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INTRODUCTION:: The study investigated the effect of untreated cardiac arrest (CA), i.e. “no-flow” time, on post-resuscitation myocardial and neurological injury, and survival in a pig model to identify an optimal duration that adequately reflects the most frequent clinical scenario. METHODS:: An established model of myocardial infarction followed by CA and cardiopulmonary resuscitation was used. Twenty-two pigs were subjected to 3 no-flow durations: short (8–10?min); intermediate (12–13?min); and long (14–15?min). Left ventricular ejection fraction (LVEF) was assessed together with thermodilution cardiac output (CO) and high sensitivity cardiac troponin T (hs-cTnT). Neurological impairment was evaluated by neurological scores, serum neuron specific enolase (NSE), and histopathology. RESULTS:: More than 60% of animals survived when the duration of CA was ≤13?min, compared to only 20% for a duration ≥14?min. Neuronal degeneration and neurological scores showed a trend towards a worse recovery for longer no-flow durations. No animals achieved a good neurological recovery for a no-flow ≥14?min, in comparison to a 56% for a duration ≤13?min (p?=?0.043). Serum NSE levels significantly correlated with the no-flow duration (r?=?0.892). Longer durations of CA were characterized by lower LVEF and CO compared to shorter durations (p?


Baldassarro V.A.,University of Bologna | Marchesini A.,Fondazione Filarete | Giardino L.,University of Bologna | Giardino L.,Fondazione Filarete | And 2 more authors.
DMM Disease Models and Mechanisms | Year: 2017

Microvascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the?-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a?-secretase inhibitor or astrocyte conditioned medium. © 2017. Published by The Company of Biologists Ltd.


Conti L.,Durham University | Conti L.,University of Milan | Nelis S.,Durham University | Zhang C.,Durham University | And 10 more authors.
Developmental Cell | Year: 2014

Plants survive adverse conditions by modulating their growth in response to a changing environment. Gibberellins (GAs) play a key role in these adaptive responses by stimulating the degradation of growth-repressing DELLA proteins. GA binding to its receptor GID1 enables association of GID1 with DELLAs. This leads to the ubiquitin-mediated proteasomal degradation of DELLAs and consequently growth promotion. We report that DELLA-dependent growth control can be regulated independently of GA. We demonstrate that when a proportion of DELLAs is conjugated to the Small Ubiquitin-like Modifier (SUMO) protein, the extent of conjugation increases during stress. We identify a SUMO-interactingmotif in GID1 and demonstrate that SUMO-conjugated DELLA binds to this motif in a GA-independent manner. The consequent sequestration of GID1 by SUMO-conjugated DELLAs leads to an accumulation of non-SUMOylated DELLAs, resulting in beneficial growth restraint during stress. We conclude that plants have developed a GA-independent mechanism to control growth. © 2014 Elsevier Inc.


Idilli A.I.,University of Milan | Morandini P.,University of Milan | Onelli E.,University of Milan | Rodighiero S.,Fondazione Filarete | And 2 more authors.
Molecular Plant | Year: 2013

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM. © 2013 The Author.


Moscatelli A.,University of Milan | Idilli A.I.,University of Milan | Rodighiero S.,Fondazione Filarete | Caccianiga M.,University of Milan
Plant Biology | Year: 2012

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.


Riboni M.,University of Milan | Galbiati M.,University of Milan | Galbiati M.,Fondazione Filarete | Tonelli C.,University of Milan | And 3 more authors.
Plant Physiology | Year: 2013

Modulation of the transition to flowering plays an important role in the adaptation to drought. The drought-escape (DE) response allows plants to adaptively shorten their life cycle to make seeds before severe stress leads to death. However, the molecular basis of the DE response is unknown. A screen of different Arabidopsis (Arabidopsis thaliana) flowering time mutants under DE-triggering conditions revealed the central role of the flower-promoting gene GIGANTEA (GI) and the florigen genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) in the DE response. Further screens showed that the phytohormone abscisic acid is required for the DE response, positively regulating flowering under long-day conditions. Drought stress promotes the transcriptional upregulation of the florigens in an abscisic acid- and photoperiod-dependent manner, so that early flowering only occurs under long days. Along with the florigens, the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 is also up-regulated in a similar fashion and contributes to the activation of TSF. The DE response was recovered under short days in the absence of the floral repressor SHORT VEGETATIVE PHASE or in GI-overexpressing plants. Our data reveal a key role for GI in connecting photoperiodic cues and environmental stress independently from the central FT/TSF activator CONSTANS. This mechanism explains how environmental cues may act upon the florigen genes in a photoperiodically controlled manner, thus enabling plastic flowering responses. © 2013 American Society of Plant Biologists. All Rights Reserved.


Cartelli D.,University of Milan | Ronchi C.,University of Milan | Maggioni M.G.,University of Milan | Rodighiero S.,Fondazione Filarete | And 2 more authors.
Journal of Neurochemistry | Year: 2010

Dysfunction of the microtubule (MT) system is an emerging theme in the pathogenesis of Parkinson's disease. This study was designed to investigate the putative role of MT dysfunction in dopaminergic neuron death induced by the neurotoxin 1-methyl-4-phenylpiridinium (MPP+). In nerve growth factor-differentiated PC12 cells, we have analyzed post-translational modifications of tubulin known to be associated with differently dynamic MTs and show that MPP+ causes a selective loss of dynamic MTs and a concomitant enrichment of stable MTs. Through a direct live cell imaging approach, we show a significant reduction of MT dynamics following exposure to MPP+ and a reorientation of MTs. Furthermore, these alterations precede the impairment of intracellular transport as revealed by changes in mitochondria movements along neurites and their accumulation into varicosities. We have also analyzed activation of caspase 3 and mitochondrial injury, well-known alterations induced by MPP+, and found that they are noticeable only when MT dysfunction is already established. These data provide the first evidence that axonal transport impairment and mitochondrial damage might be a consequence of MT dysfunction in MPP+-induced neurodegeneration, lending support to the concept that alterations of MT organization and dynamics could play a pivotal role in neuronal death in Parkinson's disease. © 2010 International Society for Neurochemistry.


Verderio C.,CNR Institute of Neuroscience | Matteoli M.,CNR Institute of Neuroscience | Matteoli M.,Fondazione Filarete
Brain Research Reviews | Year: 2011

ATP accomplishes important roles in brain, where it functions as neurotransmitter or co-transmitter, being stored and released either as single mediator or together with other neuromodulators. In the last years, the purinergic system has emerged as the most relevant mechanism for intercellular signalling in the nervous system, affecting communication between many types of neurons and all types of glia. In this review, we will focus on recently reported data which describe the role of ATP in bidirectional signalling between neurons and different populations of glial cells, in both peripheral and central system. © 2010 Elsevier B.V.


Patent
Fondazione Filarete | Date: 2012-05-31

A method is described for producing a microfluidic device (19), which comprises the phases of producing a three-dimensional template (15) of geometry equal to the channelings that is desired to obtain in the device; inserting the template in the desired position into a mould (16), keeping it suspended by at least one of its end; coating said template by immersion in (or deposition of) a material in the liquid phase (or dissolved or dispersed in a solvent) capable of solidifying by means of a chemical reaction or physical transformation, forming a material constituting the body of the final device; and selectively removing the three-dimensional template. In a variant of the method, useful for the production of scaffolds to be inserted into the human body, a porogenic material is added to the liquid precursor or to the precursor solution, such that the material of the solid matrix is characterised by a continuous structure of pores into which it is possible to insert live cells.


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