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Aim Percutaneous coronary intervention with stent implantation (PCI-S) in patients requiring chronic oral anticoagulant therapy (OAC) is associated with an increased risk of bleeding and ischemic complications. Different randomized studies showed a significant advantage of a double antithrombotic therapy and superiority of direct oral anticoagulant (DOAC) compared with warfarin, but real-world data are limited. Aim is to evaluate the antithrombotic management and clinical outcome of patients with an indication for OAC who undergo PCI-S in a 'real-world' setting. Methods The multicentre prospective observational PERSEO (PERcutaneouS coronary intErventions in patients treated with Oral anticoagulant therapy) Registry (ClinicalTrials.gov Identifier: NCT03392948) has been designed to enrol patients requiring OAC treated by PCI-S in 25 Italian centres. A target of at least 1080 patients will be followed for 1 year and data on thromboembolic and bleeding events and changes in antithrombotic therapy will be registered. The primary end point is a combined measure of efficacy and safety outcome (NACE), including major bleeding events and major adverse cardiac and cerebral events at 1-year follow-up in patients treated with DOAC (and dual or triple antiplatelet therapy) compared with the corresponding strategies with vitamin K antagonists. A secondary prespecified analysis has been defined to evaluate NACE in dual versus triple antithrombotic therapy after hospital discharge at 1-year follow-up. Conclusion The PERSEO Registry will investigate in a 'real world' setting the safety and efficacy of DOAC versus warfarin and dual versus triple antithrombotic therapy in patients with indication for oral anticoagulant therapy who undergo PCI-S.

Background: Measuring mobility in daily life entails dealing with confounding factors arising from multiple sources, including pathological characteristics, patient specific walking strategies, environment/context, and purpose of the task. The primary aim of this study is to propose and validate a multi-task and multi-phase protocol for simulating real-world gait accounting for all these factors within a single set of observations carried out within a limited laboratory space, while ensuring minimisation of participant burden and safety.Methods: The protocol included eight motor tasks at varying speed, incline/steps, surface, path shape, cognitive demand, and included postures that may abruptly alter the participants’ strategy of walking. It was deployed in a convenience sample of 108 participants recruited from six cohort groups that included older healthy adults (HA) and participants with potentially altered mobility due to Parkinson’s Disease (PD), Multiple Sclerosis (MS), Proximal Femoral Fracture (PFF), Chronic Obstructive Pulmonary Disease (COPD) or Congestive Heart Failure (CHF). A novelty introduced in the protocol was the tiered approach to increase difficulty both within the same task (e.g., by allowing use of aids or armrests) and across tasks.Results: The protocol proved to be safe and feasible and the addition of the more complex tasks allowed a much greater spread in walking speeds to be achieved compared to standard straight walking trials. Furthermore, it allowed a relatively realistic representation of daily life relevant mobility aspects and can therefore be used for the validation of monitoring devices used in real life.Conclusions: The suitability of the protocol for measuring gait in a variety of pathological conditions suggests that it can also be used to detect changes in gait due to, for example, the onset or progression of a disease, or due to therapy.Trial registration: ISRCTN – 12246987.

Background and aims Novel genetic determinants associated with coronary artery disease (CAD) have been discovered by genome wide association studies. Variants encompassing the CELSR2- PSRC1-SORT1 gene cluster have been associated with CAD. This study is aimed to investigate the rs629301 polymorphism association with the extent of CAD evaluated by coronary angiography (CAG), and to evaluate its associations with an extensive panel of lipid and lipoprotein measurements in a large Italian cohort of 2429 patients. Methods and results The patients were collected by four Intensive Care Units located in Palermo and Verona (Italy). Clinical Records were filed, blood samples were collected, lipids and apolipoproteins (apo) were measured in separate laboratories. CAD was defined by the presence of stenotic arteries (>50% lumen diameter) by CAG. The presence of CAD was associated with the rs629301 genotype. Patients with CAD were 78% and 73% (p = 0.007) of the T/T vs. T/G + G/G genotype carriers respectively. T/T genotype was also correlated with the number of stenotic arteries, with a 1.29 (1.04–1.61) risk to have a three-arteries disease. T/T genotype correlated with higher levels of LDL-, non-HDL cholesterol, apoB, apoE and apoCIII, and lower HDL-cholesterol. Logistic Regression confirmed that rs629301was associated with CAD independently from the common risk factors, with a risk similar to that conferred by ten years of age [odds ratios were 1.43 (1.04–1.96) and 1.39 (1.22–1.58) respectively]. Conclusions rs629301 risk allele was independently associated with the extension and severity of CAD and positively with apoE and apoB containing lipoproteins.

