A current flowing in the Sb2Te3 surface states generates a net spin polarization due to spin-momentum locking, which is electrically detected as a voltage on an Fe/Al2O3 tunnel barrier detector. Measurements of this voltage as a function of current direction and detector magnetization indicate that hole spin-momentum locking follows the right-hand rule, opposite that of electron, providing direct confirmation that the chirality is indeed inverted below Dirac point. The spin signal is linear with current, and exhibits a temperature dependence consistent with the semiconducting nature of the TI film and freeze-out of bulk conduction below 100 K. Our results demonstrate that the chirality of the helical spin texture of TI surface states can be determined electrically, an enabling step in the electrical manipulation of spins in next generation TI-based quantum devices.The surface states of 3D topological insulators (TIs) exhibit a helical spin texture with spin locked at right angles with momentum. The chirality of this spin texture is expected to invert crossing the Dirac point, a property that has been experimentally observed by optical probes. Here, we directly determine the chirality below the Dirac point by electrically detecting spin-momentum locking in surface states of a p-type TI, Sb We use Fe/Al2O3 tunnel barrier contacts to detect bias current generated spin polarization. From measurements of the carrier type and sign of the spin voltage, we deduce a counter-clockwise chirality with carrier spin-momentum locking following a right-hand rule, confirming the chirality inversion of the helical spin texture of TI surface state above and below the Dirac point. The direct electrical access to the helical spin texture in the TI surface states is an enabling step in the electrical manipulation of spins in topological devices for spintronics and quantum computation applications.Here we demonstrate the determination of the chirality of the spin texture below the Dirac point using transport measurement, through the direct electrical detection of spin-momentum locking in a p-type TI, Sb2Te3 thin films 30 nm thick are grown by molecular beam epitaxy (MBE) on epitaxial graphene/SiC(0001) substrates at 275–325 °C (see Methods). A conductive nitrogen doped n-type 4H-SiC substrate (0.05 Ohms-cm) is used to facilitate the in situ scanning tunneling microscopy/spectroscopy (STM/STS) monitoring of the surface morphology and electronic structure to ensure optimal layer-by-layer spiral growth  (identified by an arrow in the inset). This indicates p-type carriers in the TI surface states, with an estimated hole concentration of ~4.7 × 1011/cm2. The p-type conductivity is as also confirmed by ex situ Hall measurements, as shown in the 2Te328Sbl growth 27. The ni.e., the Fermi level lies within the bandgap. A weakly temperature dependent plateau is seen below 100 K, characteristic of metallic behavior, and attributed to freeze-out of bulk carriers and conduction only in metallic states such as the TI surface states. These findings are consistent with earlier work on MBE grown Sb2Te3 films2829p-type conductivity is typically found and attributed to excess Sb leading to SbTe acceptor-like antisites with an activation energy of ~7 meV2Te3 single crystals synthesized by melting mixtures of Sb and Te, where much larger p-type doping is typically found with the Fermi level lying within the bulk valence bandTemperature dependent resistivity measurements show over two orders of magnitude increase in resistivity with decreasing temperature , indicat2O3, are utilized as spin sensitive probes to detect the bias current-generated spin polarization in the Sb2Te3 surface states. Such contacts have enabled electrical detection of spin current and accumulation in both semiconductors and metals2Se315161819Ferromagnet/oxide tunnel contacts, Fe/Al2Te3 films were processed into the device structures illustrated in 2Te3 mesa, with several pairs of ferromagnetic (red) detector and corresponding non-magnetic Au/Ti (yellow) reference contacts in between. The unpolarized current flowing between the two outer contacts produces a spontaneous spin polarization in the Sb2Te3 surface states throughout the channel due to spin-momentum locking. The projection of this spin onto the magnetization of the ferromagnetic detector contact is recorded as a voltage with a high-impedance voltmeter (>1 Giga-ohm). Note that no current flows through the detector contact. An in-plane magnetic field is applied to switch the magnetization of the detector contact, so that the spins generated by carriers populating the Sb2Te3 surface states are either parallel or antiparallel to the magnetization, which changes the magnitude of detector voltage. Here we define the positive current to be holes flowing from left to right along the +x axis, and the positive magnetic field to be pointing in the +y direction.MBE grown Sb2Se3 using the same detector contacts2O3), which are only sensitive to the orientation of the spin regardless of the source, this indicates that in Transport measurements were carried out in a closed cycle cryostat equipped with an electromagnet . 2Te3 surface states is locked in a counterclockwise helicity to the hole momentum, demonstrating that chirality in spin-momentum locking is indeed inverted below the Dirac point.Another hallmark of the current generated spin in the TI surface states due to spin-momentum locking is that the spin orientation can be changed by reversing the current direction, as shown in ΔV = V(M) − V(M−), produced when the detector magnetization reverses  depends linearly upon the applied bias current that flows in the Sb2Te3 film, as shown in ΔV is determined from the data as illustrated in the inset. This behavior is consistent with a spin polarization generated by a bias current, and also consistent with earlier theoretical workThe change in detector voltage, V(M) − V(M−)] measured at ±1 mA is shown in Te antisites in the semiconducting TI bulk, which shunts increasing fractions of the current, and dilutes the portion that flows through the surface states. By increasing the total bias current from ±1 mA to ±3.7 mA at 175 K, a clear hysteretic step-like behavior is again seen  are directly related to the current and spin polarization by [V(M) − V(M−)] = IbRBPFM (p. Mu), (bold case denotes a vector) where Ib is the (hole) bias current in the +x direction, RB is the ballistic resistance of the channel, and PFM is the transport spin polarization of the FM detector metal. Mu is a unit vector along the detector magnetization M, and p is the degree of spin polarization induced per unit current by both spin-momentum locking in TI Dirac surface states and Rashba spin-orbit coupling in the two-dimensional electron or hole gas that may form on the surface due to band bending.The linear behavior of the spin signal is consistent with the theoretical work by Hong 2Te3 is much more stable against adsorbate-induced band bending and shifting of the Fermi level2Se3322Te3 surface, these states can exhibit Rashba-type spin-orbit splitting with spins in-plane and also at approximately right angles to the carrier momentum, and therefore may also contribute to the spin signal measured. However, these states exist as spin-split pairs with opposite spin orientation at each momentum, therefore the resulting current-induced spin densities tend to cancel. Hence the net spin polarization should be dominated by that from the TI surface state, as predicted by model calculationsV(M) − V(M−)] that we measure . Even though temperature dependent resistivity measurements suggest freeze out of bulk carriers at lower temperatures  ~ 0.4, and kF ~ 0.07 Å−1 p| ~ 0.15 from the data in Alternatively, earlier work has shown robust and well-defined QW states due to confinement in MBE grown Sb2Te3, where the carrier type is confirmed by Hall measurement, using Fe/Al2O3 tunnel barrier contacts to detect bias current generated spin polarization, our results indicate a counter-clockwise chirality with carrier spin-momentum locking following a right-hand rule, confirming that the chirality is indeed