The restoration of voluntary muscle activity in posttraumatic paraplegia in both animal experiments and other clinical applications requires reproducibility of a technically-demanding microsurgical procedure, limited by physicians’ understanding of Brunelli’s spinal cord grafting paradigm. The insufficient clinical investigation of the long-term benefits of the CNS-PNS graft application warrants additional inquiry.The objective of this study is to explore the potential benefits of the first replicated, graft-induced neuroregeneration of denervated skeletal muscle regarding long-term clinical outcomes and to investigate the effect of Cerebrolysin on neuromodulation.A randomized study evaluating 30 rats, approved by the National Animal Ethics Advisory Committee was performed. The medication was administered postoperatively. For 14 days, 12 rats received Cerebrolysin (serum), 11 received NaCl 0.9% (shams), and 7 were controls. For microsurgery, the lateral corticospinal tract T10 was grafted to the denervated internal obliquus abdominal muscle. On day 90, intraoperative proof of reinnervation was observed. On day 100, 15 rats were euthanized for fixation, organ removal, and extensive histology-morphology examination, and the Wei-Lachin statistical procedure was employed.After an open revision of 16 rats, 8 were CMAP positive. After intravenous Vecuronium application, two  out of two rats showed an incomplete compound muscle action potential (CMAP) loss due to glutamatergic and cholinergic co-transmission, while two others showed a complete loss of amplitude.Cerebrolysin medication initiated larger restored muscle fiber diameters and less scarring. FB+ neurons were not observed in the brain but were observed in the Rexed laminae.Brunelli’s concept was successfully replicated, demonstrating the first graft induced existence of cholinergic and glutamatergic neurotransmission in denervated grafted muscles. Statistics of the histometric count of muscle fibers revealed larger fiber diameters after Cerebrolysin.Brunelli’s CNS-PNS experimental concept is suitable to analyze graft-neuroplasticity focused on the voluntary restoration of denervated skeletal muscles in spinal cord injury. Neuroprotection by Cerebrolysin is demonstrated. In 1981, Giorgio Brunelli (GB) began his experimental research in a rat model “to connect the cephalad cord (in which regrowing axons are present) to peripheral nerves (in which axon growth can advance) to overcome the non-permissiveness of the spinal cord after severance”. In 1994, he used his paradigm of direct neurotization of denervated skeletal muscle via presynaptic motoneurons in nonhuman primates (Macaca fascicularis) to finally demonstrate “new voluntary innervation of some hip muscles that after laminectomy T8 to T11 had been directly connected by sural nerve grafts from the lateral bundle of the spinal cord.” His ultimate target was “to search for a new microsurgical concept for functional restoration in human paraplegics that can provide human paraplegics at least a few elementary voluntary movements as to stand up and ambulate with the aid of walkers” -4. Only To date, a replication of GB’s concept for CNS-PNS graft either in animals or human restorative SCI surgery has not been reported elsewhere in the literature, as it was not recommended for clinical use -10,11. GThis second report is focused on the evidence of peripheral nerve graft (PNG) induced regeneration of denervated internal oblique abdominal muscle (IOAM) fibers, as the authors were ultimately interested in using GB’s paradigm for a new alternative microsurgical concept in restorative microsurgery of human brachial plexus and SCI lesions. Based on the literature reports on beneficial neuroprotective pharmacological neuromodulation, GB’s study design was extended as a prospective randomized, double-blinded, placebo-controlled trial (RCT) using Cerebrolysin® (CERE) medication postoperatively -18.Secondary data analyses of evidence from our previously reported graft-induced neuroplasticity in rats after replicating Brunelli's CNS-PNS concept were included ,2,11. BrWe previously provided a summary of all pharmaceuticals and biochemical products used, the applied dosages, routes of administration, and products in more detail . For sedFrequent sudden deaths of unknown objective pathology that are known from the investigation of Brunelli's concept in animal research occur, but these deaths occur less frequently after the introduction of postoperative intensive care management. A fatal outcome is registered in the daily protocol, analyzed together by the veterinarian toxicologist, pharmacologist, and pathologist, and reported to the government department of animal research.Eleven materials and equipment used in restorative microsurgery were as follows: Leica Wild M-690 Surgical Microscope; Biomed atraumatic bipolar electro muscle and nerve-stimulator; Ethilon monofil 9-0, 10-0 (not absorbable), Vicryl 3-0 (absorbable); maniple wound closure staple gun AZ 35 W 35 B 6,9 mm, H 3.6; mechanical animal ventilator  – 0.7); intra and postoperative warm circulating water blanket to prevent hypothermia and to maintain the rats' body temperature within the physiological range -22,24,25First operation: CNS-PNS graft procedure in the prone position, four steps  21-23]:-23:21-231.Harvesting of the right sural nerve graft (SNG);2.Skin incision, medial longitudinal 50 mm, microsurgical preparation and electrical identification of IOAM – NEZ ;3.Laminectomy from T10 to L1;4.At T10, incision of the dura mater and arachnoid membrane was performed dorsally ; preparation of the right SC dorsolateral bundle for the free-hand stitch incision (1.5 to 2 mm deep) directed toward the lateral cortical spinal tract and introduction of the proximal PNG end, fixed at the arachnoid membrane by one stitch 9-0 atraumatic; end to end adaptation of the graft end to the cut distal IOAM nerve stump, next to its NEZ, fixed by one atraumatic stitch 9-0. After hemostasis and wound closure, blinded medication was injected intraperitoneally in the supine position before ICU observation.Second operation at day 90: Open microsurgical check of graft's intact extradural position and IOAM nerve adaptation; CAMP assessment of neurodegeneration ,27. DepeThird  operatioAfter peripheral nerve grafting, no single histopathological measure can capture the multidimensional nature of neuroplasticity. Therefore, we chose the highly efficient nonparametric Wei-Lachin procedure  with a multidimensional approach to evaluate the assessments statistically. We are well aware that the small sample sizes render parametric statistics sensitive to outliers  28-30].-30.28-30Standard procedures were performed for all samples. Five-micron sections were cut from each block using a rotary microtome , stained with hematoxylin and eosin (HE) and Masson-trichrome (MT), and examined under an Olympus BX51 microscope. Images were collected by an Olympus SP 350 digital camera (Olympus) and Olympus Cell B morphometric program for counting and measurements. Graft and graft-dependent secondary histology-morphology changes from T10 to L1 SC segments (approximately 5 mm) were identified in each case: description, measurement, and diagnoses within the SC, NG and surrounding area, e.g., type of regeneration, atrophy, vascularization, traumatic lesion, infection, and secondary lesions. The IOAM atrophy and regeneration of both sides were measured to be compared and statistically evaluated -39. We dAnalysis and indirect immunofluorescence assessment of VGlut2 allowed for a more profound discussion on graft-induced central plasticity ,11,40-43GB's microsurgical connection of CNS-PNS by PNG successfully replicated normal histology-morphology, demonstrating the correct positioning of the implanted sural NG within the right CST. The RCS protocol with additional postoperative IP medication and intensive care was adapted to a local veterinarian's ICU and to optimal housing, as both contributed to improved postoperative animal survival. Nevertheless, during three months of follow-up, sudden mortality occurred in 14 of 29 animals at different times without showing specific pathophysiological symptoms and signs or warning behavioral abnormalities; corpse findings in postmortem review were uneventful , 21-25. The total brain, SC, the graft, and right and left IOAM were analyzed in regard to graft-induced histology-morphology and pathology by CC. The specimens of IOAM from both sides were kept in Lübeck to assess graft-induced regeneration after denervation by comparative histometric muscle fiber analysis (ML).To aid in identifying the IOAM nerve, the graft, and neuroregeneration of the grafted IOAM nerve were examined using intraoperative EMG (CAMP). Uncertainty about positive functional neuroregeneration in cases of negative EMG stimulation, when commonly related to technical problems, does not contradict positive regeneration, as positive regeneration was subsequently demonstrated by histometric measurement of grafted muscle fiber diameters. CAMP positive recordings were observed in 8 animals at the second operation, demonstrating the achieved extent of graft-induced muscle regeneration. Positive CAMP signals are necessary to analyze the type of neurotransmitter: 4 out of 8 CAMP+ rats received Vecuronium IV during artificial ventilation; two specimens showed the expected complete block of electrical stimulation, while two others showed a partial loss of CAMP+ amplitudes. The latter result strongly suggests mixed-type cholinergic/glutamatergic co-neurotransmission.A total CAMP blockage is equated with glutamatergic  reinnervation (Brunelli's paradigm) -4. This RCT experiments demonstrated the beneficial effect of postoperative CERE medication on functional neuroregeneration regarding larger IOAM fascicle diameters after atrophy compared with the two other cohorts when the statistical evaluation was unblinded. Neuroprotection by CERE was demonstrated by histology-morphology analysis, revealing less secondary scar formation around the tip of the graft and more intense axonal sprouting and vascularization next to the implanted graft in CST, inside the regenerated graft, and outside the SC -39 Figu.Postoperative temporary neurological deficit was more or less pronounced as the typical sequence of surgical manipulations of the SC during the grafting procedure in all 29 rats. Animals recovering from functional impairments showed good functional movement and neurobehavioral activity over time by demonstrating regular food intake and resumption of climbing, running, and playing ,40,41,44The histology – morphology revealed a final CNS-PNS connection by describing and explaining in detail all aspects of interest in this research, e.g., individual microsurgical free-hand positioning of NG within and next to the SC and of the graft itself inside and outside of the SC as well as its adaptation at the IOAM-NEZ, together with all diagnosed primary and secondary surgical lesions related to the given RCT design ,31-39. DFigure 7. AStatistical evaluation (Wei-Lachin procedure) of the specimens for IOAM fiber diameters were analyzed and identified by the histometric assessment. PNG-induced functional neuroregeneration was evident in