Editing the plastid genome of recalcitrant plant species.

Transformation of the chloroplast genome offers key advantages over traditional methods for generating transgenic plants, but this approach is limited to a few plant species. Nakazato et al. have developed a novel technique that will help to extend the technology to other plant species that are recalcitrant to current tissue culture-based chloroplast transformation protocols.

Homozygous mutations in C14orf39/SIX6OS1 cause non-obstructive azoospermia and premature ovarian insufficiency in humans.

Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

Constructing Nano-Interlayer Inhibiting Interfacial Degradation toward High-Voltage PEO-Based All-Solid-State Lithium Batteries.

The interfacial instability between PEO-based solid electrolyte (SPE) and high-voltage cathode materials inhibits the longevity of high-energy-density all-solid-state polymer lithium metal batteries (ASSPLBs). Herein, for the first time it is demonstrated, that contact loss caused by gas generation from interfacial side reactions between the high-voltage cathode and solid polymer electrolyte (SPE) can also arise in ASSPLBs. To alleviate the interfacial side reactions, a LiNb0.6Ti0.5O3 (LNTO) layer is well coated on LiNi0.83Co0.07Mn0.1O2 (NCM83), denoted as (CNCM83). The LNTO layer with low electronic conductivity reduces the decomposition drive force of SPE. Furthermore, Ti and Nb in the LNTO layer spontaneously migrate inside the NCM83 surface to form a strong Ti/Nb─O bond, stalling oxygen evolution in high-voltage cathodes. The interfacial degradation phenomena, including SPE decomposition, detrimental phase transition and intragranular cracks of NCM83, and void formation between cathode and SPE, are effectively mitigated by the LNTO layer. Therefore, the growth rate of interfacial resistance (RCEI) decreases from 37.6 Ω h-0.5 for bare NCM83 to 2.4 Ω h-0.5 for CNCM83 at 4.2 V. Moreover, 4.2 V PEO-based ASSPLBs achieve impressive cyclability with high capacity retention of 135 mAh g-1 (75%) even after 300 cycles at 0.5 C.

Impact of Asian and Black Donor and Recipient Ethnicity on the Outcomes After Deceased Donor Kidney Transplantation in the United Kingdom.

Patients of Asian and black ethnicity face disadvantage on the renal transplant waiting list in the UK, because of lack of human leucocyte antigen and blood group matched donors from an overwhelmingly white deceased donor pool. This study evaluates outcomes of renal allografts from Asian and black donors. The UK Transplant Registry was analysed for adult deceased donor kidney only transplants performed between 2001 and 2015. Asian and black ethnicity patients constituted 12.4% and 6.7% of all deceased donor recipients but only 1.6% and 1.2% of all deceased donors, respectively. Unadjusted survival analysis demonstrated significantly inferior long-term allograft outcomes associated with Asian and black donors, compared to white donors. On Cox-regression analysis, Asian donor and black recipient ethnicities were associated with poorer outcomes than white counterparts, and on ethnicity matching, compared with the white donor-white recipient baseline group and adjusting for other donor and recipient factors, 5-year graft outcomes were significantly poorer for black donor-black recipient, Asian donor-white recipient, and white donor-black recipient combinations in decreasing order of worse unadjusted 5-year graft survival. Increased deceased donation among ethnic minorities could benefit the recipient pool by increasing available organs. However, it may require a refined approach to enhance outcomes.

Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation.

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

A critical look on CRISPR-based genome editing in plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.

Engineering microalgae through chloroplast transformation to produce high-value industrial products.

The last few years have seen an ever-increasing interest in the exploitation of microalgae as an alternative platform to produce high-value products such as biofuels, industrial enzymes, therapeutic proteins, including antibodies, hormones, and vaccines. Due to some unique attractive features, engineering of the chloroplast genome provides a promising platform for the production of high-value targets because it allows manipulation of metabolic processes in ways that would be impossible, or at least prohibitively difficult through traditional approaches. Since its initial demonstration in 1988 in Chlamydomonas reinhardtii, genetic tools have been developed, which have made it possible to produce high-value molecules in different species. However, the commercial application of microalgae as production platform is hindered by many factors like poor biomass, low product yields, and costly downstream processing methodologies. In this review, we discuss the potential of microalgae to use as an alternative production platform for high-value targets using chloroplast transformation technology.

The evolution of donation after circulatory death renal transplantation: a decade of experience.

