"Clean" genome editing in grapevine (Vitis ssp.)

In recent years new plant breeding techniques (NPBT), and in particular genome editing via Crispr/Cas9, emerged as breakthrough tools for the genetic improvments of agricultural species, allowing to precisely modify specific genes in shorter time compared to traditional breeding and without altering the genetic heritage of cultivars. Grapevine, the most economical valuable fruit crop in the world, may receive a major benefit from NPBT since viticulture is based on a few elite varieties. However, to date the European Coummission (EC) has not yet deliberated on the legal status of the NPBT products, whether they should or should not be covered by GMO legislation (Directive 2001/18). Waiting for the EC decision, we applied the Crispr/Cas9 system in grapevine for the inactivation of the VvMLO7 gene which plays a key role in susceptibility to powdery mildew. Our "clean" strategy aims at leaving in th eplant genome the minimal trace of exogenous DNA. It used the classical Agrobacterium tumefaciens (A.t.) to introgress Cas9, the sgRNA and the selection marker gene nptIIand allow removing the T-DNA cassette from the grapevine genome once the targeted mutations have been obtained. To this purpose, the Flp recombinase gene under the control of a heat-shock inducible promoter has been integrated in the T-DNA as well as its recognition sites (FRT), placed next to the A.t. left and right borders. NptII- and Cas9- positive lines of 'Chardonnay', 'Thompson seedless' and 'Microvine' were analyzed by next generation sequencing in order to assess the induced mutations in the target sites. Subsequently, the site-specific removal of the T-DNA cassette was evaluated in the heat-treated lines by quantifying nptII copy number with Real-time PCR method. The effect of powdery mildew infection on VvMLO7 edited plants in currently under evaluation

Evaluation of structural and functional changes of tight junctions in acute graft-versus host disease (GVHD)

