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The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of the most widely adopted methods for viability assessment, which is also used in evaluating drug cytotoxicity, is the resazurin-based assay. This assay exploits the ability of living cells to convert resazurin into fluorescent resorufin, providing a reliable indicator of metabolic activity. By measuring this conversion, cell viability can be estimated. Resazurin assay is extensively used for evaluating cytotoxic effects on various cell lines, including A549 cells, thereby bridging the gap between in vitro experimentation and drug development. However, frequent data inconsistencies in pre-clinical drug screening highlight the critical need for standardization to ensure reliability and reproducibility. This manuscript addresses these challenges by describing the optimization of resazurin-based viability assays for A549 cells in both 2D cultures and 3D fibrin gel models. By optimizing this test, the study aims to enhance the reliability of cytotoxicity results and introduces a new standard operating procedure, thus providing consistent results with minimal measurement uncertainty. This standardization is crucial for advancing drug screening and ensuring robust research findings.

Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

RNA-dependent RNA polymerases (RDRs) are key players in the antiviral defence mediated by RNA silencing in plants. RDR6 is one of the major components of the process, regulating the infection of certain RNA viruses. To better clarify its function against DNA viruses, we analyzed the effect of RDR6 inactivation (RDR6i) in N. benthamiana plants on two phloem-limited begomoviruses, the bipartite Abutilon mosaic virus (AbMV) and the monopartite tomato yellow leaf curl Sardinia virus (TYLCSV). We observed exacerbated symptoms and DNA accumulation for the New World virus AbMV in RDR6i plants, varying with the plant growth temperature (ranging from 16 °C to 33 °C). However, for the TYLCSV of Old World origin, RDR6 depletion only affected symptom expression at elevated temperatures and to a minor extent; it did not affect the viral titre. The accumulation of viral siRNA differed between the two begomoviruses, being increased in RDR6i plants infected by AbMV but decreased in those infected by TYLCSV compared to wild-type plants. In situ hybridization revealed a 6.5-fold increase in the number of AbMV-infected nuclei in RDR6i plants but without egress from the phloem tissues. These results support the concept that begomoviruses adopt different strategies to counteract plant defences and that TYLCSV evades the functions exerted by RDR6 in this host.

Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th–September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 ± 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.

DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection with TYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd infection promotes a strong hypermethylation of TYLCSV DNA, thus supporting a mechanistic link with the antagonism of the viroid on the virus in co-infected tomato plants. These results describe the interaction between two nuclear-replicating pathogens and show that they differentially interfere with DNA methylation pathways.

Ricania speculum is an Asian planthopper that was accidentally imported into Europe from the Far East. The planthopper was first observed in Italy in 2009 (Genoa province, Liguria) and is now considered established. The current range of R. speculum in Italy includes Liguria, Tuscany, Veneto, Piedmont and Latium. Ricania speculum is polyphagous and has been observed on wild and cultivated plants that are herbaceous or woody. This phloem feeder frequently feeds on Vitis spp. (cultivated and wild grapevines) and Clematis vitalba plants that are hosts of Flavescence dorée phytoplasma (FDp), a quarantine phloem-limited bacterial pathogen and a major threat to viticulture of several European regions. The aim of this work was to assess if R. speculum could act as a vector of FDp, thereby affecting disease epidemiology. To explore the role of R. speculum in FDp transmission, nymphs were allowed to feed on FDp-infected Vicia faba plants to estimate acquisition efficiency and successively transferred onto Vitis vinifera, V. faba and C. vitalba test plants to determine transmission ability. Ricania speculum was unable to transmit FDp; some individuals acquired the phytoplasma but supported a very low level of pathogen multiplication, compared with the competent vector. Consistent with transmission results, FDp was detected only in one out of 51 tested salivary gland samples. According to our results, the role of R. speculum in FD epidemiology is expected to be negligible.

Flavescence dorée (FD) is a serious disease of grapevine, spread in Europe and caused by phytoplasmas. They are uncultivable bacteria, transmitted from plant to plant by hemipteran insects (mainly leafhoppers) and classified according to their genetic traits. Two different phytoplasma strains are associated with the disease, namely FD-C and FD-D. The former outcompetes the latter during the infection of an experimental plant host (periwinkle), although the latter is more abundant in vineyards. Mixed infections are rare in the field. Here, competition between FD-C and FD-D pathogen strains was investigated during the infection of the laboratory insect vector Euscelidius variegatus (Hemiptera: Cicadellidae). Although insects were forced to acquire both strains, single infection, irrespective of the strain type, was more frequent than expected, probably due to competition among strains. Management of the disease mainly relies on the use (compulsory in some European areas) of insecticides, with evident undesirable effects on the environment and public health. Deciphering mechanisms regulating the epidemiology of FDp strains may pave the way towards the integrated management of the disease, such as by fine-tuning the treatments and identifying mild suppressor strains to outcompete the severe ones. Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

RNA-dependent RNA polymerases (RDRs) are key players in the antiviral defence mediated by RNA silencing in plants. RDR6 is one of the major components of the process, regulating the infection of certain RNA viruses. To better clarify its function against DNA viruses, we analyzed the effect of RDR6 inactivation (RDR6i) in N. benthamiana plants on two phloem-limited begomoviruses, the bipartite Abutilon mosaic virus (AbMV) and the monopartite tomato yellow leaf curl Sardinia virus (TYLCSV). We observed exacerbated symptoms and DNA accumulation for the New World virus AbMV in RDR6i plants, varying with the plant growth temperature (ranging from 16 °C to 33 °C). However, for the TYLCSV of Old World origin, RDR6 depletion only affected symptom expression at elevated temperatures and to a minor extent; it did not affect the viral titre. The accumulation of viral siRNA differed between the two begomoviruses, being increased in RDR6i plants infected by AbMV but decreased in those infected by TYLCSV compared to wild-type plants. In situ hybridization revealed a 6.5-fold increase in the number of AbMV-infected nuclei in RDR6i plants but without egress from the phloem tissues. These results support the concept that begomoviruses adopt different strategies to counteract plant defences and that TYLCSV evades the functions exerted by RDR6 in this host.

Flavescence dorée (FD) is a threat for wine production in the vineyard landscape of Piemonte, Langhe-Roero and Monferrato, Italy. Spread of the disease is dependent on complex interactions between insect, plant and phytoplasma. In the Piemonte region, wine production is based on local cultivars. The role of six local grapevine varieties as a source of inoculum for the vector Scaphoideus titanus was investigated. FD phytoplasma (FDP) load was compared among red and white varieties with different susceptibility to FD. Laboratory-reared healthy S. titanus nymphs were caged for acquisition on infected plants to measure phytoplasma acquisition efficiency following feeding on different cultivars. FDP load for Arneis was significantly lower than for other varieties. Acquisition efficiency depended on grapevine variety and on FDP load in the source plants, and there was a positive interaction for acquisition between variety and phytoplasma load. S. titanus acquired FDP with high efficiency from the most susceptible varieties, suggesting that disease diffusion correlates more with vector acquisition efficiency than with FDP load in source grapevines. In conclusion, although acquisition efficiency depends on grapevine variety and on FDP load in the plant, even varieties supporting low FDP multiplication can be highly susceptible and good sources for vector infection, while poorly susceptible varieties may host high phytoplasma loads.

The rat ErbB2 (rErbB2) protein is a 185‐kDa glycoprotein belonging to the epidermal growth factor‐related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2‐pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ‐HT expression vector as 6X His tag fusions. All rErbB2 variants (72–74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2⁺ mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.