both independently assessed measurements (ML and CC)  11,17,1,42-44. AIndirect immunofluorescence of VGluT2 was used in two CAMP+ cases to diagnose graft-induced neurotransmitters in PNG and IOAM specimens . AB. To Replication of GB's concept shows the capacity of spinal VGlutT2 neurons to regenerate PNG and to produce terminals that reinnervated the IOAM skeletal muscle (GB paradigm) that correlates with the Western blot analyses of coexisting acetylcholine and glutamate neurotransmitters shown both within the SC and the regenerated IOAM nerve. The co-neurotransmission is in contrast to GB's paradigm, and we did not reveal any retrograde FB+ tracer fluorescence within the brainstem or brain. When our intraoperative FB+ application (during the second operation) was different from GB's technique, the application was consistent with the labeling techniques used by M. Schwab and coworkers ,44. ThisIrrespective of GB's promising scientific reports, the paradigm for partial functional restoration remains controversial without additional external proof. Every novel medical concept in restorative surgery, such as here for spinal cord repair, should be first replicated, validated, and discussed in light of GB's report. Clinical fMRI performance of client G during her brain functional diagnostic 15 yers after GB's grafting procedure in Italy "has upset all the doctors present because they saw a patient with a broken medulla and a client that pedaled, extended knees and abducted her hips in following the radiologist's commands." The car accident occurred at the time of her wedding, and she got divorced soon after her SCI and had to learn to manage life on her own. Over the years, she has been in personal, friendly contact with GB and his wife, Luisa Monini Brunelli, Professor of Orthopedics, who assisted in the grafting operation in 2000. The patient, unfortunately, has refused any state of the art professional neurological/neurophysiological/neuro-radiological follow-up examination and external medical audit. However, the assessment of the surgical results by an objective third party is mandatory to objectively evaluate the first experimental of GB's microsurgical concept regarding neuroregeneration in traumatic thoracic paraplegia (the operation was performed by GB and KvW). Her detailed fMRI evaluation by a third party at the Department of Radiology, Münster UKM, will be published in the near future in conjunction with her first professional external neurological expert assessment. This paper focuses on understanding the special role of the rubrospinal tract in the functional regeneration of denervated muscles after PNS-CNS grafting. Second, the role of intensive neurorehabilitation for the functional outcome is stressed to induce regeneration and to maintain partial restoration of skeletal muscle groups after successful grafting when translated into human SCI repair. Aspects of subjective wellbeing and personal HRQOL following SCI are still underestimated ,56,60,61Prof. Andrea Falini from Milano wrote in her original medical report of GB's first fMRI when analyzing her voluntary muscle activities by obeying different simple motor tasks both physically and/or imaginary:" Having given the patient orders during her only fMRI studies to do certain movements, for example, tasks such as abduction or extension of hip muscles and then to "think" only. In conclusion, the preliminary report of data analysis, which we tried to carry out by automatically identifying the membership of the activation clusters to cortical areas defined according to atlases implemented in the processing software. We had the impression that the motor stimulus of the right lower limb produced a rather typical activation in the left paracentral lobule and in the premotor area, with some clusters in the right frontal operculum (area 44?), and perhaps temporal left, which deserve attention." Another external audit of the total examination following our strict criteria and performed by an expert in functional neuroradiology at Münster UKM, Germany, is forthcoming and will be discussed in more detail in a future medical report that will focus on brain reorganization after SCI and restoration of muscle functioning ,47.Numerous surgical, medical, and biochemical strategies that have been based on experimental trials are recommended and used clinically in human SCI rehabilitation to foster the restoration of voluntary muscle function and beneficial personal HRQOL after traumatic spinal cord disruption with permanent paraplegia ,60, 61. Current clinical treatment strategies are targeted at promoting axon regeneration and outgrowth beyond the scar formation in SC avulsion. The strategies are all based on the preserved central plasticity of a functioning upper SC. Neuroplasticity is the ability of the nervous system to respond to intrinsic or extrinsic stimuli by building and reorganizing its structure, function, and connections ,45,51,53In accordance with Dopkin's demand and the statement of Papastefanski and Matas, new strategies are needed in neuroscience to improve the transparency of experimental descriptions by uniform reporting. This first replication of Brunelli's grafting procedure in a rat model provides new insight into graft-induced central neuroplasticity and neuroregeneration of denervated skeletal muscles, and the cortical-rubrospinal tract has shown to be crucial in neuroregeneration, which was first discussed as the fundamental structure by GB in his CNS-PNS concept and is now also confirmed in stroke rehabilitation. The actual evidence of acetylcholine/glutamatergic co-transmission in the grafted muscle must be replicated in a more extensive series, including pharmacological neuromodulation and additional behavioral outcome assessments by extended follow-up controls. SCI paraplegics may receive GB's clinical restorative microsurgery when presenting CNS-PNS graft-induced glutamatergic/cholinergic co-transmission, which will open a new discussion of the targeted aspects of neuroregeneration and neurorehabilitation. Eighteen years of follow-up and actual fMRI examination after GB's grafting of incomplete thoracic SCI provide evidence that the client was able to voluntarily stand and walk using tripod sticks with good personal physical-medical and mental-social quality of life (HRQOL). However, this type of experimental surgery requires the client's explicit consent with regular neurological/neuropsychological and social follow-up examination and an external audit, including fMRI, at least four years after the intervention. Over the last two decades, we have learned from our numerous clients in restorative microsurgery and in scientific discussions with other specialists and rehab doctors worldwide that these recommendations are best for the paraplegic patient's neurorehabilitation.The authors confirm that there are no conflicts of interest.


Genome Biology and Evolution has turned ten years old. Founded by Bill Martin  and Takashi Gojobori  as the first open access journal to be owned by an academic society, the Society for Molecular Biology and Evolution, the journal has gone from strength to strength and now receives over 500 manuscripts a year, of which about 60% get published. These vary tremendously in terms of subject matter, organismal focus and methodological approach, but they are bound by a common theme: to understand evolution using genome-scale data.Drosophila and Lamarckian theory, highlighting the breadth of the journal. However, as with all journals, most papers receive far fewer citations than these, and we take pride in publishing excellent quality manuscripts from emerging fields.GBE has published ground-breaking and controversial science over the last ten years. Our most highly cited article is a well-reasoned critique, by Dan Graur  and colleagues, of the ENCODE project’s claim that most of the human genome is functional. Among our other top cited manuscripts are those that focus on transposable elements (i.e. the mobilome), archaeal genomes, plastid origins, non-protein-coding RNAs, With the combination of decreasing costs of sequencing and increasing availability of open source analytical tools, we expect the field of genome evolution to expand geographically. Currently, a large proportion of the papers we publish come from North America, Western Europe and East Asia, but we are proud of the fact that we published papers from 45 countries in 2018. As part of our strategy to increase submissions from around the world, we are aiming to appoint members of the editorial board from countries that currently have smaller communities of researchers studying genome evolution.Laura A. Katz and Adam Eyre-Walker. Laura works on phylogenomics and genome evolution in microbial eukaryotes, and Adam on adaptive evolution and the distribution of fitness effects. We have published a number of articles in GBE and GBE’s sister journal MBE, been on the editorial board of the journals, and we are long-term members of SMBE.Following the lead set by its parent society SMBE, the journal, under the leadership of Bill Martin, has done much to promote women, and approximately half the editorial board is female. It therefore seems fitting, that after ten years at the helm, Bill Martin has stepped down as Editor-in-Chief and been replaced by not one but two Editors-in-Chief, We look forward to continuing to develop the journal, and we are eager to hear suggestions from the community. We welcome analyses of genomic-scale data, theoretical advancements, and method development. We have recently updated the description of article types, and, though we anticipate that most of the manuscripts will be full length articles, there are also opportunities to submit shorter letters, genome reports of data that will be useful to the community, and software/method updates. We are also looking to commission additional reviews and perspectives, the latter allowing you to engage in controversy in our field. We welcome suggestions for debates, which might take the form of two opposing perspectives. In short, manuscripts that help us understand evolution at the genome level are encouraged in a variety of shapes and forms.GBE is an open access journal, published by Oxford University Press, a not-for-profit publisher. GBE relies on author publication charges to fund its editorial and publishing operations. Any profit that the journal makes is ploughed back into the community through the Society of Molecular Biology and Evolution. However, as a society-owned journal, we believe it is important to publish science irrespective of the ability to pay, and hence we will consider applications for waivers on a case-by-case basis.Although GBE has had a Twitter account in the past, it has remained underused. However, that is set to change. We have taken on Casey McGrath, who did her undergraduate work with Laura and her PhD with Michael Lynch, as our social media and highlights writer. She has established Facebook and Instagram accounts and will be populating these, along with Tweets, to increase the profile of the papers that GBE publishes. You can follow us @GenomeBiolEvol.We look forward to receiving your submissions and suggestions as we guide the journal over the coming years.


