Functional characterization of a novel PRRT2 variant found in a Portuguese patient with hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare autosomal-dominant form of migraine with aura. Three disease-causing genes have been identified for FHM: CACNA1A, ATP1A2 and SCN1A. However, not all families are linked to one of these three genes.PRRT2 variants were also commonly associated with HM symptoms; therefore, PRRT2 is hypothesized as the fourth gene causing FHM. PRRT2 plays an important role in neuronal migration, spinogenesis, and synapse mechanisms during development and calcium-dependent neurotransmitter release. We performed exome sequencing to unravel the genetic cause of migraine in one family, and a novel PRRT2 variant (c.938C > T;p.Ala313Val) was identified with further functional studies to confirm its pathogenicity. PRRT2-A313V reduced protein stability, led to protein premature degradation by the proteasome and altered the subcellular localization of PRRT2 from the plasma membrane (PM) to the cytoplasm. We identified and characterized for the first time in a Portuguese patient, a novel heterozygous missense variant in PRRT2 associated with HM symptoms. We suggest that PRRT2 should be included in the diagnosis of HM.

Evaluation of urinary cysteinyl leukotrienes as biomarkers of severity and putative therapeutic targets in COVID-19 patients.

BACKGROUND: Cysteinyl leukotrienes (CysLT) are potent inflammation-promoting mediators, but remain scarcely explored in COVID-19. We evaluated urinary CysLT (U-CysLT) relationship with disease severity and their usefulness for prognostication in hospitalized COVID-19 patients. The impact on U-CysLT of veno-venous extracorporeal membrane oxygenation (VV-ECMO) and of comorbidities such as hypertension and obesity was also assessed. METHODS: Blood and spot urine were collected in "severe" (n = 26), "critically ill" (n = 17) and "critically ill on VV-ECMO" (n = 17) patients with COVID-19 at days 1-2 (admission), 3-4, 5-8 and weekly thereafter, and in controls (n = 23) at a single time point. U-CysLT were measured by ELISA. Routine markers, prognostic scores and outcomes were also evaluated. RESULTS: U-CysLT did not differ between groups at admission, but significantly increased along hospitalization only in critical groups, being markedly higher in VV-ECMO patients, especially in hypertensives. U-CysLT values during the first week were positively associated with ICU and total hospital length of stay in critical groups and showed acceptable area under curve (AUC) for prediction of 30-day mortality (AUC: 0.734, p = 0.001) among all patients. CONCLUSIONS: U-CysLT increase during hospitalization in critical COVID-19 patients, especially in hypertensives on VV-ECMO. U-CysLT association with severe outcomes suggests their usefulness for prognostication and as therapeutic targets.

Non-coding variants in VAMP2 and SNAP25 affect gene expression: potential implications in migraine susceptibility.

Migraine is a common and complex neurological disease potentially caused by a polygenic interaction of multiple gene variants. Many genes associated with migraine are involved in pathways controlling the synaptic function and neurotransmitters release. However, the molecular mechanisms underpinning migraine need to be further explored.Recent studies raised the possibility that migraine may arise from the effect of regulatory non-coding variants. In this study, we explored the effect of candidate non-coding variants potentially associated with migraine and predicted to lie within regulatory elements: VAMP2_rs1150, SNAP25_rs2327264, and STX1A_rs6951030. The involvement of these genes, which are constituents of the SNARE complex involved in membrane fusion and neurotransmitter release, underscores their significance in migraine pathogenesis. Our reporter gene assays confirmed the impact of at least two of these non-coding variants. VAMP2 and SNAP25 risk alleles were associated with a decrease and increase in gene expression, respectively, while STX1A risk allele showed a tendency to reduce luciferase activity in neuronal-like cells. Therefore, the VAMP2_rs1150 and SNAP25_rs2327264 non-coding variants affect gene expression, which may have implications in migraine susceptibility. Based on previous in silico analysis, it is plausible that these variants influence the binding of regulators, such as transcription factors and micro-RNAs. Still, further studies exploring these mechanisms would be important to shed light on the association between SNAREs dysregulation and migraine susceptibility.

A review of migraine genetics: gathering genomic and transcriptomic factors.

Migraine is a common and complex neurologic disorder that affects approximately 15-18% of the general population. Although the cause of migraine is unknown, some genetic studies have focused on unravelling rare and common variants underlying the pathophysiological mechanisms of this disorder. This review covers the advances in the last decade on migraine genetics, throughout the history of genetic methodologies used, including recent application of next-generation sequencing techniques. A thorough review of the literature interweaves the genomic and transcriptomic factors that will allow a better understanding of the mechanisms underlying migraine pathophysiology, concluding with the clinical utility landscape of genetic information and future consideration to creating a new frontier toward advancing the field of personalized medicine.

