Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.

CD34 Protein: Its expression and function in inflammation.

CD34 has spear-headed the field of basic research and clinical transplantation since the first reports of its expression on hematopoietic stem cells (HSCs). Expressed in mice, humans, rats and other species, CD34 has been used for more than 40 years as a hematopoietic stem and progenitor cell marker. It was later found that muscle satellite cells and epidermal precursors can also be identified with the aid of CD34. Despite the usefulness of CD34 as a marker of HSCs, its overall purpose in animal physiology has remained unclear. This review recaptures CD34 structure, evolutionary conservation, proposed functions, and role in lung inflammation, to describe current research findings and to provide guidance for future studies on CD34.

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

Effects of Different Irrigation Regimes, Nitrogen Levels and Storage Conditions on Volatiles of 'Gala' Apple.

With a characteristic flavour and aroma, "Maçã de Alcobaça" are apples produced in the western region of the mainland of Portugal. Given the known influence of pre-harvest cultural techniques and post-harvest conservation methods on fruit quality, this work evaluated the effect of cultural factors and conservation methods on the volatile profile of 'Gala' apples. Tests were carried out during four seasons (2018 to 2021) in two 'Gala' apple orchards (F and S) maintained with different irrigation rates and nitrogen fertilisation [normal irrigation and normal nitrogen (Control, NINN), normal irrigation and excess nitrogen (NIEN), excess irrigation and normal nitrogen (EINN), excess irrigation and excess nitrogen (EIEN)], and under three storage conditions [Controlled Atmosphere + 1-methylcyclopropene (CA+1-MCP), Dynamic Controlled Atmosphere (DCA) and DCA+1-MCP]. The intact fruit volatiles were isolated by headspace solid-phase microextraction (HS-SPME) and analysed by Gas Chromatography with Flame Ionisation Detection and Gas Chromatography-Mass Spectrometry at harvest (T0) and after 8 months of storage (T8). HS-SPME volatiles from 'Gala' apples, obtained at T0 in control conditions, were characterised by trans,trans-α-farnesene dominance (36-69%), followed by hexyl acetate (5-23%) and hexyl hexanoate (3-9%). The four irrigation and nitrogen treatments did not evidence main changes in the apple volatile profile. Instead, storage conditions changed the ratio between compounds; previously undetected compounds attained high percentages and decreased the intensity of the dominant compounds in the control conditions. Although all storage conditions tested changed the volatile profile and emanation intensity, the effect was more accentuated in storage for 8 months with DCA+1-MCP.

IR780 loaded gelatin-PEG coated gold core silica shell nanorods for cancer-targeted photothermal/photodynamic therapy.

Gold core silica shell (AuMSS) nanorods present excellent physicochemical properties that allow their application as photothermal and drug delivery agents. Herein, AuMSS nanorods were dual-functionalized with Polyethylene glycol methyl ether (PEG-CH3 ) and Gelatin (GEL) to enhance both the colloidal stability and uptake by HeLa cancer cells. Additionally, the AuMSS nanorods were combined for the first time with IR780 (a heptamethine cyanine molecule) and its photothermal and photodynamic capacities were determined. The obtained results reveal that the encapsulation of IR780 (65 µg per AuMSS mg) increases the photothermal conversion efficiency of AuMSS nanorods by 10%, and this enhanced heat generation was maintained even after three irradiation cycles with a NIR (808 nm) laser. Moreover, the IR780-loaded AuMSS/T-PEG-CH3 /T-GEL presented ≈2-times higher uptake in HeLa cells, when compared to the non-coated counterparts, and successfully mediated the light-triggered generation of reactive oxygen species. Overall, the combination of photodynamic and photothermal therapy mediated by IR780-loaded AuMSS/T-PEG-CH3 /T-GEL nanorods effectively promoted the ablation of HeLa cancer cells.

Patterns of adult and youth inpatient admissions before and after the COVID-19 pandemic in a psychiatric ward: an observational study.