Orientador: Erich Vinicius de PaulaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: INTRODUÇÃO: A doença do enxerto contra o hospedeiro aguda (DECHa) é a maior causa de morbidade e mortalidade precoce no transplante de células tronco hematopoéticas (TCTH). A quebra da barreira epitelial intestinal na fase inicial do TCTH é reconhecida como um evento fisiopatológico crítico para a DECHa. Esta quebra permite a translocação de antígenos da luz intestinal para submucosa e estimula a ativação de linfócitos T do doador, os quais atuam como efetores da resposta imune responsável pela DECHa. O epitélio intestinal mantém sua função de barreira através de complexos juncionais, entre os quais as junções tight (JT). Estes complexos multiproteicos dinâmicos regulam a passagem de solutos, mediante a ativação de vias se sinalização por estímulos do meio externo. Nos últimos anos, as interações proteicas e os estímulos responsáveis pela regulação funcional das JT foram descritos detalhadamente, permitindo a exploração destes mecanismos nas doenças em que a quebra da barreira epitelial apresenta papel relevante. Apesar destes avanços, aspectos fundamentais da fisiopatologia da DECHa, tais como os mecanismos celulares e moleculares que permitem a translocação de antígenos do lúmen intestinal, e os mecanismo efetores finais que resultam em diarreias profusas, ainda não foram elucidados. Neste contexto, este trabalho teve como objetivo de explorar alterações estruturais e funcionais da barreira epitelial intestinal na DECHa com foco em proteínas das junções tight. Paralelamente, utilizamos o mesmo modelo para estudar o efeito do heme, uma molécula que atua como ativador da resposta imune inata ou DAMP ("Danger associated molecular pattern") em outros contextos inflamatórios, sobre a barreira epitelial intestinal. RESULTADOS: Inicialmente, a análise retrospectiva das biopsias de 25 pacientes com DECHa GI permitiu caracterizar a associação da gravidade clínica dessa condição com a perda acentuada de criptas intestinais, mas não foi suficiente para avaliação da estados das JT. A avaliação mais detalhada destas estruturas no contexto da DECHa GI foi realizada através da imunofluorescência com marcação para a proteína da JT ZO-1, e por microscopia eletrônica, que evidenciaram alterações compatíveis com perda da integridade destas junções por ambas metodologias. Em seguida, demonstramos que o soro de pacientes com DECHa GI colhido após o condicionamento retardou a recuperação da integridade da barreira epitelial tendo como referência o efeito do soro colhido antes do condicionamento sobre esta mesma barreira. Além disso, o soro causou mudanças na localização da proteína ZO-1, que representa um elemento fundamental das JT. No que diz respeito aos efeitos do heme sobre a integridade da barreira epitelial intestinal, pudemos demonstrar que o heme foi capaz de induzir uma redução duradoura da integridade da barreira epitelial, e que heme e LPS possuem efeito sinérgico de quebra da barreira epitelial nestas células. CONCLUSÃO: Neste estudo, descrevemos as alterações histológicas mais frequentemente observadas em pacientes com DECHa GI, e apresentamos evidências morfológicas e funcionais da quebra da barreira epitelial intestinal como um dos mecanismos envolvidos com a fisiopatologia da DECHa. Paralelamente, também demonstramos o efeito do heme sobre a barreira epitelial intestinal, bem como sua sinergia com o LPSAbstract: INTRODUCTION: Acute host graft disease (GVHD) is the major cause of early morbidity and mortality in hematopoietic stem cell transplantation (HSCT). The breakdown of the intestinal epithelial barrier in the early phase of HSCT is recognized as a critical pathophysiological event for GVHD. This break allows the translocation the lumen of the intestinal to submucosal and stimulates the activation of donor T lymphocytes, which act as effectors of the immune response responsible for the GVHD. The intestinal epithelium maintains its barrier function through junctional complexes, among which the tight junctions (TJ). These dynamic multiprotein complexes regulate the passage of solutes, through the activation of pathways and signaling by stimuli from the external environment. In recent years, the protein interactions and the stimuli responsible for the functional regulation of TJ have been described in detail, allowing the exploration of these mechanisms in diseases in which the breakdown of the epithelial barrier plays a relevant role. Despite these advances, fundamental aspects of the pathophysiology of GVHD, such as the cellular and molecular mechanisms that allow the translocation of intestinal lumen antigens, and the final effector mechanisms that result in profuse diarrhea, have not yet been elucidated. In this context, this work aimed to explore structural and functional alterations of the intestinal epithelial barrier in the GVHD with a focus on tight junction proteins. In parallel, we used the same model to study the effect of heme, a molecule that acts as an activator of the innate immune response or DAMP (Danger associated molecular pattern) in other inflammatory contexts, on the intestinal epithelial barrier. RESULTS: Initially, the retrospective analysis of the biopsies of 25 patients with intestinal GVHD allowed characterizing the association of the clinical severity of this condition with the marked loss of intestinal crypts, but it was not enough to evaluate the TJ states. The more detailed evaluation of these structures in the context of the intestinal GVHD was performed by immunofluorescence with labeling for TJ ZO-1 protein, and by electron microscopy, which evidenced alterations compatible with loss of the integrity of these joints by both methodologies. Next, we demonstrate that serum from patients with intestinal GVHD harvested after conditioning delayed the recovery of epithelial barrier integrity based on the effect of serum harvested prior to conditioning on this same barrier. In addition, the serum caused changes in the localization of the ZO-1 protein, which represents a fundamental element of the TJ. Regarding the effects of heme on the integrity of the intestinal epithelial barrier, we could demonstrate that heme was able to induce a lasting reduction in the integrity of the epithelial barrier, and that heme and LPS have a synergistic effect of breaking the epithelial barrier in these cells. CONCLUSION: In this study, we describe the histological changes most frequently observed in patients with intestinal GVHD, and present morphological and functional evidence of intestinal epithelial barrier breakage as one of the mechanisms involved with the pathophysiology of GVHD. In parallel, we also demonstrated the effect of heme on the intestinal epithelial barrier, as well as its synergy with LPSDoutoradoPatologia ClinicaDoutora em Ciências Médicas01-P-3368/2017  CAPE

Universal testing for COVID-19 in patients undergoing cancer treatment during the second outbreak in Brescia

Background: The impact of coronavirus disease 2019 (COVID-19) has been overwhelming on patients with cancer, who may be at higher risk of developing severe disease. During the second COVID-19 outbreak in Italy, we planned universal microbiologic screening for patients scheduled for antineoplastic treatment. Methods: All patients with planned active treatment at Brescia University Radiation Oncology Department were screened for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with repeated nasopharyngeal swabs (NPS) from October 31, 2020. Treatment continuation, suspension, or delay was modulated for patients testing positive according to clinical presentation. Results: From October 31, 2020, to February 6, 2021, 636 patients were enrolled and 1243 NPS were performed, of which 28 (2.25%) were positive. The infection rate was 2.52%; 81.3% of the patients with a positive NPS were asymptomatic, 2 had mild disease, and 1 severe disease that led to death. All patients already on treatment with mild or asymptomatic COVID-19 carried on the therapy with no or minimal delay. Median delay for patients with infection detected before treatment start was 16.5 days. Conclusions: Detected incidence of COVID-19 was lower during the second outbreak in our patients (2.52% vs 3.23%), despite the extensive testing schedule, and substantiates the high rate of asymptomatic infections and the low mortality among patients with COVID-19 (6.3% vs 38.5% during the first outbreak). Universal SARS-CoV-2 screening for all patients with planned treatment might allow early identification of patients with COVID-19, resulting in timely management that could improve clinical outcomes and prevent spread of the infection

