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Abstract Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.

The DOWNY MILDEW RESISTANCE 6 (DMR6) protein is a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, involved in salicylic acid (SA) metabolism. SA is recognized as an abiotic stress-tolerance enhancer, and in tomato the inactivation of DMR6 was found increase its level and induce disease-resistance against several pathogens. By applying the CRISPR/Cas9 technique, we generated Sldmr6-1 tomato mutants and tested their tolerance to drought as well as to Late Blight. Wild-type tomato cultivar ‘San Marzano’ and its Sldmr6-1 mutants were subjected to water deprivation for 7 days. WT plants exhibited severe wilting, while T2 Sldmr6-1 mutants showed turgid leaves and maintained higher Soil Relative Water Content. Eco-physiological measurements highlighted that Sldmr6-1 mutants adopted a water saving behavior reducing transpiration rate by decreasing stomatal conductance. Under drought stress the assimilation rate decreased as well, resulting in no alteration of the CO2 concentration in the sub-stomatal chamber and increasing the Water Use Efficiency. Furthermore, in Sldmr6-1 mutants the drought stress induced up-regulation of the anti-oxidant related genes SlAPX and SlGST as well as down-regulation of SlCYP707A2 gene, which is involved in ABA catabolism. At last we highlighted, for the first time in tomato, that Sldmr6-1 mutants exhibited a reduced susceptibility to Phytophthora infestans, the causal agent of Late Blight.

Objective: Recent advancements in augmented reality led to planning and navigation systems for orthopedic surgery. However little is known about mixed reality (MR) in orthopedics. Furthermore, artificial intelligence (AI) has the potential to boost the capabilities of MR by enabling automation and personalization. The purpose of this work is to assess Holoknee prototype, based on AI and MR for multimodal data visualization and surgical planning in knee osteotomy, developed to run on the HoloLens 2 headset. Methods: Two preclinical test sessions were performed with 11 participants (eight surgeons, two residents, and one medical student) executing three times six tasks, corresponding to a number of holographic data interactions and preoperative planning steps. At the end of each session, participants answered a questionnaire on user perception and usability. Results: During the second trial, the participants were faster in all tasks than in the first one, while in the third one, the time of execution decreased only for two tasks (“Patient selection” and “Scrolling through radiograph”) with respect to the second attempt, but without statistically significant difference (respectively $p$ = 0.14 and $p$ = 0.13, $p < 0.05$ ). All subjects strongly agreed that MR can be used effectively for surgical training, whereas 10 (90.9%) strongly agreed that it can be used effectively for preoperative planning. Six (54.5%) agreed and two of them (18.2%) strongly agreed that it can be used effectively for intraoperative guidance. Discussion/Conclusion: In this work, we presented Holoknee, the first holistic application of AI and MR for surgical planning for knee osteotomy. It reported promising results on its potential translation to surgical training, preoperative planning, and surgical guidance. Clinical and Translational Impact Statement - Holoknee can be helpful to support surgeons in the preoperative planning of knee osteotomy. It has the potential to impact positively the training of the future generation of residents and aid surgeons in the intraoperative stage.

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

Abstract Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis-acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them (PPO6, 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.

Bone segmentation and 3D reconstruction are crucial for total knee arthroplasty (TKA) surgical planning with Personalized Surgical Instruments (PSIs). Traditional semi-automatic approaches are time-consuming and operator-dependent, although they provide reliable outcomes. Moreover, the recent expansion of artificial intelligence (AI) tools towards various medical domains is transforming modern healthcare. Accordingly, this study introduces an automated AI-based pipeline to replace the current operator-based tibia and femur 3D reconstruction procedure enhancing TKA preoperative planning. Leveraging an 822 CT image dataset, a novel patch-based method and an improved segmentation label generation algorithm were coupled to a Combined Edge Loss UNet (CEL-UNet), a novel CNN architecture featuring an additional decoding branch to boost the bone boundary segmentation. Root Mean Squared Errors and Hausdorff distances compared the predicted surfaces to the reference bones showing median and interquartile values of 0.26 (0.19–0.36) mm and 0.24 (0.18–0.32) mm, and of 1.06 (0.73–2.15) mm and 1.43 (0.82–2.86) mm for the tibia and femur, respectively, outperforming previous results of our group, state-of-the-art, and UNet models. A feasibility analysis for a PSI-based surgical plan revealed sub-millimetric distance errors and sub-angular alignment uncertainties in the PSI contact areas and the two cutting planes. Finally, operational environment testing underscored the pipeline’s efficiency. More than half of the processed cases complied with the PSI prototyping requirements, reducing the overall time from 35 min to 13.1 s, while the remaining ones underwent a manual refinement step to achieve such PSI requirements, performing the procedure four to eleven times faster than the manufacturer standards. To conclude, this research advocates the need for real-world applicability and optimization of AI solutions in orthopedic surgical practice.

