Assessment and Comparison of Microplastic Contamination in Atlantic Navigation Routes with Known Uncertainty.

Microplastics (MP) have been found in various environments worldwide. However, not many studies focus on the open ocean due to logistical restraints. Between January and May 2020, the NRP Sagres sampled 123 linear paths of subsurface water of the Atlantic Ocean, passing by Cape Verde, the east coast of South America, and the west coast of Africa. The water was sampled through the ship's water system. The membranes were analyzed by the Hydrographic Institute of Portugal and the Norwegian Institute for Water Research by micro-FTIR. The contamination levels were reported with uncertainty, for 99% confidence level (CL), normalized for filtered water volume and the distance traveled during sampling. Uncertainties were calculated through a detailed ″bottom-up″ evaluation. MP were found in about a third of the stations (48 out of 123), and most of those stations (43 out of 48) presented concentrations below 1 m-3 km-1. The sites where higher concentrations were registered were the port of the island of Santiago (Cape Verde) ((5.9 ± 5.2) m-3 km-1), the Guanabara Bay in Rio de Janeiro (Brazil) ((41 ± 27) m-3 km-1), and close to South Africa ((4.9 ± 2.4) m-3 km-1). Most MP found were polyamide, polyester, polyethylene, ethylene vinyl acetate, and poly(methyl methacrylate). The estimated contamination levels cannot be directly compared with information obtained in other studies due to differences in how MP were determined and the unknown uncertainty of their measured values. This article presents a relevant and reliable contribution to understanding the MP distribution in the Atlantic Ocean.

Bottom-Up Evaluation of the Uncertainty of the Quantification of Microplastics Contamination in Sediment Samples.

The quantification and comparison of microplastic contamination of sediments are affected by sample heterogeneity and the systematic and random effects affecting sample analysis. The quantification and combination of these components in the measurement uncertainty allows the objective interpretation of analysis results. This work presents the first detailed evaluation of the uncertainty of microplastic contamination quantification in sediments. The random and systematic effects affecting microplastic counts are modeled by the Poisson-lognormal distribution with inputs estimated from duplicate sediment analysis and the analysis of sediments spiked with microparticles. The uncertainty from particle counting was combined with the uncertainty from the determination of the dry mass of the analytical portion by the Monte Carlo method. The developed methodology was implemented in a user-friendly spreadsheet made available as the Supporting Information. The contamination of sediment samples collected in various inland Portuguese waters was determined, ranging from [0; 160] to [361; 2932] kg-1 for a 99% confidence level, and compared by assessing if the difference between contamination levels is equivalent to zero for the same confidence level. Several samples proved to have metrologically different microplastic contamination. This work represents a contribution to the objectivity of the assessment of environmental contamination with microplastics.

Optimization of River Sampling: Application to Nutrients Distribution in Tagus River Estuary.

The assessment of river water pollution trends is affected by the seasonal variation of river conditions, the variability of pollution sources, the heterogeneity of pollutants distribution, the representativeness/uncertainty of sampling, and the uncertainty of sample analysis. This work presents a methodology to model the uncertainty of river water sampling based on available information about the spatial distribution of the studied parameter in the river. The uncertainty from "single sampling" (SS) or by production of a composite sample by mixing m subsamples collected randomly (RS) or in a line that crosses the sampling circle (LS) was studied. This methodology was applied to the determination of nutrients (NO x, NO2, PO4, and SiO2) in an area of the Tagus River estuary with a range of about 350 m. This methodology can be applied to the determination of the mean value of other parameters in other river areas requiring a previous study of system heterogeneity. The spatial distribution of nutrients in the studied river area was characterized from the analysis of 10 samples collected at known geographical coordinates. The system heterogeneity was described by a three-dimensional ( x, y, z) surface with x and y variables for samples positions and z variable representing the measured nutrient levels. The randomization of this surface for the uncertainty of coordinates and repeatability of nutrient concentration measurement, using Monte Carlo simulations, allowed estimation of the uncertainty of the three sampling strategies: SS, RS, and LS. The uncertainty from RS and LS is equivalent and significantly smaller than that from SS when at least three subsamples are mixed in the composite sample. The sampling relative standard uncertainty ranged from 0.31% to 4.4%, producing nutrient concentration estimates in the river area with a relative expanded uncertainty from 5.9% to 10% with approximately 95% confidence level (coverage factor of 2). The used spreadsheet is available as Supporting Information.

