Robotic Approach to Ureteral Endometriosis: Surgical Features and Perioperative Outcomes

Introduction: Surgical treatment of ureteral endometriosis is necessary to relieve urinary symptoms of obstruction and to preserve renal function. Which surgical approach to ureteral endometriosis should be considered the most appropriate is debated, due to the lack of scientific evidence. The aim of the present study is to assess the feasibility and to describe the perioperative outcomes of minimally invasive treatment of deep ureteral endometriosis using robotic assistance, highlighting the technical benefits and the limits of this approach. Method: A case-series including 31 consecutive patients affected by high-stage endometriosis including ureteral endometriosis using robotic assistance in our Department between November 2011 and September 2017. Results: All procedures were successfully completed by robotic technique, resulting in full excision of the parametrial nodules involving the ureter. Mean operating time was 184.8 ± 81min. Mean hospital stay was 4.02 ± 3 days. Perioperative complications occurred in five patients and 4 out of 5 involved the urinary tract. Conclusions: Robotic surgery for deep infiltrating endometriosis of the ureter was feasible and allowed complete resection of ureteral nodules in all cases. No intraoperative complications arose, but a non-negligible rate of urinary tract complications was detected. This calls for a careful assessment of the benefits and specific risks associated with the use of robotic surgery for the treatment of deep infiltrating endometriosis of the ureter

Free and poly-methyl-methacrylate-bounded BODIPYs: photodynamic and antimigratory effects in 2D and 3D cancer models

Several limitations, including dark toxicity, reduced tumor tissue selectivity, low photostability and poor biocompatibility hamper the clinical use of Photodynamic therapy (PDT) in cancer treatment. To overcome these limitations, new PSs have been synthetized, and often combined with drug delivery systems, to improve selectivity and reduce toxicity. In this context, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) have recently emerged as promising and easy-to-handle scaffolds for the preparation of effective PDT antitumor agents. In this study, the anticancer photodynamic effect of newly prepared negatively charged polymethyl methacrylate (nPMMA)-bounded BODIPYs (3@nPMMA and 6@nPMMA) was evaluated on a panel of 2D- and 3D-cultured cancer cell lines and compared with free BODIPYs. In particular, the effect on cell viability was evaluated, along with their ability to accumulate into the cells, induce apoptotic and/or necrotic cell death, and inhibit cellular migration. Our results indicated that 3@nPMMA and 6@nPMMA reduce cancer cell viability in 3D models of HC116 and MCF7 cells more effectively than the corresponding free compounds. Importantly, we demonstrated that MDA-MB231 and SKOV3 cell migration ability was significantly impaired by the PDT treatment mediated by 3@nPMMA and 6@nPMMA nanoparticles, likely indicating the capability of this approach to reduce metastatic tumor potential

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

FM19G11-Loaded Gold Nanoparticles Enhance the Proliferation and Self-Renewal of Ependymal Stem Progenitor Cells Derived from ALS Mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression

Correlati infiammatori in pazienti con endometriosi: analisi degli outcome clinici nella Procreazione Medicalmente Assistita

