Not Only RET but NF1 and Chromosomal Instability Are Seen in Young Patients with Sporadic Medullary Thyroid Carcinoma.

Context: Genetic analysis of sporadic medullary thyroid carcinoma (MTC) has revealed somatic variants in RET, RAS, and occasionally other genes. However, around 20% of patients with sporadic MTC lack a known genetic driver. Objective: To uncover potential new somatic or germline drivers, we analyze a distinct cohort of patients with sporadic, very early-onset, and aggressive MTC. Methods: Germline and somatic DNA exome sequencing was performed in 19 patients, previously tested negative for germline RET variants. Results: Exome sequencing of 19 germline samples confirmed the absence of RET and identified an NF1 pathogenic variant in 1 patient. Somatic sequencing was successful in 15 tumors revealing RET variants in 80%, predominantly p.Met918Thr, which was associated with disease aggressiveness. In RET-negative tumors, pathogenic variants were found in HRAS and NF1. The NF1 germline and somatic variants were observed in a patient without a prior clinical diagnosis of neurofibromatosis type 1, demonstrating that the loss of heterozygosity of NF1 functions as a potential MTC driver. Somatic copy number alterations analysis revealed chromosomal alterations in 53.3% of tumors, predominantly in RET-positive cases, with losses in chromosomes 9 and 22 being the most prevalent. Conclusion: This study reveals that within a cohort of early-onset nonhereditary MTC, RET remains the major driver gene. In RET-negative tumors, NF1 and RAS are drivers of sporadic MTC. In addition, in young patients without a RET germline mutation, a careful clinical evaluation with a consideration of germline NF1 gene analysis is ideal to exclude Neurofibromatosis type 1 (NF1).

Parental exposure to antidepressants has lasting effects on offspring? A case study with zebrafish.

Fish have common neurotransmitter pathways with humans, exhibiting a significant degree of conservation and homology. Thus, exposure to fluoxetine makes fish potentially susceptible to biochemical and physiological changes, similarly to what is observed in humans. Over the years, several studies demonstrated the potential effects of fluoxetine on different fish species and at different levels of biological organization. However, the effects of parental exposure to unexposed offspring remain largely unknown. The consequences of 15-day parental exposure to relevant concentrations of fluoxetine (100 and 1000 ng/L) were assessed on offspring using zebrafish as a model organism. Parental exposure resulted in offspring early hatching, non-inflation of the swimming bladder, increased malformation frequency, decreased heart rate and blood flow, and reduced growth. Additionally, a significant behavioral impairment was also found (reduced startle response, basal locomotor activity, and altered non-associative learning during early stages and a negative geotaxis and scototaxis, reduced thigmotaxis, and anti-social behavior at later life stages). These behavior alterations are consistent with decreased anxiety, a significant increase in the expression of the monoaminergic genes slc6a4a (sert), slc6a3 (dat), slc18a2 (vmat2), mao, tph1a, and th2, and altered levels of monoaminergic neurotransmitters. Alterations in behavior, expression of monoaminergic genes, and neurotransmitter levels persisted until offspring adulthood. Given the high conservation of neuronal pathways between fish and humans, data show the possibility of potential transgenerational and multigenerational effects of pharmaceuticals' exposure. These results reinforce the need for transgenerational and multigenerational studies in fish, under realistic scenarios, to provide realistic insights into the impact of these pharmaceuticals.

Anti-SSA Ro52 and anti-Ro60 autoantibodies: association with clinical phenotypes.

