Primate-specific isoform of Nedd4-1 regulates substrate binding via Ser/Thr phosphorylation and 14-3-3 binding.

Nedd4 (Nedd4-1) is an E3 ubiquitin ligase involved in crucial biological processes such as growth factor receptor signaling. While canonical Nedd4-1 comprises a C2-WW(4)-HECT domain architecture, alternative splicing produces non-canonical isoforms that are poorly characterized. Here we characterized Nedd4-1(NE), a primate-specific isoform of Nedd4-1 that contains a large N-terminal Extension (NE) that replaces most of the C2 domain. We show that Nedd4-1(NE) mRNA is ubiquitously expressed in human tissues and cell lines. Moreover, we found that Nedd4-1(NE) is more active than the canonical Nedd4-1 isoform, likely due to the absence of a C2 domain-mediated autoinhibitory mechanism. Additionally, we identified two Thr/Ser phosphoresidues in the NE region that act as binding sites for 14-3-3 proteins, and show that phosphorylation on these sites reduces substrate binding. Finally, we show that the NE region can act as a binding site for the RPB2 subunit of RNA polymerase II, a unique substrate of Nedd4-1(NE) but not the canonical Nedd4-1. Taken together, our results demonstrate that alternative splicing of the ubiquitin ligase Nedd4-1 can produce isoforms that differ in their catalytic activity, binding partners and substrates, and mechanisms of regulation.

Residues of veterinary antibiotics in manures from pig and chicken farms in a context of antimicrobial use reduction by implementation of health and welfare plans.

The use of antibiotics in food-producing animals can induce the presence of residual substances in manure, which are then released into the environment and may contribute to soil and groundwater contamination. During the on-farm implementation of strategies to improve animal health and welfare in chicken and pig farms, the consequences of antibiotic use were evaluated in terms of the occurrence and levels of antibiotic residues in manure. A set of 35 broiler farms from Cyprus, Greece, the Netherlands and 40 pig farms from France and Italy provided a total of 350 manure samples. The primary objective was to develop a specific LC/MS/MS method capable of quantifying antibiotic residues in both types of manure. The method was able to detect fifteen antibiotics belonging to nine classes, with validated limits of quantification of 10-20 µg/kg, and accuracies ranging from 81% to 138%. With the exception of amoxicillin, which was never detected in any manure, all antibiotics used were detected in manure from treated animals with typical concentrations ranging from 10 to 99198 µg/kg for both chickens and pigs. The occurrence of residual antibiotics was higher in chicken than in pig manure, especially for fluoroquinolones and doxycycline which were detected in 89% and 100% of the chicken manure, respectively, and in 28% of the pig manure. The impact of the health plans on the antibiotic load manure was assessed by measuring for each farm the ratio of the sum of all antibiotic concentrations measured after and before the implementation of the plan. The results showed that, in addition to the frequency of treatments, the class of antibiotic used is an important factor to consider as it strongly influences the stability/instability of the compounds, i.e. their ability to persist in the manure of food-producing animals.

Flood damage inspection and risk indexing data for an inventory of bridges in Central Greece.

This dataset is related to the research paper entitled "Bridge-specific flood risk assessment of transport networks using GIS and remotely sensed data" published in the Science of the Total Environment. It provides the information necessary for the reproduction of the case study that was used for the demonstration and validation of the proposed risk assessment framework. The latter integrates indicators for the assessment of hydraulic hazards and bridge vulnerability with a simple and operationally flexible protocol for the interpretation of bridge damage consequences on the serviceability of the transport network and on the affected socio-economic environment. The dataset encompasses (i) inventory data for the 117 bridges of the Karditsa Prefecture, in Central Greece, which were affected by a historic flood that followed the Mediterranean Hurricane (Medicane) Ianos, in September 2020; (ii) results of the risk assessment analysis, including the geospatial distribution of hazard, vulnerability, bridge damage, and associated consequences for the area's transport network; (iii) an extensive damage inspection record, compiled shortly after the Medicane, involving a sample of 16 (out of the 117) bridges of varying characteristics and damage levels, ranging from minimal damage to complete failure, which was used as a reference for validation of the proposed framework. The dataset is complemented by photos of the inspected bridges which facilitate the understanding of the observed bridge damage patterns. This information is intended to provide insights into the response of riverine bridges to severe floods and a thorough base for comparison and validation of flood hazard and risk mapping tools, potentially useful for engineers, asset managers, network operators and stakeholders involved in decision-making for climate adaptation of the road sector.

Analysis of Antimicrobials in Muscle and Drinking Water in Terms of Reducing the Need of Antimicrobial Use by Increasing the Health and Welfare of Pig and Broiler.

Antimicrobial residues may pose harmful effects on the health of consumers. At the same time, an adequate quality of drinking water for animals is one of the important element to ensure animal welfare and food without antibacterials. The presented study is aimed at estimating the residue levels of antibacterial compounds, such as penicillins, cephalosporin, macrolides, tetracyclines, quinolones, sulphonamides, aminoglycosides, diaminopirymidines, pleuromutilines and lincosamides in meat and on-farm drinking water samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), as a part of a surveillance system on pig and broiler farms within the project Healthy Livestock. A total of 870 samples of muscle from pig and broiler, as well as 229 water samples were analysed for antibiotic residues. Samples were collected from farms in EU countries in two steps, before and after implementation of a tailor-made health plan. In muscle samples, the detected concentrations of doxycycline in the post-intervention step (15.9-70.8 µg/kg) were lower than concentrations in the pre-intervention step (20.6-100 µg/kg). In water samples, doxycycline in an average concentration of 119 µg/L in the pre- and 23.1 µg/L in the post-intervention step, as well as enrofloxacin at concentrations of 170 µg/L in the pre- and 1.72 µg/L in the post-intervention step were quantified. Amoxicillin was only present before intervention. The obtained results confirm the effectiveness of the intervention actions. The concentrations of antibiotics in muscles and water were lower after implementation of a health plan on the farms.

