Other fluid inclusion populations include N2-CO2, NaCl-H2O ± N2 and CO2 ± N2-NaCl-H2O, but no hydrocarbons were detected. The host rocks are part of the Wudaogou Group metamorphic series, which mainly consist of Ca-rich mica schist. Subhedral sulfide minerals occur in early disseminated W-mineralized quartz veins, or have partially replaced early scheelite. ThN2 and ThN2-H2O indicate N2 fluid-trapping from 315 °C to 410 °C and from 80 MPa to 350 MPa. Oxygen and hydrogen isotopic data  suggest that the mineralizing fluids were composed of mixed magmatic and metamorphic water, N2-rich inclusions (δ15N = −0.5‰ to 1.4‰) indicate fluid-rock interaction with metamorphic rocks. The N2-rich fluid was closely associated with scheelite precipitation. During thermal decomposition under high oxygen fugacity conditions, which occurred synchronously with metamorphism and magmatic activity, large amounts of N2 were liberated from NH4+-micas, which then accumulated in the parent fluid of the quartz scheelite veins.Nearly pure N In silicates, under conditions of elevated temperatures, low water activity, and high oxygen fugacity, N2 is favorably released from minerals containing ammonium 5.Nitrogen is the dominant constituent of the atmosphere and a key component in the biosphere. High-density CO6. In sedimentary-type deposits, tungsten (W) is liberated as a result of exhalative processes, forming stratiform scheelite6, in which mineralizing fluids containing CH4 and N2 are generally attributed to in situ organic matter6. Several examples of scheelite deposits related to metamorphic hydrothermal fluids are reported to contain small amounts of N28. These types of scheelite deposits are of interest to the geologist, because they are related to Au deposits9. Furthermore, Gibert8 found that the addition of only 5% N2 would decrease the solubility of scheelite in micaschists from 40 ppm to less than 3 ppm W, indicating that N2 may drive scheelite participation.Scheelite containing nitrogen is most commonly recognized to be traced from sedimentary rocks2 (>88 mol%) was identified in fluid inclusions in W-mineralized quartz veins, which is absent of hydrocarbons. Previous studies have shown that NaCl-H2O and CO2 fluid inclusions are common in ore-forming fluids; however, the presence of N2-rich fluids in ore deposits is rarely reported. This study aims to identify the origin of the W-mineralizing fluid and the formation of the N2-rich fluid inclusions by combining petrography, Raman microspectroscopy, H, O and N isotope geochemistry, and microthermometry on fluid inclusions. Results from this study will improve our understanding of how N2 modulate scheelite mineralization.Nearly pure N10 and 266–249 Ma, respectively11. The Wudaogou Group is subdivided into the lower Madida Formation, the middle Yangjingou Formation, and the upper Xiangfangzi Formation12. The rocks from these formations are composed of muscovite, K-feldspar, amphibolite, plagioclase, quartz, and small amounts of cordierite, andalusite, rutile and kaolinite13.The Yangjingou scheelite is the largest identified scheelite deposit in the Yanbian belt, NE China. It is located between the Cuihongshan and Sanjiazi scheelite deposits in the Heilongjiang-Jilin metallogenic belt, 8 km south of the Xiaoxinancha Au deposit, approximately 25 km east of the Russian border Fig. . In thisThe Yangjingou scheelite deposit Fig.  is locat14 and early Indosinian Yangjingou granodiorite (249.4 ± 2.7 Ma)15 have locally deformed rocks of the Wudaogou Group. Based on the Yangjingou granodiorite chemical signature, a transition from I- to S-type magmatism is observed, interpreted as a result of subduction16. Yanshanian intrusives, including monzonitic granodiorite (178.5–197 Ma)17 and dioritic porphyry dykes (120.73–157.27 Ma)17 were later emplaced within zones of pre-existing structural weakness. Previous studies have revealed a close association between Indosinian magmatism and W mineralization in the Yangjingou deposit18. Furthermore, Shan19 and Hu17 suggest that the dikes present in our study area are genetically related to an unexposed granite intrusion.Several generations of magmatic intrusions, including late Hercynian granodiorite (267 ± 1 Ma)The scheelite ore bodies are mainly hosted in Ca-rich mica schists. The deposit can be subdivided into a southern and a northern ore block, separated by a N-W-trending fault Fig. . In the Mineralization in the southern ore block is structurally controlled by normal faults striking 300–350° and dipping 60–75° Fig. . The wid3) grade of the mine is 0.22% to 1.5%, reaching up to 5.25%, with accessory Au (0.49–0.79 g/t), Cu, Mo, Fe, and P19. Scheelite mineralization predominantly occurred via micro-vein dissemination and thin vein mineralization  capable of temperature measurements from −196 to 600 °C. The accuracy between 31 °C and −100 °C is better than ±0.2 °C and better than 5 °C at >300 °C. The heating/freezing rate is generally 0.2–1 °C/min but is reduced to 0.1 °C/min close to the phase transformation point. The FLUIDS−1, and the laser beam diameter 1–2 μm. A detailed description of the measurement procedure was provided by Wopenka24. The measurements reveal that the major gaseous fluid inclusion components are N2 and CO2. A large proportion of the inclusions show only a single strong band at 2328.2–2328.3 cm−1, indicating pure N2, which is the first time pure N2 inclusions have been observed in the W deposit  relative to the Vienna Standard Mean Ocean Water (VSMOW) standard, with precisions of ±2‰ for δD and ±0.2‰ for δ18O at the 1σ level. δ18Oquartz was measured in order to calculate the δ18OH2O values of hydrothermal fluids using the following equation:The oxygen and hydrogen isotopic compositions of primary fluid inclusions in the main stage (oxide stage) of the disseminated scheelite quartz veins were analysed at the analytical laboratory of Beijing Research Institute of Uranium Geology (BRIUG), China, using a MAT253-type mass spectrometer. Grains (40–60 nm) were handpicked to ensure the absence of mineral impurities. Oxygen was liberated from quartz for isotopic analyses via quantitative reaction with BrF25.where T represents the homogenization temperature of the fluid inclusions−3 Pa). N2 was then directly released from fluid inclusions by heating the quartz samples in a high-purity quartz cell to temperatures above 600 °C. The released N2 was then separated using a chromatographic column cold trap26. Nitrogen isotopic compositions of the samples are reported using δ notation, where δ15Nsample (‰) = [(15N/14N) sample/(15N/14N) standard−1] ×1000. The results are reported in per mil (‰) relative to atmospheric N227, and the precision is ±1‰ at the 1σ level.Seven quartz samples from scheelite quartz veins were selected for N isotopic analysis. In order to exclude atmospheric interference, samples were degassed for 15 minutes under vacuum  and phase transition during total homogenization, four types of fluid inclusions have been identified in quartz crystals from the scheelite paragenetic quartz veins: pure N2 fluid inclusions (Type I), CO2-N2 fluid inclusions (Type II), CO2 ± N2-NCl-H2O inclusions (Type III) and aqueous two-phase VL inclusions  fluid inclusions ) and are closely related to scheelite deposits, but have been rarely reported. At low temperatures, bubbles nucleate at −162 °C~−182 °C. The final homogenization of the liquid phase Th(L) occurs between −151 °C~−168 °C, although a few Type I fluid inclusions homogenize to the gas phase at −150.3 °C  inclusions >S+L+G->L+G->L(G) , showing homogenization as the final phase transition or S+L+G->S+L(G)->L(G)  showing sublimation as the final phase transition30. Bubbles of nitrogen nucleate between −178 and −159 °C  to a liquid (gas) of is between −163.2 and −147.5 °C. Critical homogenization can be observed around −147 °C and the total homogenization (ThCO2) is characterized by sublimation to liquid (gas) phase from −86~−69 °C. The ThN2 of H-type inclusions to the liquid (gas) phase coincided with critical homogenization and occurred between −149 and −148 °C. The total homogenization (ThCO2) to liquid (gas) varied from −60.8 to −45.6 °C with the TmCO2 between −60.9 and −60.7 °C. Raman spectroscopy shows that the inclusions are composed of 42–79 mol% N2, and 21–58 mol% CO2 with molar volumes between 30 and 65 cm3/mole, in agreement with microthermometry results  inclusions contain liquid and gas at room temperature and have been subdivided into Type IIIa and Type IIIb populations. Type IIIa inclusions contain gas volume fractions of <50%, where most are between 10 and 30%  inclusions contain CO2 gas, CO2 liquid, and a saline solution at room temperature , and WO4−2 complexes in W-mineralizing fluid. The presence of NH4+ in the solution stabilizes the W polyacid, thereby increasing its compatibility in migrating fluids36. The stability of NH4+ (substituting for K+) widely depends on the metamorphic and oxygen fugacity conditions37. Nevertheless, under metamorphic conditions, NH4+-bearing minerals may release N2 (350–600 °C) or NH3 (650–700 °C) at higher oxygen fugacity and concentrate in inclusions. The following equation illustrates the production of N2 under oxidizing conditions38:Under alkaline conditions, W is transported in the form of H2+, which results in the chemical equilibration of the mineralizing fluid with the host rock and has been recorded at a range of pressures and temperatures32. The addition of the non-polar volatile N2 via water–rock interactions in mica may decrease scheelite solubility in common metamorphic assemblages. N2 has been demonstrated to dominate changes in the distribution of W aqueous species and increase coefficient activity8. In other words, an increase in the N2 content in the mineralizing fluid may promote scheelite precipitation.The main cause for scheelite precipitation is an increase in the activity of Ca2O-CO2-N2-NaCl-bearing fluids with low to moderate salinity at moderate to high temperatures. The only volatile components in this fluid are CO2 and N2; hydrocarbons  have not been detected. From the vx plot  have lower molar volumes than those richer in N2(42-150 cm3/mol). This has been observed in many metamorphic areas29 indicating that CO2 and N2 may not have the same origin38. The homogenization temperatures of N2 (ThN2) and H2O ± N2-NaCl (ThH2O) constrain the pressure-temperature (P–T) conditions of trapping to 315–410 °C at 80–350 MPa  the interaction of magmatic fluids with NH4+-rich host rocks, and/or mixing with metamorphic fluids in equilibrium with metasedimentary lithologies 7.The stable isotopic