Genealogical tracing of Olea europaea species and pedigree relationships of var. europaea using chloroplast and nuclear markers.

BACKGROUND: Olive is one of the most cultivated species in the Mediterranean Basin and beyond. Despite being extensively studied for its commercial relevance, the origin of cultivated olive and the history of its domestication remain open questions. Here, we present a genealogical and kinship relationships analysis by mean of chloroplast and nuclear markers of different genera, subgenus, species, subspecies, ecotypes, cultivated, ancient and wild types, which constitutes one of the most inclusive research to date on the diversity within Olea europaea species. A complete survey of the variability across the nuclear and plastid genomes of different genotypes was studied through single nucleotide polymorphisms, indels (insertions and deletions), and length variation. RESULTS: Fifty-six different chlorotypes were identified among the Oleaceae family including Olea europaea, other species and genera. The chloroplast genome evolution, within Olea europaea subspecies, probably started from subsp. cuspidata, which likely represents the ancestor of all the other subspecies and therefore of wild types and cultivars. Our study allows us to hypothesize that, inside the subspecies europaea containing cultivars and the wild types, the ancestral selection from var. sylvestris occurred both in the eastern side of the Mediterranean and in the central-western part of Basin. Moreover, it was elucidated the origin of several cultivars, which depends on the introduction of eastern cultivars, belonging to the lineage E1, followed by crossing and replacement of the autochthonous olive germplasm of central-western Mediterranean Basin. In fact, our study highlighted that two main 'founders' gave the origin to more than 60% of analyzed olive cultivars. Other secondary founders, which strongly contributed to give origin to the actual olive cultivar diversity, were already detected. CONCLUSIONS: The application of comparative genomics not only paves the way for a better understanding of the phylogenetic relationships within the Olea europaea species but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance and parentage inside olive cultivars, opening new scenarios for further research such as the association studies and breeding programs.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Impact of High Light Intensity and Low Temperature on the Growth and Phenylpropanoid Profile of Azolla filiculoides.

Exposure to high light intensity (HL) and cold treatment (CT) induces reddish pigmentation in Azolla filiculoides, an aquatic fern. Nevertheless, how these conditions, alone or in combination, influence Azolla growth and pigment synthesis remains to be fully elucidated. Likewise, the regulatory network underpinning the accumulation of flavonoids in ferns is still unclear. Here, we grew A. filiculoides under HL and/or CT conditions for 20 days and evaluated the biomass doubling time, relative growth rate, photosynthetic and non-photosynthetic pigment contents, and photosynthetic efficiency by chlorophyll fluorescence measurements. Furthermore, from the A. filiculoides genome, we mined the homologs of MYB, bHLH, and WDR genes, which form the MBW flavonoid regulatory complex in higher plants, to investigate their expression by qRT-PCR. We report that A. filiculoides optimizes photosynthesis at lower light intensities, regardless of the temperature. In addition, we show that CT does not severely hamper Azolla growth, although it causes the onset of photoinhibition. Coupling CT with HL stimulates the accumulation of flavonoids, which likely prevents irreversible photoinhibition-induced damage. Although our data do not support the formation of MBW complexes, we identified candidate MYB and bHLH regulators of flavonoids. Overall, the present findings are of fundamental and pragmatic relevance to Azolla's biology.

Nocturnal gibberellin biosynthesis is carbon dependent and adjusts leaf expansion rates to variable conditions.

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA4 levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA4 by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

Botrytis cinerea induces local hypoxia in Arabidopsis leaves.

Low oxygen availability often is associated with soil waterlogging or submergence, but may occur also as hypoxic niches in otherwise aerobic tissues. Experimental evidence assigns a role in Botrytis cinerea resistance to a group of oxygen-unstable Ethylene Response Factors (ERF-VII). Given that infection by B. cinerea often occurs in aerobic organs such as leaves, where ERF-VII stability should be compromised, we explored the possibility of local leaf hypoxia at the site of infection. We analyzed the expression of hypoxia-responsive genes in infected leaves. Confocal microscopy was utilized to verify the localization of the ERF-VII protein RAP2.12. Oxygen concentration was measured to evaluate the availability of oxygen (O2 ). We discovered that infection by B. cinerea induces increased respiration, leading to a drastic drop in the O2 concentration in an otherwise fully aerobic leaf. The establishment of a local hypoxic area results in stabilization and nuclear relocalization of RAP2.12. The possible roles of defence elicitors, ABA and ethylene were evaluated. Local hypoxia at the site of B. cinerea infection allows the stabilization of ERF-VII proteins. Hypoxia at the site of pathogen infection generates a nearly O2 -free environment that may affect the stability of other N-degron-regulated proteins as well as the metabolism of elicitors.