BACKGROUND: This study compared long-term outcomes of renal transplantation from donors following donation after circulatory death (DCD) with those following donation after brain death (DBD) from one of the largest centres in the UK. METHOD: Recipients of renal transplants from deceased donors between 2002 and 2014 were identified from a prospectively maintained database. Outcomes were compared between DCD (468) and DBD (905) donors and between standard criteria donors (SCDs) and extended criteria donors (ECDs). RESULTS: Graft survival (GS) and patient survival (PS) from DCD and DBD donors were comparable up to 10 years (GS: 61 versus 55%, P = 0.780; PS: 78 versus 71%, P = 0.285, respectively). Graft function was comparable after 3 months. GS and function were worse in the ECD groups, with no difference between EC-DBD and EC-DCD. PS in the ECD groups was worse than the SCD groups and PS in the EC-DCD group was worse than in the EC-DBD group. DCD donors were an independent risk factor for delayed graft function. Post-operative complications and EC-DCD donation were independent risk factors for reduced GS and PS. CONCLUSION: This study supports the use of DCD renal grafts with comparable long-term survival and function to DBD grafts. The use of EC-DCD grafts is justified in selected recipients and provides acceptable function and survival advantages over dialysis.

The impact of changing practice on improved outcomes of paediatric renal transplantation in the United Kingdom: a 25 years review.

This review reports the outcomes of paediatric renal transplantation in the United Kingdom over the last 25 years. UK Transplant Registry data on 3236 paediatric renal transplants performed between 1 January 1992 and 31 December 2016 were analysed. Significant improvements in human leucocyte antigen (HLA) matching have been achieved; 84% of recipients received 000 or favourable (0 DR and 0 or 1 B) mismatched kidneys in 2016 compared with 27% in 1992. The median waiting time has increased from 126 days in 1999 to 351 days in 2016. Tacrolimus replaced ciclosporin in most immunosuppressive regimens after 2002. Renal transplant outcome has improved significantly, mainly because of a reduction in early graft loss. One-year donation after brain death renal allograft survival for those transplanted from 2012 to 2016 was 98%, compared with 72% for those transplanted from 1987 to 1991. Renal allograft survival for first kidney only transplants at 1, 5, 10, 20 and 25 years were 89%, 79%, 65%, 42% and 33% respectively. Superior survival with living donor was maintained throughout the study period with 25-year graft survival at 33% compared with 31% from deceased donor (P < 0.0001). Changes in immunosuppression regimens, improvements in HLA matching and a reduction of cold ischaemia time may in part explain the improvements in graft survival.

Alleviation of chromium toxicity in maize by Fe fortification and chromium tolerant ACC deaminase producing plant growth promoting rhizobacteria.

Chromium (Cr) is becoming a potential pollutant with the passage of time. Higher intake of Cr does not only affect the productivity of crops, but also the quality of food produced in Cr polluted soils. In the past, foliar application of Fe is widely studied regarding their potential to alleviate Cr toxicity. However, limited information is documented regarding the combined use of PGPR and foliar Fe. Therefore, the current study was conducted to screen Cr tolerant PGPR and examine effect of foliar Fe with and without Cr tolerant PGPR under Cr toxicity (50 and 100 mg kg-1) in maize (Zea mays) production. Out of 15, two Cr tolerant PGPR were screened, identified (Agrobacterium fabrum and Leclercia adecarboxylata) and inoculated with 500 µM Fe. Results confirmed that Agrobacterium fabrum + 500 µM Fe performed significantly best in improving dry weight of roots and shoot, plant height, roots and shoot length and plant leaves in maize under Cr toxicity. A significant increase in chlorophyll a (51.5%), b (55.1%) and total (32.5%) validated the effectiveness of A. fabrum + 500 µM Fe to alleviate Cr toxicity. Improvement in intake of N (64.7%), P (70.0 and 183.3%), K (53.8% and 3.40-fold) in leaves and N (25.6 and 122.2%), P (25.6 and 122.2%), K (33.3% and 97.3%) in roots of maize at Cr50 and Cr100 confirmed that combined application of A. fabrum with 500 µM Fe is a more efficacious approach for alleviation of Cr toxicity and fortification of Fe comparative to sole foliar application of 500 µM Fe.

Renal transplant from infant and neonatal donors is a feasible option for the treatment of end-stage renal disease but is associated with increased early graft loss.

