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In the design and assessment of precast concrete beams with a slab cast on top, namely concrete composite beams, engineers still face in practice unsolved shear-related issues, such as the contribution to shear strength of the slab, the concrete strength to be considered in shear formulations or the influence of the interface between concretes in the shear behaviour. In the present research, 69 shear tests were performed on monolithic and composite beams, with rectangular or T-shaped cross-section, with or without transverse reinforcement and with different concrete qualities, to experimentally analyse the issues mentioned above. The study of the shear transfer mechanisms at failure led to formulating a model for explaining the observed results. Based on this model, a shear strength predictive formulation for concrete composite beams with web reinforcement has been developed, which has been verified with the experimental results from this research and 24 additional tests from the literature. This formulation provides accurate predictions compared to the shear strength formulations of current codes EC2, MC-10 and ACI 318-19. The proposed model lays the foundations for the future development of a user-friendly formulation for calculating the shear strength of concrete composite beams.

Lysosomes maintain cellular homeostasis by clearing macromolecules and recycling their content to support the cell's needs. Lysosomal dysfunction is also a common feature in many human diseases. However, the mechanisms underlying the recycling of many essential metabolites remain unknown. The authors use a creative CRISPR-Cas9 screen to identify proteins involved in choline export from lysosomes to the cytosol. They identified SPNS1, a lysosomal transmembrane protein essential for cell growth under choline-depleted conditions. Targeted lipidomics on isolated lysosomes and isotope tracing revealed that SPNS1 transports lysophosphatidylcholine from lysosomes into the cytosol, where it is re-esterified into phosphatidylcholine. This work defines a new pathway for lysosomal phospholipid egress and provides a reliable new screening method for the identification of novel lysosomal pathways. This preprint has been assigned the following badges: New Hypothesis, New Methods, Cross-Validation. Read the preprint on bioRxiv ( Scharenberg et al., 2022 ): https://doi.org/10.1101/2022.11.27.517422 .

Microtubule detyrosination, the post-translational removal of a tyrosine residue on α-tubulin, marks a stable and rare subpopulation of microtubules. Vasohibin (VASH) is the main enzyme responsible for α-tubulin detyrosination and it binds to α-tubulin on polymerized microtubules. However, the dynamics underlying detyrosination remain unknown. The authors use quantitative super-resolution microscopy, single molecule tracking, and computational modeling to explore microtubule detyrosination. VASH engages with microtubules in a stochastic fashion and detyrosination depends on VASH concentration on single microtubules. As VASH accumulation requires microtubule stability, detyrosination occurs via a stabilization-detyrosination feedback mechanism. This work provides important and novel mechanistic insight into microtubule detyrosination and serves as an example for studying other microtubule post-translational modifications essential for cellular homeostasis.

Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.

Genetic variation at the leucine-rich repeat kinase 2 ( LRRK2 ) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2 + lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Protein coding mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.

Ruptured lysosomes are cleared by lysophagy, a selective form of macroautophagy. Thus lysophagy protects cells against lysosomal cell death, a form of cell death caused by the release of lysosomal proteases to the cytosol from ruptured lysosomes. However, the mechanism underlying this process is still incomplete. Using HeLa cells, rat primary neurons, and human neurons, the authors show that SQSTM1/p62 is a key lysophagy adaptor that forms phase-separated condensates. Mechanistically, p62 oligomerizes but remains in liquid phase to ensure the formation of an autophagosome along the ruptured lysosome. The liquidlike state of p62 is maintained by the small chaperone HSP27. Amyotrophic lateral sclerosis (ALS)-associated mutations in p62 prevent its localization and disrupt lysophagy. These results demonstrate a new role of p62 in lysophagy and implicate lysophagy in the pathobiology of ALS.

