Canopy Architectural Characteristics of Ten New Olive (Olea europaea L.) Genotypes and Their Potential for Cultivation in Super-High-Density Orchards.

In recent years, there has been growing interest in olive genotypes (Olea europaea L.) suitable for super-high-density (SHD > 1200 trees/hectare) orchards. To date, only a few cultivars are considered fitting for such cultivation system. In this study, the first results on the architectural characteristics of the canopy of ten new olive genotypes are presented. Their suitability for SHD orchards was evaluated and compared with the cultivar 'Arbequina', which is considered suitable for SHD olive orchards and, for this reason, was used as the control. Several canopy measurements were taken, and some architectural parameters, such as branching frequency, branching density, and branch diameter/stem diameter ratio were calculated. The branching frequency value was greater than 0.20 in 'Arbequina' and in only four of the genotypes. The branching density in five genotypes was similar to 'Arbequina'. 'Arbequina' had the lowest value for the branch diameter/stem diameter ratio, and only three genotypes had similar values. These initial results showed that only one genotype has all canopy architectural characteristics comparable to those of the cv. 'Arbequina'. Further studies are needed to evaluate the production traits of these new genotypes and complete their characterization.

Dodine an effective alternative to copper for controlling Venturia oleaginea, the causal agent of pea-cock eye disease, in highly infected olive trees.

A trial was carried out in central Italy in an olive orchard of cultivar Moraiolo, highly infected by Venturia oleaginea. The aim of the investigation was to evaluate the effects of autumn and spring applications of copper oxychloride or dodine to control the disease. Non treated trees were used as the control. The effects of the fungal attacks on leaves and inflorescence development confirmed the high susceptibility of the cultivar Moraiolo to the disease. The results show that in trees heavily infected, but with most of the infected leaves at the early stage of the disease (asymptomatic phase), treatments with dodine had a curative effect, with consequent reduction in the appearance of symptomatic leaves and defoliation with respect to the control or copper-treated trees. The use of dodine against the autumnal attacks of V. oleaginea allowed most of the old leaves to be maintained until the new ones had formed, which is important for the growth processes during the early part of the growing season. Overall, the results indicate that to efficiently control the pathogen using copper compounds, treatments must start soon after the beginning of the attack and be repeated in order to maintain the infection at a low level. Dodine can be efficiently used if there is a great increase in infected leaves. The use of dodine to solve particular situations and not for normal repeated use is regulated by the fact that in some countries, Italy included, protocols for integrated pest management allow only one dodine treatment/year.

Cultivar ideotype for intensive olive orchards: plant vigor, biomass partitioning, tree architecture and fruiting characteristics.

In order to achieve higher and earlier yield, modern olive orchards are increasingly intensified, with tree densities up to > 1500 trees hectare-1. With increasing tree densities, individual-tree canopy volume must be proportionally reduced. Not all cultivars are adaptable to high and very high orchard densities, because of excessive vigor and/or insufficient bearing when the canopy is pruned to a small volume. However, what makes an olive cultivar suitable for intensive and super intensive orchards is not clear. Recently, few studies have addressed this topic, suggesting that tree architecture and early bearing are essential traits. Yet, what architectural and productive features are important, how they work and whether they are interrelated remains elusive. This review summarizes and interprets the literature on olive, as well as the more abundant literature available for other fruit species, aiming to provide a comprehensive knowledge framework for understanding how tree architectural characteristics, plant vigor, and fruiting vary across olive genotypes, and how they are interconnected. It is concluded that, among the architectural characteristics, greater branching and smaller diameters of woody structures are particularly important features for cultivar suitability to intensive and super intensive olive orchards. Greater branching allows to produce more fruiting sites in the small volume of canopy allowed in these systems. It also reduces investments in woody structures, liberating resources for fruiting. Additional resources are liberated with smaller structure diameters. Greater branching also increases resources by increasing biomass partitioning into leaves (i.e. the photosynthetic organs), relative to wood. Since yield is affected by the competition for resources with vegetative growth, reducing resource investments in woody structures and/or increasing resource directly, increases yield. Yield, in turn, depresses vegetative growth, reducing vigor and the need for pruning. High yields also produce short shoots which have relatively greater investments in leaf mass and area, and lower in the woody stem, making them more suitable than long shoots to support concurrent fruit growth. This single framework of interpretation of how the different architectural and fruiting characteristics work and interact with one-another, will provide guidance for cultivar selection and breeding for intensive and super intensive olive orchards.

Avelumab internalization by human circulating immune cells is mediated by both Fc gamma receptor and PD-L1 binding.