OBJECTIVES: Anti-SSA autoantibodies can be differentiated according to their antigenic target proteins as anti-Ro60 (60 kDa) or anti-Ro52 (52 kDa). Anti-SSA(Ro60) antibodies are clearly associated with connective tissue diseases (CTD), but the clinical significance of anti-SSA(Ro52) antibodies remains unclear. The aim of the present study was to analyse the disease phenotype of patients with anti-Ro52 and/or anti-Ro60 antibodies. METHODS: A multicentre, cross-sectional study was carried out of positive anti-Ro52 and/or Ro60 antibodies patients followed at 10 Rheumatology centres from January 2018 until December 2021. Patients were categorised into 3 groups: group 1 (Ro52+/Ro60-); group 2 (Ro52-/Ro60+); group 3 (Ro52+/Ro60+). Antinuclear antibodies were evaluated by indirect immunofluorescence assay and further screened for anti-extractable nuclear antigen (ENA) antibodies. Demographicsand clinical data were compared between the 3 groups, by patients' medical chart review. Univariate analysis was performed and subsequently logistic regression was used to identify intergroup differences and calculate the odds ratio with a 95% confidence interval (95% CI). RESULTS: We included 776 patients [female: 83.1%; median age: 59 (46-71) years]. Groups 1, 2, and 3 comprised 31.1%, 32.6%, and 36.3% of the patients, respectively. Anti-Ro52 antibody alone was more frequently associated with non-rheumatic diseases, older age, and men (p<0.05). Among patients with CTD, the diagnosis of systemic lupus erythematosus is 3 and 2 times more prevalent in groups 2 and 3, respectively, than in group 1 [OR 2.8 (95% CI 1.60, 4.97), p<0.001; OR 2.2 (95% CI 1.28, 3.86), p<0.01]. In group 2, the diagnosis of undifferentiated CTD is more frequent than in the other groups. Group 1 was more frequently associated with inflammatory myositis than group 2 [OR 0.09 (95% CI 0.01, 0.33), p<0.001] or group 3 [OR 0.08 (95% CI 0.01, 0.29), p<0.001]. Group 1 was also more frequently associated with arthritis (p<0.01), interstitial lung disease (p<0.01), and myositis (p<0.01). CONCLUSIONS: Anti-Ro52+ antibody alone is frequently found in patients with non-rheumatic diseases. In addition, anti-Ro52+ antibody is also prevalent in patients with CTD and associated with clinical phenotypes that are different from anti-Ro60+ antibody.

Phytoremediation by trees as a nature-based solution for mitigating metal contamination in urban soils.

Trace metals in the environment are important pollutants affecting human health, particularly in urban areas worldwide. Phytoremediation as a nature-based solution (NBS) and environmentally friendly technology may decrease high concentrations of trace metals in urban soils, protecting public health (especially children) and contributing to urban sustainability. This study examined trace metal contamination of urban soils and trees in six cities in the Republic of Srpska (RS), Bosnia and Herzegovina (BiH) and investigated the potential of selected tree species for phytoremediation as a NBS for metal-polluted urban soils. Contamination of urban soils was assessed by quantifying the concentrations of 11 trace metals (B, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn). To estimate phytoremediation potential of urban tree species, concentration and bioconcentration factor of the 11 metals were quantified in leaves of three common and abundant tree species: Aesculus hippocastanum L. (horse chestnut), Platanus acerifolia Willd. (plane), and Tilia sp. (lime). The results showed that trace metal concentrations in leaf samples did not exceed toxicity threshold guideline values. Further assessments are needed to establish the true potential of the three species as NBS for urban soils.

Sub-chronic exposure to paroxetine disrupts ecologically relevant behaviours in fish.