There is scarce knowledge on what are the actual water requirements of persimmon trees (Diospyros kaki). Research on this topic is required to better adjust irrigation water supplies to the actual crop water needs. This study aimed to determine the transpiration coefficients (K) of persimmon ‘Rojo Brillante’ trees and assess how transpiration and K are influenced by environmental factors and the atmospheric demand under Mediterranean climatic conditions. This was done over the course of three growing seasons by means of sap flow measurements calibrated with whole canopy gas exchange chambers. Irrigation was applied aiming to avoid water limiting conditions for trees. This was verified by monitoring the trees’ water status across the growing seasons. Sap flow measurements were linearly and highly correlated (p < 0.001) with canopy transpiration (E) obtained with the whole canopy chambers, but either overestimated or underestimated it. E increased from April until June-July in response to canopy development and increased midday canopy light interception. There was a positive linear relationship between daily E, reference evapotranspiration (ET) and solar radiation. However, a non-linear relationship was obtained between E and vapor pressure deficit (VPD). A positive non-linear relationship (p < 0.01) was observed between K (E / ET in non-limited water conditions) and VPD, illustrating the low stomatal regulation that persimmon trees exhibit in response to air dryness. K varied over the growing seasons following the ET pattern although showing comparatively a slighter decrease during the last part of the growing season. From June to September, K of persimmon ‘Rojo Brillante’ trees with a midday photosynthetically active radiation (PAR) intercepted of ~30 % ranged from 0.45 to 0.56. A strong relationship was obtained between K and percentage of midday intercepted PAR that can be used to estimate K of persimmon ‘Rojo Brillante’ trees with a range of ground canopy covers. This information is useful for deriving specific water use models for persimmon trees to be implemented in decision support systems. The monthly transpiration coefficients here provided will be useful for optimizing irrigation scheduling decisions in persimmon orchards and therefore irrigation water management., This research was funded by the Valencian Institute of Agricultural Research (IVIA), Spain, and “Denominación de origen Kaki Ribera del Xuquer”, Spain, via “Proyecto Integral Caqui”. Complementary funds from the project Preciriego grant number RTC-2017-6365-2 funded by AEI-Feder, Spain, are also acknowledged.

Resolving the molecular structure of the brain requires mapping specific proteins and cellular structures with a level of detail beyond the capabilities of conventional light microscopy. Electron microscopy enables visualization at the ultrastructural level, but there are limited methods for localizing specific proteins. The authors present pan-expansion microscopy of tissue (pan-ExM-t), a new method that combines ∼24-fold expansion microscopy with fluorescent pan-staining of proteins and immunostaining of specific protein targets. The authors visualize the structure of brain slices at the synaptic level with outstanding detail using a standard confocal microscope. This new method enhances the ability to visualize the nanoarchitecture of the brain and the subcellular localizations of specific proteins.

Loss-of-function mutations in VPS13C cause familial Parkinson’s disease (PD) and increase the risk to develop the sporadic form of the disease. However, the underlying disease mechanisms remain unclear. It has been previously established that VPS13C tethers lysosomes with the endoplasmic reticulum (ER) and promotes lipid interchange between both organelles. This study provides a cellular role of VPS13C, specifically regulating the cGAS/STING pathway and contributing to the innate immune response. The authors generate VPS13C knockout HeLa cells and use confocal microscopy and biochemical approaches to show loss of VPS13C leads to altered lysosome lipid composition and mitochondrial DNA leakage. Understanding how VPS13C preserves cellular homeostasis is an exciting discovery for scientists working on neurodegeneration and for cell biologists interested in lysosome-to-mitochondria cross-talk.

Growing consumer awareness about the environmental impact of their food purchase decisions means having to create labels that better communicate sustainability aspects. The aim of this study is to explore consumers’ responses to “sustainable irrigation” (SI)-labeled wine. To this end, the effect of two label factors, SI claims (no SI info, logo, and text), and their position (front- vs. back-labels) on consumer choice, reasons for choice, perceived sustainability, and willingness-to-pay is determined. Moreover, we determine, for the first time, for SI claims, the relationship between consumer choice and paid attention. Our results reveal that almost 90% of the 408 consumers participating in this study show an interest in the SI-labeled wines. The main reason for choosing the SI-labeled wines rather than the control (no SI info-label) was the following: ‘I think it’s more environmentally friendly’, with an increase of two points on a nine-point sustainability perception scale. Consumers prefer the logo-label to the text-label, mainly because they find it more attractive, and a close relationship between paid attention and product choice probability is determined. The vast majority of consumers are willing to pay an extra cost of 15% or more for SI-labeled wine vs. the control. These results have relevant implications for the industry because they show that the SI logo is a useful way to draw consumers’ attention to the sustainability of irrigation practices and positively affect their choice. Our findings indicate that this way of differentiating the product in the market can contribute to compensating the economic cost of implanting SI practices.

Rab GTPases are key regulators of vesicle-mediated transport and are proposed to play a crucial role in the pathobiology of Parkinson's disease. As membrane trafficking seems to be a relevant pathway altered in Parkinson' disease, understanding the role of Rab GTPases in the disease progression could open a window for therapeutic interventions. In this review, we focus on the recent advances on the role of Rab GTPases in the biology of two main proteins involved in Parkinson's disease: LRRK2 and α-synuclein, given that mutations in their genes (LRRK2 and SNCA) cause familial and sporadic Parkinson's disease.