inverted below the Dirac point. The spin signal arising from spin-momentum locking of the surface states is linear with current, and exhibits a temperature dependence consistent with the semiconducting nature of the TI film. With the efficient spin-momentum driven spin transfer torque2Se3, the direct electrical access to the spin texture of the helical TI surface states is an enabling step towards realizing next generation TI-based spintronics devices.In summary, we demonstrate that the chirality of the helical spin texture of a topological insulator can be determined using transport measurement, by electrically detecting the spin-momentum locking of the TI surface states. In the p-type TI, Sb2Te3 films was carried out on epitaxial graphene/SiC(0001) substrates in an ultrahigh vacuum (UHV) system (base pressure ~1 × 10−10 Torr) that integrates two MBE chambers and a low temperature (5–300 K) scanning tunneling microscope (STM). As-received nitrogen-doped 4H-SiC(0001) substrates were first etched in H2/Ar atmosphere in a separate vacuum chamber at ~1500 °C to remove polishing damage. They were then transferred to the MBE system and annealed in UHV in a Si flux (~0.1 ML/min) at 950 °C to produce a (3 × 3) reconstructed surface, and further annealed at temperatures 1000–1300 °C without Si flux to grow epitaxial graphene2Te3, the substrate was held at 275–325 °C, and Sb and Te were supplied via separate Knudsen cells at 460 and 250 °C, respectively. In situ STM imaging is used to monitor surface morphology and electronic structure and ensure optimal layer-by-layer spiral growth, as shown in 11/cm2. These values represent an upper bound due to the upward shift of the Dirac point accommodating charges induced by the electric field between the STM tip and sampleThe growth of Sb2O3 contacts were formed on the air-exposed surface of a 20 nm thick Sb2Te3 film in a separate MBE system as follows. A 0.7 nm layer of polycrystalline Al was first deposited by MBE, and then oxidized in 200 Torr O2 for 20 min in the presence of UV light in the load-lock chamber. This step was then repeated for a total Al2O3 thickness of 2 nm. The sample was then transferred under UHV to an interconnected metals MBE chamber, where 20 nm of polycrystalline Fe was deposited at room temperature from a Knudsen cell.The Fe/Al2 to 80 × 80 μm2, with adjacent contact separation ranging from 45 to 200 μm. Ion milling was used to pattern the Sb2Te3 mesa. Large Ti/Au contacts were deposited by lift-off in an electron beam evaporator as non-magnetic reference contacts and bias current leads. The Fe contacts were capped with 10 nm Ti/100 nm Au, and bond pads for wire bonded electrical connections are electrically isolated from the SiC using 100 nm of Si3N4.The samples were processed into the device structures illustrated in 2Te3 mesa, and the voltage on the detector contact was recorded as a function of the in-plane magnetic field applied orthogonal to the electron bias current direction in the TI.Transport measurements were performed in a closed cycle cryostat equipped with an electromagnet . An unpolarized bias current was applied through the outer Ti/Au contacts on the opposite ends of the SbHow to cite this article: Li, C. H. et al. Electrical Detection of the Helical Spin Texture in a p-type Topological Insulator Sb2Te3. Sci. Rep.6, 29533; doi: 10.1038/srep29533 (2016).


COPD-6™ is a lung function testing device for a rapid pre-spirometry testing to screen-out at-risk individuals not having COPD and indicating those at risk. The aim of this study was to validate COPD-6™ lung function testing (index test) in general practice in discriminating patients with COPD out of the population at risk - smokers/ex-smokers with no previous diagnosis of COPD, using measurements at tertiary care as reference standard.Consecutive 227 subjects  with no previous diagnosis of COPD, aged 52.5 (SD 6.8) years from 26 general practitioners (GPs) were recruited, lung function tested with COPD-6™, referred to the tertiary institution for repeated COPD-6™ testing followed by spirometry with a bronchodilator , examination, and pulmonologist consultation for the diagnosis and severity of COPD.P < 0.001) for the diagnosis of COPD when lung function was measured using COPD-6™ in GP’s office with a specificity of 100 %  but a very low sensitivity of 32.56 % . Significant agreement for forced expiratory volume in 1 s measured at GP’s office and at lung function lab was found  but not for other measured parameters .COPD was diagnosed in 43 subjects (18.9 %), with an AUC of 0.827 . Based on our results lung function testing with COPD-6™ can substitute spirometry testing in cases where it is not readily available to the patient/physician taken into account that the traditional FEVNCT01550679 Registered 28 September 2014, retrospectively registeredClinicalTrials.gov Identifier  COPD is one of the leading causes of morbidity and mortality worldwide . Existin1/FVC <0.70 is required to make a diagnosis of COPD in a clinical context of the disease [Spirometry with the documented post-bronchodilator FEV disease . Unavail disease –11.6 instead of FVC could underestimate the later; (2) no post-bronchodilator testing; (3) no flow-volume curve presentation; (4) a single criterion for fixed airflow limitation as defined in GOLD initiative (FEV1/FEV6 < 0.70) thus possibly producing a significant over-diagnosis in elderly (>70 years of age) according to The Global Lung Function Initiative data [COPD-6™  is a cheap, simple lung function testing device approved as a rapid pre-spirometry testing tool to screen-out the at-risk individuals who do not have COPD and indicate those that may be at risk. It is a simple device, easy to learn how to operate, having the readout without the risk of false COPD negatives, thus focusing spirometry resources on a smaller population with most of the risk. Four major problems could arise from using COPD-6™ as a screening device instead of spirometry: (1) using FEVive data .Having all that in mind, we wanted to explore the diagnostic accuracy of COPD-6™ in a population of smokers/ex-smokers with a significant exposure to cigarette smoke, with no previous diagnosis of COPD, in a general practice setting comparing it to the ‘gold standard’, spirometry conducted in a lung function laboratory at the tertiary care level  by experienced staff with special training. This is a population with expected 25 % of undiagnosed COPD cases in which such case identification is recommended . So, thehttps://clinicaltrials.gov/ct2/show/NCT01550679. The study was approved by the local ethics committee and conducted according to the Declaration of Helsinki and other relevant international and national laws. The patients were approached by their GPs during any (unrelated to respiratory problems) visit to their office if they were smokers or ex-smokers of the predefined age group for the study together with the prescreening for inclusion/exclusion criteria using a structured interview. Eligible patients were given the Informed consent document with enough time to read it and to discuss any relevant issues regarding the study before they signed the written consent. They were informed about the prospective nature of the study and their right to withdraw their consent and claim the withdrawal of all gathered data and destroying all biological samples at any time without any explanation, obligation or consequence from their side. All participants signed the written consent before starting any procedure for the study.This prospective cohort study was a part of broader research project (Early detection of COPD patients in GOLD 0 (smokers) population – MARKO project). The whole protocol of the MARKO project can be found at The consecutive patients from 26 GPs (representing the same number of GP offices) were recruited based on inclusion/exclusion criteria. We decided on consecutive patients based on the limited number of insured persons under the care by each GP (approx. 