Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains

This work was supported by grants from Cancer Research UK (C434/A13067), the Wellcome Trust (098391/Z/12/Z) and Biotechnology and Biological Sciences Research Council (BB/J016004/1).The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis.PostprintPeer reviewe

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Class II MHC Self-Antigen Presentation in Human B and T Lymphocytes

Human CD4[superscript +] T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4[superscript +] T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.National Institutes of Health (U.S.) (grant P01AI039671)National Institutes of Health (U.S.) (P01AI045757

Advanced Technologies for Oral Controlled Release: Cyclodextrins for oral controlled release

Cyclodextrins (CDs) are used in oral pharmaceutical formulations, by means of inclusion complexes formation, with the following advantages for the drugs: (1) solubility, dissolution rate, stability and bioavailability enhancement; (2) to modify the drug release site and/or time profile; and (3) to reduce or prevent gastrointestinal side effects and unpleasant smell or taste, to prevent drug-drug or drug-additive interactions, or even to convert oil and liquid drugs into microcrystalline or amorphous powders. A more recent trend focuses on the use of CDs as nanocarriers, a strategy that aims to design versatile delivery systems that can encapsulate drugs with better physicochemical properties for oral delivery. Thus, the aim of this work was to review the applications of the CDs and their hydrophilic derivatives on the solubility enhancement of poorly water soluble drugs in order to increase their dissolution rate and get immediate release, as well as their ability to control (to prolong or to delay) the release of drugs from solid dosage forms, either as complexes with the hydrophilic (e.g. as osmotic pumps) and/ or hydrophobic CDs. New controlled delivery systems based on nanotechonology carriers (nanoparticles and conjugates) have also been reviewed

DNA vaccination for prostate cancer: key concepts and considerations

While locally confined prostate cancer is associated with a low five year mortality rate, advanced or metastatic disease remains a major challenge for healthcare professionals to treat and is usually terminal. As such, there is a need for the development of new, efficacious therapies for prostate cancer. Immunotherapy represents a promising approach where the host’s immune system is harnessed to mount an anti-tumour effect, and the licensing of the first prostate cancer specific immunotherapy in 2010 has opened the door for other immunotherapies to gain regulatory approval. Among these strategies DNA vaccines are an attractive option in terms of their ability to elicit a highly specific, potent and wide-sweeping immune response. Several DNA vaccines have been tested for prostate cancer and while they have demonstrated a good safety profile they have faced problems with low efficacy and immunogenicity compared to other immunotherapeutic approaches. This review focuses on the positive aspects of DNA vaccines for prostate cancer that have been assessed in preclinical and clinical trials thus far and examines the key considerations that must be employed to improve the efficacy and immunogenicity of these vaccines

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Partitioning of Minimotifs Based on Function with Improved Prediction Accuracy

Background: Minimotifs are short contiguous peptide sequences in proteins that are known to have a function in at least one other protein. One of the principal limitations in minimotif prediction is that false positives limit the usefulness of this approach. As a step toward resolving this problem we have built, implemented, and tested a new data-driven algorithm that reduces false-positive predictions. Methodology/Principal Findings: Certain domains and minimotifs are known to be strongly associated with a known cellular process or molecular function. Therefore, we hypothesized that by restricting minimotif predictions to those where the minimotif containing protein and target protein have a related cellular or molecular function, the prediction is more likely to be accurate. This filter was implemented in Minimotif Miner using function annotations from the Gene Ontology. We have also combined two filters that are based on entirely different principles and this combined filter has a better predictability than the individual components. Conclusions/Significance: Testing these functional filters on known and random minimotifs has revealed that they are capable of separating true motifs from false positives. In particular, for the cellular function filter, the percentage of known minimotifs that are not removed by the filter is,4.6 times that of random minimotifs. For the molecular function filter this ratio is,2.9. These results, together with the comparison with the published frequency score filter, strongly suggest tha