Kidney transplants from young pediatric donors are uncommonly performed in the UK. Published literature of kidney transplant from donors weighing less than 5 kg is sparse. We present our initial experience of en bloc kidney transplantation (EKT) from donors weighing less than 20 kg, including neonatal donors. All recipients undergoing EKT from donors under 20 kg at our center from January 2005 to October 2016 were included. Donor and recipient details were recorded from a prospective database. Electronic patient records were examined for follow-up data. Of 30 EKTs included, 15 were from ≤5 kg donors and 15 from >5 kg donors (median weight 3.4 and 12.7 kg, respectively). One-year graft survival for ≤5 kg and >5 kg donors for EKT was 86.7% and 93.3% (P = 0.85), respectively. Progressive improvement in estimated GFR (eGFR) was noted in both donor categories through first-year posttransplant but in the ≤5 kg donor category significant improvement was seen at 12 months compared to 3 months after transplantation (median eGFR 37.3 vs 70.0 mL/min/1.73 m2 , P = 0.03). Two early graft losses were attributable to early vascular complications and one graft loss due to primary nonfunction. Our data show that kidney transplantation from such donors is a feasible option at centers with experience of EKT, albeit with increased risk of early graft loss.

Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers.

Extra-hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self-defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro-carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier-forming differentiated states in vitro. However, ethoxyresorufin O-deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP-DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain-of-function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle-invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into "luminal" and "basal" groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over-expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1-activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over-expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP-function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

Bilateral adrenal hemorrhage in a neonatal kidney donor.

EKT from neonatal donors remains rare despite successful outcome being reported. The surgical aspects of neonatal abdominal organ recovery remain unfamiliar to the vast majority of abdominal organ recovery teams and renal transplant surgeons. BAH is not uncommon in newborn babies suffering distress in the perinatal period. BAH is often also associated with RVT and will impact on utilization of kidneys for transplantation. We present a case of a neonatal kidney donor with massive BAHs discovered at the time of organ recovery. This made the procurement challenging. Both kidneys were recovered en bloc with pancreas and the liver with aorta and inferior vena cave as vascular conduits. The kidneys were successfully implanted into an adult recipient with good function at 1-year follow-up. Association between adrenal hemorrhage and RVT needs to be considered before utilizing such kidneys. This case exemplifies successful outcome after careful assessment and transplantation of such kidneys.

Genetic manipulations in crops: Challenges and opportunities.

An alarming increase in the human population necessitates doubling the world food production in the next few decades. Although a number of possible biotechnological measures are under consideration, central to these efforts is the development of transgenic crops to produce more food, and the traits with which plants could better adapt to adverse environmental conditions in a changing climate. The emergence of new tools for the introduction of foreign genes into plants has increased both our knowledge and the capacity to develop transgenic plants. In addition, a better understanding of genetic modifications has allowed us to study the impact that genetically modified crop plants may have on the environment. This article discusses different techniques routinely used to carry out genetic modifications in plants while highlighting challenges with them, which future research must address to increase acceptance of GM crops for meeting food security challenges effectively.

Challenges and perspectives in commercializing plastid transformation technology.

Plastid transformation has emerged as an alternative platform to generate transgenic plants. Attractive features of this technology include specific integration of transgenes-either individually or as operons-into the plastid genome through homologous recombination, the potential for high-level protein expression, and transgene containment because of the maternal inheritance of plastids. Several issues associated with nuclear transformation such as gene silencing, variable gene expression due to the Mendelian laws of inheritance, and epigenetic regulation have not been observed in the plastid genome. Plastid transformation has been successfully used for the production of therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various agronomic traits including resistance to biotic and abiotic stresses. However, these demonstrations have usually focused on model systems such as tobacco, and the technology per se has not yet reached the market. Technical factors limiting this technology include the lack of efficient protocols for the transformation of cereals, poor transgene expression in non-green plastids, a limited number of selection markers, and the lengthy procedures required to recover fully segregated plants. This article discusses the technology of transforming the plastid genome, the positive and negative features compared with nuclear transformation, and the current challenges that need to be addressed for successful commercialization.

Non-HLA-matched 3rd party vascular allograft in renal transplant may lead to sensitization against donor HLA.

