  within a scanning absorption spectrum (350–700 nm), and the contents were determined using to the following formula:All assays were performed in 5 replicates and the results are expressed in milligrams per gram of fresh weight (mg/g FW).−2.s−1). Each step lasted 10 s. Then, the relative electron transport rate (rETR) and effective photosynthetic yield (Y(II)) of photosystem II (PSII) values under each light intensity were measured. The RLCs were fitted using the empirical equation determined by Platt et al. in order to determine parameters α (photosynthetic rate in light-limited region of RLC), rETRmax (maximum relative electron transport rate), and Ek (minimum saturating irradiance) Photosynthetic efficiency was determined using a portable pulse amplitude modulated (PAM) fluorometer . Samples were dark-adapted under the respective culture treatments for about 20 min before measurement. The Fv/Fm was determined in 5 replicates. No step was repeated on the same tissue sample during the same time period. Then RLCs were measured. The samples were exposed to a light intensity gradient , antioxidant enzymatic activity, and non-enzyme antioxidant activity were analyzed. The activity of the enzyme nitrate reductase (NR), which has a close relationship with nutrient absorption, was estimated simultaneously. Samples (0.2–0.3 g) were ground in liquid nitrogen and extracted using 1.5 ml of 50 mmol/l potassium phosphate buffer (pH7.0) containing 0.1 mmol/l EDTA (Na2 ethylenediaminetetraacetic acid). Extracts were centrifuged at 4000 rpm for 15 min and a crude enzymatic extraction was used for lipid peroxidation, antioxidant enzymatic activity, and non-enzymatic antioxidant assay.On the 7As an indicator of oxidative stress, lipid peroxidation was studied as malondialdehyde (MDA) formation. The MDA content was measured using thiobarbituric acid (TBA). Then 0.2 ml of extract was homogenized in 1 ml of 10% trichloroacetic acid (TCA). Homogenates were centrifuged at 4000 rpm and 4°C for 10 min. To each 2 ml aliquot of the supernatant, 2 ml of 0.6% TBA was added. The mixtures were heated at 95°C for 40 min and then quickly cooled in an ice bath. After centrifugation at 4000 rpm and 4°C for 10 min, the absorbance of the supernatant was recorded at 532 nm. Results are expressed as n mol MDA per mg of total soluble protein (n mol MDA/mg pro).Total levels of soluble protein per g of fresh-weight thallus  and total levels of soluble protein of the crude extract for antioxidant enzyme activities  were determined using a commercial protein assay . Protein content was determined spectrophotometrically at 595 nm and concentrations were calculated relative to bovine serum albumin as a standard . Total antioxidant ability (T-AOC) was determined using a commercial assay kit .SOD was measured using the xanthine oxidase-cytochrome c reduction method Total glutathione  content was measured using 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB). Half-milliliter quantities of extract were extracted with 2 ml of 5% (v/v) 5-sulfosalicylic acid containing 1% (w/v) polyvinylpyrrolidone 40. Homogenates were centrifuged at 4000 rpm at 4°C for 10min. One milliliter of supernatant was mixed with 100 μl of 6 mM DTNB, 175 μl distilled water, and 25 μl of 266 U/ml glutathione reductase. The rates of increase in absorbance at 420 nm were quantified using a standard curve. Enzyme activities were expressed as mg GSH per mg of total soluble protein (mg GSH/mg pro.)2 concentration over time. Enzyme activities are expressed as units per mg of mg fresh weight thallus (U/mg).The activity of nitrate reductase (NR) was estimated using the assay method described by Young et al. The environmental parameters were tested using a one-way ANOVA with the date as a fixed factor (factor “month”); laboratorialdata were tested using a two-way ANOVA with culture treatment (factor “developing phase”), and species (factor “species”) were treated as fixed factors. All values cited in this paper were obtained from fully independent samples. Data were initially examined with Levene's test for homogeneity and the Shapiro-Wilk test for normality. The Student-Newman-Keuls post-hoc multiple comparison test and Duncan post-hoc test were used if ANOVA indicated a significant effect. Differences between treatment means were considered significant at α<0.05. Data in the present study were analyzed using IBM SPSS Statistics 19 −2.s−1), and a minimum at the end of December (monthly mean of 241 μmol photons.m−2.s−1). SSS ranged from 28.9–31.4PSU and averaged at 30.7PSU, but its tendencies were quite different from those of temperature and irradiance; the lowest values were observed in September and the highest in March.Year-round variations in SST, SSS, and PAR from November 2009 to March 2011 are shown in 16, 85 = 128.3, P<0.0005), SSS , and PAR . Changes in these three factors appeared very regular during different months , but values were found to decrease over time. After post-hoc multiple comparison tests, DIN concentrations from April–May were found to be significantly higher than that from June–July (Dissolved inorganic nitrogen (DIN) concentrations in the water column ranged from 21.9 to 8.2 μmol/L from April to July, 2010. There were no highly significant differences in DIN concentrations between months , but the difference among the treated groups was not obvious, especially in the long-term (7days) experiments  , especially