ARGONAUTE1 and ARGONAUTE4 Regulate Gene Expression and Hypoxia Tolerance.

In plants, hypoxia can be induced by submergence, and the lack of oxygen impairs mitochondrial respiration, thus affecting the plant's energy status. Hypoxia has major effects on gene expression; these changes induce key responses that help meet the needs of the stressed plant. However, little is known about the possible role of RNA signaling in the regulation of gene expression under limited oxygen availability. Here, we report the contribution of ARGONAUTE1 (AGO1) to hypoxia-induced gene regulation in Arabidopsis (Arabidopsis thaliana). Submergence induced changes in levels of the microRNAs miR2936 and miR398, but this had no obvious effects on their putative target mRNA levels. However, we found that ago1-27 plants are intolerant to submergence and transcriptome analysis identified genes whose regulation requires functional AGO1. Analysis of mutants affected in various branches of RNA signaling highlighted the convergence of AGO1 signaling with the AGO4-dependent RNA-directed DNA methylation (RdDM) pathway. AGO4-dependent RdDM represses the expression of HOMOLOG OF RPW8 4 (HR4) and alters its response to submergence. Remarkably, methylation of the second exon of HR4 is not only reduced in ago4-1 but also in plants overexpressing a constitutively stable version of the oxygen sensor RELATED TO APETALA2 12 (RAP2.12), indicating convergence of oxygen signaling with epigenetic regulation of gene expression. Therefore, our results identify a role for AGO1 and AGO4 RNA-silencing pathways in low-oxygen signaling in Arabidopsis.

Characterization of Differentially Expressed Genes under Salt Stress in Olive.

Climate change, currently taking place worldwide and also in the Mediterranean area, is leading to a reduction in water availability and to groundwater salinization. Olive represents one of the most efficient tree crops to face these scenarios, thanks to its natural ability to tolerate moderate salinity and drought. In the present work, four olive cultivars (Koroneiki, Picual, Royal de Cazorla and Fadak86) were exposed to high salt stress conditions (200 mM of NaCl) in greenhouse, in order to evaluate their tolerance level and to identify key genes involved in salt stress response. Molecular and physiological parameters, as well as plant growth and leaves' ions Na+ and K+ content were measured. Results of the physiological measurements showed Royal de Cazorla as the most tolerant cultivar, and Fadak86 and Picual as the most susceptible ones. Ten candidate genes were analyzed and their complete genomic, CDS and protein sequences were identified. The expression analysis of their transcripts through reverse transcriptase quantitative PCR (RT-qPCR) demonstrated that only OeNHX7, OeP5CS, OeRD19A and OePetD were upregulated in tolerant cultivars, thus suggesting their key role in the activation of a salt tolerance mechanism.

Gene Regulation and Survival under Hypoxia Requires Starch Availability and Metabolism.

Plants respond to hypoxia, often caused by submergence, by expressing a specific set of genes that contribute to acclimation to this unfavorable environmental condition. Genes induced by low oxygen include those encoding enzymes for carbohydrate metabolism and fermentation, pathways that are required for survival. Sugar availability is therefore of crucial importance for energy production under hypoxia. Here, we show that Arabidopsis (Arabidopsis thaliana) plants require starch for surviving submergence as well as for ensuring the rapid induction of genes encoding enzymes required for anaerobic metabolism. The starchless pgm mutant is highly susceptible to submergence and also fails to induce anaerobic genes at the level of the wild type. Treating wild-type plants under conditions inducing sugar starvation results in a weak induction of alcohol dehydrogenase and other anaerobic genes. Induction of gene expression under hypoxia requires transcription factors belonging to group VII ethylene response factors (ERF-VII) that, together with plant Cys oxidases, act as an oxygen-sensing mechanism. We show that repression of this pathway by sugar starvation occurs downstream of the hypoxia-dependent stabilization of ERF-VII proteins and independently of the energy sensor protein kinases SnRK1.1 (SNF1-related kinase 1.1).

Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains.

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.

Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3.

The Role of Immersive Virtual Reality Interventions in Pediatric Cerebral Palsy: A Systematic Review across Motor and Cognitive Domains.

