Inferring upstream regulatory genes of FOXP3 in human regulatory T cells from time-series transcriptomic data.

The discovery of upstream regulatory genes of a gene of interest still remains challenging. Here we applied a scalable computational method to unbiasedly predict candidate regulatory genes of critical transcription factors by searching the whole genome. We illustrated our approach with a case study on the master regulator FOXP3 of human primary regulatory T cells (Tregs). While target genes of FOXP3 have been identified, its upstream regulatory machinery still remains elusive. Our methodology selected five top-ranked candidates that were tested via proof-of-concept experiments. Following knockdown, three out of five candidates showed significant effects on the mRNA expression of FOXP3 across multiple donors. This provides insights into the regulatory mechanisms modulating FOXP3 transcriptional expression in Tregs. Overall, at the genome level this represents a high level of accuracy in predicting upstream regulatory genes of key genes of interest.

Plastics in biogenic matrices intended for reuse in agriculture and the potential contribution to soil accumulation.

The spread of biogenic matrices for agricultural purposes can lead to plastic input into soils, raising a question on possible consequences for the environment. Nonetheless, the current knowledge concerning the presence of plastics in biogenic matrices is very poor. Therefore, the objective of the present study was a quali-quantitative characterization of plastics in different matrices reused in agriculture as manures, digestate, compost and sewage sludges. Plastics were quantified and characterized using a Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in Attenuated Total Reflectance mode. Our study showed the presence of plastics in all the investigated samples, albeit with differences in the content among the matrices. We measured a lower presence in animal matrices (0.06-0.08 plastics/g wet weight w.w.), while 3.14-5.07 plastics/g w.w. were measured in sewage sludges. Fibres were the prevalent shape and plastic debris were mostly in the micrometric size. The most abundant polymers were polyester (PEST), polypropylene (PP) and polyethylene (PE). The worst case was observed in the compost sample, where 986 plastics/g w.w. were detected. The majority of these plastics were compostable and biodegradable, with only 8% consisting of fragments of PEST and PE. Our results highlighted the need to thoroughly evaluate the contribution of reused matrices in agriculture to the plastic accumulation in the soil system.

Unveiling the multilevel impact of four water-soluble polymers on Daphnia magna: From proteome to behaviour (a case study).

The ubiquitous presence of water-soluble polymers (WSPs) in freshwater environments raises concerns regarding potential threats to aquatic organisms. This study investigated, for the first time, the effects of widely used WSPs -polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), and polyethylene glycol (PEG)- using a multi-level approach in the freshwater biological model Daphnia magna. This integrated assessment employed a suite of biomarkers, evaluation of swimming behaviour, and proteomic analysis to investigate the effects of three environmentally relevant concentrations (0.001, 0.5, and 1 mg/L) of the tested WSPs from molecular to organismal levels, assessing both acute and chronic effects. Our findings reveal that exposure to different WSPs induces specific responses at each biological level, with PEG being the only WSP inducing lethal effects at 0.5 mg/L. At the physiological level, although all WSPs impacted both swimming performance and heart rate of D. magna specimens, PAA exhibited the greatest effects on the measured behavioural parameters. Furthermore, proteomic analyses demonstrated altered protein profiles following exposure to all WSPs, with PVA emerging as the most effective.

To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

Monthly variability of floating plastic contamination in Lake Maggiore (Northern Italy).

The monitoring of plastics in freshwater ecosystems has witnessed a significant increase in recent years, driven by the awareness that approximately 80 % of marine plastic litter originates from terrestrial sources transported to the seas through lakes and rivers. Consequently, it is imperative to develop monitoring plans that offer a comprehensive understanding of plastic contamination in these aquatic environments, given their seasonal variations in hydrochemical characteristics and anthropogenic sources. Historically, most global lake monitoring campaigns have been limited to one-time or, at most, seasonal sampling. In this context, the primary objective of the present study was to assess the quantitative and qualitative monthly variations of floating plastics in Lake Maggiore, a large European lake with high ecological and economic significance. Twelve transverse transects were conducted from January to December 2022 using a Manta-net with a 100 µm mesh. Characterization of each plastic particle was performed using a µ-Fourier Transform Infrared Spectroscope (µFT-IR). The results revealed relatively low levels of contamination in Lake Maggiore when compared with other lakes worldwide exclusively from a secondary origin. However, a considerable heterogeneity was observed, both quantitatively and qualitatively. Notably, we identified a 13-fold difference between the minimum (0.02 plastics/m3 in September) and maximum (0.29 plastics/m3 in December) concentrations of plastics, accompanied by significant variations in polymer composition. Our monitoring underscored the necessity of also considering the temporal variation as a potential factor influencing plastic contamination in a lake. Moreover, frequent sampling emerged as a crucial requirement to accurately gauge the extent of plastic pollution, yielding robust and valuable data essential for effective environmental management.