under the bloom culture treatment (P<0.05). The concentration of chlorophyll b was more affected by experimental treatments than that of chlorophyll a in both the short-term and long-term scenarios  . The chlcenarios . HoweverP<0.0005). The Fv/Fm of U. prolifera was significantly higher than that of U. intestinalis, especially during the bloom culture treatment (P<0.05). The maximum initial values of Fv/Fm occurred under bloom conditions in both the short- and long-term experiments. For U. intestinalis, this decrease was observed when it was exposed to pre-bloom culture treatment in the short-term experiments, but results of other treatments showed little change (P>0.05). In addition, both species showed higher Fv/Fm values in long-term experiments than in short-term experiments under the pre-bloom and bloom conditions, but almost no changes were observed under post-bloom conditions . However, the effects of culture treatment (developing phase) were slight (P>0.05).Y (II) and rETR are two different indicators of rapid light respond curves (RLCs). These were measured to determine photosynthetic performance. Y (II) decreased with set light intensity gradient, and Y (II) of reatment  and 6. Mreatment , and 6. U. prolifera and U. intestinalis thallus, were clearly higher in the long-term experiments than in short-term experiments (P<0.0005), and α only showed significant differences across species but not phase of development (P>0.05). These values also showed almost the same photosynthetic physiological status, like Fv/Fm and Y (II). These parameters of U. prolifera thallus were significantly more pronounced than those of U. intestinalis in both the short-term and long-term experiments. The maximum value appeared under the bloom culture conditions, as Fv/Fm did (pre-bloom and bloom conditions but not under post-bloom conditions(Like Y (II), rETR showed a clear pattern of photosynthetic performance, and RLCs appeared similar to a traditional oxygen-based photosynthesis-irradiance (P–I) curve  and 8. Ieriments  and 8.. v/Fm did . The rETonditions.P<0.01). Only GSH showed no difference in the combined effect of species and phase of development .Biochemical parameters  showed highly significant differences across treatments (U. prolifera only under pre-bloom and post-bloom conditions (P<0.05), providing a measure of lipid oxidative damage of the treatments. This effect was slighter in U. intestinalis (P>0.05), showing no difference across the three culture treatments.MDA concentrations showed statistically significant increases in the U. prolifera than U. intestinalis (P<0.0005), and the TSP of both species was significantly lower under pre-bloom and post-bloom culture treatments (P<0.05). T-AOC as an estimator of total antioxidant capacity in defense system had a lower value in U. prolifera than U. intestinalis. It also showed a marked decrease under the post-bloom conditions . U. prolifera under pre-bloom and post-bloom culture treatments trended to have higher antioxidant enzyme activities than under bloom conditions . However, U. intestinalis did not show such changes . These two enzymes were found to have more sensitive responses in U. prolifera than in U. intestinalis under all three sets of conditions.The activities of SOD and Gpx in nditions  and 13, U. prolifera than in U. intestinalis . Like the antioxidant enzymes SOD and Gpx, GSH showed more activity in U. prolifera under pre-bloom and post-bloom conditions than under bloom conditions, but this was not true in U. intestinalis.The activity of GSH, as the major endogenous non-enzyme antioxidant, was higher in stinalis , especiabloom and post-bloom conditions than under pre-bloom conditions  and by Bischof et al (U. lactuca). However, the reason for this is still unknown. The slow response of pigments (chlorophylls) to stress relative to that of other physiological parameters may have contributed to these results. Pigment content usually takes several days to weeks to acclimate. This has been recognized in previous reports on UlvaThe pigments did not show pronounced changes in concentration with respect to culture treatment, especially under long-term conditions . SimilarU. prolifera had a higher photosynthetic capacity than U. intestinalis. The ratio of dark-adapted Fv/Fm and maximum Y (II) in U. prolifera was significantly higher than that of U. intestinalis, especially under bloom conditions. This suggested that U. prolifera had greater photosynthetic efficiency. This conclusion is further evidenced by the observations of RLCs. U. prolifera showed a steadier and slower decline in Y (II) over the course of the RLCs than U. intestinalis did  responses . However, under the post-bloom treatment, U. prolifera did not show higher Fv/Fm, rETRmax, or Ek values during short-term culture. This indicated that recovery of photosynthesis in both species was inhibited by combined environmental factors under post-bloom conditions. The thinner and more filamentous thallus morphology may also have partly contributed to this inhibition. This morphology exposes more surface area to stress. Our biochemical results provided another explanation, discussed below. In this way, the reason why green tides caused by U. prolifera diminish is that it cannot adapt to post-bloom stress with high SST, high PAR, and low SSS either in the laboratory or in the more complex post-bloom environment that occurs in the field. Judging