HIV-1 Tat protein vaccination in mice infected with Mycobacterium tuberculosis is safe, immunogenic and reduces bacterial lung pathology.

BACKGROUND: The therapeutic HIV-1 Tat protein vaccine is in advanced clinical development. Tuberculosis, the main AIDS co-infection, is highly endemic in areas where AIDS prevention through vaccination is needed. However, safety and immunogenicity of Tat vaccination in the course of Mycobacterium tuberculosis (Mtb) infection is still unknown and it prevents the possibility to administer the vaccine to Mtb-infected individuals. We addressed the interplay and effects of Tat vaccination on Mtb infection in immunocompetent mice. METHODS: C57BL/6 mice were vaccinated or not with Bacillus Calmette-Guerin (BCG), the current tuberculosis vaccine, and after 5 weeks were infected with Mtb by intravenous route. The Tat protein was injected intradermally at 1, 2 and 4 weeks after Mtb challenge. Eight weeks after Mtb infection, all mice were sacrificed, and both the degree of pathology and immune responses to Mtb and Tat were evaluated. As additional control, some mice were either vaccinated or not with BCG, were not challenged with Mtb, but received the Tat protein. Statistical significances were evaluated by one-way or two-way ANOVA and Tukey's multiple comparisons post-test. RESULTS: In the lungs of Mtb-infected mice, Tat-vaccine did not favour Mtb replication and indeed reduced both area of cellular infiltration and protein levels of Interferon-γ, Chemokine (C-C motif) ligand-4 and Interleukin-1ß, pathological events triggered by Mtb-infection. Moreover, the protection against Mtb infection conferred by BCG remained good after Tat protein treatment. In spleen cells of Mtb-infected mice, Tat vaccination enhanced Mtb-specific Interferon-γ and Interleukin-17 responses, which may have a protective role. Of note, Mtb infection reduced, but did not suppress, the development of anti-Tat antibodies, required for Tat vaccine efficacy and the titer of anti-Tat IgG was potentiated by BCG vaccination in Mtb-free mice. In general, Tat treatment was well tolerated in both Mtb-infected and Mtb-free mice. CONCLUSIONS: Tat protein vaccine, administered in Mtb-infected mice with a protocol resembling that used in the clinical trials, was safe, immunogenic, limited the lung Mtb-associated immunopathology and did not abrogate the protective efficacy of BCG. These data provide preliminary evidence for a safe use of Tat vaccine in people vaccinated with BCG and/or suffering from tuberculosis.

Mycobacterium tuberculosis PstS1 amplifies IFN-γ and induces IL-17/IL-22 responses by unrelated memory CD4+ T cells via dendritic cell activation.

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN-γ and, to a lesser extent, of IL-17 by CD4(+) T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag-unrelated CD4(+) T-cell responses. Here we demonstrate that PstS1, a 38 kDa-lipoprotein of Mtb, promotes Ag-independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4(+) and CD8(+) memory T cells, amplifies secretion of IFN-γ and IL-22 and induces IL-17 production by effector memory cells in an Ag-unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α(-) subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL-6, IL-1ß and, to a lower extent, IL-23. IL-6 secretion by PstS1-stimulated DCs was required for IFN-γ, and to a lesser extent for IL-22 responses by Ag85B-specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.

The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Exploring the Benefits of Nutritional and Chemical Characteristics of Touriga Nacional and Arinto Varieties (Vitis vinifera L.).