Background: In recent years, new technologies have been applied in cerebral palsy. Among these, immersive virtual reality is one with promising motor and cognitive effects along with the reduced costs of its application. The level of immersion of the subject in the illusional world gives the feeling of being a real part of the virtual environment. This study aims to investigate the safety and the efficacy of immersive virtual reality in children affected by cerebral palsy. Methods: PubMed, Embase, Cochrane Database of Systematic Reviews, RehabData, and Web of Science were screened up to February 2023 to identify eligible clinical studies. Results: Out of 788, we included 15 studies involving CP patients. There was high heterogeneity in the outcomes considered, and the results showed non-inferiority to conventional therapy and initial additional benefits in comparison with conventional rehabilitation. Conclusions: Immersive virtual reality emerges as a pivotal technological tool in rehabilitation, seamlessly integrating with conventional therapy within CP rehabilitation programs. Indeed, it not only enhances motivation but significantly increases children's functional capacity and abilities.

Anomalous drainage of the right gonadal vein.

The present research aims to present and describe an unusual and rare anatomical variation in relation to the drainage of the right gonadal vein. This anatomical knowledge is crucial as it assists in the work of surgeons and health professionals in general. The dissection occurred in the anterior wall of the abdomen and, through observational analysis, an anatomical variation was found in the drainage of the right gonadal vein in a human cadaver, obtained by anonymous donation, male gender and without predetermined clinical characteristics, ethnicity, and age, belonging to the Padre Albino University Center collection. This research was approved by the Research Ethics Committee under protocol 12923919.8.0000.5430. The drainage of right gonadal vein is this variant occurs anastomosed with an innominate venous trunk that empties into the inferior vena cava. Furthermore, the presence of an accessory right renal vein is also noticed, which anastomoses with the innominate venous trunk and with the right renal vein, through a vein suggestively called interrenal, differing from the anatomical normality described in the literature. This variation is supposed to occur due to flaws in the development of the embryo, which generate venous changes in the origins of the right gonadal vein. Acknowledging the existence of it is relevant when performing surgical procedures in the region, as it differs from the most frequent anatomy found in the human population. The rare drainage of the right gonadal vein through an innominate trunk to the inferior vena cava and its importance is highlighted in this article.

Bioactive Compound Profiling of Olive Fruit: The Contribution of Genotype.

The health, therapeutic, and organoleptic characteristics of olive oil depend on functional bioactive compounds, such as phenols, tocopherols, squalene, and sterols. Genotype plays a key role in the diversity and concentration of secondary compounds peculiar to olive. In this study, the most important bioactive compounds of olive fruit were studied in numerous international olive cultivars during two consecutive seasons. A large variability was measured for each studied metabolite in all 61 olive cultivars. Total phenol content varied on a scale of 1-10 (3831-39,252 mg kg-1) in the studied cultivars. Squalene values fluctuated over an even wider range (1-15), with values of 274 to 4351 mg kg-1. Total sterols ranged from 119 to 969 mg kg-1, and total tocopherols varied from 135 to 579 mg kg-1 in fruit pulp. In the present study, the linkage among the most important quality traits highlighted the scarcity of cultivars with high content of at least three traits together. This work provided sound information on the fruit metabolite profile of a wide range of cultivars, which will facilitate the studies on the genomic regulation of plant metabolites and development of new olive genotypes through genomics-assisted breeding.

[Structured implementation and high adherence to the ERAS program in colorectal surgery in two operating units of the ASUR Marche.] / Implementazione strutturata ed elevata aderenza al programma ERAS in chirurgia colorettale in due unità operative dell'ASUR Marche.