composition of a vein provides direct information regarding the transport processes during vein formationows Fig. : 1) a do18O and δD of the Yangjingou scheelite deposit indicate that the magmatic component of the fluid played a more dominant role in this deposit compared to the Nyakabingo deposit, in which the mineralizing fluid was metamorphic , demonstrating that the fluid was derived from the mantle. However, compared with magmatic hydrothermal deposits in this area  the mineralizing fluid of magmatic origin, mixed with low-δD meteoric waters. Because W-mineralized in the presence of Ca-mica schist host rock coupled with N2-rich fluid inclusions in the Yangjingou deposit, the first explanation is preferred.Low δhic Fig. . Earlierrea Fig. , the Yan37. N2 has been shown to be released during metamorphism and the breakdown of NH4+-bearing minerals, such as biotite, cordierite, and white mica43. During metamorphism, isotopically “light” N is preferentially fractionated into metamorphic fluids46, and the δ15N values of fluid inclusions in quartz veins in low-grade metamorphic rocks yield values ranging from −3 to 5‰31. Similarly, the W-mineralized quartz veins in the Yangjingou deposit feature low δ15N values  trapping of a volatile phase separated from its parent H2O–NaCl–CO2–N2-rich fluid, 2) mixing of a magmatic–hydrothermal CO2-H2O-rich fluid with N2 released via the thermal decomposition of NH4+-bearing minerals, or 3) CO2–N2 degassing of the mantle.There are three mechanisms by which N2 is released during magmatic differentiation49, producing a distinct δ13C signature17. Meanwhile, the Wudaogou Group contributed Ca2+ to the system, facilitating the precipitation of Ca(WO)4. Within this metamorphic environment, N occurs as NH4+, which substitutes for K+ in detrital phases such as K-feldspar, authigenic clays and illite. With increasing pressure and temperature, both feldspar and clays participate in low-grade metamorphic reactions, and illite gradually transforms into micas50. During this transformation, a large amount of carbon is released in the form of CO2 due to decarbonization during progressive metamorphism. Moreover, the reported content of CO2 in schist (<0.25%) is notably lower than that in clay and shale (2.5–5%)36. Thus, the contribution of organic matter to the mineralizing fluid was negligible. No CH4 was identified in the W-mineralizing fluid, which may be due to the high oxygen fugacity51. Subsequently, Ca2+ from the Ca-rich mica schist country rocks was incorporated into the mineralizing fluid. The P–T conditions of the Yangjingou deposit (315–410 °C at 110–350 MPa) are lower than greenschist-facies P–T conditions (350–550 °C at 150–1100 MPa)52, indicating that scheelite mineralization likely occurred in the later stages of Wudaogou Group metamorphism. Additionally, the δ15N values of the W-mineralized quartz veins show that water–rock interaction occurred under sub-greenschist-facies conditions. However, minor mantle N2 participation cannot be completely ruled out.The stable isotope data in this study indicate that the mineralizing fluid was of a mixed magmatic and metamorphic origin. Magmatic melt inclusion studies have shown that CO16, synchronous with Wudaogou Group metamorphism. A/CNK (1.1–1.18) and Na2O/K2O (2.45–7.06)16 major element values are characteristic of alkali series I- to S-type transient magmatism. The volatile component of the melts was influenced by pressure, which affected the volatile solubility in the magma during ascent. With decreasing pressure, gas was liberated from the magma53, resulting in an increasing volume of CO2. CO2 dissolves relatively readily in intermediate-alkaline magmas, leading to more widespread and more rapid emplacement of mineralizing fluids46. Under these conditions, the diffusion of the W-rich mineralizing fluid was accelerated along interformational structures, causing the fluid to migrate to and rapidly expand into the host rocks of the Wudaogou Group. Only minor N2 (<0.25 mol%)54 was identified in fluid inclusions in the Xiaoxinancha Cu-Au porphyry deposit (123.35 ± 0.8 Ma)42, which is younger than the deposits in the Wudaogou Group and other stratigraphic units. The high concentration of N2 observed in the scheelite deposits cannot simply be attributed to organic-rich sedimentary rocks. Findings from this study demonstrate that N2-rich fluids are closely related to the degree of metamorphism and magmatic activity.Yangjingou scheelite mineralization is related to the intrusion of granodiorites at 249 ± 2.7 Ma2 ± CO2 fluid was a product of late-stage Wudaogou Group metamorphism, which caused H2O–NaCl–CO2-bearing magmatic–hydrothermal fluids containing HWO4− and MWO4− to migrate through fractures and pre-existing structural weaknesses via fluid circulation, thereby promoting interaction with metamorphic wall rocks , particularly under greenschist-facies conditions, NH4+ was released via continuous metamorphism or thermal decomposition 50. Simultaneously (~249 Ma), the circulation of a magmatic–hydrothermal W-mineralizing fluid under high oxygen fugacity conditions transferred heat (~400 °C) to the surrounding metamorphic rock and broke down micas in the host rock51. Within an oxidizing environment at this temperature range, large amounts of N2 were released from the NH4+-bearing host rocks and stabilized into fluid43. The addition of sufficient amounts of N2 to the mineralizing fluid increased activity coefficients and catalysed the saturation of Ca2+ and WO42–, greatly decreasing the scheelite solubility and accelerating scheelite precipitation. Moreover, saturated fluids, driven by advection, ascended into the surrounding country rock, resulting in alteration  and the incorporation of H2O into new minerals, leaving N2 ± CO2 trapped in quartz inclusions. Thus, the N2-rich fluid represents W-mineralizing fluid evolution and is indicative of W fluid saturation and scheelite precipitation.Thus, we assume that the Ncks Fig. . An init2 (>88 mol%) fluid inclusions have been identified in W-bearing quartz veins in the Yangjingou deposit.Nearly pure NFour fluid inclusion populations have been identified in the deposit:2-rich inclusions  N2 = −151 to −168 °C, Th(V)N2 = −150.3 °C, 42–150 cm3/mole).•Type I: N2 ± CO2 inclusions .•Type II: N2O inclusions .•Type III: NaCl–H2 ± N2–NaCl–H2O inclusions .•Type IV: CO3.2O–CO2 fluids were magmatic–hydrothermal in origin, and the N2 was derived from the breakdown of NH4+-bearing minerals during alteration.The fluid inclusions were trapped at 315–410 °C and 80–350 MPa. The NaCl–H4.Hydrogen and oxygen isotopic data indicate that the mineralizing fluids were of a mixed magmatic and metamorphic origin, and the nitrogen isotopic data from the W-mineralized quartz veins suggest that water–rock interaction occurred under low greenschist-facies conditions.5.2-rich fluid was intimately associated with the scheelite precipitation. During alteration under high oxygen fugacity conditions, N2 was released from NH4+-bearing minerals. The N2-rich fluid represents the W-mineralizing fluid evolution, following W saturation and scheelite precipitation. Therefore, areas that have experienced regional metamorphism (orogenesis) and exhibit similar geologic histories to the Yanbian area in NE China are likely candidates for scheelite precipitation as observed in the Yangjingou deposit. In this study, we demonstrate that scheelite precipitation can be genetically related to almost pure N2 fluid inclusions. The presence of the almost pure N2 fluid inclusions in these types of localities therefore can be used as a proxy for scheelite precipitation and thus, the presence of W ± Au deposits.The N


Residency programs face many challenges in educating learners. The millennial generation’s learning preferences also force us to reconsider how to reach physicians in training. Social media is emerging as a viable tool for advancing curricula in graduate medical education.The authors sought to understand how social media enhances a residency program’s educational mission.While chief residents in the 2013-2014 academic year, two of the authors  maintained a Twitter feed for their academic internal medicine residency program. Participants included the chief residents and categorical internal medicine house staff.At the year’s end, the authors surveyed residents about uses and attitudes toward this initiative. Residents generally found the chief residents’ tweets informative, and most residents  agreed that Twitter enhanced their overall education in residency.Data from this single-site intervention corroborate that Twitter can strengthen a residency program’s educational mission. The program’s robust following on Twitter outside of the home program also suggests a need for wider adoption of social media in graduate medical education. Improved use of data analytics and dissemination of these practices to other programs would lend additional insight into social media’s role in improving residents’ educational experiences. The current learning environment for internal medicine residency contains potential obstacles to learning: duty hour restrictions, training at multiple sites, increasingly complex patients, and pressures for clinical productivity and timely discharge. Moreover, our trainees have evolved; the so-called millennial generation may require more interactive learning experiences and may favor technology-driven learning platforms . The vasWe tested the use of a novel social media initiative—specifically, a Twitter feed—to enhance the education of our residents and the mission of our internal medicine residency program. A recent systematic review explored the role of social media in medical education; results of social media initiatives were largely mixed . These iTo our knowledge, no residency program has reported on its experience or outcomes in using social media to advance the program’s educational mission. Herein we describe our implementation strategy for using Twitter and report on its reception among residents. We specifically sought to understand Twitter’s value as an extension of our teaching conferences and as a more general educational resource for residents.We conducted our educational intervention in a single internal medicine residency program at an academic medical center over one academic year. The intervention was led by the chief residents, and residents voluntarily participated on Twitter at their convenience.Our program includes 95 categorical residents. During the intervention, our residents rotated through three hospitals on two campuses. This project represents a pilot curricular