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor-mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development.

Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

Effects of cultivar, fruit presence and tree age on whole-plant dry matter partitioning in young olive trees.

This study investigated the effects of cultivar, fruit presence and tree age on whole-plant partitioning of dry matter and energy equivalents (i.e., glucose equivalents). Young trees of two cultivars characterized by different vigor (i.e., Arbequina, low vigor and Frantoio, high vigor) were either completely deflowered from 2014 to 2017 or never, providing two contrasting levels of cumulated reproductive growth over the following 4 years. Total vegetative dry matter growth over the 4 years was assessed by destructive samplings (whole tree). Plant growth was inversely correlated to reproductive efforts, with Arbequina producing more and growing less than Frantoio. Deflowered trees grew similarly across cultivars, although deflowered Arbequina grew statistically less than deflowered Frantoio by the fourth year, due to abundant flower production. Total reproductive (flowers + fruit) and vegetative biomass production were the same for all cultivars and treatments. Arbequina had a greater distribution of dry matter in directly productive structures (current and one-year-old shoots) and in leaves. This allows it to increase the number of current and following-year production sites, and to save in the resources invested in non-productive sinks (roots, trunk and branches), thus liberating resources for reproductive growth. Greater investments in leaves allow it to intercept more light and thus to increase assimilation. Increased assimilation and increased partitioning towards productive structures, and decreased competition by non-productive structures might contribute to explain the greater early bearing attitude of this cultivar.

A novel monovalent FGFR1 antagonist: Preclinical safety profiles in rodents and non-human primates.

Blocking Fibroblast Growth Factor Receptor 1 (FGFR1) is an attractive therapeutic option for treatment of cancer subtypes with amplification and over-expression of FGFR1. Selective targeting of FGFR1 can be achieved using an antibody-based approach, as small molecule inhibitors may not discriminate between FGFR1, 2, 3 and 4 due to their highly homologous kinase domain. However, development of classical bivalent FGFR1 directed antibodies has failed due to non-tolerated body weight decreases in preclinical species. M6123 is a novel IgG-like monovalent FGFR1 specific binder with enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) effector function and inhibits tumor growth significantly in FGFR1-dependent human xenograft models without reduced body weight in tumor-bearing mice. Toxicology studies reported here characterized the safety profile of M6123 in mouse, rat, and monkey. There were significant differences among animal species under similar M6123 exposure levels. Rats showed metastatic mineralization with an imbalance in serum phosphate at low doses, while mineralization was not found in mice or monkeys, even though hyperphosphatemia was detected in mice. Subtle differences in calcium/phosphate homoeostasis feedback loops may trigger the susceptibility to mineralization among animal species; nevertheless, the exact mechanism remains unknown. Monkeys showed marked, but reversible, decreases in peripheral blood NK cells and neutrophils. The latter was associated with considerably increased neutrophilic infiltrates in the liver sinusoids and red pulp of the spleen. These effects in monkeys are likely related to the enhanced ADCC activity of M6123. Overall, M6123 showed a superior safety profile in animals compared to bivalent FGFR1 antagonists or pan-FGFR inhibitors.

Identification and characterization of M6903, an antagonistic anti-TIM-3 monoclonal antibody.

T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint that regulates normal immune responses but can be exploited by tumor cells to evade immune surveillance. TIM-3 is primarily expressed on immune cells, particularly on dysfunctional and exhausted T cells, and engagement of TIM-3 with its ligands promotes TIM-3-mediated T cell inhibition. Antagonistic ligand-blocking anti-TIM-3 antibodies have the potential to abrogate T cell inhibition, activate antigen-specific T cells, and enhance anti-tumor immunity. Here we describe M6903, a fully human anti-TIM-3 antibody without effector function and with high affinity and selectivity to TIM-3. We demonstrate that M6903 blocks the binding of TIM-3 to three of its ligands, phosphatidylserine (PtdSer), carcinoembryonic antigen cell adhesion-related molecule 1 (CEACAM1), and galectin 9 (Gal-9). These results are supported by an atomic resolution crystal structure and functional assays, which demonstrate that M6903 monotherapy enhanced T cell activation. This activation was further enhanced by the combination of M6903 with bintrafusp alfa, a bifunctional fusion protein that simultaneously blocks the transforming growth factor-ß (TGF-ß) and programmed death ligand 1 (PD-L1) pathways. M6903 and bintrafusp alfa combination therapy also enhanced anti-tumor efficacy in huTIM-3 knock-in mice, relative to either monotherapy. These in vitro and in vivo data, along with favorable pharmacokinetics in marmoset monkeys, suggest that M6903 as a monotherapy warrants further pre-clinical assessment and that M6903 and bintrafusp alfa may be a promising combination therapy in the clinic.