Development and implementation of a risk assessment tool for broiler farm biosecurity and a health intervention plan in the Netherlands, Greece, and Cyprus.

Preventing pathogens from entering and spreading on farms is the first step in reducing health problems. For this study a BiosEcurity Assessment Tool was developed to identify strengths and weaknesses in biosecurity on broiler farms, which was used as a starting point to formulate tailor-made health plans to improve broiler health and reduce antimicrobial use. Farms were divided into 3 separate areas according to associated biosecurity risk; high disease risk external areas (red zone), medium risk service areas (orange zone), and the clean and highly secure access-restricted green zone. In the Netherlands, Cyprus, and Greece, 13, 15, and 7 broiler houses were monitored for 4 production cycles (2 preintervention and 2 postintervention cycles). At the start of the study the BiosEcurity Assessment Tool assessment was performed and a health plan was made in consultation with the veterinarian. After the second cycle a start was made with the implementation of the health plan. Overall, the biosecurity level in the green and orange zones were significantly higher in the Netherlands compared to Greece and Cyprus, but there was no difference for the red zone or the transition zones between the countries. The interventions in the health plans were mostly directed towards those measures that could be implemented in the short term and with low costs in the green zone. In Cyprus a decrease in antimicrobial use was found postintervention. This was not the case in Greece and the Netherlands. In Cyprus and Greece footpad lesion improved after interventions were implemented, although this may have been an effect of season. In Dutch farms no improvement was detected, but both antimicrobial use and footpad lesions were lower at the start of the study compared to Cypriot and Greek farms. In conclusion, the BEAT shows to be a promising tool to assess biosecurity risks on broiler farms. The biosecurity assessment in combination with the farm specific health plans could contribute to antimicrobial reduction on broiler farms.

Bridge-specific flood risk assessment of transport networks using GIS and remotely sensed data.

A novel framework for the expedient assessment of flood risk to transportation networks focused on the response of the most critical and vulnerable infrastructure assets, the bridges, is developed, validated and applied. Building upon the recent French guidelines on scour risk (CEREMA, 2019), this paper delivers a thorough methodology, that incorporates three key, risk parameters: (i) the hydrodynamic loading, a hazard component of equal significance to scour, for the assessment of hazard; (ii) the correlation of select scour indicators with a new index relating to flow velocity, a primary measure of the adverse impacts of flow-structure interaction, enabling a more accurate and automated, assessment of bridge susceptibility to scour; (iii) the use of a new, comprehensive indicator, namely the Indicator of Flood Hazard Intensity (IFHI) which incorporates, in a simple yet efficient way, the key parameters controlling the severity of flood impact on bridges, namely flow velocity, floodwater height, flow obstruction, and sediment type. The framework is implemented for the analysis of flood risk in a case study area, considering an inventory of 117 bridges of diverse construction characteristics, which were affected by a major flood that impacted Greece in September 2020. The reliability of the method is validated against an extensive record of inspected and documented bridge damages. Regional scale analysis is facilitated by the adoption of the Multi-Criteria Decision-Making method for flood hazard indexing, considering geomorphological, meteorological, hydrological, and land use/cover data, based on the processing of remotely sensed imagery and openly available geospatial datasets in GIS.

Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2.

Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.

Peptide-based sequestration of the adaptor protein Nck1 in pancreatic ß cells enhances insulin biogenesis and protects against diabetogenic stresses.

One feature of diabetes is the failure of pancreatic ß cells to produce insulin, but the molecular mechanisms leading to this failure remain unclear. Increasing evidence supports a role for protein kinase R-like endoplasmic reticulum kinase (PERK) in the development and function of healthy pancreatic ß cells. Previously, our group identified the adaptor protein Nck1 as a negative regulator of PERK. Indeed, we demonstrated that Nck1, by directly binding PERK autophosphorylated on Tyr561, limits PERK activation and signaling. Accordingly, we found that stable depletion of Nck1 in ß cells promotes PERK activation and signaling, increases insulin biosynthesis, and improves cell viability in response to diabetes-related stresses. Herein, we explored the therapeutic potential of abrogating the interaction between Nck and PERK to improve ß-cell function and survival. To do so, we designed and used a peptide containing the minimal PERK sequence involved in binding Nck1 conjugated to the cell-permeable protein transduction domain from the HIV protein TAT. In the current study, we confirm that the synthetic TAT-Tyr(P)561 phosphopeptide specifically binds the SH2 domain of Nck and prevents Nck interaction with PERK, thereby promoting basal PERK activation. Moreover, we report that treatment of ß cells with TAT-Tyr(P)561 inhibits glucolipotoxicity-induced apoptosis, whereas it enhances insulin production and secretion. Taken together, our results support the potential of sequestering Nck using a synthetic peptide to enhance basal PERK activation and create more robust ß cells.

PERK leads a hub dictating pancreatic ß cell homoeostasis.

In humans, the pathogenesis of diabetes is characterised by two major pancreatic ß cell defects: a reduction in ß cell mass and the failure of ß cells to produce enough insulin. Over the past two decades, multiple studies involving cell cultures, animal models and human subjects have established the importance of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) in the adaptive functional capacity of pancreatic ß cells during embryonic development and into adulthood. In this review, we will highlight major findings identifying PERK as a crucial player in ß cell physiology and in diabetes.