The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

Plant-microbe interactions in the apoplast: Communication at the plant cell wall.

The apoplast is a continuous plant compartment that connects cells between tissues and organs and is one of the first sites of interaction between plants and microbes. The plant cell wall occupies most of the apoplast and is composed of polysaccharides and associated proteins and ions. This dynamic part of the cell constitutes an essential physical barrier and a source of nutrients for the microbe. At the same time, the plant cell wall serves important functions in the interkingdom detection, recognition, and response to other organisms. Thus, both plant and microbe modify the plant cell wall and its environment in versatile ways to benefit from the interaction. We discuss here crucial processes occurring at the plant cell wall during the contact and communication between microbe and plant. Finally, we argue that these local and dynamic changes need to be considered to fully understand plant-microbe interactions.

Trend and Seasonality of Hip Fractures in Catalonia, Spain: Exploring the Influence of Climate.

To describe the secular trend and seasonality of the incidence of hip fracture (HF) and its relationship with climatic variables during the period 2010-2019 in Catalonia in people aged ≥ 65 years. The results were analyzed by sex, age groups (65-74, 75-84, and ≥ 85), and types of fracture (extracapsular and intracapsular). Data on sex, age, type of fracture, year, and month of hospitalization of patients admitted with a diagnosis of HF between January 1, 2010 and December 31, 2019 were collected. Crude and standardized HF incidence (HFi) rates were obtained. Data on the monthly mean of climatological variables (temperature, insolation, icy days, rain, relative humidity, atmospheric pressure, and wind force) were obtained from the network of meteorological stations in Catalonia. Time series analytical statistics were used to identify trends and seasonality. Linear regression and a seasonal autoregressive integrated moving average (ARIMA) were used to analyze the relationship of each climatic parameter with fracture rates. In addition, generalized additive models were used to ascertain the best predictive model. The total number of HF episodes was 90,149 (74.1% in women and 25.9% in men). The total number of HFs increased by 6.4% between 2010 and 2019. The median age (SD) was 84.5 (7.14) and 54% of patients were ≥ 85 years of age. Extracapsular fractures were the most common (55%). The standardized incidence rates decreased from 728.1/100,000 (95% CI 738.6-769.3) to 624.5/100,000 (95% CI 648.7-677.0), which represents a decrease of 14.2% (p < 0.05). The decline was greater at older ages. There were seasonal variations, with higher incidences in autumn (27.2%) and winter (25.7%) and lower rates in summer (23.5%) and spring (23.6%). Seasonality was more pronounced in elderly people and men. In the bivariate regression analysis, high temperatures and greater insolation were negatively associated with the HF rate, while the number of icy days, rainy days, and high relative humidity were associated with a higher incidence of fractures in all age groups and sexes. In the regression analysis using the seasonal ARIMA model, only insolation had a consistently significant association with overall HFi, after adjusting by trend and other climatic parameters. While the global number of HFs grew in Catalonia due to increases in the elderly population, the standardized HF rate decreased during the years 2010-2019. There was a seasonal trend, with predominance in the cold months and correlations with climatic parameters, especially with insolation.

Pathogen-induced pH changes regulate the growth-defense balance in plants.

Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall-related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation-based proton pump activation in response to the fungus Fusarium oxysporum immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. In addition, pH seems to influence cellulose structure. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth-defense balance.