Autosomal dominant disorders present unique challenges, as therapeutics must often distinguish between healthy and diseased alleles while maintaining high efficiency, specificity, and safety. For this task, CRISPR/Cas remains particularly promising. Various CRISPR/Cas systems, like homology-directed repair, base editors, and prime editors, have been demonstrated to selectively edit mutant alleles either by incorporating these mutations into sgRNA sequences (near the protospacer-adjacent motif ["near the PAM"]) or by targeting a novel PAM generated by the mutation ("in the PAM"). However, these probability-based designs are not always assured, necessitating generalized, mutation-agnostic strategies like ablate-and-replace and single-nucleotide polymorphism editing. Here, we detail recent advancements in CRISPR therapeutics to treat a wide range of autosomal dominant disorders and discuss how they are altering the landscape for future therapies.

The causal agent of mal secco disease is the fungus

CRISPR/Cas9 genome editing is a modern biotechnological approach used to improve plant varieties, modifying only one or a few traits of a specific variety. However, this technology cannot be easily used to improve fruit quality traits in citrus, due to the lack of knowledge of key genes, long juvenile stage, and the difficulty regenerating whole plants of specific varieties. Here, we introduce a genome editing approach with the aim of producing citrus plantlets whose fruits contain both lycopene and anthocyanins. Our method employs a dual single guide RNA (sgRNA)-directed genome editing approach to knockout the fruit-specific β-cyclase 2 gene, responsible for the conversion of lycopene to beta-carotene. The gene is targeted by two sgRNAs simultaneously to create a large deletion, as well as to induce point mutations in both sgRNA targets. The EHA105 strain of Agrobacterium tumefaciens was used to transform five different anthocyanin-pigmented sweet oranges, belonging to the Tarocco and Sanguigno varietal groups, and ‘Carrizo’ citrange, a citrus rootstock as a model for citrus transformation. Among 58 plantlets sequenced in the target region, 86% of them were successfully edited. The most frequent mutations were deletions (from -1 to -74 nucleotides) and insertions (+1 nucleotide). Moreover, a novel event was identified in six plantlets, consisting of the inversion of the region between the two sgRNAs. For 20 plantlets in which a single mutation occurred, we excluded chimeric events. Plantlets did not show an altered phenotype in vegetative tissues. To the best of our knowledge, this work represents the first example of the use of a genome editing approach to potentially improve qualitative traits of citrus fruit.

Traditional techniques of construction using natural and locally available materials are nowadays raising the interest of architects and engineers. Clayey soil is widely present in all continents and regions, and where available it is obtained directly from the excavation of foundations, avoiding transportation costs and emissions due to the production of the binder. Moreover, raw earth is recyclable and reusable after the demolition, thanks to the absence of the firing process. The rammed earth technique is based on earth compressed into vertical formworks layer by layer to create a wall. This material owes its strength to the compaction effort and due to its manufacture procedure exhibits layers resembling the geological strata and possessing high architectural value. The hygroscopic properties of rammed earth allow natural control of the indoor humidity, keeping it in the optimal range for human health. Stabilization with lime or cement is the most common procedure to enhance the mechanical and weather resistance at once. This practice compromises the recyclability of the earth and reduces the hygroscopic properties of the material. The use of different natural stabilizers, fibers, and natural polymers by-products of the agriculture and food industry, can offer an alternative that fits the circular economy requirements. The present study analyses the mechanical strength of an Italian earth stabilized with different local waste and recycled materials that do not impair the final recyclability of the rammed earth.

This study aims to check the compatibility of a selection of waste and recycled biopolymers for rammed earth applications in order to replace the more common cement-based stabilization. Five formulations of stabilized rammed earth were prepared with different biopolymers: lignin sulfonate, tannin, sheep wool fibers, citrus pomace and grape-seed flour. The microstructure of the different formulations was characterized by investigating the interactions between earth and stabilizers through mercury intrusion porosimetry (MIP), nitrogen soprtion isotherm, powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The unconfined compressive strength (UCS) was also evaluated for all stabilized specimens. Three out of five biopolymers were considered suitable as rammed earth stabilizers. The use of wool increased the UCS by 6%, probably thanks to the combined effect of the length of the fibers and the roughness of their surfaces, which gives a contribution in binding clay particles higher than citrus and grape-seed flour. Lignin sulfonate and tannin increased the UCS by 38% and 13%, respectively, suggesting the additives' ability to fill pores, coat soil grains and form aggregates; this capability is confirmed by the reduction in the specific surface area and the pore volume in the nano- and micropore zones.