Several factors such as recipient age, BMI, serum cratinine, and positive history of dialysis are important in predicting graft survival among kidney transplant recipients. One factor affecting the transplant outcomes is donors and recipients gender, which is usually ignored.A total of 1113 kidney transplant recipients were studied in this retrospective cohort study. Several factors were taken into account for graft survival and outcomes such as donors and recipients gender and age in addition to common recipient factors like cratinine, eGFR, BMI, and positive history of dialysis.The most successful transplant based on donor-recipient gender was observed in male donor to male recipient, and then male donor to female recipient. In female transplant recipients, level of serum cratinine and eGFR, positive dialysis history before transplant, and low donor hemoglobin level can be considered as good prognostic factors recommended for kidney transplant survival.Our results suggested gender matching for kidney transplant. Only in some exceptional conditions, male donor to female recipient kidney transplant may be successful and female donors to male recipients are not suggested, especially in aged patients with the history of dialysis. For most patients with end-stage renal disease (ESRD), the only treatment strategy is renal transplantation. Kidney transplantation has become a better cost-effective alternative to dialysis as a result of several improvements in early graft survival and long-term graft function. The first kidney transplantation was performed about half a century ago in which the transplant was performed from a live donor to his identical twin. After that, the concept of living and non-living unrelated donor resulted in increasing the number of organ donation in ESRD, and the number of kidney transplantations has escalated over the last ten years. More than two thousand kidney transplants have been reported in Iran, among which 50% were deceased (brain death) cases.Several factors may have an impact on patients and/or graft survival, and also on transplant outcomes. Donor factors such as age, female gender, brain death of cerebrovascular cause, and prolonged criteria donor status had a noteworthy effect on the renal graft function –3. Both In this study, we aimed to investigate the impact of gender match between kidney donor and recipients. Moreover, we had the purpose of evaluating the predictive markers of graft survival.In this retrospective cohort study, among 2000 graft recipient patients who were registered in transplant center of Shariati Hospital transplant data bank from 2002 to 2018, 1113 renal graft recipients were chosen for our research. All selected patients were similar in being living donors; however, they were different in having the same donor gender and relative or non-relative donor. Patients with positive cross-match, incompatible blood group, age < 18 years old, multiple organ transplantation history, positive chronic viral B, C hepatitis, HIV, pregnancy, and diabetes were excluded from the study. In addition, patients with positive history of liver diseases such as Cirrhosis, Autoimmune hepatitis (including Gamma globulin serum & FANA), primary biliary cirrhosis, biliary obstruction, hemochromatosis, alpha-1- antitrypsin deficiency, Wilson’s disease , Chronic disabling diseases , and known cancers were excluded from the study.Delayed graft function was defined by reduction in urine volume (≤400 ml/24 h) or requiring dialysis one week after graft receipt. The biopsy was done for patients who were at the risk of rejection with more than 30% increase in their basic levels of cratinine. Anti-rejection medication was Methylprednisolone and on the occasion of its resistance, Anti-thymocyte globulin (ATG) was the alternative. Transplant rejection was defined as dialysis need for more than 30 days, death, or graft excision. Patients included in this study were categorized into four distinct groups as group 1 , group 2 , group 3 , and finally group 4 . Clinical data such as duration of dialysis before kidney transplantation, serum cratinine, and eGFR (Estimated Glomerular Filtration Rate) for transplant recipients were considered. In addition, some demographic information belonged to both recipients and donors like age, BMI in both donor, and recipient and donor’s hemoglobin were considered. Graft rejection, needing dialysis again, and death were checked for several factors using multiple logistic regression models.p-value less than 0.05 was considered as significant. To determine the factors having an independent impact on graft survival, a Cox proportional hazard analysis was utilized.Actuarial survival was assessed by Kaplan-Meier test, and the log derivation of the survival percentage was employed for the half-life predicting of grafts and/or patients. Differences in survival were done through log-rank test, and 3/min in the first month, 62.0 ± 21.4 cm3/min after six months, and 62.15 ± 21.1 cm3/min by passing one years from transplant. These decreasing trends in the amount of serum creatinine and the increasing trend in serum eGFR were meaningful . Table The average creatinine levels (mean and standard deviation (SD)) were 1.65 ± 1.46 mg/dl, 1.63 ± 1.29 mg/dl, 1.37 ± 0.69 mg/dl and 1.37 ± 0.66 mg/dl for one week, one month, six months, and one year post-transplant, respectively. The mean eGFR was 58.2 ± 22.2 cmSide effects and complications after surgery were just seen in 4.6% of the patients. Graft rejection was reported in about 51 cases (4.6%) of graft recipients, and 130 (11.7%) of the patients were made to have dialysis after transplantation. There were 41 (3.7%) post-transplant death cases. Surgery Consequences, side effects, graft rejection, dialysis return, and death caused by transplant in recipients are presented in Table Based on the results of Kaplan-Meier test, the survival percentage was 97.3, 95.7, 95.1, 94.8, 94.4, and 93.7% one year, two, three, four, five, and ten years’ post-transplant, respectively , delayed graft function (DGF) , recipients age , primary serum level of creatinine and eGFR , and donor hemoglobin levels  are critical elements of graft rejection. Based on the results shown in Table Predictive factors for mortality, graft rejection, and back to dialysis in both donor and recipient genders were age (esp. in transplant recipients), serum creatinin, eGFR, level of donor hemoglobuline, history, and duration of dialysis before kidney transplant. The result of multiple logistic regression analysis of graft rejection, dialysis return, and death recipients are presented in Table The multiple regression models showed that kidney transplant recipients with the history of dialysis, older age, higher Creatinine/eGFR, and higher hemoglobin levels in their donors are more susceptible to the return of dialysis after transplantation. In addition, higher Creatinine/eGFR, BMI and older age of recipients (≥35 years) can decrease the risk of transplant survival, and also increase the risk of death in graft recipients. Figure Our observational study indicated that gender match between kidney donor and recipient is an important graft survival determinant. More than sex match, transplant survival was dependent on younger age of recipient and having the history of dialysis before transplant. Several factors such as the incidence of post-transplant hyperglycemia, its cardiovascular disease as the Pre-transplant characteristics, and particularly, the glycemia during the first month post-transplant identified patients with the risk of post-transplant diabetes, suggested by some studies can change the long-term survival in renal transplant recipients with graft function . For exaPeter Stenvinkel et al. suggested that as inflamed female’s patients have a better outcome compared to inflamed males, because sex hormones may have important cardioprotective effects that limit the effect of inflammation on vascular injury in female patients with end-stage renal disease (ESRD). This can be the reason that female recipients, even from male donors, showed higher transplant outcomes in comparison to male recipients . Additional studies must conclude whether sex- and age-specific immunosuppressant is warranted for kidney graft recipients –15. DespOur data indicated that women are good donors on the basis of hemoglobin; however, on the basis of contradictory gender, we conclude that men are better donors. In female donors, individuals with lower hemoglobin are considered to be more appropriate as the donor than women with higher hemoglobin (within group), and in men with lower hemoglobin compared to higher hemoglobin. Time of being in waiting list of transplantation and duration of dialysis can be confounders such as the donors and recipients age. Our data suggested that in addition to recipients’ age and gender matching as well as positive history of dialysis can be a prognostic element of transplant survival. Several previous studies pointed out the natural history of permanent renal dysfunction and severe liver failure in liver transplant recipients that can be helpful in the progress of non-nephrotoxic immunosuppressive regimens for high-risk liver transplant recipients , 22.. The advantage of living-donor versus cadaveric-donor transplantation can be the reason for waiting time. Cadaveric renal transplant recipient with an end-stage renal disease time ≤ 6 months has the equivalent graft survival of living donor transplant recipients compared to those waiting on dialysis waiting list for ≥2 years . In fact, increased time on dialysis before kidney transplantation is linked with the reduction in survival of transplant recipients.A paired donor kidney analysis indicated that waiting time on dialysis can be the strongest modifiable risk factor for renal transplant outcomes . The adv≥2 years . In factBy far, the most successful transplants, based on donor-recipient gender, were seen in male donors to male recipients, and then male donors to female recipients. Contradictory, the most unsuccessful transplant was observed when the donor was female and the recipient was male. In female transplant recipients, the level of serum cratinine, and eGFR, positive dialysis history before transplant, and low donor hemoglobin levels can be good prognostic factors in kidney transplant survival. By Judging these results based on hemoglobin yields, if we take Gender into account, we get inconsistent results. Therefore, further studies are needed to complete this section.”


In December 2016, the executive committee of the European Society for Cognitive Psychology (ESCoP) faced a momentous decision about whether to continue sponsoring and working on a subscription-based journal we did not fully control. We decided unanimously to abandon our journal, which was owned by its publisher, and instead establish an open-access journal focused on cognitive psychology and cognitive neuroscience. We saw an emerging need for an open-access journal with fair pricing, which embraced modern standards of transparency and openness about research methods while maintaining the quality and rigor expected from a traditional peer-reviewed journal. The Journal of Cognition, now ESCoP’s official journal, was born.https://www.journalofcognition.org/collections/special/the-confluence-of-sound-and-cognition/), organized by John Marsh and Rob Hughes, and another focusing on pupillometry , organized by Sebastian Mathôt. We featured a review article by Jan Theeuwes . We have published our first completed Registered Report . A portion goes to our publisher for essential services, a portion goes to editors (who are paid a per-manuscript honorarium regardless of whether they decide to accept or reject the paper), and some of the fee is intended to defray processing charges for articles from institutions that cannot afford to pay fees. Furthermore, ESCoP members receive a discount when publishing their own work. Editorial board members, all of whom have committed to review incoming papers within their area of expertise promptly and constructively, earn a further discount for everyone at their institution. We continuously accept nominations for well-qualified editorial board members willing to take up this commitment to benefit their institution. Any profit ESCoP receives from article processing charges will be spent to promote cognitive psychology in Europe .ESCoP envisioned that a shift toward open-access publishing was coming, and Journal of Cognition is already fully compliant with Plan S. All of our content is published under a Creative Common License. All of our content is made publicly available as soon as it is produced. Our maximum article processing fee, €1150 per article, is both competitive  and is transparently explained on our website .If you are a cognitive scientist, especially if you are based in Europe, join ESCoP . Proposals should include working titles of papers that will be submitted for consideration from 5–8 corresponding authors from a variety of institutions.Consider proposing a special collection to the Journal of Cognition. The article processing charges for articles chosen for an approved special collection are defrayed by ESCoP. You may propose a collection directly to the editor by email . You can follow us on Twitter (@JCgntn) or Facebook or join our mailing list (http://eepurl.com/dhdAtX) for updates about new content.Read our content regularly (Talk to representatives at your university about fair open access, and lobby if necessary for provision for covering article processing charges at your institution.journalofcognition.org.Register as a reviewer for Journal of Cognition by clicking on the “Become a Reviewer” button at Many thanks to all the authors, reviewers, and editors who contributed to the making of the inaugural volume of the Journal of Cognition. We hope that even more of you will be inspired to help us continue to make Journal of Cognition a success as we get to work on Volume 2 in 2019.