BACKGROUND: The current pandemic situation led to deep changes not only in social relationships, but also on clinical care and access to healthcare facilities. The authors aimed to understand whether this context affected the main characteristics of psychiatric hospitalizations, measured by admissions in a portuguese acute psychiatric ward. METHODS: Retrospective data collection of all patients admitted in Centro Hospitalar Psiquiátrico de Lisboa, in two different time periods: pre-COVID-19 (march 11th, 2019 to march 10th, 2020, n = 1845) and COVID-19 (march 11th, 2020 to march 10th, 2021, n = 1278); comparing the number of total admissions, compulsory ones, age, sex, median days of admission, median days to readmission and diagnosis at discharge. Distribution of disorders in both groups, as well as in compulsory admissions were also evaluated. The same comparisons were evaluated in the 15-25-year-old patient group. RESULTS: Statistical significance was found regarding total number of admissions (reduction of around 30.7%), as well as compulsory ones (reduction of 14%, although the relative frequency had increased), days of admission and distribution between admissions (with lower reductions regarding dementias, schizophrenia and affective disorders, while substance use disorders and intellectual disabilities presented reductions of over 50%), with no differences between gender, median age, previous admissions or readmissions. Distribution between compulsory admissions did not present differences before and during COVID periods. For patients between 15 and 25 years of age, statistical significance was found regarding total number of compulsory ones (94 versus 44, p-value = 0.01), and in all groups of diagnoses (all with p-value = 0.001). CONCLUSIONS: While there was a general reduction in the overall number of patients admitted, in the most severe conditions (compulsory admissions and schizophrenia diagnosis) did not present such a reduction. Difficulties in social, clinical and family networks can explain the reduction of the time to readmission. Future research could show whether there is a rebound increase number of admissions in the other diagnoses. TRIAL REGISTRATION: The current study was approved by the hospital's scientific and ethics committees (CCP number 0060/2021 and CES 09/2021).

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Bio-Based Sensors for Smart Food Packaging-Current Applications and Future Trends.

Intelligent food packaging is emerging as a novel technology, capable of monitoring the quality and safety of food during its shelf-life time. This technology makes use of indicators and sensors that are applied in the packaging and that detect changes in physiological variations of the foodstuffs (due to microbial and chemical degradation). These indicators usually provide information, e.g., on the degree of freshness of the product packed, through a color change, which is easily identified, either by the food distributor and the consumer. However, most of the indicators that are currently used are non-renewable and non-biodegradable synthetic materials. Because there is an imperative need to improve food packaging sustainability, choice of sensors should also reflect this requirement. Therefore, this work aims to revise the latest information on bio-based sensors, based on compounds obtained from natural extracts, that can, in association with biopolymers, act as intelligent or smart food packaging. Its application into several perishable foods is summarized. It is clear that bioactive extracts, e.g., anthocyanins, obtained from a variety of sources, including by-products of the food industry, present a substantial potential to act as bio-sensors. Yet, there are still some limitations that need to be surpassed before this technology reaches a mature commercial stage.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

Overview of stimuli-responsive mesoporous organosilica nanocarriers for drug delivery.

The application of nanomaterials is regarded nowadays as a highly promising approach for overcoming the limitations of the currently available cancer treatments, contributing for the creation of more effective, precise, and safer therapies. In the last years, organosilica nanoparticles arisen as alternatives to the most common mesoporous silica nanoparticles. The organosilica nanoparticles combine the advantages of the mesoporous silica, such as structural stability and mesoporous structure, with the increased biocompatibility and biodegradability of organic materials. Therefore, the variety of organic bridges that can be incorporated into the silica matrix allowed the development of new and exciting compositions, properties, and functions for improving the therapeutic effectiveness of the anticancer nanomedicines. In this review, the strategies that have been explored to create stimuli-responsive organosilica-based drug delivery systems are highlighted, describing the practical approaches and mechanisms controlling the drug release. Additionally, the organosilica nanoparticles surface modifications aimed for increasing the blood circulation time and the tumor targeting are also described.

Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids.

3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration.

Multiscale land use impacts on water quality: Assessment, planning, and future perspectives in Brazil.

Brazil contains the largest volume of freshwater of any nation in the world; however, this essential natural resource is threatened by rapid increases in water consumption and water quality degradation, mainly as a result of anthropogenic pressures. Declining water quality has become an increasingly more significant global concern as economic activities and human populations expand and climate change markedly alters hydrological cycles. Changes in land-use/land-cover (LULC) pattern have been recognized as a major driver of water quality degradation, however different LULC types and intensities affect water quality in different ways. In addition, the relationships between LULC and water quality may differ for different spatial and temporal scales. The increase in deforestation, agricultural expansion, and urban sprawl in Brazil highlights the need for water quality protection to ensure immediate human needs and to maintain the quality of water supplies in the long-term. Thus, this manuscript provides an overview of the relationships between LULC and water quality in Brazil, aiming at understanding the effects of different LULC types on water quality, how spatial and temporal scales contribute to these effects, and how such knowledge can improve watershed management and future projections. In general, agriculture and urban areas are the main LULCs responsible for water quality degradation in Brazil. However, although representing a small percentage of the territory, mining has a high impact on water quality. Water quality variables respond differently at different spatial scales, so spatial extent is an important aspect to be considered in studies and management. LULC impacts on water quality also vary seasonally and lag effects mean they take time to occur. Forest restoration can improve water quality and multicriteria evaluation has been applied to identify priority areas for forest restoration and conservation aiming at protecting water quality, but both need further exploration. Watershed modelling has been applied to simulate future impacts of LULC change on water quality, but data availability must be improved to increase the number, locations and duration of studies. Because of the international nature of watersheds and the consistent relationships between land use and water quality in Brazil, we believe our results will also aid water management in other countries.

Right Ventricular Outflow Tract Pseudo Aneurysm After Reconstruction With Small Intestinal Submucosal Cormatrix Patch A Word Of Caution.

Tetralogy of Fallot is very prevalent with correction techniques well standardized. Whenever infundibular incisions are needed, patch reconstruction seems mandatory. Recently, the small intestinal submucosal (CorMatrix, MAC's Medical Group,) patch was introduced, with optimal results in pre-clinical studies. However, clinical results do not match its pre-clinical promises, particularly when used in right ventricular outflow tract and pulmonary artery reconstructions. We describe a case of Tetralogy of Fallot for which small intestinal submucosal (CorMatrix) patch was used as a trans-annular patch, with development of a massive pseudo-aneurysm.

Arterial Switch Operation Variables Predicting Reoperation.

OBJECTIVES: Jatene surgery or arterial switch is performed at our institution since the late nineties. We reviewed our results to identify the main causes of reoperation and, more importantly, to determine what variables predict the need for reoperation. METHODS: In this retrospective analysis were included all the 91 patients with d-TGA who underwent an arterial switch operation at our institution between 1995 and 2016. RESULTS: Mean follow-up was 10 years (range 5-25 years). Seventy-one percent of patients had simple TGA and 29% had complex TGA. The need of reoperation was 21% (n=19 patients). Right ventricle outflow tract obstruction was the main indication for reoperation (58%). The overall mortality was 9.9%. The gender (P= 0.8), diagnosis (simple or complex TGA) (P= 0,5) or the existence of palliative surgeries (P=0.9) were unable to predict the need for reoperation. The presence of anomalous coronary pattern was the only variable reaching statistical significance (P < 0.05), both in univariate and multivariate analysis. CONCLUSIONS: In our series, the main indication for reoperation after arterial switch operation was right ventricle outflow tract obstruction and the only predictive variable was the presence of anomalous coronary pattern.

Comparative genome analysis of Phyllosticta citricarpa and Phyllosticta capitalensis, two fungi species that share the same host.

