MAF1: a new target of mTORC1.

Yeast and mammalian MAF1 are both regulated by the TOR (target of rapamycin) pathway. However, the exact mechanisms of regulation diverge at TOR, with yeast Maf1 phosphorylated mainly by the TORC1 (TOR complex 1) substrate Sch9 kinase and mammalian MAF1 by mTORC1 (mammalian target of rapamycin complex 1) itself. Sch9 phosphorylation of yeast Maf1 regulates Maf1 localization, but it is less clear whether phosphorylation of human MAF1 regulates its localization. Replacement of phosphosites with alanine decreases Pol III (RNA polymerase III) transcription, but the effect is much more pronounced for human MAF1 than for the yeast protein. In both cases, Pol III repression can be further increased by rapamycin treatment or, in mammalian cells, serum starvation, suggesting that the TOR pathway controls another aspect of Pol III transcription that is closely linked to MAF1, as it depends on the presence of MAF1.

Cytomegalovirus serology and replication remain associated with solid organ graft rejection and graft loss in the era of prophylactic treatment.

BACKGROUND: Cytomegalovirus (CMV) replication has been associated with more risk for solid organ graft rejection. We wondered whether this association still holds when patients at risk receive prophylactic treatment for CMV. METHODS: We correlated CMV infection, biopsy-proven graft rejection, and graft loss in 1,414 patients receiving heart (n=97), kidney (n=917), liver (n=237), or lung (n=163) allografts reported to the Swiss Transplant Cohort Study. RESULTS: Recipients of all organs were at an increased risk for biopsy-proven graft rejection within 4 weeks after detection of CMV replication (hazard ratio [HR] after heart transplantation, 2.60; 95% confidence interval [CI], 1.34-4.94, P<0.001; HR after kidney transplantation, 1.58; 95% CI, 1.16-2.16, P=0.02; HR after liver transplantation, 2.21; 95% CI, 1.53-3.17, P<0.001; HR after lung transplantation, 5.83; 95% CI, 3.12-10.9, P<0.001. Relative hazards were comparable in patients with asymptomatic or symptomatic CMV infection. The CMV donor or recipient serological constellation also predicted the incidence of graft rejection after liver and lung transplantation, with significantly higher rates of rejection in transplants in which donor or recipient were CMV seropositive (non-D-/R-), compared with D- transplant or R- transplant (HR, 3.05; P=0.002 for liver and HR, 2.42; P=0.01 for lung transplants). Finally, graft loss occurred more frequently in non-D- or non-R- compared with D- transplant or R- transplant in all organs analyzed. Valganciclovir prophylactic treatment seemed to delay, but not prevent, graft loss in non-D- or non-R- transplants. CONCLUSION: Cytomegalovirus replication and donor or recipient seroconstellation remains associated with graft rejection and graft loss in the era of prophylactic CMV treatment.

Physical activity modulates heat shock protein-72 expression and limits oxidative damage accumulation in a healthy elderly population aged 60 90 years

BACKGROUND: Reactive oxygen species production increases during aging, whereas protective mechanisms such as heat shock proteins (HSPs) or antioxidant capacity are depressed. Physical activity has been hypothesized to provide protection against oxidative damage during aging, but results remain controversial. This study aimed to investigate the effect of different levels of physical activity during aging on Hsp72 expression and systemic oxidative stress at rest and in response to maximal exercise. METHODS: Plasma antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC), thiobarbituric acid-reactive species (TBARS), advanced oxidized proteins products (AOPP), and Hsp72 expression in leukocytes were measured before and after maximal exercise testing in 32 elderly persons (aged 73.2 years), who were assigned to two different groups depending on their level of physical activity during the past 12 months (OLow = moderate to low level; OHigh = higher level). RESULTS: The OHigh group showed higher aerobic fitness and TEAC (both representing 120% of OLow values) as well as lower oxidative damage (50% of OLow values) and Hsp72 expression. Exercise led to a lower increase in oxidative damage in the OHigh group. Aerobic fitness was positively correlated with TEAC and negatively with lipid peroxidation (TBARS). Hsp72 expression was negatively correlated with TEAC but positively correlated with TBARS levels. CONCLUSIONS: The key finding of this study is that, in people aged 60 to 90 years, long-term high level of physical activity preserved antioxidant capacity and limited oxidative damage accumulation. It also downregulated Hsp72 expression, an adaptation potentially resulting from lower levels of oxidative damage.

Lentiviral vectors: a powerful tool to target astrocytes in vivo.