Forest restoration methods, seasonality, and penetration resistance does not influence aboveground biomass stock on mining tailings in Mariana, Brazil.

The restoration methods applied on the areas affected by the Fundão tailings dam collapse have a high priority in Mariana region. We evaluated the effect of different restoration methods and site preparation techniques, depth and seasonality on penetration resistance of tailings, and how these predictors affect tree aboveground biomass in areas affected by the Fundão dam collapse in Mariana, Brazil. No significant differences in penetration resistance and aboveground biomass between treatments were observed, but significant differences were observed between seasonal periods. The main univariate model explained the significant effects of depth and seasonality, mainly by a negatively wet effect on penetration resistance. According to the best models (univariate and multivariate) were those that had depth as a predictor. This study showed how penetration resistance can be an indicator to select the best period for restoration process in areas affected by the collapse of the Fundão dam, but no limit to the aboveground biomass recovery on tailing.

Inflammatory Bowel Disease - Non-biological treatment.

Nowadays, non-biological treatments remain valuable approaches among the therapeutic armamentarium of inflammatory bowel disease (IBD). Mesalamine is the core treatment of mild­to­moderate ulcerative colitis (UC) and corticosteroids are crucial for the induction of remission of moderate­to­severe flares in both UC and Crohn's disease (CD). Even approaches as cyclosporine, tacrolimus, azathioprine, methotrexate, and surgery still have a nuclear position as strategies to induce and/or maintain remission in IBD. Due to their particularities and to the accumulated evidence, each of these strategies conquered peculiar roles in the overall IBD strategy, all of them contributing to better outcomes. This review emphasizes the particular roles that non-biological treatments gained over time: recent mesalamine formulations to increase adhesion rates, higher doses of 5-ASA for high-risk patients, MMX technology to improve drug release and attain higher bowel concentrations, cyclosporine as a bridge to vedolizumab, tacrolimus as a potential alternative to thiopurines or infliximab, azathioprine in combination therapy with infliximab and dubious in monotherapy, and surgery as a mean to a "better end".

Spatiotemporal-based clusters as a method for dengue surveillance.

OBJECTIVES: To develop and demonstrate the use of a new method for epidemiological surveillance of dengue. METHODS: This was a retrospective cohort study using data from the Health Department of São José do Rio Preto (São Paulo, Brazil). The geographical coordinates were obtained using QGIS™ (Creative Commons Corporation, Mountain View, California, United States), based on patient addresses in the dengue notification system of the Government of Brazil. SaTScan™ (Martin Kulldorff, Boston, Massachusetts, United States) was then used to create a space-time scan analysis to find statistically significant clusters of dengue. These results were plotted and visualized using Google Earth™ mapping service (Google Incorporated, Mountain View, California, United States). RESULTS: More clusters were detected when the maximum number of households per cluster was set to 10% (11 statistically significant clusters) rather than 50% (8 statistically significant clusters). The cluster radius varied from 0.18 - 2.04 km and the period of time varied from 6 days - 6 months. The infection rate was more than 0.5 cases/household. CONCLUSIONS: When using SaTScan for space-time analysis of dengue cases, the maximum number of households per cluster should be set to 10%. This methodology may be useful to optimizing dengue surveillance systems, especially in countries where resources are scarce and government programs have not had much success controlling the disease.

Hereditary transthyretin amyloidosis: a myriad of factors that influence phenotypic variability.

Hereditary transthyretin-related amyloidosis (ATTRv amyloidosis) is a rare and progressively debilitating disease characterized by the deposition of transthyretin (TTR) amyloid fibrils in various organs and tissues, most commonly in the heart and peripheral nerves. This pathological deposition can lead to significant organ dysfunction and, ultimately, organ failure. ATTRv amyloidosis exhibits a broad range of clinical presentations, from purely neurological symptoms to purely cardiac manifestations, as well as mixed phenotypes which result from both neurological and cardiac implications. This wide phenotypical spectrum realistically challenges disease diagnosis and prognosis, especially in individuals without or with an unknown family history. Multiple factors are thought to contribute to this variability, including genetic, epigenetic, and even environmental influences. Understanding these factors is crucial, as they can significantly affect disease expression and progression. This review aims to summarize each of these contributing factors, to help elucidate the current knowledge on the phenotypical variability of ATTRv amyloidosis.