The importance of CXCR4 expression in tumor stroma as a potential biomarker in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the main causes of cancer mortality in the world. A characteristic feature of this cancer is that a large part of the tumor volume is composed of a stroma with different cells and factors. Among these, we can highlight the cytokines, which perform their function through binding to their receptors. Given the impact of the CXCR4 receptor in the interactions between tumor cells and their microenvironment and its involvement in important signaling pathways in cancer, it is proposed as a very promising prognostic biomarker and as a goal for new targeted therapies. Numerous studies analyze the expression of CXCR4 but we suggest focusing on the expression of CXCR4 in the stroma. METHODS: Expression of CXCR4 in specimens from 33 patients with PDAC was evaluated by immunohistochemistry techniques and matched with clinicopathological parameters, overall and disease-free survival rates. RESULTS: The percentage of stroma was lower in non-tumor tissue (32.4 ± 5.2) than in tumor pancreatic tissue (67.4 ± 4.8), P-value = 0.001. The level of CXCR4 expression in stromal cells was diminished in non-tumor tissue (8.7 ± 4.6) and higher in tumor pancreatic tissue (23.5 ± 6.1), P-value = 0.022. No significant differences were identified in total cell count and inflammatory cells between non-tumor tissue and pancreatic tumor tissue. No association was observed between CXCR4 expression and any of the clinical or pathological data, overall and disease-free survival rates. Analyzing exclusively the stroma of tumor samples, the CXCR4 expression was associated with tumor differentiation, P-value = 0.05. CONCLUSIONS: In this study, we reflect the importance of CXCR4 expression in the stroma of patients diagnosed with PDAC. Our results revealed a high CXCR4 expression in the tumor stroma, which is related to a poor tumor differentiation. On the contrary, we could not find an association between CXCR4 expression and survival and the rest of the clinicopathological variables. Focusing the study on the CXCR4 expression in the tumor stroma could generate more robust results. Therefore, we consider it key to develop more studies to enlighten the role of this receptor in PDAC and its implication as a possible biomarker.

A primary cell wall cellulose-dependent defense mechanism against vascular pathogens revealed by time-resolved dual transcriptomics.

BACKGROUND: Cell walls (CWs) are protein-rich polysaccharide matrices essential for plant growth and environmental acclimation. The CW constitutes the first physical barrier as well as a primary source of nutrients for microbes interacting with plants, such as the vascular pathogen Fusarium oxysporum (Fo). Fo colonizes roots, advancing through the plant primary CWs towards the vasculature, where it grows causing devastation in many crops. The pathogenicity of Fo and other vascular microbes relies on their capacity to reach and colonize the xylem. However, little is known about the root-microbe interaction before the pathogen reaches the vasculature and the role of the plant CW during this process. RESULTS: Using the pathosystem Arabidopsis-Fo5176, we show dynamic transcriptional changes in both fungus and root during their interaction. One of the earliest plant responses to Fo5176 was the downregulation of primary CW synthesis genes. We observed enhanced resistance to Fo5176 in Arabidopsis mutants impaired in primary CW cellulose synthesis. We confirmed that Arabidopsis roots deposit lignin in response to Fo5176 infection, but we show that lignin-deficient mutants were as susceptible as wildtype plants to Fo5176. Genetic impairment of jasmonic acid biosynthesis and signaling did not alter Arabidopsis response to Fo5176, whereas impairment of ethylene signaling did increase vasculature colonization by Fo5176. Abolishing ethylene signaling attenuated the observed resistance while maintaining the dwarfism observed in primary CW cellulose-deficient mutants. CONCLUSIONS: Our study provides significant insights on the dynamic root-vascular pathogen interaction at the transcriptome level and the vital role of primary CW cellulose during defense response to these pathogens. These findings represent an essential resource for the generation of plant resistance to Fo that can be transferred to other vascular pathosystems.

Educating EVs to Improve Bone Regeneration: Getting Closer to the Clinic.

The incidence of bone-related disorders is continuously growing as the aging of the population in developing countries continues to increase. Although therapeutic interventions for bone regeneration exist, their effectiveness is questioned, especially under certain circumstances, such as critical size defects. This gap of curative options has led to the search for new and more effective therapeutic approaches for bone regeneration; among them, the possibility of using extracellular vesicles (EVs) is gaining ground. EVs are secreted, biocompatible, nano-sized vesicles that play a pivotal role as messengers between donor and target cells, mediated by their specific cargo. Evidence shows that bone-relevant cells secrete osteoanabolic EVs, whose functionality can be further improved by several strategies. This, together with the low immunogenicity of EVs and their storage advantages, make them attractive candidates for clinical prospects in bone regeneration. However, before EVs reach clinical translation, a number of concerns should be addressed. Unraveling the EVs' mode of action in bone regeneration is one of them; the molecular mediators driving their osteoanabolic effects in acceptor cells are now beginning to be uncovered. Increasing the functional and bone targeting abilities of EVs are also matters of intense research. Here, we summarize the cell sources offering osteoanabolic EVs, and the current knowledge about the molecular cargos that mediate bone regeneration. Moreover, we discuss strategies under development to improve the osteoanabolic and bone-targeting potential of EVs.