The functional conservation of important selective serotonin reuptake inhibitor (SSRI) targets in non-target organisms raises concerns about their potential adverse effects on the ecosystems. Although the environmental levels of SSRIs like paroxetine (PAR) have risen, the knowledge regarding the effects of long-term exposure to PAR is limited. This study investigated the impact of sub-chronic exposure (21 days) to two sub-lethal concentrations of PAR (40 and 400 µg/L) on the behaviour of adult zebrafish in different scenarios: basal activity (under dark and light conditions), stress response (evoked by sudden light transitions) and stress response recovery. A new framework was employed for the integrative study of fish's swimming performance based on their innate ability to respond to light shifts. Several swimming-associated parameters (e.g., total swimming distance, time of inactivity, swimming angles) and thigmotaxis were monitored for an integrated analysis in each scenario. Data revealed reduced swimming activity, impaired behavioural response to stress and alterations in stress recovery of PAR-exposed fish. An anxiolytic effect was particularly noticeable in fish basal swimming activity in the dark at 400 µg/L and in the behavioural response to stress (from dark to light) and stress recovery (from light to dark) for organisms exposed to 40 µg/L. The detected PAR-induced behavioural modifications suggest a disruption of brain glucocorticoid signalling that may have implications at the individual level (e.g., changing behavioural responses to predators), with potential repercussions on the population and community levels. Therefore, the applied protocol proved sensitive in detecting behavioural changes induced by PAR.

Desenvolvimento e validação de software web de apoio à gestão da assistência pré-natal / Desarrollo y validación de software web de apoyo a la gestión de la atención prenatal / Development and validation of web software to support prenatal care management

Resumo Objetivo Descrever o processo de desenvolvimento e validação de um software web de apoio à gestão da assistência pré-natal. Métodos Pesquisa de produção tecnológica, realizada entre março e agosto de 2022, com base em princípios e técnicas do método Scrum. As funcionalidades do software web, com acesso e funções específicas a três diferentes usuários (Administrador, Gestor e Visitante), foram desenvolvidas de forma colegiada entre desenvolvedores, pesquisadores, gestores e profissionais da saúde, a partir de necessidades práticas relacionadas à gestão da assistência pré-natal. Resultados O software web é uma ferramenta de apoio à gestão da assistência pré-natal, com potencial para gerar infográficos que denotem intercorrências gestacionais e que permitem, de forma ágil e segura, à tomada de decisões estratégicas loco-regionais. Conclusão O processo de desenvolvimento e validação do software web de apoio à gestão da assistência pré-natal deu-se de forma colegiada entre desenvolvedores, pesquisadores, gestores e profissionais da saúde, a partir de necessidades práticas relacionadas à gestão da assistência pré-natal. A ferramenta foi desenvolvida de modo a possibilitar acesso e funções específicas a três diferentes usuários (Administrador, Gestor e Visitante), com vistas à tomada de decisões rápidas e seguras.

Resumen Objetivo Describir el proceso de desarrollo y validación de un software web de apoyo a la gestión de la atención prenatal. Métodos Investigación de producción tecnológica, realizada entre marzo y agosto de 2022, con base en principios y técnicas de la metodología Scrum. Las funcionalidades del software web, con acceso y funciones específicas para tres usuarios (administrador, gestor y visitante), se desarrollaron de forma colegiada entre desarrolladores, investigadores, gestores y profesionales de la salud, a partir de necesidades prácticas relacionadas con la gestión de la atención prenatal. Resultados El software web es una herramienta de apoyo a la gestión de la atención prenatal, con potencial para generar infográficos que indiquen complicaciones gestacionales y que permitan tomar decisiones estratégicas locorregionales de forma ágil y segura. Conclusión El proceso de desarrollo y validación del software web de apoyo a la gestión de la atención prenatal se realizó de forma colegiada entre desarrolladores, investigadores, gestores y profesionales de la salud, a partir de necesidades prácticas relacionadas con la gestión de la atención prenatal. La herramienta se desarrolló a fin de permitir el acceso y funciones específicas para tres diferentes usuarios (administrador, gestor y visitante), con el objetivo de tomar decisiones rápidas y seguras.