by to its photosynthetic plasticity, U. prolifera has more capacity for photosynthesis under pre-bloom and bloom conditions, creating ephemeral blooms in the field.After 7 days of stress treatments, esponses , Table 3bloom) and stress conditions . Many case studies on species of the genus Ulva have used the production of reactive oxygen species to indicate the influence of environmental stress on their growth Ulva species are sensitive to stress conditions Every species has its own ideal irradiance, temperature, and salinity. When any of these factors stray beyond a suitable range, photosynthesis and growth become limited. Culture conditions that incorporate combined environmental factors, as in this study, can be divided into two parts: suitable conditions  and non-enzyme antioxidants (GSH) increased in response to damage. U. prolifera showed significantly lower SOD and GSH levels under post-bloom conditions than under pre-bloom conditions  was significantly lower than under pre-bloom stress conditions . The comparatively low level of PS II activity in U. prolifera following stress treatments implies the generation of enhanced ROS, because previous study had shown blockage of photosynthetic electron transport, which would lead to overproduction of ROS U. prolifera, the lowest pigment concentration was observed under the post-bloom culture treatment in both short- and long-term scales, but this was not true of U. intestinalis  corresponded to the higher degree of lipid peroxidation (MDA) in is study –14. Undestinalis . The Fv/U. prolifera bloom formation because U. prolifera has a higher nitrogen utilization capacity than U. intestinalis does , nutrient utilization absorption related proteins (such as NR), proteins related to the defense system (such as antioxidant enzyme and HSP), and so on. Among these proteins, only NR was clearly found to contribute to TSP in this study. This compound appears in very high concentrations, especially under the bloom culture treatment.Another biochemical analysis showed that the concentration of total soluble protein (TSP) in Ulva species are known to have a high nitrogen requirements for optimal growth U. prolifera over U. intestinalis from May to June. In this way, higher nitrogen utilization capacity indicates higher biomass. Therefore, U. prolifera, unlike U. intestinalis, can form blooms when the other factors (like the environment) are suitable.rbcL, rbcS encoding for the large and small rubisco subunit, and psbA encoding for the D1 protein in PSII) U. prolifera and U. intestinalis stresses has remained unclear. Linking physiological responses to stress with their genetic bases, particularly using current genomic and proteomic approaches, would certainly improve the understanding of stress responses  of these macroalgal species. Few studies on the factors that regulate genes  in macroalgae exposed to stress are available. In our subsequent experiments, quantitative real time RT-PCR was found to be an efficient means of performing comparative transcriptional analysis. This technique will be used to investigate whether stress responses occur in U. prolifera and determine how the defense systems of these macroalgal species are regulated under stress conditions. These qRT-PCR results will further confirm the findings reported in the present study.Photosynthetic protein may also have a correlation with TSP. This is because the concentration of TSP has a pattern similar to that of Fv/Fm, which was here used as indicator of photosynthetic capacity. The decrease may also be affected by ROS. ROS has recently been identified as an important component in certain signal transduction pathways conclusions: (1) The method used in this study provides a maximum estimate of the physiological responses to environmental changes, maximum replication of natural processes, and comparison of results found in the laboratory. This allows us to develop explanations of how U. prolifera can form green tides in the Yellow Sea under natural conditions. (2) Differences of ecophysiological traits in photosynthesis and defense system of these two co-occurring Ulva macroalgae under combined abiotic conditions correlated with their different natural processes: U. prolifera was found to be more sensitive to dynamic environments, especially to harsh or changing environmental conditions, characterizing their purely opportunistic strategy. U. intestinalis is more stable, but this is largely due to the stress tolerance provided by antioxidant system. (3) The ecological success of U. prolifera is inextricably linked to its higher capacity, its plasticity, and its nutrient absorption and assimilation systems. Once conditions become favorable, U. prolifera seems to be able to successfully compete with U. intestinalis for nutrients and space.From the present study we can draw three key


Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture.  The eukaryotic flagellum (synonymous with motile cilium) is a biological machine that drives fluid movement across epithelial surfaces and propulsion of single cells. Biophysical principles underlying flagellar motility, namely dynein-dependent sliding and bending of adjacent microtubules in the axoneme, are universally conserved, as are the basic arrangements of axonemal sub-structures Trypanosoma brucei is a uniflagellated protozoan parasite that causes significant human mortality and limits economic development across sub-Saharan Africa. Without treatment, the human disease, known as African sleeping sickness, is fatal T. brucei flagellum functions in cell morphogenesis and host-parasite interactions T. brucei flagellum exhibits an unusual, bihelical beat, in which helical waves of alternating handedness propagate from flagellum tip to base and drive cell movement with the flagellum tip leading T. brucei flagellum architecture that impact flagellum waveform. Most notably the T. brucei flagellum contains a structure known as the paraflagellar rod (PFR) that is attached along the length of the axoneme T. brucei biology and disease, the three-dimensional (3D) structural organization of the axoneme and PFR, particularly their relationships to one another, has not been defined.The T. brucei. Our studies provide the first quantitative description of the T. brucei axonemal repeating unit and provide insight into flagellum assembly and function. Given the importance of the flagellum to T. brucei biology and infection, our findings are relevant to understanding the mechanism of disease pathogenesis and development of novel therapeutics.Studies of flagellar ultrastructure in trypanosomes have traditionally used TEM techniques and freeze-fracture techniques that rely on interpreting 3D structure from 2D projection images We collected and analyzed a total of 439 tomographic datasets containing flagella  from botUltrastructure was well-preserved in all specimens, as indicated by the presence of all major axonemal structures, including the 9+2 axoneme, outer and inner arm dyneins, nexin links, radial spokes and central pair complex; together with axoneme-PFR connectors and the proximal, intermediate and distal zones of the PFR . AsymmetComposition and arrangement of the trypanosome axonemal repeating unit has not previously been defined. Digital slices of tomograms were used to define the trypanosome axonemal repeat unit and determine architectural spacing of core substructures . These nLongitudinal views of the PFR-axoneme interface revealed mass densities that repeat every 57.2 nm  along the length of the PFR , 2. DigiElectron dense filaments provide a physical connection between the PFR and axoneme in the region between outer doublets (OD) 4, 5, 6 and 7 Transverse views of the OD-PFR interface showed a radial organization of connectors to OD4, 5 and 6. In most cases one connector appeared to be  attached directly to the outer arm dynein and the others attached to the A tubule, near the A tubule-B tubule (At-Bt) interface , and the B tubule (5.3 and 6.3) . BecauseAxoneme-PFR connectors were examined in further detail using sagittal and coronal views of digital slices from tomograms of stained samples and sagittal view of digital slices from frozen samples. . These aThin (1nm) transverse digital slices  showed tCoronal digital slices through 3 nm of sample thickness revealed distinctive patterns of density in the proximal, intermediate and distal zones . Slices Perspective views of tomograms  indicateSurface renderings of segmented tomogram volumes provided 3D views of PFR structure, including conformational changes that mark the interface between PFR zones . The proT. brucei flagellum and identify connections from the PFR to dynein motors that drive axonemal motility using dual-axis ET and cryo ET. Robustness of the data is evidenced by identical dimensions of major structural landmarks in frozen-hydrated samples and stained, embedded samples. Our findings provide a structural foundation for considering molecular mechanisms of PFR assembly and flagellar motility in trypanosomes.We define the axoneme and PFR repeat units of the ChlamydomonasT. brucei has three spokes per repeat . DNase I (Worthington) was added at 0.25 mg/ml to remove mitochondrial DNA. Following a 10 minute incubation at room temperature, samples were incubated on ice for 30 minutes, and flagellar skeletons were collected by centrifugation at 16,000xg for 10 minutes. Flagellar skeleton pellets were subsequently transferred to a fresh tube and washed twice with PMN buffer. This procedure allowed for one-step fractionation to obtain flagellar skeletons and improved sample preservation Procyclic 29-13 10 cell equivalents/ml were mixed with protein-A gold (10nm) purchased from Cell Microscopy Center of University Medical Center Utrecht at a 70∶1 v/v mixing rate. A 3 µl droplet of the gold-containing flagellar skeleton sample was applied onto 200 mesh Quantifoil 3∶1 µm holey-carbon coated grids, blotted by filter paper, and plunge-frozen in ethane cooled by liquid nitrogen.For stained and embedded samples, flagellar skeletons were fixed in 1% glutaraldehyde and 1% paraformaldehyde in 0.1M sodium cacodylate buffer containing 1% tannic acid at pH 7.2, post-fixed in 1% osmium tetroxide with 1.5% potassium ferrocyanide. Samples were dehydrated using an ascending acetone series, followed by infiltration and embedding in Eponate 12  as described in 2 TF20, operated at 200kV as previously described Batchtomography software from FEI. Each series was taken with a defocus of −3 µm from −70° to +70° tilt along alpha and beta tilt axes, with 2° increments at the lower tilt angles (range±40°) and 1° increments above +40° and below −40°. Dual axis data sets were achieved by taking single tilt data sets at several points of the grid before manually rotating the grid by 90° and taking the second tilt series at the same locations.Electron