Environmental degradation leads to an unsustainable food system. In addition to this issue, the consumption of foods that improve people's health and well-being is recommended. One of the alternatives is undoubtedly the use of by-products of winemaking, namely in the form of grape pomace flour (GPF). To verify the benefits of using the Touriga Nacional and Arinto (Vitis vinifera L.) flour varieties, analytical determinations were made to identify and quantify different components. In terms of nutritional characterization, the Touriga Nacional GPF showed results that indicate better nutritional quality than the Arinto GPF. The Touriga Nacional and Arinto samples had protein contents of 10.13% and 8.38%, polyunsaturated fatty acids of 6.66% and 5.18%, soluble dietary fiber of 14.3% and 1.7%, and insoluble dietary fiber of 55.1% and 46.4%, respectively. The anthocyanins, proanthocyanidins, and flavonols presented in samples were detected by HPLC-DAD/ESI-MS. Atomic absorption spectrometry revealed elevated concentrations of certain elements in Touriga Nacional compared to Arinto, with the former showing higher levels of aluminum (130 mg/kg) and iron (146 mg/kg) against the latter's Al (120 mg/kg) and Fe (112 mg/kg) content. GPF could become a valuable ingredient due to its nutritional quality and high content of various polyphenols.

Mycobacterium tuberculosis antigen 85B modifies BCG-induced antituberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis has developed strategies not only to evade host immunity but also to manipulate it for its survival. We investigated whether Mycobacterium tuberculosis exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host antituberculosis immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin impaired the protection elicited by vaccination, causing a more severe infection when mice were challenged with Mycobacterium tuberculosis. Ag85B administration reduced Bacillus Calmette-Guérin-induced CD4 T-cell activation and IFN-γ, CCL-4, and IL-22 production in response to Mycobacterium tuberculosis-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses, and activation of regulatory and exhaustion pathways, including programmed death ligand 1 expression on macrophages. All this resulted in impaired intracellular Mycobacterium tuberculosis growth control by systemic immunity, both before and after the Mycobacterium tuberculosis challenge. Interestingly, Mycobacterium tuberculosis infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mycobacterium tuberculosis in both unvaccinated and Bacillus Calmette-Guérin-vaccinated mice. Our data suggest that Mycobacterium tuberculosis can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and is commonly found in the lungs of the Mycobacterium tuberculosis-infected host, our findings may advance the understanding on the mechanisms of Mycobacterium tuberculosis pathogenesis and immune evasion.

Evaluation of temporal trends and correlations of physical-chemical parameters in vast oceanic areas robust to information uncertainty.

The physical-chemical monitoring of vast oceanic areas aims at assessing the status and evolution of the environmental resource for its exploration, protection and/or better understanding. However, the interpretation of monitoring data is affected by ocean seasonality and heterogeneity, and by the quality of sampling and characterization tools used to study the environment. All these factors contribute to the uncertainty of collected information that should be expressed in determined parameter values or trends. A trend of a studied parameter quantified by values difference is significant if the observed absolute value of the difference is larger than their expanded uncertainty. The correlation of studied parameters, useful for their interpretation, is equality affected by the mentioned sources of uncertainty. This work describes the metrologically sound evaluation of trends and correlations of physicochemical parameters of vast oceanic areas where all uncertainty sources affecting the information are considered by simulating their complex impact by the Monte Carlo Method. The described methodology was successfully used to study the impact of summer upwelling in an 800 km2 coastal area offshore two large cities in Portugal. Nutrients, conductivity, salinity and temperature trends and correlations are distinguished from system heterogeneity, sampling and sample analysis uncertainty for a 99% confidence level.

Long-term variation of dissolved metals and metalloid in the waters of an Atlantic mesotidal estuary (Sado Estuary, Portugal).

Estuaries have long been preferred sites of human settlement due to the benefits regarding proximity to fresh water and the ocean. As such, these environments have been subject to increasing anthropogenic pressures, resulting in issues of pollution and contamination. However, since the second half of the 20th century an environmental concern has reflected in the development of legislation, monitoring programmes and measures to diminish and control those impacts. The study presented herein integrates metals and metalloid concentrations from surface water samples obtained in a long-term monitoring programme (1986-2020) conducted in the Sado Estuary. The results obtained show a decrease and stabilisation of the concentrations of elements (between 81 % for Pb and 11 % for As in the average concentrations, between 83 % for Pb and 11 % for Cd in the median concentrations, and an increase of 1 % in the As median values). Nevertheless, high concentrations were still observed in the stations closest to the industrial area and the main freshwater to confluence with the estuary. Despite the efforts in improving the environmental quality of the Sado Estuary, possible effects in native species such as cuttlefishes and oysters are still a possibility, particularly in the stations where higher concentrations were registered, as well as close to nurseries as a result of trace metal transport through currents and tides.