1700), and relatively low response rate for a public health campaigns in our country. Inclusion criteria were: written consent; smokers/ex-smokers of either sex, aged 40–65 years with a smoking history of at least 20 pack-years ; with no previous diagnosis of COPD. Exclusion criteria were: any clinically relevant chronic disease significantly affecting QoL at the time of the first visit ; immunosuppressive therapy; preceding acute respiratory disease 4 weeks before the visit; hospitalization for any reason during past 3 months; myocardial infarction, cerebrovascular infarction or transient ischemic attack during past 6 months; diagnosis of asthma; and an inability to perform the diagnostic protocol. Exclusion criteria were introduced not to exclude patients having comorbidities common in COPD, but to exclude the ones that represent acute/subacute clinical states/disorders or states of recovery from major clinical disorders representing an absolute or relative contraindication for spirometry or significantly influencing the diagnostic process or an already present diagnosis of respiratory disorder .1/FVC <0.70) [All GPs went through short small groups training and were provided with the COPD-6™ devices. GPs were not extensively trained in spirometry or assessed for their skill level because we wanted that the measurements would be performed as close as possible to the regular real-life clinical situation where GPs scarcely use these measurements. After the examination at the GP’s office and lung function testing with COPD-6™ (index test), patients were referred after 2–4 weeks to one of the tertiary institutions to a designated team consisting of a pulmonologist, research nurse and lung function laboratory technician. Standard diagnostic workup consisted of repeated COPD-6™ lung function testing followed by spirometry with a bronchodilator , history, physical examination and specialist consultation. Patients with no previous diagnosis of COPD were chosen to avoid bias coming from a previous knowledge of a diagnosis but allowing a significant subsample of subject with COPD (according to previous studies up to 25 % of subjects in this population has an undiagnosed COPD) . To avoi1 in L), forced expiratory volume in 6 s (FEV6 in L), FEV1/FEV6 ratio (%), and lung age (years) and as % of predicted values for FEV1, FEV6 and FEV1/FEV6 according to prediction equations already in the device calculated according to sex, age and height. The device uses the pre-specified cut-off levels for visually suggesting the preliminary diagnosis of COPD (FEV1/FEV6 ratio of <0.7) and assesses the severity according to GOLD initiative [Lung function measurements using COPD-6™ were performed according to the manufacturer’s recommendations and ATS/ERS guidelines . Measure1, forced expiratory capacity (FVC), FEV1/FVC ratio were together with sex, age and height entered into the Excel sheet  for all subjects and using predicted values equation from Quanjer, % of predicted was calculated in a single act using Excel [Spirometry was performed using computerized pneumotachographs  using the same procedure at all clinical sites  according to ATS/ERS guidelines . The besng Excel . Tertiarhttps://www.medcalc.org; 2015). Minimal sample size of 70 subjects (14 positive and 56 negative) was calculated for the expected area under the curve (AUC) of 0.8 with a statistical power of 95 % (beta 0.05) and alpha of 0.05. Categorical data was presented as absolute and relative (%) numbers. Continuous variables were presented as mean and standard deviations (SD). Categorical data was compared between subgroups using chi-square (χ2) test and continuous variables using Student’s t-test or Mann-Whitney U test and analysis of variance (ANOVA). The criteria to use Student’s t-test and ANOVA were checked and fulfilled before the tests were performed. Agreement between lung function measurement methods was conducted using Bland-Altman statistics and plots. Utility of FEV1/FEV6 measured using COPD-6™ at GP’s office (index test) for diagnosing COPD was analyzed comparing it to the reference standard using receiver operator curve (ROC) analysis and data was presented as AUC together with sensitivity, specificity and positive and negative predictive values together with 95 % confidence intervals (CIs). P < 0.05 was used as statistically significant for all analyses.Data analysis was performed using STATISTICA version 12  and MedCalc Statistical Software version 15.8  or between smokers and ex-smokers . Cross-tabulation of an index test positivity against the reference standard is presented in Table t = 1.139, P = 0.256), presence of comorbid disorders (55.5 %) and chronic treatment , BMI  or smoking habit . Also no clustering of COPD diagnosis, demographics or smoking habit data was evident for different GPs . In Table Out of 326 consecutive prescreened smokers of eligible age and smoking history, 227 (69.6 %) subjects 115 women) at risk for COPD (185 smokers and 42 ex-smokers) aged 52.5 (SD 6.8) years were included in this study  being significantly different to spirometry . Using the usual (pre-specified) threshold of <0.7 for FEV1/FEV6 for the diagnosis of COPD for the lung function (COPD-6™) values measured at GP’s offices gave the highest specificity of 100 %  but a very low sensitivity of 32.56 %  with a PPV of 100 %  and NPV of 86.38 % .ROC curve analyses of FEV1/FEV6 that gave a specificity of 88.95 %  with a sensitivity of 70.97 % , a PPV of 52.38 % , NPV of 94.71 % . The highest NPV  was achieved with a cut-off value of 0.85 with the negative likelihood value of 0.26 .Exploratory analyses using the change in threshold gave somewhat better overall results with a change of threshold to ≤0.78 for FEV1 values measured using COPD-6™ at GP’s offices showed small differences with the same measurement at tertiary care and no clinically relevant differences when compared with spirometric measurement and postbronchodilator one  showed clinically non-relevant difference  when compared to reference measures with a trend of increasing the difference with larger values  when compared to reference measures  at tertiary care hospitals with a trend of increasing the difference with lower values  COPD-6™ showed moderate accuracy with high specificity but low sensitivity for COPD; (2) COPD-6™ could be with certain restrictions reliably used in GP’s offices; (3) in the population at risk for COPD, there was a substantial number (18.9 %) of undiagnosed patients. Our data comparing COPD-6™ measurements with a reference standard (tertiary care COPD diagnosis) shows that COPD-6™ can be used with enough accuracy in screening for COPD on a primary care level. Exploratory analyses with the change in threshold showed the possible improvement in the accuracy, but this data needs additional confirmatory studies to be conducted using these values as a pre-specified ones. Although it is our opinion that based on substantial number of undiagnosed patients screening for COPD in a population at risk is valuable, this opinion needs further corroboration based on the studies arguing benefits coming from this effort.Spirometry is the basis for diagnosing COPD but primary care providers, who first meet patients with respiratory symptoms, do not always have access , time or1/FEV6 measurements were not compared between GP’s offices and lung function laboratory [1/FEV6 ratios were suggested with different sensitivity and specificity that didn’t meet the criteria to establish the diagnosis of COPD [1/FEV6 ratio <0.7 measured at GP’s office has a specificity of 100 % with positive predictive value of 100 % indicating that a patient had a COPD and can be diagnosed in accordance to other diagnostic criteria with no further lung function testing needed. These results were good but significantly worse than spirometry. The same was the case for the FEV1/FEV6 ratio measured by COPD-6™ at a lung function lab at tertiary care hospitals using highly experienced staff. The reason behind it, lies in the lack of real-time visual control, present during spirometry, thus underestimating the real value of FEV6 (a surrogate measure for FVC). This produced a systematic bias overestimating the real value of FEV1/FEV6 ratio. This points out that additional training