tomography was performed in an FEI Technai G2 TF20, operated at 200kV, and FEI Titan Krios, operated at 300kV using a Gatan energy filter 2002 and 2kx2k CCD camera. Focus and tracking positions were located along the holder tilt axis 5–7 µm away from the exposure area, to prevent exposing the region of interest during focus and tracking. Single tilt data sets from 70° to +70° were taken at a 2° intervals. The defocus used was −5 µm. The dosage of cryo-samples was adjusted so that the total accumulated electron dose for each entire tilt series is between 70–100 e/A2.CryoET data sets were taken using low dose imaging protocols using FEI Technai GeTomo  eTomo to create a single final tomogram. To reduce image noise, the tomograms were first filtered using the median function of the clip IMOD program and subsequently low-pass filtered to 40 Å resolution. Z-axis adjustment was made on the stained samples to correct for shrinkage according to IMOD protocols (3Dmod users guide). Segmentation and image processing were conducted using Amira 5.2.0 (Visage Imaging).Images in each tomography tilt series were aligned and combined to generate 3D tomograms using Segmentation is based upon distinct characteristics of the data, the density of stain and structural morphology. Regions of interest (ROI) were selected (using the “brush” tool), and data within the ROI displayed using automatic functions (“thresholding” and “smoothing”). Different colors in the volumes were the result of displaying several ROI  together. Videos were generated using the demo-maker and movie-maker functions on Amira. Videos of tilt series and tomograms were generated using 3Dmod  and VideoMach  was used to combine images into a video.Measurements to quantify dimensions in the flagellum were made using the Amira measuring tool of frozen and stained samples. Measurements were confirmed by independently applying Fourier transform to axoneme and PFR images from frozen flagellar skeletons . Spatial frequencies were calculated along the horizontal plane  of the power spectrum using the bright spots as markers. Images used for the diffraction data were taken with the TF20 and Titan Krios microscopes and boxed to contain only axoneme or PFR structures . All anaFigure S1Imaging of stained versus frozen-hydrated samples. (A). Schematic diagram comparing image interpretation for stained and cryo (frozen-hydrated) data. Stain accumulates around and within structures, sharply delineating edges in the final images. There is no stain in the frozen/cryo samples, thus electrons are deflected by the sample itself. (B) Both stained and frozen-hydrated samples are tilted to create a series of projection images. Weighted back-projection of aligned images creates a 3D tomogram. (C) Tomogram volumes can be viewed in x, y and z planes.(TIF)Click here for additional data file.Figure S2Bihelical arrangement of spoke triplets around the central pair microtubules. (A) Cross-sectional tomogram slice (≈50nm thick) shows a transverse view of the flagellum skeleton. Color scheme for spoke heads is based on outer doublet number as shown on the left, with outer doublet numbering as shown in (TIF)Click here for additional data file.Figure S3Serial tomogram slices of a frozen flagella skeleton showing the axoneme and PFR repeat. (A–E) Longitudinal section tomogram slices (≈1nm thick) show the axoneme and PFR repeat. (A) OD7-PFR connectors are in sync with periodic PFR densities. (B) OD6-PFR connectors are attached to periodic PFR densities in the proximal zone (Prox. PFR). Longitudinal fibrils are present in the intermediate (Int. PFR) and distal  PFR zones. (C) Spoke triplets are arranged along the axoneme between the central pair microtubules and the outer doublets. (C–E) Distinct bands of periodic densities can be observed in the PFR (arrows). Scale bars are 100nm.(TIF)Click here for additional data file.Figure S4CS slices showing contiguous densities extending from proximal through to distal zones of the PFR. Transverse views of the flagellum skeleton. Slice thickness is indicated in each panel. The 70-nm thick slice shows the proximal (P), intermediate (I) and distal (D) PFR zones. Parallel linear densities are (white arrow) visible in the intermediate zone. Thin slices (10nm and 1nm) show densities extending across the PFR (white arrows). The x-y position of the proximal and distal segments of these contiguous densities displays variability in sequential thin z-slices. See also (TIF)Click here for additional data file.Figure S5TEM image showing the relative positions of laths in each PFR zone. TEM image (∼200-nm thick) is of the same sample shown in 4C. Colored arrows indicate the position and direction of laths in the proximal (gold), intermediate (red) and distal (purple) zones. Proximal and distal laths are offset from one another by 24nm. Compass shows sample orientation, as described in (TIF)Click here for additional data file.Figure S6Digital slice from tomograms showing filaments in the PFR intermediate zone.  Sagittal tomogram slices (≈3nm thick) from a stained (A) and frozen (B) sample showing the 3 PFR zones, proximal (gold), Intermediate (red) and distal (purple). Filaments are observed in the intermediate zone parallel to the long axis of the flagellum (red arrows) and obliquely orientated, forming a connection from proximal through distal zones (blue arrows). Scale bar is 50nm.(TIF)Click here for additional data file.Video S1Aligned tilt series of stained flagella skeletons.(AVI)Click here for additional data file.Video S2Serial Z slices through a tomogram volume. The frozen-hydrated flagella skeleton Click here for additional data file.Video S3Serial Z slices through a tomogram volume. Cross-section of flagella skeleton, Click here for additional data file.Video S4Surface rendering of segmented volume. Segmented volume Click here for additional data file.Video S5Three-dimensional graphical model of the PFR.(AVI)Click here for additional data file.Video S6Motility of a procyclic-form T. brucei cell.(WMV)Click here for additional data file.