Determination of microplastic contamination levels and trends in vast oceanic sediment areas with uncertainty.

Small plastic particles, designated as microplastics, are known vehicles of several contaminants desorbed from their surface after being ingested by marine organisms. The monitoring of the levels and trends of microplastics in oceanic areas is essential to identify relevant threats and respective sources whose management should be improved to protect the environmental resources. However, the assessment of contamination trends in large oceanic areas is affected by contamination heterogeneity, sampling representativeness, and the uncertainty of collected sample analyses. Only contamination variations not justifiable by system heterogeneity and their characterisation uncertainty are meaningful and should be taken seriously by the authorities. This work describes a novel methodology for the objective identification of meaningful variation of microplastic contamination in vast oceanic areas by the Monte Carlo simulation of all uncertainty components. This tool was successfully applied to the monitoring of the levels and trends of microplastic contamination in sediments from a 700 km2 oceanic area from 3 km to 20 km offshore Sesimbra and Sines (Portugal). This work allowed concluding that contamination has not varied between 2018 and 2019 (difference of mean total microplastic contamination between -40 kg-1 and 34 kg-1) but that microparticles made of PET are the major type of studied microplastics (in 2019, mean contamination is between 36 kg-1 and 85 kg-1). All assessments were performed for a 99 % confidence level.

Statistically sound comparison of standardized and simulation methods for oil spill source identification in real spill scenarios.

The comparison of oil patterns of a spill (Sp) and suspected spill source (SS) samples is based on ratios between correlated GC-MS signals of oil-discriminating compounds, i.e., diagnostic ratios (DR). The Student's t statistics (S-t) and a maximum relative difference (SC), proposed in standard methods, have been used for DR comparison due to their simplicity. An alternative methodology based on Monte Carlo Method (MCM) simulations of correlated signals, capable of accurately defining DR comparison criteria, proved that S-t and SC assumptions regarding DR normality and precision are frequently not valid, affecting comparison reliability. The performance of the approaches was accurately compared from independent signals of the same oil sample from a perfect match between Sp and SS. The present study describes the comparison of the approaches in real oil spill scenarios reproduced in International Round Robin Tests. Since as the number of compared DR increases, also rises the probability of not all equivalent DR being actually considered equivalent, the decision of oil pattern equivalence was based on two comparisons of independent sets of Sp and SS signals. The risk of true oil standard equivalency claims is compared for the three oil spill scenarios studied, which are different considering oil types, DR sets and spill weathering. The ability of the approaches to distinguish the Sp sample from an oil sample known not to be the source of the spill was also assessed. The MCM based on two independent DR comparison trials was the only one consistently producing fingerprint comparison risks of correct equivalence claims larger than 98 %. MCM also performed better in distinguishing different oil patterns. It was concluded that comparing >22 DR does not change the risk of correct oil pattern equivalence assessment significantly. The complexity of the MCM approach is overcome by using user-friendly and validated software.

Metrologically sound comparison of trace-metal levels in sea cucumber tissues from different species and habitats.

Sea cucumbers are indicators of metal contamination in sea bottoms due to their low mobility and feeding behaviour. Comparing contaminations of specimens from different locations, habitats, and/or organs allows understanding of contamination processes and differences. However, the interpretation of these data is affected by the variability of contamination levels in specimens, the uncertainty of tissue analyses, and the complex correlation of mass fractions estimated by using the same calibration of the used instrumental method of analysis. This work presents a novel tool for the sound comparison of contamination levels of biota where all mentioned factors are considered to produce reliable and undisputable information on the studied system. The Monte Carlo simulation of uncertainty components, affecting the determination of mean contamination levels observed in selected types of tissues, allowed simulating mean contamination differences and determining if these are meaningful. This tool was used to assess the levels of Cd, Cu, Ni and Pb of animals collected in different locations of Sesimbra-Portugal. It was concluded that specimens that selectively consume macroalgae have larger contamination levels than animals feeding on sediment. The gut is the most contaminated organ suggesting intake from feeding is dominant. Three of the analysed animals have Pb mass fractions larger than a maximum admissible value for human consumption of 3 mg kg-1 with a probability larger than 2.5%.