M14 from a mutant pool induced by 60Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB, which encodes pyruvate orthophosphate dikinase . Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant  In cereal endosperm, starch is synthesized in amyloplasts and forms starch granules. Mature seeds of cereals such as rice, maize and wheat contain large amounts of starch. It provides a substrate for seed germination and seedling growth. In addition, endosperm starch makes an important contribution to the human diet throughout the world .d-glucopyranosyl units, whereas amylopectin is a highly branched macromolecule [ISA1 produces a sugary endosperm and accumulates abundant phytoglycogen instead of normal starch granules [amylose-extender (ae) mutant lacking BEIIb produces a white-core endosperm and alters the fine structure of amylopectin [Starch consists of two major components, amylose and amylopectin. Amylose is a linear polymer made of α-1,4-linked molecule . The enzmolecule . Mutatiogranules ,6. The alopectin .FLO2 (FLOURY ENDOSPERM2), encoding a tetratricopeptide repeat motif-containing protein, affects the starch quality by regulating the expression of starch synthesis-related genes [FLO6 contains a CBM48 domain in the C-terminal and regulates the starch synthesis and starch granule development by interacting with ISA1 [FLO7 influences the amylose content and amylopectin structure in the outer endosperm [FLO8, encoding the UDP-glucose pyrophosphorylase 1 (Ugp1), modulates the starch synthesis and structure [FLOURY genes, other genes can also cause floury endosperm phenotype after loss of function, such as Grain Incomplete Filling1 (GIF1) encoding a cell wall invertase, Fructose-6-phosphate 1-phosphotransferase (PFP) encoding a fructose-6-phosphate 1-phosphotransferase β subunit, and Glutelin Precursor Accumulation3 (GPA3) encoding a plant-specific kelch-repeat protein [In addition, some mutations in genes that are not directly involved in starch metabolism also affect starch synthesis. ed genes ,9. FLO6 ith ISA1 . A domaindosperm . FLO8, etructure . Besides protein ,14,15. T4 plants is most well known as a photosynthetic enzyme, which catalyzes the formation of CO2 acceptor phosphoenolpyruvate from pyruvate [4 species, contains three PPDK genes, 4ppdkZm1C, cyppdkZm1 and cyppdkZm2 [cyPPDKZm1 can be regulated by the O2 (Opaque2) gene [3 tissues, PPDK is either not present or at very low abundance [4-type chloroplastic PPDK (chOsPPDKB) and cytosolic PPDK (cyOsPPDKB) [GUS) gene driven by cytosolic OsPPDKB promoter is conducted in rice. The GUS activity shows a high level in developing kernels, but a low level in the flowers and roots [OsPPDKB shows a white-core endosperm, indicating the essential role of OsPPDKB in modulating the carbon flow for starch and lipid synthesis [cyOsPPDKB mutant Suweon 542 with a milky-white floury endosperm has been identified and characterized [OsPPDKB mutation on starch development, starch physicochemical properties and starch metabolism are still unknown.Pyruvate orthophosphate dikinase  catalyzes the interconversion of pyruvate, ATP, and Pi with phosphoenolpyruvate, AMP, and PPi . PPDK inpyruvate . The actpyruvate . Maize, yppdkZm2 . During e2) gene . In mostbundance . In riceOsPPDKB) ,23. A cond roots . The T-Dynthesis . Recentlcterized . HoweverM14. Map-based cloning and complementation test revealed that the mutant phenotype was caused by OsPPDKB, which is a new allele of FLO4 [M14 and its wild type (WT) were investigated and compared. Our data showed a vital importance of OsPPDKB in starch metabolism and structure in rice endosperm development.In the present study, we characterized a floury endosperm mutant,  of FLO4  from ricM14 with a floury endosperm phenotype was identified from a 60Co-irradiated mutant pool of japonica rice variety Kitaake, and displayed no visible differences from WT plants at the vegetative stage. The brown rice of M14 exhibited an opaque endosperm when compared with WT seeds  was prepared into semi-thin sections. I2-KI staining displayed that amyloplasts in the outer endosperm of M14 and WT developed well and filled with densely packed, polyhedral starch granules  (M14 (13.8%) compared with WT (15.8%) , and their thermal parameters are shown in M14 starch were all about 2 °C lower than those of WT starch. The pasting properties of WT and M14 starches determined by rapid visco analyzer (RVA) are presented in M14 starch had significantly lower peak viscosity, hot viscosity, and final viscosity than WT starch. In conclusion, the physicochemical characteristics of M14 starch changed significantly compared with WT starch.The seeds of WT had a starch content of 79.1%, whereas the  (73.6%) A. The am (15.8%) B, but pr (15.8%) C. The am with WT D. The th2 population was produced from reciprocal crosses between WT and M14. All F1 seeds displayed WT phenotype, and the F2 population exhibited a 3:1 segregation ratio . Therefore, the floury phenotype in the M14 was controlled by a recessive nuclear gene. In order to identify the mutation locus controlling the M14 phenotype, the approach of map-based cloning was adopted. An F2 mapping population with approximately 5000 progeny was created by crossing the M14 with the wide-compatibility indica variety Dular. For bulk-segregant analysis (BSA), two DNA pools were generated from 10 F2 floury endosperm and 10 normal endosperm individuals, respectively. These two pools were genotyped with 170 selected insertion-deletion (InDel) markers for rough mapping. The M14 mutation locus was initially mapped between InDel marker IZ5-1 and IZ5-4 on the long arm of chromosome 5. On this basis, 854 recessive individuals were selected and detected by the two pairs of primers to screen recombinants. To further narrow down the locus, six new InDel markers were developed. The number of recombinants gradually decreased as primers were closer to the target gene    A. The gee (PPDK) B. Amino e (PPDK) B. The exndosperm .OsPPDKB gene contains two transcripts, chOsPPDKB and cyOsPPDKB, and the cyOsPPDKB expressed at a high level in developing kernels [Os05g0405000 was responsible for the M14 mutation, the pUbi:cyOsPPDKB was introduced into the M14 to complement the floury phenotype. Positive transgenic lines expressing pUbi:cyOsPPDKB were identified by PCR analysis  contains only a white-core endosperm in the center of seeds [Starch, as the main storage material, occupies most of the volume of rice endosperm . Defectshenotype . Despite content A,B than nt  in maize is seedling lethal. One hypothesis is that other genes with a similar function in photosynthesis might offset the loss caused by mutation in the chOsPPDKB gene in M14. Unlike rice, there are no significant differences in amylose content and the fine structure of amylopectin in maize PPDK-deficient transgenic seeds [M14, the amylose content and amylopectin structure changed significantly  participation in the process of gluconeogenesis and provision of hexose for starch synthesis, (ii) providing pyruvate for the synthesis of lipids, and (iii) regulating the sucrose synthase by controlling the homoeostasis of PPi ,35,36. T2 mutant . This reynthesis . The devynthesis . PPDK isynthesis ,40. The ynthesis . Indeed, reduced . Therefojaponica rice variety Kitaake were irradiated with 60Co-γ ray at the dose of 300 Gy. Mature grains of M1 plant were harvested respectively and dehusked with a rice huller to screen endosperm mutants. A floury endosperm mutant M14 was identified. The M14 and an indica variety Dular were hybridized to produce F2 population for map-based cloning. The WT and M14 were planted at the experimental farm of Yangzhou University in May and harvested in September.The grains of M14 kernels at 9 DAF were fixed overnight in 2% (v/v) glutaraldehyde. The samples were dehydrated in an alcohol series, embedded in LR White resin, and sectioned using an ultrathin microtome . Semi-thin sections were dried at 40 °C, stained with I2-KI, and observed with a light microscope .The samples were prepared following the method as described previously . BrieflyMature rice grains were air-dried and dehulled. Brown rice was further ground with a mortar and passed through a 0.15 mm sieve. The total starch content in the flour was determined with a total starch assay kit . The amylose content was measured by using an iodine-potassium iodide colorimetric method as described previously . The cha2 population of M14 (japonica) and Dular (indica) for map-based cloning. One-hundred-and-seventy genome-wide InDel markers showing polymorphism were selected . The first-strand cDNA was reverse transcribed by priming with oligo (dT18) based on HiScript 1st Strand cDNA Synthesis Kit . The qRT-PCR was performed on a CFX Connect real-time PCR system  using AceQ qPCR SYBR Green Master Mix Kit . The PCR procedure was carried out using the following program: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The rice Actin1 was employed as an endogenous control. Quantitative gene expression was analyzed from three biological replicates by the 2Ct−ΔΔ method, where ΔΔCt =  M14 −  WT [OsPPDKB and Actin1 were listed in Total RNA from various tissues of WT and tin1) WT . The priOsPPDKB (chOsPPDKB and cyOsPPDKB) were cloned in the pCAMBIA1305-GFP vector to create 35s::chOsPPDKB-GFP and 35s::cyOsPPDKB-GFP. The two binary vectors were transformed into Nicotiana benthamiana leaves following the method as described previously [The two transcripts of eviously . Laser sM14 at 9 DAF were harvested, and the embryo and pericarp were removed subsequently. Proteins were extracted following the method as described previously [In order to separate the endosperm tissue, the developing kernels of WT and eviously , and traeviously .M14 was isolated from Kitaake grains irradiated with 300 Gy 60Co-γ ray. Map-based cloning analysis showed that the floury endosperm phenotype was controlled by cyOsPPDKB. Abundant single and abnormal starch granules were observed in M14 endosperm. Compared with WT, amylopectin short branch-chains with DP 6–12 increased, but intermediate branch-chains with DP 13–50 decreased in M14. The M14 starch had a significantly lower gelatinization temperature and peak, hot and final viscosities. The chloroplast-localized PPDK highly expressed in leaf and leaf sheath and the cytoplasm-localized PPDK exhibited a high expression in developing endosperm. The expression of starch synthesis-related genes was significantly altered in M14. The substitution of single amino acid in M14 affects the catalysis of PPDK on the reaction of Pyruvate to PEP, leading to the change of starch metabolism and structure through regulating the expression of starch synthesis-related enzymes in rice endosperm. This study contributed to an understanding of the role of PPDK in starch metabolism and structure and provided some information for quality breeding of rice.A floury endosperm mutant


Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for “Bimodal Starch Granule”. Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed. Several additional criteria distinguish starches depending on their botanical origins. They include the size of the granules (from 0.1 to up to 100 µm in diameter) or their shapes which can be ovoid, ellipsoidal, spherical, angular or lenticular5. Whilst our knowledge of the enzymatic reactions leading to starch synthesis has strongly improved during the last two decades, factors determining starch granules shape and size remain to be identified. Most starch accumulating organs contain one type of granule shape the size distribution of which is usually unimodal  with the notable exception of Festucoideae species such as wheat (Triticum aestivum), barley (Hordeum vulgare) or rye . In these plants, starch granule size distribution is bimodal8; large disk-like granules  and smaller but spherical granules  accumulate in the endosperm9. Synthesis of the two types of starch granules is not simultaneous, as the B-granules appear several days after the large A-granule during grain filling11. While the majority of plants accumulates storage granules with specific size ranges and shape in sink tissues, the starch built in the source organs  is unlikely to be identifiable to plant species as its shape is not specific and is likely to be determined by the space available at the site of synthesis12. In the unicellular green microalgae Chlamydomonas reinhardtii, different types of starch can be produced depending on growth conditions. Indeed, under unrestricted growth conditions, cells divide actively and accumulate photosynthetic (transitory) starch granules resembling the one found in higher plants source organs while a storage-like starch is synthesized under stress conditions, which harbors by many criteria the characteristics of endosperm storage starch16. While actively dividing, Chlamydomonas cells accumulate starch mostly around the pyrenoid, a specialized intraplastidial structure composed of aggregated ribulose-1,5-biphosphate carboxylase/oxygenase responsible for CO2 fixation. In such growth conditions, most starch is found organized as a sheath surrounding the pyrenoid17. In contrast, when subjected to a stress such as nitrogen starvation (-N), starch granules are massively accumulating in the stroma. After few days in these unfavorable conditions, starch granules are almost filling the cells15. This massive accumulation correlates with the loss of photosynthetic activity and disorganization of the thylakoid membranes18. The pyrenoid is hardly detected and no starch sheath can be detected around the remaining pyrenoid-like structure suggesting that it has been either degraded and/or modified15. Transitory and storage-like starches can also be distinguished by the structure of amylopectin (amylopectin chain length distribution (CLD) is different) and the amylose content (higher in storage-like starch)15. Even if both pyrenoidal and storage starches are synthesized by the same set of enzymes, differences in structure and composition reveals different enzymatic contributions in the processes. For instance, the study of a KO mutant of Chlamydomonas defective for one of the plastidial isoforms of starch-phosphorylase showed that this enzyme is required for correct storage starch synthesis but had no function in transitory starch synthesis19.Starch is the major storage polysaccharide in plants and accumulates in the form of insoluble granules inside the plastids of the plant cells. Starch is composed of two types of α-glucan polymers, amylose and amylopectin, which represent approximately 98–99% of the granule dry weight. The ratio of the two polymers varies according to the botanical origin of the starch but is generally comprised between 20–30% (dry weight) of amylose and 70–80% of amylopectin. Amylopectin consists of short linear chains of glucose units linked by α-1,4 glycosidic bonds and contains around 5% of branches at the α-1,6 position. Amylose is composed of long linear glucose chains containing less than 1% of branch points. The highly ordered organization of glucose chains in amylopectin confers specific physicochemical properties to this polysaccharide. Starch is semi-crystalline but the allomorphic type and degree of crystallinity vary depending of the botanical and/or genetic sourcesChlamydomonas reinhardtii mutant carrying a small deletion on chromosome 2 induced by insertional mutagenesis. This mutant has a reduced rate of starch degradation and a modified amylopectin structure. When grown under nitrogen starvation, this mutant has a phenotype similar to that reported in cereals endosperm accumulating two populations of starch granules with distinct sizes. The first population contains starch granules similar to that of the wild-type while the second is composed of abnormally large granules with irregular shapes. Because of this original phenotype, the mutant strain was named bsg1 for “bimodal starch granule distribution”. Genetic and functional complementation experiments allowed us to identify a candidate BSG1 gene that was confirmed by the phenotypic characterization of a second mutant allele. Structural characterizations of both mutant granule populations and electron microscopy observations suggest different origins of the two types of starch granules. Possible function of the BSG1 protein leading to this unique phenotype of the mutant is discussed.In this work, we have isolated and characterized a Chlamydomonas reinhardtii wild-type strain 137C to identify new components of the starch degradation machinery20. From this mutant library, we have selected one mutant with an abnormal starch phenotype which was selected as a putative starch catabolism mutant20. Surprisingly, this mutant accumulated less starch than the wild type after a 5-day period in condition of massive starch accumulation. Indeed, this mutant showed a lighter color than the wild type when cell patches on Petri dishes were stained with iodine vapors . Thus, after a 24-h period of degradation, the mutant still contains 76 ± 14% the initial starch amount (measured at the end of the massive accumulation period) while the wild type contains 42 ± 6% of starch. As suggested by iodine staining and surprisingly, even if starch degradation was impaired in the strain, it accumulated half as much starch as the wild type when grown under nitrogen starvation  PCR on bsg1-1 genomic DNA allowed the localization of one insertion of the cassette into chromosome 2. We were able to amplify by PCR a fragment containing 87 bp corresponding to the end of the paromomycin cassette and 446 bp of the flanking genomic sequence when the genomic DNA purified from the mutant was used. This flanking sequence located at the 3′ end of the inserted resistance gene, matches to the 11 last coding nucleotides of the Cre02.g091750 locus and the following genomic region . It is well known that insertional mutagenesis can lead to multiple insertions of the resistance cassette in the genome and to chromosomic rearrangements such as deletions23. Using specific primers annealing either to Cre02.g091750 or to the adjacent loci, the bsg1-1 mutant was shown to carry a deletion of approximatively 15 kbp at the insertion site , Cre02.g091850 (annotated as a methylglutaconyl-CoA hydratase) and Cre02.g091750  genes were deleted. Glucan, water dikinases (GWD) and phosphoglucan, water dikinases (PWD) are key enzymes of the initial steps of starch degradation25. These starch phosphorylating enzymes are required for efficient starch degradation in leaves and share a common catalytic mechanism in which a conserved histidine residue is involved in the transfer of the β-phosphate group of ATP to the glucan28. The homology of the protein encoded by the Cre02.g091750 locus with dikinases only implied a restricted part of the polypeptide (from amino acids 72 to 150) corresponding to a carbohydrate-binding domain (CBM20) at the N-terminal extremity of the protein and sharing 29% identity (65% similarity) with the CBM module found in the Arabidopsis PWD (At5g26570). Moreover, this homology didn’t cover the part of PWD containing the essential catalytic histidine residue. When the complete protein sequence was used to try to find homologs in higher plants, all attempts failed. The only homologs were identified in microalgae close relatives to Chlamydomonas such as Volvox although homology with the Chlamydomonas protein was low . The lack of expression of Cre02.g091750 in the bsg1-1 mutant was confirmed by RT-PCR performed on total RNAs samples prepared from either log-phase growing-cells or cells submitted to nitrogen starvation for 1 hour , two segregants were resistant to paromomycin and two sensitive indicating that only one functional copy of the mutational paromomycin cassette has been inserted in the genome of the mutant. The consequence of the insertion of the paromomycin cassette at the Cre02.g091750 locus was checked in all seven tetrads. A mendelian segregation was obtained with two spores carrying the wild type allele and two carrying the chromosomal deletion . Second, the starch degradation phenotype was scored in the progeny. The segregation pattern fitted that expected for a single mendelian trait  annotated as PWD. We suspected that the starch phenotype of the bsg1-1 mutant was the consequence of the specific lack of this gene. We have tested this hypothesis by reintroducing a wild-type copy of this gene in the mutant. To this end, the 3.7 kbp region between the start and the stop codon of the gene Cre02.g091750 was amplified and cloned under the strong PSAD (a subunit of photosystem I) promoter into the pSLHyg plasmid containing the Aph7″ gene conferring hygromycin resistance29. The transformants were selected on TAP plates containing both paromomycin and hygromycin. Starch of twenty transformed strains resistant for both paromomycin and hygromycin was analyzed for particle size distribution. Eighteen of them displayed a unimodal starch granule distribution with peak size of 0.75 ± 0.03 µm . Furthermore, the complemented strains still degraded starch with a rate similar to that of the original mutant and not the wild type strain . To resolve these discrepancies, we characterized the phenotype of a second insertional mutant at the same locus. The LMJ.RY0402.102699 strain from the CLiP library contains a copy of the paromomycin cassette inserted into its penultimate exon  at the Cre02.g091750 locus. We performed a complete phenotypic characterization of this strain and its wild-type counterpart (CC5325) to discriminate between the phenotypes due to a defect in BSG1 and additional phenotypes that could be the consequence of the deletion of the neighboring genes evidenced in the original bsg1-1 mutant strain.While a normal starch granule distribution was observed in the complemented strains expressing a wild-type copy of the xon Fig. . At the ain Fig. . This stPCR Fig. . Thus, tbsg1-2 mutant accumulated similar starch amounts than the wild type CC5325 in both growth conditions tested .The max values recorded for these two strains in both conditions  was similar to that of the bsg1-1 mutant starch obtained from cells cultivated in mixotrophic conditions  close to the value recorded for starch produced by the mutant in mixotrophic conditions . Cells were visualized 8, 24 and 120 h after the switch. When starved of nitrogen source, Chlamydomonas cells engage in a scavenging mechanism that yields to the formation of non-photosynthetic cells with disorganized thylakoid membranes and in which the pyrenoid is slowly degraded. In the wild type, the pyrenoidal starch sheath was unchanged after 8 h of starvation .Since the large starch granules in both ion Fig.  but becaion Fig. . In the ion Fig. . Conversion Fig.  in the bion Fig. . Few newion Fig. . After 2ion Fig. . After 5ion Fig. . The TEMChlamydomonas reinhardtii mutant strain, produced by insertional mutagenesis with a paromomycin resistance cassette. This mutant was selected for reduced starch degradation . However, the most striking phenotype of this mutant stands on the accumulation of two starch granule populations of different sizes when the cells were grown under nitrogen starvation. Cosegregation analysis performed on tetrads arising from a cross between the original bsg1-1 mutant and a wild type reference strain revealed a segregation as a single mendelian trait and cosegregation between this deletion and the defect in starch degradation, the modified amylopectin structure and the bimodal starch granules distribution