innovation. Regarding ethical approval, we have obtained Institutional Review Board (IRB) exemption for this study.We selected Twitter for our platform based on its efficiency, propensity for publicity, its rapid growth, and its novelty. First, a well-curated Twitter feed can offer succinct, timely educational material for busy residents, including program-specific announcements, educational materials, and discussion points on the latest medical headlines. Next, because websites are static and require active maintenance, they do not promote learner engagement or the interactivity that Twitter offers. Finally, a Twitter feed improves the visibility of a program’s academic activities, including publicly highlighting the quality and diversity of teaching conferences. We could also promote residents’ publications, research posters, teaching awards, and community service work. This publicity may also represent a powerful recruitment tool. We launched our @Medchiefs Twitter account in July 2013 while two of the authors  served as chief residents and the third author (VA) served as associate program director for the University of Chicago Internal Medicine Residency Program. Only the chief residents had the account’s password. We linked our Twitter account to Facebook, and thus all of our tweets were also simultaneously broadcasted to our followers’ Facebook feeds. We also embedded the Twitter feed directly into our chief residents’ website . BecauseWe estimate that the chief residents dedicated 30 to 60 minutes daily to actively maintaining our feed. This time included reviewing other users’ posts, messaging followers, and queueing up tweets for later dissemination. Additionally, we often tweeted in real time during conferences, such as weekly Department of Medicine Grand Rounds.By searching specific “hashtags,” Twitter users can quickly find tweets or users within specific areas of interest. When possible, we applied hashtags to conference tweets such as #grandrounds. After reaching out to other programs on Twitter and reviewing hashtag trends, we settled on the nascent #AMReport to designate tweets from our internal medicine morning report. We also created a program-specific hashtag, #UCIMR, to encourage our residents to reference our program online.We loosely adhered to our own “rule of thirds” when tweeting; a balanced number of tweets pertained to resident education, recognition of our program’s academic activities, and outward promotion.Tweets about our morning report constituted the greatest number of indexed tweets; a total of 31% of our tweets related to the morning report conference. Tweets were used to broadcast teaching points from our morning report and summarize key features of the case.In addition, tweets from teaching conferences linked to relevant medical literature discussed, and occasionally shared, educational radiographs, computerized tomography (CT) scans, or pathology slides from interesting cases. Any patient-related materials that were shared were stripped of all identifiers to avoid Health Insurance Portability and Accountability Act (HIPAA) violations.Because resident-prepared morning report cases were reviewed by chief residents in advance, “tweetable” teaching points were prescheduled for broadcasting during the conference via Hootsuite, a third-party social media application. Using this approach prevented chief residents from being distracted during the conference and also offered the opportunity to reflect on the most valuable teaching points for each case.We also tweeted relevant news for our residents by discriminating which medical headlines would apply most to our residents. In addition, we highlighted institution-specific accomplishments and examples of our faculty physicians being recognized in national and global forums.We recognized that our Twitter feed might also buttress our recruitment efforts. By having applicants follow us during the recruitment season, we could continually highlight our robust academic activities and dedication to resident education. As such, during the 2013-2014 interview season, we distributed flyers promoting our Twitter feed to all applicants on their interview day.At the end of the 2013-2014 academic year, we conducted a brief 14-item survey on our residents’ use patterns with our Twitter account. Our survey asked residents how frequently they viewed the Twitter feed, how informative they found tweets, and how valuable they found the intervention. Our IRB determined this survey-based study to be exempt.Both Twitter and third-party companies Hootsuite and Simply Measured provide a number of metrics on followers and online activities. These metrics include online influence of followers, approximate locations of followers, and popularity of individual tweets. This online data was retrieved on July 23, 2014, approximately one year after the inception of our account.In our first year, we amassed a following of more than 1000 Twitter users; we grew our global following by 3 users per day. At the end of the 2013-2014 academic year, we identified at least 27.1% (35/129) of our current residents, preliminary interns, and chief residents among our Twitter followers. We also published over 1000 tweets, or about 3 tweets per day. Our messages generated a total of 782 retweets.medicine, medical, health, and physician. Only 35% of our 1091 followers access Twitter from our program’s time zone; this data suggests that most our followers have no formal connection to our home institution.The largest segment of our followers consists of health professionals as evidenced by the four most common keywords in our followers’ profiles: Our survey on attitudes and use of the Twitter feed was completed by 61 of 95 of our categorical medicine residents (64% response rate). Of 61 responding residents, 33 (54%) residents reported having a Twitter account. However, the majority of our residents  reported using Facebook or our website as their preferred way of accessing our Twitter feed. Most residents  reported reading our tweets at least once per week, but only 11% (7/61) of our residents accessed the feed daily. The majority of our respondents  agreed that our Twitter feed enhanced their education in residency.Tweets about our morning report were well received with 84% (51/61) of our residents reporting morning report tweets as occasionally or often informative. Most residents  agreed that our morning tweets captured the major teaching points. Interestingly, our tweets about medical news and the latest literature garnered greater interest, with 87% (53/61) of our residents finding these tweets informative.Additional survey data on our intervention is found in Maintaining a residency-run Twitter feed appears to have educational value both internally and externally. Our survey data demonstrates that Twitter was a frequently accessed and appreciated educational resource for residents. Moreover, our robust following outside of our home institution suggests a greater need for resident-directed educational resources in social media.Though maintaining a Twitter feed requires an additional investment of a chief resident’s time, we believe social media’s strengths in reaching learners justify the effort. Although the educational cornerstone of our residency program has been live teaching conferences, residents have competing priorities that limit attendance. For example, in 2013-2014, our average resident was present for only 48% of conferences they were eligible to attend . Giving residents an alternate means of receiving educational material thus represents an important service.We opted for voluntary participation in our intervention for at least two practical reasons. Most importantly, we wanted to respect residents’ limited time. Additionally, we wanted to use Twitter as an adjunct to—and not a substitute for—our more traditional educational offerings. We felt that compulsory participation could potentially detract from participation in other valuable and mandatory curricula including live conferences and Web-based learning modules.Our work has some important limitations. Most notably, this intervention occurred at a single site in a single residency program; thus, our results may not be generalizable to all types of medical education settings. Additionally, we did not directly compare Twitter to other educational interventions and thus cannot comment on social media’s relative value in supporting our program’s curricula. Our survey data also relies on self-report of frequency of use rather than actual quantification of use. Finally, we did not directly examine Twitter’s ability to promote knowledge acquisition and retention.Concerns about professional boundaries and patient privacy might deter residency programs from engaging in social media. Fortunately, guidelines on professional use of social media can be found in a position paper by the American College of Physicians and the Federation of the American Colleges , and othAs program leaders, chief residents are well positioned to role model the use of social media and understand the responsibility and care required to manage a medium prone to misconduct. For example, while we occasionally posted interesting images from case conferences or teaching services, all images were completely deidentified and posted asynchronously to the patient’s hospitalization. Likewise, when tweeting educational materials and original articles from journals, we maintained compliance with fair use by sharing only small parts of published works.To develop a better understanding of the impact of our program’s Twitter account, we would like to expand the use of data analytics in the coming academic years. Tools embedded in Twitter or offered by third parties allow for meaningful data extraction including the times of day that followers interface with our account and the popularity of specific tweets. These data may better inform future programs on how to best use social media to reach residents.Analysis of tweets by hashtags would provide additional insight on the content and potential uses of Twitter in resident education. Hashtags likely represent the easiest way to standardize tweets across residency programs. Though other authors have recently analyzed the use of academic conference-specific hashtags on Twitter ,8, we arAlthough recent systematic reviews have relied on low-level outcomes to assess social media’s role in medical education and higher education , we beli


The second grant number from the funder the Polish National Science Center is missing. The second grant number is: DEC-2015/19/B/NZ5/02208.