[Autoimmune/inflammatory syndrome induced by adjuvants. Case report]. / Síndrome autoinmunitario/inflamatorio inducido por adyuvantes. Reporte de caso.

Background: The indiscriminate application of substances for aesthetic purposes, such as silicone in breast implants, leads to the production of common local signs such as inflammation, skin irregularities, edema, erythema, vascular neoformations, and ulcers, which can evolve into general symptoms such as fever, asthenia, weakness, arthralgia or activate the immune system abnormally, causing the appearance of autoimmune diseases. This set of signs and symptoms is called adjuvant-induced autoimmune/inflammatory syndrome. Clinical case: We present the case of a 50-year-old woman with a history of silicone-based breast implants who spontaneously developed a hemorrhagic coagulopathy, type A acquired hemophilia was documented, that is, autoantibodies against coagulation factor VIII. Thanks to the work of a multidisciplinary team, it is possible to successfully diagnose and treat the patient with bridging agents, implant removal and management of associated symptoms. Conclusion: the importance of knowing the pathology is recognized, which, although it is rare, when it occurs has a high mortality rate if it is not diagnosed and treated on time.

Introducción: la aplicación de sustancias con fines estéticos de forma indiscriminada, como es el caso de la silicona en los implantes mamarios, llevan a la producción de signos locales comunes como: inflamación, irregularidad en la piel, edema, eritema, neoformaciones vasculares y úlceras, que pueden evolucionar a síntomas generales como la fiebre, astenia, adinamia, artralgias o a activar, de manera anómala, el sistema inmunitario, causando la aparición de enfermedades autoinmunitarias. A este conjunto de signos y síntomas se le denomina síndrome autoinmunitario/inflamatorio inducido por adyuvantes. Caso clínico: presentamos el caso de una mujer de 50 años con antecedente de implantes mamarios a base de silicona que desarrolla, de manera espontánea, una coagulopatía hemorrágica, se documenta hemofilia tipo A adquirida, es decir, autoanticuerpos contra el factor VIII de la coagulación. Gracias al trabajo de un equipo multidisciplinario se consigue diagnosticar y tratar de manera exitosa a la paciente con agentes de puente, remoción de los implantes y manejo de los síntomas asociados. Conclusión: se reconoce la importancia de conocer la patología que, si bien es rara, cuando se presenta tiene alta tasa de mortalidad si no se diagnostica y trata a tiempo.

Increased oyster aquaculture in the Sado Estuary (Portugal): How to ensure ecosystem sustainability?

Aquaculture is one of the fastest growing sectors in the world. However, this may come with a cost, as increasing aquatic production is likely to impose changes in the environment. To ensure ecosystem sustainability, it is essential to think on this larger scale. This study aims to use the Delft3D model suite to evaluate the ecological carrying capacity for bivalve production in the Sado Estuary (Portugal), under present and future conditions (2050). Scenarios for increased oyster production resulted in reductions of chlorophyll a associated with increased nutrient concentrations. In the most extreme production scenario, which considered an increase of 100 ha in production area, a predicted decrease of 90 % in phytoplankton biomass was observed. Climate change (CC) was incorporated as an increase in sea level and water temperature, as well as a reduction in river flow. Under present oyster production conditions, CC revealed contrasting patterns, i.e. an increase in chlorophyll a concentrations and a reduction in nutrients. These results suggest that CC has a positive effect in counteracting the impacts of increased oyster production, however further research is necessary. All scenarios point to reduced dissolved oxygen concentrations, highlighting the need to monitor this parameter. Given the difficulty in defining what are unacceptable impacts to the ecosystem it would be prudent to include a socio-ecological framework in the future, in order to integrate ecosystem services and the perception of local stakeholders.

Development and validation of statistically sound criteria for the match of unweathered GC-MS fingerprints in oil spill forensics.