but not to the low starch content phenotype. As no statistical difference could be observed between the amount of starch between the wild type and the mutant strains in the meiotic progeny of the cross, this phenotype cannot be linked to the mutation but appears to be dependent of the genetic background of the strains. This was confirmed by the bsg1-2 mutant strain that accumulates similar amounts of starch than the isogenic wild-type reference strain CC5325.In this work, we have isolated a ion Fig. . The strion Fig. . It alsoCre02.g091750 locus encodes a predicted protein of 792 amino acids for which no orthologous gene can be identified in higher plant genomes. The few proteins sharing low homology with the Chlamydomonas polypeptide was only found in phylogenetically close microalgae such as Volvox. The restricted distribution of this protein family may suggest an involvement in a metabolic pathway specific to microalgae. Concerning the defect in starch mobilization and the abnormal starch structure (high amylopectin λmax) recorded in the original bsg1-1 mutant, we did not observe the restoration of a wild-type phenotype in the complemented strains. Moreover, these phenotypes were not recorded in the bsg1-2 mutant strain in which only the Cre02.g091750 gene is deleted. It seems likely these phenotypes are the consequence of the deletion of one of the adjacent genes of the Cre02.g091750 locus. A good candidate could be Cre02.g091850 (annotated as a methylglutaconyl-CoA hydratase and putatively involved in the leucine degradation pathway) as this kind of mutation may affect the whole cellular metabolism and consequently affect starch metabolism. The responsibility of this gene could be tested by introducing back a wild-type copy in the bsg1-1 mutant strain.Functional complementation experiments allowed us identifying the gene responsible for the unusual starch granules sizes distribution. The BSG1 in starch metabolism is not fully uncovered, its importance in the process leading to the switch from pyrenoidal to storage starch metabolism is clearly demonstrated in this work. This protein contains a putative transit peptide of 50 amino acids as determined by ChloroP and a carbohydrate binding module extending from amino-acids 72 to 150 (CBM20). We have also found (by the use of three independent methods37) consistent evidences for the presence of a coiled-coil domain downstream the CBM20 into the BSG1 protein sequence. The strength of the predictions were in all cases strong and involved the same region extending from residue 302 to 380 (Supplementary Table 38. Two PTST homologs (PTST2 and PTST3) were identified in Arabidopsis thanks to their sequence similarity with PTST39. The phenotype of the corresponding single and double mutants suggested an important role of both proteins in starch metabolism39. PTST2 and 3 were proposed as interacting with soluble starch synthase 4 (SS4) which is a major factor controlling the initiation of starch granule synthesis42. In rice, FLO6 (a PTST2 ortholog) was proposed to interact with the debranching enzyme ISA1 and to influence granule morphology and grain starch content43. More recently, proteins containing coiled-coil domain typically involved in protein/protein interactions44 but without CBM were shown as mediating interactions between proteins required for correct starch synthesis. This is the case for PII1/MRC and MFP1 that were shown as essential for normal starch granule initiation in Arabidopsis46. Furthermore, with regard to sequence/structure relationships and carbohydrate binding, the CBM48 family is closely related to the CBM20 family that is found in the Chlamydomonas protein47. Finally, BSG1 was recently shown to be one of the 190 proteins present in the Chlamydomonas pyrenoid proteome48 suggesting a role in pyrenoidal starch metabolism. The large starch granules found in the bsg1 mutants grown under nitrogen starvation have a high λmax of the iodine-amylopectin complex that is characteristic of pyrenoidal starches  and 137C (mt− nit1 nit2). Standard media47 and growth conditions have been previously described49. Briefly, all experiments unless specified were carried out in continuous light (40 µE m−2 s−1) in the presence of acetate at 24 °C in liquid cultures that were shaken without air or CO2 bubbling. Late-log phase cultures were inoculated at 105 cells mL−1 and harvested at 2–3.106 cells mL−1. Nitrogen-starved cultures were inoculated at 5.105 cells mL−1 and were harvested after 5 days at a final density of 1 to 2.106 cells mL−1. For starch degradation kinetics experiments, the cells harvested after 5 days under nitrogen starvation were collected in sterile conditions and concentrated ten times by resuspension in TMP (TAP medium without acetate) liquid medium before incubation in the dark for 4, 8 and 24 h. Starch was purified from these samples and the amount of remaining polysaccharide was assayed and compared to the initial starch amount assayed before the switch to darkness. The 2 steps iodine screening procedure for identification of starch degradation mutants on solid medium has been described elsewhere20. The bsg1-1 mutant was isolated after nuclear genome transformation of the wild-type strain 137C using a paromomycin cassette as described in20. The bsg1-2 mutant (LMJ.RY0402.102699) together with its background strain CC5325 were purchased from the CLiP collection at https://www.chlamylibrary.org 50. For tetrads analyses, the bsg1-1 mutant was crossed with the wild-type strain 37 (strain 37 is a Chlamydomonas reinhardtii segregant with a genetic background derived from the wild-type reference) and the meiotic progeny was analyzed in 7 complete tetrads. Auxotrophy and mating type of each progeny were determined to ensure that segregation occurred correctly in each tetrad.The two wild-type strains of 13. Separation of starch polysaccharides by gel permeation chromatography on a Sepharose CL-2B column was performed as in51. Student’s t test were carried out using the online tool available at http://www.physics.csbsju.edu/stats/t-test.html.A full account of amyloglucosidase assays, starch purification and λmax (maximum wavelength of the iodine polysaccharide complex) measurements can be found inAll oligonucleotides used in this study are described and listed in Supplementary Table 52. Thermal asymmetric interlaced (TAIL) PCR was used to obtain the DNA sequence flanking the paromomycin cassette insertion site using the primers and cycling parameters previously described20. Homology searches were performed using the Phytozome website53. Total RNA from Chlamydomonas was extracted following an established protocol54. An aliquot of 1 μg of total RNA was used for reverse transcription (RT)-PCR analysis using the One Step RT PCR-Kit (Qiagen) following the manufacturer’s instructions. The complete Chlamydomonas Cre02.g091750 locus was amplified as a 3.7 kb fragment using the Dynazyme EXT DNA polymerase (Thermo Fisher scientific) and was subsequently cloned using the TOPO TA Cloning Kit (Invitrogen). This genomic DNA fragment was transferred into pSLHyg19 in the EcoRV site downstream of the PSAD promoter and 5′-UTR sequence. For functional complementation, the mutant strain was transformed either with an empty pSLHyg vector conferring resistance to hygromycin (20 μg ml−1) or with the same vector containing the Chlamydomonas gene, using the glass bead transformation method55. The identification of the deletion in chromosome 2 in the meiotic progeny was performed through PCR amplification of a 1078 bp fragment of the Cre02.g091750 locus using the BSGF and BSGR primers  were purified from nitrogen starved cultures of each strain and filtered through a 0.8 µm pore size filter. The flow-through containing only small granules and the retaining fraction highly enriched in large granules were checked in a multisizer 4 Coulter-counter (Beckman). Purified starch granules (approximatively 1 µg) from nitrogen starved cultures in 10 mL of IsoFlow Sheath (Beckman Coulter) were analyzed in a multisizer 4 Coulter-counter (Beckman) with a 20 µm aperture tube. The Multisizer software was set to determine the size of 30,000 particles ranging from 0.5 to 5 µm. 300 bins are logarithmically spaced between 0.5 and 5 µm (X-axis) and the size frequency distribution was expressed as relative percentage of total amount (Y-axis).Small and large starch granule populations produced by both The cells were fixed in suspension with glutaraldehyde, post-fixed with osmium tetroxide and embedded in Epon resin. 70 nm-thin sections were cut with a diamond knife in a Leica UC6 ultramicrotome and post-stained with uranyl acetate and lead citrate. The sections were collected on carbon-coated copper grids and observed with an FEI-Philips CM200 transmission electron microscope operating at 200 kV. Digital images were recorded with a TVIPS TemCam F216 camera.Droplets of purified starch granule suspensions were allowed to dry on freshly cleaved mica. The specimens were coated with Au/Pd and secondary electron images were recorded with an FEI Quanta 250 scanning electron microscope equipped with a field emission gun and operating at 2 kV.Supplementary information


Measures of the functional and structural properties of blood vessels can be used to assess preclinical stage of vascular disorders. Recent experimental and population studies show that arterial stiffening precedes development of high blood pressure, and can be used to predict future cardiovascular events. Arterial stiffness was also shown to be reversible in several experimental models of various conditions. Since reversing arterial stiffness could prevent development of hypertension and other clinical conditions, understanding the biological mechanisms of arterial stiffening and investigating potential therapeutic interventions to modulate arterial stiffness are important research topics. For research and application in general clinical settings, it is an important step to develop reliable devices and a standardized arterial stiffness measurement protocol. The walls of large arteries, especially the aorta, lose elasticity over time, and this process results in increased arterial stiffness. Arterial stiffening, at least in part, reflects gradual fragmentation and loss of elastin fibers and accumulation of stiffer collagen fibers in the arterial wall . IncreasAlthough the causality between increased arterial stiffness and hypertension is complex because of many confounding factors , recent studies in humans and animals suggest that increased arterial stiffness can precede hypertension. For example, several research projects funded by the NHLBI  – the NIH  Institute focused on supporting cardiovascular research – had examined the temporal and causal relationship between arterial stiffness and hypertension . StudiesBoth human and animal studies have suggested that arterial stiffness is reversible. In a murine model of diet-induced obesity, the increased pulse wave velocity  in obese mice fed a high fat/high sucrose diet (HFHS) for 5 months was reduced to normal after returning obese mice to standard chow for 2 months  studied Using an aging rat model , Steppan et al.  studied In humans, short-term aerobic exercise (3 months) reduced arterial stiffness in older adults (> 65 years) with type 2 diabetes and might thereby lower the risk of cardiovascular morbidity and mortality . A recenArterial stiffness is an important arterial phenotype and an excellent indicator of cardiovascular morbidity and mortality . It is a> 50 years) of the Framingham Heart Study and have found that maintaining HVA beyond age 70 is extremely challenging. With rapid population aging, it will be important in the future to explore the possibility of prevention or reversal of arterial stiffness as a potential therapeutic strategy to control hypertension and/or hypertension-related diseases. In this regard, the European Society of Hypertension and the European Society of Cardiology published a guideline in 2013 to suggest the measurement of arterial stiffness as a way of evaluating hypertensive patients at high cardiovascular risk [Both arterial stiffness and hypertension are positively associated with aging. Studies from animals and humans suggest that arterial stiffness can be reversible under certain conditions Fig. . Niirane