Abstract Objective To describe the development and validation process of a web software to support the management of prenatal care. Methods Technological development research, carried out between March and August 2022, based on principles and techniques of the Scrum method. The functionalities of the web software, with access and specific functions for three different users (Administrator, Manager and Visitor), were developed jointly by developers, researchers, managers and health professionals, based on practical needs related to the management of prenatal care. Results The web software is a tool to support the management of prenatal care, with the potential to generate infographics that denote gestational intercurrences and that allow, in an agile and safe way, the making of local-regional strategic decisions. Conclusion The process of development and validation of the web software to support the management of prenatal care was carried out jointly by developers, researchers, managers and health professionals, based on practical needs related to the management of prenatal care. The tool was developed in order to allow access and specific functions to three different users (Administrator, Manager and Visitor), with a view to making quick and safe decisions.

Cycling versus swapping strategies in psoriatic arthritis: results from the rheumatic diseases Portuguese register.

OBJECTIVE: To compare the 2-year retention rate between a second tumor necrosis factor alpha inhibitor (TNFi) and secukinumab (SEK) or ustekinumab (UST), in Psoriatic Arthritis (PsA) patients with previous inadequate response to their first TNFi. METHODS: Prospective longitudinal cohort study with a follow-up period of 2 years using the Nationwide Portuguese Reuma.pt database. Patients with a clinical diagnosis of PsA who also fulfill the CASPAR classification criteria, with previous treatment failure to a first-line TNFi and having started a second biotechnological drug (TNFi, SEK or UST) were included. The Cycling group was defined as switching from a first TNFi to a second TNFi, and the Swapping group as switching from a first TNFi to SEK or UST. Sociodemographic data, disease characteristics, disease activity scores and physical function at baseline and after 6, 12 and 24 months were recorded. Cox-proportional hazards regression was used to compare retention rates between Cycling and Swapping groups. To obtain a predictor model of 2-year discontinuation, a multivariable Cox regression model was performed. RESULTS: In total, 439 patients were included, 58% were female, with a mean age (standard deviation) of 49 (12) years. Globally, 75.6% initiated a second TNFi (Cycling group), and 24.4% started SEK/UST (Swapping group). The retention rates after 6, 12 and 24 months were 72%/66%/59% in the Cycling group; and 77%/66%/59% in the Swapping group. There were no significant differences in retention rates between both strategies (HR: 1.06, 95% CI 0.72-1.16). After 2 years of follow-up, 34.4% of patients discontinued their second biologic, mainly due to inefficacy (72.8%), with no differences found between groups. Baseline treatment with glucocorticoids was the only predictor of discontinuation after 2 years of follow-up (HR:1.668, 95% CI 1.154-2.409). CONCLUSIONS: After failure of a first TNF inhibitor, Cycling and Swapping strategies result in similar retention rates suggesting that both are acceptable in the management of patients with psoriatic arthritis.

Plant functional traits explain long-term differences in ecosystem services between artificial forests and natural grasslands.

Declining ecosystem services have prompted numerous studies aiming at developing more sustainable management practices for vegetation restoration. Advances in functional ecology indicate that the sustainable management of afforestation ecosystems should be performed based on plant functional traits, which provides pivotal knowledge for long-term sustainable vegetation restoration. Currently, the mechanism of how plant functional traits affect long term ecosystem services in restored areas is still unclear. This study investigates plant functional traits and the associated ecosystem services from artificial forestlands (Robinia pseudoacacia, Caragana korshinskii) and natural grasslands following different durations of vegetation restoration (10, 20, 30 and 40 years) in the Danangou watershed, a loess hilly-gully region in the Loess Plateau, China. The results showed that 1) the water conservation services of artificial forestlands first decreased and then increased over time, whereas the soil conservation service had an opposite trend; in turn, natural grassland led to a consistent increase in soil conservation and carbon sequestration services over time. 2) Artificial forestlands had greater soil conservation and carbon sequestration services than natural grassland but had lower water conservation services. 3) Leaves had a greater impact on carbon sequestration and water conservation services than did root length and root biomass density. 4) Root biomass density had a greater effect on soil conservation services than did leaf carbon content and soil organic matter. 5) Leaf carbon content, specific root length, and root biomass density had significant effects on the trade-off value between any two ecosystem services with increasing time after restoration of artificial forestland. 6) Specific leaf area had a greater effect on the trade-off values among the three services than did the other functional traits in the natural grassland. In arid ecosystems, natural grasslands are the best restoration strategy given their higher water conservation services. However, in soil erosion-affected areas, restoration through artificial forestlands is more appropriate. To mitigate the trade-offs between ecosystem services, it is recommended that artificial forestlands be thinned before the leaf carbon content, specific root length, and root biomass density reach a maximum (i.e., mature forestland).

