Strategy to isolate, in vivo, a eukaryotic replication origin for the analysis of its asscoiated proteins

In all organisms the DNA content is precisely and efficiently replicated. DNA replication starts at specific regions called replication origins, where a preReplicative Complex (pre-RC) is assembled at the end of the G2/M phase, maldng chromatin competent for replication. At the transition between G1 and S phases the activation of two conserved protein kinases triggers the transition from pre-RC into the active form, the Replicative Complex (RC). Replication origins are not fired at the same time during S phase, thus we can define origins as “early” and “late” according to their time of activation. During the past years our knowledge about DNA replication has improved due to the identification of novel proteins. Despite a large body of work, it remains possible that additional factors remain unidentified. The discovery of new proteins by genetic means is difficult due to the presence of redundant mechanisms and to their requirement for cell survival. Thus the aim of my project is to develop an unbiased assay to study, in Saccharomyces cerevisiae, the proteome of a single eukaryotic replication origin and identify novel proteins involved in DNA replication. Because the chromosomal environment profoundly influences the behaviour of replication origins (such as timing, efficiency), it is therefore of interest to analyse the proteomics of replication origins in their endogenous context. Taking advantage of the pSRl recombination system, I constructed two plasmids (pLTl/RsiteA and pLT2/RsiteB) that led to the integration of recombination sites into the yeast genome and the excision in vivo of the late replication origin ARS1413. These plasmids, allowing the excision of any flanking region, represent important genetic tools. The resulting episome has been isolated from the genome by density gradient purification in order to analyze by mass spectrometry the protein associated with the specific replication origin. This technique can be used to study the proteins involved in different steps of DNA replication. In fact cells arrested in G1 with a-factor will provide the proteome of the pre-RC while cells treated with HU will represent the proteome of late origins that are inhibited by the S-phase checkpoint

H3 K36 Methylation Helps Determine the Timing of Cdc45 Association with Replication Origins

Replication origins fire at different times during S-phase. Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin. Activation of replication origins involves the conversion of a pre-replicative complex to a replicative complex. A pivotal step during this conversion is the binding of the replication factor Cdc45, which associates with replication origins at approximately their time of activation in a manner partially controlled by histone acetylation.Here we identify histone H3 K36 methylation (H3 K36me) by Set2 as a novel regulator of the time of Cdc45 association with replication origins. Deletion of SET2 abolishes all forms of H3 K36 methylation. This causes a delay in Cdc45 binding to origins and renders the dynamics of this interaction insensitive to the state of histone acetylation of the surrounding chromosomal region. Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states. Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins.We propose a model in which K36me1 together with histone acetylation advance, while K36me3 and histone deacetylation delay, the time of Cdc45 association with replication origins. The involvement of the transcriptionally induced H3 K36 methylation mark in regulating the timing of Cdc45 binding to replication origins provides a novel means of how gene expression may affect origin dynamics during S-phase

Innate Lymphoid Cells: Expression of PD-1 and Other Checkpoints in Normal and Pathological Conditions

Innate lymphoid cells (ILCs) belong to a family of immune cells. Recently, ILCs have been classified into five different groups that mirror the function of adaptive T cell subsets counterparts. In particular, NK cells mirror CD8+ cytotoxic T cells while ILC1, ILC2, ILC3, and Lymphoid tissue inducer (LTi)-like cells reflect the function of CD4+T helper (Th) cells (Th1, Th2, and Th17 respectively). ILCs are involved in innate host defenses against pathogens and tumors, in lymphoid organogenesis, and in tissue remodeling/repair. In recent years, important molecular inducible checkpoints (PD-1, TIM3, and TIGIT) were shown to control/inactivate different immune cell types. The expression of many of these receptors has been detected on NK cells and subsets of tissue-resident ILCs in both physiological and pathological conditions, including cancer. In particular, it has been demonstrated that the interaction between PD-1+ immune cells and PD-L1/PD-L2+ tumor cells may compromise the anti-tumor effector function leading to tumor immune escape. However, while the effector function of NK cells in tumor is well-established, limited information exists on the other ILC subsets. We will summarize what is known to date on the expression and function of these checkpoint receptors on NK cells and ILCs, with a particular focus on the recent data that reveal an essential contribution of the blockade of PD-1 and TIGIT on NK cells to the immunotherapy of cancer. A better information regarding the presence and the function of different ILCs and of the inhibitory checkpoints in pathological conditions may offer important clues for the development of new immune therapeutic strategies