Il contributo si propone di offrire una panoramica sullo stato del fumetto 2.0 – vengono così definite quelle forme di fumetto che hanno luogo su blog e social network – attraverso l’analisi della sua lingua. Prospettiva che porta a individuare degli elementi di continuità del fumetto 2.0 con quello tradizionale; a dar conto di variabili che collocano il fumetto digitale nel solco del più ampio fenomeno del fumetto d’autore; e a dare un primo sguardo alle problematiche derivate dal passaggio del medium fumetto dalla carta al pixel. Come campione esemplificativo, e certo non esaustivo, del fumetto 2.0 si sono quindi scelte le opere di quattro autori la cui presenza sul web ha seguito tempi e modalità differenti, nell’ordine: Leo Ortolani, Zerocalcare, Sio e Fumettibrutti.

Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry “Berkeley”, and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

Tomato (Solanum lycopersicum L.) is one of the most widely grown vegetables in the world and is impacted by many diseases which cause yield reduction or even crop failure. Breeding for disease resistance is thus a key objective in tomato improvement. Since disease arises from a compatible interaction between a plant and a pathogen, a mutation which alters a plant susceptibility (S) gene facilitating compatibility may induce broad-spectrum and durable plant resistance. Here, we report on a genome-wide analysis of a set of 360 tomato genotypes, with the goal of identifying defective S-gene alleles as a potential source for the breeding of resistance. A set of 125 gene homologs of 10 S-genes (PMR 4, PMR5, PMR6, MLO, BIK1, DMR1, DMR6, DND1, CPR5, and SR1) were analyzed. Their genomic sequences were examined and SNPs/indels were annotated using the SNPeff pipeline. A total of 54,000 SNPs/indels were identified, among which 1300 were estimated to have a moderate impact (non-synonymous variants), while 120 were estimated to have a high impact (e.g., missense/nonsense/frameshift variants). The latter were then analyzed for their effect on gene functionality. A total of 103 genotypes showed one high-impact mutation in at least one of the scouted genes, while in 10 genotypes, more than 4 high-impact mutations in as many genes were detected. A set of 10 SNPs were validated through Sanger sequencing. Three genotypes carrying high-impact homozygous SNPs in S-genes were infected with Oidium neolycopersici, and two highlighted a significantly reduced susceptibility to the fungus. The existing mutations fall within the scope of a history of safe use and can be useful to guide risk assessment in evaluating the effect of new genomic techniques.

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars (‘MoneyMaker’ and ‘San Marzano’) and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 ‘MoneyMaker’ gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

Cerebral cavernous malformation (CCM) or cavernoma is a major vascular disease of genetic origin, whose main phenotypes occur in the central nervous system, and is currently devoid of pharmacological therapeutic strategies. Cavernomas can remain asymptomatic during a lifetime or manifest with a wide range of symptoms, including recurrent headaches, seizures, strokes, and intracerebral hemorrhages. Loss-of-function mutations in KRIT1/CCM1 are responsible for more than 50% of all familial cases, and have been clearly shown to affect cellular junctions, redox homeostasis, inflammatory responses, and angiogenesis. In this study, we investigated the therapeutic effects of multidrug-loaded lipid nanoemulsions in rescuing the pathological phenotype of CCM disease. The pro-autophagic rapamycin, antioxidant avenanthramide, and antiangiogenic bevacizumab were loaded into nanoemulsions, with the aim of reducing the major molecular dysfunctions associated with cavernomas. Through Western blot analysis of biomarkers in an in vitro CCM model, we demonstrated that drug-loaded lipid nanoemulsions rescue antioxidant responses, reactivate autophagy, and reduce the effect of pro-angiogenic factors better than the free drugs. Our results show the importance of developing a combinatorial preventive and therapeutic approach to reduce the risk of lesion formation and inhibit or completely revert the multiple hallmarks that characterize the pathogenesis and progression of cavernomas.