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHO(C110R)/hRHO(WT) humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHO(C110R)/hRHO(WT) patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

Among Citrus species, lemon is one of the most susceptible to mal secco disease, a tracheomycosis caused by the mitosporic fungus Plenodomus tracheiphilus, which induces chlorosis followed by leaf drop and progressive desiccation of twigs and branches. Severe infection can cause the death of the plant. Since no effective control strategies are available to efficiently control the pathogen spread, host tolerance is the most desirable goal in the struggle against mal secco disease. To date, both traditional breeding programs and biotechnological techniques were not efficient in developing novel varieties coupling tolerance to mal secco with optimal fruit quality. Furthermore, the genetic basis of host resistance has not been fully deciphered yet, hampering the set-up of marker-assisted selection (MAS) schemes. This paper provides an overview of the biotechnological approaches adopted so far for the selection of mal secco tolerant lemon varieties and emphasizes the promising contribution of marker-trait association analysis techniques for both unraveling the genetic determinism of the resistance to mal secco and detecting molecular markers that can be readily used for MAS. Such an approach has already proved its efficiency in several crops and could represent a valuable tool to select novel lemon varieties coupling superior fruit quality traits and resistance to mal secco.

Hemp-lime is one of the nature-based solutions developed since the early 2000s to reduce the huge environmental impact of the building sector; it is produced mixing lime with hemp shives produced by the scutching of industrial hemp plants. The production of such material determines the recovery and enhancement of by-products of agricultural cultivation in accordance with the principles of circular economy. Hemp-lime is a carbon negative material mainly due to the utilization of vegetal aggregates and to the carbonation reaction, characteristic of lime, through which carbon dioxide sequestration takes place. In this research, the behavior of a wall built in prefabricated hemp-lime blocks has been investigated focusing on the mortar joints and on the determination of their eventual seismic and wind bracing contribution in a building. Starting from the determination of mechanical properties of the blocks, a finite element numerical analysis of the hemp-lime wall system has been developed. The results show that mortar joints exert a stiffening action in a hemp-lime system as opposite to other building techniques where joints are generally weak parts. Although the mechanical properties of the hemp-lime wall are generally less performing than those offered by a traditional wall (brick, stone) the bracing effect is appreciable for residential buildings in moderate seismicity areas due to the reduction of stress states in the load-bearing elements as frame or pillars.

This paper is focused on the experimental characterization of the mechanical behaviour of hemp-lime block. To this purpose two different mechanical test are considered. After a preliminary geometric characterization of blocks and fibers a series of compression tests were carried out to characterize the elastic Young modulus and the compressive strength (UCS). Subsequently, a series of two direction stress path tests were carried out to describe the limit state behaviour of the material in term of internal friction angle and cohesion.

Peels are major byproducts of the juice processing industry. Citrus peels are composed of two tissues, namely the flavedo (outer layer) and the albedo (inner layer). Here, flavedo and albedo were obtained from the peel of grapefruit cv. ‘Star ruby’, lemon cv. ‘Akragas’, and sweet orange cvs. ‘Tarocco’, ‘Sanguinello’ and ‘Moro’. Freeze dried flavedo and albedo were investigated for their functional properties, and subjected to enzyme-assisted extraction with pectinase and cellulase (concentration of 6 U g−1 DW) to extract the phenolic compounds. Albedo showed superior physicochemical characteristics than flavedo. Albedo from ‘Tarocco’ exhibited the maximum water holding capacity (10.9 ± 0.48 g/g). Grapefruit albedo revealed the highest water swelling (13.4 ± 2.62 mL/g) and oil binding (6.31 ± 2.62 g/g) capacities. As regards the enzyme-assisted extraction of phytochemicals, both enzymes enhanced the extraction of phenolics (p

According to the different classification methods, the genus Opuntia belongs to the Cactaceae family native to the Americas, and encompasses several species ranging from 75 to 250. Many of these species are diffused, and some of them are cultivated in arid and semi-arid areas. The most economically important species is O. ficus-indica L. (Mill.), but the genus includes other important edible species (from diploid to octoploid), which are spread, wild, or cultivated in many regions worldwide. Opuntia species are gaining popularity in different areas, mainly arid and semi-arid, both for fruit production for human consumption and mainly for animal feeding using the tender and mature green part of the whole plant (cladodes). The taxonomic classification of the genus is hampered by several factors: the lack of reliable morphological descriptors, the recurrent intra- and intergeneric hybridization, the frequent polyploidy, and the phenotypic variation displayed by the genotypes under different ecological conditions. Due to the ambiguity of the different taxonomic hypotheses formulated based on morphological traits, in the last years, several DNA-based studies, relying on different categories of molecular markers, have been carried out in order to better assess variability level within the main cultivated species (O. ficus-indica) and to assess relationships and origin of the main species.