Acute Fulminant Cerebral Edema in a Child With Suspected Meningoencephalitis.

Acute fulminant cerebral edema (AFCE) is a recently identified encephalitis type associated with significant morbimortality. Described as rare, limited data exists on its early detection and treatment. This paper describes a case of AFCE that progressed to unresponsive intracranial hypertension. A previously healthy four-year-old boy presented with fever, myalgias, and neurological symptoms. Diagnostic assessments showed cerebrospinal fluid abnormalities, and despite medical interventions, his condition deteriorated rapidly and developed severe cerebral edema and herniation within 24 hours. A decompressive craniectomy was attempted to decrease intracranial pressure, without success. This case emphasizes the urgency of early AFCE recognition and effective management strategies given its severe prognosis, aiming to improve understanding and spur further research.

Spontaneous reporting of adverse reactions associated with the COVID-19 vaccine in health care professionals: A descriptive observational study conducted in a Portuguese hospital.

Background: The coronavirus disease 2019 (COVID-19) was classified as a pandemic in March 2020 by the World Health Organization. The Pfizer-BioNTech COVID-19 vaccine was the first to be authorized in the European Union, based on data from phase 1, 2, and 3 clinical trials of limited duration. Concerns have been raised regarding the vaccine's safety profile. Some of the adverse drug reactions (ADRs) associated with vaccines may not have been identified during clinical trials. This study aimed to identify ADRs associated with the Pfizer-BioNTech vaccine in health care professionals at a Portuguese tertiary university hospital. Methods: The data used in this analysis consist of ADRs reported through a spontaneous notification system from vaccines administered between December 27, 2020, and January 31, 2021. ADRs were categorized according to the MedDRA terminology. Results: A total of 8,605 Pfizer-BioNTech vaccines were administered to 4568 health care professionals. ADRs were reported among 520 of the vaccines, with an incidence of 13.56% in women and 5.31% in men. The mean age of the population reporting ADRs was 41.52 years, with a standard deviation of 9.83 years. The most frequent ADRs were myalgia (n = 274), headache (n = 199), pyrexia (n = 164), injection site pain (n = 160), fatigue (n = 84), nausea (n = 81), chills (n = 65), lymphadenopathy (n = 64), and arthralgia (n = 53). Hypersensitivity reactions occurred in 15 health care professionals, with no anaphylactic reactions observed. A total of four Important Medical Events were observed, which consisted of two cases of syncope, one case of sudden hearing loss, and one case of transverse myelitis. Conclusion: The vaccine was well-tolerated among the study participants. Reactogenicity was greater after the second dose. The incidence of ADRs was higher in women and individuals aged between 40 to 49 years. Systemic adverse reactions were most frequently reported. Systematic monitoring of ADRs of COVID-19 vaccines in real-life context is essential for a more robust establishment of its safety profile.

Effects of a lycopene-layered double hydroxide composite administration in cells and lungs of adult mice.

Lycopene is a natural compound with one of the highest antioxidant activities. Its consumption is associated with lower risks in lung cancer and chronic obstructive pulmonary disease, for example. Experimentally, a murine model demonstrated the ingestion of lycopene, which reduced the damage in lungs caused by cigarette smoke. Since lycopene is highly hydrophobic, its formulations in supplements and preparations for laboratory assays are based on oils, additionally, bioavailavility is low. We developed a lycopene layered double hydroxide (Lyc-LDH) composite, which is capable of transporting lycopene aqueous media. Our objective was to evaluate the cytotoxicity of Lyc-LDH and the intra-cellular production of reactive oxygen species (ROS) in J774A.1 cells. Also, in vivo assays were conducted with 50 male C57BL/6 mice intranasally treated with Lyc-LDH 10 mg/kg (LG10), Lyc-LDH 25 mg/kg (LG25) and Lyc-LDH 50 mg/kg (LG50) during five days compared against a vehicle (VG) and control (CG) group. The blood, bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed. The results revealed that Lyc-LDH composite attenuated intracellular ROS production stimulated with lipopolysacharide. In BALF, the highest doses of Lyc-LDH (LG25 and LG50) promoted influx of macrophages, lymphocytes, neutrophils and eosinophils compared to CG and VG. Also, LG50 increased the levels of IL-6 and IL-13, and promoted the redox imbalance in the pulmonary tissue. On the contrary, low concentrations did not produce significative effects. In conclusion, our results suggest that intranasal administration of high concentrations of Lyc-LDH induces inflammation as well as redox status changes in the lungs of healthy mice, however, results with low concentrations open a promising way to study LDH composites as vehicles for intranasal administration of antioxidant coadjuvants.