should be provided to heath care personnel at GP’s office to understand and recognize this possible measurement bias.Other studies evaluating the use of COPD-6™ differ in many aspects from our study. The population at risk was not strictly defined , the patboratory . The res of COPD , 21. Bas1/FEV6 ratio >0.85 measured at GP’s office had a NPV of 95.74 % and was reasonable to conclude that a patient was not suffering from COPD and further testing was indicated to reveal the reasons for respiratory symptoms. A strategy of using this two cut-off values (FEV1/FEV6 < 0.7 to diagnose and >0.85 to rule-out COPD) could increase the number of patients diagnosed at GP’s offices and treated according to the current guidelines. A confirmatory study validating these thresholds should be done using these cut-off values as a pre-specified goals. If this thresholds are confirmed, only patients with the FEV1/FEV6 ratio between the 0.7 and 0.85 should be referred for further lung function testing and (sub)specialist evaluation. Our results were based on the study population from 40 to 65 years of age so in older populations recommendations from Global Lung Initiative (GLI) to use lower limit of normal (LLN) for FEV1/FVC (different from a fixed criterion of <0.7) should be taken into account to prevent over diagnosing COPD in older population [Otherwise, our exploratory analysis showed that FEVpulation .Diagnosing COPD is important because it was shown that undiagnosed patients with COPD have increased mortality , morbidi1/FEV6 should be revised to LLN according to GLI recommendations [1/FEV6 < 0.7 – treatment should be started according to the current guidelines in an uncomplicated patient or referred to specialist for further assessment if a patient is severe or has significant comorbidities; (2) FEV1/FEV6 ≥ 0.7 and ≤0.85 – refer to pulmonologist for further consultation; (3) >0.85 should consider alternative diagnosis than COPD. Using such an algorithm in regular GP practice will allow GPs to make an early COPD diagnosis or a proper specialist referral thus producing more appropriate use of resources, cost savings and task shifting. These effects are based on much broader accessibility and lower price of services of general practice and limited resources on the secondary/tertiary care level considering the number of smokers in general population (up to 30 % of adults) and COPD patients (up to 10 % of adults). Substantial positive effects can be expected based on this broad accessibility regarding early diagnosis of COPD allowing early preventive interventions (quitting smoking) [Based on the results of our study we could recommend the clinical algorithm Fig.  for the ndations ): (1) FEsmoking) . Althougsmoking) .Fig. 4Prn = 43, 18.9 %), allowing us to properly evaluate the accuracy of the tested device. Also GPs were not extensively trained for the use of the tested device and were not aware to be part of the study thus allowing us to make conclusions that can be generalized to a regular real life clinical setup. Using a “gold standard” to make a COPD diagnosis on the referent level (tertiary care) as a reference standard and evaluation of lung function measurements conducted by GPs against spirometry conducted by experienced staff provides us with an objective evaluation of the tested device and GPs performance. The possible weaknesses of our study are based on recruitment process that may not represent the actual general practice population thus possibly diminishing generalizability of our results, although our analysis showed that there was no clustering present regarding the characteristics of patients recruited by different GPs and the proportion of undiagnosed COPD patients was comparable to other studies. Also the age range of our study participants (40–65 years of age) does not allow us to generalize our results outside this age range thus leaving the most questionable population regarding the diagnostic criteria out of our focus (>70 years of age). A bias could be present because there was no formal panel diagnosis of COPD done, but the diagnosis was done using the harmonized criteria by experienced pulmonologists . So for our data to become more generalizable our research needs to be conducted in a broader population  using also two threshold values that were found out in our exploratory analyses (FEV1/FEV6 < 0.7 and >0.85). The strengths of our study are based on the significant number of indicative COPD patients diagnosed through the diagnostic process  of undiagnosed patients with COPD in a population at risk (smokers/ex-smokers) in our study points to the fact that active case finding should be instituted in such a population. Based on the results of our study lung function testing with COPD-6™ can in a significant part substitute spirometry in cases where it is not readily available to the patient/physician. Results of lung function testing with COPD-6™ performed at GP’s offices and in lung function laboratory were comparable, so there is a possibility to establish the diagnosis of COPD and start adequate treatment in a GP’s office in a substantial number of patients at risk. This could be based on two cut-of values for FEV


Our results suggest that Arctic aerosols are derived from a combination of the long-range transport of terrestrial organics and local sea-to-air emission of marine organics, with an estimated contribution from the latter of 8.7–77% (mean 45%).Organic aerosols are ubiquitous in the earth’s atmosphere. They have been extensively studied in urban, rural and marine environments. However, little is known about the fluorescence properties of water-soluble organic carbon (WSOC) or their transport to and distribution in the polar regions. Here, we present evidence that fluorescent WSOC is a substantial component of High Arctic aerosols. The ratios of fluorescence intensity of protein-like peak to humic-like peak generally increased from dark winter to early summer, indicating an enhanced contribution of protein-like organics from the ocean to Arctic aerosols after the polar sunrise. Such a seasonal pattern is in agreement with an increase of stable carbon isotope ratios of total carbon δCTC from  Atmospheric aerosols have been recognized to play an important role in regulating regional and global climate4Water-soluble organic carbon (WSOC) is a major fraction of atmospheric aerosols. WSOC contains aromatic rings or aliphatic structures with carboxyl, hydroxyl, carbonyl or methoxy functional groups. The chemical nature of 10–20% (by mass) of WSOC has been resolved at a molecular level, and found to include mono- and di-carboxylic acids, sugar compounds, amino acids, and polar organic marker compounds (e.g. 2-methyltetrols and pinic acid) from the photooxidation of biogenic volatile organic compounds (BVOCs)567810Fluorescence techniques (e.g. synchronous scan and excitation-emission matrix spectroscopy) have been widely used to investigate the sources and optical properties of dissolved organic matter (DOM) or humic substances in aquatic environments121314161218111921222426The Arctic atmosphere was once considered as one of the most pristine environments on earth. However in the 1950s, two pilots flying over the North American Arctic noticed a widespread haze that was subsequently found to be observable every winter and early spring4δThe objective of this study was to investigate the EEM properties of WSOC in Arctic aerosols collected at Alert, in the Canadian High Arctic , in orde−3 (mean 253ng m−3) and 40.7–300ng m−3 (186ng m−3), respectively (−3 (5250ng m−3). OC and WSOC made up only a small fraction of the aerosol mass , suggesting that mineral sea salts dominate High Arctic aerosols. Their fractions were higher in February and late March, and lower in early summer. The decrease in the concentrations of OC and WSOC (29The concentrations of organic carbon (OC) and WSOC in the Alert aerosols ranged from 73.4–387ng mectively . The aerand WSOC  from Febδ2931δChesselet et al.