Lemon (Citrus limon (L.) Burm. f.) is an evergreen tree belonging to the genus Citrus. The fruits are particularly prized for the organoleptic and nutraceutical properties of the juice and for the quality of the essential oils in the peel. Herein, we report, for the first time, the release of a high-quality reference genome of the two haplotypes of lemon. The sequencing has been carried out coupling Illumina short reads and Oxford Nanopore data leading to the definition of a primary and an alternative assembly characterized by a genome size of 312.8 Mb and 324.74 Mb respectively, which agree well with an estimated genome size of 312 Mb. The analysis of the transposable element (TE) allowed the identification of 2878 regions on the primary and 2897 on the alternative assembly distributed across the nine chromosomes. Furthermore, an in silico analysis of the microRNA genes was carried out using 246 mature miRNA and the respective pre-miRNA hairpin sequences of Citrus sinensis. Such analysis highlighted a high conservation between the two species with 233 mature miRNAs and 51 pre-miRNA stem-loops aligning with perfect match on the lemon genome. In parallel, total RNA was extracted from fruit, flower, leaf, and root enabling the detection of 35,020 and 34,577 predicted transcripts on primary and alternative assemblies respectively. To further characterize the annotated transcripts based on their function, a gene ontology and a gene orthology analysis with other Citrus and Citrus-related species were carried out. The availability of a reference genome is an important prerequisite both for the setup of high-throughput genotyping analysis and for functional genomic approaches toward the characterization of the genetic determinism of traits of agronomic interest.

Mal secco is one of the most severe diseases of citrus, caused by the necrotrophic fungus Plenodomus tracheiphilus. With the main aim of identifying candidate genes involved in the response of citrus plants to “Mal secco”, we performed a de novo transcriptome analysis of rough lemon seedlings subjected to inoculation of P. tracheiphilus. The analysis of differential expressed genes (DEGs) highlighted a sharp response triggered by the pathogen as a total of 4986 significant DEGs (2865 genes up-regulated and 2121 down-regulated) have been revealed. The analysis of the most significantly enriched KEGG pathways indicated that a crucial role is played by genes involved in “Plant hormone signal transduction”, “Phenylpropanoid biosynthesis”, and “Carbon metabolism”. The main findings of this work are that under fungus challenge, the rough lemon genes involved both in the light harvesting and the photosynthetic electron flow were significantly down-regulated, thus probably inducing a shortage of energy for cellular functions. Moreover, the systemic acquired resistance (SAR) was activated through the induced salicylic acid cascade. Interestingly, RPM1 interacting protein 4, an essential positive regulator of plant defense, and BIR2, which is a negative regulator of basal level of immunity, have been identified thus representing useful targets for molecular breeding.

To evaluate the safety and efficacy of the Orsiro sirolimus-eluting stent (Biotronik) in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention (pPCI). Specific drug-eluting stent (DES) platforms might influence pPCI success rate in the mid-to-long term. Orsiro, a hybrid sirolimus DES with thin struts and a biodegradable polymer, may potentially cause less stent malapposition, stent-induced inflammation, and mechanical damage, improving clinical outcomes.We retrospectively enrolled all patients who received 1 or more Orsiro DES in the target vessel of pPCI at 9 Italian centers from January 2012 to March 2016. The primary endpoint was a device-oriented composite endpoint (DOCE) of cardiac death, any myocardial infarction clearly attributable to the intervention culprit vessel (TVMI), and ischemic-driven target-lesion revascularization (ID-TLR) at 1-year follow-up. Secondary endpoints were: (1) DOCE at 6-month and 3-year follow-up; (2) any definite/probable stent thrombosis; and (3) any major bleeding.The study cohort comprised 353 patients. At 1-year follow-up, we observed a 3.7% cumulative incidence of DOCE, consisting of 11 cardiac deaths (3.1%), 2 TVMIs (0.6%), and 2 ID-TLRs (0.6%). There was only 1 definite stent thrombosis (0.3%) and 8 bleedings (2.4%). Kaplan-Meier analysis showed DOCE-free survival rates of 96.6% at 6 months, 96.3% at 1 year, and 93.8% at 3 years.Our findings support the real-world safety and efficacy of the Orsiro stent for pPCI.

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. &times, limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = &minus, 0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.