Comparison of plastic pollution between waters and sediments in four Po River tributaries (Northern Italy).

The monitoring of plastic contamination in freshwaters is still pioneering in comparison with marine environments, and few studies analyzed the distribution of these pollutants in both aqueous and bottom compartments of continental waters. Therefore, the aim of this study was the comparison of plastic pollution in both waters and sediments of four Po River tributaries (Ticino, Adda, Oglio and Mincio Rivers), which outflow from the main Italian sub-alpine Lakes, in order to establish the strengths and weaknesses of both matrices. The main results pointed out a heterogeneous plastic contamination, with the lowest values in Ticino (0.9 ± 0.5 plastics/m3 in waters and 6.8 ± 4.5 plastics/kg dry weight - d.w. - in sediments) and the highest in Mincio (62.9 ± 53.9 plastics/m3 in waters and 26.5 ± 13.3 plastics/kg d.w in sediments), highlighting a plastic amount in sediments four times higher than waters. Plastic pollution, mainly due to microplastics, was associated principally to a domestic input in both waters and sediments of Ticino and Adda Rivers, as well as in sediments of Oglio, while an industrial pollution was found in waters and sediments of Mincio and Oglio waters. Our data clearly highlighted as the monitoring of both matrices provide complementary information for a holistic risk assessment of these emerging contaminants in freshwaters: the aqueous matrix provides an instantaneous picture of contamination, while sediments the history of pollution.

Plastic Contamination in Seabass and Seabream from Off-Shore Aquaculture Facilities from the Mediterranean Sea.

We characterized the presence of plastics in different organs of the gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) from some off-shore aquaculture facilities of the Mediterranean Sea. Plastics were detected in 38% of analyzed fish. Higher contamination was observed in fish from Turkey and Greece with respect to Italy, without significant differences between the geographical areas. Plastics accumulated mostly in the gastrointestinal tract and, to a lower extent, in the muscle, which represents the edible part of fish. Based on the particle detected, a maximum amount of 0.01 plastic/g wet weight (w.w.) can occur in muscles, suggesting a low input for humans through consumption. A large portion of the particles identified was represented by man-made cellulose-based fibers. The characterization of the polymeric composition suggests that plastics taken up by fish can have land-based and pelagic origins, but plastics can be introduced also from different aquaculture practices.

Assessment of behavioural effects of three water-soluble polymers in zebrafish embryos.

The water-soluble polymers (WSPs) are widely used in many industrial applications and are present in several commonly used products due to their physical-chemical characteristics: as their name indicates, despite being synthetic polymers, they are able to solubilize in water. Because of this peculiar property, both the qualitative-quantitative evaluation in aquatic ecosystems and their potential (eco)toxicological effects have been neglected until now. The aim of this study was to evaluate the possible effects of three of the most widely used WSPs as polyacrylic acid (PAA), polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) on the swimming behaviour of zebrafish (Danio rerio) embryos after the exposure to different concentrations (0.001, 0.5, 1 mg/L). The exposure lasted from the eggs' collection up to 120 h post fertilization (hpf) also using three different light intensity (300 lx, 2200 lx, 4400 lx) to better evaluate any effects related to different gradients of light/dark transitions. In order to analyze individual behavioural changes in embryos, their swimming movements were tracked and a number of parameters for locomotion and directionality were quantified. The main results showed that all three WSPs resulted in significant (p ≤ 0.05) variations in different movement parameters, suggesting a possible toxicity scale: PVP > PEG â‰« PAA.

Single-cell transcriptomics of NRAS-mutated melanoma transitioning to drug resistance reveals P2RX7 as an indicator of early drug response.

Treatment options for patients with NRAS-mutant melanoma are limited and lack an efficient targeted drug combination that significantly increases overall and progression-free survival. In addition, targeted therapy success is hampered by the inevitable emergence of drug resistance. A thorough understanding of the molecular processes driving cancer cells' escape mechanisms is crucial to tailor more efficient follow-up therapies. We performed single-cell RNA sequencing of NRAS-mutant melanoma treated with MEK1/2 plus CDK4/6 inhibitors to decipher transcriptional transitions during the development of drug resistance. Cell lines resuming full proliferation (FACs [fast-adapting cells]) and cells that became senescent (SACs [slow-adapting cells]) over prolonged treatment were identified. The early drug response was characterized by transitional states involving increased ion signaling, driven by upregulation of the ATP-gated ion channel P2RX7. P2RX7 activation was associated with improved therapy responses and, in combination with targeted drugs, could contribute to the delayed onset of acquired resistance in NRAS-mutant melanoma.