Risk Stratification in Older Intensively Treated Patients With AML

\ua9 2024 by American Society of Clinical Oncology.PURPOSE AML is a genetically heterogeneous disease, particularly in older patients. In patients older than 60 years, survival rates are variable after the most important curative approach, intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (allo-HCT). Thus, there is an urgent need in clinical practice for a prognostic model to identify older patients with AML who benefit from curative treatment. METHODS We studied 1,910 intensively treated patients older than 60 years with AML and high-risk myelodysplastic syndrome (HR-MDS) from two cohorts (NCRIAML18 and HOVON-SAKK). The median patient age was 67 years. Using a random survival forest, clinical, molecular, and cytogenetic variables were evaluated in an AML development cohort (n = 1,204) for association with overall survival (OS). Relative weights of selected variables determined the prognostic model, which was validated in AML (n = 491) and HR-MDS cohorts (n = 215). RESULTS The complete cohort had a high frequency of poor-risk features, including 2022 European LeukemiaNet adverse-risk (57.3%), mutated TP53 (14.4%), and myelodysplasia-related genetic features (65.1%). Nine variables were used to construct four groups with highly distinct 4-year OS in the (1) AML development, (2) AML validation, and (3) HR-MDS test cohorts ([1] favorable: 54% \ub1 4%, intermediate: 38% \ub1 2%, poor: 21% \ub1 2%, very poor: 4% \ub1 1%; [2] 54% \ub1 9%, 43% \ub1 4%, 27% \ub1 4%, 4% \ub1 3%; and [3] 54% \ub1 10%, 33% \ub1 6%, 14% \ub1 5%, 0% \ub1 3%, respectively). This new AML60+ classification improves current prognostic classifications. Importantly, patients within the AML60+ intermediate- and very poor-risk group significantly benefited from allo-HCT, whereas the poor-risk patients showed an indication, albeit nonsignificant, for improved outcome after allo-HCT. CONCLUSION The new AML60+ classification provides prognostic information for intensively treated patients 60 years and older with AML and HR-MDS and identifies patients who benefit from intensive chemotherapy and allo-HCT

Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs

Oncological outcome and patient satisfaction with skin-sparing mastectomy and immediate breast reconstruction: a prospective observational study

<p>Abstract</p> <p>Background</p> <p>The management of early breast cancer (BC) with skin-sparing mastectomy (SSM) and immediate breast reconstruction (IBR) is not based on level-1 evidence. In this study, the oncological outcome, post-operative morbidity and patients' satisfaction with SSM and IBR using the latissimus dorsi (LD) myocutaneous flap and/or breast prosthesis is evaluated.</p> <p>Methods</p> <p>137 SSMs with IBR (10 bilateral) were undertaken in 127 consecutive women, using the LD flap plus implant (n = 85), LD flap alone (n = 1) or implant alone (n = 51), for early BC (n = 130) or prophylaxis (n = 7). Nipple reconstruction was performed in 69 patients, using the trefoil local flap technique (n = 61), nipple sharing (n = 6), skin graft (n = 1) and Monocryl mesh (n = 1). Thirty patients underwent contra-lateral procedures to enhance symmetry, including 19 augmentations and 11 mastopexy/reduction mammoplasties. A linear visual analogue scale was used to assess patient satisfaction with surgical outcome, ranging from 0 (not satisfied) to 10 (most satisfied).</p> <p>Results</p> <p>After a median follow-up of 36 months (range = 6-101 months) there were no local recurrences. Overall breast cancer specific survival was 99.2%, 8 patients developed distant disease and 1 died of metastatic BC. There were no cases of partial or total LD flap loss. Morbidities included infection, requiring implant removal in 2 patients and 1 patient developed marginal ischaemia of the skin envelope. Chemotherapy was delayed in 1 patient due to infection. Significant capsule formation, requiring capsulotomy, was observed in 85% of patients who had either post-mastectomy radiotherapy (PMR) or prior radiotherapy (RT) compared with 13% for those who had not received RT. The outcome questionnaire was completed by 82 (64.6%) of 127 patients with a median satisfaction score of 9 (range = 5-10).</p> <p>Conclusion</p> <p>SSM with IBR is associated with low morbidity, high levels of patient satisfaction and is oncologically safe for T(is), T1 and T2 tumours without extensive skin involvement.</p