Modern cancer therapies aim at targeting tumour-specific alterations, such as mutations or neo-antigens, and maximal treatment efficacy requires that targeted alterations are present in all tumour cells. Currently, treatment decisions are based on one or a few samples per tumour, creating uncertainty on whether alterations found in those samples are actually present in all tumour cells. The probability of classifying clonal versus sub-clonal alterations from multi-region profiling of tumours depends on the earliest phylogenetic branching event during tumour growth. By analysing 181 samples from 10 renal carcinoma and 11 colorectal cancers we demonstrate that the information gain from additional sampling falls onto a simple universal curve. We found that in colorectal cancers, 30% of alterations identified as clonal with one biopsy proved sub-clonal when 8 samples were considered. The probability to overestimate clonal alterations fell below 1% in 7/11 patients with 8 samples per tumour. In renal cell carcinoma, 8 samples reduced the list of clonal alterations by 40% with respect to a single biopsy. The probability to overestimate clonal alterations remained as high as 92% in 7/10 renal cancer patients. Furthermore, treatment was associated with more unbalanced tumour phylogenetic trees, suggesting the need of denser sampling of tumours at relapse. Recent advances in next-generation sequencing have led to the widespread identification of somatic changes in the genomes of a large number of tumours, raising the hope to transform cancer therapy based on patient-specific data2345678910The success of this therapeutic strategy however, relies on selecting the correct targets in each patient81213131516However, intra-tumour heterogeneity and sampling bias complicate the correct classification of truly clonal and sub-clonal alterations. Independent multi-region profiling of spatially distinct tumour samples increases the information on individual tumours and allows the reconstruction of phylogenetic trees18192021222324252319222324f and 1 − f. The sizes of these fractions are the result of potentially complicated processes, e.g. clonal selection, immune system escape or random drift. If we were to sample from both sides of the tree, all alterations that appear clonal in both samples will also be truly clonal in the whole tumour. But if we only sample from either side, we will misclassify a fraction of sub-clonal alterations as clonal, see i independent spatially separated tumour samples, the probability to sample from both sides of the tree isLet us consider the complete phylogenetic tree of a tumour. Each leaf of this tree is a cancer cell. Leaves are separated by bifurcations representing cell divisions prone to inheritable alterations, which could be single nucleotide polymorphisms, gene duplications, translocations or any other genomic change. Alterations that are in the trunk of the tree must be present in all cells of the tumour, if we neglect unlikely events of back mutations. The first bifurcation divides the tumour into two populations of fraction f determines the shape of this curve, see pf(i = 1) = 0). Including more samples i to the analysis increases the probability to classify truly clonal alterations correctly. The probability increases fastest for trees in which the first bifurcation splits the tumour population approximately in half (f = 1/2). These are often referred to as ‘balanced’ phylogenetic trees, and are often, but not always, consistent with neutral growth i. Two tumour samples have a probability of 50% to correctly classify all truly clonal alterations and the probability increases to 99% for 8 independent samples. However, the probability increases more slowly in unbalanced tumours, e.g. in cases of strong on-going sub-clonal selection during tumour growth or as a result of treatment. For example, if one side of the tree is 5 times larger compared to the other side, two independent tumour samples result in a probability of 28% to correctly classify all alterations and increases to 73% for 8 independent samples  after adjusting for finite sampling (see f = 0.5), half of the subsample combinations would recover the exact minimal list of clonal alterations. For unbalanced tumours (f < 0.5) fewer combinations of subsamples will recover the minimal list of alterations. This procedure is then continued for all possible combinations of subsamples. Comparing the shape of the universal curve (8) to the actual information gain from the data allows assigning an empirical balancing factor f to a tumour. Each tumour-specific balancing factor provides a rational of whether the current number of tumour samples is sufficient, or if additional sampling is necessary to ascertain the identity of truly clonal alterations in that particular patient. In addition, the value of f would determine whether it makes sense to sequence additional parts of the tumour, if the expected information gain from each sample is very small.The balancing factor et al.18mm3 and thus each sample contained ~108 cells. The protein coding region of the genome (exome) was sequenced with a depth of >70x for all samples, allowing the identification of clonal mutations within each single bulk sample with high precision.First, we tested if the information on clonal alterations gained from multi-region sequencing data falls onto the theoretically predicted universal curve (8). We evaluated ten cases of multi-region sequenced clear cell renal carcinoma  recently published by Gerlinger Intra-tumour heterogeneity was high in all 10 tumours. The number of coding mutations identified within a single sample ranged from 9 to 76 across tumours, see f = 0.5) in only two tumours, see f = 0.35), while 7 tumours appeared to be highly unbalanced (f < 0.01). In the latter cases, distinct clonal expansions were likely driven by selection, supporting the original findings of the authors of on-going clonal selection and convergent evolution in the majority of the patients analysed18Strikingly, the universal curve (8) describes the information gain from additional samples very well in all 10 cases and we can assign balancing factors to all 10 tumours. We found balanced phylogenetic trees , or the percentage of uniquely detected mutations defined as clonal across all samples of a single tumour . The mutational load of a tumour is the result of many potentially interacting factors, e.g. the age of a patient or the intrinsic  mutation rate. Furthermore the majority of mutations are likely neutral passengers or provide only a weak selective advantage to the tumour and correlations might be masked by treatment induced selection biases. This suggests that a sampling strategy based on mutational diversity alone may not be optimal. As we show, the change of diversity across independent tumour samples is the variable of interest.We note that the balancing factor f for all possible combinations of subsets with a minimum of 4 samples. For example, all combinations of 6 out of 12 tumour samples yield 924 independent estimates for f. The distributions of values for f are summarised in 2327f).We then tested the robustness of our estimates by applying our analysis to a subset of tumour samples. We inferred the balancing factor before initiation of any therapy may simplify the identification of truly clonal abnormalities that could be the targets of therapy. Future studies are needed to test this observation further. It will also be important to stratify patients for potentially other confounding factors, such as tumour size, tumour stage, and the spatial distribution of tumour samples.Interestingly, 6/7 unbalanced tumours received treatment before resection (and sequencing) and all 7 cases developed metastatic disease. In contrast, 2/3 balanced tumours were treatment naive at the time of sequencing and the only 2 tumours without metastatic disease  were bal4 cells) of 11 treatment naive colorectal tumours  previously published in ref. f ≈ 0.5), two cases show slightly unbalanced trees (f = 0.19 and f = 0.3) and four cases have unbalanced trees (f < 0.01). Based on this data, two samples have a median probability of 58% (95% CI of 38 to 75%) to overestimate the number of clonal copy number changes. Overall, these results support previous observations of largely a single clonal expansion in a majority of colorectal tumours that would lead to more balanced phylogenetic trees19Next, we tested if the information on copy number changes also follows our theoretical prediction (8). We reevaluated copy number changes in multiple single crypts . Balanced tumours (f ≈ 0.5) acquired fewer sub-clonal copy number changes  compared to unbalanced tumours. This is in contrast to the mutational burden in renal cancer patients, where we could not observe a similar correlation. There are several potential reasons for this observation. All colon cancer samples were treatment naive. Copy number changes occur less frequently compared to mutations and do not accumulated with age in healthy tissues. Furthermore it seems plausible that a larger fraction of copy number changes is under selection (either positive or negative), whereas the majority of mutations are likely neutral passengers. The balancing estimates on all possible combinations of tumour samples yield results similar to the mutational burden in renal cancer . However, we observed a strong positive correlation between the balancing factor We note that our analysis does not depend on the detailed effects of selection, i.e. whether selection acts on copy number changes, mutations or epigenetic alterations. Changes in tree balance caused by any type of fitness advantage could potentially be detected. Moreover, the evolutionary mechanisms that generate balanced or unbalanced trees can be arbitrarily complexf ≪ 0.5) may be difficult and perhaps less cost-effective, posing new challenges. However, in many cases the required number of independently sequenced samples appears surprisingly manageable.Accumulating evidence indicates that future personalised treatment strategies of human malignancies must be based on information from multi-region profiling of tumours825Our approach is independent of any threshold that is often imposed from a statistical analysis of the distribution of mutations identified in a tumour. Our analysis also suggests that the optimal time to perform genome profiling in tumours is at the time of diagnosis since therapy appears to introduce strong selection that may interfere with the identification of the therapeutically relevant truly clonal mutations or immune therapeutic targets8t (e.g. at diagnosis). Each leaf of this