To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line.Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a mutant were more resistant to digestion by pancreatic α-amylase, and the sbe1a mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the sbe1a mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination.Endosperm starch granules from the in vivo. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited.The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch  The starch granule is a highly-ordered structure with alternating crystalline and amorphous growth rings ,2. StarcStarch-branching enzyme (SBE) plays an important role in starch biosynthesis by introducing branch points, the α-1,6 linkages in starch. Boyer and Preiss  identifiIn vitro biochemical analyses have documented that the SBEI and SBEII isoform activities are not identical [in vivo, as starch biosynthesis occurs in the presence of starch synthases and debranching enzymes. Studies have suggested that multi-protein starch synthesizing complexes exist, and that interactions within these complexes could modulate the intricate structure of a developing starch granule [in vivo remains to be addressed.dentical ,16, but  granule -33. Whetin vivo function of an SBE may be gained from the study of sbe mutants deficient in one or more SBE isoform activities. The maize amylose extender (ae) mutant, which is deficient in SBEIIb, has a profound effect on starch structure, leading to an increased amylose proportion and a reduced branching density of endosperm amylopectin [sbe2a mutant showed that deficiency of the SBEIIa isoform decreased plant fitness and resulted in lower kernel yield, but there was minimal effect on kernel starch properties [sbe1a mutation) on starch molecular size and on chain length distribution after debranching [sbe1a endosperm starch to pancreatic α-amylase digestion, using the AOAC procedure (2002.02) to determine enzyme-resistant starch (RS), indicated that sbe1a mutant endosperm starch had a greater resistance to digestion [sbe1a mutant. The objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line.Insight into a possible lopectin ,33-35. Moperties ,36. Prevranching ,37. Subsigestion . We reasSbe1a starch and mutant sbe1a starch were fractionated from mature kernels. The maize sbe1a mutant contains a Mu transposon in the 14th exon of the Sbe1a gene, and was previously shown to be null for the expression of SBEI transcript and protein [sbe1a starch (data not shown). To study the molecular fine structure, β-amylolysis and subsequent isoamylase and pullulanase debranching were applied to both the amylopectin and amylose fractions from Wt and sbe1a. Despite a similar chain length (CL) profile observed for both fractions from the two genotypes , before and after pullulanase addition Figure  online. a Figure  online. a Figure .in vitro method (AOAC 2002.02) is used for determination of the RS level. This method was modified to allow study of both the digestion rate and the extent of digestion [p = 0.000), but no effect of biological replication (p = 0.334). The RS value was higher in the sbe1a mutant starch (13.2%) than in the Wt starch (1.6%) from measures of 3 biological replications (p < 0.05).Starch hydrolysis is an important feature of starch function both in the plant and when the plant is used for human food. Hydrolysis of starch ingested as food can vary both with respect to the rate and the extent of digestion by pancreatic α-amylase. In the human digestive tract, the undigested starch that reaches the colon is termed RS; the level of RS is a measure of the extent of digestion by this enzyme. An official igestion ,42. F-te0 y(the limit of digestion as determined using the model) was found in sbe1a than for Wt . For graphic illustration of the digestion time-course, curves for one biological replication for each genotype are shown in Figure sbe1a mutant plants in native form and after the 16 h in vitro digestion with pancreatic α-amylase for determination of the RS value. Prior to digestion, native starch granules from Wt and sbe1a had similar morphology .Light micrographs of iodine-stained native granules are shown in Figure sbe1a mutant has a lower susceptibility to pancreatic α-amylase, we suspected that the sbe1a endosperm starch might be less readily utilized during kernel germination. To study the effect of sbe1a on kernel germination, starch utilization and coleoptile growth during germination of Wt and sbe1a mutant kernels were examined.As endosperm starch from the sbe1a genotypes were germinated, demonstrating no differences in germination rate. The coleoptile length of each genotype was measured daily over 11 days  [sbe1a samples   would hattached) , a slow s Figure , suggestsbe1a and Wt are presented to account for