Citrus fruit quality and scion productivity are influenced by the choice of rootstock. We aimed to evaluate the effect of rootstocks on yield and fruit quality of Mandared, a triploid pigmented mandarin. To do so, we established a rootstock field trial on a high pH soil (8.6) in which Mandared was grafted onto 11 rootstocks. These included some standard rootstocks, such as trifoliate orange ((Poncirus trifoliata (L.) Raf.), Troyer citrange (Citrus sinensis (L.) Osb. × P. trifoliata), Swingle citrumelo (Citrus paradisi Macf. × P. trifoliata), and C35 citrange (C. sinensis × P. trifoliata), as well as new releases from the Council for Agricultural Research and Economics (CREA, Acireale, Italy) and the University of California Riverside (UCR). The cumulative yield was measured over five consecutive years, while fruit quality was analyzed for two years. The trees on C35, C57 (Citrus sunki Hort. ex. Tan. × P. trifoliata), and C22 (C. sunki × P. trifoliata), started to set fruits one year earlier than the others. The trees on C57 provided some of the highest cumulative yields and canopy volumes. The production of Mandared grafted onto C57 was double that of Mandared grafted onto Troyer, while Mandared grafted onto C35 and C22 resulted in the best yield efficiency. The trees on Swingle and C57 significantly reduced the pre-harvest fruit drop, to which Mandared is particularly sensitive. However, grafting Mandared onto Swingle resulted in the highest variation among replicates, probably due to its high sensitivity to iron chlorosis. Most of the fruit quality parameters, such as fruit size, total soluble solids (TSS), and acidity were not significantly different among the rootstock treatments. However, fruits produced by Mandared grafted onto C22 had one of the highest rates of anthocyanin accumulation. The results indicate that C57, C35, and C22 were the most suitable rootstocks for Mandared in South-Eastern Sicily.

Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3&prime, hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3′-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

The origin, development, and characteristics of the principal citrus scion varieties in commercial production are described. As citrus moved from its site of origin, the primitive, ancestral forms were selected resulting in the wide range of varieties and selections of varieties currently available today. Selection criteria differed according to local preference (traditions, customs, beliefs, etc.), and the perception of fruit quality differs according to regional preferences. Most citrus varieties in commercial production were developed through the selection of natural mutations, e.g., pummelos, grapefruit, lemons, limes, oranges, and many mandarin types, although induced mutations by irradiation have resulted in several seedless citrus varieties, whereas numerous mandarin hybrids are either man-made or naturally occurring.

Citrus are fruit crops that are widely cultivated in Southern Italy. The phytochemical profile of citrus depends on the species, the tissue and the ripening stage. This study aimed to perform a comprehensive analysis of the phenolic compounds and carotenoids from four citrus species cultivated in the region of Sicily, Southern Italy, between and within the different tissues and the ripening stages. The selected species were pummelo [C. maxima (Burm. ex Rumph.) Merr.] cv. ‘Chandler’, lemon [C. limon (L.) Burm. f] cv. ‘Akragas’, and sweet orange [C. sinensis (L.) Osbeck] cvs ‘Tarocco’ and ‘Washington navel’. Fruits were harvested four times every five weeks from Sep 2018 to Jan 2019. At any stage, flavedo and albedo showed the highest contents of phytochemicals. Elevated levels of phenolic compounds, e.g., narirutin, were found in the flavedo of unripe lemon and ‘Tarocco’ orange (54.8 ± 8.15 and 248 ± 30.3 mg kg−1 dw, respectively). Narirutin decreased significantly (p

This chapter describes traditional strategies used by breeding programs around the world, and how they have resulted in improved citrus varieties. The first part examines scion breeding objectives and the advantages/disadvantages of important techniques including somatic mutants, nucellar selection, diploid and polyploid hybridization, and mutation breeding. Seedlessness remains an enormous challenge for breeders and the available methods are discussed within the context of having to simultaneously maintain or improve a host of other critical traits. The second part describes the main steps leading to the release of improved rootstocks. The importance of hybridization with citrus relatives to introgress useful genes both in scions and rootstocks breeding programs is also emphasized. New diseases and increasing consumer expectations present additional challenges for breeding programs and more efficient phenotyping and molecular markers are required.

The recent discovery of a Roman ceramics manufacturing workshop at Montelabate (Perugia, Italy), in use from the first century BC until the late-fourth to fifth centuries AD, offers a unique opportunity to study the technical processes for producing Roman amphorae. Ancient and modern clays were sampled and analysed; they do not differ significantly, supporting the hypothesis of the exploitation of the rich local clay source that allowed a continuity of production. Characterization of the clays was performed using geotechnical methods (Atterberg limits and size distribution) and by thermogravimetric and differential thermogravimetric analysis, Fourier-transform infrared spectroscopy, X-ray diffraction and X-ray fluorescence analyses. The material was suitable for pottery making with the addition of calcite and quartz sand temper. Production waste and discarded materials as well as good-quality products were also analysed with the same methodology. It is therefore possible to reconstruct the ancient technology by defining the recipe for the production of the amphorae and their firing temperature on the basis of the decomposition of clay materials and the presence of newly formed minerals.