Murine Animal Models in Osteogenesis Imperfecta: The Quest for Improving the Quality of Life.

Osteogenesis imperfecta is a rare genetic disorder characterized by bone fragility, due to alterations in the type I collagen molecule. It is a very heterogeneous disease, both genetically and phenotypically, with a high variability of clinical phenotypes, ranging from mild to severe forms, the most extreme cases being perinatal lethal. There is no curative treatment for OI, and so great efforts are being made in order to develop effective therapies. In these attempts, the in vivo preclinical studies are of paramount importance; therefore, serious analysis is required to choose the right murine OI model able to emulate as closely as possible the disease of the target OI population. In this review, we summarize the features of OI murine models that have been used for preclinical studies until today, together with recently developed new murine models. The bone parameters that are usually evaluated in order to determine the relevance of new developing therapies are exposed, and finally, current and innovative therapeutic strategies attempts considered in murine OI models, along with their mechanism of action, are reviewed. This review aims to summarize the in vivo studies developed in murine models available in the field of OI to date, in order to help the scientific community choose the most accurate OI murine model when developing new therapeutic strategies capable of improving the quality of life.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

Adhesion G protein-coupled receptor, ELTD1, is a potential therapeutic target for retinoblastoma migration and invasion.

BACKGROUND: Prognosis for pediatric metastatic Retinoblastoma (Rb) is poor and current therapies are limited by high systemic toxicity rates and insufficient therapeutic efficacy for metastatic Rb. Tumor dissemination to the brain is promoted by the heterogeneous adhesive and invasive properties of Rb cells within the tumor. In this study we evaluate, for the first time, the expression, and roles of the ELTD1 and GPR125 adhesion G protein-coupled receptors (GPCRs) in Rb cell migration, viability and invasion. METHODS: We characterized the RNA expression of adhesion-GPCRs in 64 Rb tumors compared to 11 fetal retinas using the database from the Childhood Solid Tumor Network from St Jude Children's Research Hospital. The role of ELTD1 and GPR125 in Rb were investigated ex vivo by microarray analysis, in vitro by cell viability, Western blot and migration assays, in addition to imaging of the subcellular localization of the GPCRs. To elucidate their role in vivo we utilized siRNA technology in an established Rb orthotopic xenograft murine model. RESULTS: Our investigation demonstrates, for the first time, that ELTD1 but not GPR125, is significantly increased in Rb tumors compared to fetal retinas. We utilized established the Rb cell lines Y79 and Weri-Rb-1, which represent an aggressive, metastatic, and non-metastatic phenotype, respectively, for the in vitro analyses. The studies demonstrated that ELTD1 is enriched in Weri-Rb-1 cells, while GPR125 is enriched in Y79 cells. The measured differences extended to their subcellular localization as ELTD1 labeling displayed punctate clusters in cell-to-cell adhesion sites of Weri-Rb-1 cells, while GPR125 displayed a polarized distribution in Y79 cells. Lastly, we demonstrated the lack of both adhesion receptors does not affect Rb cell viability, yet inhibition of ELTD1 decreases Y79 cell migration in vitro and invasion in vivo. CONCLUSION: Taken together, our data suggest that ELTD1, is a potential target to prevent extraocular Rb. The results within establish ELTD1 as a potential therapeutic target for metastatic Rb.

Current development of alternative treatments for endothelial decompensation: Cell-based therapy.