L’endometriosi è una patologia benigna cronica e ricorrente, estrogeno dipendente, che colpisce circa il 10% delle donne in età fertile ed è spesso associata a dolore pelvico cronico ed infertilità. È stato stimato che il 30-50% nelle donne con infertilità presenti endometriosi e che essa coinvolga il 10-25% delle donne che ricorrono alla Procreazione Medicalmente Assistita (PMA). I meccanismi per cui questa patologia determini infertilità, oltre al sovvertimento anatomico della pelvi, sono molti ed ancora non sono stati del tutto chiariti. L’endometriosi sembra essere il risultato di una alterazione del sistema immunitario associata ad infiammazione cronica e ad una iperproduzione di prostaglandine, citochine e chemochine. La transizione epitelio-mesenchimale (EMT) è un processo attraverso cui le cellule epiteliali perdono la tipica organizzazione polarizzata, acquisendo la motilità delle cellule mesenchimali. Si ritiene che questi cambiamenti siano prerequisiti essenziali per lo sviluppo delle lesioni endometriosiche. Dato che il processo dell’EMT è principalmente studiato a livello oncologico, e che l’eziopatogenesi dell'endometriosi rimane controversa, pochi studi hanno fino ad ora indagato il fenomeno dell’EMT in tale patologia. Su queste basi, lo scopo di tale lavoro è stato quello di investigare i correlati del processo EMT nell’ambito dell’endometriosi nelle pazienti infertili. In particolare, l’analisi dei biomarker e la correlazione con i dati clinici si è focalizzata sui pathway collegati all’EMT di tipo 2, che spesso si verifica in risposta a lesioni infiammatorie. Il nostro è uno studio preliminare prospettico in cui sono stati analizzati parametri clinici e biologici di 14 pazienti affette da endometriosi che si sono sottoposte ad un ciclo di stimolazione ovarica controllata per infertilità presso il Centro di Procreazione Medicalmente Assistita del Dipartimento di Medicina Clinica e Sperimentale dell’Università di Pisa da Ottobre 2018 a Giugno 2019. Esse sono state confrontate con 12 pazienti non affette da endometriosi, che si sono rivolte al nostro centro per infertilità di origine tubarica o maschile. Al momento del pick-up ovocitario, per ciascuna paziente è stato effettuato un prelievo ematico ed un prelievo del liquido follicolare “puro” da almeno un follicolo. Il liquido follicolare è stato centrifugato per eliminare le cellule residue e conservato -80°C fino al momento del dosaggio, così come il siero ottenuto per centrifugazione da sangue intero. Il gruppo di controllo ha mostrato valori medi di Ormone Anti-Mulleriano (AMH) e conta dei follicoli antrali (AFC) significativamente maggiori rispetto al gruppo delle pazienti con endometriosi (AMH 3.02±1.22 vs 1.43±0.73, p < 0.05; AFC 15.20±3.83 vs 9.54±2.76, p < 0.05) ed ha utilizzato una media della dose totale di gonadotropine significativamente inferiore (2010±801.7 vs 2970±878.8, p<0.05). I risultati hanno mostrato inoltre una migliore qualità embrionaria nei controlli piuttosto che nelle pazienti con endometriosi, con una differenza statisticamente significativa del numero medio di embrioni di grado I (1.67±1.03 vs 0.42±0.65; p < 0.05). L’analisi del siero ha evidenziato un aumento significativo, rispetto alle pazienti del gruppo di controllo, dei livelli di transforming-growth factor ß1 (TGF-ß1), il principale mediatore del fenomeno dell’EMT. Si sono riscontrati inoltre livelli significativamente più alti di NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), un fattore di trascrizione responsabile dell’amplificazione del segnale infiammatorio. Un trend di aumento, seppur non significativo, si riscontra nel siero delle pazienti per l’hypoxia inducible factor-1alpha (HIF-1alpha), la cui stabilità ed espressione viene in genere indotta da TGF-ß1. Inoltre, il siero delle pazienti ha presentato un aumento significativo dei marker associati al fenomeno dell’EMT rispetto ai controlli: si sono riscontrati infatti aumentati livelli di N-caderina (indice di un fenotipo mesenchimale) e diminuiti livelli di E-caderina (marker di un fenotipo epiteliale) rispetto ai controlli. In linea con questi dati, le pazienti reclutate nello studio mostrano una diminuzione significativa rispetto ai soggetti di controllo dei livelli sierici di interleuchina-10 (IL-10), riconosciuta tra le principali citochine anti-infiammatorie. Nel complesso tali dati suggeriscono un coinvolgimento sistemico del processo infiammatorio nelle pazienti con endometriosi, caratterizzato da un aumento di molecole pro-infiammatorie e da una diminuzione dei mediatori anti-infiammatori. Come evidenziato nel siero, anche nel fluido follicolare delle pazienti si è riscontrato un aumento significativo dei marker associati al fenomeno dell’EMT rispetto ai controlli. Nel complesso, tali dati evidenziano il coinvolgimento del fenomeno dell’EMT a livello del fluido follicolare, che potrebbe essere correlato all’invasività cellulare a livello delle lesioni endometriosiche. Sorprendentemente, non si evidenziano significative differenze tra pazienti e controlli nei livelli di TGF-ß1, come di HIF-1alpha, nel fluido follicolare. Tali dati necessiteranno tuttavia di conferma su un campione statistico più ampio