The Role of Pain Inflexibility and Acceptance among Headache and Temporomandibular Disorders Patients.

Temporomandibular disorders (TMD) and headache are complex. This study aims to assess the association between TMD, headache, and psychological dimensions such as psychological inflexibility and pain acceptance. The sample consisted of 120 participants following a non-probabilistic convenience sampling strategy through a direct invitation to the patients attending our facilities and their relatives (n = 61 diagnosed with headache, n = 34 diagnosed with TMD-headache, n = 25 control group). Diagnostic Criteria for Temporomandibular Disorders (DC-TMD), International Classification of Headache Disorders (ICHD-3 beta version), Chronic Pain Acceptance Questionnaire (CPAQ-8), and Psychological Inflexibility in Pain Scale (PIPS) were used as assessment tools. One-way ANOVA, multiple regression analysis (MRA), and the Johnson-Neyman approach were run by IBM SPSS, version 27 (IBM® Company, Chicago, IL, USA). The significance level was 0.05. One third of our sample presented with headache with TMD. Females were predominant. Males with headache, no systemic disease, less pain severity but higher frequency, living longer with the disease and having sensitive changes, showed higher pain acceptance. When headache occurs with TMD, women with higher education, no headache family history, less pain, and no motor changes showed higher pain acceptance. Patients with both conditions are more liable to have chronic pain and pain inflexibility. Pain intensity and willingness explain 50% of the psychological inflexibility in the headache group. In our sample, individuals suffering from both conditions show greater pain inflexibility, implicating more vivid suffering experiences, leading to altered daily decisions and actions. However, further studies are needed to highlight this possible association.

A High Methylation Level of a Novel -284 bp CpG Island in the RAMP1 Gene Promoter Is Potentially Associated with Migraine in Women.

Migraine is a complex neurovascular disorder affecting one billion people worldwide, mainly females. It is characterized by attacks of moderate to severe headache pain, with associated symptoms. Receptor activity modifying protein (RAMP1) is part of the Calcitonin Gene-Related Peptide (CGRP) receptor, a pharmacological target for migraine. Epigenetic processes, such as DNA methylation, play a role in clinical presentation of various diseases. DNA methylation occurs mostly in the gene promoter and can control gene expression. We investigated the methylation state of the RAMP1 promoter in 104 female blood DNA samples: 54 migraineurs and 50 controls. We treated DNA with sodium bisulfite and performed PCR, Sanger Sequencing, and Epigenetic Sequencing Methylation (ESME) software analysis. We identified 51 CpG dinucleotides, and 5 showed methylation variability. Migraineurs had a higher number of individuals with all five CpG methylated when compared to controls (26% vs. 16%), although non-significant (p = 0.216). We also found that CpG -284 bp, related to the transcription start site (TSS), showed higher methylation levels in cases (p = 0.011). This CpG may potentially play a role in migraine, affecting RAMP1 transcription or receptor malfunctioning and/or altered CGRP binding. We hope to confirm this finding in a larger cohort and establish an epigenetic biomarker to predict female migraine risk.

Gut bacterial microbiome composition and statin intake-A systematic review.

Recently, the gut microbiome has become an important field of interest. Indeed, the microbiome has been associated to numerous drug interactions and it is thought to influence the efficacy of pharmacologic treatments. Although statins are widely prescribed medications, there remains considerable variability in its therapeutic response. In this context, we aimed to investigate how statins modulate the gut microbiome and, reversely, how can the microbiome influence the course of anti-hypercholesterolemic treatment. We conducted a systematic review by searching four online databases, in accordance with PRISMA guidelines. Studies addressing gut microbiome changes following statin treatment and those assessing statins' response and associating it with patients' microbiome were included. Due to the limited number of results, we decided to include studies enrolling both humans and animals. We summarized information from three human and seven animal studies and aimed to assess the influence of gut microbiome composition on statin response (Outcome 1) and to evaluate the impact of statin treatment on the gut microbiome (Outcome 2). An association between a certain microbiome composition that promoted the lipid-lowering effect of statins was found. However, what kind of microorganisms and how they can exert this effect remains uncertain. Furthermore, statins might have a role in the modulation of the gut microbiome, but then again, it is still unknown whether this change is directly caused by the drug or another metabolic mechanism. Even though gut microbiota may have several potential therapeutic implications, its use as a personalized predictive biomarker requires further studies.