Strategies for addressing needle debris: A scoping review of needle debris and discarded drug paraphernalia associated with substance use.

BACKGROUND AND AIMS: Needle debris and discarded drug paraphernalia can pose risks to people who use drugs (PWUD) and other members of the community. The research question guiding our scoping review was, "What is the scope of literature around discussions of and interventions for needle debris associated with drug use in a community setting"? METHODS: The review was guided by the Joanna Briggs Institute methodology. Reporting was in accordance with the PRISMA scoping review extension. Searches in August of 2022 on Medline, CINAHL, PsycINFO, EMBASE, and Social Services Abstracts were completed. Quantitative and qualitative study designs were included. Grey literature was excluded. Extracted data included disposal initiatives and factors influencing disposal practices. RESULTS: The databases combined search total was 3074. A total of 1115 duplicates were removed. Inclusion and exclusion criteria resulted in 72 studies full-text studies reviewed. Nineteen articles met all requirements. Studies from multiple continents resulted in similar themes. Two main themes were identified: needle disposal challenges for PWUD and disposal initiatives. Disposal challenges related to legal barriers and law enforcement influence, the varying definitions of 'safe disposal' and perspectives of PWUD. Disposal initiatives stemmed from community-based initiatives, pharmacy contributions and the perceptions and attitudes of community members. CONCLUSIONS: Needle debris is a complex phenomenon highly impacted by the threat of persecution from law enforcement. PWUD need various disposal methods in proximity to their injecting location to avoid fear of prosecution from residual substances on the syringes.

Proof of evidence of changes in global terrestrial biomes using historic and recent NDVI time series.

Climate change affects plant dynamics and functioning of terrestrial ecosystems. This study aims to investigate temporal changes in global vegetation coverage and biomes during the past three decades. We compared historic annual NDVI time series (1982, 1983, 1984 and 1985) with recent ones (2015, 2016, 2017 and 2018), captured from NOAA-AVHRR satellite observations. To correct the NDVI time series for missing data and outliers, we applied the Harmonic Analysis of Time Series (HANTS) algorithm. The NDVI time series were decomposed in their significant amplitude and phase given their periodic fluctuation, except for ever green vegetation. Our findings show that the average NDVI values in most biomes have increased significantly (F-value<0.01) by 0.05 ndvi units over during the past three decades, except in tundra, and deserts and xeric shrublands. The highest rates of change in the harmonic components were observed in the northern hemisphere, mainly above 30° latitude. Worldwide, the mean annual phase reduced by 9° corresponding to a 9 days shift in the beginning of the growing season. Annual phases in the recent time series reduced significantly as compared to the historic time series in the five major global biomes: by 14.1, 14.8, 10.6, 9.5, and 22.8 days in boreal forests/taiga; Mediterranean forests, woodlands, and scrubs; temperate conifer forests; temperate grasslands, savannas, and shrublands; and deserts, and xeric shrublands, respectively. In tropical and subtropical biomes, however, changes in the annual phase of vegetation coverage were not statistically significant. The decrease in the level of phases and acceleration of growth and changes in plant phenology indicate the increase in temperature and climate changes of the planet.

Soil erodibility for water and wind erosion and its relationship to vegetation and soil properties in China's drylands.