The morphological and functional diversity of astrocytes, and their essential contribution in physiological and pathological conditions, are starting to emerge. However, experimental systems to investigate neuron-glia interactions and develop innovative approaches for the treatment of central nervous system (CNS) disorders are still very limited. Fluorescent reporter genes have been used to visualize populations of astrocytes and produce an atlas of gene expression in the brain. Knock-down or knock-out of astrocytic proteins using transgenesis have also been developed, but these techniques remain complex and time-consuming. Viral vectors have been developed to overexpress or silence genes of interest as they can be used for both in vitro and in vivo studies in adult mammalian species. In most cases, high transduction efficiency and long-term transgene expression are observed in neurons but there is limited expression in astrocytes. Several strategies have been developed to shift the tropism of lentiviral vectors (LV) and allow local and controlled gene expression in glial cells. In this review, we describe how modifications of the interaction between the LV envelope glycoprotein and the surface receptor molecules on target cells, or the integration of cell-specific promoters and miRNA post-transcriptional regulatory elements have been used to selectively express transgenes in astrocytes.

Managing 'resistance': is adherence a target for treatment?

PURPOSE OF REVIEW: Adherence to preventive measures and prescribed medications is the cornerstone of the successful management of hypertension. The role of adherence is particularly important when treatments are not providing the expected clinical results, for example, in patients with resistant hypertension. The goal of this article is to review the recent observations regarding drug adherence in resistant hypertension. RECENT FINDINGS: Today, the role of drug adherence as a potential cause of resistant hypertension is largely underestimated. Most studies suggest that a low adherence to the prescribed medications can affect up to 50% of patients with resistant hypertension.A good adherence to therapy is generally associated with an improved prognosis. Nonetheless, adherence should probably not be a target for treatment per se because data on adherence should always be interpreted in the view of clinical results. In our opinion, the availability of reliable data on drug adherence would be a major help for physicians to manage patients apparently resistant to therapy. SUMMARY: The actual development of new drugs for hypertension is slow. Thus, focusing on drug adherence to the drugs available is an important way to improve blood pressure control in the population. More emphasis should be put on measuring drug adherence in patients with resistant hypertension to avoid costly investigations and treatments.

Valved stents for transapical pulmonary valve replacement.

OBJECTIVES: Pulmonary valve insufficiency remains a leading cause for reoperations in congenital cardiac surgery. The current percutaneous approach is limited by the size of the access vessel and variable right ventricular outflow tract morphology. This study assesses the feasibility of transapical pulmonary valve replacement based on a new valved stent construction concept. METHODS: A new valved stent design was implanted off-pump under continuous intracardiac echocardiographic and fluoroscopic guidance into the native right ventricular outflow tract in 8 pigs (48.5 +/- 6.0 kg) through the right ventricular apex, and device function was studied by using invasive and noninvasive measures. RESULTS: Procedural success was 100% at the first attempt. Procedural time was 75 +/- 15 minutes. All devices were delivered at the target site with good acute valve function. No valved stents dislodged. No animal had significant regurgitation or paravalvular leaking on intracardiac echocardiographic analysis. All animals had a competent tricuspid valve and no signs of right ventricular dysfunction. The planimetric valve orifice was 2.85 +/- 0.32 cm(2). No damage to the pulmonary artery or structural defect of the valved stents was found at necropsy. CONCLUSIONS: This study confirms the feasibility of direct access valve replacement through the transapical procedure for replacement of the pulmonary valve, as well as validity of the new valved stent design concept. The transapical procedure is targeting a broader patient pool, including the very young and the adult patient. The device design might not be restricted to failing conduits only and could allow for implantation in a larger patient population, including those with native right ventricular outflow tract configurations.

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish.

Formation of the long range Dpp morphogen gradient.

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.

FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4.

Toll-like receptor 4 (Tlr4) has a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a Tlr4-induced gene. Here we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide. FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of Tlr4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.

Retinal degeneration progression changes lentiviral vector cell targeting in the retina.

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.

Assessment of needs, health-related quality of life, and satisfaction with care in breast cancer patients to better target supportive care.

BACKGROUND: This study assessed whether breast cancer (BC) patients express similar levels of needs for equivalent severity of symptoms, functioning difficulties, or degrees of satisfaction with care aspects. BC patients who did (or not) report needs in spite of similar difficulties were identified among their sociodemographic or clinical characteristics. PATIENTS AND METHODS: Three hundred and eighty-four (73% response rate) BC patients recruited in ambulatory or surgery hospital services completed the European Organisation for Research and Treatment of Cancer Quality of Life questionnaire (EORTC QLQ)-C30 quality of life [health-related quality of life (HRQOL)], the EORTC IN-PATSAT32 (in-patient) or OUT-PATSAT35 (out-patient) satisfaction with care, and the supportive care needs survey short form 34-item (SCNS-SF34) measures. RESULTS: HRQOL or satisfaction with care scale scores explained 41%, 45%, 40% and 22% of variance in, respectively, psychological, physical/daily living needs, information/health system, and care/support needs (P < 0.001). BC patients' education level, having children, hospital service attendance, and anxiety/depression levels significantly predicted differences in psychological needs relative to corresponding difficulties (adjusted R(2) = 0.11). Medical history and anxiety/depression levels significantly predicted differences in information/health system needs relative to degrees of satisfaction with doctors, nurses, or radiotherapy technicians and general satisfaction (adjusted R(2) = 0.12). Unmet needs were most prevalent in the psychological domains across hospital services. CONCLUSIONS: Assessment of needs, HRQOL, and satisfaction with care highlights the subgroups of BC patients requiring better supportive care targeting.