Currently, electrospinning membranes for vascular graft applications has been limited, due to random fiber alignment, to use in mandrel-spun, straight tubular shapes. However, straight, circular tubes with constant diameters are rare in the body. This study presents a method to fabricate curved, non-circular, and bifurcated vascular grafts based on electrospinning. In order to create a system capable of electrospinning membranes to meet specific patient needs, this study focused on characterizing the influence of fiber source, electrical field collector position (inside vs. outside the mandrel), and the motion scheme of the mandrel (rotation vs. rotation and tilting) on the vascular graft membrane morphology and mechanical properties. Given the extensive use of poly(ε-caprolactone) (PCL) in tubular vascular graft membranes, the same material was used here to facilitate a comparison. Our results showed that the best morphology was obtained using orthogonal sources and collector positioning, and a well-timed rotation and tilting motion scheme. In terms of mechanical properties, our bifurcated vascular graft membranes showed burst pressure comparable to that of tubular vascular graft membranes previously reported, with values up to 5126 mmHg. However, the suture retention strength shown by the bifurcated vascular graft membranes was less than desired, not clinically viable values. Process improvements are being contemplated to introduce these devices into the clinical range. The generation of tubular membranes for vascular applications is commonly performed via electrospinning, as the basic manufacturing process for a resorbable or inert inner membrane. However, producing vascular graft devices with complex geometries is challenging. The use of resorbable materials for a curving, bifurcated graft is potentially beneficial to pediatric patients with congenital heart defects as well as pancreatitis patients undergoing resection of the pancreaticoduodenal junction. Congenital cardiac anomalies are the most common birth defect, affecting nearly 1% of all live births, with approximately 25% of patients with congenital heart disease having critical defects. The most common critical congenital heart defects are ventricular and atrial septal defects, which are two of a limited number of defects which may be monitored in certain patients . A significant percent of the morbidity and mortality of pediatric cardiac surgery arises from the fact that the inert synthetic conduits and patches frequently used to repair congenital defects are susceptible to thromboembolism, stenosis, ectopic calcification, and infection . Tissue ®) and polyethylene terephthalate  and polyurethane (PU) ) to determine how the electrospun fibers adhered to the shape of mandrel, c) membrane thickness (t[μm]) of the resultant vascular graft membrane, and d) burst pressure reached by the bifurcated vascular graft membrane. The vascular graft membrane was divided into regions to analyze how the electrospun fibers were distributed over the mandrel. The procedures are detailed next.The following measurements were used to characterize the samples: a) fiber diameter, b) corner profile fidelity (FA scanning electron microscope (SEM) EVOMA25  was used to characterize the vascular graft membrane morphology. The coating procedure was conducted using gold. Images were acquired using conventional SEM operating at an accelerated voltage of 5 kV with a working distance of 9 mm. The images were analyzed using ImageJ software  to determine the diameter of fibers and alignment. Fiber diameter measurements were conducted at 200 random positions. Distribution and average diameter were computed and reported. The alignment analysis was achieved using ImageJ software.CP). This indicator was defined as the percentage of projected area without fibers, relative to the total area in a picture taken from a fixed angle on regions R5 and R6, as defined in CP indicated a better vascular graft membrane that fits more closely to the surface of the mandrel shape, and this translated to a bifurcated shape of better quality.In order to characterize how closely the vascular graft membrane followed (adhered to) the surface of the mandrel, we defined the following indicator: corner profile fidelity (FCP [%]. Images were then measured in ImageJ. The samples were photographed in the same position they were in when extracted from the positioning mechanism (see CP [%] was determined. The results are expressed as the mean ± standard error. The corner profile fidelity was measured while the mandrel was still inside. The procedure was as follows: a) draw a square around region R5 with “rectangle” tool on ImageJ to measure the resulting area to establish the reference total area (As); b) delineate the area free of fibers, inside the previously drawn rectangle, and register the measurement (Ai) with “polygon selections” tool; and c) compute FCP [%] on R5 as An optical microscope USB2-MICRO-250X , with 0x–250x magnification range, was used to visualize the corner profile’s fidelity to the mandrel surface, Fnism see . Based oAfter removal of the mandrel, membrane thickness (t) in each region was measured with a digital micrometer  with a resolution of ±1 μm see . For conUsing the bifurcated electrospun vascular graft membranes and an angioplasty balloon, pressurization to burst was achieved and the pressure at bursting was measured see . The meaFor this test, samples were cut out of the bifurcated membrane to generate a flat rectangular shape. Therefore, the thickness of these samples corresponded to the average thickness in regions R1, R2 and R3. Characterization, to determine the force necessary to pull a suture from the vascular graft membrane, was carried out using a universal testing machine Instron 3365 , according to the International ISO 7198:2016 for Cardiovascular implants and extracorporeal systems -- Vascular prostheses -- Tubular vascular grafts and vascular patches, with a 5 N load cell under physiological conditions  and a teTo assess the in vitro cytotoxicity of the produced mats, the International ISO 10993-5 for Biological evaluation of medical devices was followed. Samples of PCL mats were soaked in 70% ethanol for 30 min, followed by washing, twice, in sterile PBS. Then, the samples were exposed to UV light for 15 min, 30 min prior to cell seeding.2 in an incubator , Moriguchi, Japan) for 24 h to allow attachment. The culture medium was replaced every day and the cells were trypsinized upon reaching a confluence of 50%. Cell proliferation was assessed using MTT assay. Human fibroblasts  were placed into sterile of 96-well plates with low glucose Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS  and 1% penicillin–streptomycin . The cultures were kept at 37 °C and 5% CO3. Next, 100 µL of medium without cells and 100 µL of 1% Triton X-100 in PBS were added to the wells and were used as negative and positive controls, respectively. The microtiter plates were incubated for 96 h in an incubator with 5% CO2 at 37 °C. After incubation, 20 µL of MTT were added to the test wells and allowed to react for 6 h at 37 °C and 5% CO2. Afterwards, the medium was discarded and replaced with 150 µL of dimethyl sulfoxide. After gently shaking for 10min, the optical density (OD) was determined by reading the absorbance at 500 nm on a Biotek Synergy 2 microplate reader . This method was based on the conversion of MTT -2,5-diphenyltetrazolium bromide) into formazan, which determined the mitochondrial activity. Fibroblasts were seeded, in triplicate, in 96-well culture microtiter plates at a density of 3 × 10GraphPad Prism Software Version 8.0  was used for statistical analysis. Data were expressed as mean ± standard deviation (SD). Univariate statistical comparisons were made with a Student’s t-test. Multivariate statistical analysis was carried out using ANOVA and Tukey’s tests. A p-value of p < 0.05 was considered statistically significant.Four groups of mandrels were electrospun under different conditions, as indicated in After imaging and analysis, the mean observed fiber diameter for single axis rotation-only average fiber diameter was 290.2 ± 104.6 nm and 60° alignment fiber around 3000 measured fibers, while a motion scheme that involved rotational and longitudinal motion resulted in an average fiber diameter of 276.8 ± 78.5 nm and around –90°. Almost 6000 fibers were measured, as shown in CP to show that the electrospun membrane properly fit the given shape of the mandrel. There was no significant difference between region R5 and R6 . Under both motion schemes there was a statistically significant difference between R5 and R6 compared to at least one of the other regions. The fact that R5 and R6 had thinner membranes was thought to be due to their location at the corner of the mandrel, where deposition of electrospun fibers tended to be more difficult.Our data revealed that all bifurcated scaffolds showed viability higher than 86%, which confirmed that PCL bifurcated scaffolds presented cytocompatibility (cell viability higher than 50%).The work presented here demonstrates that bifurcated vascular grafts can be constructed via electrospinning by combining an internal electrical field collector design and a 2-DOF positioning mechanism, for the mandrel. The addition of a second longitudinal axis for tilting of the mandrel improved the corner profile fidelity of the vascular graft membrane. However, it was not closely adherent in either case, suggesting that a more direct, perhaps robotically-assisted, approach to controlling needle tip orientation and mandrel surface was needed to ensure closer apposition. Alternatively, ejection pressure, mandrel speed, and/or needle tip distance to the mandrel may also improve apposition.CP), and membrane thickness. The average fiber diameter obtained in these experiments was 276.8 and 290.2 nm. These diameters were thinner but comparable to the low end of those previously reported, as shown in CP) indicator there was a clear benefit to having a combination of rotational and longitudinal motion. In the case of region R6, this benefit was not statistically significance. The need for better apposition of the vascular graft membrane to the mandrel, especially in regions R5 and R6, suggested that the relationship of speed, fiber alignment, and mechanical properties could be further explored, as suggested by Fukunishi et al. [Vascular graft membrane morphology was evaluated through fiber diameter, corner profile fidelity  tests showed that the proposed approach yielded acceptable results. For the rotational and longitudinal motion approach, an average BPS of 5126.6 ± 1074.57 mmHg was obtained for the bifurcated vascular grafts. This may have correlated with other measured characteristics such as fiber diameter. However, that variable was not studied. These levels of strength were comparable to those reported for straight tubular electrospun vascular graft membranes that did not change diameter see . These vIn the context of Y-grafts, we identified the following major functional specifications: (a) corner profile fidelity, (b) burst pressure strength, and (c) suture retention strength. These functional specifications correlated to the basic properties of the electrospun fibers and their corresponding membranes, which were from the complete array of process parameters. ® 200  photocurable resin. The values obtained for bifurcated vascular graft membrane thickness and fiber diameter with rotational scheme motion (BLSP_CC_R), 171.22 ± 26.09µm and 276.8 ± 78.5nm respectively, and rotational and longitudinal scheme motion (BLSP_CC_RL), 119.58 ± 24.74 µm and 290.2 ± 104.6nm respectively, were comparable to similar studies with tubular vascular graft membranes (see This study aimed to characterize the basic impact of spinning one versus two long axes of vascular graft membrane thickness and the resulting properties of the electrospun vascular graft membranes see . These vanes see . In termIn this study we identified the potential applications of bifurcated constructs such as vascular grafts for use in the Fontan procedure see , or panc