Non-Contact Resonant Ultrasound Spectroscopy (NC-RUS) has emerged as a powerful tool to determine plant water status in a non-destructive, non-invasive and rapid way. In this study, ultrasonic parameters directly obtained from experimental measurements in the field using NC-RUS - such as resonant frequency (fres), velocity (v) and Q-factor - were evaluated as potential water stress indicators in Citrus plants. The experiments were carried out in two-year-old mandarin trees (Citrus clementina Hort. ex Tan. ‘Clemenules’) grown in pots in an open greenhouse where two different groups of plants were tested: a Control group (full irrigation) and a Drought Stress group (DS) whose irrigation was withdrawn during 7 days, followed by a 16 days recovery period. Soil water content, leaf water potential (Ψleaf) and the considered ultrasonic parameters were measured in the same leaves. fres detected changes between control and DS at day 7 without irrigation. Conversely, v showed differences after day 3, which were statistically significant at day 7, enabling discrimination between C and DS groups. Hence, Q-factor was the ultrasonic parameter that showed statistically significant differences between C and DS groups at days 3 and 7. Consequently, Signal Intensity in Q during the drought treatment showed a similar evolution to Ψleaf, although with slightly lower performance. However, Q-factor sensitivity excels Ψleaf at each day studied. Finally, a linear correlation (R2=0.57) between Ψleaf and Q-factor of all experimental data measured in DS group plants along the drought treatment was found. In conclusion, the ultrasonic parameters obtained using NC-RUS and in particular the Q-factor, demonstrated to be potential new water stress indicators in Citrus trees, with the novelty of being non-destructive, non-invasive and rapid. Future work should explore its suitability for its use in irrigation scheduling for Citrus trees.

[EN] Composite concrete members without web reinforcement are often used in precast construction. The contribution of the cast-in-place concrete topping slab to vertical shear strength has been traditionally disregarded. However, significant cost savings can result from designing and assessing these structures if this contribution is considered. This paper presents the experimental study of a series of 21 monolithic and composite (precast beam and cast-in-place slab) specimens without web reinforcement, and with rectangular and T-shaped cross-sections, failing in shear. The vertical shear strength was analysed by the following test variables: cross-section shape, the existence of an interface between different aged concretes, strengths of the two concretes and the differential shrinkage effect. From these experimental tests, it was concluded that the slab contributed to shear strength, the use of high-strength concrete slightly increased specimens' shear strength and the differential shrinkage did not reduce shear strength. Specimens' failure modes were analysed based on their shear transfer mechanisms, noticing that the arching action in the slab was considerable after critical shear crack formation. The vertical shear strength experimental results were well predicted by the codes' formulations (Eurocode 2, Model Code 2010 and ACI 318-19) when composite beam depth was taken for the calculations instead of beam depth. Codes significantly underestimated the horizontal shear strengths of the composite specimens., This research has been supported by: the Spanish Ministry of Science and Innovation through Research Project BIA2015-64672-C4-4-R and RTI2018-099091-B-C21-AR; the Regional Government of Valencia through Project AICO/2018/250; the European Union with FEDER funds. The experimental programme was developed in the Laboratory of Concrete of the Institute of Concrete Science and Technology (ICITECH) of the Universitat Politècnica de València (UPV) with concrete supplied by Caplansa. The Spanish Ministry of Science and Innovation supported Lisbel Rueda through grant BES-2016-078010.

Genetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to enhanced lifetime risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2-positive lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing expression of ARL8B and SKIP or by knockdown of the motor adaptor protein JIP4 blocked recruitment and phosphorylation of Rab10 by LRRK2. Conversely, overexpression of the Rab7 effector protein RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our novel findings suggest LRRK2 can be activated at multiple cellular membranes including lysosomes, and that lysosomal positioning further provides regulation of some Rab substrates likely via differential phosphatase activity in nearby cellular compartments.

The Parkinson disease kinase LRRK2 translocates to the lysosomal membrane triggering lysosomal sorting through JIP4., Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson’s disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane–rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