Utilização do target costing na determinação do preço de venda: O caso da Alvo, s.a.

Com a crescente competitividade no mundo empresarial, os preços dos produtos passaram a exercer um papel fundamental na expansão e sobrevivência das empresas. Consequentemente, hoje em dia é o mercado que determina o preço de venda de um produto, devendo a empresa produzir ao menor custo possível para garantir o retorno financeiro desejado. O objectivo do trabalho é verificar se os procedimentos de Target Costing podem ser aplicados nas Pequenas e Médias Empresas industriais, em São Vicente, cuja actividade também se destina à produção de produtos alimentícios. Como produto teste foi selecionado o produto A, confecçionado pela empresa Alvo, SA. Para atingirmos os objectivos foram utilizadas várias técnicas e métodos de pesquisa, tais como: levantamento bibliográfico, entrevista, conversas informais, questionário, levantamento de dados nos documentos financeiros da empresa ALVO, SA. Para entendermos e aplicarmos o processo de Target Costing recorreu-se à literatura do mesmo. Foi aplicado um questionário para ver a percepção dos clientes da empresa objecto de estudo, quanto ao preço que considerariam ideal pagar por cada quilograma do produto A adquirida. A entrevista realizada com o director geral da ALVO, SA acompanhada com os dados obtidos do departamento de contabilidade serviram como um meio de conhecer a empresa e o seu funcionamento, realçando informações sobre a fixação do preço venda dos seus produtos, a gestão de custos, entre outros. Antes de testar, através de um caso prático, a aplicação do Target Costing, verificou-se, primeiramente, sua aplicação em termos teóricos, testando os seus princípios e premissas para o produto A, na Empresa ALVO, SA. Como resultado constactou-se que os procedimentos de Target Costing podem ser aplicados, tanto na teoria como na prática, nas Pequenas e Médias Empresas industriais, em São Vicente, cuja actividade se destina a produção do produto A. With the increasing competition in the business world, the prices of products have come to play a key role in the expansion and survival of businesses. Consequently, today the selling price of a product is determined by the market so companies should produce at the lowest possible cost to ensure the desired financial return. The purpose of this paper work is to verify if the Target Costing’s procedure can be applied in small and medium business enterprises in São Vicente, whose activity is production of food. For that, product A was selected for tests. In order to achieve these objectives, some techniques and research methods like bibliographic analysis, interviews, informal conversations, questionnaires and analysis of the financial documents of ALVO, SA that is the subject of this case study, were utilized. With the intention of understanding and applying the Target Costing process we also resorted to a detailed reading of related bibliography. A questionnaire was applied in order to know the customers’ opinions about the ideal price for each kilogram of product A. The interview with the managing director of ALVO, SA, combined with the data obtained in the accounting department, was also used as a way to know the company, and it ’s functioning, highlight ing items such as: selling price format ion, cost management, among other aspects. Before testing, through a practical case study, the use of the Target Costing, the theoretical application was firstly verified by testing its principles and assumptions. Secondly, the application of the Target Costing’s process was shown step by step concerning product A at ALVO SA company. As a result we came to the conclusion that procedures used with Target Costing can be applied, in theory and in practice, in small or medium-sized industrial enterprises, in São Vicente, where product A is being manufactured.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

Retrovirus-induced target cell activation in the early phases of infection: the mouse mammary tumor virus model.

Mouse mammary tumor virus (MMTV) infects B lymphocytes and expresses a superantigen on the cell surface after integration of its reverse-transcribed genome. Superantigen-dependent B- and T-cell activation becomes detectable 2 to 3 days after infection. We show here that before this event, B cells undergo a polyclonal activation which does not involve massive proliferation. This first phase of B-cell activation is T cell independent. Moreover, during the first phase of activation, when only a small fraction of B cells is infected by MMTV(SW), viral DNA is detected only in activated B cells. Such a B-cell activation is also seen after injection of murine leukemia virus but not after injection of vaccinia virus, despite the very similar kinetics and intensity of the immune response. Since retroviruses require activated target cells to induce efficient infection, these data suggest that the early polyclonal retrovirus-induced target cell activation might play an important role in the establishment of retroviral infections.