First comparative assessment of contamination by plastics and non-synthetic particles in three bivalve species from an Italian sub-alpine lake.

This study aimed to compare the contamination from plastics and non-synthetic particles in the three freshwater bivalve mollusks Unio elongatulus, (native) and Corbicula fluminea and Dreissena polymorpha (invasive), collected in Lake Maggiore, the second greatest Italian lake. Organisms were collected from eight sites located throughout the lake, during three years (2019-2021). The quali-quantitative characterization of particles has been carried out using a Fourier Transform Infrared Microscope System (µFT-IR). Results showed that both plastics and non-synthetic particles released in the water are taken up by bivalves, even though low intake-up to 6 particles/individuals-were measured for all the three species. Microfibers of both synthetic (polyester, polyamide) and natural (cellulose) origin represented the particles mostly ingested by bivalves. A significant decrease of particle loads was observed in 2020 with respect to 2019 and 2021, significantly different for D. polymorpha and U. elongatulus, suggesting a transient reduction of the particle release in the lake in this year. Our findings highlight the need to improve the understanding of the mechanisms of uptake and clearance of these contaminants by filter feeding organisms, and their adverse consequences in realistic environmental conditions.

Packaging contaminants in former food products: Using Fourier Transform Infrared Spectroscopy to identify the remnants and the associated risks.

Food waste and feed-food competition can be reduced by replacing traditional feed ingredients such as cereals, with former food products (FFPs) in livestock diets. These foodstuffs, initially intended for human consumption, are recovered, mechanically unpacked, and then ground. Despite this simple and inexpensive treatment, packaging contaminants (remnants) are often unavoidable in the final product. To maximize the exploitation of FFPs and to minimize the associated risks, packaging remnants need to be quantified and characterized. This study tested the efficacy of the Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in identifying packaging remnants in 17 FFP samples collected in different geographical areas. After a visual sorting procedure, presumed packaging remnants were analyzed by µFT-IR. The results showed significant differences (p < 0.05) between the FFPs in terms of the total number of foreign particles found (plastics, cellulose and aluminum remnants, ranging from 4 to 19 particles per 20 g fresh matter), and also regarding the number of cellulose and aluminum particles. These data clearly demonstrate the need for sensitive instruments that can characterize the potential contaminants in the FFPs. This would then help to reduce the overestimation of undesirable contaminants typical of simple visual sorting, which is currently the most common method.

Ensemble classification combining ResNet and handcrafted features with three-steps training.

Objective.This work presents an ECG classifier for variable leads as a contribution to the Computing in Cardiology Challenge/CinC Challenge 2021. It aims to integrate deep and classic machine learning features into a single model, exploring the proper structure and training procedure.Approach.From the initial 88 253 signals, only 84 210 were included. Low quality and unscored recordings were excluded. Three different database subsets of 40 365 recording each were created by dividing in three normal sinus rhythm and sinus bradycardia recordings. Each subset was used to train a different model with shared architecture integrated as an ensemble to provide the final classification through major voting. Models contained a deep branch composed of a modified ResNet with dilation convolutional layers and squeeze and excitation Block that took as input windowed ECG signals. This was concatenated with a wide branch that integrated 20 cardiac rhythm features into a fully connected 3-layered network. Three different training steps were studied: just the deep branch (D), wide integration and training (D+W), and a final fine tuning of the deep branch posterior to wide training (D+W+D).Main Results.Results obtained in a local test set formed by a stratified 12.5% split of the given full dataset were presented for 2-lead and 12-lead models. The best training method was the 3-step D + W + D procedure obtaining a challenge metric of 0.709 and 0.677 for 12 and 2-lead models respectively.Significance.Integration of handcrafted features and deep learning model not only may increase the generalization capacity of the network but also provide a path to add explicit information into the classification decision process. To the best of our knowledge this is the first work studying the training procedure to properly integrate both types of information for ECG signals classification.

Surviving in a changing ocean. Tolerance to acidification might affect the susceptibility of polychaetes to chemical contamination.