[EN] Continuous reinforced concrete (RC) beams may develop significant plastic rotations to enable the redistribution of bending moments. These rotations occur at plastic hinges, which are subject to high shear forces. The influence of rotations on the shear strength for statically determined beams without shear reinforcement failing in shear after yielding of the flexural reinforcement has already been experimentally verified. However, this influence has not been studied in continuous members with shear reinforcement. An innovative tests system has been specially designed to develop shear failures before and after yielding of the flexural reinforcement in both statically determinate and indeterminate structures. Nine beams (9000 mm long, 250 mm wide, 450 mm high) with a shear reinforcement of phi 8/30 (rho(w) = 0.13%) and different longitudinal reinforcement ratios were tested under different load and support conditions. The shear strength provided by shear reinforcement and that provided by the other mechanisms of resistance (shear strength provided by concrete) for each specimen were calculated based on the critical shear crack width measurements performed by Digital Image Correlation (DIC). Bending rotation and crack rotation along the development length of the critical shear crack were also obtained by DIC. Based on the test results, the shear strength provided by concrete was studied in relation to the bending rotation and the average crack width in reinforced concrete beams with shear reinforcement. It was confirmed that the shear strength provided by concrete decreased with increasing both bending rotations and crack widths. The shear strength values predicted by different design codes (ACI 318-19, Eurocode 2 and Model Code 2010) were compared with the test results, and showed that these formulations did not properly capture the loss of shear strength caused by bending rotation., This research was funded with grants from the Spanish Ministry of Economy and Competitiveness to Research Project BIA2015-64672-C4-4-R. The experimental programme was developed in the Laboratory of Concrete of the Institute of Concrete Science and Technology (ICITECH) of the Universitat Politecnica de Valencia (UPV), with concrete supplied by Caplansa. Andrea Monserrat was supported by the Conselleria d'Educacio, Investigacio, Cultura i Esport of the Generalitat Valenciana (Order 6/2015, DOCV no. 7615 15.09.2015) with European Regional Development Funds (ERDF) allocated by the EU.

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

Purpose IORT with mobile linear accelerators is a well-established modality where the dose rate and, therefore, the dose per pulse are very high. The constancy of the dosimetric parameters of the accelerator has to be checked daily. The aim of this work is to develop a phantom with embedded detectors to improve both accuracy and efficiency in the daily test of an IORT linac at the surgery room. Methods The developed phantom is manufactured with transparent polymethyl methacrylate (PMMA), allocating 6 parallel-plate chambers: a central one to evaluate the on-axis beam output, another on-axis one placed at a fixed depth under the previous one to evaluate the energy constancy and four off-axis chambers to evaluate the flatness and symmetry. To analyse the readings a specific application has been developed. Results For all chambers and energies, the mean saturation and polarization corrections were smaller than 0.7%. The beam is monitored at different levels of the clinical beam. Output, energy constancy and flatness correlate very well with the correspondent values with the complete applicator. During the first six months of clinical use the beam dosimetric parameters showed excellent stability. Conclusions A phantom has been developed with embedded parallel plate chambers attached to the upper applicator part of an IORT linac. The phantom allows a very efficient setup reducing the time to check the parameters. It provides complete dosimetric information (output, energy and flatness) with just one shot and using ionization chambers with minimum saturation effect, as this highly pulsed beam requires.

[EN] The accurate estimation of plant water needs is the first step for achieving high crop water productivity. The main objective of the work was to develop an irrigation scheduling procedure for mandarin orchards under Mediterranean conditions based on replacing the amount of consumed water using reference values of soil moisture according to different phenological periods. The proposed methodology includes a definition part where the threshold values were established relating the trees' stem water potential and the volumetric soil water content measured with Frequency Domain Reflectometry probes. A second part includes the steps for standardizing measurements from capacitance probes by using the LEACHM hydrological simulation model to take into account the sensor-to-sensor variations. Finally, an extrapolation procedure based on soil water retention curves was used for adapting critical soil water content thresholds to different soil conditions. Field evaluations were made in a citrus orchard located in eastern Spain during two seasons. Standardize critical soil water contents were: i) 24 % vol. for post-harvest, bloom - fruit set and phase III of fruit growth; ii) 27 % vol. for phase I of fruit growth, and iii) 29 % vol. for phase II of fruit growth with average daily air vapour pressure deficit values ranging between 0.2 - 0.4; 0.9-1.1 and 1.1-1.3 kPa, respectively. When implemented in the orchard, the sensor-based strategy resulted in water saving of 26 % respect to a control treatment, irrigated using the standard FAO-56 approach, without significant differences in yield and increasing the crop water productivity by 33 %. In conclusion, we suggest that the determination and use of the critical soil water content is a useful tool for scheduling irrigation. The proposed standardization and extrapolation methodology allows the irrigation strategy to be applied to other mandarin orchards under similar climatic conditions., This experiment was funded by European project WEAM4i Water & Energy Advanced Management for Irrigation, grant agreement 619061 and FEDER-MINECO projects EASYRIEGO IPT-2012-0950-310000, RISUB IPT-2012-0480-310000 and RIEGOTEL RTC-2016-4972-2. M.A. Martinez-Gimeno acknowledges the financial support received from the Spanish Ministry of Education, Culture and Sports (MECD) program Formacion Profesorado Universitario (FPU). Juan G. Perez-Perez also gratefully acknowledges the post-doctoral contract in the `Ramon y Cajal' program, supplied by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). Authors would like to thank M. Jorda, C. Albert, F. Sanz and A. Yeves for the support on installation and maintenance of the equipment. Thanks also to Prof. G. Provenzano (University of Palermo) for his critical comments and suggestions.