The investigation of an oil spill's origin frequently relies on determining the equivalence of oil component patterns in samples from the contaminated environment and suspected oil source. This comparison benefits if based on the ratio of the abundance of unweathered characteristic components of the oil product, Diagnostic Ratios, DR. Replicate determinations of DR from one sample are used to set limits for the second sample's DR. The composition equivalence of oil patterns in both samples is indicated if all compared DR are statistically equivalent with a high confidence level. Some studies define DR limits assuming their normality and using Student's t statistics (S-t). However, since the ratio of correlated abundances can be not normally distributed, this criterion can drive to more false comparisons than predicted by the test confidence level. This work developed a computational tool for the reliable description of the non-normal distribution of the DR based on the Monte Carlo Method (MCM), aiming to allow the accurate control of the confidence of DR comparison. This work concluded that S-t defines 95% or 98% confidence limits with probabilities of falsely rejecting samples equivalence, φ, that can be up to 4.3% higher than predicted by the confidence level of the S-t test (i.e., 5% and 2%). The fragilities of the S-t limits significantly reduce the probability (1-θ) of two samples with the same oil producing equivalent values of all compared DR. For the studied 69 DR from unweathered components, the (1-θ) for 98% confidence level limits, set by the MCM and S-t from triplicate injections of one sample, are 94.8% and 91.7%, respectively. These values are below the confidence level (P) defined for each DR because DR are correlated with a correlation coefficient lower than 1. The (1-θ) can be increased to above P by using MCM limits and accepting composition equivalence if at least one of two sample extract injections produces values within limits set from the other sample's replicate injection. The validated user-friendly MS-Excel file used to set and access comparison criteria is made available as Supplementary Material and was checked experimentally. However, it is not feasible to estimate model confidence exclusively from experimentation because it would require too much independent analysis.

Optimisation of the uncertainty of oil spill identification from replicate comparative analyses: Comparison of standardised and novel identification methods.

Oil spill identifications involve the comparison of oil fingerprints between the oil spill and suspected oil sources, defined by ratios between the abundances of oil-discriminating compounds, Diagnostic Ratios (DR). The normalised Nordtest and EN 15522-2 methodologies use Student's t statistic (S-t) or a maximum relative difference (SC) to compare mean DR from replicate sample analysis. While the S-t method assumes the normality of DR distribution, the SC method is based on controlled DR dispersion. However, when false, the assumptions and approximations adopted can lead to low true identification rates. This work presents a novel computational tool for the statistically sound oil spill identification that allows following requirements defined by EN 15522-2, the comparison of replicate DR determinations, and the use of different DR sets and formats. The tool uses the Monte Carlo Method (MCM) to describe the probability distribution of the difference of mean DR, allowing estimating the probability of the true acceptance of fingerprints equivalence. The studied methods were applied to the comparison of signals from the same oil and to a real scenario reproduced in an International Round Robin Test. The methods were compared considering the probabilities of true acceptance of oil patterns equivalence based on a single, γ, or various, δ, DR. The MCM method performs identifications with γ equivalent to the defined confidence level for the comparison, P. Since the various DR studied are not perfectly correlated, the δ is below P. The number of replicate analyses performed and the DR considered in the comparison affect identification performance. The S-t produces comparison criteria with a γ lower than P. The SC criteria for duplicate analysis is associated with a δ lower than the obtained by the MCM. A user-friendly MS-Excel spreadsheet is available to perform oil pattern comparisons using various methods and conditions.

Microplastics contamination in sediments from Portuguese inland waters: Physical-chemical characterisation and distribution.