While a number of therapeutic options to control the progression of human immunodeficiency virus (HIV-1) now exist, a broadly effective preventive vaccine is still not available. Through detailed structural analysis of antibodies able to induce potent effector cell activity, a number of Env epitopes have been identified which have the potential to be considered vaccine candidates. These antibodies mainly target the gp120 Cluster A region which is only exposed upon viral binding to the target cell with epitopes becoming available for antibody binding during viral entry and fusion and, therefore, after the effective window for neutralizing antibody activity. This review will discuss recent advances in the structural characterization of these important targets with a special focus on epitopes that are involved in Fc-mediated effector function without direct viral neutralizing activities. According to the World Health Organization, HIV-1 has infected more than 70 million people, resulting in over 35 million deaths, including 1 million in the last year alone . AntibodEnv employs a number of strategies to avoid immune system recognition including heavy glycosylation, high sequence variability, and conformational masking ,6,7,8,9.Another obstacle in mounting an effective neutralizing humoral response is related to the error prone replication of HIV . The amiAccording to basic definition, neutralization is the loss of virus infectivity caused by antibody binding and therefore neutralizing antibodies must recognize and bind at the Env epitopes/sites that are directly engaged in the functional mechanism of viral entry and spread . HoweverRecent and accumulating evidence points toward conformational epitopes within the constant region 1 and 2 (C1-C2) of the gp120 domain as being major Env targets for antibodies capable of potent ADCC without direct neutralization activity, as measured using TZM-bl assays ,30,31. TAt least three distinct epitope targets can be assigned within the Cluster A region  by cross-competition with the mAbs A32 and C11 . The A32The core of the HIV-1 gp120 glycoprotein is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain contains the CD4 binding site and is exposed in the prefusion, closed HIV trimer while the inner domain is occluded and packs against gp41 inside the trimer . The innThese initial mappings enabled basic epitope assignments, the precise and exact location of the A32 epitope region could only be defined once the structures of complexes formed between the A32-like antibodies and an appropriate Env antigen became available. Currently, these include crystal structures of complexes formed between CD4-triggered gp120 cores and antigen binding fragments (Fabs) of four antibodies specific for the A32 region: three A32-like antibodies  and the prototype antibody A32 itself . mAbs N5H affinity maturation and ADCC activity for antibodies targeting the A32 region epitopes [The gp120 surface which contributes the A32 epitope region in the CD4-triggered Env is relatively flat, glycan-free, and moderately electronegative [epitopes . Conversepitopes ,56,57,58Similar to the A32 region, the C11 epitope region was initially mapped by antibody cross-competition and mutagenesis analyses. These studies placed the C11 epitopes at the gp41 interactive region of gp120 within the C1, C5, and the seven-stranded-β-sandwich . sandwich . N12-i3 The only structure that has been determined to date of an antibody in complex with gp120 that displaces both A32 and C11 in competitive ELISAs is the macaque antibody JR4 . The strThe Cluster A epitopes become available for antibody recognition as Env transition state structures are exposed following engagement of the Env trimer with host receptors during viral entry ,33 and oIn contrast, the nascent C11 epitope region is located at the bottom of the prefusion trimer, proximal to the viral membrane and is directly involved in interactions with helices α6, α7, α8, and α9, and β-strand β27 of gp41. In addition, the N- terminus of gp120, which in the C11-bound state will form the 8th strand of the eight-stranded β-sandwich, is held by a triple-tryptophan clasp consisting of tryptophans 623, 628, and 623 of gp41 , the A32In contrast, all evidence indicates that the C11 epitope region only becomes exposed during the final stages of viral entry, after the dissociation of gp41 from gp120 and is, thus, indicative of a late entry stage. The N- and C-termini of gp120 are integral elements in stabilizing the Env trimer in its closed, prefusion conformation and are engaged in a gp41 clasp . Only upADCC and other Fc-mediated effector cell functions have been implicated in protection from HIV infection as well as in the control of infection in a few elite HIV controllers ,71,72,7350 value using target cells (CEM-NKr-CCR5+) sensitized with a saturating dose of HIV-1Ba-L isolate gp120 in comparison to N5-i5, A32, and other A32-like antibodies. The analysis of the 2.2c Fab complex structure confirmed that 2.2c largely overlapped with N5-i5 in binding to the CD4-triggered gp120, but it recognized its epitope with some key differences. Specifically, 2.2c bound almost exclusively to mobile layer 1 of gp120 in the C1 region with no contacts to α1-helix and only a few contacts to the C2 region. In addition, the antibody approached the antigen from a slightly different angle and bound by swapping heavy and light chain relative to N5-i5. Binding analysis indicated that the mode of 2.2c attachment did not change the affinity of 2.2c for antigen, as compared to N5-i5, when measured by the SPR with antibody immobilized on sensor chip (affinity format). However, it did impair the ability of 2.2c to effectively bind to the antigen immobilized on the sensor chip (avidity format) and by inference, to recognize Env epitopes on the target cell. In addition, based on the inferred models of immune-complexes formed by 2.2c and N5-i5 at the target-effector cell interface, the Fc domain of 2.2c was orientated much closer to the putative target cell membrane than N5-i5, potentially impeding the effector cell access and the Fc-Fcγ receptor interaction [Structural analysis has mapped the Cluster A region epitopes at the molecular level, defined the exact antibody contact sites on gp120 and described their three-dimensional shapes. Although most of the antibodies targeting these sites are uniformly able to mediate potent ADCC against targets exposed on trimeric Env after CD4 engagement, there is only one known example of an antibody which targets the A32 epitope region but is capable of only moderate ADCC. Comparative structural and functional studies of this antibody, known as 2.2c, to the potent ADCC mediator N5-i5, which targets an overlapping epitope structure in the A32 binding site, shed new light onto the role of fine epitope specificity and the mode of antibody attachment in the ADCC mechanism. In an RFADCC assay , 2.2c waeraction . H and VL domains swapped in variants of 2.2c and N5-i5 in which Fc domain orientations were interconverted. Surprisingly, the swapped variant of 2.2c which preserved the binding properties of wild type 2.2c but had the Fc orientation similar to that of N5-i5 showed an improved activity, but not to the levels seen for N5-i5 and other Cluster A antibodies. Conversely, the swapped variant of N5-i5 fully preserved the potent ADCC activities of the wild-type antibody. These findings strongly suggested there are two major determinates contributing to ADCC effectiveness to the A32 region epitopes; the ability of an antibody to form multivalent interactions with epitopes on the target cell surface (antibody-antigen crosslinking) and the Fc orientation in the immune complex. Although these observations were made for an antibody pair specific for the A32 epitope, these findings can be generalized and applied to other epitope targets indicating a dominant role for the precise epitope targeting and the mode of antibody attachment in ADCC responses. The hypothesis of obstruction to effector function due to limited effector cell access to the Fc portion was confirmed in a set of experiments with the VIf orientation and accessibility of Fc (for interaction with effector cells) in the immune complex does contributes to the effectiveness of ADCC, as suggested above, the C11 epitopes of the Cluster A region should be optimal targets. As shown in + T cell responses [H) chain somatic mutation (0.5 to 1.5%) and mediated cross-clade ADCC activity against HIV-1 isolates of clades represented in the immunogen (Clade B and CRF01 AE) as well as Clade C, which was not represented in the vaccine [The recent vaccination strategy tested in the ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial, which showed an estimated vaccine efficacy of 31.2% in preventing HIV-1 acquisition, is of particular importance in the development of a HIV vaccine to induce protection in humans . It suggesponses ,80. The esponses . While Vesponses ,83, the esponses . Moreoveesponses . Clusteresponses . Furtheresponses . These a vaccine . Moreove vaccine . The RV1With regard to studies in non-human primates (NHP), the evidence for a role of Cluster A epitopes in vaccine induced protection is even stronger. ADCC measured using the rapid fluorometric antibody-dependent cellular cytotoxicity assay (RFADCC)  and antiIt is clear that a pressing need for a preventative HIV vaccine exists and that we are closer than ever to overcoming the many obstacles required to create an effective therapy. The revelation that a degree of HIV protection could be achieved in the absence of broadly neutralizing antibodies in the RV144 vaccine trial spurred interest into the role of non-neutralizing antibodies and the epitopes to which they bind, which results in effector cell mediated killing of infected cells. Structural analysis of the gp120 cluster A region of the HIV envelope has greatly enhanced our understanding of the importance of antibodies generated to epitopes within this region. Through detailed analysis of isolated antibodies in complex with CD4 and gp120, the A32 region has been defined as a discontinuous site involving mobile layers 1 and 2 in the C1–C2 regions of gp120 in its CD4-bound state and C11 region as epitopes mapped exclusively within the eight-stranded β-sandwich of the inner domain of the C1-C2 regions of gp120 centered around the N-terminal eighth-strand. These two epitope regions are non-overlapping and are emblematic of two different steps in the process of CD4-induced opening of the trimer with C11 marking the late stages of trimer disassembly, most likely after complete gp120 detachment. This detailed map of the vital antibody binding epitopes will be invaluable for the development of future vaccine candidates for both the prevention and clearance of HIV.