Background: Cystic pancreatic lesions (CPLs) are being identified increasingly, and some benefit from surgical treatment. With the increasing use of robotic-assisted surgery (RAS) for neoplasms of the pancreas, the aim of the present comparative study is to establish whether the RAS offered any advantages over conventional open surgery (OS) in the management of CPLs. Patients and Methods: Twenty-seven out of 37 robot-assisted left-sided pancreatectomy (LSP) performed between January 2010 and April 2017 were carried out for CPLs. The surgical outcome and histopathology were compared retrospectively with a control group of 27 patients who had undergone open LSP for CPLs, selected using a one-to-one case-matched methodology (OS-Group) from the prospectively collected institutional database. Results: The spleen was preserved in a significantly higher percentage of patients in the RAS-group (63% vs. 33.3%,P < 0.05). There was no difference in the post-operative course (pancreatic fistula and morbidity) between the two groups. The median post-operative hospital stay was significantly shorter in the RAS-group: 8 days (range 3–25) versus 12 days (range 7–26) in the OS-group (P < 0.01). No conversion to open approach was reported in the RAS-group. Conclusions: Robotically assisted LSP is a safe and effective procedure. It is accompanied by a significantly higher spleen preservation rate compared to the open approach. In addition, because of the reduced trauma, RAS incurred a shorter post-operative hospital stay and faster return to full recovery, particularly important in patients undergoing surgery for relative indications. However, these benefits of RAS for LSP require confirmation by prospective randomised controlled studies.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The ‘new genomic techniques’ (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR–Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: ‘San Marzano’ (SM) and ‘Oxheart’ (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a −7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (−7 bp and −2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.

Capsicum annuum L. is one of the most cultivated Solanaceae species, and in the open field, water limitation leading to drought stress affects its fruit quality, fruit setting, fruit size and ultimately yield. We identified stage-specific and a common core set of differentially expressed genes, following RNA-seq transcriptome analyses of a breeding line subjected to acute drought stress followed by recovery (rewatering), at three stages of plant development. Among them, two NAC transcription factor (TF) genes, i.e., CaNAC072 and CaNAC104, were always upregulated after drought stress and downregulated after recovery. The two TF proteins were observed to be localized in the nucleus following their transient expression in Nicotiana benthamiana leaves. The expression of the two NACs was also induced by NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, suggesting that CaNAC072 is an early, while CaNAC104 is a late abiotic stress-responsive gene. Virus-induced gene silencing (VIGS) of CaNAC104 did not affect the pepper plantlet’s tolerance to drought stress, while VIGS of CaNAC072 increased drought tolerance. Heterologous expression of CaNAC072 in Arabidopsis thaliana as well as in plants mutated for its homolog ANAC072 did not increase drought stress tolerance. This highlights a different role of the two NAC homologs in the two species. Here, we discuss the complex role of NACs as transcriptional switches in the response to drought stress in bell pepper.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.

In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta.