this difference in β-amylase action on DP 4 stubs  hindered hydrolysis of residual A chains of DP 4 to DP 2 due to steric constraint, and 2) more residual B chains with DP 4 due to incidence of short internal segments , a higher proportion of long chains of DP ≥ 100 was observed in sbe1a to Wt, more of DP 2 and DP 3 chains are not debranched by isoamylase in β-LD from sbe1a amylose  longer internal chains and 2) more A chains attached by branch points close to each other. This evidence can be used to create an overall model for amylose branching patterns of sbe1a and Wt , and a slowly-digested substrate (S2) [S1 and S2 and the reaction rate constants for these two components. The S1 components of Wt and sbe1a starch were 85.9% and 59.8% respectively. This suggests that the sbe1a mutation altered the starch structure and this resulted in less rapidly-digested component. Consistent with our results, Ao et al. [S1) and an increased proportion of SDS .The kinetic model is based on the presence of two general types of starch substrate: a rapidly-digested substrate (ate (S2) ,42. The o et al.  found thsbe1a appear similar in size, shape, degree of birefringence, and morphology, as described in a previous report for wx and sbe1a wx granules [sbe1a, many digested granules had maintained some birefringence in the peripheral area of the granules, which indicates that the center of the digested sbe1a granules is either gone or no longer crystalline enough to show birefringence. The presence of a hollow interior in the digested sbe1a granules was confirmed by SEM  and light microscopy (LM), were employed to observe granular structure before and after RS digestion by pancreatic α-amylase. Native starch granules from Wt and granules . PolarizM Figure , indicatsbe1a RS showed variations in morphology, from small fragments to hollow granules. The difference in digestion of individual granules may be due to differences in heterogeneity in granule structure, as a higher proportion of relatively dark-stained granules were observed in sbe1a than in Wt native starch . In order to compare starch material within a highly similar genetic background, homozygous Sbe1a/Sbe1a (i.e. Wt) and sbe1a/sbe1a mutant siblings were identified from a single segregating population derived from seeds of selfed Sbe1a/sbe1a plants to obtain ears for endosperm analysis. Genotyping of Wt and sbe1a mutant plants followed Blauth et al. [sbe1a mutant plants were self-pollinated to produce ears for endosperm analysis, and are segregated from a BC4F3 population backcrossed by Blauth et al. [Maize plants of Wt and h et al. . The deth et al. ,37. Starh et al. . Starch h et al. .M, pH 6.0) was added. The mixture was heated in a boiling water bath for 10 min and cooled to 50°C. A 200-μL aliquot of a β-amylase  solution  was added, and the samples were incubated at 50°C with constant agitation (200 strokes/min). At approximately 10 min, 30 min, 1 h, 2 h, 6 h, and 24 h, a 0.5-mL aliquot of sample was removed and heated in a boiling water bath for 10 min to stop the reaction. The procedures for precipitating β-dextrins and debranching β-dextrins by successive action of isoamylase  and pullulanase  were the same as used previously for β-LDs) [β-Dextrins were prepared by the method of Xia and Thompson  with slir β-LDs) ,40.The preparation and debranching of β-LDs followed the procedures in Klucinec and Thompson  with sliin vitro RS determination  was employed, which was scaled-down and modified for direct analysis of the digestion supernatant for total carbohydrate [g, 10 min), the supernatant was analyzed in duplicate for total carbohydrate using the phenol sulfuric acid method [sbe1a mutant endospermes from three separate plants  were subjected to triplicate pancreatic α-amylase digestion, for determining the RS values.The official method for ohydrate . The modd method . The perFor determination of digestion time-course, the starch samples were digested as described above. An aliquot was removed at approximately 30 sec, 3 min, 6 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, 10 h, 13 h, and twice at 16 h, and added to 1 volume of ethanol/EDTA solution to ensure immediate deactivation of the enzymes. After centrifugation the supernatants were analyzed for total carbohydrate as described above.Digestion time-course was analyzed following the method developed by Rees  to obtaiy0 is the y-value that the model asymptotically approaches, S1 and S2 are the concentrations of the two different substrate components, and k1 and k2 are the reaction rate constants for the decay of the two different components. The units for y0, S1, and S2 were % of initial starch, and the units for the rate constants were min-1. After running the regression program, the software gives three possible completion status messages depending on how well the model fits the data:where y is % NDS, x is the time, (1) Converged, tolerance satisfied.(2) Converged, tolerance satisfied. Parameter may not be valid. Array numerically singular on final iteration.