Drylands with fragile socio-ecological systems are vulnerable to soil erosion. China's drylands face the dual threat of water (WAE) and wind erosion (WIE). To mitigate soil erosion in drylands, China has implemented numerous ecological restoration measures. However, whether vegetation and soil have different effects on soil erodibility for water erosion (soil erodibility, K) and wind erosion (soil erodible fraction, EF) in drylands is unclear, hindering decision makers to develop suitable ecological restoration strategies. Here, we conducted a large-scale belt transect survey to explore the spatial variation of K and EF in China's drylands, and examined the linear and nolinear effects of aridity (aridity index), vegetation (fractional vegetation cover and below-ground biomass), and soil properties (bulk density, total nitrogen, and total phosphorus) on K and EF. The results showed in China's drylands that the K ranges from 0.02 to 0.07, with high values recorded in the northern Loess Plateau and the eastern Inner Mongolia Plateau. The EF ranges from 0.26 to 0.98, and shows longitudinal zonation with higher values in the east and lower values in the west. Aridity has a negative linear effect on K and an inverse U-shaped nonlinear effect on EF. Aridity can affect K and EF by suppressing vegetation growth and disrupting soil properties. However, K and EF had different responses to some vegetation and soil variables. K and EF had opposite relationships with soil bulk density, and EF was significantly affected by fractional vegetation cover, while K was not. Overall, the effects of aridity and soil properties on soil erodibility were more pronounced than those from vegetation, whose effect on soil erodibility was limited. This study provides relevant information to support reducing soil water and wind erosion by highlighting the hotspot areas of soil erodibility, relevant for implementing vegetation restoration and soil conservation measures in drylands.

Are early and young life stages of fish affected by paroxetine? A case study with Danio rerio.

Paroxetine (PAR) is a selective serotonin reuptake inhibitor (SSRI) antidepressant increasingly detected in surface waters worldwide. Its environmental presence raises concerns about the potential detrimental effects on non-target organisms. Thus, this study aimed to increase knowledge on PAR's potential environmental impacts, assessing the effects of commercial formulation (PAR-c) and active ingredient (PAR-a) on fish. Therefore, the short-term exposure effects of PAR-c and PAR-a were assessed on zebrafish (Danio rerio) embryos/larvae to determine the most toxic formulation [through median lethal (LC50) and effective concentrations (EC50)]. PAR-c and PAR-a induced morphological abnormalities (scoliosis) in a dose-dependent manner from 96 hours post-fertilization onwards, suggesting the involvement of a fully functional biotransformation system. As PAR-c exhibited higher toxicity, it was selected to be tested in the subsequent stage (juvenile stage), which was more sensitive (lower LC50). PAR-c significantly decreased fish swimming activity and disrupted fish stress response. Overall, the results highlight the ability of PAR-c to adversely affect fish swimming performance, an effect that persisted even after exposure ceases (21-day depuration), suggesting that PAR-c may impair individual fitness.

Using Glyphosate on Guarana Seedlings in the Amazon.

The seed yield of guarana (Paullinia cupana H.B.K. var. sorbilis) is affected by weeds. Management is difficult for Amazon farmers and ranchers, owing to the hot and humid climate prevailing in the region, which makes mechanical control inefficient and leads farmers to the decision to use herbicides. Herbicide damage to this species is unknown. The objective of this study was to evaluate glyphosate damage to the development and quality of guarana seedlings. The treatments consisted of glyphosate doses at concentrations of 0, 126, 252, 540, 1080, 2160 and 3240 g a.e. ha-1 and were evaluated for 60 days, in two applications. Analyses were performed for biometrics, seedling development, anthracnose and Injury characteristics. Glyphosate caused symptoms of Injury in all doses applied, but lower doses did not interfere with seedling growth and development. There was a correlation between anthracnose severity and increased glyphosate dose. When applied correctly, glyphosate can be an integrated weed management tool for use in guarana crops.

Identifying knowledge gaps in understanding the effects of selective serotonin reuptake inhibitors (SSRIs) on fish behaviour.

Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants increasingly prescribed to treat patients with clinical depression. As a result of the significant negative impact of the COVID-19 pandemic on the population's mental health, its consumption is expected to increase even more. The high consumption of these substances leads to their environmental dissemination, with evidence of their ability to compromise molecular, biochemical, physiological, and behavioural endpoints in non-target organisms. This study aimed to provide a critical review of the current knowledge regarding the effects of SSRI antidepressants on fish ecologically relevant behaviours and personality-dependent traits. A literature review shows limited data concerning the impact of fish personality on their responses to contaminants and how such responses could be influenced by SSRIs. This lack of information may be attributable to a lack of widely adopted standardized protocols for evaluating behavioural responses in fish. The existing studies examining the effects of SSRIs across various biological levels overlook the intra-specific variations in behaviour and physiology associated with different personality patterns or coping styles. Consequently, some effects may remain undetected, such as variations in coping styles and the capacity to handle environmental stressors. This oversight could potentially result in long-term effects with ecological implications. Data support the need for more studies to understand the impact of SSRIs on personality-dependent traits and how they may impair fitness-related behaviours. Given the considerable cross-species similarity in the personality dimensions, the collected data may allow new insights into the correlation between personality and animal fitness.

The role of nature reserves in conservation effectiveness of ecosystem services in China.

Establishing nature reserves (NRs) is a common method to avoid biodiversity loss and degradation of ecosystem services (ESs). The evaluation of ESs in NRs and the exploration of associated influencing factors are the basis for improving ESs and management. However, the ES effectiveness of NRs over time remains questionable, namely due to the heterogeneity of landscape characteristics inside and outside of NRs. This study (i) quantifies the role of 75 NRs in China in maintaining ESs (i.e., net primary production (NPP), soil conservation, sandstorm prevention and water yield) from 2000 to 2020, (ii) reveals the trade-offs/synergies, and (iii) identifies the main influencing factors of the ES effectiveness of NRs. The results show that more than 80% of NRs had positive ES effectiveness, which was greater in older NRs. For different ESs, effectiveness over time increases for NPP (E_NPP), soil conservation (E_SC) and sandstorm prevention (E_SP) but declines for water yield (E_WY). There is a clear synergistic relationship between E_NPP and E_SC. Moreover, the effectiveness of ESs is closely correlated with elevation, precipitation, and perimeter area ratio. Our findings can provide important information to support site selection and management of reserves to improve the delivery of critical ecosystem services.

GC-MS Silylation Derivative Method to Characterise Black BIC® Ballpoint 2-Phenoxyethanol Ratio Evaporation Profile-A Contribution to Ink Ageing Estimation.

One of the major challenges in forensic document analysis is estimating the age of ink deposition on a manually written document. The present work aims to develop and optimise a methodology, based on the evaporation of 2-phenoxyethanol (PE) over time, that can contribute to ink age estimation. A black BIC® Crystal Ballpoint Pen was purchased in a commercial area, and ink deposition began in September 2016 over 1095 days. For each ink sample, 20 microdiscs were subjected to n-hexane extraction in the presence of an internal standard (ethyl benzoate) followed by derivatisation with a silylation reagent. A gas chromatography-mass spectrometry (GC/MS) method was optimised for PE-trimethylsilyl (PE-TMS) to characterise the ageing curve. The developed method presented good linearity between 0.5 and 50.0 µg mL-1, as well as limits of detection and quantification of 0.026 and 0.104 µg mL-1, respectively. It was possible to characterise PE-TMS concentration over time, which reveals a two-phase decay behaviour. First, there was a substantial decline between the 1st and the 33rd day of deposition, followed a by a stabilisation of the signal, which allowed to detect the presence of PE-TMS up to 3 years. Two unknown compounds were also present and allowed to identify three dating time frames for the same ink stroke: (i) between time 0 and 33 days, (ii) between time 34 and 109 days, and (iii) more than 109 days. The developed methodology allowed to characterise the behaviour of PE over time and to establish a relative dating of three-time frames.