BACKGROUND: An Enhanced Recovery After Surgery (ERAS) program in colorectal surgery is able to significantly reduce the morbidity rates and postoperative hospital stay (LOS) related to the intervention. However, it is not clear what modalities and levels of implementation are necessary to achieve these results. The purpose of this work is to analyze the methods and results of the first year of implementation of the program in two centers of the Agenzia Sanitaria Unica Regionale (ASUR) Marche. MATERIALS: After a structured implementation pathway, characterized by the creation of a core team, field training, internal courses and coaching, the details of 196 consecutive cases of patients submitted to colorectal resection over a one-year period in two surgical units of the ASUR Marche were prospectively loaded in a database, considering over 50 variables including adherence to the individual items of the ERAS program. The primary outcomes were: overall and major morbidity, mortality and anastomotic dehiscence rates; secondary outcomes were: LOS, re-admission and re-intervention rates. The results of primary endpoints were evaluated by univariable and multivariable analyses with logistic regression and, thereafter, according to ERAS item adherence rate. RESULTS: After a median (interquartile range, IQR) follow-up of 40 (32-94) days, we recorded complications in 72 patients (overall morbidity 36.7%), major morbidity in 14 patients (7.1%), 6 deaths (mortality 3.1%), an anastomotic dehiscence in 9 cases (4.9%), median (IQR) overalll LOS 5 (3-7) days, 10 readmissions (5.1%) and 13 reoperations (6.7%). The mean adherence rate to the items of the ERAS program was 85.4%, showing a significant dose-effect curve for overall morbidity, major morbidity, anastomotic leakage and for overall LOS. DISCUSSION: The ERAS implementation methods in this project led to a high adherence (>80%) to the program items. All the results showed a significant improvement compared to the previous pre-implementation period and according to the adherence to program items rate.

Arabidopsis phenotyping reveals the importance of alcohol dehydrogenase and pyruvate decarboxylase for aerobic plant growth.

Alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) are key to the establishment of the fermentative metabolism in plants during oxygen shortage. Most of the evidence that both ADH and PDC are required for plant tolerance to hypoxia comes from experiments performed by limiting oxygen in the environment, such as by exposing plants to gaseous hypoxia or to waterlogging or submergence. However, recent experiments have shown that hypoxic niches might exist in plants grown in aerobic conditions. Here, we investigated the importance of ADH and PDC for plant growth and development under aerobic conditions, long-term waterlogging and short-term submergence. Data were collected after optimizing the software associated with a commercially-available phenotyping instrument, to circumvent problems in separation of plants and background pixels based on colour features, which is not applicable for low-oxygen stressed plants due to the low colour contrast of leaves with the brownish soil. The results showed that the growth penalty associated with the lack of functional ADH1 or both PDC1 and PDC2 is greater under aerobic conditions than in hypoxia, highlighting the importance of fermentative metabolism in plants grown under normal, aerobic conditions.

Cytokines IL-17 and IL-22 in the host response to infection.

Cytokines IL-17 and IL-22 play pivotal roles in host defense against microbes and in the development of chronic inflammatory diseases. These cytokines are produced by cells that are often located in epithelial barriers, including subsets of T cells and innate lymphoid cells. In general, IL-17 and IL-22 can be characterized as important cytokines in the rapid response to infectious agents, both by recruiting neutrophils and by inducing the production of antimicrobial peptides. Although each cytokine induces an innate immune response in epithelial cells, their functional spectra are generally distinct: IL-17 mainly induces an inflammatory tissue response and is involved in the pathogenesis of several autoimmune diseases, whereas IL-22 is largely protective and regenerative. In this review, we compare IL-17 and IL-22, describing overlaps and differences in their cellular sources as well as their regulation, signaling, biological functions and roles during disease, with a focus on the contribution of these cytokines to the gut mucosal barrier during bacterial infection.

Pathogenic E. coli exploits SslE mucinase activity to translocate through the mucosal barrier and get access to host cells.

SslE is a zinc-metalloprotease involved in the degradation of mucin substrates and recently proposed as a potential vaccine candidate against pathogenic E. coli. In this paper, by exploiting a human in vitro model of mucus-secreting cells, we demonstrated that bacteria expressing SslE have a metabolic benefit which results in an increased growth rate postulating the importance of this antigen in enhancing E. coli fitness. We also observed that SslE expression facilitates E. coli penetration of the mucus favouring bacteria adhesion to host cells. Moreover, we found that SslE-mediated opening of the mucosae contributed to the activation of pro-inflammatory events. Indeed, intestinal cells infected with SslE-secreting bacteria showed an increased production of IL-8 contributing to neutrophil recruitment. The results presented in this paper conclusively designate SslE as an important colonization factor favouring E. coli access to both metabolic substrates and target cells.

The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

Methyl carnosate, an antibacterial diterpene isolated from Salvia officinalis leaves.

Ethanolic extracts of Salvia officinalis leaves demonstrated antibacterial activity against Bacillus cereus. Fractionation of the extracts led to the isolation of the most active antibacterial compound, which, from spectroscopic and LC-MS evidence, was proved to be the diterpene, methyl carnosate.