The geochemical evolution of olivine from primitive magmatic skarn environments has been studied by atmospheric pressure experiments carried out at 1250, 1200, and 1150 °C under QFM oxygen buffering conditions. The starting materials were three synthetic basalts (i.e., meltMg#78, meltMg#75, and meltMg#72) doped with variable amounts of CaCO3, in order to reproduce the natural concentration levels of CaO-rich magmas interacting with the skarn rock shells. Results from decarbonation experiments evidence that the crystallization of Fo-CaO-rich, NiO-poor olivines is more favored at higher temperatures when primitive basaltic magmas assimilate increasing amounts of carbonate materials. The number of large size Ca cations entering olivine crystal lattice is proportional to the amount of Ca-O-Si bonds available in the melt. Due to differences between Fe2+ and Mg cation radii, the Ca-Fe2+ substitutions into M2 crystallographic site are more facilitated than Ca Mg ones, thus enhancing the forsterite component in olivine. The partitioning behavior of Ni, Mg, Fe2+, Mn, and Ca between olivine and melt has been also investigated to better understand cation redistribution mechanisms at the magma‑carbonate reaction zone. In this context, some partitioning models from the literature have been refined to more accurately quantify the geochemical evolution of primitive skarn systems. Under the effect of CaCO3 assimilation, the partitioning of divalent cations, can be parameterized as a function of temperature, bulk composition (mostly, CaO and MgO contents in both olivine and melt) and melt structure (expressed as the number of non-bridging oxygens per tetrahedrally coordinated cations). Conversely, the exchange partition coefficients between Fe2+/Ca/Mn/Ni and Mg do not vary significantly as a function of temperature and meltMg#, due to the limited influence of these parameters on the melt structure. In turn, cation exchange reactions are primarily controlled by the strong depolymerizing effect of CaCO3 assimilation that increases the number of structural sites critically important to accommodating network-modifying cations in the melt phase. The comparison between cumulates and magmatic skarns from the Colli Albani Volcanic District (Italy) and experiments from this study provides quantitative constraints on the geochemical evolution of olivine phenocrysts and their melt inclusions as a function of carbonate assimilation.

Fruits of sixteen Tarocco sweet orange (Citrus sinensis [L.] Osbeck) clonal selections were compared for their long-period cold storage aptitude, for fresh fruit consumption. Selections were evaluated, for two consecutive seasons, at harvest (T0) and after 30 (T30), 60 (T60) days of storage at 5 ± 1 °C and 85–90% relative humidity (RH), followed by 7 days of shelf-life at 20±2 °C (T60+7). The following parameters were evaluated: fruit weight, peel and pulp color, initial and residual deformation, elasticity, firmness, juice yield, TSS, pH, TA, total anthocyanin content, and for each control: weight loss and physiological disorders (chilling injury and aging). The selections showed significant variability regarding the physiological disorders, especially at T60+7 with a range of 0.0–0.6 for chilling injury index and a range of values of 0–63% for aging. At T60+7 the weight loss range was between 10.0–14.3%. Regarding overall qualitative parameters, relevant changes were observed for firmness which decreased during the storage, with values ranging from 2.58-5.28 (T0) to 2.56–4.41 (T60+7), and for the anthocyanins which increased from a range of 1.67–39.71 (T0) to 30.47–114.86 mg/L (T60+7), with different values depending on the clonal selections. Based on our results, the clonal selections that showed a better aptitude to long-period cold storage were Gangi, 2B-12–9, Pedalino and Amantea, together with the reference cultivars Meli and Messina. These clones resulted particularly tolerant to chilling injury and aging, they maintained the firmness and the most relevant qualitative traits (TSS and TA) and showed the best fruit external appearance after one week of shelf-life (T60+7).

Sweet oranges are an important source of ascorbic acid (AsA). In this study, the content of AsA in the juice and leaves of four orange clonal selections, different in terms of maturity time and the presence/absence of anthocyanins, was correlated with the transcription levels of the main genes involved in the biosynthesis, recycling, and degradation pathways. Within each variety, differences in the above pathways and the AsA amount were found between the analysed tissues. Variations were also observed at different stages of fruit development and maturation. At the beginning of fruit development, AsA accumulation was attributable to the synergic action of l-galactose and Myo-inositol, while the l-gulose pathway was predominant between the end of fruit development and the beginning of ripening. In leaves, the l-galactose pathway appeared to play a major role in AsA accumulation, even though higher GalUr isoform expression suggests a synergistic contribution of both pathways in this tissue. In juice, the trend of the AsA content may be related to the decrease in the transcription levels of the GME, GDH, MyoOx, and GalUr12 genes. Newhall was the genotype that accumulated the most AsA. The difference between Newhall and the other varieties seems to be attributable to the GLDH, GalUr12, APX2, and DHAR3 genes.