Plastics are the major constituent of waste accumulated in inland waters and subsequently transferred to the ocean. The smaller plastic particles, typically obtained from the fragmentation of larger pieces, are vehicles for food chain accumulation of plastic components and contaminants sorbed to these particles through their ingestion by small organisms. The monitoring of the level and trends of the contamination by microplastics is essential to determine the relevance and potential sources of this contamination necessary to define strategies to reduce this threat. This work presents microplastic contamination levels and trends of sediments of four Portuguese inland waters, namely Ria de Aveiro, Ria Formosa, Mira river, and Mondego river, between 02/2019 and 09/2020. The contamination is classified considering the type of polymer and size, shape, and colour of particles. Polymers are identified by micro-ATR-FTIR with true and false identification rates larger and lower than 95% and 5%, respectively. Duplicate analysis results are used to quantify contamination heterogeneity subsequently applied to assess if a specific contamination trend is not meaningful for a 99% confidence level. The analytical procedure is described in detail to clarify the scope of the analysis. Tests' quality is controlled by following strict quality control measures. Results from sixty-three sediment samples proved the ubiquitous presence of microplastic (MP) in these inland waters with contamination levels ranging between 20 MP kg-1 and 1090 MP kg-1, excluding six samples not contaminated with these particles. Overall, more than 86% of the microplastics were fragments lower than 1000 µm, and 33% were identified as polyethylene or polypropylene. A large diversity of microplastic colours was observed. For the Mondego River and Ria de Aveiro locations monitored for consecutive years, no significant variations of microplastic contamination were observed for a 99% confidence level.

The M. tuberculosis phosphate-binding lipoproteins PstS1 and PstS3 induce Th1 and Th17 responses that are not associated with protection against M. tuberculosis infection.

The M. tuberculosis phosphate-binding transporter lipoproteins PstS1 and PstS3 were good immunogens inducing CD8(+) T-cell activation and both Th1 and Th17 immunity in mice. However, this antigen-specific immunity, even when amplified by administration of the protein with the adjuvant LTK63 or by the DNA priming/protein boosting regimen, was not able to contain M. tuberculosis replication in the lungs of infected mice. The lack of protection might be ascribed with the scarce/absent capacity of PstS1/PstS3 antigens to modulate the IFN-γ response elicited by M. tuberculosis infection during which, however, PstS1-specific IL-17 secreting cells were generated in both unvaccinated and BCG-vaccinated mice. In spite of a lack of protection by PstS1/PstS3 immunizations, our results do show that PstS1 is able to induce IL-17 response upon M. tuberculosis infection which is of interest in the study of anti-M. tuberculosis immunity and as potential immunomodulator in combined vaccines.

Microplastics identification by infrared spectroscopy - Evaluation of identification criteria and uncertainty by the Bootstrap method.

The assessment of microplastic contamination in an environmental compartment involves identifying and counting microplastics in a representative fraction of the compartment. Microplastics can be identified by µFTIR spectroscopy where spectra are manually examined for characteristic polymer bands or by an automatic comparison of particle spectrum with reference spectra of polymers. The automatic spectra comparison can involve calculating a correlation coefficient, CC, between particle and reference spectra where a minimum correlation above which identification is adequately reliable should be defined. Correlation can be calculated from original or transformed signals, such as taking the first derivative, and by using unweighted or weighted CC. Weighted CC can highlight the spectral features more relevant to distinguish polymers. This work describes a methodology for setting the minimum CC, P5¼P, associated with a true positive result rate, TP, of 95% and for checking if this threshold allows identifications with a false positive result rate, FP, not greater than 5%. This methodology was successfully applied to the use of various CC determined from original or transformed spectra for the identification of polyethylene, PE, and polypropylene, PP, microplastics in river sediments by µFTIR. The analytical portions of sediments were digested with H2O2 and microplastics separated from the remaining particles by density using a saturated NaCl solution. Pearson's, Spearman's and Alternative unweighted and weighted correlation coefficients were studied. The P5¼P was estimated by the Bootstrap method that resamples spectra CC between a reference material and microparticle of the same polymer collected from the environment. This resampling allows simulating CC distribution required to estimate its 5th percentile (i.e. P5¼P). The FP was estimated from the probability of a particle not from the same polymer type of the reference material producing a CC greater than P5¼P. Some unweighted and weighted CC determined from original or transformed spectra were successfully used to identify PE or PP particles in river sediments. More particle spectra need to be collected to ensure performance is assessed from a representative diversity of aged polymers with different additives. The spreadsheets used for CC calculations and Bootstrap simulations are made available and can be used for the validation of the identification of other polymer types by µFTIR or ATR-FTIR spectroscopy.