δδStable carbon isotopic composition δC has beeIn order to understand the general transport characteristics of air masses recorded at the sampling site, we conducted a ten-day backward Lagrangian Particle Dispersion Modeling (LPDM) analysis using the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model35−3) but higher concentrations in dark winter. It is not surprising given that biomass-burning emissions are one of the most significant sources for atmospheric HULIS10The time series measurements of OC and WSOC  showed aThe winter maxima of OC, WSOC, and organic species can be explained by the concept of the Arctic acting as a cold sink during winter to receive long-range transported aerosols and their precursors transported from their emission regions in the mid-latitudes in Eurasia and North America. Under stagnant conditions with the lack of solar radiation in winter, aerosol removal rates are minimizedRousseau et al.Fluorescent aerosol particles may also contain primary biological aerosol particles, which are ubiquitous and comprise a variety of particle types such as bacteria, algae, pollen, fungal spores, plant debris and biopolymers 2425Fu et al.In the Earth’s atmosphere, SOA formed by the photooxidation of BVOCs is believed to be more abundant than directly emitted POA. Recent field and laboratory studies have demonstrated that non-biological particles such as biogenic SOA from the photooxidation of isoprene and monoterpenes can also emit a strong fluorescence26The temporal variations of 2-methyltetrols , the iso20:0) are a major fraction of marine aerosols , while HMW fatty acids (>C20:0) mainly originate from terrestrial higher plants. The concentration ratios of LMW to HMW fatty acids in High Arctic aerosols showed an increased trend from February to May  to humic-like fluorescence (peak A) in the WSOC fraction  from dary to May , rangingy to May ; the raty to May .It has been recognized that in the marine atmosphere, organic enrichment is strongly associated with decreasing size of particlesHawkins and RussellFacchini et al.In fact, ocean surface waters are enriched with small particulate materials including phytoplankton, algae, bacteria, viruses, fragments of larger organisms and organic detritusδNarukawa et al.δ3132δδ3 plants range from −24 to −37‰2931323352δfmarine and fcontinental are the fractions of marine and continental carbon, and δmarine and δcontinental are the reported isotopic values for marine and continental carbon. Based on the previous studies29δBased on ionic composition and stable carbon isotope ratios δC of Arct−3 (mean 133ngC m−3) and continental carbon concentration from 21.7 to 510ngC m−3 (210ngC m−3). The relative contribution of marine carbon clearly increased from winter to early summer  during the Alert2000 campaigny summer , indicatZsolnay et al.14152 = 0.60) between HIX and stable carbon isotope ratios was found in the Arctic samples  was collected weekly by filtering air through precombusted  quartz fiber filters using a high-volume sampler in the Canadian High Arctic at Station Alert 82.5°N, 62.3°W) from February 19 to June 10, 1991. The surface air temperature (mean) ranged from −33.5°C in February to −1.9°C in June during the sampling period. Each filter was stored in a precombusted glass bottle with a Teflon-lined screw cap in darkness at −20°C until analysis. Based on the reanalysis for dicarboxylic acids in 2010 of one aerosol filter sample (QFF 298) collected in mid-May during this campaign, and a comparison with the results obtained in 1994 for the same sample, 2.3°W froDing et al.We conducted backward particle release simulations using the Lagrangian dispersion model Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT), which was developed in the Air Resource Laboratory of the National Oceanic and Atmospheric Administration (NOAA), USA2) of filter samples were extracted by 20mL of organic-free Milli-Q water under ultrasonication for 20 min. The water extracts were then passed through a syringe filter  and analyzed for water-soluble organic carbon (WSOC) using a TOC analyzer .An aliquot (ca. 10cm2) of the filter samples were extracted by organic-free Milli-Q water under ultrasonication for 15 min. The water extracts were then passed through a syringe filter . Excitation-emission matrix (EEM) fluorescence was measured using a fluorometer . All fluorescence spectra were acquired in the S/R mode with instrumental bias correction. Inner filter correction−1). Based on the ranges of Coble−1 m−3. In addition, the humification index , index of recent autochthonous contribution  and fluorescence index (FI) (ratio of Ex/Em = 370/470nm to Ex/Em = 370/520nm) were calculated from each EEM14An aliquot (ca. 7cm2) of each quartz fiber filter sample were analyzed for total aerosol carbon content using an elemental analyzer (EA) . The filter punch was rounded using a pair of flat tipped tweezers, placed into a tin cup, and then caked into a ball. Before use, the tin cup was washed with acetone under ultrasonication to remove organic and other contaminants. Carbon was converted to CO2 in the combustion furnace (1020°C). Stable carbon isotopic analyses were conducted using the same EA interfaced  to isotope ratio mass spectrometer (IRMS) . The isotopic compositions (δ12C. Vienna Pee Dee Belemnite (13C) was used as the isotope standard. Samples were analyzed in duplicate and averaged concentrations and isotopic ratios are reported here after the blank correction. Reproducibility of TC measurement was within 2%. Analytical error in the carbon isotope ratios was within 0.2‰.Small punches (3.14cmsitions δC were de2) was cut into pieces and extracted three times with dichloromethane/methanol  under ultrasonication for 10 min. The solvent extracts were concentrated by a rotary evaporator, and then blown down to dryness with pure nitrogen gas. The extracts were then reacted with 50μL of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylsilyl chloride and 10μL of pyridine at 70°C for 3 h. After reaction, the derivatives were diluted using 140μL of n-hexane with 1.43ng μL−1 of the internal standard  prior to gas chromatography/mass spectrometry (GC/MS) injection. GC/MS analyses were performed on a Hewlett-Packard model 6890 GC coupled to Hewlett-Packard model 5973 mass-selective detector (MSD). The GC was equipped with a split/splitless injector and a DB-5MS fused silica capillary column . The GC oven temperature was programmed to increase from 50°C (2 min) to 120°C at 15°C min−1 and then to 300°C at 5°C min−1 with a final isotherm hold at 300°C for 16 min. Helium was used as the carrier gas at a flow rate of 1.0mL min−1. The sample was injected on a splitless mode with the injector temperature at 280°C. The mass spectrometer was operated on the electron impact (EI) mode at 70eV and scanned from 50 to 650 Da. Data were processed with the Chemstation software. Individual compounds were identified by comparison of mass spectra with those of authenticated standards or literature data. Field and laboratory blank filters were treated as the real samples for quality assurance. Target compounds reported here were not detected in the blanks. Relative standard deviations of the concentrations based on duplicate analysis were <10%.The dataset of molecular marker compounds and detailed measurement methods have been reported elsewhere5


Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRβ (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRβ did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRβ-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies. We showed that the effects of the thyroid hormones on skin proliferation are mediated through interactions with TRα and TRβ and that both receptor genes contribute to attain a normal proliferative response Most actions of the thyroid hormones are mediated by binding to nuclear receptors (TRs) that belong to the superfamily of nuclear receptors and act as ligand dependent transcription factors. TRs are encoded by two different genes, and TRα1 and TRβ are the main thyroid hormones binding isoforms With age mice lacking TRs show an obvious hair phenotype, with a less thick fur and occasional hair tail loss . NonetheTo examine the contribution of the TRα and TRβ receptors to hair growth, we first analyzed by immunohistochemistry their expression in the skin of Wt mice. As shown in To analyze the role of the ligand on hair growth, we also conducted experiments in hypothyroid mice. As shown in To further analyze the role of TRs in hair growth, we next conducted a chronic experiment in which animals were depilated once a week for 21 weeks . In Wt aWe next analyzed whether wound healing was also altered in the absence of TRs. With this aim, skin wounds were made in Wt and TR-deficient mice and the healing process was followed. A slower rate of healing was apparent in TR KO mice , and difAfter wounding, a rapid proliferation of the epithelial cells immediately next to the migrating epithelial tongue is observed. At 10 days after wounding, epidermal proliferation was significantly lower in the epidermis of TR-deficient mice, as reflected by the reduced number of BrdU-positive keratinocytes in the transitional compartment adjacent to the wound edge . In addiThe healing process involves both hyperproliferation of keratinocytes in the wound borders that feed the epithelial tongue, as well as their migration until wound closure is attained Both clinical and experimental data indicate that thyroid hormones have a stimulatory effect on hair growth Analysis of the phenotypes of mice in which TRα1 or TRβ have been individually inactivated Interestingly, a point mutation of the TRα gene with a proline to histidine substitution (P398H) is sufficient to affect hair growth. Mice with this mutation frequently presented delayed hair growth and, in some cases, the mice had no body hair for up to eight weeks of age The phenotype of mice devoid of TRs is not always identical to that found in hypothyroid mice, since the unliganded receptors can act as repressors of gene expression in vitro their migratory behavior using scratch wound closure assays in primary cultures and keratinocyte migration assays from skin explants. The results obtained demonstrated that the delayed re-epithelialization observed in TR KO mice was not secondary to a decreased keratinocyte migratory capacity, since they rather exhibited increased migration in vitro. Because the re-epithelialization rate is determined by both proliferation and migration of keratinocytes, these data suggest that retarded re-epithelialization in the absence of TRs is due to a decrease in keratinocyte proliferation.Our data also revealed that cutaneous wounds heal more slowly and show reduced collagen deposition in animals lacking TRs. This is in agreement with the finding that thyroid hormone administration accelerates wound healing in mice in vitro migratory capacity of the TR KO keratinocytes, as well as their reduced proliferation obtained in primary cultures The increased Hair follicle stem cells are responsible for maintaining hair cycling Regulation of gene expression by nuclear receptors plays an important role on the skin. The important role of the glucocorticoid (GR), androgen (AR), and estrogen (ER) receptors is well known, but non-steroid receptors, which heterodimerize with the retinoid X receptors (RXR) are also involved in skin proliferation, hair growth and wound healing All animal work was done in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/2005), with approval of the Ethics Committee of the Consejo Superior de Investigaciones Científicas (CSIC) were approved by the Subcommittee on Bioethics of the Consejo Superior de Investigaciones Científicas with the approval reference number BFU2011-28058. All efforts were made to minimize suffering. Anesthetics were used as indicated.−/−/TRβ−/− double knockout (KO) mice and the corresponding wild-type TRα+/+/TRβ+/+ (Wt) animals have been described previously −/−/TRβ−/−(KO), TRα1−/−TRβ+/+ (KOα1), TRα1+/+TRβ−/−  as described vs. the total number of follicles was determined from at least 10 slides/animal of each experimental group . In serial depilation experiments, 4 mice/genotype were depilated once a week during 21 weeks. In order to label proliferating cells in hair follicles, 1 h prior sacrifice mice were injected ip with bromodeoxyuridine .Animals were housed in temperature (22±2°C) and light  controlled conditions and had free access to food and water. TRα1t = 0 h) and every 24 h to calculate distance. Magnification 100×.Primary keratinocytes were isolated from newborn KO and wild-type mice as previously described 2 concentration to avoid fibroblast growth. Explants were cultured with medium alone or in the presence of epidermal growth factor . To assess keratinocyte outgrowth, phase-contrast micrographs of explants were taken at day 6. Micrographs were scanned and outgrowth was quantitated counting the number and size of keratinocyte foci. Foci were classified into three groups: containing less than 50 cells, containing between 50 and 100 cells and containing more than 100 cells. The vast majority of cells were keratinocytes, as identified by immunodetection with K5.Skin explants were prepared from 2-day-old pups as described previously Mice were injected intraperitoneally with a mixture of 20 mg/kg ketamine and 100 µg/kg of medetomidine. Once the mice were adequately anesthetized, four 4 mm wounds were punched in the depilated dorsal skin. Animals (3–4/group) were sacrificed 2 and 10 days after wounding. Biopsies containing the wounded tissue and the surrounding skin were harvested, fixed with paraformaldehyde and embedded in paraffin. H&E staining was performed on 4 µm serial sections, measuring every 10 sections the distance between the unwounded dermis. The percentage of re-epithelialization was calculated measuring from the wound center the distance between the unwounded dermis, considering this length as the initial wound. The length of the epithelial tongues migrating from the unwounded skin was also measured, and this value was considered as the re-epithelialized surface.2/animal of each experimental group. The mean percentage of proliferating over total follicular cells was then estimated. Immunofluorescence was carried out on deparaffinized sections. TRα (600-401-A38 Rockland), TRβ (Affinity BioReagents ABR PA1-213A) and keratin 14  antibodies were used at a 1/100 dilution. Slides were incubated with the antibodies at 4°C overnight and then with anti-rabbit-TexasRed  or anti-mouse FITC  secondary antibodies at room temperature for 1 h. Sections were examined for fluorescence in a Zeiss Axioplan2 microscopy.Skin sections (4 µm) were stained with H&E or processed for immunohistochemistry or immunofluorescence. For frozen sections (5 µm), fresh skin samples were embedded in OCT (TissueTech) and kept frozen. Picrosirius red staining was performed for detection of collagen fibers t test when only two groups were compared. In the figures differences between wild-type and TR KO mice is indicated as *p<0,05; **p<0,01 and *** p<0,001.Differences between KO and wild-type animals were assessed by ANOVA followed by the Bonferroni correction, or with the