The present study aimed to investigate the effects of alcoholic fermentation on the physicochemical and phytochemical characteristics of the tissues flavedo and albedo from five citrus fruits, namely grapefruit (Citrus paradisi), lemon (Citrus limon), and sweet oranges (Citrus sinensis) cvs. ‘Tarocco’, ‘Washington navel’, and ‘Moro’. The tissues were fermented with pure and mixed cultures of Saccharomyces cerevisiae, Brettanomyces bruxellensis, and Torulaspora delbrueckii. Flavedo and albedo increased the water holding capacity (WHC) and oil binding capacity (OBC) when subjected to fermentation. The treatment with B. bruxellensis resulted the most effective at enhancing WHC and OBC, e.g., lemon flavedo achieved the highest WHC (9.67 ± 0.15 g/g; P

The taxonomic classification of the genus Opuntia, which includes several cultivated species, is complicated mainly because of inadequate morphological descriptors, the common intra- and inter-generic hybridization and the relationships between phenotypic variation and ecological conditions. The phylogenetic relationships among 72 cultivated genotypes, either selected for fruit or forage production, and wild accessions belonging to approximately 15 different species of Opuntia were inferred using cytoplasmic markers. Previous studies indicated as the most important cultivated accessions for fruit production have a polyphyletic origin but polyploidy hampered their clear phylogenetic assignment based on nuclear markers. Cytoplasmic markers are considered helpful for their maternal inheritance and for overcoming the multiple gene copy problem in polyploid phylogenetics already reported in the Opuntia genus. In particular, we combined in this work capillary electrophoresis for newly designed cpSSRs and high resolution melting for SNV analyses to identify chloroplast (ndhF-rpl32, psbJ-petA, atpB-rbcL, matK, ycf1) and mitochondrial (rpl5) DNA markers in a selected group of genotypes. The results revealed the presence of polymorphisms in the predicted cpSSRs and SNVs and clearly evidenced that most of the studied genotypes were closely related to Opuntia ficus-indica (L.) Miller. Plastid markers identified 11 chlorotypes and 8 unique genotypes. Interestingly the analysis evidenced a multiple maternal phylogeny for the fleshy fruit varieties classified as O. ficus-indica. These results allow questioning of the reliability of the current classification based on morphological parameters and reveal the narrow genetic base of the most common cultivated opuntias for fruit production, while forage genotypes evidenced greater variability.

In spite of the increasing diffusion of tunnel boring machines, conventional tunnelling is still preferred for economic reasons in case of short tunnels, unconventional cross sections or irregular tunnel trajectories. In conventional tunnelling, the mechanical response of the tunnel front is a main concern and, when tunnels are excavated in cohesive soils, this is dominated by the time factor, related to geometry, to the mean excavation rate and to the hydro-mechanical properties of the materials involved. This is particularly evident during excavation standstill: front displacements progressively increase with time and, in many cases, the system response under long-term conditions becomes unstable. In conventional tunnelling, a common technique employed to improve the system response (under both short- and long-term conditions) is the installation of fibreglass tubes within the advance core. In this paper, the mechanical response of both unreinforced and reinforced deep tunnel fronts in cohesive soils is experimentally analysed. In particular, the results of a series of 1g small-scale tests, taking into account both the influence of the excavation rate (the unloading time) on the system response and the evolution with time of the tunnel face displacements, induced by a rapid reduction in the horizontal stress applied on the tunnel face, are reported.

Objectives: This retrospective analysis sought to evaluate 1-month outcomes and therapy effectiveness of a population of patients treated with MitraClip therapy. We describe in this article the preliminary results of primary effictiveness endpoint.Background: Percutaneous Mitral Repair is being developed to treat severe mitral regurgitation (MR),with increasing real-world cases of functional MR(FMR). In the EVEREST(Endovascular Valve Edge-to-Edge Repair Study)II trial,percutaneous device showed superior safety but less reduction in MR at 1year. 4-year outcomes from EVEREST II trial showed no difference in the prevalence of moderate-severe and severe MR or mortality at 4years between surgical mitral repair and percutaneous approach.Methods: We analysed retrospectively collected data from one center experience in Italy enrolled from January2011 to December2016. The study included 62patients[mean age74±11years, 43 men(69%)] with MR of at least grade3+. Most of patients had functional MR, were in New York Heart Association(NYHA) functional class III or IV,with a large portion(78%) of mild-to-moderate Tricuspid Regurgitation(TR). One or more clips were implanted in 67procedures(62 patients). Results and Conclusions: Severity of MR was reduced in all successfully treated patients,54(90%) were discharged with MR≤2+(primary effictiveness endpoint). Clinical 1-month follow-up data showed an improvement in NYHA functional class (42patients (70%) in NYHA class I-II). 60 of 62 (97 %) successfully treated patients were free from death and mitral valve surgery at 1-month follow-up. MitraClip therapy reduces functional MR with acute MR reduction to