BACKGROUND: Citrus are among the most important crops in the world. However, there are many diseases that affect Citrus caused by different pathogens. Citrus also hosts many symbiotic microorganisms in a relationship that may be advantageous for both organisms. The fungi Phyllosticta citricarpa, responsible for citrus black spot, and Phyllosticta capitalensis, an endophytic species, are examples of closely related species with different behavior in citrus. Both species are always biologically associated and are morphologically very similar, and comparing their genomes could help understanding the different lifestyles. In this study, a comparison was carried to identify genetic differences that could help us to understand the biology of P. citricarpa and P. capitalensis. RESULTS: Drafts genomes were assembled with sizes close to 33 Mb for both fungi, carrying 15,206 and 14,797 coding sequences for P. citricarpa and P. capitalensis, respectively. Even though the functional categories of these coding sequences is similar, enrichment analysis showed that the pathogenic species presents growth and development genes that may be necessary for the pathogenicity of P. citricarpa. On the other hand, family expansion analyses showed the plasticity of the genome of these species. Particular families are expanded in the genome of an ancestor of P. capitalensis and a recent expansion can also be detected among this species. Additionally, evolution could be driven by environmental cues in P. citricarpa. CONCLUSIONS: This work demonstrated genomic differences between P. citricarpa and P. capitalensis. Although the idea that these differences could explain the different lifestyles of these fungi, we were not able to confirm this hypothesis. Genome evolution seems to be of real importance among the Phyllosticta isolates and it is leading to different biological characteristics of these species.

Microneedle-based delivery devices for cancer therapy: A review.

Macroscale delivery systems that can be locally implanted on the tumor tissue as well as avoid all the complications associated to the systemic delivery of therapeutics have captured researchers' attention, in recent years. Particularly, the microneedle-based devices can be used to efficiently deliver both small and macro-molecules, like chemotherapeutics, proteins, and genetic material, along with nanoparticle-based anticancer therapies. Such capacity prompted the application of microneedle devices for the development of new anticancer vaccines that can permeate the tumor tissue and simultaneously improve the effectiveness of therapeutic agents. Based on the promising results demonstrated by the microneedle systems in the local administration of anticancer therapeutics, this review summarizes the different microneedle formulations developed up to now aimed for application on cancer therapy (mphasizing those produced with polymers). Additionally, the microneedles' general properties, type of therapeutic approach and its main advantages are also highlighted.

Comparison of two pharmacological prophylaxis strategies for venous thromboembolism in spinal cord injury patients: a retrospective study.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To compare the cost and incidence of venous thromboembolism (VTE) and bleeding between two different VTE pharmacological prophylaxis strategies in individuals with spinal cord injury: one based on motor impairment (Protocol 1) and the other based on time from the lesion and presence of associated risk factors for VTE (Protocol 2). SETTING: A tertiary rehabilitation hospital in Brazil. METHODS: We retrospectively reviewed a total of 1475 charts of individual admissions: 814 individuals received pharmacological prophylaxis according to Protocol 1 and 661 according to protocol 2. These cohorts were compared with respect to age, time and level of injury, length of stay, AIS classification, type of injury, and occurrence of VTE and major bleeding. The number of prescribed doses of enoxaparin and expenditures associated with enoxaparin during each period were evaluated. RESULTS: The median lesion time was 3 years. The risk-based strategy drastically reduced the average monthly use of enoxaparin by 75% and the 12-month enoxaparin expenditure by $119,930.33, without increasing the risk of VTE. The incidence density of thromboembolic events was 0.55/10,000 patient-days, and all events occurred in individuals receiving prophylaxis according to Protocol 1. CONCLUSIONS: Time from injury and risk of VTE-based protocol for indication of pharmacological prophylaxis drastically reduced costs. No difference in occurrence of VTE was observed.

Multiple Wire-Mesh Sensors Applied to the Characterization of Two-Phase Flow inside a Cyclonic Flow Distribution System.