Current treatment for corneal endothelial dysfunction consists in the replacement of corneal endothelium by keratoplasty. Owing to the scarcity of donor corneas and the increasing number of transplants, alternative treatments such as cell-based therapies are necessary. In this article, we highlight the biological aspects of the cornea and the corneal endothelium, as well as the context that surrounds the need for new alternatives to conventional keratoplasty. We then review some of those experimental treatments in more detail, focusing on the development of the in vitro and preclinical phases of two cell-based therapies: tissue-engineered endothelial keratoplasty (TE-EK) and cell injection. In the case of TE-EK graft construction, we analyse the current progress, considering all the requirements it must meet in order to be functional. Moreover, we discuss the inherent drawbacks of endothelial keratoplasties, which TE-EK grafts should overcome in order to make surgical intervention easier and to improve the outcomes of current endothelial keratoplasties. Finally, we analyse the development of preclinical trials and their limitations in terms of performing an optimal functional evaluation of cell-based therapy, and we conclude by discussing early clinical trials in humans.

Sucrose-induced Receptor Kinase 1 is Modulated by an Interacting Kinase with Short Extracellular Domain.

Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1, qsk1, and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins.

Cannabinoid Receptors and Ligands: Lessons from CNS Disorders and the Quest for Novel Treatment Venues.

The potential use of cannabinoids for therapeutic purposes is at the forefront of cannabinoid research which aims to develop innovative strategies to prevent, manage and treat a broad spectrum of human diseases. This chapter briefly reviews the pivotal role of the endocannabinoid system in modulating the central nervous system and its roles on neurodegenerative diseases and brain disorders. Ligand-induced modulation of cannabinoid 1 and 2 receptors to modulate immune response, decrease neurodegeneration and pain are aspects that are also discussed.

Cutting Edge Endogenous Promoting and Exogenous Driven Strategies for Bone Regeneration.

Bone damage leading to bone loss can arise from a wide range of causes, including those intrinsic to individuals such as infections or diseases with metabolic (diabetes), genetic (osteogenesis imperfecta), and/or age-related (osteoporosis) etiology, or extrinsic ones coming from external insults such as trauma or surgery. Although bone tissue has an intrinsic capacity of self-repair, large bone defects often require anabolic treatments targeting bone formation process and/or bone grafts, aiming to restore bone loss. The current bone surrogates used for clinical purposes are autologous, allogeneic, or xenogeneic bone grafts, which although effective imply a number of limitations: the need to remove bone from another location in the case of autologous transplants and the possibility of an immune rejection when using allogeneic or xenogeneic grafts. To overcome these limitations, cutting edge therapies for skeletal regeneration of bone defects are currently under extensive research with promising results; such as those boosting endogenous bone regeneration, by the stimulation of host cells, or the ones driven exogenously with scaffolds, biomolecules, and mesenchymal stem cells as key players of bone healing process.

Generation of Mesenchymal Cell Lines Derived from Aged Donors.

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1.

The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.

Multi-pathway human exposure risk assessment using Bayesian modeling at the historically largest mercury mining district.

The largest mercury (Hg) mining district in the world is located in Almadén (Spain), with well-known environmental impacts in the surrounding ecosystem. However, the impact of mercury on the health of the inhabitants of this area has not been documented accordingly. This study aims to carry out a probabilistic human health risk assessment using Bayesian modeling to estimate the non-carcinogenic risk related to Hg through multiple exposure pathways. Samples of vegetables, wild mushrooms, fish, soil, water, and air were analyzed, and adult residents were randomly surveyed to adjust the risk models to the specific population data. On the one hand, the results for the non-carcinogenic risk based on Hazard Quotient (HQ) showed unacceptable risk levels through ingestion of Hg-contaminated vegetables and fish, with HQ values 20 and 3 times higher, respectively, than the safe exposure threshold of 1 for the 97.5th percentile. On the other hand, ingestion of mushrooms, dermal contact with soil, ingestion of water, dermal contact with water and inhalation of air, were below the safety limit for the 97.5th percentile, and did not represent a risk to the health of residents. In addition, the probabilistic approach was compared with the conservative deterministic approach, and similar results were obtained. This is the first study conducted in Almadén, which clearly reveals the high levels of human health risk to which the population is exposed due to the legacy of two millennia of Hg mining.