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Chronic Mg 2+ deficiency is the underlying cause of a broad range of health dysfunctions. As 25% of body Mg 2+ is located in the skeletal muscle, Mg 2+ transport and homeostasis systems (MgTHs) in the muscle are critical for whole-body Mg 2+ homeostasis. In the present study, we assessed whether Mg 2+ deficiency alters muscle fiber characteristics and major pathways regulating muscle physiology. C57BL/6J mice received either a control, mildly, or severely Mg 2+ -deficient diet (0.1%; 0.01%; and 0.003% Mg 2+ wt/wt, respectively) for 14 days. Mg 2+ deficiency slightly decreased body weight gain and muscle Mg 2+ concentrations but was not associated with detectable variations in gastrocnemius muscle weight, fiber morphometry, and capillarization. Nonetheless, muscles exhibited decreased expression of several MgTHs ( MagT1 , CNNM2 , CNNM4 , and TRPM6 ). Moreover, TaqMan low-density array (TLDA) analyses further revealed that, before the emergence of major muscle dysfunctions, even a mild Mg 2+ deficiency was sufficient to alter the expression of genes critical for muscle physiology, including energy metabolism, muscle regeneration, proteostasis, mitochondrial dynamics, and excitation-contraction coupling.

Purpose: To evaluate safety and efficacy of a mesh reinforcement following stoma reversal to prevent stoma site incisional hernia (SSIH) and differences across the prostheses used., Methods: A systematic search of PubMed/MEDLINE, EMBASE, SCOPUS and Cochrane databases was conducted to identify comparative studies until September 2020. A meta-analysis of postoperative outcomes and a network meta-analysis for a multiple comparison of the prostheses with each other were performed., Results: Seven studies were included in the analysis (78.4% ileostomy and 21.6% colostomy) with a total of 1716 patients with (n = 684) or without (n = 1032) mesh. Mesh placement was associated with lower risk of SSIH (7.8%vs18.1%, OR0.266,95% CI 0.123-0.577, p < 0.001) than no mesh procedures but also with a longer operative time (SMD 0.941, 95% CI 0.462-1.421, p < 0.001). There was no statistically significant difference in terms of Surgical Site infection (11.5% vs 11.1%, OR 1.074, 95% CI 0.78-1.48, p = 0.66), seroma formation (4.4% vs 7.1%, OR 1.052, 95% CI 0.64-1.73, p = 0.84), anastomotic leakage (3.7% vs 2.7%, OR 1.598, 95% CI 0.846-3.019, p = 0.149) and length of stay (SMD - 0.579,95% CI - 1.261 to 0.102, p = 0.096) between mesh and no mesh groups. Use of prosthesis was associated with a significant lower need for a reoperation than no mesh group (8.1% vs 12.1%, OR 0.332, 95% CI 0.119-0.930, p = 0.036). Incidence of seroma is lower with biologic than polypropylene meshes but they showed a trend towards poor results compared with polypropylene or biosynthetic meshes., Conclusion: Despite longer operative time, mesh prophylactic reinforcement at the site of stoma seems a safe and effective procedure with lower incidence of SSIH, need for reoperation and comparable short-term outcomes than standard closure technique. A significant superiority of a specific mesh type was not identified.

Chemoresistance causes cancer relapse and metastasis, thus remaining the major obstacle to cancer therapy. While some light has been shed on the underlying mechanisms, it is clear that chemoresistance is a multifaceted problem strictly interconnected with the high heterogeneity of neoplastic cells. We utilized two different human cell lines, i.e., LoVo colon cancer and promyelocytic leukemia HL60 cells sensitive and resistant to doxorubicin (DXR), largely used as a chemotherapeutic and frequently leading to chemoresistance. LoVo and HL60 resistant cells accumulate less reactive oxygen species by differently modulating the levels of some pro- and antioxidant proteins. Moreover, the content of intracellular magnesium, known to contribute to protect cells from oxidative stress, is increased in DXR-resistant LoVo through the upregulation of MagT1 and in DXR-resistant HL60 because of the overexpression of TRPM7. In addition, while no major differences in mitochondrial mass are observed in resistant HL60 and LoVo cells, fragmented mitochondria due to increased fission and decreased fusion are detected only in resistant LoVo cells. We conclude that DXR-resistant cells evolve adaptive mechanisms to survive DXR cytotoxicity by activating different molecular pathways.

Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated information. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent (>65% of the cities), followed by mephedrone (>25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualitative and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Prenatal exposure to valproate (VPA), an antiepileptic drug, has been associated with fetal valproate spectrum disorders (FVSD), a clinical condition including congenital malformations, developmental delay, intellectual disability as well as autism spectrum disorder, together with a distinctive facial appearance. VPA is a known inhibitor of histone deacetylase which regulates the chromatin state. Interestingly, perturbations of this epigenetic balance are associated with chromatinopathies, a heterogeneous group of Mendelian disorders arising from mutations in components of the epigenetic machinery. Patients affected from these disorders display a plethora of clinical signs, mainly neurological deficits and intellectual disability, together with distinctive craniofacial dysmorphisms. Remarkably, critically examining the phenotype of FVSD and chromatinopathies, they shared several overlapping features that can be observed despite the different etiologies of these disorders, suggesting the possible existence of a common perturbed mechanism(s) during embryonic development., Competing Interests: RF formerly held a leadership position in TeratOmic Consulting LLC. This now dissolved organization provided expert consulting support in birth defects litigation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parodi, Di Fede, Peron, Viganò, Grazioli, Castiglioni, Finnell, Gervasini, Vignoli and Massa.)

Drug-eluting bioresorbable scaffolds represent the last frontier in the field of angioplasty and stenting to treat coronary artery disease, one of the leading causes of morbidity and mortality worldwide. In particular, sirolimus-eluting magnesium-based scaffolds were recently introduced in clinical practice. Magnesium alloys are biocompatible and dissolve in body fluids, thus determining high concentrations of magnesium in the local microenvironment. Since magnesium regulates cell growth, we asked whether high levels of magnesium might interfere with the antiproliferative action of sirolimus. We performed in vitro experiments on rabbit coronary artery endothelial and smooth muscle cells (rCAEC and rSMC, respectively). The cells were treated with sirolimus in the presence of different concentrations of extracellular magnesium. Sirolimus inhibits rCAEC proliferation only in physiological concentrations of magnesium, while high concentrations prevent this effect. On the contrary, high extracellular magnesium does not rescue rSMC growth arrest by sirolimus and accentuates the inhibitory effect of the drug on cell migration. Importantly, sirolimus and magnesium do not impair rSMC response to nitric oxide. If translated into a clinical setting, these results suggest that, in the presence of sirolimus, local increases of magnesium concentration maintain normal endothelial proliferative capacity and function without affecting rSMC growth inhibition and response to vasodilators.

Background: The spread of the SARS-CoV2 virus, which causes COVID-19 disease, profoundly impacted the surgical community. Recommendations have been published to manage patients needing surgery during the COVID-19 pandemic. This survey, under the aegis of the Italian Society of Endoscopic Surgery, aims to analyze how Italian surgeons have changed their practice during the pandemic., Methods: The authors designed an online survey that was circulated for completion to the Italian departments of general surgery registered in the Italian Ministry of Health database in December 2020. Questions were divided into three sections: hospital organization, screening policies, and safety profile of the surgical operation. The investigation periods were divided into the Italian pandemic phases I (March-May 2020), II (June-September 2020), and III (October-December 2020)., Results: Of 447 invited departments, 226 answered the survey. Most hospitals were treating both COVID-19-positive and -negative patients. The reduction in effective beds dedicated to surgical activity was significant, affecting 59% of the responding units. 12.4% of the respondents in phase I, 2.6% in phase II, and 7.7% in phase III reported that their surgical unit had been closed. 51.4%, 23.5%, and 47.8% of the respondents had at least one colleague reassigned to non-surgical COVID-19 activities during the three phases. There has been a reduction in elective (> 200 procedures: 2.1%, 20.6% and 9.9% in the three phases, respectively) and emergency (< 20 procedures: 43.3%, 27.1%, 36.5% in the three phases, respectively) surgical activity. The use of laparoscopy also had a setback in phase I (25.8% performed less than 20% of elective procedures through laparoscopy). 60.6% of the respondents used a smoke evacuation device during laparoscopy in phase I, 61.6% in phase II, and 64.2% in phase III. Almost all responders (82.8% vs. 93.2% vs. 92.7%) in each analyzed period did not modify or reduce the use of high-energy devices., Conclusion: This survey offers three faithful snapshots of how the surgical community has reacted to the COVID-19 pandemic during its three phases. The significant reduction in surgical activity indicates that better health policies and more evidence-based guidelines are needed to make up for lost time and surgery not performed during the pandemic.

Targeted therapy improves outcomes in BRAF V600-mutant metastatic melanoma with active brain metastases. We present the case of a patient with rapid brain disease progression upon temporary targeted therapy discontinuation and unusual rapid disease response upon treatment resumption. This report presents a 78-year-old woman with metastatic BRAF V600E positive melanoma (bladder and brain localizations). The patient started first-line dabrafenib and trametinib with good tolerability and evidence of complete response (CR). After 8 months of maintained CR, the patient took a drug holiday for 14 days. Brain MRI performed after treatment pause showed extensive disease progression, whereas extracranial staging was negative. The patient was asymptomatic: she restarted targeted therapy and underwent evaluation for whole-brain radiotherapy. Brain computed tomography scan and subsequent MRI performed to plan radiotherapy showed brain CR after only 10 days of targeted therapy resumption. The patient continued treatment, and radiotherapy indication was withheld. Repeated brain MRI confirmed maintained CR. Treatment with dabrafenib and trametinib is ongoing with excellent tolerability. Rapid intracranial progression is a well-known finding after discontinuation of combined targeted therapy in the case of extracranial progressive disease. This is the first report of documented disease progression upon temporary treatment discontinuation for reasons other than toxicity, with an unusual response after retreatment. Caution should be used in tailoring treatment during targeted therapy, allowing pauses for reasons other than toxicity. Strict adherence to treatment is paramount to guarantee disease control., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

There is considerable concern around the use of new psychoactive substances (NPS), but still little is known about how much they are really consumed. Analysis by forensics laboratories of seized drugs and post-mortem samples as well as hospital emergency rooms are the first line of identifying both 'new' NPS and those that are most dangerous to the community. However, NPS are not necessarily all seized by law enforcement agencies and only substances that contribute to fatalities or serious afflictions are recorded in post-mortem and emergency room samples. To gain a better insight into which NPS are most prevalent within a community, complementary data sources are required. In this work, influent wastewater was analysed from 14 sites in eight countries for a variety of NPS. All samples were collected over the 2019/2020 New Year period, a time which is characterized by celebrations and parties and therefore a time when more NPS may be consumed. Samples were extracted in the country of origin following a validated protocol and shipped to Australia for final analysis using two different mass spectrometric strategies. In total, more than 200 were monitored of which 16 substances were found, with geographical differences seen. This case study is the most comprehensive wastewater analysis study ever carried out for the identification of NPS and provides a starting point for future, ongoing monitoring of these substances., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Magnesium (Mg) is essential to skeletal muscle where it plays a key role in myofiber relaxation. Although the importance of Mg in the mature skeletal muscle is well established, little is known about the role of Mg in myogenesis. We studied the effects of low and high extracellular Mg in C2C12 myogenic differentiation. Non-physiological Mg concentrations induce oxidative stress in myoblasts. The increase of reactive oxygen species, which occurs during the early phase of the differentiation process, inhibits myoblast membrane fusion, thus impairing myogenesis. Therefore, correct Mg homeostasis, also maintained through a correct dietary intake, is essential to assure the regenerative capacity of skeletal muscle fibers.

New psychoactive substances (NPS), often designed as (legal) substitutes to conventional illicit drugs, are constantly emerging in the drug market and being commercialized in different ways and forms. Their use continues to cause public health problems and is therefore of major concern in many countries. Monitoring NPS use, however, is arduous and different sources of information are required to get more insight of the prevalence and diffusion of NPS use. The determination of NPS in pooled urine and wastewater has shown great potential, adding a different and complementary light on this issue. However, it also presents analytical challenges and limitations that must be taken into account such as the complexity of the matrices, the high sensitivity and selectivity required in the analytical methods as a consequence of the low analyte concentrations as well as the rapid transience of NPS on the drug market creating a scenario with constantly moving analytical targets. Analytical investigation of NPS in pooled urine and wastewater is based on liquid chromatography hyphenated to mass spectrometry and can follow different strategies: target, suspect and non-target analysis. This work aims to discuss the advantages and disadvantages of the different data acquisition workflows and data exploration approaches in mass spectrometry, but also pays attention to new developments such as ion mobility and the use of in-silico prediction tools to improve the identification capabilities in high-complex samples. This tutorial gives an insight into this emerging topic of current concern, and describes the experience gathered within different collaborations and projects supported by key research articles and illustrative practical examples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The purpose of this study was to evaluate the effect of 3D visualization applied to laparoscopic appendectomy (LA) performed by young surgeons (YS). We considered both operative features and clinical outcomes, aiming to highlight the benefits that this technology could bring to novice surgeons and their laparoscopic training., Methods: All the surgical procedures were performed by residents who had performed less than 20 appendectomies prior to the beginning of the study under the supervision of an expert surgeon. At the time of enrolment patients were randomized into two arms: Experimental arm (EA): laparoscopic appendectomy performed with laparoscopic 3D vision technology. Control arm (CA): laparoscopic appendectomy performed with the "standard" 2D technology. The primary endpoint was to find any statistically significant difference in operative time between the two arms. Differences in conversion rate, intra-operative complications, post-operative complications and surgeons' operative comfort were considered as secondary endpoints., Results: We randomized 135 patients into the two study arms. The two groups were homogeneous for demographic characteristics, BMI and ASA scores. The characteristics of clinical presentation and anatomical position showed no significant difference. The operative time was longer in the CA (57.5 vs. 49.6 min, p = 0.048, 95% CI). In the subgroup of complicated appendicitis, this trend toward inferior operative time was confirmed without reaching statistical significance (2D = 60 min, 3D = 49.5 min, p = 0.082 95% CI). No intra-operative complications were observed in either group. The conversion rate was 5.6% (4 patients) in the 2D group and 4.6% (3 patients) in 3D group., Conclusion: The utilization of 3D laparoscopy was associated with reduction in operative time without influencing other parameters, in particular without altering the safety profile of the procedure.

Sewage sludge is a by-product of wastewater treatment processes, and may be employed in agriculture as a fertilizer or in forestry for land reclamation. It is an important source of nutrients but its reuse can arouse concern on account of the wide range of contaminants that are retained and may persist during treatments. Information on the emerging contaminants (ECs) in sewage sludge in Italy is limited. The present study developed and applied a reliable analytical method for the analysis of 44 ECs in sewage sludge. ECs were extracted by accelerated solvent extraction followed by a clean-up step on solid-phase cartridges. High performance liquid chromatography coupled to mass spectrometry was used for analysis. The ECs, selected on the basis of their use and documented presence in the environment, were 42 pharmaceuticals belonging to 12 therapeutic categories and 2 perfluorinated substances. The method performance was good, with recoveries higher than 70%, good repeatability (<20%) and sensitivity in the low ng g -1 range, allowing measurement of the analytes selected. The method was applied for analysis of sludge from 12 wastewater treatment plants in Italy. The most abundant compounds were antibiotics, anti-inflammatories and antihypertensives and ranged up to 5 μg g -1 (ciprofloxacin). Seasonal differences were found for some antibiotics and anti-inflammatory drugs as well as some differences - in terms of concentration - with other European countries. This is the first Italian study to investigate the presence of a large number of ECs in sewage sludge and the results may be useful to drive future regulatory actions.

Background: Magnesium deficiency contributes to atherogenesis partly by promoting the dysfunction of endothelial cells, which are critical in vascular homeostasis and diseases. Since EDF-1 and PPARγ regulate crucial endothelial activities, we investigated the modulation of these proteins involved in lipogenesis as well the deposition of lipids in human endothelial cells cultured in different concentrations of magnesium., Methods: Human endothelial cells from the umbilical vein were cultured in medium containing from 0.1 to 5 mM magnesium for 24 h. The levels of EDF-1 and PPARγ were visualized by Western blot. Reactive oxygen species (ROS) were measured by DCFDA. Lipids were detected after O Red Oil staining., Results: Magnesium deficiency leads to the accumulation of ROS which upregulate EDF-1. Further, PPARγ is increased after culture in low magnesium, but independently from ROS. Moreover, lipids accumulate in magnesium-deficient cells., Conclusions: Our results suggest that magnesium deficiency leads to the deposition of lipids by inducing EDF-1 and PPARγ. The increase in intracellular lipids might be interpreted as an adaptive response of endothelial cells to magnesium deficiency.

Smoking cigarettes and drinking coffee are common habits in today's society. However, it is not easy to get up-to-date information on smoking prevalence and caffeine consumption as it is usually obtained from population surveys. To overcome this limitation and complement epidemiological information, we employed wastewater-based epidemiology (WBE) to gain a picture of the consumption of cigarettes and caffeine per day per person in Italy. A nationwide study was conducted by measuring two urinary metabolites of nicotine (cotinine and trans-3'-hydroxycotinine) and a caffeine metabolite (1,7-dimethyluric acid) in untreated wastewater from 16 cities. The spatial profiles of use depended mostly on the specific population habits in each city, not on the geographical area or the population size. The patterns of consumption were stable over the week, except in Milan where the use of both substances decreased on Sundays, probably because there were no commuters. In Milan, the use of nicotine decreased from 2013 to 2015. There was a significant relationship between smoking and consumption of products containing caffeine (mainly coffee), thus in cities where more cigarettes were smoked, more caffeine was drunk. These results are generally in accordance with findings from epidemiological studies, but provide some additional local profiles of use and closely follow changes over the years. This information could be useful for healthcare professionals and policy-makers to monitor progress towards the reduction of prevalence in tobacco use, and set up new health campaigns., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.

Background: Italy experienced one of the world's severest COVID-19 outbreak, with Lombardy being the most afflicted region. However, the imposed safety measures allowed to flatten the epidemic curve and hence to ease the restrictions and inaugurate, on the 4th of May 2020, the Italian phase (P) 2 of the pandemic. The present survey study, endorsed by CODRAL and AIRO-L, aimed to assess how radiotherapy (RT) departments in Lombardy have dealt with the recovery., Materials and Methods: A questionnaire dealing with the management of pandemic was developed online and sent to all CODRAL Directors on the 10th of June 2020. Answers were collected in full anonymity one week after., Results: All the 33 contacted RT facilities (100%) responded to the survey. Despite the scale of the pandemic, during P1 14 (42.4%) centres managed to safely continue the activity (≤ 10% reduction). During P2, 10 (30.3%) centres fully recovered and 14 (42.4%) reported an increase. Nonetheless, 6 (18.2%) declared no changes and, interestingly, 3 (9.1%) reduced activities. Overall, 21 centres (63.6%) reported suspected or positive cases within healthcare workforce since the beginning of the pandemic. Staff units were quarantined in 19 (57.6%) and 6 (18.2%) centres throughout P1 and P2, respectively. In the two phases, about two thirds centres registered positive or suspected cases amongst patients., Conclusion: The study revealed a particular attention to anti-contagion measures and a return to normal or even higher clinical workload in most RT centres in Lombardy, necessary to carry out current and previously deferred treatments.

The contamination by pharmaceuticals products (PPs) in the marine environment is particularly relevant where wastewater treatment of urban areas on land is lacking. However, the number of studies focused on description of sources and fate of PP molecules in the marine environment remains still limited. In this study, the occurrence of 46 PPs was investigated in the marine and coastal-marine system (waters and sediments) of Augusta Bay (central Mediterranean Sea). This area is highly affected by industrial pollution and urban discharges (without wastewater treatment) and thus represents a 'natural laboratory' for exploring dynamics of multi-mixture contaminants in the marine environment. The study area is also part of the sub-region 'Central Mediterranean Sea' of the Marine Strategy Framework Directive and therefore offers an important reference site for exploring the distribution modes of PPs in the central Mediterranean Sea. In this work, samples of seawater, sediment, untreated wastewater, and marine receiving water were analysed using mass spectrometry with a target analysis for PPs and a suspect screening analysis for the presence of other contaminants. PPs concentration ranges were: 2426-67,155 ng/L for untreated wastewaters, 550-27,889 ng/L for marine receiving waters and 12-281 ng/L for seawaters. The highest concentrations were measured for the antibiotics, anti-inflammatories, cardiovascular and antihypertensive therapeutic classes. Likewise, sediments collected from untreated wastewater sewers resulted more contaminated. Ionic, non-ionic surfactants and personal care products were the most abundant compounds found in waters and sediments by suspect screening analysis. The risk associated with PPs contamination for aquatic organisms was relatively high in samples of marine receiving waters of the bay (with a risk quotient value up to 33,599). The levels of PPs in seawater and sediment compartments were generally not hazardous (RQ < 0.01), except for estrone with a calculated RQ = 2775., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sub-optimal adherence to pharmacological therapy is one of the main reasons for poor effectiveness, reducing the patient's quality of life and affecting health-care economics. This study investigated the possibility for a wastewater-based epidemiology approach to assess the overall adherence of a population to some pharmacological therapies in a defined area. We selected specific active ingredients and their main urinary metabolites (biomarkers) according to the best practice protocol available and we measured them for the first time in urban wastewater. We conducted this first case study in Italy, considering the whole country and two Italian regions by sampling for five days six cities from north to south. Liquid chromatography tandem mass spectrometry was used to measure the biomarkers in raw wastewater, developing and validating specific analytical methods. We used concentrations measured in influent wastewater, together with a careful review of the excretion profile of the active ingredients selected, to back-calculate consumption (measured consumptions - MC) which were then compared with consumptions calculated from medical prescriptions (expected consumptions - EC) reported annually by the Italian Medicine Agency. In general there was a rough correlation between MC and EC, with ratios sometimes close to unity and always within a 0.3-3.0 range. Interpretation of the results suffers some biases, which are case-by-case discussed in detail, but inclusion of measurements of the urinary metabolites together with the active ingredients gave a fuller understanding of the results., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Consumption of alcohol and new psychoactive substances (NPS) in a population or during special events (music festivals) is usually monitored through individual questionnaires, forensic and toxicological data, and drug seizures. However, consumption estimates have some biases due mostly to the unknown composition of drug pills for NPS and stockpiling for alcohol. The aim of this study was to evaluate for the first time the real use of alcohol and the occurrence of NPS in Slovakia by wastewater-based epidemiology (WBE). Urban wastewater samples were collected from nine Slovak cities over two years (2017-2018) and during three music festivals. The study included about 20% of the Slovak population and 50 000 festival attendees. The urinary alcohol biomarker ethyl sulfate (EtS) and thirty NPS were analyzed by liquid chromatography tandem mass spectrometry (LC - MS/MS). EtS concentrations were used for estimating the per capita alcohol consumption in each city. The average alcohol consumption in the selected cities and festivals in 2017-2018 ranged between 7 and 126 L/day/1000 inhabitants and increased during the weekends and music festivals. Five NPS belonging to the classes of synthetic cathinones (mephedrone, methcathinone, buphedrone and pentedrone) and phenethylamines (25-iP-NBoMe) were found in the low ng/L range. Methcathinone was the most frequently detected NPS, while the highest normalized mass load corresponded to mephedrone (3.1 mg/day/1000 inhabitants). Wastewater-based epidemiology can provide timely information on alcohol consumption and NPS occurrence at the community level that is complementary to epidemiology-based monitoring techniques (e.g. population surveys, police seizures, sales statistics)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The consumption of psychoactive substances is considered a growing problem in many communities. Moreover, new psychoactive substances (NPS) designed as (legal) substitutes to traditional illicit drugs are relatively easily available to the public through e-commerce and retail shops, but there is little knowledge regarding the extent and actual use of these substances. This study aims to gain new and complementary information on NPS and traditional illicit drug use at six music festivals across Europe by investigating wastewater and pooled urine. Samples were collected, between 2015 and 2018, at six music festivals across Europe with approximately 465.000 attendees. Wastewater samples were also collected during a period not coinciding with festivals. A wide-scope screening for 197 NPS, six illicit drugs and known metabolites was applied using different chromatography-mass spectrometric strategies. Several illicit drugs and in total 21 different NPS, mainly synthetic cathinones, phenethylamines and tryptamines, were identified in the samples. Ketamine and the traditional illicit drugs, such as amphetamine-type stimulants, cannabis and cocaine were most abundant and/or frequently detected in the samples collected, suggesting a higher use compared to NPS. The analyses of urine and wastewater is quick and a high number of attendees may be monitored anonymously by analysing only a few samples which allows identifying the local profiles of use of different drugs within a wide panel of psychoactive substances. This approach contributes to the development of an efficient surveillance system which can provide timely insight in the trends of NPS and illicit drugs use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pesticides offer many benefits for humanity and agriculture, but at the same time pose a potential risk to human health because of their widespread use and high biological activity. Human biomonitoring (HBM) studies are the main tool to investigate human exposure to pesticides and other chemicals, but face limitations such as sampling biases, long time to complete and high costs. Wastewater-based epidemiology (WBE) is an alternative approach that is centered on the chemical analysis of biomarkers of (pesticide) exposure in urban wastewater. The present study used WBE to assess human exposure to selected classes of pesticides, triazines, pyrethroids and organophosphates, in Norway. Untreated wastewater samples were collected from four cities, covering approximately 20% of the Norwegian population. The highest population weighted mass loads (mg/day/1000 inhabitants) were for alkyl phosphates and the lowest for triazines. Some differences were observed for the two metabolites, 2-isopropyl-6-methyl-4-pyrimidinol (IMPY) and 3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (DCCA), which were higher in the rural city of Hamar. WBE figures were comparable with HBM findings for the specific metabolite of chlorpyrifos and chlorpyrifos methyl (3,5,6-trichloro-2-pyridinol; TCPY) but were different for the alkyl phosphates. Pyrethroid intake was calculated and was lower than the acceptable daily intake in all the cities, indicating low risk for human health. This is the most extensive WBE study performed to date to assess national human exposure to pesticides. This study demonstrated that WBE has the potential to be a useful complementary biomonitoring tool for assessing population-wide exposure to pesticides, overcoming some of the limitations of HBM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Methamphetamine (METH) is a central nervous system stimulant drug whose use has increased in the last few years worldwide. After the ingestion of even a single dose, METH is excreted by the organism and enters the aquatic ecosystems, whereby concentrations up to hundreds of ng/L were measured in both sewage and surface waters. Although the environmental concentrations are currently quite low, the high biological activity of METH might cause adverse effects towards non-target organisms. However, to date the information on METH toxicity towards aquatic organisms is limited. Thus, the present study aimed at investigating biochemical and behavioral effects induced by METH exposure towards the Cladoceran Daphnia magna. A 21-days exposure to two environmental concentrations of METH (50 ng/L and 500 ng/L) was performed. At selected time points (7, 14 and 21 days) the amount of pro-oxidant molecules, the activity of antioxidant enzymes (SOD, CAT, GPx) and levels of lipid peroxidation (LPO) were measured as oxidative stress-related endpoints. Changes in swimming activity and reproductive output were assessed as behavioral endpoints. METH exposure affected the oxidative status of D. magna specimens at both tested concentrations, although no oxidative damage occurred. Although METH did not modulate the swimming activity of D. magna, a significant, positive effect on reproductive output, in terms of number of offspring was found. Our results showed that low concentrations of METH might represent a threat for D. magna, affecting the health status of this aquatic species at different level of biological organization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) was applied for the first time in seven cities across Europe with the aim of estimating quinolones consumption via the analysis of human urinary metabolites in wastewater. This report is also the first pan-European study focussed on the enantiomeric profiling of chiral quinolones in wastewater. By considering loads of (fluoro)quinolones in wastewater within the context of human stereoselective metabolism, we identified cities in Southern Europe characterised by both high usage and direct disposal of unused ofloxacin. In Northern European cities, S-(-)-ofloxacin loads were predominant with respect to R-(+)-ofloxacin. Much more potent, enantiomerically pure S-(-)-ofloxacin was detected in wastewaters from Southern European cities, reflecting consumption of the enantiomerically pure antibiotic. Nalidixic acid, norfloxacin and lomefloxacin were detected in wastewater even though they were not prescribed according to official prescription data. S,S-(-)-moxifloxacin and S,S-(-)-moxifloxacin-N-sulphate were detected in wastewater due to metabolism of moxifloxacin. For the first time, average population-normalised ulifloxacin loads of 22.3 and 1.5 mg day -1 1000 people -1 were reported for Milan and Castellón as a result of prulifloxacin metabolism. Enrichment of flumequine with first-eluting enantiomer in all the samples indicated animal metabolism rather than its direct disposal. Fluoroquinolone loads were compared with qnrS gene encoding quinolone resistance to correlate usage of fluoroquinolone and prevalence of resistance. The highest daily loads of the qnrS gene in Milan corresponded with the highest total quinolone load in Milan proving the hypothesis that higher usage of quinolones is linked with higher prevalence of quinolone resistance genes. Utrecht, with the lowest quinolones usage (low daily loads) had also one of the lowest daily loads of the qnrS gene. However, a similar trend was not observed in Oslo nor Bristol where higher qnrS gene loads were observed despite low quinolone usage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factor family, well known for its role in the homeostasis of the cardiovascular system. Recently, the human BDNF Val66Met single nucleotide polymorphism has been associated with the increased propensity for arterial thrombosis related to acute myocardial infarction (AMI). Using cardiac magnetic resonance imaging and immunohistochemistry analyses, we showed that homozygous mice carrying the human BDNF Val66Met polymorphism (BDNF Met/Met ) undergoing left anterior descending (LAD) coronary artery ligation display an adverse cardiac remodeling compared to wild-type (BDNF Val/Val ). Interestingly, we observed a persistent presence of pro-inflammatory M1-like macrophages and a reduced accumulation of reparative-like phenotype macrophages (M2-like) in the infarcted heart of mutant mice. Further qPCR analyses showed that BDNF Met/Met peritoneal macrophages are more pro-inflammatory and have a higher migratory ability compared to BDNF Val/Val ones. Finally, macrophages differentiated from circulating monocytes isolated from BDNF Met/Met patients with coronary heart disease displayed the same pro-inflammatory characteristics of the murine ones. In conclusion, the BDNF Val66Met polymorphism predisposes to adverse cardiac remodeling after myocardial infarction in a mouse model and affects macrophage phenotype in both humans and mice. These results provide a new cellular mechanism by which this human BDNF genetic variant could influence cardiovascular disease.

In a neuropsychological assessment, each test aims at measuring a single cognitive function. However, test performance depends on an interconnected system of cognitive functions and individual characteristics. For a better understanding of cognitive deficits, it is fundamental to recognize this complexity and study the relationships between test performances. This study aims to evaluate complexity in neuropsychological assessment through network analysis (NA) in 165 healthy older adults, 191 patients with Alzheimer's disease (AD), and 129 patients with vascular encephalopathy (VaE). NA is a flexible method to explore different domains where many variables are correlated with each other, and the relationships are key for understanding the domains. We included general aspects of individual differences (i.e., age, sex, years of education) and the raw scores of a clinically used neuropsychological battery in the network. Healthy subjects showed a segregated pattern, suggesting a good specificity of each test in measuring a specific cognitive function. Moreover, the scores were related to age and education. In the patient groups, the identified patterns changed in a consistent manner, showing less specificity and new relationships between the various tests, thereby reducing the impact of age and education on the performance. In particular, AD patients showed worse performance on the tests but also a different balance between different tasks, suggesting a reorganization of the cognitive system and not a mere decline. These results provide a new perspective in looking at the complexity of cognitive function assessment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this work we evaluated the contamination of the water cycle in Como Bay by measuring 38 selected pharmaceuticals in two main wastewater treatment plant in Switzerland and in Italy, two influents (River Breggia and Cosia), lake water (epilimnion and hypolimnion), as well as potable water. The collection of comparative information on the presence and environmental fate of these substances contributes to set specific environmental quality standard (EQS). The results presented show that the contamination of the lake reflects national health policies, which deeply influence the usage of chemicals. The outcomes of this study give an overall picture of contamination in the area, showing that concentrations of the measured compounds are generally low and under the commonly adopted ecotoxicological and toxicological thresholds. Only in a few cases did the contamination appear to be noteworthy, for some of the most persistent compounds (antibiotic macrolides, diclofenac, irbesartan, carbamazepine and dihydrocarbamazepine, bezafibrate, furosemide and hydrochlorothiazide). Some concern can be also be raised for the presence of antibiotics (clarithromycin) in drinking water, although at very low levels, due to the problem of antibiotic resistance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A correct magnesium (Mg 2+ ) intake is essential for bone health. In particular, Mg 2+ deficiency inhibits the proliferation of osteoblast-like SaOS-2 cells by increasing nitric oxide (NO) production through the upregulation of inducible NO synthase. At the moment, little is known about the expression and the role of TRPM7, a channel/enzyme involved in Mg 2+ uptake, and MagT1, a Mg 2+ selective transporter, in SaOS-2 cells. Here, we demonstrate that TRPM7 is not modulated by different extracellular concentrations of Mg 2+ and its silencing exacerbates growth inhibition exerted by low Mg 2+ through the activation of inducible NO synthase and consequent accumulation of NO. Moreover, MagT1 is upregulated in SaOS-2 cultured in high Mg 2+ and its silencing inhibits the growth of SaOS-2 cultured in media containing physiological or high Mg 2+ , without any modulation of NO production. We propose that TRPM7 and MagT1 are both involved in regulating SaOS-2 proliferation through different mechanisms.

Fishes are exposed to mixtures of different classes of steroids, but ecotoxicological implications are not sufficiently known. Here, we systematically analyze effects of different combinations of steroid mixtures in zebrafish embryos to assess their joint activities on physiology and transcriptional alterations of steroid-specific target genes at 96 and 120 h post fertilization. In binary mixtures of clobetasol propionate (CLO) with estradiol (E2) or androstenedione (A4), each steroid exhibited its own expression profile. This was also the case in mixtures of 5-, 8-, and 13-different classes of steroids in exposure concentrations of 10-10,000 ng/L. The transcriptional expression of most genes in different mixtures was steroid-specific except for genes encoding aromatase ( cyp19b ), sulfotransferase ( sult2st3 ), and cyp2k22 that were induced by androgens, progestins, and glucocorticoids. Marked alterations occurred for sult2st3 in binary mixtures of CLO + E2 and CLO + A4. Glucocorticoids increased the heart rate and muscle contractions. In mixtures containing estrogens, induction of the cyp19b transcript occurred at 10 ng/L and protc from the anticoagulation system at 100 ng/L. Our study demonstrates that steroids can act independently in mixtures; the sum of individual steroid profiles is expressed. However, some genes, including cyp19b , sult2st3 , and cyp2k22 , are regulated by several steroids. This joint effect on different pathways may be of concern for fish development.

Mycotoxins are among the compounds of most concern for human health because of their common presence in food and their proven toxicity for human health. Human biomonitoring (HBM) studies, foodstuff analysis and dietary surveys are usually used to assess human exposure, but they are costly and time-consuming. Wastewater-based epidemiology (WBE) can complement the established exposure monitoring tools. The aim of this study was to develop a new WBE application for assessing human exposure to mycotoxins by measuring specific biomarkers in raw wastewater. Eleven substances were analysed by liquid chromatography-tandem mass spectrometry in wastewater from four cities in Italy and Spain. Only deoxynivalenol (DON) and fumonisines were found, DON being the most abundant, in accordance with HBM studies. DON intake, back-calculated by WBE, was lower than the established Health-Based Guidance values, indicating moderate exposure in the population, with no risks for human health. WBE intake estimates were very close to those reported in HBM studies, validating WBE for estimating DON intake in the population. This study describes a promising WBE approach to complement HBM studies and assess the human intake of specific classes of mycotoxins, thus helping to identify risks for human health., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sixty-nine dyads of patients with Alzheimer's disease and primary caregivers have been followed up for 1 year to evaluate cognitive (Mini-Mental State Examination), functional (Instrumental Activities of Daily Living), and behavioral (Neuropsychiatric Inventory) decline of patient in relation to burden (Caregiver Burden Inventory), stress (Relative Stress Scale), anxiety (State-Trait Anxiety Inventory Y), and depression (Beck Depression Inventory) reported by the caregivers. After 1 year of observation, cognitive and functional scores worsened while behavioral problems remained unchanged and relatively mild in patients. After 1 year, caregivers' scores of scales of anxiety and depression decreased significantly, while stress scores remained unchanged and burden slightly increased. In our opinion, the unexpected improvement in psychological situation of caregivers may be mainly due to educational interventions focused on knowledge of the disease with a particular attention directed toward emotional support and individual needs.

Conceptual knowledge is supported by multiple semantic systems that are specialized for the analysis of different properties associated with object concepts. Various types of semantic association between concrete concepts-categorical (CA), encyclopedic (EA), functional (FA), and visual-encyclopedic (VEA) associations-were tested through a new picture-to-picture matching task (semantic association task, SAT). Forty individuals with Alzheimer's disease (AD), 13 with behavioral variant of frontotemporal dementia (bv-FTD), 6 with primary progressive aphasia (PPA), and 37 healthy participants were tested with the SAT. Within-group comparisons highlighted a global impairment of all types of semantic association in bv-FTD individuals but a disproportionate impairment of EA and FA, with relative sparing of CA and VEA, in AD individuals. Single-case analyses detected dissociations in all dementia groups. Conceptual knowledge can be selectively impaired in various types of neurodegenerative disease on the basis of the specific cognitive process that is disrupted.

Background and Aims: Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data., Design: Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017., Setting and Participants: Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries., Measurements: Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ 9 -tetrahydrocannabinol (11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol) were measured in wastewater using liquid chromatography-tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world-wide, and temporal trends were discerned at European level by comparing 2011-13 drug loads versus 2014-17 loads., Findings: Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge., Conclusions: The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data., (© 2019 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

This study used wastewater-based epidemiology (WBE) to investigate the lifestyle of the inhabitants of Malé, the capital of the Republic of Maldives. Raw wastewater 12-h composite samples were collected from nine pumping stations serving the city area - thus representative of the whole Malé population. Samples were analysed by liquid chromatography coupled to mass spectrometry for estimating the profile of use of a large number of substances including illicit drugs, alcohol, caffeine, tobacco and pharmaceuticals. The illicit drugs most used were cannabis (THC) and heroin (700 and 18 g/day), with lower consumption of cocaine and amphetamines (0.1-1.2 g/day). It is important to note that the consumption of cannabis in Malé was comparable to that measured in other countries, while the consumption of heroin was higher. Among cathinones, mephedrone was detected at the highest levels similar to other countries. Consumption of alcohol, which is not allowed in Maldives, was found (1.3 L/day/1000 inhabitants), but at a low level compared with other countries (6-44 L/day/1000 inhabitants), while the consumption of caffeine and tobacco was generally in line with reports from other countries. Unique information on pharmaceuticals use was also provided, since no official data were available. Human lifestyle was evaluated by applying for the first time the full set of WBE methodologies available in our laboratory. Results provided valuable epidemiological information, which may be useful for national and international agencies to understand population lifestyles better, including illicit drug issues, and for planning and evaluation of drug prevention programs in Malé., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Exposure to real or simulated microgravity is sensed as a stress by mammalian cells, which activate a complex adaptive response. In human primary endothelial cells, we have recently shown the sequential intervention of various stress proteins which are crucial to prevent apoptosis and maintain cell function. We here demonstrate that mitophagy contributes to endothelial adaptation to gravitational unloading. After 4 and 10 d of exposure to simulated microgravity in the rotating wall vessel, the amount of BCL2 interacting protein 3, a marker of mitophagy, is increased and, in parallel, mitochondrial content, oxygen consumption, and maximal respiratory capacity are reduced, suggesting the acquisition of a thrifty phenotype to meet the novel metabolic challenges generated by gravitational unloading. Moreover, we suggest that microgravity induced-disorganization of the actin cytoskeleton triggers mitophagy, thus creating a connection between cytoskeletal dynamics and mitochondrial content upon gravitational unloading., (© 2019 Federation of American Societies for Experimental Biology.)

N-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) seem to prevent endothelial dysfunction, a crucial step in atherogenesis, by modulating the levels of vasoactive molecules and by influencing Na,K-ATPase activity of vascular myocytes. The activity of endothelial Na,K-ATPase controls the ionic homeostasis of the neighboring cells, as well as cell function. However, controversy exists with respect to the vascular protective effect of EPA and DHA. We argue that this dispute might be due to the use of different concentrations of EPA and DHA in different studies. Therefore, this study was designed to define an optimal concentration of EPA and DHA to investigate endothelial function. For this purpose, human endothelial cells were exposed for 24 h to different concentrations of DHA or EPA (0-20 μM) to study membrane fluidity, peroxidation potential and Na,K-ATPase activity. EPA and DHA were linearly incorporated and this incorporation was mirrored by the linear increase of unsaturation index, membrane fluidity, and peroxidation potential. Na,K-ATPase activity peaked at 3.75 μM of EPA and DHA and then gradually decreased. It is noteworthy that DHA effects were always more pronounced than EPA. Concluding, low concentrations of EPA and DHA minimize peroxidation sensitivity and optimize Na,K-ATPase activity.

Bee pollen loads generally have a homogeneous and monospecific pollen content and assume a typical form and color, due to the typical bee foraging habits, thus having a typical composition related to the botanical origin. The present study aims to characterize bee pollen loads belonging to different botanical species using morphological, spectroscopic and color properties and to find relationships between these variables. IR spectra analysis allowed to have a reliable picture of the components present in the different samples; color and granulometry permits a visual identification of pollen load belonging to different species. Multivariate analysis enabled differentiation among the botanical origin of most of the bee pollen samples, grouping them according to the family and the genus and confirming the possibility to use IR and color measurements for the evaluative analysis and classification of bee pollen samples, to promote the consumption of this bee product as functional food.

The number of new psychoactive substances (NPS) on the recreational drug market has increased rapidly in the last years, creating serious challenges for public health agencies and law enforcement authorities. Epidemiological surveys and forensic analyses to monitor the consumption of these substances face some limitations for investigating their use on a large scale in a shifting market. The aim of this work was to develop a comprehensive and flexible screening approach for assessing the presence of NPS in urban wastewater by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Almost 200 substances were selected as "priority NPS" among those most frequently and recently reported by the Early Warning Systems (EWS) of different agencies and were included in the screening. Wastewater samples were collected from several cities all over Europe in 2016 and 2017, extracted using different solid-phase cartridges and analysed by LC-HRMS. The screening workflow comprised three successive analytical steps and compounds were identified and confirmed following specific criteria from the current guidelines. Thirteen NPS were identified at different confidence levels by using analytical standards or information from libraries and literature, and about half of them were phenethylamines. As far as we know, this is the first time that four of them (i.e. 3,4-dimethoxy-α-pyrrolidinovalerophenone, para-methoxyamphetamine, 2-phenethylamine and α-methyltryptamine) have been found in urban wastewater. The proposed screening approach was successfully applied in the largest NPS European wastewater monitoring, providing an innovative and easily adapted procedure for investigating NPS. In the light of current challenges and specific future research issues, this approach may complement epidemiological information and help in establishing measures for public health protection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Illicit drugs and their metabolites have been identified as emerging aquatic pollutants. Cocaine (COC) is one of the most used illicit drug worldwide. After human consumption, COC enters the aquatic ecosystems, where it is commonly detected in ng L -1 concentration range. Although a number of studies have shown that the exposure to environmental concentrations of COC can induce diverse biochemical, molecular and histological effects on aquatic organisms, the information of COC-induced behavioral alterations is scant. Thus, the present study aimed at exploring both biochemical and behavioral effects induced by the exposure to two environmental concentrations (50 ng L -1 and 500 ng L -1 ) of COC on the freshwater cladoceran Daphnia magna. Specimens were exposed to selected COC concentrations for 21 days and the effects on the oxidative status, including the amount of reactive oxygen species and the activity of antioxidant (SOD, CAT and GPx) and detoxifying (GST) enzymes, and swimming activity were investigated after 7, 14 and 21 days of treatment, while effects on reproductive success was assessed after 21-days only.. Exposure to COC induced an overproduction of reactive oxygen species and a modulation of the activity of defense enzymes. Moreover, COC affected the swimming behavior and altered the reproductive success of treated specimens. Our results highlighted that environmental concentrations of COC can cause adverse effects at different levels of the biological hierarchy in a zooplanktonic species, confirming the potential threat due to this illicit drug for the aquatic community., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) can give valuable light on the extent and actual use of new psychoactive substances (NPS). In this work, a fully validated methodology for the simultaneous determination of illicit drugs and NPS in wastewater by solid-phase extraction followed by UHPLC-MS/MS has been developed. The small sample volume (5 mL) required for analysis is of high interest, especially when performing large sampling campaigns involving many locations of different geographical origin, as it has been performed in the past. The method was applied to wastewater samples from different European locations and permitted the simultaneous monitoring of conventional drugs and NPS. Cocaine, amphetamine, MDMA, methamphetamine and ketamine were found in all wastewater samples, and several NPS (dipentylone, butylone, mephedrone, methedrone and methylone) were observed in some of the samples monitored. It is noteworthy that dipentylone was detected in wastewater for the very first time. Furthermore, a detailed comparison of micro liquid chromatography (μLC) and UHPLC, both coupled to tandem mass spectrometry, in terms of sensitivity and reproducibility has been made for the first time in the application field of WBE. An average increase factor of 14 (mass normalized data) was observed in sensitivity for μLC-MS/MS. The overall method performance was also compared (un-normalized data), and an average increase sensitivity factor of 4.5 was observed for μLC-MS/MS. However, large deviations in retention time (up to 0.4 min) affected the reproducibility and robustness of the μLC-MS/MS method when it was applied to wastewater analysis. Although in this work μLC-MS/MS was strongly influenced by the amount of matrix loaded in the separation device, its enhanced sensitivity and promotion of green chemistry (faster analysis time and less solvent consumption) allow to expect improved future applications, especially when analytes are present at very low concentrations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The core knowledge about biomineralization is provided by studies on the advanced phases of the process mainly occurring in the extracellular matrix. Here, we investigate the early stages of biomineralization by evaluating the chemical fingerprint of the initial mineral nuclei deposition in the intracellular milieu and their evolution toward hexagonal hydroxyapatite. The study is conducted on human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days, exploiting laboratory X-ray diffraction techniques and cutting-edge developments of synchrotron-based 2D and 3D cryo-X-ray microscopy. We demonstrate that biomineralization starts with Zn-hydroxyapatite nucleation within the cell, rapidly evolving toward hexagonal hydroxyapatite crystals, very similar in composition and structure to the one present in human bone. These results provide experimental evidence of the germinal role of Zn in hydroxyapatite nucleation and foster further studies on the intracellular molecular mechanisms governing the initial phases of bone tissue formation., Competing Interests: The authors declare no competing financial interest.

Drug resistance remains a major obstacle in cancer treatment. Because mitochondria mediate metabolic reprogramming in cancer drug resistance, we focused on these organelles in doxorubicin sensitive and resistant colon carcinoma cells. We employed soft X-ray cryo nano-tomography to map three-dimensionally these cells at nanometer-resolution and investigate the correlation between mitochondrial morphology and drug resistance phenotype. We have identified significant structural differences in the morphology of mitochondria in the two strains of cancer cells, as well as lower amounts of Reactive oxygen species (ROS) in resistant than in sensitive cells. We speculate that these features could elicit an impaired mitochondrial communication in resistant cells, thus preventing the formation of the interconnected mitochondrial network as clearly detected in the sensitive cells. In fact, the qualitative and quantitative three-dimensional assessment of the mitochondrial morphology highlights a different structural organization in resistant cells, which reflects a metabolic cellular adaptation functional to survive to the offense exerted by the antineoplastic treatment.

The magnesium transporters TRPM7 and MagT1 are overexpressed in osteoblastogenesis. We have shown that silencing either TRPM7 or MagT1 accelerates the osteogenic differentiation of human bone mesenchymal stem cells. Here we demonstrate that the simultaneous downregulation of TRPM7 and MagT1 inhibits cell growth and activates autophagy, which is required in the early phases of osteoblastogenesis. In TRPM7/MagT1 downregulating cells the expression of two transcription factors required for activating osteogenesis, i.e. RUNX2 and OSTERIX, is induced more than in the controls both in the presence and in the absence of osteogenic stimuli, while COL1A1 is upregulated in co-silencing cells as much as in the controls. This explains why we found no differences in calcium deposition. We conclude that one of the two transporters should be expressed to accelerate osteogenic differentiation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Culture of human endothelial cells for 10 d in real microgravity onboard the International Space Station modulated more than 1000 genes, some of which are involved in stress response. On Earth, 24 h after exposure to simulated microgravity, endothelial cells up-regulate heat shock protein (HSP) 70. To capture a broad view of endothelial stress response to gravitational unloading, we cultured primary human endothelial cells for 4 and 10 d in the rotating wall vessel, a U.S. National Aeronautics and Space Administration-developed surrogate system for benchtop microgravity research on Earth. We highlight the crucial role of the early increase of HSP70 because its silencing markedly impairs cell survival. Once HSP70 up-regulation fades away after 4 d of simulated microgravity, a complex and articulated increase of various stress proteins (sirtuin 2, paraoxonase 2, superoxide dismutase 2, p21, HSP27, and phosphorylated HSP27, all endowed with cytoprotective properties) occurs and counterbalances the up-regulation of the pro-oxidant thioredoxin interacting protein (TXNIP). Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after spaceflight. We conclude that HSP70 up-regulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them toward the acquisition of a novel phenotype that maintains cell viability and function through the sequential involvement of different stress proteins.-Cazzaniga, A., Locatelli, L., Castiglioni, S., Maier, J. A. M. The dynamic adaptation of primary human endothelial cells to simulated microgravity.

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D₃ (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.