tree is a cell. Assume there are N leaves and therefore N − 1 bifurcations in the tree. By definition, alterations present in the trunk of this tree are truly clonal and thus are present in all cells of the tumour. The first bifurcation splits the tumour into two subpopulations, the “left” side with proportion f, and the “right” side with proportion 1 − f. If we were to take a single tissue sample, many alterations carried by this subpopulation would likely not be truncal. If we took a second tissue sample, we would increase our chance to identify truly clonal alterations. In this case, we have three possibilities: with probability f2 we have two tissue samples from one side, with probability (1 − f)2 we have two tissue samples from the other side, and with probability 2f(1 − f) we have one tissue sample from each side. Only in this last case, the alterations common to both samples would represent the true set of truncal  alterations and consequently must be present in all cells of the tumour. With n independent samples, the probability p to have picked both sides of the tumour becomesLet us consider the complete phylogenetic tree of a tumour at a certain time n samples. A single sample never provides the full information, as pf(1) = 0 for n = 1. The expected gain of information with an additional sample n + 1 isresulting in a non-linear dependence of the probability to find the true set of clonal mutations through f = 0.5 and the expected gain of information from sample n to sample n + 1 isFor example consider the case of a perfectly balanced tree , implying that different parts of the tree have grown at radically different rates, e.g. due to clonal selection. In this case, pf → 0(n) ≈ nf and the remaining uncertainty decreases linearly in n. For sufficiently small n, the gain of information by an additional tumour sample becomes incrementalHowever, one “side” of the tumour could be very small with f → 0). Although, many samples are needed to infer all true clonal alterations in this situation, the clonal alterations of the extremely dominant and tumour-driving side are of practical interest and again fewer samples may suffice. However, very small ancient sub-clones might drive tumour relapse, as is for example observed in certain leukaemias31In this case, many tumour samples are required to reach a high level of confidence of finding all true clonal alterations. However, a very slowly growing side contributes very little, if at all, to the overall aggressiveness of the tumour, especially if this side virtually vanishes  and an exponential (f → 1/2) gain of confidence with additional samples n.which lies between a linear (pf(n) to find all clonal alterations from n independent tumour samples. This method allows us to infer the balancing factor f of a tumour with respect to the first bifurcation and thus to estimate the expected gain of information with respect to truly clonal alterations by including additional tumour samples in the analysis:Here we propose a method to calculate the probability n samples of a tumour.(i) Collect n samples and determine all alterations.(ii) Analyse the n tumour samples.(iii) Take the intersection of all alterations of all n − 1 tumour samples.(iv) Take the intersection of all alterations of all possible combinations of 1 to (v) Calculate the probability that the alteration identified in step (iii) and (iv) coincide.n samples.By definition, this probability approaches 1 for the combination of all To allow a comparison with n is the maximal number of available samples and i = 1,…,n denotes possible sub-samples. The only free parameter of this equation is f. Thus fitting f, see for example f.Here, Our algorithm is sensitive to misclassified mutations, e.g. mutations not found in a subset of samples due to normal contamination or limitations of sequencing depth . Those are inevitable problems in multi-region sequencing studies, leading to a few mutations that seem to contradict the phylogenetic history of these tumours, the so-called “homoplasy” events. Standard phylogenetic reconstruction algorithms, such as Maximum Parsimony, discard those, hence we filtered the few homoplasy events present in a small subset of renal patients (3/10) from our analysis.How to cite this article: Werner, B. et al. Detecting truly clonal alterations from multi-region profiling of tumours. Sci. Rep.7, 44991; doi: 10.1038/srep44991 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are ‘always on' or irreversible ‘turn on' modality, resulting in poor signal-to-noise ratios or even ‘false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics. A number of nanomaterials for dual diagnostic and therapeutic application have a number of limitations including poor signal-to-noise ratio. Here, the authors developed dual stimuli-responsive and reversibly activatable nanoprobes for tumour targeting and fluorescence-guided photothermal therapy. Theranostics, which integrates both diagnostic and therapeutic functions into a single platform, has potential to propel the biomedical field towards personalized medicine123568181415161718212223248212223D-glucose transported protein (GLUT), one of important nutrient transporters, presents particularly high-concentration in cancer cells3738in vivo tumour-targeted imaging and therapy3640Tumour microenvironment, especially the acidic microenvironment, is a universal feature of solid tumours, regardless of tumour types or development stages2627293031333435in vivo tumour-targeting and specific imaging-guided precision photothermal therapy. We use gold nanorods (AuNRs) and asymmetric cyanine as two building units for the theranostic probe. Asymmetric cyanine serves as both the tumour-specific imaging probe and auxiliary photothermal agent due to its reversible pH-responsive near-infrared absorption and fluorescence222N–GPLGVRGC–SH) serves as the linker between the AuNRs and the asymmetric cyanine near-infrared probe to build MMPs/pH synergistically and reversibly activatable theranostic nanoprobe. Further conjugation of glycosyl endows the nanoprobe active tumour-targeting ability and good biocompatibility. The as-prepared theranostic nanoprobe not only exhibits precision tumour-targeted imaging with ultra-high specificity and negligible background, but also possesses ultra-strong photothermal effect without obvious side-effect, holding great promising for theranostic application.Herein, we report a strategy to design and fabricate dual-stimuli synergistically and reversibly activatable multifunctional nanoprobes for et al.2N–GPLGVRGC–SH , and the presence of new characteristic peaks of the –CONH– stretching vibration of Pep–Acy and the –CH–O–CH– vibration of Glu-SH  in the FT-IR spectra of Pep-Acy/Glu@AuNRs confirm the successful functionalization of Pep-Acy and Glu (We then characterized the prepared Pep-Acy/Glu@AuNRs by transmission electron microscopy (TEM), Zeta potential analysis, dynamic light scattering, Fourier transform infrared (FT-IR), ultraviolet–vis-near-infrared absorption (UV-vis-NIR) and fluorescence spectroscopy. The as-prepared Pep-Acy/Glu@AuNRs presents well-dispersed and homogenous core–shell structure . The ins and Glu .The prepared Pep-Acy/Glu@AuNRs gave two characteristic absorption bands in UV-vis-NIR spectra at pH 7.4 . One abs4546Conjugation of Pep-Acy to AuNRs led to prominent fluorescence quenching of Pep-Acy  due to tin vitro test due to its high sensitivity. The fluorescence signal recovery of Pep-Acy/Glu@AuNRs was closely correlated with the concentration of MMP-13 in an acidic microenvironment , slightly higher than Pep-Acy/@AuNRs (ca. 80%), whereas the CTAB@AuNRs showed remarkable cytotoxicity even at much lower concentrations. These results demonstrate that the modification strategy in our design greatly improved the biocompatibility of AuNRs.The above results encouraged us to apply the Pep-Acy/Glu@AuNRs for cell imaging. Both MMPs positive  and negative cells  were employed as model cells. The expression levels of MMPs including MMP-2, MMP-9 and MMP-13 in SCC-7 cells are much higher than those in 293T cells, as verified by western blotting and quantitative MMPs ELISA . We firsWe then studied the cell internalization and the imaging performance of Pep-Acy/Glu@AuNRs in living cells. SCC-7 cells incubated with Pep-Acy/Glu@AuNRs at pH 6.0 displayed remarkable fluorescence, about 2.7-fold increase of mean fluorescence intensity (MFI) compared to that in glucosamine pre-treated SCC-7 cells (GLUT receptor was blocked) or the SCC-7 cells incubated with Pep-Acy@AuNRs although the same concentration of nanoprobe (as Au) was applied . Besidesin vitro MMP/pH synergistic fluorescence activation and cell imaging encouraged us to investigate whether Pep-Acy/Glu@AuNRs is good for in vivo precision tumour-targeting imaging (L-tumour and R-tumour) into two groups (Group A and B). For both Group A and B, a dramatic fluorescence signal appeared in the untreated R-tumour from 2 h after the intravenous injection of Pep-Acy/Glu@AuNRs. The fluorescence signal gradually increased with the maximum at ca. 4 h, indicating 4 h is the most appropriate time point for the follow-up in vivo photothermal therapy. Besides, the fluorescence signal was clearly identified up to at least 12 h. For comparison, MMPs inhibitor (for Group A) or NaHCO3 (for Group B) was intratumorally administered to the L-tumour 30 min before the intravenous injection of Pep-Acy/Glu@AuNRs to change the tumour microenvironment. For Group A, only a slight fluorescence signal appeared in the MMPs inhibitor-treated L-tumour after 6 h, and the fluorescence signal in L-tumour was always significantly weaker than that of in R-tumour during the whole process, while for Group B almost no fluorescence signal appeared in the NaHCO3-treated L-tumour and the normal tissues during the whole process. The above results confirm that the fluorescence activation of Pep-Acy/Glu@AuNRs was specifically governed by MMPs enzyme and acidic microenvironment simultaneously. The results also show that Pep-Acy/Glu@AuNRs has great potential to guide the stimulus to occur in the accurate location and the best time.The inspiring results of the  imaging . For thiin vivo biodistribution of the nanoprobe in the mice . The treatment group refers to the mice treated with Pep-Acy/Glu@AuNRs under 808 nm laser irradiation (0.6 w cm−2 for 10 min) at 4 h post-injection, guided by the in vivo imaging results, while three control groups include untreated mice, the mice injected with PBS subjected to laser exposure, and the mice injected with Pep-Acy/Glu@AuNRs without laser irradiation. On 808 nm laser irradiation, the temperature of tumours