Resource investments in reproductive growth proportionately limit investments in whole-tree vegetative growth in young olive trees with varying crop loads.

It has long been debated whether tree growth is source limited, or whether photosynthesis is adjusted to the actual sink demand, directly regulated by internal and environmental factors. Many studies support both possibilities, but no studies have provided quantitative data at the whole-tree level, across different cultivars and fruit load treatments. This study investigated the effect of different levels of reproductive growth on whole-tree biomass growth across two olive cultivars with different growth rates (i.e., Arbequina, slow-growing and Frantoio, fast-growing), over 2 years. Young trees of both cultivars were completely deflowered either in 2014, 2015, both years or never, providing a range of levels of cumulated reproductive growth over the 2 years. Total vegetative dry matter growth over the 2 years was assessed by destructive sampling (whole tree). Vegetative growth increased significantly less in fruiting trees, however, the total of vegetative and reproductive growth did not differ significantly for any treatment or cultivar. Vegetative growth over the 2 years was closely (R2 = 0.89) and inversely related to reproductive growth across all treatments and cultivars. When using data from 2015 only, the regression improved further (i.e., R2 = 0.99). When biomass was converted into grams of glucose equivalents, based on the chemical composition of the different parts, the results indicated that for every gram of glucose equivalent invested in reproductive growth, vegetative growth was reduced by 0.73-0.78 g of glucose equivalent. This indicates that competition for resources played a major role in determining tree growth, but also that photosynthesis was probably also enhanced at increasing fruit load (or downregulated at decreasing fruit load). The leaf area per unit of trunk cross sectional area increased with deflowering (i.e., decreased with reproductive growth), suggesting that water relations might have limited photosynthesis in deflowered plants, which had much greater canopies. Net assimilation rate (NAR) increased with reproductive growth and decreased with plant size. Net assimilation rate was also negatively correlated with the leaf area per unit of trunk cross sectional area, suggesting that water relations might have contributed to decreasing NAR at increasing plant size.

Fruit production and branching density affect shoot and whole-tree wood to leaf biomass ratio in olive.

The amount of shoot stem (i.e., woody part of the shoot) dry matter per unit shoot leaf dry matter (i.e., the shoot wood to leaf biomass ratio) has been reported to be lower in short shoots than in long ones, and this is related to the greater and earlier ability of short shoots to export carbon. This is important in fruit trees, since the greater and earlier carbon export ability of shoots with a lower wood to leaf biomass ratio improves fruit production. This ratio may vary with cultivars, training systems or plant age, but no study has previously investigated the possible effect of fruit production. In this study on two olive cultivars (i.e., Arbequina, with low growth rate, and Frantoio, with high growth rate) subject to different fruit production treatments, we found that at increasing fruit production, shoot length and shoot wood to leaf biomass ratio were proportionally reduced in the new shoots growing at the same time as the fruit. Specifically, fruit production proportionally reduced total new-shoot biomass, length, leaf area and average shoot length. With decreasing shoot length, shoot diameter, stem mass, internode length, individual leaf area and shoot wood to leaf biomass ratio also decreased. This may be viewed as a plant strategy to better support fruit growth in the current year, given the greater and earlier ability of short shoots to export carbon. Moreover, at the whole-tree level, the percentage of total tree biomass production invested in leaves was closely correlated with branching density, which differed significantly across cultivars. By branching more, Arbequina concentrates more shoots (thus leaves) per unit of wood (trunk, branches and root) mass, decreasing wood to leaf biomass ratio at the whole-tree level. Therefore, while, at the shoot level, shoot length determines shoot wood to leaf biomass ratio, at the canopy level branching density is also an important determinant of whole-tree wood to leaf biomass ratio. Whole-tree wood to leaf biomass ratio is likely to affect the canopy's ability to export carbon (i.e., towards fruits), as shoot wood to leaf biomass ratio affects the carbon export ability of the shoot.

Phosphorylation of phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) in the flesh of fruits.

This study determined whether phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) are phosphorylated in the flesh of a range of fruits. This was done by incubating fruit flesh with 32P[P] (where 32P[P] = 32PO43-), then PEPCK and PEPC were immunoprecipitated from extracts using specific antisera. The incorporation of 32P[P] into these enzymes was then determined by autoradiography of SDS-PAGE gels. Both enzymes were subject to phosphorylation in vivo in the flesh of grape, tomato, cherry and plum. PEPCK was also subject to phosphorylation in vivo in developing grape seeds. Proteolytic cleavage of PEPCK showed that it was phosphorylated at a site(s) located on its N-terminal extension. Potentially phosphorylation of these enzymes could contribute to the coordinate regulation of their activities in the flesh of fruits and in developing seeds.