En este estudio se presentan los últimos datos experimentales en campo en hojas de Clemenules x carrizo. Se comparan los datos tomados con la tradicional cámara de presión y con la novedosa técnica de Espectroscopía Ultrasónica Resonante mediante sensores sin contacto (NC-RUS). Las plantas fueron sometidas a tres tratamientos de riego: T100, plantas regadas al 100% de la evapotranspiración de cultivo (ETc); T50, riego al 75% de la ETc; y T25, riego al 50% de la ETc. Al Ministerio de Ciencia e Innovación por el contrato postdoctoral ‘Juan de la Cierva’ de L. Fariñas (FJCI-2017-34759). A la Conselleria de Educación, Investigación Cultura y Deporte de la Comunitat Valenciana por el proyecto GV/2019/037.

Increasing water-use efficiency (WUE) is mandatory for more sustainable agricultural land use. Here we studied during three consecutive years in a nectarine (Prunus persica L.) orchard the effects of placing 2 × 40 L coconut fiber substrate (S) bags per tree on the agronomic performance and WUE. Additionally, there were two regimes of watering, control (C), fulfilling 100% of the estimated crop evapotranspiration (ETc) and a deficit (D) irrigation treatment, replacing the 65% of the ETc. Root colonization of the substrate, changes in the soil and substrate water content and temperature, tree water status (midday stem water potential), shoot growth (trunk diameter and tree volume), fruit yield and fruit quality compounds, were determined. Coconut substrate was colonized by the roots after two months from its installation, and after three years root dry weight in the substrate averaged 172 and 155 g per tree for the control and deficit irrigation treatments, respectively. During the first year, tree growth was enhanced in the C irrigation, while freezing temperatures during fruit set almost totally removed all fruits. In the second year, probably the abundant rainfall disguised the effect of the substrate on the yield parameters. However, this year and the next, deficit irrigation reduced fruit mean weight and delayed fruit harvest. In the third year, the substrate in the deficit irrigation treatment had a positive effect on fruit yield by increasing the number of fruits per tree in comparisons with deficit irrigation trees without substrate. The combination of deficit irrigation and substrate increased the WUE over the rest of the treatments but with no clear improvements in the tree water status. The results suggest that nectarine trees with deficit irrigation and substrate could be benefiting from a more efficient use of nutrients that further increases fruit set or prevent fruit drop.

Irrigation systems aimed to optimize water use efficiency in agriculture have become essential due to the increasing water limitations that agriculture is currently facing. Assessment of crop responses to different irrigation systems and strategies are therefore encouraged to find the most efficient options for each specific case. The main objective of this study was to assess the performance of a citrus crop under a surface (SI) and subsurface drip irrigation (SSI) with 7 (SI7, SSI7) or 14 emitters (SI14, SSI14) per plant, as well as a third SS treatment (SSIA), identical to SSI7 but equipped with and additional drip line buried between the tree rows. Evaluations were made in terms of yield, fruit composition, irrigation water productivity (IWP) and water savings. Results showed that, on average, water savings were 23.0% in the SSI treatment compared to the SI treatment without significant differences in either yield or fruit composition. IWP was higher in SI14, SSI14 and SSIA than in SI7 and SSI7 treatments. SSIA was the treatment with the lowest irrigation volumes and the highest yield and compared to SI7 allowed, over the three years, water savings in the range between 22.4 and 27.9%. Results from this study illustrate that there is opportunity to substantially save water in citrus production and that further research in this direction is needed to contribute to better optimize the water resources in agriculture.