C1QA and C1QC modify age-at-onset in familial amyloid polyneuropathy patients.

OBJECTIVES: Transthyretin (TTR) familial amyloid polyneuropathy (FAP) (OMIM 176300) shows a variable age-at-onset (AO), including within families. We hypothesized that variants in C1QA and C1QC genes, might also act as genetic modifiers of AO in TTR-FAP Val30Met Portuguese patients. METHODS: We analyzed DNA samples of 267 patients (117 families). To search for variants, all exons and flanking regions were genotyped by automated sequencing. We used generalized estimating equations (GEEs) to take into account the non-independency of AO among relatives. Intensive in silico analyses were performed, using various software to assess miRNAs target sites, splicing sites, transcription factor binding sites alterations, and gene-gene interactions. RESULTS: Two variants for C1QA gene, GA genotype of rs201693493 (P < 0.001) and CT genotype of rs149050968 (P < 0.001), were significantly associated with later AO. In silico analysis demonstrated, that rs201693493 may alter splicing activity. Regarding C1QC, we found three statistically significant results: GA genotype of rs2935537 (P = 0.003), GA genotype of rs201241346 (P < 0.001) and GA genotype of rs200952686 (P < 0.001). The first two were associated with earlier AO, whereas the third was associated with later-onset. INTERPRETATION: C1QA was associated with later onset, whereas C1QC may have a double role: variants may confer earlier or later AO. As found in a study in Cyprus, we confirmed the role of complement C1Q genes (and thus of inflammation) as modulator of AO in Portuguese patients with TTR-FAP Val30Met.

Using an apex predator for large-scale monitoring of trace element contamination: Associations with environmental, anthropogenic and dietary proxies.

Understanding the levels and drivers of contamination in top predators is important for their conservation and eventual use as sentinels in environmental monitoring. Therefore, metals and trace elements were analyzed in feathers of Bonelli's eagles (Aquila fasciata) from southern Portugal in 2007-2013, where they are believed to be exposed to a wide range of contamination sources such as agricultural land uses, urban areas, active and abandoned mines and a coal-fired power plant. We focused on concentrations of aluminum (Al), arsenic (As), copper (Cu), chromium (Cr), mercury (Hg), lead (Pb), selenium (Se) and zinc (Zn), as these contaminants are potentially associated with those sources and are known to pose a risk for terrestrial vertebrates. Stable isotope values of nitrogen (δ15N: 15N/14N), carbon (δ13C: 13C/12C) and sulphur (δ34S: 34S/32S) were used as dietary proxies to control for potential effects of prey composition on the contamination pattern. The spatial distribution of potential contamination sources was quantified using geographic information systems. Concentrations of Hg in the southern part of the study area were above a reported toxicity threshold for raptors, particularly in territories closer to a coal-fired power plant at Sines, showing that contamination persisted after a previous assessment conducted in the 1990s. Hg and Se levels were positively correlated with δ15N, which indicates biomagnification. Concentrations of As, Cr, Cu, Pb and Zn were generally low and unrelated to mining- or industrial activities, indicating low environmental background concentrations. Al was found at higher concentrations in the southernmost areas of Portugal, but this pattern might be related to external soil contamination on feathers. Overall, this study indicates that, among all elements studied, Hg seems to be the most important contaminant for Bonelli's eagles in southern Portugal, likely due to the power plant emissions and biomagnification of Hg in terrestrial food webs.

Correction to: Progress on bringing together raptor collections in Europe for contaminant research and monitoring in relation to chemicals regulation.

The correct affiliation of Sabrina Lo Brutto is shown in this paper.

Genomic imbalances defining novel intellectual disability associated loci.

BACKGROUND: High resolution genome-wide copy number analysis, routinely used in clinical diagnosis for several years, retrieves new and extremely rare copy number variations (CNVs) that provide novel candidate genes contributing to disease etiology. The aim of this work was to identify novel genetic causes of neurodevelopmental disease, inferred from CNVs detected by array comparative hybridization (aCGH), in a cohort of 325 Portuguese patients with intellectual disability (ID). RESULTS: We have detected CNVs in 30.1% of the patients, of which 5.2% corresponded to novel likely pathogenic CNVs. For these 11 rare CNVs (which encompass novel ID candidate genes), we identified those most likely to be relevant, and established genotype-phenotype correlations based on detailed clinical assessment. In the case of duplications, we performed expression analysis to assess the impact of the rearrangement. Interestingly, these novel candidate genes belong to known ID-related pathways. Within the 8% of patients with CNVs in known pathogenic loci, the majority had a clinical presentation fitting the phenotype(s) described in the literature, with a few interesting exceptions that are discussed. CONCLUSIONS: Identification of such rare CNVs (some of which reported for the first time in ID patients/families) contributes to our understanding of the etiology of ID and for the ever-improving diagnosis of this group of patients.