Phosphoenolpyruvate carboxykinase, pyruvate orthophosphate dikinase and isocitrate lyase in both tomato fruits and leaves, and in the flesh of peach and some other fruits.

In this study the occurrence of a number of enzymes involved in gluconeogenesis was investigated in both tomato fruits and leaves during their development and senescence and in some other fruits. The enzymes studied were phosphoenolpyruvate carboxykinase (PEPCK), pyruvate orthophosphate dikinase (PPDK) and glyoxysomal isocitrate lyase (ICL). PPDK was detected in the ripe flesh of tomato, and much smaller amounts were detected in the flesh of both peach and pepper, whereas it was not detected (not present or at very low abundance) in the other fruits which were investigated (apricot, aubergine, blackberry, blueberry, cherry, grape, plum, raspberry and red current). By contrast PEPCK was present in the flesh of all the fruits investigated. Very small amounts of ICL were detected in ripe tomato flesh. PEPCK was present in the skin, flesh, locular gel and columella of tomato fruit, and in these its abundance increased greatly during ripening. PPDK showed a similar distribution, however, its abundance did not increase during ripening. PEPCK was not detected in tomato leaves at any stage of their development or senescence. The content of PPDK g(-1) fresh weight (FW) increased in tomato leaves as they matured, however, it declined during their senescence. In tomato leaves the content of ICL g(-1) FW increased until the mid-stage of development, then decreased as the leaf matured, and then increased during the latter stages of senescence. In the flesh of tomato fruits the contents of PPDK and PEPCK g(-1) FW decreased during senescence. The results suggest that in fruits other than tomato the bulk of any gluconeogenic flux proceeds via PEPCK, whereas in tomato both PEPCK and PPDK could potentially be utilised. Further, the results indicate that the conversion of pyruvate/acetyl-CoA to malate by the glyoxylate cycle, for which ICL is necessary, is not a major pathway utilised by gluconeogenesis in fruits under normal conditions of growth. Finally, the results contribute to our understanding of the role of several enzymes in the senescence of both leaves and fruits.

Effect of agronomical practices on carpology, fruit and oil composition, and oil sensory properties, in olive (Olea europaea L.).

We examined whether some agronomical practices (i.e. organic vs. conventional) affect olive fruit and oil composition, and oil sensory properties. Fruit characteristics (i.e. fresh and dry weight of pulp and pit, oil content on a fresh and dry weight basis) did not differ. Oil chemical traits did not differ except for increased content of polyphenols in the organic treatments, and some changes in the acidic composition. Sensory analysis revealed increased bitterness (both cultivars) and pungency (Frantoio) and decreased sweetness (Frantoio) in the organic treatment. Fruit metabolomic analysis with HRMAS-NMR indicated significant changes in some compounds including glycocholate, fatty acids, NADPH, NADP+, some amino acids, thymidine, trigonelline, nicotinic acid, 5,6-dihydrouracil, hesanal, cis-olefin, ß-D-glucose, propanal and some unassigned species. The results suggest that agronomical practices may have effects on fruit composition that may be difficult to detect unless a broad-spectrum analysis is used.

Tissue size and cell number in the olive (Olea europaea) ovary determine tissue growth and partitioning in the fruit.

The relationship between tissue size and cell number in the ovary and tissue size in the fruit, was studied in eight olive (Olea europaea L.) cultivars with different fruit and ovary size. All tissues in the ovary increased in size with increasing ovary size. Tissue size in the fruits correlated with tissue size in the ovary for both mesocarp and endocarp, but with different correlations: the mesocarp grew about twice as much per unit of initial volume in the ovary. Tissue size in the fruit also correlated with tissue cell number in the ovary. In this case, a single regression fitted all data pooled for both endocarp and mesocarp, implying that a similar tissue mass was obtained in the fruit per initial cell in the ovary, independent of tissues and cultivars. Tissue relative growth from bloom to harvest (i.e. the ratio between final and initial tissue size) differed among cultivars and tissues, but correlated with tissue cell size at bloom, across cultivars and tissues. These results suggest that in olive, tissue growth and partitioning in the fruit is largely determined by the characteristics of the ovary tissues at bloom, providing important information for plant breeding and crop management.