Human natural killer cells and other innate lymphoid cells in cancer: Friends or foes?

Innate lymphoid cells (ILC) including NK cells (cytotoxic) and the recently identified "helper" ILC1, ILC2 and ILC3, play an important role in innate defenses against pathogens. Notably, they mirror analogous T cell subsets, regarding the pattern of cytokine produced, while the timing of their intervention is few hours vs days required for T cell-mediated adaptive responses. On the other hand, the effectiveness of ILC in anti-tumor defenses is controversial. The relevance of NK cells in the control of tumor growth and metastasis has been well documented and they have been exploited in the therapy of high risk leukemia in the haploidentical hematopoietic stem cell transplantation setting. In contrast, the actual involvement of helper ILCs remains contradictory. Thus, while certain functional capabilities of ILC1 and ILC3 may favor anti-tumor responses, other functions could rather favor tumor growth, neo-angiogenesis, epithelial-mesenchymal transition and metastasis. In addition, ILC2, by secreting type-2 cytokines, are thought to induce a prevalent pro-tumorigenic effect. Finally, the function of both NK cells and helper ILCs may be inhibited by the tumor microenvironment, thus adding further complexity to the interplay between ILC and tumors

What’s in the bee nest holes? A single aggregation of Megachile parietina reveals and helps to fill up Eltonian shortfalls

Megachile (Chalicodoma) parietina (Geoffroy, 1785) is a Palearctic solitary bee included in the Red List of some central European Countries. Females build durable nests, reused year after year, by mixing soil with a salivary secretion. Like for most solitary bees, the resources contained within M. parietina nests attract several other insects which exploit pollen supplies or feed on the immature brood. These associated insects have mainly been studied for mantained bees and considered for their effect on the host reproductive success.A very large nesting aggregation of M. parietina in Central Tuscany has been studied for three consecutive years. We have identified 32 associated insect species, which certainly are an underestimate of the species present. Among the identified species, only eight had been previously reported for M. parietina. All the species were classified both according to the specificity for the host taxon (Chalicodoma, Megachilidae, Anthophila, Hymenoptera, Others) and to the ecological relationship (cleptoparasites, parasitoids, predators of larvae, food commensal, scavengers, and occasional nest users).This highlighted both the richness of the ecological network within the nesting aggregation and the value of studying these nesting sites to fill Eltonian shortfalls, i.e. the deficiency in ecology knowledge, of bees and their associated fauna.Implications for insect conservation.We suggest that, besides their role in pollination, large and stable bee nesting sites increase the local insect biodiversity, and that attention should be paid to their conservation within actions aimed to support populations of wild pollinators

Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer

TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells

BackgroundIn early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s).MethodsSP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs.ResultsSPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals.ConclusionsTLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID‐19 subjects

Secondo Protocollo di Implementazione Misure per il contrasto e il contenimento della diffusione del virus Sars-CoV-2