Blood oranges have been cultivated in Italy since the early 19th century. Growers and breeders were able to identify and collect many somatic mutants, which differed in their major pomological traits, such as fruit size and firmness, pulp and peel pigmentation, and ripening period. Recently, in the framework of a genetic resource conservation program, most of the accessions collected at CREA in recent decades were planted in a single block to evaluate their characteristics. The presence of many selections in the same block, of the same age and grafted on the same rootstock is extremely important for a reliable characterization of the pomological characters, particularly pigmentation, which is strongly influenced by both the environment and the rootstock. The collection consists of 88 genotypes, including old lines and nucellar selections. Some commercial Tarocco, Moro and Sanguinello varieties were also included in the collection to facilitate comparison among clones. Moreover, the pomological features of 20 elite Tarocco clones cultivated nearby in the same experimental station were used as a further reference. The pomological characterization, based on 13 traits and performed in two consecutive years, revealed significant variability. Some genotypes showed strong year-to-year variability, mainly in fruit pigmentation, probably due to different climatic conditions during winter. Similarly, other traits such as fruit size, total soluble solids and acidity also varied among and within the varietal groups. These findings have a potential utility in identifying promising clones, which might fulfil the growing market needs such as higher anthocyanin content and longer shelf life.

The building sector generates around 5-8% of greenhouse gas emissions (GHG)[1] and the disposal of C&D waste at the end-of-life is a high environmental cost. The raw earth is a sustainable construction material with low embedded energy, available locally. It is the most ancient technique of construction, studied in recent years to reduce the environmental life cycle impact of buildings. Clay is responsible for the earth plastic behaviour and represents the binder that keeps together silt and sand grains. Earth sets through drying without chemical reactions, so it could be reinserted into the nature. At the same time, earthen constructions do not withstand weathering and develop lower mechanical performances compared with those which exploit hydraulic binders. We investigated the possibility of improving these characteristics by stabilizing earthen products with the addition of small amounts of lime preserving clay as eco-friendly binder and the full end-of-life recyclability. Four earths with different binder properties – two kaolin clays, one illite clay and a smectite – were characterized and analysed at the lab scale. The change of the Atterberg limits on adding lime was determined. The clay modification and potential pozzolanic reactions with lime were measured by X-ray diffraction. Two earths were selected to be tested as plasters, adding sand and lime in order to reduce shrinkage and increase water resistance. Cracking and adhesion tests were run for all mixtures. The results show decreasing performances for all the plasters mixtures stabilized with lime, especially in the presence of an expandable clay fraction. The water resistance is improved for the stabilized mixtures that require less sand to reduce cracking and swelling. Lime is better used as a surface finish and plaster, not as stabilizer, because reacting with clay it increases the plastic limit and raises the water demand to obtain a plastic and workable mixture., Academic Journal of Civil Engineering, Vol 37 No 2 (2019): Special Issue - ICBBM 2019

Pain management is one of the main challenges in addressing the improved care of hospitalized newborns. The administration of oral sucrose with and without non-nutritive suction has been proposed as a nonpharmacological intervention to relieve procedural pain in newborns. The effects have not yet been well characterized. The aim of this study is to investigate, using skin conductance algesimeter (SCA) pain monitor index, the effects of 24% sucrose solution on pain perception during capillary and arterial blood sampling. It is a prospective, randomized controlled study: sucrose versus placebo. Sucrose was given orally to infants who were submitted to arterial or capillary sampling. The SCA was measured during, and for 3 min before and after the intervention. Fifty-six infants were enrolled: 31 in the sucrose group and 25 in the placebo group. SCA showed that the measurement of peaks per second of pain during and 3 min after the procedures was lower in the sucrose group than the placebo group and that this difference was statistically significant (

Somatic mutations are a cause of intraspecific diversity in many fruit crops, including citrus. In the case of sweet orange [Citrus sinensis (L.) Osbeck], intraspecific variability is determined only by somatic mutations. Tools for clonal fingerprinting are required by breeders and nurserymen and have important implications for traceability. With the aim of studying genomic variability and identifying mutational events responsible for varietal diversification, we deep-resequenced 22 accessions including navel, common and blood oranges using an Illumina platform. We also resequenced an induced mutant of Tarocco 'Scire D2062' obtained using two cycles of gamma ray irradiation at 40 Gy. A robust and reliable set of single nucleotide polymorphisms (SNPs), structural variants (SVs) and indels, specific of each accession or common to varietal groups, was identified both in natural and induced mutants. A subset of SNPs, transposable element insertions and small indels (2 to 18 base pairs) was validated by Sanger sequencing, PCR amplification and high resolution melting analysis, confirming the results of the bioinformatics analysis. Moreover, to identify a reliable marker set for traceability of specific cultivars, we collected leaf and juice samples from many Italian citrus growing areas and used a KASP platform for their fingerprinting. These tools will be useful to prove true-to-typeness of specific sweet orange cultivars.