Avaliação das ementas e referências bibliográficas de disciplinas de Odontologia Legal no Brasil ­ uma análise documental / Evaluation of menu and bibliographical references of Legal Dentistry disciplines in Brazil ­ a documental analysis

Nesta pesquisa avaliou-se o plano de ensino de 25 faculdades do Brasil, sendo 5 faculdades por região para melhor representatividade do país. Um instrumento de coleta de dados foi construído para extração das informações necessárias, a saber: ementa e referências bibliográficas. Os dados obtidos por esta pesquisa foram tabulados em planilhas utilizando o programa Microsoft Office Excel. Inicialmente, cada frase das ementas foi transcrita para uma entrada de linha da planilha e forma associadas a 10 categorias de sentido. Foram transpostas para uma ementa provisória e proposta uma versão de ementa final. Já as referências bibliográficas foram descritas em linhas das planilhas, individualmente, e após a inclusão de todas foi feito um ranqueamento por frequência de citação. Em termos quantitativos esta pesquisa evidenciou que, as componentes curriculares de Odontologia Legal, abordam mais frequentemente em suas ementas os conteúdos relacionados às Ciências Forenses e ao Direito, sendo estas duas categorias responsáveis por 51,4% (121) das frases das ementas avaliadas e, Jorge Paulete Vanrell, é o autor mais frequentemente referenciado (80%) com sua obra Odontologia Legal e Antropologia Forense

In this research, the teaching plan of 25 colleges in Brazil was evaluated, with 5 colleges per region for better representation of the country. A data collection instrument was built to extract the necessary information, namely: menu and bibliographic references. The data obtained by this research were tabulated in spreadsheets using the Microsoft Office Excel program. Initially, each sentence of the menus was transcribed into a line entry in the spreadsheet and was associated with 10 categories of meaning. They were transposed to a provisional menu and a version of the final menu was proposed. The bibliographic references were described in lines of the spreadsheets, individually, and after the inclusion of all, a ranking was performed by citation frequency. In quantitative terms, this research showed that the Forensic Dentistry disciplines more frequently address content related to Forensic Sciences and Law in their menus, these two categories being responsible for 51.4% (121) of the sentences of the evaluated menus and, Jorge Paulete Vanrell is the author most frequently referenced (80%) with his work Odontologia Legal e Antropologia Forense

The role of conservative versus innovative nesting behavior on the 25-year population expansion of an avian predator.

Species ranges often change in relation to multiple environmental and demographic factors. Innovative behaviors may affect these changes by facilitating the use of novel habitats, although this idea has been little explored. Here, we investigate the importance of behavior during range change, using a 25-year population expansion of Bonelli's eagle in southern Portugal. This unique population is almost exclusively tree nesting, while all other populations in western Europe are predominantly cliff nesting. During 1991-2014, we surveyed nest sites and estimated the year when each breeding territory was established. We approximated the boundaries of 84 territories using Dirichlet tessellation and mapped topography, land cover, and the density of human infrastructures in buffers (250, 500, and 1,000 m) around nest and random sites. We then compared environmental conditions at matching nest and random sites within territories using conditional logistic regression, and used quantile regression to estimate trends in nesting habitats in relation to the year of territory establishment. Most nests (>85%, n = 197) were in eucalypts, maritime pines, and cork oaks. Nest sites were farther from the nests of neighboring territories than random points, and they were in areas with higher terrain roughness, lower cover by agricultural and built-up areas, and lower road and powerline densities. Nesting habitat selection varied little with year of territory establishment, although nesting in eucalypts increased, while cliff nesting and cork oak nesting, and terrain roughness declined. Our results suggest that the observed expansion of Bonelli's eagles was facilitated by the tree nesting behavior, which allowed the colonization of areas without cliffs. However, all but a very few breeding pairs settled in habitats comparable to those of the initial population nucleus, suggesting that after an initial trigger possibly facilitated by tree nesting, the habitat selection remained largely conservative. Overall, our study supports recent calls to incorporate information on behavior for understanding and predicting species range shifts.