rose rapidly and reached about 50 °C within 6 min in the treatment group  for specific response to cathepsin B (CtsB), a tumour-specific enzyme overexpressed in various malignant tumours25482-Acy@AuNRs synergistically governed by CtsB and pH  was incubated with p-aminophenol mercuric acid (1 mM) in TCNB buffer  at 37 °C for 2 h to obtain the activated MMPs for further use. Subsequently, various types of activated MMPs or different concentration of activated MMP-13 was added to the solution of Pep-Acy/Glu@AuNRs, respectively. After incubation at 37 °C for 2 h, the solution was adjusted to pH 6.0 to measure the fluorescence spectra. For comparison, the same amount of the activated MMP-13 solution in the presence or absence of WAY 170523 (MMP-13 inhibitor) was added to the solution of Pep-Acy/Glu@AuNRs, and incubated at 37 °C for another 2 h. Two aliquots of the resulting mixture were adjusted to pH 6.0 and 7.4 respectively to monitor the fluorescence spectra change.Multiple types of recombinant human MMPs  (100 μg ml−1) and Pep-Acy (1 × 10−5 M) at pH 6.0 were separately collected in 1.5 ml Eppendorf tubes, then irradiated with a 808 nm laser at various power densities  for 10 min. PBS (0.01 M) and Pep-Acy solution (1 × 10−5 M) at pH 7.4 irradiated with a 808 nm laser at 0.6 w cm−2 for 10 min were chosen as control. The thermal images and the temperature changes of each solution were recorded real-time on a FLIR E50 thermal camera.The 0.5 ml solutions of CTAB@AuNRs, Pep-Acy/Glu@AuNRs with the same amount of Au , and not tested for mycoplasma. SCC-7 and 293T cells were cultured respectively in DMEM/high-glucose medium and Roswell park memorial institute (RPMI)-1640 medium with 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37 °C under a 5% humidified atmosphere. Standard MTT assay was carried out to evaluate the cytotoxicity and  therapy .−1) and incubated for an additional 24 h. After that, the cells were collected, washed twice with cold PBS and counted. Two aliquots of the cells were separately re-suspended into 500 μl PBS and digested with 500 μl aqua regia for flow cytometry analysis and inductively coupled plasma mass spectrometry (ICP-MS) measurement. Each experiment group was done in triplicate.To quantify the cell internalization of Pep-Acy/Glu@AuNRs, SCC-7 cells were cultured in 90 mm petri-dished until the cells reached at the end the logarithmic phase. The original medium was then replaced by fresh culture medium containing Pep-Acy/Glu@AuNRs  and further incubated for 12 h. As a control to verify the specificity of MMPs enzyme, SCC-7 cells were seeded into another 12-well plate and pre-treated with MMPs inhibitor 1 h before incubating with Pep-Acy/Glu@AuNRs. The cells were then rinsed three times with PBS, fixed with 1 ml of 4% formaldehyde for 15 min, washed twice with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) solution for 5 min. After thorough rinsing with PBS, the cells were finally soaked in PBS with pH 7.4 and 6.0 to simulate the normal biological fluid pH and tumour acidic microenvironment, respectively. The confocal fluorescence images were carried out on Leica TCS sp8 confocal laser scanning microscope. To evaluate the MMPs and pH dependent cell imaging quantitatively, flow cytometry analysis was performed in the same way.For MMPs and pH dependent cell imaging, both SCC-7 and 293T cells were seeded separately in a 12-well plate at a density of 5 × 106 cells in 50 μl PBS) into the selected positions of the nude mice. In vivo imaging and photothermal therapy were carried out about 8 days later when the tumour reached about 6 mm in diameter.All animals operations were performed in compliance with the guidelines of Tianjin Committee of Use and Care of Laboratory Animals, and all experimental protocols were approved by the Animal Ethics Committee of Nankai University. Female BALB/C nude mice with 5–6 weeks old were purchased form Vital River Laboratories . SCC-7 tumour-bearing mice models were established by subcutaneously injecting a suspension of SCC-7 cells  were randomly divided into two groups (two mice each group). MMPs inhibitor or NaHCO3 was intratumorally administered to the L-tumour of each experiment mouse 30 min before the intravenous injection of Pep-Acy/Glu@AuNRs . NIR fluorescence imaging was then conducted on an IVIS Lumina II in vivo imaging system at different points in time. During the whole process, mice were anesthetized with isoflurance gas to minimize suffering. After imaging, all the mice were killed, and the tumour tissue and main organs containing lung, spleen, kidney, heart and liver were excised and digested in aqua regia for one week. The resulting solution was filtered and diluted. The content of Au in the above solution was determined by ICP-MS for monitoring the biodistribution of the nanoprobe.For in vivo photothermal therapy, the SCC-7 tumour-bearing mice were randomly divided into four groups (three mice each group). Group A: Pep-Acy/Glu@AuNRs with 808 nm laser irradiation; Group B: PBS with 808 nm laser irradiation; Group C: Pep-Acy/Glu@AuNRs without laser irradiation; Group D: untreated. 180 μl of 120 μg ml−1 Pep-Acy/Glu@AuNRs in PBS was intravenously injected into the mice of Group A and C, respectively, and the same amount of PBS was injected into the mice of Group B via a tail vein. After 4 h, the mice of Group A and B were irradiated with 808 nm laser at 0.6 w cm−2 for 10 min, and the temperature increase of tumour tissues was monitored by infrared thermal camera. Furthermore, the tumour volume and body weight of each mouse were recorded every two days for 14 days. The tumour volume was calculated as length × width2 × 0.5, length and width are the greatest longitudinal diameter and the greatest transverse diameter, respectively, which were measured by a vernier caliper. To reflect the tumour growth intuitively, the relative tumour volume for each mouse was calculated as the tumour volume at different time/the original tumour volume before treatment. After the whole therapeutic process, all the mice were killed, the main organs including heart, liver, spleen, lung and kidney were collected and fixed with 4% formaldehyde for hematoxylin and eosin (H&E) staining in order to analyse the potential side effect of Pep-Acy/Glu@AuNRs. The H&E staining and histological sections were performed in a single-blind fashion at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin.For Data supporting the findings of this study are available within the article and its How to cite this article: Zhao, X. et al. Dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy. Nat. Commun.8, 14998 doi: 10.1038/ncomms14998 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Methods, and Supplementary References


Physiologically, it is only expressed in the brain, but it was found overexpressed in more than 70 % of tumours of diverse origin. Furthermore, as a plasma membrane protein, it is easily accessible to extracellular interventions. In this study we analysed the feasibility of the anti-Kv10.1 monoclonal antibody mAb62 to target tumour cells in vitro and in vivo and to deliver therapeutics to the tumour. Using time-domain near infrared fluorescence (NIRF) imaging in a subcutaneous MDA-MB-435S tumour model in nude mice, we showed that mAb62-Cy5.5 specifically accumulates at the tumour for at least 1 week in vivo with a maximum intensity at 48 h. Blocking experiments with an excess of unlabelled mAb62 and application of the free Cy5.5 fluorophore demonstrate specific binding to the tumour. Ex vivo NIRF imaging of whole tumours as well as NIRF imaging and microscopy of tumour slices confirmed the accumulation of the mAb62-Cy5.5 in tumours but not in brain tissue. Moreover, mAb62 was conjugated to the prodrug-activating enzyme β-D-galactosidase . The β-gal activity of the mAb62-β-gal conjugate was analysed in vitro on Kv10.1-expressing MDA-MB-435S cells in comparison to control AsPC-1 cells. We show that the mAb62-β-gal conjugate possesses high β-gal activity when bound to Kv10.1-expressing MDA-MB-435S cells. Moreover, using the β-gal activatable NIRF probe DDAOG, we detected mAb62-β-gal activity in vivo over the tumour area. In summary, we could show that the anti-Kv10.1 antibody is a promising tool for the development of novel concepts of targeted cancer therapy.The K Despite intense medical and research efforts during recent decades, cancer is still far from being curable and remains a leading cause of death worldwide. Consequently, there is still a substantial need to improve the existing and to develop alternative strategies to detect and to treat cancer.Several trends can be observed in cancer research, each of extreme importance for both the improvement of current tumour diagnostics and the development of novel therapeutic interventions: (1) basic research for better understanding of molecular mechanisms underlying cancer biology, (2) identification and extensive characterisation of novel molecular targets and/or tools and (3) development of increasingly sophisticated therapy concepts.v10.1; Eag1) voltage-gated potassium channel is a promising target. In contrast to its restricted distribution in normal healthy tissue, Kv10.1 is significantly overexpressed in many tumour cell lines and in a variety of solid tumours from different histological origins such as breast, colon or cervix  were shown to not only specifically induce apoptosis of tumour cells, but also to sensitise them for chemotherapeutic agents . Cells were cultured in RPMI 1640 medium with GlutaMAX supplemented with 10 % fetal calf serum .Total RNA was extracted with the RNeasy mini kit (Qiagen) following the manufacturer’s recommendations; 5 µg of total RNA was used for cDNA synthesis with SuperScript (Invitrogen).Real-time PCR was performed with 100 ng cDNA in a LightCycler 480 (Roche) as previously described  generated in our group  resulting in mAb62-β-gal was performed by Squarix GmbH .The coupling of mAb62 with β-The 96-well plates were coated overnight at 4 °C with 500 ng of h1x antigen , permeabilised for 10 min with 0.5 % Triton X-100 in phosphate-buffered saline (PBS) and washed three times with PBS. Then cells were blocked for 10 min with 10 % BSA in PBS and incubated with mAb62 (5 µg in 200 µl of 10 % BSA) in PBS for 2 h at room temperature, washed three times each for 10 min with 0.1 % Tween 20 in PBS and stained with anti-mouse Alexa Fluor 488 antibody  for 30 min at room temperature. Finally, cells were washed three times for 10 min with 0.1 % Tween 20 in PBS, rinsed with PBS and mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen).For staining of living cells, the medium was