CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: Murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers

Investigating molecular alterations to profile short- and long-term recurrence-free survival in patients with primary glioblastoma

Glioblastoma (GB) is the most aggressive type of primary brain tumor. Despite the progress in recent years regarding the diagnosis and treatment of GB, the recurrence rate remains high, due to the infiltrative and dispersive nature of the tumor, which typically results in poor patient prognosis. In the present study, 19 formalin-fixed, paraffin-embedded GB samples were selected from patients with GB tumors. The samples were classified into a short or long recurrence-free survival (RFS) group, based on the time of first recurrence of the disease in the patients. The 19 samples were molecularly characterized for mutations in the isocitrate dehydrogenase 1 (IDH1) gene, amplification of the epidermal growth factor receptor (EGFR) gene, presence of the EGFR variant III, and methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Then, the expression of 84 genes involved in cell-cell and cell-matrix interactions, and that of 84 microRNAs (miRNAs) associated with brain cancer, was profiled. In addition, a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma was also performed. Differences in the expression levels of a number of genes were detected across the short and long RFS groups. Among these genes, 5 in particular were selected, and a 5-genes combination approach was developed, which was able to differentiate between patients with short and long RFS outcome. The high levels of sensitivity and precision displayed by this 5-genes combination approach, which were confirmed with a cross-validation method, provide a strong foundation for further validation of the involvement of the aforementioned genes in GB in a larger patient population. In conclusion, the present study has demonstrated how the expression pattern of miRNAs and mRNAs in patients with GB defines a particular molecular hallmark that may increase or reduce the aggressive behavior of GB tumors, thus influencing the survival rates of patients with GB, their response to therapy and their tendency to suffer a relapse

CXCL12α/SDF‐1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo. Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro. These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Cancer astrocytes have a more conserved molecular status in long recurrence free survival (RFS) IDH1 wild-type glioblastoma patients: New emerging cancer players

Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes

ANKRd44 gene silencing: a putative role in trastuzumab resistance in HER2-like breast cancer

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance

Investigation on the molecular mechanisms underlying response to neoadjuvant therapy with Trastuzumab in HER-2 positive human breast cancer patients: ANKRD44 gene, as a new molecular factor involved in drug resistance

HER-2 receptor is overexpressed in 20-30 % of breast cancer (BC) and it has been chosen to be an effective therapeutic target of Trastuzumab, a humanized monoclonal antibody, approved by FDA. The mechanism by which this drug exerts its antitumor activity is not fully understood. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. An understanding of Trastuzumab resistance mechanisms would be a helpful tool in the development of rational drug combinations to circumvent resistance and allow better selection of patients likely to respond. Through whole-exome sequencing on 12 primary FFPE biopsies of HER2+/hormone receptor negative (ER-/PgR-) BC patients specifically selected for different response to the neoadjuvant therapy with Trastuzumab, we identify 18 informative gene mutations which selectively discriminate patients according to their response to the therapy. Among these genes, we identified ANKRD44 gene as a possible cofactor in establishing Trastuzumab resistance. By silencing ANKRD44 gene in HER-2+ breast cancer cells (BT474) we obtained a Trastuzumab-partially resistant human breast cancer cell line (shANK) in which NF-kβ is constitutively activated through the TAK1/Akt pathway. The activation of this pathway promotes also an increased glycolysis rate in resistant cells confirmed by the up-regulation of LDHB protein and an increase in TROP2 protein expression which is well known to be associated with more aggressive cancer phenotypes. Together these results identify ANKRD44 gene as a cofactor for Trastuzumab resistance but also as a possible predictive marker for the response to the neoadjuvant therapy with Trastuzumab. Moreover, it supports NF-kβ inhibition as a strategy for improving cell sensitivity to Trastuzumab

Identificazione di interattori funzionali della Poli(ADP-ribosil)polimerasi-1 mediante uno screening genetico in Saccharomyces cerevisiae