Rhizophora mucronata (Lam.) forest were first girdled and then cut. Another set of five plots at the same site served as controls. Treatment induced significant, rapid subsidence (−32.1±8.4 mm yr−1 compared with surface elevation changes of +4.2±1.4 mm yr−1 in controls). Subsidence in treated plots was likely due to collapse and decomposition of dying roots and sediment compaction as evidenced from increased sediment bulk density. Sediment effluxes of CO2 and CH4 increased significantly, especially their heterotrophic component, suggesting enhanced organic matter decomposition. Estimates of total excess fluxes from treated compared with control plots were 25.3±7.4 tCO2 ha−1 yr−1 (using surface carbon efflux) and 35.6±76.9 tCO2 ha−1 yr−1 (using surface elevation losses and sediment properties). Whilst such losses might not be permanent (provided cut areas recover), observed rapid subsidence and enhanced decomposition of soil sediment organic matter caused by small-scale harvesting offers important lessons for mangrove management. In particular mangrove managers need to carefully consider the trade-offs between extracting mangrove wood and losing other mangrove services, particularly shoreline stabilization, coastal protection and carbon storage.The importance of mangrove forests in carbon sequestration and coastal protection has been widely acknowledged. Large-scale damage of these forests, caused by hurricanes or clear felling, can enhance vulnerability to erosion, subsidence and rapid carbon losses. However, it is unclear how small-scale logging might impact on mangrove functions and services. We experimentally investigated the impact of small-scale tree removal on surface elevation and carbon dynamics in a mangrove forest at Gazi bay, Kenya. The trees in five plots of a  Trends in mangrove loss are alarming, with an estimated 30–50% of forests lost over the past half century Few studies have directly measured the impacts of tree removal on below-ground carbon storage and surface elevation. Two studies report on the effects of hurricanes Mangrove forests of the WIO region are utilized by the local communities for construction and fuelwood 2 emitted from the sediment shortly after girdling is assumed to be primarily from organic matter decomposition e.g., We used a controlled experiment to explore the impacts of tree harvesting on: a) surface elevation dynamics; b) the main processes affecting changes in surface elevation i.e., sediment accretion, sediment properties such as bulk density, %C and sediment moisture, and organic matter decomposition; and c) sediment surface C efflux in a natural mangrove forest, here at Gazi bay, Kenya. The C flux from forest floors comes from root (autotrophic) respiration and sediment/soil organic matter decomposition (heterotrophic respiration) −1, while the treatment plots showed a subsidence of −51.3±12.0 mm, at a mean rate of −32.1±8.4 mm yr−1  for final sample values was calculated by applying the equation above, while setting the value of root biomass to zero A), obtained by subtracting the values for RH from the measured total sediment respiration (RS), contributed a mean (±95% CI) of 41.5±11.8% to RS at the final sampling. Across the entire sampling period, RA contributed an average of 40.5±7.0% to RS, which was not significantly different from that obtained at the final sampling . The partitioning of the components of total RS in the control plots allowed a comparison of the treated plot CO2 fluxes (where autotrophic R was zero by definition) and control plot RH. Sediment respiration of the treated plots was higher than control plots RH throughout the treatment period (during both girdling and cutting) by 0.6 to 3.7 g CO2–C m−2 d−1  over a period of 530 days after treatment. When only RH in control plots was considered, the net additional C loss amounted to 36.7±10.7 t CO2 ha−1, with a mean rate of 25.3±7.4 t CO2 ha−1 yr−1.The net belowground carbon losses due to treatment estimated from sediment surface flux data amounted to 14.2±10.3 tCO−1, respectively . The trend in decomposition rate in the treated plots indicated that it was increasing with time, while the rate in the control plots remained constant between 156 and 267 days.After 267 days root-bags in the treated plots had lost significantly more mass than those in the controls, with rates of 0.19±0.02 and 0.16±0.03% dry weight loss day13C signature to that of the sediment carbon  of 524.1±88.8 vs. 488.4±68.6 tC ha−1, respectively, but the difference was not significant due to the very large small-scale variability in sediment properties.The plant roots had a similar δt carbon . Treated−1Tree death induced rapid and significant subsidence. This was likely due to rapid decomposition of fine roots exacerbated by the absence of new root growth, as shown by the significant difference in root bag decomposition. In addition, sediment compaction and the collapse of arenchymatic tissues due to consolidation of air spaces (as shown by the increase in bulk density), and the significant loss of sediment moisture caused by the treatment, may also have contributed. Rates of sediment accretion did not differ significantly between control and treated plots hence the subsidence was not due to enhanced erosion. The rate of subsidence was surprisingly high given the relatively small scale of the treatments; massive hurricane damage, which led to ‘peat-collapse’ in Honduran mangroves, caused elevation losses only around ∼0.3 as fast as those recorded here  2 ha−1 yr−1) 2 ha−1 yr-1 during the girdled and cut periodsrespectively) and by ∼1.5 years the C losses induced by treatment began to drop. A similar pattern was recorded in clear-cut mangroves in Belize in which the C emissions in disturbed areas declined with time -1, respectively) with most of the difference occurring after the first set of root bags were retrieved . This was probably due to enhanced sediment surface temperatures in the cut plots due to canopy removal, since the treated plots experienced increases in sediment surface temperatures of 0.8 to 5.9°C compared to the control plots. Therefore, these results highlight the potential impact of physico-chemical changes on C losses in cut forests, which are separate from and additional to the losses from root death per se.Interestingly, the C loss of around 25.3±7.4 t CO2 cannot account for below-ground, lateral flows of carbon in dissolved inorganic carbon (DIC) Although our estimates suggest high rates of below-ground C loss caused by tree death (similar to those seen following much larger impacts such as hurricanes) they are likely to represent an underestimate, since surface fluxes of CO4 emissions were significantly enhanced during the girdled period, possibly because of the addition of easily fermentable substrates −2 d−1), did not exceed those reported for a number of pristine mangrove forests worldwide, 0.01–5.0 mmol m−2 d−12 collected shortly after girdling showed significant 13C-depletion . Study of lightning damage in Florida mangroves, creating forest gaps larger than our experimental plots, showed even more subsidence (of up to 61 mm), but also suggests that undisturbed forests can recover Kenyan mangroves are being lost at a rate of 0.7% cover per year A new approach towards the sustainable management of mangrove forests uses payments for ecosystem services (PES) schemes such as the proposed REDD+ programme o 25′ S and 4o 27′ S; 39o 50′ E and 39o 50′ E), ∼55 km south of Mombasa, Kenya. Gazi Bay is a creek system with a total area of 615 ha mangrove forest Rhizophora mucronata (Lam), Ceriops tagal (Perr.) C. B. Robinson and Avicennia marina (Forsk.) Vierh. The mean annual precipitation of Gazi bay ranges from 1000–1600 mm The study was carried out at Gazi Bay (4Rhizophora mucronata dominated forest (specific location: 4°24′32″S 39°31′23″E). In October 2009, five plots (one from each pair) were randomly allocated to ‘treatment’ and all the trees within them were girdled at ∼20 cm above the highest prop root. Girdling is a method that has been used in a number of terrestrial forests to estimate the contribution of root respiration to total sediment gas flux. The rationale is to prevent the flow of carbohydrates from the tree canopy to their roots (thus stopping root respiration) whilst leaving the above-ground components relatively undisturbed; trees may retain foliage for many months after girdling Five pairs of 12 m×12 m plots were established in March 2009 at high water mark (height above mean sea-level: 2.91±0.01 m) within a Surface elevation dynamics were monitored using surface elevation stations, consisting of two stainless steel rods (6 mm by 1 m) and a horizon marker (kaolin) set up in a 20 cm×20 cm quadrat in each plot. The rods were installed leaving a height of 20 cm above the ground and at opposite corners of the quadrat such that measurements would be made diagonally across it. Height measurements vertically from the ground surface to the heights of the rods were made at seven clearly marked points along a wooden board placed across the rods. Subsequent measurements were made at the same points along the wooden board. All data were averaged to give a single measurement per plot per time. Sediment accretion was determined from measurements of height above