replaced by fresh culture medium containing 10 µg/ml mAb62-Cy5.5 and cells were left at 37 °C for another 3 h. Subsequently, cells were washed with PBS, fixed in 4 % PFA/PBS and mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen).Cells were imaged with an Axiovert 200 M fluorescence microscope (Carl Zeiss) equipped with a NIR-sensitive ORCA-ER digital camera (Hamamatsu) with the following filter settings: 365 ± 12.5 nm excitation and a 445 ± 25 nm emission filter for DAPI, 450–490 nm excitation and 515–565 nm emission filter for Alexa Fluor 488 and 640 ± 15 nm excitation and 690 ± 25 nm emission filter for Alexa-Fluor 647. Image generation and processing were performed with the AxioVision Rel.4.6 and ImageJ software Collins , respect5 of AsPC-1 or MDA-MB-435S cells was plated in 96-well plates and allowed to attach for 24 h. The medium was then replaced with 50 µl of staining buffer  containing 2.5 µg of mAb62-β-gal and the cells were incubated with the conjugate for 30 min on ice. After four washing steps, each by immersing the entire plate for 1 min into a tray containing 1 l of washing buffer , 100 µl of freshly prepared substrate solution containing 0.1 % NaN3, 1 % BSA, 2 mM MgCl2 and 2 mg/ml chlorophenol red-β-d-galactopyranoside  in PBS was added to each well and the increase of absorbance at 570 nm over time was measured immediately in a Wallac 1420 Victor2 Microplate Reader (Perkin Elmer). Five wells were analysed per measurement.1 × 10nu/nu mice (Winkelmann) were used for the in vivo imaging experiments. Animals were handled according to German ethics regulations for animal experimentation and all protocols were approved by the administration of Lower Saxony, Germany.Female athymic nude NMRI-Fox17 MDA-MB-435S or 1 × 106 AsPC1 cells suspended in 100 µl of PBS were subcutaneously transplanted into the right flank of the mice. Mice were inspected twice weekly for tumour development, loss of body weight and general condition. All animals tolerated the procedure well. At the end of the experiment animals were euthanized by isoflurane overdose and cervical dislocation.Mice were anaesthetised with 1.8–2.0 % inhaled isoflurane and either 1 × 10To reduce the fluorescence background, mice received chlorophyll-reduced GLP Nafag 890 food (Provimi Kliba AG) for ~1 week before NIRF imaging. All in vivo analyses were preceded by native scans.Mice were anaesthetised with 1.8–2.0 % inhaled isoflurane and injected intravenously (i.v.) via the tail vein with either 25 µg of mAb62-Cy5.5, 25 µg of mAb62-Cy5.5 together with 50 µg of unlabelled mAb62 or 1.2 µg of Cy5.5 (Amersham) in 150 µl NaCl 0.9 %. Animals were scanned with the Optix MX2 (ART) time-domain imaging system in vivo at the indicated times. At the end of experiments, mice were sacrificed and the excised organs were imaged ex vivo.Cy5.5 fluorescence was obtained with an excitation at 670 nm in combination with a 700-nm long-pass emission filter. Scans were performed with a 1.5 mm (whole-body scans and organ scans) or 1.0 mm  raster and a point integration time of 0.7 s. Data were analysed with OptiView software (ART).Unspecific signals were identified by lifetime measurement. Lifetimes (LTs) that deviated from measured values of the ligand-dye conjugates in vitro were considered as background, which was removed from intensity images by setting lifetime gates , Molecular Probes/Thermofisher] in 150 µl of 0.9 % NaCl/DMSO (50/50). Mice were repetitively scanned over ~3.5 h with the Optix MX2 directly after injection using a 635 nm excitation laser and a 670 ± 20 nm emission filter, 1.0–1.5 mm raster and a point integration time of 0.7 s.For in vivo measurement of β-gal activity  into 100 µm-thick sections. The tissue sections were then mounted onto microscope slides and imaged with the flatbed scanner Odyssey NIRF imager (LI-COR Biosciences) using 700 nm settings.t test using the PAST programme  mAb62 was obtained by immunisation using a fusion protein h1x that contains the pore-forming loop and the tetramerisation domain of Kv10.1 expression were detected in the MDA-MB-435S cells, whereas no Kv10.1 expression was found in the AsPC-1 cells.As shown in Fig. v10.1 high-expressing MDA-MB-435S but not on AsPC1 cells , binding of mAb62-Cy5.5 to the h1x epitope was reduced in a concentration-dependent manner. While the lowest concentration of 0.04 µg showed no effect on the average fluorescence intensity, the binding to h1x could be almost completely blocked with 4 µg of unlabelled mAb62.For in vivo imaging, the antibody was labelled with Cy5.5, resulting in mAb62-Cy5.5. Binding of the conjugate to the h1x antigen was analysed in an immunoassay, in which the fluorescence intensity upon binding of the Cy5.5-labelled antibody to an h1x antigen-coated plate was measured in the Odyssey NIRF imaging system. As shown in Fig. v10.1-expressing cells. For this purpose, living cells were incubated with mAb62-Cy5.5 for 3 h in cell culture medium. As shown in Fig. v10.1-expressing MDA-MB-435S cells, whereas no mAb62-Cy5.5-derived fluorescence was detected in the control AsPC-1 cells that showed no Kv10.1 expression in real-time PCR. In MDA-MB-435S cells the most intense fluorescence signals were detected as foci, which probably correspond to the internalised probe.Further, we analysed whether mAb62-Cy5.5 conjugate specifically binds to K3, mice were injected with 25 µg of mAb62-Cy5.5 through the tail vein and imaged 24 h later. As shown in Fig. v10.1-expressing MDA-MB-435S tumours was detected by Optix MX2 in vivo. Interestingly, AsPC-1 tumour-bearing mice also showed an increase of the fluorescence intensity over the tumour area after probe injection, although the signals were much weaker. This fluorescence can be explained by the presence of the probe in the circulating blood and/or by the unspecific accumulation of the antibody in the tumour due to leaky vessels. However, some upregulation of Kv10.1 expression within the AsPC-1 tumour tissue cannot be excluded.In order to characterise the capability of mAb62 for in vivo tumour imaging and/or targeting, MDA-MB-435S and AsPC-1 cells were subcutaneously transplanted into nude mice. After the tumours reached volumes of ~200 mmv10.1-expressing MDA-MB-435S tumour at 24 h was much higher than the one detected over the control AsPC1 tumour.Twenty-four hours after probe injection mice were killed and the excised tumours were scanned ex vivo Fig. b. As expNext, we characterised the binding and distribution pattern of mAb62-Cy5.5 in MDA-MB-435S tumour-bearing mice. Representative data for ventral and dorsal scans obtained at 24 h after probe administration are shown in Fig. Twenty-four hours after application of mAb62-Cy5.5 strong fluorescence over the tumour could be detected in whole-body scans. Moreover, the fluorescence intensity over the whole body increased, including the area over the stomach and liver as well as the area over the head, visible on the dorsal scans. The lifetime of 1.6 ns measured over the tumour was comparable to the probe’s lifetime measured in vitro  and confirms the specificity of the signal detected in vivo. The lifetime of the fluorescence measured over the background was longer (1.4–1.6 ns) in comparison to that of the prescans  of mAb62 (blocking experiment) or 1.2 µg of free Cy5.5 fluorophore was injected into MDA-MB-435S tumour-bearing mice. Time-domain fluorescence imaging of the tumour areas was performed over time, before (prescan), 4 h after as well as 1, 2, 3, 4, 7, 9 and 14 days after probe application. As shown in the representative images of the tumour areas in Fig. Mice were killed and tumour as well as liver, spleen, kidney and brain was extracted and scanned ex vivo with the Optix MX2. As shown in Fig. v10.1 is physiologically expressed in the brain, it was important to analyse whether mAb62-Cy5.5 is able to pass the blood-brain barrier and therefore detectable on brain tissue slices. Tumours and brains of mice injected with mAb62-Cy5.5 were thus embedded in paraffin and slices were scanned with the Odyssey imaging system  and analysed its binding as well as measured the specific β-gal activity at the tumour cells in vitro and at the tumour site in vivo.Finally, in order to study the feasibility of the mAb62 to deliver diagnostic tools or therapeutics to the tumour, we conjugated the antibody with the drug-activating enzyme, β-v10.1-expressing MDA-MB-435S and control AsPC-1 cells were incubated for 30 min in vitro with mAb62-β-gal as well as with the non-conjugated mAb62 as a negative control. After careful washing of the unbound conjugate, cleavage of the β-gal substrate CPRG to chlorophenol red (570 nm absorbance) by the bound β-gal conjugate was measured. A positive reaction was only detected on the MDA-MB-435S cells incubated with the mAb62-β-gal construct  using the β-gal substrate DDAOG, which shifts its excitation maximum from 465 to 646 nm upon cleavage to DDAO. In the first step, mAb62-β-gal was applied to MDA-MB-435S tumour-bearing mice and allowed to bind to the tumour for 24 h, the time when the mAb62-Cy5.5 achieved the highest tumour accumulation. Subsequently, we administered DDAOG i.v. and measured the fluorescence intensity of the DDAO over the tumour area over ~3.5 h. As shown in Fig. Finally, the in vivo activity of mAb62-β-gal was measured in MDA-MB-435S tumour-bearing mice  as the one of the mAb62-Cy5.5 measured in vitro (1.5 ns). The fact that the same lifetime was also measured all over the body, except the gastrointestinal tract, is most likely due to the probe still present in the circulation and not yet completely cleared from the blood. This is not surprising as whole antibodies such as the mAb62 are known to have relatively long circulation times (Wang et al. v10.1 is physiologically expressed in the central nervous system, and because previous immunohistochemistry experiments (Hemmerlein et al. The most likely source of harmful effects from tools targeting ion channels is the direct influence they could have on the physiology of excitable tissue. As Kv10.1-expressing cells in vitro and accumulates within Kv10.1-expressing tumours, but also retains its enzymatic activity on tumour cells, both in vitro and in vivo. Therefore, mAb62 conjugated to the enzyme β-D-galactosidase could represent a tool for tumour-specific therapies such as the antibody-directed enzyme prodrug therapy (ADEPT; Fig. In a proof-of-concept experiment we showed that the mAb62-β-gal construct can specifically deliver drug-activating enzyme to the tumour. We confirmed that the mAb62-β-gal conjugate not only specifically binds to KHere we show the use of fluorescence imaging to evaluate the specificity of the antibody binding or the activity of the antibody-enzyme conjugate at the tumour site. Additional anatomical imaging, such as CT, could be considered for anatomical correlation as has been previously shown (Alves et al. v10.1 or blocking Kv10.1 channel function have already been proposed for therapeutic purposes (Pardo et al. v10.1 current, was shown to not only reduce the growth of cancer cells in vitro, but also tumour progression in vivo in subcutaneous MDA-MB-435S xenografts and in human pancreatic cancer explants (Gomez-Varela et al. v10.1, was reported to inhibit growth of Kv10.1-expressing cancer cells in vitro and in vivo (Downie et al. Antibodies and small molecule compounds targeting Kv10.1, while small molecule drugs more often target intracellular molecules.Both, monoclonal antibodies and small molecules are promising tools for cancer targeting, but the individual advantages and disadvantages of each have to be taken into consideration (Imai and Takaoka Ion channels are especially interesting targets for therapeutic interventions as they are easily accessible from the extracellular milieu. The monoclonal antibody used in our study may not only be a valuable tool for ADEPT, but can also be used for the targeted delivery of toxic compounds to the tumours, like radioligands or bacterial toxins Fig. . These s