Nell’uomo, il gene PARP1 che codifica per l’enzima nucleare Poli(ADP-ribosil)polymerase-1, gioca un ruolo chiave in un’ampia varietà di processi che avvengono all’interno del nucleo della cellula, quali la trascrizione, l’integrità del genoma e mantiene la corretta struttura della cromatina. È stato osservato che mutazioni o alterazioni dell’espressione o attività di questa proteina sono spesso associate a difetti nella differenziazione cellulare e all’insorgenza di tumori. Infatti, gli inibitori di PARP sono usati come coadiuvanti nelle terapie antitumorali e antinfiammatorie. La resistenza agli inibitori di PARP che si manifesta in alcuni pazienti, non è stata ancora studiata in maniera approfondita. Scopo di questa tesi è identificare interattori funzionali di PARP1 utilizzando un sistema di screening genetico in S. cerevisiae. Per prima cosa, abbiamo determinato gli effetti dell’espressione di PARP1 in ceppi di lievito Saccharomyces cerevisiae sia aploidi che diploidi utilizzando il plasmide pYES2 che possiede un promotore inducibile per il galattosio a monte del gene PARP1 che ci consente così di indurne l’espressione in terreno di coltura contenente galattosio. Esperimenti di Western Blot dimostrano che il lievito è capace di esprimere PARP1. Abbiamo confermato che l’espressione di questo gene induce un’inibizione della crescita dei ceppi presi in esame. Per provare quindi che questo fenotipo sia dovuto all’attività polimerasica della proteina PARP1, abbiamo trattato le cellule esprimenti PARP1 con un inibitore specifico della sua attività enzimatica, il 6(5-H)-Phenantridinone (PHE). Abbiamo fatto quindi crescere le cellule in terreno contenente galattosio, così da far indurre l’espressione della proteina, assieme a concentrazioni scalari di tale inibitore. Dopo 24 ore di incubazione a 30° C in agitazione, abbiamo notato un significativo incremento della crescita di quelle colture alle concentrazioni più elevate di concentrazione di PHE. Abbiamo sfruttato questo particolare fenotipo per identificare possibili interattori funzionali di PARP1 utilizzando un sistema di screening genetico costituito da un “pool” di 4,741 ceppi di lievito diploidi ognuno dei quali porta una specifica delezione di un gene non essenziale fiancheggiata da due sequenze specifiche di DNA (“barcode”) che ci consentiranno di identificare quale è il gene deleto in questione. Abbiamo quindi trasformato questo pool di cloni con il plasmide pYES2-PARP1 che permette l’espressione della proteina solo in galattosio e contiene il gene di lievito URA3 come marcatore di selezione. Quindi abbiamo piastrato le cellule trasformate su terreno privo di uracile e contenente galattosio così da indurre l’espressione del gene PARP1. Le colonie cresciute porteranno una delezione di un gene che favorisce il ripristino della crescita. Questo significa che il gene in questione può essere coinvolto nella modulazione o regolazione dell’attività di PARP1. I cloni cresciuti in galattosio sono in totale 150. Da ognuno di loro abbiamo estratto il DNA genomico e amplificato per PCR una regione di 600 coppie di basi contenente il “barcode”. L’analisi delle sequenze ha permesso di identificare 110 geni che potrebbero rappresentare dei possibili interattori funzionali di PARP1. I dati sono in corso di studio per determinare gli omologhi nell’uomo e valutare se questi nuovi geni abbiano un ruolo nella resistenza agli inibitori di PARP. Studi futuri si baseranno nell’identificare quali tra questi interattori potranno essere testati come nuovi target terapeutici con lo scopo finale di poter mettere appunto delle terapie personalizzate e quindi meno nocive per i pazienti

Small Sat Mission for Maritime Surveillance, based on SAR, VDES/AIS and COMINT/ELINT Integrated Solutions

The recent growth of Security services needs in the Maritime Operations market is providing a major pull demand of Integrated Solutions for Maritime Surveillance able to provide several different applications and added value services for: · Maritime accidents investigations; · Piracy and armed robbery at sea; · Terrorist threats in maritime environment; · Maritime Safety (including Search and Rescue), · Maritime Security and prevention of pollution caused by ships; · Fisheries control; · Marine environment monitoring and disaster prevention; · Sea Border Control; · Maritime Defence operations. To respond accordingly to the above strong needs, new coming Integrated Solutions for Maritime Security & Safety shall be based on Small and Micro Satellite Constellation Systems with flexible and modular Spacecraft configurations able to host new high performing space communication and sensor payload technologies and suitable mission performances in terms of responsiveness (revisit time), resilience and processing distributed in the space. Small and Micro-satellite platforms result to be very effective and useful to respond to the abovementioned requirements when designed and optimised to host relevant mission payload (P/L) technologies such as: SAR, VDES/AIS, COMmunication INTelligence (COMINT)/ ELectronic INTelligence (ELINT). A small Synthetic Aperture Radar (SAR) payload represents a key space sensor capability to provide the acquisition of high resolution satellite imagery in a given Area of Interest (AoI) in maritime environment for ship and oil-spill detection and for elaborating on ground further products of Data Elevation Model (DEM), displacement monitoring and change detection. The VDES/AIS payload in VHF band facilitate the AIS identification, position reporting and tracking as well as an interoperable digital data exchange between ships, between ships and shore, between shore and ships and between ship and satellite. VDES in ITU roadmap is intended to be a globally available digital data exchange system dedicated to maritime safety, security and the protection of the maritime environment. The COMINT/ELINT payload will cope with the major needs to frequently acquire signals with relevant sensitivity over a given AoI and this can be obtained with a satellite constellation flying over the specific AoI. A COMINT/ELINT payload would consist of receiving antennas able to acquire the electromagnetic and communication signals in specific bands, pre-processed with an on-board digital processing unit (or received & forwarded to ground in pass-through mode) and thereafter transmitted on ground for further analyses, data fusion and investigations. Finally, this paper will provide an overview of integrated solutions with a Federated Fragmented Satellite System (FFSS) architecture based on small satellite constellations exploiting, in a distributed way, different payload capabilities with SAR, VDES/AIS and COMINT/ELINT technologies providing to a Maritime Surveillance ground segment the data coming from the different sources elaborated to create added-value information in order to collect as much as possible a complete Maritime Operational Picture