Postharvest deficit irrigation (DI) strategies have been proven to advance bloom, harvest dates and economic return in loquat ‘Algerie’ trees because an early harvest results in higher loquat fruit prices. This fact poses the question of whether postharvest DI strategies could have a similar effect on more precocious cultivars than cv. Algerie, providing thus a more profitable option to farmers. In this work, the response of an early- and intermediate-maturing loquat cultivars (cv. Cardona and Algerie, respectively) to a summer early (DIearly) and late (DIlate) DI strategy was assessed in two parallel studies during three consecutive years. The effects of the DIearly and DIlate strategies on bloom date, percentage of fruit picked at harvest per picking date and yield of both loquat varieties were studied. Moreover, fruit quality of cv. Cardona at harvest was also assessed. Plant water status was monitored by midday stem water potential (Ψstem) measurements. Results showed that DIearly and DIlate strategies advanced bloom in both cultivars although a higher effect was observed with the DIearly than with the DIlate treatment. Water restrictions did not increase the percentage of fruit picked at any of the picking dates and did not affect yield or fruit quality in the early-maturing cultivar. In ‘Algerie’ trees, a higher percentage of fruit was generally picked during the first picking dates in the DIearly and DIlate treatments than in the control although differences were only statistically significant during the last experimental season. Yield was significantly higher in ‘Algerie’ DIlate trees than in control or DIearly trees two out of the three experimental seasons. Overall, results showed that the DIearly and DIlate strategies tested here did advance bloom in the early-maturing cultivar but did not have an effect on the percentage of fruit picked per picking date at harvest. Nevertheless, the fact that substantial water savings (>30%) were obtained with no detrimental effect on yield presents the use of postharvest DI strategies as an interesting option to be followed in early-maturing cultivars for a more efficient crop production.

[EN] Compressed reinforcement buckling in concrete columns can reduce ductility in structural elements. In order to avoid this, design codes propose maximum required tie spacing. Nonetheless, they do not incorporate the positive effect of concrete fibers in their formulation, whose capability of delaying buckling has been proved. For this reason, recommendations for maximum required tie spacing for elements made with concrete, with or without steel fibers, and with normal or high strength, are proposed in this article. In order to achieve this, the mixed model proposed by Pereiro-Barceló and Bonet was extended to consider elements made of HSC with and without steel fibers thanks to the results of an experimental campaign of HSC columns with and without fibers, under monotonic loading. In these tests, the buckling critical load in compressed reinforcement was experimentally determined in all the columns. In addition, a comparison of the proposed transverse concrete separation with respect to the recommendations proposed by the main existing codes was made., The research presented herein forms part of a research undertaken at the Concrete Science and Technology Institute (ICITECH) of the Universitat Politecnica de Valencia (UPV). The authors are sincerely grateful to the Spanish Ministry of Economy and Competitiveness for the help provided through Project BIA2012-32645, and to the European Union for the financial support obtained from FEDER funds. The authors wish to thank the Spanish Ministry of Education, Culture and Sport for Grant FPU12/01451.

34 Pags.- 7 Tabls.- 1 Fig. The definitive version is available at: https://www.sciencedirect.com/science/journal/03783774, A decades-long policy for irrigation Telemetry and Remote Control (TM/RC) systems in Spain has led to installations for approximately 260 Water Users Associations (WUAs), with a total estimated area of 1.0 M ha of agricultural land. This is believed to be the largest deployment of such technologies in the world. These systems have been installed in financial cooperation between public administrations and WUAs. This paper set out to characterize these systems, assess the causes for their individual success or failure, consider their future evolution and support policy updates. A survey with 110 questions was addressed to 84 WUAs distributed throughout the country. Further, an interview with four questions was addressed to 24 selected stakeholders intervening in irrigation TM/RC projects: from policy makers to farmers. The results provide a detailed overview of these systems in Spain, characterizing the WUAs in which they are installed, their technological traits, their maintenance, the problems they face in their daily operation, their current use, the factors limiting wider use, and the willingness of the WUAs to continue bearing the costs to use TM/RC features in the future. A large majority of TM/RC systems are regularly used to improve WUA water and energy management, and receive proper maintenance. However, in 15% of WUAs, farmers are not satisfied with the TM/RC system, and in 19% of the WUAs the TM/RC system cannot operate half of the hydraulic valves connected to them. We found that early technology applications failed more than recent applications, evidencing a process of technological maturity. The standardization of TM/RC systems, adaptation of the system to WUA specificities, training of WUA personnel, and a sufficiently long guarantee period stood as critical variables for success in the implementation of these technologies., This work was supported by the Spanish State Research Agency (Agencia Estatal de Investigación) of the Ministry of Economy and Competitiveness (MINECO) of the Government of Spain, grant numbers: AGL2013-48728-C2-1-R, AGL2014-59747-C2-1- R, AGL2014-59747-C2-2-R, RTC-2015-3453-2 and RTC-2016-4972-2.