This study aimed to assess the combined effects of ocean acidification (OA) and pollution to the polychaete Syllis prolifera inhabiting the CO2 vent system of the Castello Aragonese (Ischia Island, Italy). We investigated the basal activities of antioxidant enzymes in organisms from the acidified site and from an ambient-pH control site in two different periods of the year. Results showed a limited influence of acidified conditions on the functionality of the antioxidant system. We then investigated the responsiveness of individuals living inside the CO2 vent compared to those from the control to face exposure to acetone and copper. Results highlighted a higher susceptibility of organisms from the vent to acetone and a different response of antioxidant enzymes in individuals from the two sites. Conversely, a higher tolerance to copper was observed in polychaetes from the acidified-site with respect to controls, but any significant oxidative stress was induced at sublethal concentrations.

Ecological Impact of End-of-Life-Tire (ELT)-Derived Rubbers: Acute and Chronic Effects at Organism and Population Levels.

Considering the large amount of tires that reach the end of life every year, the aim of this study was the evaluation of both acute and chronic effects of end-of-life-tire (ELT)-derived rubber granules (ELT-dg) and powder (ELT-dp) on a freshwater trophic chain represented by the green alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the teleost Danio rerio (zebrafish). Adverse effects were evaluated at the organism and population levels through the classical ecotoxicological tests. Acute tests on D. magna and D. rerio revealed a 50% effect concentration (EC50) > 100.0 mg/L for both ELT-dg and ELT-dp. Chronic exposures had a lowest observed effect concentration (LOEC) of 100.0 mg/L for both ELT-dg and ELT-dp on P. subcapitata grow rate and yield. LOEC decreased in the other model organisms, with a value of 9.8 mg/L for D. magna, referring to the number of living offspring, exposed to ELT-dg suspension. Similarly, in D. rerio, the main results highlighted a LOEC of 10.0 mg/L regarding the survival and juvenile weight parameters for ELT-dg and a LOEC of 10.0 mg/L concerning the survival and abnormal behavior in specimens exposed to ELT-dp. Tested materials exhibited a threshold of toxicity of 9.8 mg/L, probably a non-environmental concentration, although further investigations are needed to clarify the potential ecological impact of these emerging contaminants.

Are "liquid plastics" a new environmental threat? The case of polyvinyl alcohol.

Despite the pollution induced by plastics become a well-known and documented problem, bringing many countries to adopt restrictions about their production, commercialization and use, the impact of another emerging category of synthetic polymers, represented by the Water-Soluble Polymers (WSPs), also known as "liquid plastics", is overlooked by scientific community. WSPs are produced in large quantities and used in a wide plethora of applications such as food packaging, pharmaceuticals and personal care products, cosmetics and detergents, with a consequent continuous release in the environment. The aim of this study was the investigation of the possible toxicity induced by polyvinyl alcohol (PVA), one of the main produced and used WSPs, on two freshwater model organisms, the crustacean Daphnia magna and the teleost Danio rerio (zebrafish). We evaluated the effects of solubilized standard PVA powder and PVA-based commercial bags for carp-fishing, at 3 different concentrations (1 µg/L, 0.5 mg/L and 1 mg/L), through the exposures for 14 days of D. magna (daphnids; age < 24 h) and for 5 days of zebrafish embryos (up to 120 h post fertilization - hpf). As acute effects we evaluated the immobilization/mortality of specimens, while for chronic toxicity we selected several endpoints with a high ecological relevance, as the behavioural alteration on swimming performance, in real-time readout, and the activity of monoamine oxidase (MAO), a neuro-enzyme with a potential implication in the organism movement. The results showed the lack of significant effects induced by the selected substances, at all tested concentrations and in both model organisms. However, considering the wide plethora of available WSPs, other investigations are needed to provide the initial knowledge of risk assessment of these compounds contained in some consumer products.

Can COVID-19 pandemic change plastic contamination? The Case study of seven watercourses in the metropolitan city of Milan (N. Italy).

The more or less extensive lockdowns, quarantines, smart working and the closure of numerous recreational or personal care activities due to the COVID-19 pandemic have not only heavily changed the habits and behaviors of all of us, but have also had consequences on the release of some types of pollutants. The aim of this study was to evaluate the possible changes due to the indirect effects of the pandemic in the contamination of plastic mixtures sampled in 9 sites of the main watercourses of the metropolitan city of Milan (N. Italy), which is one of the major industrialized and urbanized areas in Italy. To achieve this goal, we carried out two sampling campaigns, the first one carried out in November 2019, before the arrival of the SARS-CoV-2 virus in Italy, the second in November 2020, during a severe regional lockdown that coincided with other restrictions imposed at the national level. The main results showed a difference in contamination of plastics between the two samplings, not so much due to a quantitative variation, but certainly qualitative. We obtained non-homogeneous data with respect to changes in the number of plastics sampled in the different waterbodies, while it was evident that the plastics' contamination has shifted from a primary and industrial origin to one due to a secondary origin of the sampled plastics, linked especially to the fragmentation of common use objects, or deriving from synthetic garments.