Questo documento è da intendersi come protocollo di implementazione delle attività nel corso della attuale fase dell’emergenza pandemica e, con lo “Addendum al Documento di Valutazione dei Rischi dedicato al rischio biologico derivante da Sars-CoV-2, protocollo di sicurezza anti contagio, misure di prevenzione e protezione, formazione e informazione”, le Linee guida operative per i lavoratori e le lavoratrici dello “Istituto Nazionale di Astrofisica” Misure per il contrasto e il contenimento della diffusione del virus Sars-CoV-2 e il Protocollo di implementazione MAB (Musei Archivi Biblioteche) dell’INAF, Misure per il contrasto e il contenimento della diffusione del virus Sars-CoV-2, dei quali costituisce parte integrante, contiene misure per il contrasto e il contenimento della diffusione del virus Sars-CoV-2 per ogni Struttura di Ricerca INAF - Istituto Nazionale di Astrofisica e per la sede della Amministrazione Centrale, e sostituisce integralmente il “Protocollo di Implementazione Fase 2, Misure per il contrasto e il contenimento della diffusione del virus Sars-CoV-2” adottato con nota Circolare del Direttore Generale del 15 maggio 2020, numero 2482. Le disposizioni contenute nel Decreto del Presidente del Consiglio dei Ministri del 5 Agosto 2020 non si concretano in una totale “ripresa” delle attività di ricerca, ma semplicemente in un “ampliamento”, peraltro assai limitato e condizionato, delle stesse. È quindi necessario, in questa “Fase”, adottare misure che consentano, ove possibile, di svolgere le attività lavorative nella massima sicurezza. Pertanto, il Direttore Generale, d'intesa con il Presidente, il Direttore Scientifico e il Collegio dei Direttori di Struttura, ha avviato un processo volto a definire le azioni propedeutiche all’aggiornamento del “processo di implementazione” delle attività di ricerca e di laboratorio che potranno essere svolte in questa nuova “Fase”, nella consapevolezza che le stesse non devono arrecare alcun nocumento alla salute dei dipendenti dell'Ente e non devono, in alcun modo, favorire, direttamente o indirettamente, una recrudescenza della pandemia in atto, salvaguardando il bene supremo della salute pubblica, costituzionalmente tutelato, e che facciano, quindi, prevalere l'interesse generale sulle logiche puramente individualistiche (Circolare 2 maggio 2020, n. 2083, Allegato 9). Il presente documento tiene conto delle indicazioni contenute nei vari aggiornamenti dei provvedimenti Governativi e delle raccomandazioni delle Autorità Sanitarie Nazionali ed Internazionali, individua e definisce, per tutte le Strutture di Ricerca, le misure di sicurezza che dovranno essere adottate e i dispositivi da utilizzare, suscettibili di ulteriori e/o diverse implementazioni a livello locale, in ragione delle diverse peculiarità delle singole Strutture della specificità dei luoghi, delle esigenze logistiche, delle misure organizzative adottate e di eventuali aggiornamenti delle disposizioni normative. Resta inteso che in base all’evoluzione dello scenario epidemiologico, e nell’ottica della tutela della pubblica sicurezza, le misure indicate potranno essere rimodulate, anche in senso più restrittivo, e dovranno essere immediatamente applicate eventuali, future e più restrittive disposizioni governative Regionali e/o locali. Il Direttore Generale, il Direttore Scientifico e i Direttori di Struttura, ciascuno nell'ambito delle rispettive competenze, individuano idonee procedure di controllo dell'applicazione delle predette misure di sicurezza, con la collaborazione di RSPP, RLS e Medico Competente. I contenuti del documento saranno aggiornati ad ogni variazione della valutazione del rischio e delle misure di contrasto alla diffusione del Sars-CoV-19 da parte degli organi competenti. Ogni sede integra con eventuali indicazioni del Responsabile della Prevenzione e Protezione, del Medico Competente, del Rappresentante dei Lavoratori per la Sicurezza, anche in relazione all’ambiente specifico

Addendum al Documento Valutazione dei Rischi dedicato al rischio biologico derivante da Sars-CoV-2, protocollo di sicurezza anti-contagio misure di prevenzione e protezione formazione e informazione

Documento da allegare al Documento di Valutazione dei Rischi di ogni Struttura di Ricerca INAF - Istituto Nazionale di Astrofisica. Documento approvato con nota Circolare del 15 maggio 2020, numero 2482