Persimmon cultivation is gaining importance in many semi-arid areas of the world. This is the case of eastern Spain, where the rainfall regime is well below the crop water needs, and drip irrigation is typically used to control the water and nutrient supplies. This review is a summary of the current information in the literature on persimmon water and nutrient requirements. Crop responses to variable irrigation regimes and mild to severe water stress are also analyzed. It is concluded that persimmon water needs can be particularly high during summer, when the crop coefficient can reach values close or even above 1.0. In order to cope with water scarcity, regulated deficit irrigation (RDI) has been shown as a useful technique for reducing fruit drop in persimmon trees. RDI strategies during fruit ripening can accelerate fruit color development, resulting in earlier harvesting but with lower fruit weight. Information on nutrients requirement of astringent and non-astringent varieties of persimmon is also provided. Finally, new research for continuing plant-water relations studies on persimmon trees, such as the need to quantify the optimum nutrition plans under conditions of variable water supply, will be proposed.

Persimmon trees grafted on D. lotus have been found to be extensively affected by leaf necrosis in the important cropping area of Valencia (E Spain). Although this problem has been attributed to chloride toxicity, this association had not been rigorously demonstrated. In addition the chloride and salinity tolerance of persimmons grafted on D. lotus has not been characterised, and neither has the performance of this rootstock been compared to D. virginiana. Two outdoors experiments, one in pots, and another one in the field were set up and conducted for three years with trees grafted on both rootstocks. Trees in the pots experiment were arranged in five groups and irrigated with waters differing in their salt and thus, Cl−, Ca2+ and Na+ water contents. Season-end leaf contents of Na, Cl, N, P, K, Ca, Mg, S, B, Cu, Fe, Mn and Zn, toxicity symptoms and fruit yields were assessed. In the field experiment, one irrigation water quality was used, and just leaf chloride contents, water potential and fruit yields were evaluated. Field toxicity symptoms were reproduced in the pots experiment in the trees on D. lotus, but not on D. virginiana, with leaf chloride contents increasing with increasing irrigation water salinity. Leaf chloride contents were much higher than either sodium or boron, and significantly more correlated with toxicity symptoms. Comparing to chloride toxicity and salinity tolerance data from similar woody crops, D. lotus was classified as sensitive to salinity with, respectively, threshold ECe and slope of 1.2 dS m−1 and 22 dS−1 m. D. lotus rootstocks lend persimmon trees very lower ability to avoid chloride toxicity likely because of, on the one hand, its low soil exploration capacity, and, on the other hand, its low chloride exclusion capacity. In return, however, D. lotus lends persimmon trees more productivity, which still keeps for waters with slight-to-moderate chloride contents (3–4 mmol/L)., F. Visconti thanks the financial support received from the Spanish Ministry of Economy and Competitiveness (MINECO) through grant “Juan de la Cierva” (JCI-2011-11254) during the 2012–2015 triennium.

Parkinson’s disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. While most cases of PD are sporadic in nature, several inherited monogenic syndromes exist that overlap clinically and pathologically with sporadic PD. Of these, loss of function mutations in DNAJC6 , which encodes the protein Auxilin, cause an aggressive form of juvenile onset PD. Auxilin and its homologues are known to play a role in clathrin-mediated trafficking, which is crucial for cellular function in all eukaryotes and plays a specialized role in synaptic transmission in higher organisms. Auxilin is the major neuronal uncoating protein for clathrin-coated vesicles required for delivery of cargo from the plasma membrane and trans -Golgi network to intracellular destinations. However, how mutations in Auxilin cause PD is currently not understood. To address this problem, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation. When bred to homozygosity, this mutation induced neurological phenotypes that phenocopy clinical features observed in patients, including motor impairments reminiscent of bradykinesia and gait problems. Mapping the interactome of Auxilin confirmed clathrin and synaptic clathrin adaptor protein interactions and also identified novel Golgi-resident interactors. Critically, all tested pathogenic mutations in Auxilin retained clathrin adaptor protein binding but lost interaction with clathrin itself. These observations describe a mechanism by which impaired clathrin-mediated trafficking in R857G Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide novel insights for PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin. Highlights Auxilin interacts with Golgi-resident clathrin adaptor protein GGA2 Auxilin is involved with uncoating of CCVs at the Golgi and synapse Impaired clathrin-mediated trafficking underlies PD-like phenotypes in R857G Auxilin mice

