Functional characterization of iron transporter from Dictyostelium discoideum as a model of cellular iron homeostasis

Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species. The social amoeba Dictyostelium discoideum possesses several iron genes with the exception of transferrin, ferritin and TFR (Bozzaro et al., 2013; Peracino et al., 2013) and represents a model for the study of cellular iron homeostasis showing subcellular localization of iron transporters resembling that of macrophages. In particular, D. discoideum expresses the ortholog of Nramp1 transporter in phago-lysosomes and that of Nramp2 in the contractile vacuole. To better understand the function of dd Nramp1, the protein was expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical and electrophysiological techniques. To increase the surface localization of dd Nramp1, its N and C termini were replaced with the corresponding regions of the murine DMT1, which shows a high level of expression in the membrane of X. laevis oocytes (Gunshin et al., 1997). Dd Nramp1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of the murine DMT1 (Illing et al., 2012). It transports ferrous but not ferric iron and it is partially inhibited by Na+

Studio longitudinale sul benessere e le attitudini degli Studenti di Medicina e Chirurgia. Primi risultati della fase 1: Le caratteristiche degli studenti selezionati

We present the first results of a longitudinal study for the recognition and assessment of non-cognitive aspects of the candidates access to medicine course, and throughout the course. This study considered 8 CLMs equally distributed throughout the country and 980 students enrolled in the first year, in the academic year 2013-2014. Preliminary results obtained from the analysis of the questionnaire used in the research show that students expect a profession characterized by the aid for patients and socially useful, while they do not expect a low pay as well as a work activity risky for health. Students are motivated to the academic course mainly by the desire to care for others, while variously opportunistic aspects are much less relevant. Students show high capacity of self-regulation, a high level of empathy in its both aspects of propensity to engagement with others in their difficult moments, as well as of tendency to spontaneously take the perspective of others, while they are able to maintain a goal directed behavior even in the presence of suffering others. These students are basically satisfied, they show a confident and positive attitude towards life and a substantial psychological health. However, it is possible to identify a sub-group of students showing signals of psychological fragility who must be carefully monitored: their profiles will be analyzed in more detail, through in-depth interviews scheduled for the third/fourth year as part of faculties counselling services

Clinical outcomes, MRI evaluation and predictive factors of indirect decompression with lateral transpsoas approach for lumbar interbody fusion: a multicenter experience

Introduction: Evaluating the effects of indirect decompression obtained through lateral lumbar interbody fusion (LLIF) by clinical improvements and radiological parameters on MRI scans. Identifying predictors of better decompression and clinical outcome. Materials and methods: From 2016 to 2019, patients who underwent single- or double-level indirect decompression LLIF were consecutively reviewed. Radiological signs of indirect decompression were evaluated in preoperative and follow-up MRI studies and were subsequently correlated to clinical data, expressed as axial/radicular pain (VAS back/leg), index of disability (Oswestry Disability Index) and clinical severity of lumbar stenosis (Swiss Spinal Stenosis Questionnaire). Results: 72 patients were enrolled. The mean follow-up was 24 months. Differences in vertebral canal area (p < 0.001), height of the foramina (p < 0.001), thickness of the yellow ligament (p = 0.001) and anterior height of the interbody space (p = 0.02) were observed. Older age (p = 0.042), presence of spondylolisthesis (p = 0.042), presence of intra-articular facet effusion (p = 0.003) and posterior height of the implanted cage (p = 0.020) positively affected the increase of the canal area. Change in root canal area (p < 0.001), height of the implanted cage (p = 0.020) and younger age (p = 0.035) were predictive factors of root pain relief, while increased vertebral canal area (p = 0.020) and height of the interbody fusion cage (p = 0.023) positively affected the severity of clinical stenosis. Conclusions: LLIF indirect decompression showed both clinical and radiological improvements. Presence and degree of spondylolisthesis, presence of intra-articular facet effusion, age of the patient and height of the cage were predictive factors of major clinical improvements

Self-organized Vortex State in Two-dimensional Dictyostelium Dynamics

We present results of experiments on the dynamics of Dictyostelium discoideum in a novel set-up which constraints cell motion to a plane. After aggregation, the amoebae collect into round ''pancake" structures in which the cells rotate around the center of the pancake. This vortex state persists for many hours and we have explicitly verified that the motion is not due to rotating waves of cAMP. To provide an alternative mechanism for the self-organization of the Dictyostelium cells, we have developed a new model of the dynamics of self-propelled deformable objects. In this model, we show that cohesive energy between the cells, together with a coupling between the self-generated propulsive force and the cell's configuration produces a self-organized vortex state. The angular velocity profiles of the experiment and of the model are qualitatively similar. The mechanism for self-organization reported here can possibly explain similar vortex states in other biological systems.Comment: submitted to PRL; revised version dated 3/8/9

Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd− cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd− cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein

Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner.

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV