The “SNC” cultures were grown on cytokinin-free 1/2 MS (macro- and micronutrients) medium supplemented with WPM vitamins, IBA (0.05 mg l-1), and sucrose (15 g l-1). Both media (pH 5.8) were solidified with agar (7.0 g l-1). Initiation of in vitro cultures from explants taken from adult trees and young, potted plants was feasible on both media. Cultures were established from about 1 cm long nodal explants. Generally “SNC” cultures formed one well rooted, significantly longer axillary shoot with bigger leaves than “AxB” cultures, which developed significantly more shoots and big callus at the explant base. All shoots collected from “SNC” and “AxB” cultures rooted in vivo in peat mixture and developed into similar plantlets. The single-node method based on application of cytokinin-free medium is a good alternative for the axillary-branching method for micropropagation of mountain mulberry (Morus bombycis) ‘Kenmochi”’.The aim of the study was to compare two methods of micropropagation of mulberry: single-node culture (“SNC”), and axillary-branching (“AxB”). The experiments were carried out on in vitro cultures for 6 successive passages. The “AxB” cultures were propagated on modified MS medium (+ 25% Ca Morus sp.) are valuable trees or shrubs which can be used in many ways  [Bombyx mori). Mulberry-derived products are used in traditional medicine against diabetes, throat inflammations, dysentery, constipation, and helminthiasis [Morus bombycis Koidz.) deserves attention. It is designed for sericulture and is intensively cultivated in cold areas of Japan [Morus alba L.), and black mulberry (Morus nigra L.) clones. The quality of cocoons was improved as well. However, cultivar ‘Kenmochi”’ may be also considered to be an ornamental tree. It is sufficiently winter-hardy in Poland. It bears edible black/dark red slightly sour and sweet fruit. They taste better than bland white mulberry fruit. The fruit are liked by wild birds. It is worth mentioning that ‘Kenmochi”’ fruit are characterized by high phenolics content and strong radical scavenging activity and reducing power [Morus sp.) since 1970 when the first article on this topic was published [Mulberries (biomass) ,2,3. Thenthiasis ,5. It seof Japan . It was of Japan  who showng power . For theng power ,9,10,11.ng power ,15,16,17ng power ,15. Adveng power . In summng power . It is bng power . Many stublished  and someublished ,30,31,32ublished ,22,24,25ublished . As an aMorus bombycis Koidz.) ‘Kenmochi”’. First plants were kindly provided from the Institute of Pomology and Floriculture in Skierniewice (Poland) in 1990. Their vegetative progeny has been grown on “Zalesie” Campus of University of Rzeszów, Poland since 1995. Mulberry plants were micropropagated on a small scale in the university plant nursery. In the present study, the control cultures were multiplied through commonly used, standard, axillary-branching (“AxB”) method based on the application of cytokinins in initiation and multiplication stages. The modified MS [2+ and Mg2+ dose, supplemented with 6-benzylaminopurine (BA) was used for this purpose. Alternatively, the tested cultures were propagated through single-node (“SNC”) method on cytokinin-free, double-diluted MS medium which favors rhizogenesis [−2·s−1 PPFD, provided by cool white fluorescent lamps (OSRAM). Similar temperature and light conditions were used in many procedures of mulberry micropropagation [The experiments were carried out on in vitro cultures or plants of mountain mulberry . The single-node explants were put in vials filled with the same media  as used in the next experiments. However, they were additionally supplemented with PPM® (1 mL l−1). After four weeks, the whole, healthy-looking cultures were placed in jars on  media devoid of PPM®. They were grown in the same light/temperature conditions as in multiplication stage. At the end of passage, the number of growing cultures were recorded.In the case of culture initiation, the initial (single-node) explants were prepared from new spring shoots collected from adult, at least 20-year-old trees and from one-year-old nursery plants in April. The experiments were carried out on 30 and 40 explants, respectively. Before initiation, the shoots were soaked in fungicide “Funaben T” (carbendazim 20% and thiram 45%) 0.1% solution for 12 h. Then the pieces of shoots were dipped in ethanol 70% for a few seconds and sterilized in commercial “ACE lemon” 10% solution in ultrasonic bath for 15 min. After this, they were roughly rinsed with autoclaved deionized water and finally in sterile solution of citric acid and vitamin C  with ventilated polypropylene twist lids, filled with medium (60 mL). Usually, each treatment (“AxB” and “SNC”) in each subculture was represented by 4 jars (×8 explants). The exceptions were: the first subculture (3 jars × 8 explants) and the sixth one (5 jars ×8 explants). The experiments were carried out through six subsequent 6–7-week-long subcultures. In order to check whether “the switch” from “SNC” to “AxB” method is possible, the explants prepared from “SNC” cultures were grown on control “AxB” medium. Such additional treatment was also represented by 4 jars (×8 explants) in three subcultures, and 5 jars (×8 explants) in the sixth subculture. At the end of passage, the number and length of shoots and callus size was determined. The number of growing cultures and rooted explants were also recorded.® (Hydrokomplet) designed for ornamental pot plants in mist chambers (near 100% RH) with transparent plastic covers. The same temperature and light conditions were set as during in vitro propagation. The covers were gradually disclosed from the first week and removed after the third week. Then after the next week the number and size of obtained plantlets were recorded. The leaf greenness index (relative chlorophyll content) expressed in SPAD units was determined for two young but fully developed leaves of each plant using a portable KONICA MINOLTA Chlorophyll Meter SPAD-502Plus. Forty “AxB” and 60 “SNC” shoots were used in such experiment. Shoots (at least 2 cm long with shoot tip) were collected from “AxB” and “SNC” cultures. They were rooted in vivo without application of auxins and fungicides in commercial peat mixture Bella0.05 mean separation test using Statistica 9.0 computer software. Data presented as percentages  were subjected to testing on difference between two proportions. The differences were considered to be significant at α = 0.05. Collected data were subjected to an ANOVA and LSDIt was found that initiation of in vitro mulberry cultures from initial explants taken from adult trees and young, potted plants was feasible both on standard, cytokinin-containing MS (“AxB”) and on cytokinin-free ½ MS (“SNC”) media . InitiatSimilar numbers of explants placed on PGRs-free (“SNC”) and control (“AxB”) media started to grow in all 6 subcultures of mountain mulberry ‘Kenmochi”’ . All culs 5.5) and shorter internodes (1.0 cm < 1.4 cm) of obtained shoots . A clear relationship between the micropropagation method and the total length of shoots formed by cultures was not found . Nonetheless, the mean length of shoots and internodes was significantly higher in the case of “SNC” shoots than “AxB” shoots . Thus, ectively . Therefoectively . On the ectively . The shoectively , Table 8ectively . They coMorus bombycis Koidz.) ‘Kenmochi”’. The main properties, advantages, and disadvantages of two methods of micropropagation are given in Morus alba L.) clones (In summary, the “SNC” method provides at least similar propagation efficiency and quality of obtained cultures and plantlets. It is also cheaper, as the medium without cytokinins, with lowered concentration of sucrose and macronutrients, is used. Thus, the single-node method based on application of cytokinin-free medium is a good alternative for the axillary-branching method for micropropagation and/or long-term in vitro storage of mountain mulberry  clones .


Following publication of the original article , we wereAlso, in Additional Table S1, the values for Female Asthma and Control under Bronchial Brushing were swapped and the last table column should read “Bronchial biopsy” instead of “Bronchial Brushing”.Originally published Fig. 2.Corrected Fig. 2.The original article has been corrected.


Perceived stress, burnout, and poor sleep quality are high among medical students. Interventions designed to target these issues are necessary to promote the health and well-being of medical students. The purpose of this study was twofold: 1) to assess the feasibility of implementing a sunrise alarm clock intervention with medical students and 2) to evaluate the impact of the intervention on perceived stress, burnout scores, and sleep quality.We conducted a feasibility study to evaluate the efficacy of a two-week, sunrise alarm clock intervention in combination with electronic device removal at bedtime. We assessed first- and second-year medical students’ perceived stress, burnout scores, including Emotional Exhaustion, Depersonalization, and Low Sense of Personal Achievement, and sleep quality before and after the intervention. In addition, we measured smartphone addiction prior to the intervention.n = 29) identified as women, and 68.4% (n = 39) identified as white. Pre-intervention, 42.1% (n = 24) of students met criteria for smartphone addiction and 77.2% (n = 44) met criteria for poor sleep quality. In addition, 22.8% (n = 13) of participants had high emotional exhaustion, 64.9% (n = 31) high depersonalization, and 42.1% (n = 24) low sense of personal accomplishment prior to the intervention. Following the two-week intervention, participants showed improvements in emotional exhaustion , depersonalization  low sense of personal accomplishment , perceived stress , and sleep quality . The number of participants who reported poor sleep quality decreased to 41.8% (n = 23), demonstrating a significant decline (p = 0.026). Participants also improved subjective sleep quality , sleep duration , sleep latency , and sleep efficiency .A total of 57 students consented to participate, of which 55 completed both the pre- and post-assessments (3.5% attrition). The mean age of the participants was 24.8 ± 1.9 years, 50.9% (These findings suggest that the two-week sunrise alarm clock protocol with electronic device removal was effective in improving sleep quality and reducing burnout scores, and perceived stress. However, additional research comparing this intervention to a proper control group is needed to draw meaningful conclusions about the effectiveness of this intervention. Burnout is a psychological syndrome that occurs in response to chronic interpersonal stressors on the job . MaslachWhile sleep disorders are critically linked to burnout , 18, pooSeveral studies have demonstrated the deleterious effects of smartphone use on sleep . ExposurInterestingly, smartphone overuse is associated with higher burnout scores, in particular emotional exhaustion and depersonalization, in medical students . This maOne simple behavioral approach to improve sleep quality and reduce burnout is limiting exposure to smartphones and other electronic devices at bedtime and replacing them with gentle wake-up alarms. Providing an alternative alarm clock is critical given most Americans use the alarm clock feature on their smartphone devices to wake up each morning . SunriseThis feasibility study evaluated the efficacy of a sunrise alarm clock intervention in combination with electronic device removal at bedtime. The study measured indicators of medical students’ emotional well-being before and after a two-week protocol in order to assess changes in burnout, perceived stress, and sleep quality pre- and post-intervention. The University Office of Research Compliance approved the protocol (18-X-157) and all recruitment procedures and materials.First- and second-year medical students enrolled at a large medical school in the Midwest were invited to participate in a two-week intervention. Students were recruited via email using class mailing lists. The research team distributed a recruitment email on April 23, 2018 and a reminder email one week later. Interested students were instructed to schedule an in-person visit to review the study protocol, provide informed consent, and complete the pre-assessment. Participants were recruited on a rolling basis. The first participant completed their pre-assessment on April 23, 2018 and the last participant completed their post-assessment on May 28, 2018. Participants received a $10 gift card for completion of each assessment. In addition, participants kept the alarm clock from the study. Participation in the study was voluntary.Replace current alarm clock with the sunrise alarm clock received from the study.Turn off smartphone and all electronic devices at bedtime.Follow this protocol for two weeks.For the two-week protocol, all participants were provided a sunrise alarm clock and instructed to use it for their regular sleep-wake cycles instead of their current alarm clock . Next, participants were asked to turn off their smartphone and all other electronic devices at night for the entire two-week protocol. Night was defined as when the participant chose to go to bed. Below is a bulleted list of the protocol participants were asked to complete for this protocol:Participants were not asked to change any of their other behaviors or habits in this protocol.Participants completed a brief sociodemographic  and health factors  questionnaire. Note, the data collected on race followed the United States Census categories  . In addiA 22-item measure assessing Emotional Exhaustion , Depersonalization , and Low Sense of Personal Achievement . Participants scored each item on a seven-point scale, where 0 was “never,” 1 was “sometimes per year or less often,” 2 was “once a month or less often,” 3 was “several times a month,” 4 was “once a week,” 5 was “several times a week,” and 6 was “daily.” Consistent with prior research, high emotional exhaustion scores were ≥ 27, high depersonalization scores were ≥ 10, and low sense of personal accomplishment scores ≤33 , 41. TheA 4-item measure assessing psychological stress over the past month. This brief self-assessment measures the degree to which an individual perceives their lives to be stressful . Participants scored each item on a four-point scale, where 0 was “never,” 1 was “almost never,” 2 was “sometimes,” 3 was “fairly often,” and 4 was “very often.” Scores ranged from 0 to 16, with higher scores indicating a higher perception of stress. Prior studies have demonstrated internal consistency for the PSS-4 to be between 0.67–0.73 –44. The A 19-item self-reported measure assessing quality and patterns of sleep in adults . Participants provided qualitative and quantitative data describing usual sleep habits during the past month only. The scale was then scored to create a seven-component measurement, with a total score of “5 or greater” indicating poor sleep quality. In addition, individual components were calculated for pre-post analysis, including subjective sleep quality, sleep duration, sleep latency, and sleep efficiency. Subjective sleep quality was a rating of the participants’ sleep quality in the past month, ranging from 0 to 3 with ‘0 = very good’ and ‘3 = very bad’. Sleep duration was the number of hours of actual sleep participants got at night. Sleep latency was the number of minutes it took to fall asleep each night. Lastly, sleep efficiency was calculated by dividing the sleep duration by total number of hours in bed multiplied by 100. The PSQI was administered at pre- and post-intervention.A 10-item measure assessing smartphone addiction  Participants scored items on a six-point scale, where 1 was “strongly disagree,” 2 was “disagree,” 3 was “weakly disagree,” 4 was “weakly agree,” 5 was “agree,” and 6 was “strongly agree.” Internal consistency of the measure is excellent with a Cronbach’s alpha correlation coefficient of 0.91 . The cutParticipants completed the pre- and post-assessments via the online questionnaire service Qualtrics . Participants attended one on-campus visit in a confidential space to provide written informed consent and complete the pre-assessment. At the visit, participants were asked to read and sign an informed consent form prior to participation. The informed consent document explicitly informed potential participants that their responses had no bearing on academic performance, and that they could withdraw consent at any time during the study. If they consented to participate in the study, they were assigned a unique identification number and asked to complete the pre-assessment measures via the Qualtrics link on a password-protected desktop computer in the confidential space. After participants completed the two-week intervention protocol, one member of the research team distributed a Qualtrics link for the post-assessment. To link pre- and post-assessment responses, participants were instructed to enter their unique identifier for both the pre- and post-assessment via Qualtrics. Completion of each assessment lasted approximately 15 min.p-value less than 0.05, and conducted analyses in SPSS statistical software version 26.0 Basic sociodemographic characteristics of participants were assessed using descriptive statistics. Frequencies of individual question responses were also calculated. Chi-square tests, Fisher’s exact tests, independent samples t-tests, one-way analysis of variance (ANOVA), and Pearson’s correlation coefficients were used to examine differences in emotional exhaustion, depersonalization, low sense of personal accomplishment, perceived stress, sleep quality, and smartphone addiction by demographic characteristics. Specifically, we examined differences by age, gender, race, year in medical school, and BMI based on prior literature in the US that showed older age was protective from higher burnout scores, women were more likely to screen positive for depression and fatigue compared to men, non-White medical students had lower mental health scores compared to White students, burnout scores increase after year-one and year-three of medical school, and poorer sleep quality is associated with higher BMI , 47–49. n = 29) identified as women, 68.4% (n = 39) identified as white, and 66.7% (n = 38) were second-year medical students. On average, participants were 67.7 ± 4.9 in. tall and weighed 167.4 ± 36.0 pounds. The mean body mass index  was 25.8 ± 5.0. Additional demographic and health characteristics are presented in Table p = .759; 52.3% women (n = 139), χ2 = .0467, p = 0.829; 73.3% White (n = 195), χ2 = .630, p = 0.529; BMI = 24.9 ± 4.5, .630, p = 0.529) on burnout, sleep quality, and smartphone addiction [n = 495; age = 25.0 ± 2.2; 52.7% women (n = 257); 76% White (n = 376); BMI = unknown) from this institution.Of the 286 osteopathic medical students enrolled at the main campus, 57 consented to participate in the feasibility study. Two participants did not complete the post-assessment (3.5% attrition). Thus, a total of 55 participants completed both the pre- and post-assessments. The mean age of the participants was 24.8 ± 1.9 years, 50.9% (n = 13) of participants reported high emotional exhaustion, 64.9% (n = 37) high depersonalization, and 42.1% (n = 24) low sense of personal accomplishment. Only 14.0% (n = 8) participants reported high burnout scores in all three dimensions. Dimensions of burnout did not differ by age , gender , race , year in medical school , or BMI . Interestingly, participants who met criteria for smartphone addiction were more likely to report high depersonalization , but not high emotional exhaustion  or low sense of personal accomplishment . High emotional exhaustion , high depersonalization , and low sense of personal accomplishment  were associated with poorer sleep quality.Prior to the intervention, 22.8% . The effect size for all three improvements ranged between 0.275 and 0.411, indicating small to medium effects. In addition, the number of participants with high levels of emotional exhaustion , high depersonalization , and low sense of personal accomplishment  decreased from pre- to post-intervention. In addition, only 5.3% (n = 3) participants reported high burnout scores in all three dimensions post-intervention . Poorer sleep quality remained associated with high emotional exhaustion  and high depersonalization , but not low sense of personal accomplishment t = 1.338, p = 0.187) post-intervention.Burnout scores improved post-intervention in all three dimensions (see Table p = 0.171), gender , race , year in medical school , or BMI . Higher scores of perceived stress were associated with high emotional exhaustion , high depersonalization , and low sense of personal accomplishment . Similarly, higher perceived stress scores were correlated with poorer sleep quality . Higher perceived stress scores were not associated with smartphone addiction .Pre-intervention, participants reported total perceived stress scores of 6.3 ± 3.1 (see Table p = 0.001), high depersonalization , and low sense of personal accomplishment . In addition, higher perceived stress scores were correlated with poorer sleep quality .Perceived stress scores improved post-intervention  of participants reported total global sleep quality scores ≥5, indicating poor sleep quality, with mean scores of 7.3 ± 3.2. Participants rated their subjective sleep quality between fairly good and fairly bad (mean = 1.4 ± 0.7). On average, they slept 6.7 ± 0.9 h per night, taking an average of 43.5 ± 64.5 min to fall asleep, generating a sleep efficiency score of 84.9 ± 10.2%. Sleep quality did not differ by age , gender , race , year in medical school , or BMI . As stated above, sleep quality was associated with high emotional exhaustion, high depersonalization, low sense of personal accomplishment, higher perceived stress scores, and smartphone addiction .Pre-intervention, 77.2%  participants reported total global sleep quality scores ≥5, showing a significant drop pre- and post-intervention (p = 0.026). Participants also improved subjective sleep quality , sleep duration , sleep latency , and sleep efficiency . The largest magnitude of change occurred with the subjective sleep quality, with a Cohen’s d of 1.033, indicating another very large effect. As stated above, poorer sleep quality was associated with high emotional exhaustion and high depersonalization, but not low sense of personal accomplishment post-intervention. Also, higher perceived stress scores remained correlated with poorer sleep quality post-intervention. Finally, pre-intervention smartphone addiction was no longer associated with poorer sleep quality post-intervention .Global sleep quality scores improved post-intervention  of participants met criteria for smartphone addiction. Smartphone addiction did not differ by sex , race , year in medical school , or BMI . However, participants who were older were more likely to meet criteria for smartphone addiction . Smartphone addiction was not associated with higher perceived stress scores . On the other hand, smartphone addiction was associated with high depersonalization , but not emotional exhaustion  or low sense of personal accomplishment  pre-intervention. Further, participants with smartphone addiction were more like to have poorer sleep quality  before the intervention.Pre-intervention, 42.1% , high depersonalization , or low sense of personal accomplishment . Further, pre-intervention smartphone addiction was not associated with higher perceived stress scores  or poorer sleep quality .Post-intervention, pre-intervention smartphone addiction was not associated with high emotional exhaustion , emotional exhaustion , depersonalization , low sense of personal accomplishment , or sleep quality .On average, participants used the sunrise alarm clock 11.4 ± 3.4 days out of 14 days in the two-week period and turned off or removed their smartphones and other electronic devices 11.3 ± 3.4 days. Neither adherence to the sunrise alarm clock nor adherence to the smartphone protocol were correlated with the change scores for perceived stress  of participants met criteria for poor sleep quality, which was no longer associated with pre-intervention smartphone addiction. These findings suggest that the two-week sunrise alarm clock protocol with electronic device removal at bedtime was effective in improving sleep quality and reducing perceived stress and burnout in medical students. However, additional research comparing this intervention to a proper control group is needed to draw meaningful conclusions about the effectiveness of this intervention.The behavioral impacts of problematic smartphone use have been well-documented. Smartphones can be transferred easily between home and work environments. This boundary dissolution may create stress and anxiety for some people , 50. ForIn this study, smartphone addiction was associated with high depersonalization and poorer sleep quality prior to the intervention. The negative effects of nighttime media use are partly biological in nature. The human eye is most sensitive to blue light , which iMistimed light exposure may have other deleterious outcomes. While a direct link to smartphone use remains uncertain, current theories include modulation of the hypothalamic-pituitary-adrenal (HPA) axis, downregulation of melatonin, and upregulation of stress-related hormones such as glucocorticoids . FunctioLimitations of this study include data from one medical school, selection bias, self-reported data, confounding bias, and lack of a control group. Data from one medical school limits the generalizability of findings to other programs. Only students who were enrolled in pre-clinical education (year 1 and year 2) were included in the study; medical students in clinical education (year 3 and year 4) did not have the opportunity to participate. Third- and fourth-year students were excluded because they were not physically present on the medical school campus; rather, they were completing rotations at different clinical sites across the state. Future research should include medical students of all four years. Next, our findings may be susceptible to selection bias, as students who volunteered to participate may have been more willing or motivated to participate in an intervention designed to improve sleep and reduce burnout. In addition, the self-reported data collected via the pre- and post-assessments may be vulnerable to social desirability bias . To miniIn addition, the inclusion of the sunrise alarm clock introduced confounding bias to the study because the study design did not allow us to determine whether the improvements in sleep quality, perceived stress, and burnout were attributed to the removal of the smartphones at bedtime, the sunrise alarm clock, a combination of the two, or other factors. Finally, this feasibility study did not include a control group, which limits our ability to conclude whether the changes observed in the study were due to the intervention or other factors. Thus, future research must include a control group in order to draw meaningful conclusions. We plan to conduct a randomized-control trial removing smart phones and all electronic devices at bedtime and replacing those devices with a sunrise alarm clock or standard alarm clock. The aim will be to compare the effectiveness of this sunrise alarm clock intervention versus a standard alarm clock versus a control group . This three-arm parallel randomized controlled trial will determine whether the sunrise alarm clock intervention is an effective intervention and whether the sunrise alarm clock is more effective than a standard alarm clock.These findings suggest that the two-week sunrise alarm clock protocol with electronic device removal was effective in improving sleep quality and reducing perceived stress and burnout. However, additional research comparing this intervention to a proper control group is needed to draw meaningful conclusions about the effectiveness of this intervention. If these findings are confirmed via a randomized controlled trial, this intervention has the potential to improve medical students’ health and well-being.


Thiocyanate can outcompete chlorine anions and other halides  as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids  and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN− administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN− in diseases where MPO is implicated.Thiocyanate (SCN Non-smokers have saliva SCN− concentrations of 0.5–2 mM, while concentration in smokers average around 3 mM, and some smokers may have concentrations as high as 6 mM  i0.006 mM .− include glucosidic cyanogen-rich plants such as almonds, cabbage, kale, broccoli, cassava, yam, maize, sugar cane, sorghum and linseed [− is subsequently released at neutral pH to form the stable product, indole-3-carbinol  and thiosulfate (S2O32−) in the liver [2O32− and CN− is catalysed by hepatic enzymes, including mitochondrial thiosulfate sulfurtransferase (or rhodanese) and cytosolic mercaptopyruvate sulfurtransferase  and its precursor cobinamide, before being excreted through the renal system [Although SCNhe liver ,13  in a Na+-dependent process [2+ and interleukin-4 (IL-4) sensitive Cl− channels; the IL-4 sensitive SCN−/Cl− exchanged pendrin (SLC26A4) [In human bronchial epithelium, SCN process . SubsequSLC26A4) ,24,25,26− is reported to be 6–14 days [− does enter the glomerular filtrate, there is 90% reuptake and this accounts for the low urine SCN− levels found [− half-life in healthy individuals is reported to be from 1 to 5 days, and 9 days in individuals with renal insufficiency [− concentrations have been used as helpful biomarkers for exposure to tobacco or occupational smoke [The half-life of salivary SCN–14 days ,28,29,30ls found ,31. Plasal smoke .2O2) [3+) form that can undergo a 2-electron oxidation reaction with H2O2, generating the highly reactive oxy-ferryl (Fe4+ = O) heme species containing a porphyrin π-cation radical [−, Br−) and pseudohalides  to return to its native Fe3+ form [3+ state via an independent pathway involving two sequential one-electron reductions, yielding the intermediate Compound 2 (Fe4+ = O) heme species in a process coined the “peroxidase cycle” [Myeloperoxidase (MPO) is a heme homodimeric protein (~146 kDa) with functionally independent monomer units consisting of an iron protophorphyrin IX derivative located within the heavy chain of each monomer . The hem2O2) . Native  radical , also kne3+ form . This pae cycle” .− was in 1814 by German physician Gottfried Reinhold Treviranus, as he discovered a substance in human saliva that produced a blood-red colour when ferric ion was added [− had been of particular pharmacologic interest as an oral antihypertensive. Since then, multiple researchers have identified SCN− as a potentially important factor in health and disease.The earliest report on the biological significance of SCNas added . In the − plays a vital role as a substrate for human peroxidases, including MPO, LPO, salivary peroxidase, gastric peroxidase, eosinophil peroxidase and thyroid peroxidase [−-containing extracellular fluids together with H2O2 [SCNroxidase . Most ofith H2O2 . These cith H2O2 .− to generate the potent bactericidal radical, HOSCN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multi-subunit enzyme present in neutrophils and macrophages. In inflammation, activation of NADPH oxidase catalyses the reaction between oxygen and NADPH, generating superoxide anions, a process that has been coined the “oxidative burst” [2O2. Dismutation can occur spontaneously or may be catalysed by the enzyme superoxide dismutase [− with H2O2 to produce HOSCN, which is highly effective in killing microbes in these inflammatory environments through its free radical activity  such as Cl− and Br− can also be oxidised into hypohalous acids following reaction with MPO and H2O2. In addition to SCN− has a much higher specificity for MPO than Cl−, with specificity constants of 1, 60, and 730 for Cl−, Br−, and SCN−, respectively, so that SCN− is the preferred substrate for MPO [− levels are comparatively very low, with normal halide/pseudohalide ion concentrations in healthy human plasma at 100–140 mM (Cl−), 20–100 µM (Br−) and 200–250 µM (SCN−) [SCN for MPO . HoweverM (SCN−) .− has potential to become a competitive substrate for MPO when its level is elevated beyond normal plasma level, especially in secretions where SCN− levels are much higher. This is in part due to its faster reaction rate compared with Cl− and Br− [−, Cl− and Br− are 9.6 × 106 M−1s−1, 2.5 × 104 M−1s−1 and 1.1 × 106 M−1s−1, respectively [SCN and Br− . The secectively .8 M−1s−1 and 7.8 × 104 M−1s−1, respectively. Similarly, HOSCN oxidises glutathione (GSH) with a second order rate constant considerably lower than HOCl, with values of 2.5 × 104 M−1s−1 and 1.2 × 108 M−1s−1, respectively [Due to the nature and reactivity of different reactive oxygen species (ROS), HOCl is generally considered as a strong oxidant whilst HOSCN is often classified as a weak oxidant . For exaectively ,49.− is an important part of human host defence and health, with pharmacological interest dating back to 1857, where it was investigated for its clinical use as a hypotensive agent [− in fasting gastric juice specimens from human patients increased the likelihood of nitrosamine production and this was implicated as a potential contributor to gastric cancer [− were associated with lung cancer [− were significantly correlated with several diseases including cancer, chronic bronchitis, emphysema, coughing, wheezing and sleep-related conditions [− with cancer and lung diseases in a national, population-based study supported previous investigations that utilised smaller and non-representative human sample sizes and those that used animals [SCNve agent . Use in c cancer . Moreoveg cancer ,53. A monditions . The pos animals . Recent − in modulating MPO in disease activity and outcome.We now discuss the roles of SCNCardiovascular disease (CVD) is the primary cause of death worldwide, with the majority of CVD deaths related to coronary heart disease (CHD) . CHD is −/−) model of atherosclerosis [−/− mice fed a western diet [−/− mice [Porphyromonas gingivalis in mice, rabbits and pigs [The chlorinating activity of MPO is thought to be particularly detrimental during CVD, with high density lipoproteins and low density lipoproteins being vulnerable to oxidation by HOCl and impairing endothelial function via interference with nitric oxide production ,60,61. Hclerosis . Recentlern diet . Despite−/− mice , and thiand pigs .− have been shown to improve long-term survival in patients following an acute myocardial infarction [− [−/− mice fed a western diet, which have reduced atherosclerotic plaque size following 8 weeks of NaSCN treatment [− supplementation in atherosclerosis-prone Ldlr−/− mice transgenic for human MPO decreased the total plaque area with no changes to serum MPO concentrations between SCN− supplemented and control mice [High serum levels of SCNfarction . Ironicaction [− . Unlike ction [− . In thisreatment . In thisrol mice . Collect− is extensively involved in modulating the oxidative environments of various respiratory diseases, reducing the cytotoxic effect of more powerfully oxidative HOCl. For example, Xu and colleagues demonstrated the attenuation of MPO cytotoxicity by addition of SCN− to over 100 µM in the Calu-3 human lung epithelial, Neuro2a mouse neuroblastoma, human aortic endothelial cells and Min6 mouse pancreatic β cell lines [− by SCN− was demonstrated, with slight, partial and near complete inhibition achieved using 10 µM, 50 µM and 100–400 µM SCN−, respectively [SCNll lines . In the ectively . MPO actectively ,76 and dectively . Physiolectively ,78, cont−, as the anion permeability of CFTR for SCN− exceeds that of Cl− and the concentration of SCN− in the airway surface liquid is at least ~30 times its concentration in the serum [− that subsequently form HOCl in the presence of MPO, while also facilitating the formation of the effective antibacterial compound HOSCN [− in both human CF cells [− and GSH airway surface liquid levels, a finding which was reproduced in a CFTR knockout animal model [− to date.CFTR is a transmembrane receptor that functions as a chloride channel at the apical membrane of epithelial cells and mutations in CFTR results in the clinical presentation of CF . Intereshe serum ,81. Thisnd HOSCN . UnsurprCF cells ,82, as wCF cells . While nal model ,83, therP. aeruginosa airway infections, where the subsequent inflammatory response contributes to the major clinical problems associated with CF-lung tissue destruction [− through the administration of nebulised SCN− in a cystic fibrosis model using beta epithelial sodium channel (βENaC) mice [− significantly decreased airway neutrophil infiltrate by 68%, and therefore by extrapolation, neutrophil-derived MPO, as well as rebalanced GSH redox ratio in both lung tissue and the lining fluid of airway epithelium [−. Moreover, SCN− treatment had no effect on other cytokines, including C-X-C motif chemokine ligand 1, IL-1β, TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 p70. Levels of the granulocyte oxidative activity biomarker glutathione sulfonamide and glutathione disulfide were also decreased in βENaC mice [− levels in the epithelial lining fluid were decreased by 60% in the βENaC mice that were given vehicle treatment. Thus, the administration of SCN− to βENaC mice and subsequent reduction in neutrophil infiltrate may be diverting MPO-modulated oxidative activity into another biological compartment.CF patients are particularly vulnerable to chronic truction . Recent aC) mice . Compareithelium . In contNaC mice . InteresP. aeruginosa, where SCN− administration decreased levels of inflammation, bacterial burden, proinflammatory cytokines and 3-nitrotyrosine (only in infected wild-type mice) [− as compared with wild-type mice given vehicle treatment. Furthermore, βENaC mice had an 80-fold increase in bacteria relative to their wild-type counterparts, with SCN− treatment significantly reducing bacterial burden by 92% [P. aeruginosa infection suggests a consequential increase in secreted MPO, and with these data in consideration, may suggest that bacterial burden is ameliorated potentially through the microbicidal action of MPO/SCN− derived HOSCN.Both wild-type and βENaC mice were also infected with pe mice) . Bacterin by 92% . The obs− in respiratory airways, where SCN− supplementation may provide a therapeutic effect in patients with CF. The anti-inflammatory action of SCN− may potentially be attributed to a decrease in bacterial burden, as well as in the mitigation of neutrophilic infiltration, thereby diverting MPO-mediated damage away from pulmonary tissue.Taken together, the evidence suggests a protective role for SCN−, as well as hypothiocyanite (OSCN−), the conjugate base of HOSCN, holds antiviral effects that have been investigated in various in vitro influenza viral systems. OSCN− virucidal activity against the A/H1N1 2009 pandemic influenza virus has been demonstrated in vitro. Specifically, a dose-dependent effect without cytotoxicity was observed where 2 µM OSCN− administered achieved inhibition of viral replication by 50% prior to cell inoculation [2O2/SCN− system has been shown to inactivate the A/Swine/02860/2009 influenza A strain virus within both differentiated rat and human tracheobronchial epithelial cells [− or I−) [− and its chemical analogues working through the LPO/H2O2/SCN− system are beneficially implicated in inactivating numerous influenza virus strains in vitro. In light of the current global pandemic featuring the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), investigating the effects of SCN− on SARS-CoV-2 in vitro and perhaps in animal models, may be a potential avenue for further research.In addition to its antibacterial activity that combats respiratory infections, SCNculation . Moreoveal cells , and in al cells . Increas− or I−) . Neverth− levels considerably in the mucosa, one may assume that tobacco smoke can elicit antimicrobial effects; however, a plethora of evidence exists demonstrating that cigarette smoking exacerbates respiratory infections. For example, both passive and active smoking are significant risks for the development of upper respiratory tract infections, particularly otitis media [Streptococcus pneumoniae within the nasopharynx for both children and adults [Mycobacterium tuberculosis infections, resulting in the development of, and death from, active tuberculosis, though interpretation may be affected due to differences in data from these studies [As tobacco smoking increases SCNis media ,90,91 and adults . Further studies ,94,95. T studies , China [ studies , South A studies  and Mexi studies . Additio studies  and in i studies .− may offer.Profound changes in mucous production mechanisms and airway epithelial metaplastic changes can instead explain the increased susceptibility of smokers to respiratory infections. Squamous metaplasia of respiratory epithelium is common in habitual smokers, where specialised bronchial ciliated columnar epithelia is replaced with stratified squamous epithelia . This ad−/− mice [Rheumatoid arthritis (RA) is a T-cell and autoantibody-mediated autoimmune disease which results in joint damage and destruction of cartilage. Neutrophils are prominent in the pathogenesis of RA, as demonstrated in numerous RA mouse models as well as human disease . In two −/− mice ,108,109.−/− mice . Further−/− mice . Neutrop−/− mice .2+ site [2+ sites of pro-MMPs, increasing the overall activity of MMPs. Although the respective roles of HOSCN and MMP are not fully elucidated, it is well established that cigarette smoke and the associated increase in serum SCN− is linked with increased severity and incidence of RA, and that this results in increased intensity and duration of disease, so that fewer smoking patients enter full remission [− was supplemented to mimic elevated levels of SCN− in the blood, saliva, and urine of smokers [− showed pro-arthritic and proinflammatory changes when subjected to various arthritic-inducing agents.Destruction of cartilage, but not bone, is largely attributed to matrix metalloproteinase (MMP) activity in RA . Several2+ site . The MPO2+ site . While H2+ site ,115. Theemission . In a ra smokers . Rats su−) [− forms a natural homeostatic equilibrium in physiological systems, where OCN− concentration is too low to allow extensive carbamylation of protein. However, several environmental factors, such as smoking, cause a pathophysiological increase in OCN−. Increased SCN− levels from cigarette smoke drive MPO Compound 1-mediated competitive oxidation of SCN− to HOSCN and, to a lesser extent, OCN− [Carbamylation is a non-enzymatic post-translational modification, whereby amine or thiol groups transform into carbamyl groups via the presence of increased cyanate (OCN−) . OCN− font, OCN− ,119,120.nt, OCN− . Interesnt, OCN− .− is pro-arthritic via the potential pathway for MPO-derived HOSCN to activate pro-MMP at synovial sites, or the MPO/HOSCN-mediated increase in local OCN− levels, increasing overall carbamylation of local proteins.From the above, the evidence suggests that SCNTwo main conditions drive inflammatory bowel disease (IBD) where there is chronic inflammation in the gut: ulcerative colitis (UC) and Crohn’s disease. A chronic influx of leukocytes into the gut mucosa is a major pathological presentation in IBD, which is thought to result from an abnormal host immune response to otherwise harmless commensal flora . In the It is increasingly recognised that ROS generated during inflammation plays a significant role in prolonging gastrointestinal inflammatory cycles and causing gastrointestinal injury. Colonic and faecal MPO, present in polymorphonuclear leukocytes, are significantly increased and correlate with disease severity in UC patients ,129. MPO− concentrations in body fluids also exhibit reduced risk (1.7-fold) against UC and protection from the clinical symptoms including reduced flares, less need for steroids and a lower colectomy rate [− supplementation would confer protection during experimental colitis by redirection of MPO halogenation to favour HOSCN production above HOCl.Interestingly, active smokers who have significantly higher SCNomy rate . Thus, ww/v) dextran sodium sulfate (DSS) colitis model [− levels in NaSCN-supplemented mice to above IC50 inhibition levels of MPO/HOCl, as determined by HOCl-mediated oxidation of luminol. Notably, 3-Cl-Tyr was found to be comparatively lower in colonic samples of SCN−-supplemented DSS mice. This indicated a reduction in the production of HOCl, potentially diverging MPO oxidation production to HOSCN by increased presence of free SCN− ions.In our recent study, mice were supplemented with NaSCN to closely match levels in human smokers  before bis model . We obse−-supplemented groups. Interestingly, mice supplemented with DSS/SCN− showed marked upregulation in thiol synthesis markers Nrf2 and GCLC, indicating that thiol synthesis was enhanced in this group of mice and may provide an increase in antioxidant status for the colon during DSS-insult. Overall, increasing SCN− in the gut and circulation provided minimal protection against active experimental colitis.However, NaSCN supplementation did not attenuate the course of experimental murine colitis. No data were collected on mouse activity levels, colon lengths, or colonic histopathology in the SCN− supplementation has been investigated in various disease models (− appears to be protective against disease via direct modulation of MPO activity, favouring the production of the HOSCN oxidant. On the other hand, SCN− seems to be implicated in the pathogenesis of rheumatic arthritis, while there is limited evidence to support a role in IBD. Irrespective of any possible direct roles for MPO in pathogenic mechanisms, the potential therapeutic value of SCN− must be carefully considered in the context of each specific clinical condition.SCNe models . Notably


Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions. Satellite cells (SCs) are the adult stem cells of skeletal muscle. They were first identified by Mauro in 1961who named them satellite cells because of their position between the sarcolemma and the basal lamina of the fiber . HoweverProtein kinase C Theta (PKCθ) is a serine and threonine kinase belonging to the family of novel PKCs. It is mostly expressed in hematopoietic cells with high levels in T cells, where it regulates cell activation, proliferation, and survival ,8. In adWe previously showed that knock-out of PKCθ or its inhibition in a mouse model of Duchenne Muscular Dystrophy (mdx mouse) reduces muscle damage, by modulating the immune cell infiltration. This beneficial effect was mainly due to the inhibition of PKCθ in T cells, which leads to reduced inflammation ,11,12. IHere, we show that PKCθ plays a direct role in SCs activity by stimulating their symmetric division and promoting their self-renewal. Thus, in the absence of PKCθ, there is a significant increase in the quiescent SC pool following acute muscle injury. Overall, our findings suggest that, targeting PKCθ might be a potential novel approach to manipulate SC self-renewal in pathological conditions and improve regeneration.Satellite cells can exist in a quiescent state in physiological conditions, or as activated/committed progenitors during muscle regeneration . To elucWe then analyzed the localization of active PKCθ by immunofluorescence analysis of phospho-PKCθ in SCs isolated from WT mice and cultured for 24 and 72h.To identify proliferating SCs and visualize the mitotic spindle we performed α-Tubulin co-immunostaining. At 24h in culture, when SCs did not enter the cell cycle yet, phospho-PKCθ was localized on plasma membrane D. In proThese results suggest that PKCθ could be involved in the regulation of SCs division processes.+ cells. As shown in +/Ki67+) was similar between WT and PKCθ-/- mice.To understand whether SC proliferation is affected by PKCθ,we isolated SCsfrom WT and PKCθ-/- muscles, labeled them with the CFSE proliferation die, and cultured in growth medium for 72h.FACS analysis showed that there was no difference in CFSE fluorescence between WT and PKCθ-/- cells A, suggesTogether, the results obtained demonstrate that satellite cell proliferation is not regulated by PKCθ; thus, the localization of phospho-PKCθ at the centrosomes and mitotic spindle of dividing SC should reflect a role of this protein to other processes associated to cell division, rather than proliferation. Interestingly, in T cells, PKCθ promotes MTOC reorientation and cell polarization in the direction of the antigen presenting cell. This process may also promote asymmetric division, that is important for the acquisition of T cell memory . We thus+/MyoD−, or both Pax7+/MyoD+. Asymmetric division generates two cells that differ for MyoD expression, instead: one cell will be Pax7+/MyoD−, and the other will be Pax7+/MyoD+. After 48h in culture cell doublets were visible, and symmetric/asymmetric division was studied by immunofluorescence staining for Pax7 and MyoD.To investigate this possibility, we isolated single myofibers from WT and PKCθ-/- EDL muscles, and cultured them for 48h, which is the time necessary for the first cell division to occur. In this system SCs remain associated with the fiber, allowing us to study their behavior in conditions similar to their in vivo niche environment. During in vitro culture of myofibers SCs become activated, upregulate MyoD and enter cell cycle. Symmetric division generates two identical cells, which can be both Pax7+MyoD−, whereas SCs committed to differentiation were Pax7+MyoD+. We observed a significant increase in the number of symmetric SC divisions in myofibers isolated from PKCθ-/- mice, compared to WT  and Pax7 on single myofibers isolated from WT and PKCθ-/- mice, after 36h in culture, by immunofluorescence staining. At this time point, polarity proteins are segregated, but the first mitotic division has not occurred yet. In Both WT and PKCθ-/- myofibers, 66% of the SCs showed low to absent Pard3 expression. Among the Pard3 expressing cells, we found a significant increase in the percentage of cells showing symmetric distribution of Pard3 in PKCθ-/- compared to WT SCs E–G. LikeTogether, these results suggest that PKCθ regulates SC self-renewal in vitro, and its absence promotes symmetric cell division.We next sought to validate our results from the PKCθ-/- mouse model, using the C20specific inhibitor of PKCθ ,12,18,19+/MyoD− reserve cells in a dose dependent manner  muscle injury, when the muscle is regenerating or is completely regenerated, respectively. Contralateral uninjured muscle was used as control.+ cells revealed that the number of SCs per mm2 and the number of SCs per fiber was similar in PKCθ-/- and WT gastrocnemius (GA) uninjured muscles  and analyzed theSC number before and after CTX injury.+ cells per mm2, and the number of Pax7+ cells per fiber was similar in uninjured GA in C20 and vehicle treated mice. However, the number of SCs that returned to quiescence 28 days after injury, was significantly higher in mice treated with C20, compared to vehicle, with a 50% increase  cell line it was previously shown that PKCθ is recruited to the mitotic spindle and associates with centrosomes and kinetochore of dividing cells. Furthermore, in non-proliferating or differentiated MEL cells, PKCθ expression was down-regulated . Althoug+/MyoD− ‘reserve cell’ population, in vitro and in vivo. After the induction of muscle injury, the number of SCs going back to quiescence increased by 65% in PKCθ-/- mice, compared to WT, and pharmacological inhibition of PKCθ with C20 inhibitor led to similar results after injury. Notably, a 10-day treatment with C20 during the first phase of regeneration was sufficient to observe the increase of the quiescent SC pool, suggesting that a PKCθ inhibitor treatment can be useful even when administered for a short time. No alteration in immune response has been observed in PKCθ-/- regenerating muscle compared to WT, further supporting the notion that PKCθ plays an intrinsic role in SC function, through the regulation of polarity proteins distribution, as we propose.Since we detected phospho-PKCθ to the centrosomes and mitotic spindle of SCs, we reasoned that PKCθ might regulate SC polarity during division. Par-3, Par-6, and aPKC form a complex that localizes to the apical pole of the dividing stem cells, and distribute polarity proteins that direct mitotic spindle orientation ,17. We fInterestingly, it has been previously shown that, in mdx mice, the absence of dystrophin in SCs leads to the downregulation of the important regulator of Pard3 polarization, Mark2 (Par1b in Drosophila), since it confines Pard3 to the opposite side of the cell. In the absence of dystrophin, Mark2 downregulation causes a reduction of Pard3 polarization, and the number of satellite cell symmetric divisions generating self-renewing cells increases . FurtherOverall, our findings suggest that PKCθ might be a promising target in conditions where SCs show functional decline and enhanced myogenic commitment, such as aging. The functional decline in aged satellite cells is due to modifications in the satellite cell niche, such as deregulations in Wnt, Notch, and TGFβ pathways . IntrinsIn conclusion, our results identify PKCθ as an important regulator of SC self-renewal and a novel potential target to counteract satellite cell loss in pathological conditions by limiting the number of cells committed to differentiation.PKCθ-/- mice (C57BL/6J background) were originally provided by Dan Littman . In these mice, the gene encoding PKCθ Is inactivated in all cells of the body, as previously described . C57BL/6Cardiotoxin injury was performed by injecting 20 μL of Cardiotoxin from Naja Pallida , with two injections of 10 μL each, in two different areas of the GA muscle, using a 30 Gauge micro-syringe.Single myofibers were isolated from EDL muscles of 4–6-week-old mice, as described ,32,33. BSCs were prepared from hind-limb muscles of 4–8 week-old mice, as previously described . BrieflyImmunofluorescence analysis was performed as previously described . For immFor myofiber staining, myofibers were fixed in PFA 4% for 5 min RT, permeabilized in cold methanol −20 °C for 6 min, and then incubated in Glycine 1M for 10 min RT to reduce background. Blocking was performed in BSA 4%, Goat Serum 5% for 30 min RT. Primary antibodies mouse anti Pax7, rabbit anti-MyoD , and rabbit anti Pard3  were incubated O/N at 4 °C. The next day myofibers were incubated for 1h at RT in biotin anti-mouse and subsequently in secondary antibodies goat anti rabbit Alexa Fluor 488 and Streptavidin Cy3 for 1h RT, at the concentration listed before. Nuclei were counterstained with Hoechst or Topro3. Samples were analyzed under an epifluorescence Zeiss Axioskop 2 Plus microscope .Satellite cells were homogenized in ice-cold buffer containing 20 mMTris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 250 mM sucrose, 5 mMDTT, 200 mg/mL leupeptin, 10 mg/mL Aprotinin, 1 mM PMSF, and0.1% Triton X-100  and then disrupted by sonication, as previously described . The homTo prepare single cell suspension, muscles were dissected from mice and finely minced with a scalpel in a dish containing DMEM, as previously described . Next, mFor CFSE analysis isolated SCs were stained with CFSE  5 µm for 20′ at 37 °C in dark prior to culture. Samples were processed using a Dako CyAn ADP flow cytometer and acquired data were analyzed using FlowJo software version 10 .For RNA preparation from muscle, the muscles were homogenized in TissueLyser  in the presence of TRI reagent (Sigma-Aldrich). Samples were then processed as previously described . The folt tests or Anova test. Differences were considered statistically significant at the p < 0.05 level.All statistical analyses were performed using GraphPad Prism software version 6 . Quantitative data are presented as means ± SEM of at least three experiments. Statistical analysis to determine significance was performed using Student’s


Scientific Reports10.1038/s41598-018-29949-8, published online 24 August 2018Correction to: The original version of this Article contained errors.The authors identified the following error in the published version of the Article: the topmost date of core F-02, serving as the uppermost constraint of the compound age-model was incorrectly given as 3,025–2,790 cal. yrs B.P and should be 2,783–2,505 cal. yrs B.P.The authors calculated a new age-model using the correct date as the uppermost constraint. Aeolian Sand Influx (ASI) was recalculated using the corrected sedimentation rates and results were changed accordingly in all Figures. Spectral analysis and Evolutive Harmonic analyses were performed again based on the corrected time-series of ASI. Figure 3 was corrected accordingly. Coherence analyses between the authors’ and paleoceanography records were performed again. Figure 5B and 5C were corrected.Minor adjustments have been made to the text to account for the errors in the original Article.As a result of these corrections, in the Abstract,“We find that Holocene North-Atlantic storminess is dominated by robust millennial  to centennial (≈ 400 and 200-year) periodicities.”now reads:“We find that Holocene North-Atlantic storminess is dominated by robust millennial  to centennial  periodicities.”Also in the Abstract,“Finally, we demonstrate that enhanced zonal storminess activity over the North-Atlantic was the driver of centennial-scale changes in North-Atlantic oceanic circulation, while ocean dynamics most likely influenced back the atmospheric circulation at millennial time-scales.”now reads:“Finally, we demonstrate that enhanced zonal storminess activity over the North-Atlantic was the driver of millennial and centennial-scale changes in North-Atlantic oceanic circulation, while ocean dynamics most likely influenced back the atmospheric circulation at millennial time-scales.”In the Introduction,“The record extends back to the early Holocene , making it the longest continuous and high-resolution record of past storminess for North-western Europe.”now reads:“The record extends back to the early Holocene , making it the longest continuous and high-resolution record of past storminess for North-western Europe.”In the Introduction section, under the subsection ‘Stratigraphic record’,“The inferred age of the sediment in the core F06 spans from 10,100 B.P. to about 2,500 B.P. It was sampled with a mean resolution of 40 years . Core F06 can be subdivided into four main sedimentary units : (i) Bottom units I and II  are composed of well-decomposed gyttja deposits progressively gaining organic content towards the top.”now reads:“The inferred age of the sediment in the core F06 spans from 10,200 B.P. to about 2,500 B.P. It was sampled with a mean resolution of 40 years . Core F06 can be subdivided into four main sedimentary units : (i) Bottom units I and II  are composed of well-decomposed gyttja deposits progressively gaining organic content towards the top.”The original version of Figure 2 contained errors and appears below as Figure In the Introduction section, under the subsection ‘Source of sand’,“Based on these considerations, we infer that the sedimentary sequence of core F06 continuously recorded influx of aeolian sand blown downwind by storm-winds from NW to SW sectors between 10,100 and 2,500 B.P.”now reads:“Based on these considerations, we infer that the sedimentary sequence of core F06 continuously recorded influx of aeolian sand blown downwind by storm-winds from NW to SW sectors between 10,200 and 2,500 B.P.”In the Introduction section, under the subsection ‘Storminess record’,“The sedimentary record of core F06 shows repetitive fluctuations between periods of higher and lower input of aeolian sand to the site between 10,100 and 2,500 B.P. The bottom part of the sequence , is first characterized by a marked succession of two wide peaks in both total and > 125 µm ASI separated by a clear low.”now reads:“The sedimentary record of core F06 shows repetitive fluctuations between periods of higher and lower input of aeolian sand to the site between 10,200 and 2,500 B.P. The bottom part of the sequence , is first characterized by a marked succession of two wide peaks in both total and > 125 µm ASI separated by a clear low.”and in the same section,“Following the deposition of the sandy unit III  and throughout most of unit IV, we observe a stable decrease of ASI, and notably of the ASI > 125 µm before it rises again sharply around 3,000 B.P.”now reads:“Following the deposition of the sandy unit III  and throughout most of unit IV, we observe a stable decrease of ASI, and notably of the ASI > 125 µm before it rises again sharply around 3,200 B.P.”Also in this section,“The sedimentary record from Filsø can be divided into eight main periods of increased storminess (“Filsø Storm Periods” FSP) centred on 9,700–8,900 (FSP1), 8,700–8,400 (FSP2), 8,200–7,000 (FSP3), 6,600–6,700 (FSP4), 6,400–5,300 (FSP5), 5,100–4,500 (FSP6), 4,400–3,800 (FSP7) and 3,300–2,900 B.P. (FSP7) .”now reads:“The sedimentary record from Filsø can be divided into eight main periods of increased storminess (“Filsø Storm Periods” FSP) centred on 9,700–9,100 (FSP1), 8,800–8,600 (FSP2), 8,300–7,100 (FSP3), 6,900–6,700 (FSP4), 6,400–5,500 (FSP5), 5,100–4,700 (FSP6), 4,400–3,800 (FSP7) and 3,300–2,800 B.P. (FSP7) .”Also in the Introduction, under the subsection ‘A multi-scale variability of Holocene North-Atlantic storminess activity’,30,31,32,33,34,35. The ≈ 2,500-yr periodicity has been proposed to be strongly correlated with variations in the total solar irradiance34,36. The persistence of a clear and stationary ≈ 2,500-yr periodicity in our storm-record over most of the Holocene  thus bring in contention that solar forcing may have been the main driver of the functioning of the climate system and storminess activity at a ca. 2,500-yr pace over the Holocene.”“Spectral analysis of the lower frequencies shows storminess to be composed of five periodicities of ≈ 2,500, ≈ 1,300, ≈ 600, ≈ 400 and ≈ 200 years at the 99% significance level , the ≈ 2,500-yr period showing the strongest power. As higher frequencies are concerned, strong wavelengths of ≈ 600-, ≈ 400-, ≈ 300-, ≈ 200-, ≈ 130- and ≈ 80-yr periods are evidenced , demonstrating the persistence of pluri-decadal to centennial scale modulations of North Atlantic storminess during the Holocene. EHA show variable stationarity of these millennial and centennial periodicities. A relatively wide band of quasi-periodic variability containing the dominant ≈ 2,500-yr period clearly dominates the signal over most of the Holocene . A notable non-stationarity of the spectral signature nonetheless appears between ≈ 6,000 and ≈ 4,500 B.P., when the signal observes a marked excursion toward shorter periods of ≈ 1,300 years . This temporary excursion towards a ≈ 1,300-yr mode of periodicity at the mid-Holocene seems accompanied by a convergence in the high-frequencies wavelengths characterizing the storminess signal during the early-Holocene . Indeed, from ca. 10,000 to ca. 6,800–6,500 B.P, EHA show our storm record to be dominated by robust quasi-stationary ≈ 480-yr and ≈ 300-yr wavelengths . A clear shift operates around 6,800–6,500 B.P. when the ≈ 280-yr periodicity is observed to progressively vanish while the ≈ 500-yr period evolves towards a strong ≈ 400-yr one. Pluri-millennial periodicities have been repeatedly documented in Holocene palaeo-environmental studies of the North Atlantic domain. The origins of these latter have been intensively discussed with regards to external (solar) and internal (oceanic/atmospheric) variabilitynow reads:30,31,32,33,34,35 Periodicities of ca. 2000–2,500 years have been proposed to be strongly correlated with variations in the total solar irradiance34,36. The persistence of a clear and stationary ≈ 2,200-yr periodicity in our storm-record over most of the Holocene  thus bring in contention that solar forcing may have been the main driver of the functioning of the climate system and storminess activity at a ca. 2,500-yr pace over the Holocene.”“Spectral analysis of the lower frequencies shows storminess to be composed of five periodicities of ≈ 2,200, ≈ 1,350, ≈ 590, ≈ 450 and ≈ 200 years at the 90% significance level , the ≈ 2,200-yr period showing the strongest power. As higher frequencies are concerned, strong wavelengths of ≈ 600-, ≈ 500-, ≈ 450-, ≈ 280-, ≈ 200-, ≈ 150- and ≈ 80-yr periods are evidenced , demonstrating the persistence of pluri-decadal to centennial scale modulations of North Atlantic storminess during the Holocene. EHA show variable stationarity of these millennial and centennial periodicities. A relatively wide band of quasi-periodic variability containing the dominant ≈ 2,200-yr period clearly dominates the signal over most of the Holocene . A notable non-stationarity of the spectral signature nonetheless appears between ≈ 6,000 and ≈ 4,500 B.P., when the signal observes a marked excursion toward shorter periods of ≈ 1,300 years . This temporary excursion towards a ≈ 1,300-yr mode of periodicity at the mid-Holocene seems accompanied by a divergence in the high-frequencies wavelengths characterizing the storminess signal during the early-Holocene . Indeed, from ca. 10,200 to ca. 6,800 B.P, EHA show our storm record to be dominated by robust quasi-stationary ≈ 400 to 500-yr and 300-yr wavelength . A clear shift operates around 6,800–6,500 B.P. when the ≈ 280-yr periodicity is observed to progressively vanish while the ≈ 500-yr period evolves towards strong ≈ 650-yr and 450 ones. Pluri-millennial periodicities have been repeatedly documented in Holocene palaeo-environmental studies of the North Atlantic domain. The origins of these latter have been intensively discussed with regards to external (solar) and internal (oceanic/atmospheric) variability.The original version of Figure 3 was incorrect and appears below as Figure In the Introduction section, under the subheading ‘North-Atlantic storminess and ocean dynamics linkages.’,“Multi-taper coherence analyses  performed on our storm record and on the record of the abundance of G.ruber w. show the two series to share common periodicities of ≈ 1,500, ≈ 1,000, ≈ 750, ≈ 400, ≈ 300 and ≈ 200 yrs, the highest coherence estimates being obtained for the ≈ 300 and ≈ 200-yr periods .”now reads:“Multi-taper coherence analyses  performed on our storm record and on the record of the abundance of G.ruber w. show the two series to share common periodicities of ≈ 1,300, ≈ 700, ≈ 400, ≈ 240 yrs, the highest coherence estimates being obtained for the ≈ 1,300, 700 and ≈ 400-yr periods .”Also in this section,4. The opposite appears in longer periods, the slight anti-phasing observed at the ≈ 1,500, ≈ 1,000 and ≈ 760-yr period  being in favour of a bottom-up forcing of oceanic variability on atmospheric processes at millennial time-scales.”“The results of the coherence analyses between storminess and the proxy record of the extent of the STG53 nonetheless show clear positive phasings  at centennial time-scales . Such phasing suggests that changes in storminess led STG variability at centennial time-scales, in line with what is observed nowadays at inter-annual to decadal scalesnow reads:4. Smaller phases are obtained for ≈ 1,300 and 700-yr periods  being in favour of a bottom-up forcing of oceanic variability on atmospheric processes at millennial time-scales.”“The results of the coherence analyses between storminess and the proxy record of the extent of the STG53 nonetheless show clear positive phasing  at both millennial and centennial time-scales . Such phasing suggests that changes in storminess led STG variability, in line with what is observed nowadays at inter-annual to decadal scalesAlso in this section,31 .”“A nearly perfect peak to peak synchronism is also apparent between our record and the stacked record of Ice-Rafted Debris (IRD) reconstructed in the North Atlantic over the 10,000–2,500 B.P. periodnow reads:31 .”“A nearly perfect peak to peak synchronism is also apparent between our record and the stacked record of Ice-Rafted Debris (IRD) reconstructed in the North Atlantic over the 10,200–2,500 B.P. periodAlso in this section,“Multi-taper coherence analyses of our storm record and the input of IRD to the North-eastern Atlantic show the two series to be highly coherent at the 1,500-yr period with a phase lag of 35 degrees .now reads:“Multi-taper coherence analyses of our storm record and the input of IRD to the North-eastern Atlantic show the two series to be highly coherent at the 1,500-yr period with a phase lag of 47 degrees .The original version of Figure 5 was incorrect and appears below as Figure In the Conclusion,“Storminess most likely drove the activity of North-Atlantic oceanic gyres at centennial time-scales but the ocean appears to have influenced back atmospheric variability at longer millennial time-scales.”now reads:“Storminess most likely drove the activity of North-Atlantic oceanic gyres at both millennial and centennial time-scales.”In the Methods section, under the subheading ‘Statistical analyses’,“All time-series were linearly re-interpolated at 40 yrs from 2,580 to 10,100 yrs B.P. prior to the CMTM.”now reads:“All time-series were linearly re-interpolated at 40 yrs from 2,488 to 10,200 yrs B.P. prior to the CMTM.”The original version of the Supplementary Information file also contained errors.In Supp. Info 2 changes have been made to Core F06 (left-hand side) and Core F02 (right-hand side). In Core F06 (left-hand side), the date noted for F06-U on the original figure was renamed F06-6 and annotated "rejected". Date F06-4 was recalibrated. Its position alongcore, originally mistakenly marked at 176 was changed to 181 and annotated “rejected”. In Core F02 (right-hand side) the date originally noted F02-6 was modified to read 2,783  2,505 and renamed F02-4. The date originally noted F02-5 was renamed F02-3 and annotated “rejected”. All dates were re-numbered from F02-1, instead of being numbered from F02-3 in the original figure.Supp. Info 3A has been modified where the topmost date has been changed. The date at 181 cm depth was changed to match the re-calibration of date F06-4. The curve of the age-model was redrawn to match the new age-model.Supp. Info 3B has been modified so that the dates from the two cores are mixed and presented in chronological order; as they appear in the compound stratigraphic model: the younger age on top, the older at the bottom. Three new columns were added; (i) a column showing the depth of the datation in the core from which it was obtained; (ii) an accepted/rejected column so that rejected datations are more easy to find by the reader and (iii) a column showing the position of the datation in the compound age-depth model. In the rightmost column ‘Age [cal yr BP]’, the authors added the median age (in brackets) to the minimum and maximum ages given by the two-sigma probabilities. Some ages were not reported with their full error ranges in the original version of this table so all ages were recalibrated following a unique procedure under calib 7.0 software and reported with the full 95.4% 2-sigma error range to ensure coherence and repeatability. This lead to slight changes in the min–max ages, but not in the median age.These errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file. The original Supplementary Information accompanies this correction.The overall conclusions of the Article are unaffected by these corrections.Supplementary Information.


Yet only two minerals, fougerite ([Fe2+6xFe3+6(x−1)O12H2(7−3x)]2+·[(CO2−)·3H2O]2−) and mackinawite (Fe[Ni]S), are vital—comprising precipitate membranes—as initial “free energy” conductors and converters of such disequilibria, i.e., as the initiators of a CO2-reducing metabolism. The fact that wet and rocky bodies in the solar system much smaller than Earth or Venus do not reach the internal pressure (≥23 GPa) requirements in their mantles sufficient for producing bridgmanite and, therefore, are too reduced to stabilize and emit CO2—the staple of life—may explain the apparent absence or negligible concentrations of that gas on these bodies, and thereby serves as a constraint in the search for extraterrestrial life. The astrobiological challenge then is to search for worlds that (i) are large enough to generate internal pressures such as to produce bridgmanite or (ii) boast electron acceptors, including imported CO2, from extraterrestrial sources in their hydrospheres.Life cannot emerge on a planet or moon without the appropriate electrochemical disequilibria and the minerals that mediate energy-dissipative processes. Here, it is argued that four minerals, olivine ([Mg>Fe] Life being an emergent far-from-equilibrium, actively dynamic structure suggests that astrobiological exploration should focus on wet worlds where mantle convection, coupled to hydrothermal convection, resulted in strong electrochemical disequilibria commensurate with life and its emergence ,7,8,9,10A fundamental understanding of what drove life into being on our planet requires knowledge of how inorganic minerals, accreted from the solar disc, responded to physicochemical pressure and stresses induced by convection and differentiation in an early magma ocean ,8,15,16.2—or the electrons prized therefrom—degassing from heterogeneous sources deep in the mantle and at the core-mantle boundary ..9].In order to produce these fuels, redox and pH gradients that mediated life’s emergence required just six ‘must-have’ minerals  42,62].,62.42,622SiO4), bridgmanite , serpentine ]4) and pyrrhotite (Fe(1-x)S). We begin with olivine, a ubiquitous mineral contributing to the primary extrusive and intrusive basaltic or komatiitic ocean floor, crust and upper mantle —which is hidden at depth out of reach of spectroscopy. Its occurrence must be gauged experimentally from a planet’s metamorphic response to size and dehydration and as supported by seismic profiles [The four essential minerals in planetary bodies needed to produce the chemical disequilibria and materials required for life’s emergence are olivine ([Mg>Fe]a oceans ) to a myprofiles ,71. Hydrprofiles ,44,46. Oprofiles ,56,72. Tprofiles ,50,53,73profiles ,32,69.2 increase 2+·[(CO2−)·3H2O]2−) and mackinawite (Fe[Ni]S), are vital as initial “free energy” conductors and converters of such disequilibria, and are considered as the initiators of a CO2-reducing metabolism in a membrane constituted of these same minerals  that Russell and collaborators , responsible for our oxidized mantle, indicate that ringwoodite suffers a sharp density-increasing phase change to bridgmanite at depth of ~660 km which corresponds to a pressure of 21 GPa. Thus, the original magma ocean and mafic to ultramafic volcanoes would have exhaled carbon mainly as carbon dioxide to the ocean floor, which produced an ocean and atmosphere comprising mostly electron acceptors rather than donors SiOn donors  8,55,23,55,233, mosphere ,238. Themosphere ,136. Thephere 2 ,44, and , and 3, tabolism ,130.However, the wet and rocky bodies in the solar system smaller than Earth and Venus probably have more reduced mantles comprising little or no bridgmanite. This consideration could explain the predominance of methane on the moons of Jupiter and Saturn ,243,244 2 oxidizing atmospheres and therefore no obvious electrochemical disequilibria required to drive life’s emergence, nor to sustain habitability, there are caveats:While the several approaches employed here appear to converge on the limiting mineral fundaments to life’s emergence, without which there would be no early COde novo but evolves via autotrophy and heterotrophy—a point of view open to debate and astrobiological exploration [(1) AVT assumes that phototrophy cannot emerge loration ;2 atmosphere with a pressure exceeding 250 mbar is at odds with the likely high hydrogen fugacity  of the Martian mantle and the paucity of carbonate outcrop. Although such an early atmosphere has not been disproven, high emissions of H2 could also exoplain the warm temperatures at that time [(2) The assumption derived from climate modelling that Mars once had a COhat time ,247,248;2-bearing comets in our solar system [(3) Some carbon dioxide feedstock on Mars and sub-Mars sized bodies that had no shortage of volatile reductant fuels, might have been provided, and continuously so, from COr system ,249;(4) Oxidants on moons such as Europa may be generated qualitatively by relatively local high energy radiation ,251,252;2 [2 to act as a positive ‘electrode’ for life’s emergence, a possibility enhanced by the addition of nitric oxides produced by high energy coronal mass injections [(5) It is not possible to tell as yet whether the conclusions presented here are applicable to exoplanet exploration. The challenge is to demonstrate the availability of electron acceptors on these other worlds up to 1.6 Earth radii (e.g., ,253,254.2 . While sjections .


For the qualitative and quantitative assessment of the inorganic fraction, it is highlighted the use of a nuclear analytical method, instrumental neutron activation analysis, which allows having more information as possible from the sample without performing any chemical-physical pretreatment. The results have evidenced high levels of mercury , antimony (77.7 µg g−1), strontium  and zinc (103 µg g−1), whereas rare earth elements show levels similar to the literature data. Particular consideration is drawn for arsenic (414 µg g−1): the levels found in this paper (ranging between 1 and 5100 µg g−1) explain the high content of such element (as arsenates) in the aquifer, a big issue in this area.This paper shows a characterization of the organic and inorganic fraction of river waters (Tiber and Marta) and ores/soil samples collected in the Northern Latium region of Italy for evaluating the anthropogenic/natural source contribution to the environmental pollution of this area. For organic compounds, organochloride volatile compounds in Tiber and Marta rivers were analyzed by two different clean-up methods  followed by gas chromatography–electron capture detector (GC-ECD) analysis. The results show very high concentrations of bromoform (up to 1.82 and 3.2 µg L The current legislation defines pollution as any modification of the atmospheric air, due to the introduction into it of one or more substances in quantities and with characteristics such as to damage or constitute a danger to human health or to the quality of the environment, or such as to damage material assets or compromise legitimate uses of the environment ,2. AnthrNowadays, environmental contamination episodes are becoming very frequent. From an environmental point of view, a chemical characterization is really important because it helps the evaluation of the environmental impact of anthropogenic activities on urban areas and industrial and agricultural sites . The anaAmong thousands of organic compounds present in environmental matrices, halocarbons, i.e., organic chemical molecules containing at least a chemical covalent bond between a carbon atom and one (or more) halogen atoms ,12, are −3 [On the other hand, the inorganic fraction essentially regards heavy metals. This is a general term for identifying a group of metals and metalloids with an atomic density greater than 5 g cm−3 . The hum−3 ,17. Alth−3 . Their c−3  were detStarting with these considerations, the authors would like to explore how anthropogenic activities and natural sources can affect an area located in the Northern Latium, Central Italy. Organic compounds such as halocarbons and the inorganic fraction in terms of elements were determined: the first ones can be considered an index of the anthropogenic sources, whereas the second ones can be considered an index of natural sources, i.e., from the continental crust.Ore and river-waters from Tiber and Marta river samples were collected in different intensive sampling campaigns aimed at identifying the anthropogenic/natural impact of different activities in this area. Organohalogen compounds were determined by a chromatographic method, whereas the inorganic elements by means of nuclear analytical technique such as Instrumental Neutron Activation Analysis (INAA) , i.e., a2 . The water was sampled annually in the period April 2015–March 2016: particularly, 48 water samples were taken in spring-, summer- and fall-time near Viterbo (42°25′07″ N 12°06′15″ E) and Tarquinia (42°14′57″ N 11°45′22″ E) and at the mouths of the Tiber and Marta River (42°31′59.89″ N 11°55′51.49″ E), and in the Bracciano area (42°06′ N 12°11′ E). On the other hand, 110 ores were collected from 8 different areas in the North Latium region between July and August 2015: Bracciano area (42°06′ N 12°11′ E), Ceriti Mt. (42°09′03.02″ N 11°54′34.31″ E), Fate Mt. (41°24′17.89″ N 13°18′29.52″ E), Sabatini Mt. (42°10′ N 12°15′ E), Oriolo Romano (42°09′28.08″ N 12°08′16.08″ E), Solfatara basin (42°25′17″ N 11°52′19″ E), Vulsini Mt. (42°40′ N 11°55′ E), Acqua Rossa basin (42°29′ N 12°08′ E). Soil and river water samples were sampled across the Northern Latium, throughout the volcanic area between Tuscany and the sea (Tyrrhenian Sea), in an area of 6363 km3), trichloroethane (CHCl2CH2Cl), hexachloroethane (CCl3CCl3), tetrachloromethane (CCl4), chlorobenzene (C6H5Cl), trichloroethene (CCl2CHCl), dichlorobromomethane (CHBrCl2), bromoform (CHBr3), tetrachloroethene (CCl2CCl2), dibromochloromethane (CHClBr2) and tetrachloroethane (CHCl2CHCl2) , methanol (pesticide grade), and NaCl (RPE) were all from Carlo Erba . Solutions of each halocarbon standard were prepared at the following concentrations: CHCl3—7.4 g L−1; CCl3CCl3—6.70 g L−1; CCl4—0.8 g L−1; CCl2CHCl—7.3 g L−1; CHBrCl2—9.9 g L−1; CHCl2CH2Cl—8.6 g L−1; CCl2CCl2—9.7 g L−1; CHClBr2—12.2 g L−1; C6H5Cl—5.2 g L−1; CHBr3—14.5 g L−1; CHCl2CHCl2—9.6 g L−1. From these solutions, a single mixed solution containing all the compounds was prepared. The analysis of hexane extracts was performed by the breaking method . Water samples (5 L each sample) were collected at a depth of approximately 50 cm below the surface and stored in polypropylene bottles at temperatures below 4 °C.Chloroform  was used as a carrier gas and as auxiliary gas (make-up) at 15 and 35 mL min−1, respectively.A Dani gas chromatograph, mod. 86.10 HT , equipped with a Vocol capillary column “wide-bore”, 60 m × 0.75 µm × 1.5 µm film thickness (Supelco) and a programmed temperature vaporizing (PTV) injector and an electron capture detector (ECD) connected with a Shimadzu CR 3A data processor, was used for sample analysis. The PTV was set up at 50 °C for 1 min, and then increased to 240 °C at 800 °C min−1) were extracted with 500 µL of n-hexane. After 15 minutes of vigorous shaking using a magnetic stirrer and after separation, the organic extract was recovered and injected into the GC for analysis. For headspace analysis, a river water sample (110 mL) was placed in a vial, and NaCl (10 g L−1) was added. The vial was then closed with a cap containing polytetrafluoroethylene (PTFE)-coated rubber septum and kept at 50 °C for 60 min. Headspace gas-chromatographic analysis was performed by manually injecting 1.0 mL of the vial headspace from each sample by means of a 1.5-mL Hamilton gas-tight syringe (temperature kept at 50 °C).This laboratory developed different extraction procedures ,24,25,26All the values reported in tables are the average among five determinations, and the relative standard deviations (RSDs) are in the RSD range for a standard solution analyzed by both methods.12 n×cm2×s−1 for 12 h. The inorganic element evaluation was carried out using a highly specific nuclear method, i.e., INAA, which involves collecting as much information as possible without subjecting the sample to any chemical treatment ,21,34. F60Co 55.1 and a relative efficiency of 22% at 1332.5 keV. The measurements were performed and analyzed using the Silena Silgamma software produced by Silena . According to the half-life of each element [The gamma spectrometry measurements were performed by means of an HPGe detector  with an energy resolution (FWHM) of 1.75 keV 1332.5 keV, a peak/Compton ratio  element ,41, more−1 of each one) and standard reference materials  [Primary  ,43 were It should be noted that this study is appropriate and relevant for evaluating the environmental contamination in specific areas with high anthropogenic pollution. The exportability of such investigation, including the analytical methods used for achieving the results, is based on the study of the organic fraction  and an inorganic fraction  with the aim to find out the main representative compounds/metals responsible of possible environmental damages.−1 as the sum of organohalogen compounds [The determination of the organic fraction was addressed to the evaluation of halocarbons. Before performing the determinations, the analytical methodology to be used was deeply studied. This step is fundamental for the knowledge about the reliability of the data obtained. First, all the analytical parameters in terms of linearity range, limit of detection (LOD), repeatability and recoveries were evaluated. Looking at the data , it can ompounds , both meompounds . Hence, For the Tiber , the sit3, CCl3CCl3, CCl4, CHClCCl2, CHBrCl2, CHCl2CH2Cl, CCl2CCl2, CHBr2Cl, C6H5Cl, CHBr3, CHCl2CHCl2 in different samples.Finally, a sampling of drinking water performed at different points of Rome’s water supply was carried out to understand the effect of anthropogenic activities on the water quality. This issue is important because the drinking water network is very particular : the wat−1, 0.14 µg L−1, 1.86 µg L−1 and 9.91 µg L−1, respectively). This contamination could be related to the different anthropogenic activities [The results show no significant pollution problems for samples collected at Peschiera-Cittaducale and Peschiera-Castelluccia springs: if the organohalogen compounds were present, their concentration was lower than LOD reported in tivities ,49 preseIn such kind of studies where the inorganic fraction is deeply investigated by means of nuclear analytical technique (INAA), the analysis of standard reference materials (SRMs) is a fundamental and significant issue ,51,52. T76As (559.2 keV) and 122Sb (564.0 keV) and between 85Sr (514 keV) and the annihilation (511 keV); the third interference, a total overlapping between 75Se (279.6 keV) and 203Hg (279.0 keV), is more difficult and needs the energy calibration curve for evidencing the contribution due to the interfering radionuclide.Even if INAA is a well-known and validated analytical methodology, it suffers from interference  due to b−1) along with the standard deviation, the minimum and maximum levels and CV%, for each element determined in the investigated samples. Each value is the average of the element values in all the samples collected in all the areas.−1), As (up to 5100 μg g−1), Sb (up to 3400 μg g−1), Sr , Zn . In contrast, the rare earth elements (REEs), elements also considered to be very important from an economic point of view, showed levels in agreement with the literature data of the crustal composition  [−1) in the other samples. According to the element variability, expressed as coefficient of variation (cv%), it is always above 100% except for Ag, Br and Sn and ranges between 101% (Na) and 952% (Zn), showing a large distribution of the elements in the investigated areas. This difference, which describes the large distribution variability of elements in all the areas, will be discussed with different statistical approaches. Another consideration regards one of the targets of such study, i.e., the uranium: it is at 13.6 μg g−1 mean level. The mean levels (2–14 μg g−1) are quite similar to those present in the Earth’s crust (4 μg g−1).58 However, the maximum U levels reached in few samples, i.e., 400 μg g−1 (Bracciano), 43 μg g−1 (Vulsini area) and 280 μg g−1 (Oriolo Romano), permit to consider the site as a sub-economic deposit as previously proposed by other authors [The use of a nuclear technique has allowed a very deep investigation of the investigated samples. More than 40 elements were identified and quantified: among them, interesting levels were found for Hg (up to 6500 μg g μg g−1) . Further authors .−1 and 1150 μg g−1) in relation to its very low levels in the Earth’s crust (5 μg g−1) [A very important issue of this study concerns arsenic: it reaches very high concentrations throughout the samples (ranging between 90 μg g μg g−1) . This oc μg g−1) , Rome is2) among elements investigated in ores sampled in the Northern Latium locations vs. the Bracciano area is quite good (>0.5), meaning that all the samples show the same provenance and similar average composition [x is the element concentrations in the sample and in the reference, i.e., upper continental crust [n are the concentration levels of the normalizing element  in the sample and in the reference [It should be underlined that the correlation : it means that anthropogenic sources strongly affect such element values over the geochemical origin. This is particularly important for the assessment of the arsenate content in potable waters, the issue above just stressed. Important elements such as Sr, Zn and Cd show EFs between anthropogenic and natural origins: actually, their EFs are above 40 when Fe is used as a normalizing agent. Th  and U  can be considered no enriched elements. Once again, this is another confirmation that the geochemical hypothesis  of consiCn is the element concentration level in the soil dust, Bn is the geochemical background, and 1.5 is a factor taking into account the possible variations of the background data due to lithological variations [Further, for a more detailed definition of the soil, the geoaccumulation index (Igeo) has also been studied for few elements, i.e., As, Cd, Cr, Ni, Pb (determination performed by ICP-ES), Zn . This pariations .For Igeo parameter, seven classes are identified: Igeo ≤ 0, soil is uncontaminated by that element; 0 < Igeo < 1, soil uncontaminated to moderately contaminated; 1 < Igeo < 2, soil moderately contaminated; 2 < Igeo < 3, soil moderately to heavily contaminated; 3 < Igeo < 4, soil heavily contaminated; 4 < Igeo < 5, soil heavily to extremely contaminated; Igeo ≥ 5, soil extremely contaminated. As geochemical background , four diFinally, a hierarchical cluster analysis (HAC) study is performed. The correlation among elements is reported in 10), fraction believed a risk cause of dangerous effects on human health, including lung cancer, was collected onto a 37 μm filter by means of a Hydra instrument  in the Bracciano area during September 2016. The sampling time was two weeks long: the sample was subjected to the same analytical procedure described previously , and the data are reported in Finally, a simultaneous (and brief) investigation of the atmospheric particulate matter was performed. Airborne particulate matter (PM−1) whereas as shows good mobility in relation to the seasonal variability. These results do not give any significant worries, but they are indices of an upsetting situation that could change if other different sources arise.One of the major problems in the entire Italian territory is its physical vulnerability, which increases exponentially with the urban and infrastructural expansion to which our cities and the natural physical environment as a whole are subjects . The damAnthropogenic activities remain the main cause of poor air quality, but there are also natural sources of air pollution that can play a very important role in this context. Our study shows that this area is worthy of being deeply investigated. Particularly, the study addressed two important issues, i.e., the determination of halogenated volatile organic compounds and heavy metals. The investigated organic compounds are highly volatile compounds that produce negative effects on human health and contribute to the greenhouse effect. Monitoring and limiting the presence of these substances in the atmosphere is thus crucial, in particular where different types of emission sources are present . On the