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

Vaccines against toxoplasma gondii : challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

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

Correlates of Out-of-Pocket and Catastrophic Health Expenditures in Tanzania: Results from a National Household Survey.

Inequality in health services access and utilization are influenced by out-of-pocket health expenditures in many low and middle-income countries (LMICs). Various antecedents such as social factors, poor health and economic factors are proposed to direct the choice of health care service use and incurring out-of-pocket payments. We investigated the association of these factors with out-of-pocket health expenditures among the adult and older population in the United Republic of Tanzania. We also investigated the prevalence and associated determinants contributing to household catastrophic health expenditures. We accessed the data of a multistage stratified random sample of 7279 adult participants, aged between 18 and 59 years, as well as 1018 participants aged above 60 years, from the first round of the Tanzania National Panel survey. We employed multiple generalized linear and logistic regression models to evaluate the correlates of out-of-pocket as well as catastrophic health expenditures, accounting for the complex sample design effects. Increasing age, female gender, obesity and functional disability increased the adults' out-of-pocket health expenditures significantly, while functional disability and visits to traditional healers increased the out-of-pocket health expenditures in older participants. Adult participants, who lacked formal education or worked as manual laborers earned significantly less (p < 0.001) and spent less on health (p < 0.001), despite having higher levels of disability. Large household size, household head's occupation as a manual laborer, household member with chronic illness, domestic violence against women and traditional healer's visits were significantly associated with high catastrophic health expenditures. We observed that the prevalence of inequalities in socioeconomic factors played a significant role in determining the nature of both out-of-pocket and catastrophic health expenditures. We propose that investment in social welfare programs and strengthening the social security mechanisms could reduce the financial burden in United Republic of Tanzania

The bed nucleus of stria terminalis and the amygdala as targets of antenatal glucocorticoids: implications for fear and anxiety responses

Rationale: Several human and experimental studies have shown that early life adverse events can shape physical and mental health in adulthood. Stress or elevated levels of glucocorticoids (GCs) during critical periods of development seem to contribute for the appearance of neurospyschiatric conditions such as anxiety and depression, albeit the underlying mechanisms remain to be fully elucidated. Objectives: The aim of the present study was to determine the long-term effect of prenatal erxposure to dexamethasone- DEX (synthetic GC widely used in clinics) in fear and anxious behavior and identify the neurochemical, morphological and molecular correlates. Results: Prenatal exposure to DEX triggers a hyperanxious phenotype and altered fear behavior in adulthood. These behavioral traits were correlated with increased volume of the bed nucleus of the stria terminalis (BNST), particularly the anteromedial subivision which presented increased dendritic length; in parallel, we found an increased expression of synapsin and NCAM in the BNST of these animals. Remarkably, DEX effects were opposite in the amygdala, as this region presented reduced volume due to significant dendritic atrophy. Albeit no differences were found in dopamine and its metabolite levels in the BNST, this neurotransmitter was substantially reduced in the amygdala, which also presented an up-regulation of dopamine receptor 2. Conclusions: Altogether our results show that in utero DEX exposure can modulate anxiety and fear behavior in parallel with significant morphological, neurochemical and molecular changes; importantly, GCs seem to differentially affect distinct brain regions involved in this type of behaviors.This study was supported by a grant from the Institute for the Study of Affective Neuroscience (ISAN). AJR is supported by a Fundação para a Ciência e Tecnologia (FCT) grant

Resolution of inflammation: a new therapeutic frontier

Dysregulated inflammation is a central pathological process in diverse disease states. Traditionally, therapeutic approaches have sought to modulate the pro- or anti-inflammatory limbs of inflammation, with mixed success. However, insight into the pathways by which inflammation is resolved has highlighted novel opportunities to pharmacologically manipulate these processes — a strategy that might represent a complementary (and perhaps even superior) therapeutic approach. This Review discusses the state of the art in the biology of resolution of inflammation, highlighting the opportunities and challenges for translational research in this field

Torsades de pointes during laparoscopic adrenalectomy of a pheochromocytoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>Torsades de pointes is a rare but potentially lethal arrhythmia. The amount of literature available on Torsades de pointes occurring in patients with pheochromocytoma is limited, and we found no literature describing this dysrhythmia in a patient with pheochromocytoma under anesthesia.</p> <p>Case presentation</p> <p>We describe the case of a 42-year-old Caucasian woman without QT prolongation preoperatively with recurrent Torsades de pointes during laparoscopic removal of a pheochromocytoma. Torsades de pointes mainly occurs in the setting of a prolonged QT interval. This patient neither had a prolonged QT preoperatively nor was her family history suspect for a congenital long QT syndrome. Most likely, our patient had an acquired long QT syndrome, elicited by the combination of flecainide, hypomagnesemia and adrenergic stimulation during manipulation of the tumor.</p> <p>Conclusion</p> <p>We show that in the case of a surgical pheochromocytoma removal, perioperative conditions can elicit an acquired or previously unknown congenital long QT syndrome. Therefore, preoperative α- and β-blockade is advised, QT-prolonging drugs should be avoided and potassium and magnesium plasma levels should be kept at normal to high levels.</p

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