Model-based assessment of COVID-19 epidemic dynamics by wastewater analysis.

Continuous surveillance of COVID-19 diffusion remains crucial to control its diffusion and to anticipate infection waves. Detecting viral RNA load in wastewater samples has been suggested as an effective approach for epidemic monitoring and the development of an effective warning system. However, its quantitative link to the epidemic status and the stages of outbreak is still elusive. Modelling is thus crucial to address these challenges. In this study, we present a novel mechanistic model-based approach to reconstruct the complete epidemic dynamics from SARS-CoV-2 viral load in wastewater. Our approach integrates noisy wastewater data and daily case numbers into a dynamical epidemiological model. As demonstrated for various regions and sampling protocols, it quantifies the case numbers, provides epidemic indicators and accurately infers future epidemic trends. Following its quantitative analysis, we also provide recommendations for wastewater data standards and for their use as warning indicators against new infection waves. In situations of reduced testing capacity, our modelling approach can enhance the surveillance of wastewater for early epidemic prediction and robust and cost-effective real-time monitoring of local COVID-19 dynamics.

Performance of early warning signals for disease re-emergence: A case study on COVID-19 data.

Developing measures for rapid and early detection of disease re-emergence is important to perform science-based risk assessment of epidemic threats. In the past few years, several early warning signals (EWS) from complex systems theory have been introduced to detect impending critical transitions and extend the set of indicators. However, it is still debated whether they are generically applicable or potentially sensitive to some dynamical characteristics such as system noise and rates of approach to critical parameter values. Moreover, testing on empirical data has, so far, been limited. Hence, verifying EWS performance remains a challenge. In this study, we tackle this question by analyzing the performance of common EWS, such as increasing variance and autocorrelation, in detecting the emergence of COVID-19 outbreaks in various countries. Our work illustrates that these EWS might be successful in detecting disease emergence when some basic assumptions are satisfied: a slow forcing through the transitions and not-fat-tailed noise. In uncertain cases, we observe that noise properties or commensurable time scales may obscure the expected early warning signals. Overall, our results suggest that EWS can be useful for active monitoring of epidemic dynamics, but that their performance is sensitive to certain features of the underlying dynamics. Our findings thus pave a connection between theoretical and empirical studies, constituting a further step towards the application of EWS indicators for informing public health policies.

A realistic approach for the assessment of plastic contamination and its ecotoxicological consequences: A case study in the metropolitan city of Milan (N. Italy).

The study of the contamination of plastic mixtures sampled in natural environments is currently focused on their qualitative and quantitative assessment, while the evaluation of their effects on organisms is normally performed by experiments carried out at exposure conditions (size, shape, polymers) often far from the environmental ones. To improve the ecological realism, the aim of this study was to collect different plastic mixtures in 9 sampling stations located in 7 watercourses within the metropolitan city of Milan, one of the most anthropized and industrialized European areas, to evaluate both their qualitative and quantitative characteristics and, at the same time, to assess their ecotoxicological effects by exposing for 7 days some specimens of the freshwater bivalve Dreissena polymorpha to the mixtures collected in the sampling sites. The plastic characterization was performed by a Fourier-Transform Infrared spectrometer coupled with an optical microscope (µFT-IR), after several stages aimed to sample cleaning, separation of plastics and visual sorting. The possible effects caused by the plastic mixtures were carried out by the measurements of a biomarker suite to evaluate many cellular and molecular endpoints in mussel tissues. The main results showed a widespread and heterogeneous contamination of plastics in the entire metropolitan area, with contamination peaks found above all in the only two rivers of natural origin (Olona River and Lambro River) where comparable or higher values were reached than plastic concentrations measured in several European rivers. Despite this worrying contamination, the ecotoxicological data obtained after the exposures to the plastic mixtures collected in the selected water bodies showed only a mild effect on oxidative stress and on the variation of some antioxidant enzymes.

The Parkinson's-disease-associated mutation LRRK2-G2019S alters dopaminergic differentiation dynamics via NR2F1.

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.