3rd party donor vessels are often used for vascular reconstruction in organ transplantation. While current practice ensures that 3rd party vessels are blood group matched, HLA matching to the non-intended recipient is not performed. This practice potentially sensitizes the recipient and may reduce their future chance of renal transplant from a larger pool of donors. We examined our cohort of renal transplant recipients who received non-HLA-matched 3rd party vessels for the de-novo development of donor-specific HLA antibodies. Our institution's Human Tissue Authority (HTA) blood vessel registers were examined to identify stored donor vessels and their non-intended recipients. Donor vessel HLA status was cross-referenced with the recipient HLA status. Between 2004 and 2014, five patients were identified that received 3rd party non-HLA-matched vessels for vascular reconstruction during renal transplantation. Three patients (60%) subsequently developed donor-specific HLA antibodies. These data provide evidence that use of non-HLA-matched stored 3rd party vascular grafts may lead to sensitization in the recipient. Where time permits, HLA matching should be performed to avoid this allogeneic response. Laboratories monitoring DSA should be aware of any patient receiving a non-HLA-matched 3rd party vascular graft, and recipients may benefit from increased post-transplant immunological vigilance.

Transplantation of liver and kidney from donors with malignancy at the time of donation: an experience from a single centre.

Transplantation of organs from donors with malignancy poses clinical and ethical questions regarding outcome, informed consent, immunosuppression and follow-up. We review our experience of kidney and liver transplantation from such donors. Our database was complemented by data from National Health Service Blood and Transplant. All patients who received a renal or liver transplant in our institution between April 2003 and January 2014 were included. About 2546 liver and kidney transplants were performed: 71 recipients received 53 kidney and 18 liver transplants. These included 51 (36 kidney, 15 liver) CNS malignancy, and six kidneys, three ipsilateral and three contralateral with RCC. One kidney recipient developed donor-transmitted lung cancer in the transplant kidney, and one liver transplant recipient developed donor-transmitted lymphoma; both subsequently died. Seven recipients developed donor-unrelated cancer. No recipient developed cancer, whereas the donor had a CNS or RCC. The 1-, 3- and 5-year patient survival was 96%, 93.3% and 75%, respectively, for kidneys and 83.3%, 75% and 50%, respectively, for liver. Where donor malignancy was known and assessed before transplantation, judicious use of kidney and liver for transplant achieved satisfactory outcome. The risk of transmission from donors with CNS and low-grade renal malignancy remains extremely low.

Impact of the new fast track kidney allocation scheme for declined kidneys in the United Kingdom.

INTRODUCTION: A "new" fast track kidney allocation scheme (FTKAS) was implemented in the UK in 2012 for offering of previously declined kidneys. We evaluated the impact of the FTKAS in utilization of declined kidneys and outcome. METHODS: Adult renal transplant centers were surveyed. Overall utilization was evaluated using National Health Service Blood and Transplant (NHSBT) data. Outcome of FTKAS kidneys in our center was analyzed. RESULTS: Centers cited graft, patient outcome concerns, and inadequate logistical support for their non-FTKAS participation. In the first year of the scheme, 266 kidneys were offered through the FTKAS, 158 were transplanted in 10 centers (59%). In comparison, 166 kidneys were offered through previous system over five yr (2006-2011), and 65 were utilized in 59 transplants (39%). In our center, 42 kidneys were transplanted in 39 recipients. One-yr graft and patient survival were both 95%. Results were comparable to a matched group of kidney transplants during the same periods allocated via the standard scheme. CONCLUSIONS: The FTKAS has led to effective utilization of the declined kidneys with outcome comparable to kidneys allocated through the standard scheme. Non-participation based on outcome concerns is mostly subjective while logistical issues need to be addressed.

Ureteric reconstruction for the management of transplant ureteric stricture: a decade of experience from a single centre.

This study was conducted to review the outcomes of patients who had undergone surgical repair of a ureteric stricture following renal transplantation. All patients who developed a ureteric stricture and underwent ureteric reconstruction following renal transplantation, between December 2003 and November 2013, were reviewed. One thousand five hundred and sixty renal transplants were performed during the study period. Forty patients required surgical repair of a ureteric stricture (2.5%, 25 male, median age 48 [14-78]). The median time to stricture was 3 [1-149] months. 19 patients were reconstructed by reimplantation to the bladder, 18 utilized a Boari flap, two were a pre-existing ileal conduit and one was an anastomosis to a native ureter. In one patient, reconstruction was impossible and consequently an extra-anatomic stent was used. Two patients required re-operation for restricture and kinking. Median serum creatinine at 12 months following surgery was 148 [84-508] µmol/l. There was no 90-day mortality. Eleven grafts were lost at the time of this study, a median time of 11 [1-103] months after reconstruction. The incidence of ureteric stricture following renal transplant is low. Surgical reconstruction of the transplant ureter is the optimal treatment and is successful in the majority of patients.