PCOS and Assisted Reproduction Technique: Role and Relevance of Inositols

Polycystic ovary syndrome is an endocrine disorder often characterized by insulin resistance and hyperinsulinemia, especially in overweight/obese women. Among insulin sensitizers, the positive role of inositols has been increasingly established in recent years. The action of inositols not only concerns the metabolic parameters of these patients, but also the hormonal profile, resulting in beneficial effects on ovarian function. For this reason, many studies have tried to recognize their role in PCOS infertile women who underwent in vitro fertilization (IVF) procedures

Role of robotic surgery on pelvic floor reconstruction

Over the past two decades, minimally invasive surgery (MIS) abdominal surgery has increasingly been used to treat pelvic organ prolapse. Besides the several advantages associated with minimal invasiveness, this approach bridged the gap between the benefits of vaginal surgery with the surgical success rates of open abdominal procedures. The most commonly performed procedure for suspension of the vaginal apex for postoperative vaginal prolapse by robotic-assisted laparoscopy is the sacrocolpopexy. Conventional laparoscopic application of this procedure was first reported in 1994 by Nezhat et al. and had not gained widespread adoption due to lengthy learning curve associated with laparoscopic suturing. Since FDA approval of the da Vinci® robot for gynecologic surgery in 2005, minimally invasive abdominal surgery for pelvic organ prolapse has become increasingly popular, as robotic-assisted laparoscopic sacrocolpopexy is an option for those surgeons without experience or training in the conventional route. Robotic surgery has made its way into the armamentarium of POP treatment and has allowed pelvic surgeons to adapt the 'gold standard' technique of abdominal sacrocolpopexy to a minimally invasive approach with improved intraoperative morbidity and decreased convalescence. In fact, repair of pelvic organ prolapse can be performed robotically, and sometimes surgeons can feel suturing and dissection during the procedures less challenging with the assistance of the robot. However, even if robotic surgery may confer many benefits over conventional laparoscopy, these advantages should continue to be weighed against the cost of the technology. To date, as long-term outcomes, evidence about robotic sacrocolpopexy for a repair of pelvic organ prolapse are not conclusive, and much more investigations are needed to evaluate subjective and objective outcomes, perioperative and postoperative adverse events, and costs associated with these procedures. It is plausible to think that the main advantage is that robotics may lead to a widespread adoption of minimally invasive techniques in the field of pelvic floor reconstructive surgery. The following review will address the development and current state of robotic assistance in treating pelvic floor reconstruction discussing available data about the techniques of robotic prolapse repair as well as morbidity, cost and clinical outcomes

Functional involvement of the transcription factor ANAC102 in pathogenesis-related protein expression

A functional genomic approach was undertaken to investigate the role of ANAC102 in resistance to pathogens in Arabidopsis thaliana. The gene product belongs to the NAC protein family, which contains a number of transcription factors unique to plants, associated with development and stress response. No experimental research has apparently been made until now on ANAC102. Previous work in our laboratory indicates that ANAC102 expression is induced by nitric oxide (NO) treatment and in the hypersensitive reaction (HR) of A. thaliana to avirulent Pseudomonas syringae pv. tomato and to Alternaria brassicicola. Arabidopsis Col-0 insertional mutants, carrying a single homozygous T-DNA insertion in the coding region of ANAC102, were obtained. The T-DNA insertion mutants produced undetectable levels of ANAC102 transcript as assessed by Real-Time RT-PCR, even in strongly inducing conditions (treatment with NO donors). These plants were studied to understand whether, and to what extent, resistance could be compromised by the loss-offunction of ANAC102. Experiments performed included observation of macroscopic and microscopic cell death, measurements of bacterial growth in plants, Northern analysis of the expression of pathogenesis related protein-1 (PR-1) and defensin PFD1.2, both in wild type and mutant plants. Results suggest that resistance against P.s. pv. tomato and A. brassicicola in the mutants is not compromised, but also indicate that ANAC102 is an important positive regulator of PR-1 gene expression. Plants overexpressing ANAC102 have also been produced and will be analysed in relation to resistance and hypersensitive cell death