Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

International audienceThe development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein–protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity‐tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest (POI) to these enzymes allows the definition of an unbiased “proxisome,” that is, all of the proteins in interaction or in close vicinity of the POI. Here, the different proximity‐labeling tools available are described and comprehensive comparison to discuss advantages and limitations is provided

Prenatal noise stress impairs HPA axis and cognitive performance in mice

Noise stress is a common environmental pollutant whose adverse effect on offspring performance has been less studied. This study was novel in terms of using "noise" as a prenatal stress compared with physical stress to explore the effect of stress during gestation on HPA axis activation, cognitive performance, and motor coordination, as well as in investigating the effect of behavioral assessments on the corticosterone (CORT) levels. Three groups of C57BL/6 mice with a gestational history of either noise stress (NS), physical stress (PS), or no stress were examined in several behavioral tests. Plasma CORT level was significantly higher before starting the behavioral tests in NS group than the two other groups. It was significantly increased after the behavioral tests in both prenatal stressed groups relative to the controls. Stress caused anxiety-like behavior and reduced learning and memory performance in both stressed groups compared to the controls, as well as decreased motor coordination in the NS group relative to the other groups. The findings suggested that: prenatal NS severely changes the HPA axis; both prenatal stressors, and particularly NS, negatively impair the offspring's cognitive and motor performance; and, they also cause a strong susceptibility to interpret environmental experiences as stressful conditions. © 2017 The Author(s)

Propofol affords no protection against delayed cerebral ischemia in a mouse model of subarachnoid hemorrhage

Delayed cerebral ischemia (DCI) is an important contributor to poor outcomes in aneurysmal subarachnoid hemorrhage (SAH) patients. We previously showed that volatile anesthetics such as isoflurane, sevoflurane and desflurane provided robust protection against SAH-induced DCI, but the impact of a more commonly used intravenous anesthetic agent, propofol, is not known. The goal of our current study is to examine the neurovascular protective effects of propofol on SAH-induced DCI. Twelve-week-old male wild-type mice were utilized for the study. Mice underwent endovascular perforation SAH or sham surgery followed one hour later by propofol infusion through the internal jugular vein (2 mg/kg/min continuous intravenous infusion). Large artery vasospasm was assessed three days after SAH. Neurological outcome assessment was performed at baseline and then daily until animal sacrifice. Statistical analysis was performed via one-way ANOVA and two-way repeated measures ANOVA followed by the Newman-Keuls multiple comparison test with significance set a

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-β (Aβ) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer\u27s disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aβ peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aβ peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA

Inhibition of Geranylgeranyl Diphosphate Synthase is a Novel Therapeutic Strategy for Pancreatic Ductal Adenocarcinoma

Rab proteins play an essential role in regulating intracellular membrane trafficking processes. Rab activity is dependent upon geranylgeranylation, a post-translational modification that involves the addition of 20-carbon isoprenoid chains via the enzyme geranylgeranyl transferase (GGTase) II. We have focused on the development of inhibitors against geranylgeranyl diphosphate synthase (GGDPS), which generates the isoprenoid donor (GGPP), as anti-Rab agents. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abnormal mucin production and these mucins play important roles in tumor development, metastasis and chemo-resistance. We hypothesized that GGDPS inhibitor (GGDPSi) treatment would induce PDAC cell death by disrupting mucin trafficking, thereby inducing the unfolded protein response pathway (UPR) and apoptosis. To this end, we evaluated the effects of RAM2061, a potent GGDPSi, against PDAC. Our studies revealed that GGDPSi treatment activates the UPR and triggers apoptosis in a variety of human and mouse PDAC cell lines. Furthermore, GGDPSi treatment was found to disrupt the intracellular trafficking of key mucins such as MUC1. These effects could be recapitulated by incubation with a specific GGTase II inhibitor, but not a GGTase I inhibitor, consistent with the effect being dependent on disruption of Rab-mediated activities. In addition, siRNA-mediated knockdown of GGDPS induces upregulation of UPR markers and disrupts MUC1 trafficking in PDAC cells. Experiments in two mouse models of PDAC demonstrated that GGDPSi treatment significantly slows tumor growth. Collectively, these data support further development of GGDPSi therapy as a novel strategy for the treatment of PDAC

Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

A Flavonoid, Luteolin, Cripples HIV-1 by Abrogation of Tat Function

Despite the effectiveness of combination antiretroviral treatment (cART) against HIV-1, evidence indicates that residual infection persists in different cell types. Intensification of cART does not decrease the residual viral load or immune activation. cART restricts the synthesis of infectious virus but does not curtail HIV-1 transcription and translation from either the integrated or unintegrated viral genomes in infected cells. All treated patients with full viral suppression actually have low-level viremia. More than 60% of treated individuals also develop minor HIV-1 –associated neurocognitive deficits (HAND) due to residual virus and immune activation. Thus, new therapeutic agents are needed to curtail HIV-1 transcription and residual virus. In this study, luteolin, a dietary supplement, profoundly reduced HIV-1 infection in reporter cells and primary lymphocytes. HIV-1inhibition by luteolin was independent of viral entry, as shown by the fact that wild-type and VSV–pseudotyped HIV-1 infections were similarly inhibited. Luteolin was unable to inhibit viral reverse transcription. Luteolin had antiviral activity in a latent HIV-1 reactivation model and effectively ablated both clade-B- and -C -Tat-driven LTR transactivation in reporter assays but had no effect on Tat expression and its sub-cellular localization. We conclude that luteolin confers anti–HIV-1 activity at the Tat functional level. Given its biosafety profile and ability to cross the blood-brain barrier, luteolin may serve as a base flavonoid to develop potent anti–HIV-1 derivatives to complement cART

Na mira do sucesso : estratégias de combate ao insucesso escolar de alunos estrangeiros

Trabalho de projeto de mestrado, Ciências da Educação (Área de especialização Formação de Adultos), Universidade de Lisboa, Instituto de Educação, 2017Considerando a importância da integração no contexto escolar e consequentemente no sucesso educativo, o presente trabalho projeto, desenvolvido no âmbito do Mestrado em Ciências da Educação, área de especialidade em Formação de Adultos, centra-se na integração de alunos estrangeiros no ensino português, assim como na dinamização de atividades de promoção do seu sucesso escolar. Deste modo, o relatório apresenta como principais temáticas a integração de estrangeiros em Portugal, o insucesso escolar dos alunos estrangeiros e as políticas educativas de combate a este fenómeno, culminando na definição e desenvolvimento de um projeto de combate ao insucesso escolar destinado a este público. O trabalho desenvolvido junto destes jovens nos últimos nove anos e o diagnóstico elaborado no Agrupamento de Escolas João de Barros permitiu compreender a importância de intervir junto da população estrangeira, fomentando o sucesso educativo e a integração no país de acolhimento, uma vez que a taxa de retenção destes jovens está muito acima dos seus colegas autóctones.Considering the importance of integration in the school context, and consequently in educational success, this project, developed in the scope of the master’s degree in Educational Sciences, specialization in Adult Education, focuses on foreign students’ integration at the Portuguese educational system and activities that could be developed to promote their school success. Therefore, the main themes of the report are the integration of foreigners in Portugal, the lack of academic experience from foreign students and educational policies to reduce this barrier, in order to define and develop a project to combat school failure among this cases. The work developed for the last nine years with this students, the diagnostic elaborated in João de Barros Group of Schools, allowed me to understand the importance of an intervention among foreign students, to foment educational success and integration in the host country, bearing in mind that the retention rate in those cases is far above the native students’ average

SIRT1-NOX4 Signaling Axis Regulates Cancer Cachexia

Approximately one third of cancer patients die due to complexities related to cachexia. However, the mechanisms of cachexia and the potential therapeutic interventions remain poorly studied. We observed a significant positive correlation between SIRT1 expression and muscle fiber cross-sectional area in pancreatic cancer patients. Rescuing Sirt1 expression by exogenous expression or pharmacological agents reverted cancer cell-induced myotube wasting in culture conditions and mouse models. RNA-seq and follow-up analyses showed cancer cell-mediated SIRT1 loss induced NF-κB signaling in cachectic muscles that enhanced the expression of FOXO transcription factors and NADPH oxidase 4 (Nox4), a key regulator of reactive oxygen species production. Additionally, we observed a negative correlation between NOX4 expression and skeletal muscle fiber cross-sectional area in pancreatic cancer patients. Knocking out Nox4 in skeletal muscles or pharmacological blockade of Nox4 activity abrogated tumor-induced cachexia in mice. Thus, we conclude that targeting the Sirt1-Nox4 axis in muscles is an effective therapeutic intervention for mitigating pancreatic cancer-induced cachexia