Background PD is a complex polygenic disorder. In recent years, several genes from the endocytic membrane-trafficking pathway have been suggested to contribute to disease etiology. However, a systematic analysis of pathway-specific genetic risk factors is yet to be performed. Objectives To comprehensively study the role of the endocytic membrane-trafficking pathway in the risk of PD. Methods Linkage disequilibrium score regression was used to estimate PD heritability explained by 252 genes involved in the endocytic membrane-trafficking pathway including genome-wide association studies data from 18,869 cases and 22,452 controls. We used pathway-specific single-nucleotide polymorphisms to construct a polygenic risk score reflecting the cumulative risk of common variants. To prioritize genes for follow-up functional studies, summary-data based Mendelian randomization analyses were applied to explore possible functional genomic associations with expression or methylation quantitative trait loci. Results The heritability estimate attributed to endocytic membrane-trafficking pathway was 3.58% (standard error = 1.17). Excluding previously nominated PD endocytic membrane-trafficking pathway genes, the missing heritability was 2.21% (standard error = 0.42). Random heritability simulations were estimated to be 1.44% (standard deviation = 0.54), indicating that the unbiased total heritability explained by the endocytic membrane-trafficking pathway was 2.14%. Polygenic risk score based on endocytic membrane-trafficking pathway showed a 1.25 times increase of PD risk per standard deviation of genetic risk. Finally, Mendelian randomization identified 11 endocytic membrane-trafficking pathway genes showing functional consequence associated to PD risk. Conclusions We provide compelling genetic evidence that the endocytic membrane-trafficking pathway plays a relevant role in disease etiology. Further research on this pathway is warranted given that critical effort should be made to identify potential avenues within this biological process suitable for therapeutic interventions. © 2019 International Parkinson and Movement Disorder Society.

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.

In recent years many hectares planted with persimmon trees in E Spain have been diagnosed with chloride toxicity. An effect of this abiotic stress on fruit quality has been reported in different crops. However, the impact of chloride stress on persimmon fruit quality is unknown. The harvest and postharvest quality of persimmons harvested from trees that manifest different intensities of chloride toxicity foliar symptoms was evaluated herein. Our results revealed that fruits from trees under chloride stress conditions underwent chloride accumulation in the calyx, which was more marked the greater the salt stress intensity trees were exposed to. Increased chloride concentrations in the calyx stimulated ethylene production in this tissue. In the fruits affected by slight and moderate chloride stress, calyx ethylene production accelerated the maturity process, as reflected by increased fruit colour and diminished fruit firmness. In the fruits under severe chloride stress, the high ethylene levels in the calyx triggered autocatalytic ethylene production in other fruit tissues, which led fruit maturity to drastically advance. In these fruits effectiveness of CO2 deastringency treatment was not complete and fruit softening enhanced during the postharvest period. Moreover, chloride stress conditions had a marked effect on reducing fruit weight, even in slightly stressed trees.

Purpose To investigate the possible role of activating transcription factor 3 (ATF3) in retinal ganglion cell (RGC) neuroprotection and optic nerve regeneration after optic nerve crush (ONC). Methods Overexpression of proteins of interest (ATF3, phosphatase and tensin homolog [PTEN], placental alkaline phosphatase, green fluorescent protein) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs) expressing corresponding proteins. The number of RGCs and αRGCs was evaluated by immunostaining retinal sections and whole-mount retinas with antibodies against RNA binding protein with multiple splicing (RBPMS) and osteopontin, respectively. Axonal regeneration was assessed via fluorophore-coupled cholera toxin subunit B labeling. RGC function was evaluated by recording positive scotopic threshold response. Results The level of ATF3 is preferentially elevated in osteopontin+/RBPMS+ αRGCs following ONC. Overexpression of ATF3 by intravitreal injection of rAAV 2 weeks before ONC promoted RBPMS+ RGC survival and preserved RGC function as assessed by positive scotopic threshold response recordings 2 weeks after ONC. However, overexpression of ATF3 and simultaneous downregulation of PTEN, a negative regulator of the mTOR pathway, combined with ONC, only moderately promoted short distance RGC axon regeneration (200 μm from the lesion site) but did not provide additional RGC neuroprotection compared with PTEN downregulation alone. Conclusions These results reveal a neuroprotective effect of ATF3 in the retina following injury and identify ATF3 as a promising agent for potential treatments of optic neuropathies.

SUMMARY Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson’s disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD., Graphical Abstract, In Brief Mutations in LRRK2 are linked to Parkinson’s disease. However, the cellular role of LRRK2 remains elusive. Using a combination of proteomics and imaging techniques, Beilina et al. describe a function of LRRK2 mediating endosome-TGN transport by scaffolding the GARP:Syntaxin-6 interaction, suggesting a connection between GARP and LRRK2 in Parkinson’s disease.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson’s disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.