Aims: The role of minimally invasive surgery of gastrointestinal stromal tumours (GISTs) of the stomach remains uncertain especially for large and/or difficult located tumours. We are hereby presenting a single-centre series of robot-assisted resections using the da Vinci Surgical System (Si or Xi). Subjects and Methods: Data of patients undergoing robot-assisted treatment of gastric GIST were retrieved from the prospectively collected institutional database and a retrospective analysis was performed. Patients were stratified according to size and location of the tumour. Difficult cases (DCs) were considered for size if tumour was >50 mm and/or for location if the tumour was Type II, III or IV sec. Privette/Al-Thani classification. Results: Between May 2010 and February 2017, 12 consecutive patients underwent robot-assisted treatment of GIST at our institution. DCs were 10/12 cases (83.3%), of which 6/10 (50%) for location, 2/10 (25%) for size and 2/10 (25%) for both. The da Vinci Si was used in 8 patients, of which 6 (75%) were DC, and the da Vinci Xi in 4, all of which (100%) were DC. In all patients, excision was by wedge resection. All lesions had microscopically negative resection margins. There was no conversion to open surgery, no tumour ruptures or spillage and no intraoperative complications. Conclusion: Our experience suggests a positive role of the robot da Vinci in getting gastric GIST removal with a conservative approach, regardless of size and location site. Comparative studies with a greater number of patients are necessary for a more robust assessment.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

Abstract The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF−α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species.

Background: The STAR System (Ekymed SpA) is a novel multipurpose sponge developed for conventional manual laparoscopic surgery. Materials and Methods: Between December 2012 and December 2014, we successfully used the sponge in ten robot-assisted and ten direct manual laparoscopic operations to achieve haemostasis, for blunt dissections, for atraumatic lifting of solid organs, to check for bile leaks, for cleaning the surgical field thus avoiding frequent use of suction or the application of haemostatic agents. The reason of the insertion (RI), the main use (MU) and any further use (FU), once inserted, were registered for each operation and compared between the two groups. Results: The principal RI was haemostasis for minor bleeding, without differences between the two groups (P = not significant). Regard to MU, in the robotic group cleaning the surgical field was utilised more than laparoscopic group (100% vs. 60%; P = 0.03). About FU, atraumatic solid organs lifting was more frequent during robotically assisted surgery than with laparoscopy (50% vs. 0%; P = 0.01). A statistically more frequent use of the sponge was registered during standard laparoscopy for the blunt dissection (30% vs. 80%; P = 0.03). Conclusions: The STAR System was beneficial in both approaches, but it imparts added benefit during robotically-assisted laparoscopic surgery organs because of the lack of tactile feedback and because the operating surgeon is remote from the patient, and has to rely on the assisting surgeon in the sterile field for dealing with bleeding episodes, cleansing/mopping the operative field when necessary, who may not be experienced or completely proficient.

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2 and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues.For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing (VIGS) approach with the goal of assessing in vivo the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of Nicotiana benthamiana through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in N. benthamiana leaves we also established the sub-cellular localization of these three enzymes.

The recently acquired genome sequence of globe artichoke (Cynara cardunculus var. scolymus) has been used to catalog the genome's content of simple sequence repeat (SSR) markers. More than 177,000 perfect SSRs were revealed, equivalent to an overall density across the genome of 244.5 SSRs/Mbp, but some 224,000 imperfect SSRs were also identified. About 21% of these SSRs were complex (two stretches of repeats separated by

Avenanthramides (Avns), polyphenols found exclusively in oats, are emerging as promising therapeutic candidates for the treatment of several human diseases, including colon cancer. By engineering a Saccharomyces cerevisiae strain, we previously produced two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, YAvnI) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, YAvnII), which are endowed with a structural similarity to bioactive oat avenanthramides and stronger antioxidant properties. In this study, we evaluated the ability of these yeast-derived recombinant avenanthramides to inhibit major hallmarks of colon cancer cells, including sustained proliferation, migration and epithelial-mesenchymal transition (EMT). Using the human colon adenocarcinoma cell line HT29, we compared the impact of YAvns and natural Avns, including Avn-A and Avn-C, on colon cancer cells by performing MTT, clonogenic, adhesion, migration, and anchorage-independent growth assays, and analyzing the expression of EMT markers. We found that both YAvns and Avns were able to inhibit colon cancer cell growth by increasing the expression of p21, p27 and p53 proteins. However, YAvns resulted more effective than natural compounds in inhibiting cancer cell migration and reverting major molecular features of the EMT process, including the down-regulation of E-cadherin mRNA and protein levels.