Porphyry deposits are copper-rich orebodies formed by precipitation of metal sulphides from hydrothermal fluids released from magmatic intrusions that cooled at depth within the Earth’s crust. Finding new porphyry deposits is essential because they are our largest source of copper and they also contain other strategic metals including gold and molybdenum. However, the discovery of giant porphyry deposits is hindered by a lack of understanding of the factors governing their size. Here, we use thermal modelling and statistical simulations to quantify the tempo and the chemistry of fluids released from cooling magmatic systems. We confirm that typical arc magmas produce fluids similar in composition to those that form porphyry deposits and conclude that the volume and duration of magmatic activity exert a first order control on the endowment  of economic porphyry copper deposits. Therefore, initial magma enrichment in copper and sulphur, although adding to the metallogenic potential, is not necessary to form a giant deposit. Our results link the respective durations of magmatic and hydrothermal activity from well-known large to supergiant deposits to their metal endowment. This novel approach can readily be implemented as an additional exploration tool that can help assess the economic potential of magmatic-hydrothermal systems. Porphyry Copper Deposits (PCDs) are classically regarded as large upper crustal volumes of hydrothermally altered, veined and mineralized rocks centred on small appendices of magmatic rocks extruded from a larger magma body lying at greater depth112367108To explore the relative contribution of magma fertility, magma volume and rate of magma injection on the genesis and total metal endowment of PCDs, we simulated the emplacement, cooling and degassing of fluid-saturated magmatic intrusions of various volumes containing different initial concentrations of “ore ingredients”, emplaced in the crust over different timescales. In particular, we focussed on the tempo and the chemistry of the degassed fluid. Magma degassing is the process by which volatiles , together with fluid-mobile elements  initially dissolved in magmas, exsolve and migrate to shallower depths where they may form a PCD or escape to the atmosphere . The ext1ϕc) is eventually reached at which bubbles coalesce and volatiles  can escape the crystallising magmaIsobaric cooling of a thermally homogeneous volatile-saturated volume of magma leads to crystallisation and to the progressive exsolution of bubbles containing supercritical fluids . Initial1415162O and CO2 contents of 5 wt% and 200 p.p.m., respectively) and thermally homogeneous unit-volume of magma that undergoes isobaric crystallization and cooling at 250 MPa . Because crystallinity, temperature, mass fraction of fluid, and chemistry of fluid at each outgassing event depend on various factors  we contemporaneously and randomly varied all these parameters within reasonable ranges . For each thermal model, we extracted the respective volumes of magma that cool below one of the four percolation isotherms in between each time step  with an initial temperature of 900 °C. Unlike previous models that considered ‘instantaneous’ emplacement of large magma volumes101119ime step . Subsequfluid/melt < ~20) or decrease (Dfluid/melt > ~20) depending on their respective fluid/melt partition coefficientsResults show that the composition of the fluids released over time from the magmatic intrusion is largely controlled by the chemistry of the fluids outgassed at the highest temperature when magma reaches the first percolation threshold , lead content (0.005–0.08 wt%), zinc content (0.01–0.16 wt%) and sulphur content (0.1–1.1 wt%) of intermediate-density fluid inclusions (ID-FIs) measured in PCDsfluid > 2 and enough sulphur is present in the fluids to precipitate all the copper together with large amounts of pyrite and other sulphides, as typically observed in PCDs. Therefore, our results suggest that mafic magma input, proposed to critically provide the sulphur required to form PCDs2526For all simulations, the chemistry of the computed fluid matches the salinity (2–15 wt% NaCl3 yr−1) all the injected magma is cooled to solidus and all the fluids are released during the magma injection phase. At higher magma fluxes, although most outgassing occurs during injection, a significant mass (up to 30%) of the total magma remains at temperature higher than 800 °C , the volumetric cooling rates sharply decrease just after injection ceases. This is due to a combination of latent heat release  and to the cessation of displacement of the host rocks (advection) caused by the intrusion. At any given final magma volume, the ratio between the duration of fluid release and the duration of magma emplacement increases with increasing magma flux  only represent minimum values while likely providing close estimates (see Methods). Based on these durations, we estimate the magma volume and average magma flux associated with the formation of these deposits , giant  and supergiant  PCDs for which the actual copper endowment is known . The durdeposits . Overlapdeposits , we alsodeposits .3 yr−1; ref. 3) and large  plutons corresponding to large and supergiant deposits, respectively  form on timescales smaller than 100 kyr  and 30, 3) are key requirements for the genesis of the largest economic deposits . These findings are in line with global estimates of PCD formation rates  are forming a PCD at depth today (assuming magmatic system lifespans >1 Myr). Similarly, a comparable proportion of outcropping plutons may have once had a now eroded PCD on top. This reinforces the idea that PCD provinces mainly correspond to optimal erosion levels rather than more PCD-productive crustal segments32Our results confirm that almost any hydrous intermediate arc magma has the potential to exsolve fluids capable of forming a PCD. The agreement between our modelling results and the data collected from PCDs suggests that, regardless of the range of initial metal and sulphur concentrations in the magma, long durations of a magmatic episode (>1 Ma) and large intrusion volumes , −1 K−1), −3), −1), k is the heat conductivity (W m−1 K−1), L is the latent heat of fusion (J kg−1) and where T, °C) as:where  km−1, intrusion centre depth 10 km, heat conductivity 2.7 W m−1 K−1, heat capacity 1 kJ kg−1 K−1, rock density 2700 kg m−3, latent heat of fusion 350 kJ kg−1.3 yr−1) and final injection volumes . Intrusion was treated by setting the temperature within the newly intruded portion of the magma body , while the adjacent rocks were advected outward to accommodate the volume increase. However, because the model is formulated in the Eulerian framework, advection does not influence the computational mesh, which remains fixed. The velocity field (with components  km−1).Most calculations were performed assuming a horizontally expanding cylindrical magmatic system (thickness of 8 km) with new magma injection  occurring in the centre of the intrusion with a temperature of 900 °C. We also tested sensitivity to the intrusion geometry and injection temperature by comparing these results with outward inflating spheres (new magma injection in the form of spheres) with an injection temperature of 1,000 °C . We studT), we back calculated the volume of the pluton cooling through four specific isotherms (representing four different outgassing fronts) through time . Outgassing occurs once the volume fraction of fluid with respect to the melt phases reaches the percolation threshold (3) of bubble and melt at the percolation threshold where −3) are the densities of melt and fluid, respectively, 3) are the initial mass and volume of injected magma, respectively. Note that where: with 3) is the volume of crystals at the outgassing event n:where where The chlorine, copper, lead, zinc and sulphur concentrations of the melt, crystals and fluid were calculated assuming equilibrium partitioning between these phases. The concentration of element x in the melt at the outgassing event n (with where eq) was derived from the chlorine content as follows:Fluid salinity (wt% NaClwhere fluid (t) in Mt) is calculated as the sum of the fluids outgassed along the four outgassing fronts. Using For an incrementally growing magmatic system, the total mass of fluid outgassed at each time interval (m3) is the volume of melt that cooled below one of the four percolation isotherm between two time steps, extracted from the thermal model. Similarly, the total mass of element x being outgassed from the system at each time step is:where Finally, at each time step, the composition of the bulk fluid for element x is given by:The range of initial melt concentrations of chlorine, copper, lead, zinc and sulphur  was deriThe upper limit of http://earthref.org/GERM/) for plagioclase, amphibole, biotite, K-feldspar and magnetite. We have assumed that sulphur is fully incompatible in these minerals. We have calculated the bulk crystals/melt partition coefficients for Cl, Cu, Pb and Zn for various mixtures of plagioclase, amphibole, biotite, K-feldspar, quartz and magnetite. This results in values from 0 to 0.55 for  cm−3 to 0.612 g cm−3, consistent with densities estimated in natural intermediate-density fluid inclusions .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.