(3) Didn't converge, exceeded maximum number of iterations.The data were kept for further regression analysis if message 1 or 2 resulted, and were discarded if message 3 resulted.Digestion time-course analysis was performed for three biological replications per genotype. For each biological replication, two technical replications were performed. If both sets of data "converged" using the model (message 1 or 2), no further analyses were performed. If message 3 appeared, a new technical replication was done until the data "converged." The data from the two "converged" technical replications for each biological replication were combined, and the software program was run on the combined data. For all samples, the regression model fit for the combined data completed with convergence (Message 1), and generated valid parameters for analysis. Using the combined data, values for five parameters in the equation were determined for each biological replication. A mean and standard deviation of the five parameters for each genotype was then calculated, and comparisons among genotypes were made by one-way ANOVA analysis.2, 0.12% KI) was placed onto 20 μL of the dispersed sample to give a final I2 concentration of 0.04%. In order to compare birefringence between granules, the camera's automatic exposure function was turned off, and the exposure was set the same for all samples. The same sample field was examined under bright field and polarized light.Bright field and polarized light microscopy were performed using a light microscope  with an attached digital camera . 5 mg of native starch sample was mixed with 0.5 mL of deionized water in a micro-centrifuge tube. For the resistant starch samples, the supernatant was removed after centrifugation of digestion solution and 20 μL of deionized water was added to the pellets to disperse the sample. To examine the sample under the microscope, 20 μL of the dispersed sample was added to a glass slide, and a cover slip was fixed over the sample with fingernail polish. Examination of iodine-stained starch followed the method in Evans and Thompson . 20 μL osbe1a granules. Four micrographs for each genotype (Wt or sbe1a) were used for sorting. The evaluators were then given those eight micrographs, unlabeled and in randomized order, and asked to sort the granules into two categories. The proportion of dark granules for each micrograph was calculated based on the sorting results from all five evaluators, and a mean proportion was obtained for each micrograph. For each genotype, a mean was calculated from the means of the four micrographs. Comparison between two genotypes was made by one-way ANOVA.Heterogeneity of iodine staining was evaluated quantitatively by a volunteer panel. Differentially iodine-stained starch granules were classified into two categories, dark or light stained granules, and were sorted visually by five individual evaluators who were not otherwise involved in the research. The evaluators were trained to understand the difference between dark and light stained granules, by observing granules in a portion of a micrograph for A thin layer of starch sample was applied to double-sided sticky carbon tape on a specimen stub, and sputter-coated with 10 nm Au/Pd . Samples were then examined using a scanning electron microscope  at an accelerating voltage of 20 keV and at different magnification levels . For image collection, lower magnification was first employed to examine the whole view of samples, and higher magnification was then used to focus on sample areas that were representative overall.g for 10 min, and suspended in 3 mL of deionized water. For calculating the dry weight of samples, 1 mL of this suspension was dried at 70°C overnight and weighed. The remaining 2 mL of the suspension was boiled for 30 min, and the total glucan polysaccharide in the solubilized solution was quantified in triplicates, using a commercial assay kit that measures glucose released after digestion with α-amylase and amyloglucosidase . The quantified starch content was normalized against the dry weight for comparison between genotypes.A kernel germination assay was performed according to the method in Dinges et al.  with sliHX conceived of the study, performed the experiments, and drafted the manuscript. MYN participated in maize genotype breeding and discussion of experimental design and major results. DBT & MJG advised the conception of the study, experimental design, result discussion, and revised the manuscript. All authors read and approved the final manuscript.sbe1a mutant (- - -) starchChromatograms of isoamylase-debranched amylopectin and amylose fractions from Wt (----) and .Click here for filesbe1a mutant and Wt starch for the proportions of chains from debranched β-dextrins during time course of β-amylolysis of amylopectinDifference plots between . Individual plots for sbe1a mutant and Wt are presented in Figure Click here for filesbe1a mutant starchChain length distribution of isoamylase-debranched and isoamylase-plus-pullulanase-debranched β-limit dextrins from the amylopectin fraction from Wt and .Click here for filesbe1a mutant starchChain length distribution of isoamylase-debranched and isoamylase-plus-pullulanase-debranched β-limit dextrins from the amylose fraction from Wt and .Click here for filesbe1a mutantBright field (left) and polarized light (right) micrographs of residual starch after 16 h α-amylase digestion from Wt and . Arrows point to residual granules with dark center.Click here for filesbe1a mutant (right)Transmission electron micrographs of residual starch after 16 h α-amylase digestion from Wt (left) and . Scale bars represent 5 μm at the top of the graphs.Click here for filesbe1a mutant (- - -) starchChromatograms of isoamylase-debranched resistant starch from Wt (----) and Click here for file