Urografia-TC multidetettore: ruolo diagnostico nella valutazione del paziente con ematuria non traumatica

L’ematuria può originare da qualsiasi tratto dell’apparato urinario e può essere anche unico segno di patologia neoplastica (cancro del rene o della vescica). La letteratura raccomanda pertanto di sottoporre ad attenta valutazione clinico-strumentale tutti i casi di ematuria, macroscopica e microscopica. Lo scopo del presente contributo è quello di definire il ruolo diagnostico dell’urografia-TC multidetettore (uTC-MD) nella valutazione di questo sintomo e analizzarne l’impatto nel management del paziente attraverso lo studio di 181 pazienti consecutivi valutati per macro- e microematuria nel periodo compreso tra gennaio 2003 e marzo 2006

Estudo da modulação fenotípica da célula conjuntiva hepática

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Locally advanced leiomyosarcoma of the spleen. A case report and review of the literature

<p>Abstract</p> <p>Background</p> <p>Leiomyosarcomas are rare tumours, predominantly localized in the stomach, small intestine and retroperitoneum. Only one case of primary leiomyosarcoma of the spleen is described in human beings in literature.</p> <p>Case presentation</p> <p>We report a case of locally advanced primary leiomyosarcoma of the spleen in a 54 year-old woman, diagnosed only after splenectomy, performed with the suspicion of splenic haematoma.</p> <p>Conclusion</p> <p>Due to the lack of cases, no specific chemotherapy regimen has been tested to provide a longer survival.</p

Transparent carbon nanotubes promote the outgrowth of enthorino-dentate projections in lesioned organ slice cultures

The increasing engineering of carbon-based nanomaterials as components of neuro-regenerative interfaces is motivated by their dimensional compatibility with subcellular compartments of excitable cells, such as axons and synapses. In neuroscience applications, carbon nanotubes (CNTs) have been used to improve electronic device performance by exploiting their physical properties. Besides, when manufactured to interface neuronal networks formation in vitro, CNT carpets have shown their unique ability to potentiate synaptic networks formation and function. Due to the low optical transparency of CNTs films, further developments of these materials in neural prosthesis fabrication or in implementing interfacing devices to be paired with in vivo imaging or in vitro optogenetic approaches are currently limited. In the present work, we exploit a new method to fabricate CNTs by growing them on a fused silica surface, which results in a transparent CNT-based substrate (tCNTs). We show that tCNTs favour dissociated primary neurons network formation and function, an effect comparable to the one observed for their dark counterparts. We further adopt tCNTs to support the growth of intact or lesioned Entorhinal-Hippocampal Complex organotypic cultures (EHCs). Through immunocytochemistry and electrophysiological field potential recordings, we show here that tCNTs platforms are suitable substrates for the growth of EHCs and we unmask their ability to significantly increase the signal synchronization and fibre sprouting between the cortex and the hippocampus with respect to Controls. tCNTs transparency and ability to enhance recovery of lesioned brain cultures, make them optimal candidates to implement implantable devices in regenerative medicine and tissue engineering. This article is protected by copyright. All rights reserved

Investigation on MMACHC-R161Q pathological mutant from cblC disease

The cblC disease is a rare inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by combined methylmalonic aciduria and homocystinuria. The clinical consequences are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The molecular genetic cause of the disease was found in the mutations of the gene coding for MMACHC, a 282 amino acid protein that transports and processes the various forms of Cbl. Here we present the biophysical characterization of wild type MMACHC and a variant, p.R161Q, resulting from the most common missense pathological mutation found in cblC patients. By using a biophysical approach we investigated the stability of the two proteins and their ability to bind and transform the vitamin B12, and to assemble in a dimeric structure. Moreover, interesting indications about the behaviour of the proteins resulted from the Molecular Dynamics (MD) simulations. Overall, our results reveal how a biophysical approach based on the complementarity of computational and experimental methods can offer new insights in the study of the specific effects of the pathological cblC mutation and help prospecting new routes for the cblC treatment