Wire-mesh sensors are used to determine the phase fraction of gas⁻liquid two-phase flow in many industrial applications. In this paper, we report the use of the sensor to study the flow behavior inside an offshore oil and gas industry device for subsea phase separation. The study focused on the behavior of gas⁻liquid slug flow inside a flow distribution device with four outlets, which is part of the subsea phase separator system. The void fraction profile and the flow symmetry across the outlets were investigated using tomographic wire-mesh sensors and a camera. Results showed an ascendant liquid film in the cyclonic chamber with the gas phase at the center of the pipe generating a symmetrical flow. Dispersed bubbles coalesced into a gas vortex due to the centrifugal force inside the cyclonic chamber. The behavior favored the separation of smaller bubbles from the liquid bulk, which was an important parameter for gas-liquid separator sizing. The void fraction analysis of the outlets showed an even flow distribution with less than 10% difference, which was a satisfactorily result that may contribute to a reduction on the subsea gas⁻liquid separators size. From the outcomes of this study, detailed information regarding this type of flow distribution system was extracted. Thereby, wire-mesh sensors were successfully applied to investigate a new type of equipment for the offshore oil and gas industry.

Assessing the ecological status of small Mediterranean rivers using benthic macroinvertebrates and macrophytes as indicators.

Rivers are amongst the most threatened ecosystems in Europe. To prevent further degradation and to improve their ecological status, effective mitigation and restoration actions are needed. Those actions are primarily based on the precision of the ecological assessment results. This study aims to assess the ecological status of two small Mediterranean rivers-the Âncora (AR) and the Ferreira (FR) rivers-through the analysis of biological (benthic macroinvertebrates and macrophytes), physico-chemical and hydromorphological quality elements. Specific objectives were to analyse variations of biotic and abiotic parameters amongst rivers, and amongst seasons and sites within each river, to confirm adequate temporal windows to develop the monitoring surveys. Results showed that only one AR site achieved a good ecological status in spring and summer, while the other sites presented a moderate ecological status in all seasons. FR sites had a moderate to bad ecological status in all seasons. Both rivers showed high levels of nutrients, particularly during spring and summer, and were found quite altered in terms of floristic composition of the riparian communities. The riparian forest and the forbs fringe were dominated by several alien woody species and nitrophilous communities, respectively. Based on a multidisciplinary approach, this study provides an in-depth description of the ecological status of two small Mediterranean rivers located in sites of European interest, as well as a sound basis for the management of the aquatic environments. Mitigation of diffuse pollution and restoration of the riparian zones are a priority to improve their ecological status.

Assessing the ecological status of fluvial ecosystems employing a macroinvertebrate multi-taxon and multi-biomarker approach.

Biomarkers are recognised sensitive early-warning tools of biological effects in aquatic organisms. In this scope, the main aim of this study was to investigate the potential usefulness of a battery of biomarkers, evaluated in different benthic macroinvertebrate taxa, to discriminate aquatic ecosystems with different levels of ecological status and to provide further clues supporting environmental management. The study took place during the autumn of 2013 and the spring and summer of 2014, and the study cases were two Mediterranean rivers (Âncora and Ferreira rivers), differing in their ecological status. The biomarkers determined are widely employed and comprise a large set of biochemical responses: the activity of enzymes (cholinesterases, glutathione S-transferases, catalase and lactate dehydrogenase) and the levels of lipid peroxidation. They were assessed seasonally and in different macroinvertebrate taxa. Thirteen water physico-chemical parameters were also seasonally determined, and the concentration of seven organophosphorus pesticides and the percentage of 32 trace metals in sediments were determined in the spring. This is particularly useful for water management. Based on this, authorities can take actions to prevent further damage in the ecological status. Multivariate analyses showed distinct patterns of biological response for the Calopteryx spp., Chironomidae and Baetis spp. taxa. Calopteryx spp. and Chironomidae, in particular, showed distinct response patterns for the two rivers, which were fairly stable across seasons. This study sets the foundations for future cost-effective biomonitoring campaigns in Mediterranean rivers, allowing to establish historical data important to understand ecosystem evolution, as well as baseline levels of diagnostic biomarkers in informative macroinvertebrate taxa.