the horizon marker. In each quadrat, at least four sediment blocks of 2 cm×2 cm were carefully removed with a sharp knife, the height of sediment above the horizon marker was noted and the block was then carefully replaced in its original position. This approach allows the separation of total surface elevation/subsidence (which depends on both the accretion or erosion of new sediments and on below-ground processes such as root growth and expansion) from accretion/erosion 13C of OC. The concentrations of OC, total N, and δ13C values of sediment OC were measured on subsamples weighed into Ag cups, acifidied with dilute HCl to remove inorganic C, and analysed with a Thermo Flash HT elemental analyser coupled to a Thermo Delta V Advantage IRMS (Conflo IV interface). Data were calibrated with IAEA-C6, and internally calibrated acetanilide and leucine. From the sediment bulk density and OC content, the carbon density and hence the sediment carbon stocks down to 100 cm were derived for the control and treated plots.Samples for sediment physico-chemical analysis were taken from each plot during February 2010 and August 2012. A sediment core was taken in the centre of the plot using a plastic corer  in February 2010 and again in August 2012. Subsamples of this large core were taken with a small stainless steel corer  at depths of 0, 2, 4, 8, 10, 20, 30 and 40 cm (February 2010) and depths of 0, 10, 20, 30, 50 and 100 cm (August 2012). To minimize compression of the sediment the coring was done in a series of stages according to the depth profiles for sub-sampling. Sediment samples were oven-dried at 80°C to constant dry weight and bulk density was determined at six depths down to 1 m. The oven-dried samples were transferred to the Department of Earth and Environmental Sciences, KU Leuven, Belgium for analysis of % OC (organic carbon), % N and δ2, CH4) samples were collected at approximately monthly intervals at low tide during spring tides using six chambers per plot; some sampling times that were missed due to loss or damage to bags and other equipment. Each chamber was inserted ∼5 cm in to the sediment, occupying an area of 0.064 m2 with an internal volume of 0.011 m3. The samples from each chamber were taken 20 minutes after closure. Using a 60 ml syringe, at least 240 ml of gas were transferred from each chamber to labelled airtight gas-bags . A gas sample of ambient concentration was taken from each chamber before closure; ambient air concentration samples for each plot were collected in one gas bag. Linearity checks were performed by repeatedly sampling the chamber gas for periods of about 60 minutes. They showed that a linear approximation over a 20 minutes period resulted in ∼15% underestimation of the slope of gas concentration increase over time. This systematic downward bias was not corrected for. Sediment surface temperature measurements were made beside each chamber with a temperature probe inserted to ∼1 cm in to the sediment. The number of crab burrows within the area enclosed by the chamber was noted. The positions of the chambers were marked for subsequent sampling; chambers were always returned to the same sampling positions within plots. Samples for δ13C analysis of CO2 were transferred from the chambers to 12 ml pre-evacuated exetainers . Gas flux samples were analysed at the Institute of Atmospheric and Environmental Sciences, University of Edinburgh, UK. For CO2, the samples were analysed by gas chromatography (GC) using a Perkin Elmer Model 310 with a thermal conductivity detector (TCD). Concentrations of CH4 were measured using GC  equipped with a flame ionisation detector (FID) and a digital integrator. The δ13C analysis samples were transferred to the Department of Earth and Environmental Sciences, KU Leuven, Belgium and analysed using a Sercon 20–20 isotope ratio mass spectrometer (IRMS) interfaced with a cryofocussing unit.Gas flux  bags each containing ∼30 g fresh roots were buried to ∼20 cm depth at six random points within each plot. Three bags were retrieved from each plot 156 days after burial, whilst the other three were retrieved 267 days after burial. The contents of each root-bag were rinsed and oven-dried at 80°C for 24 h before weighing. The rate of root decay (% weight loss day−1) was calculated as the % weight loss divided by the number of days buried, using wet-dry weight conversion factors derived from representative samples of live roots, oven-dried at 80°C until constant dry weight.In September 2010, live roots were excavated within the 2 were log-transformed and the analysis for each gas was executed using MINITAB 14 software package. Initial analyses included time in repeated measures models, however there were multiple significant interactions preventing legitimate conclusions and so data were separated into the three experimental periods. The gas flux values for each chamber were pooled across each period for the controls and treated plots, giving six single values per period for each plot. For each period, nested ANOVA was carried out for each gas, with variation among plots (i.e. 6 chambers) nested within treatment and sediment surface temperature and crab burrows as covariates. Estimates for respired δ13C were derived from the Miller-Tans mixing model combined with geometric regression 13CS (S =  sediment respired CO2). The gas mixing models are based on the conservation of mass given as The data for COet al., 13C of the respired CO2 for each plot was analysed using two-sample t-tests.This equation describes the gas observed (obs) as coming from two sources: background atmosphere (bg) and source of respiration (s), where δ refers to the isotopic value of each component. Details of the Miller-Tans mixing model combined with geometric regression are discussed by Kayler 2 fluxes measured in each control chamber in April 2011 as the dependent variable and the live root biomass, sediment surface temperature and crab burrows for each chamber as the independent variables. The equation takes the form of y = k+a*roots +b*temperature + c*burrows; where k =  constant, a, b and c are the coefficients of the estimators. The number of crab burrows was not significant, and hence this term was omitted from the equation. The significant factors were used in estimating the autotrophic respiration from the final CO2 flux data. First, the heterotrophic respiration (RH) was calculated as the value of the ‘y’ when live root biomass  = 0, i.e. H = k+ b*temperatureR. Then the autotrophic respiration (RA) was obtained as the difference between total sediment respiration (RS) and the heterotrophic component (RH) and expressed as a percentage of RS (i.e. %RA =  (RS–RH)/RH*100). To estimate the contribution of RA across the entire sampling period, the equation was applied to the CO2 flux data, together with the sediment surface temperature for each control chamber at each sampling time. The mean RA contribution across the sampling period was then compared with that obtained from the final sampling time.To examine the autotrophic contribution to sediment fluxes, stepwise multiple regressions (forward and backward elimination) were performed, with the final COe =  treatment induced C emissions, n =  number of measurements, m =  individual measurements and t =  time difference between any two consecutive measurements.The total additional C emissions due to treatment (treatment-induced C emissions) were estimated as the area under the response curve based on the trapezoidal rule −3) was calculated as the product of bulk density (BD) and % organic C of the sediment. Thereafter, the sediment carbon stocks down to a depth of 100 cm for each treatment were calculated as the product of carbon concentration and the depth and expressed as t C ha−1: CS = CC * (100+Ec), where, CS =  sediment C stocks, CC =  C concentration and Ec =  elevation change. Since the control plots gained 1.1 cm and the treated plots lost 5.1 cm in surface elevation (seec = 101.1 and 94.9 cm for control and treated plots, respectively.The difference in the rates of root decomposition in the control and treated plots was tested using a two-sample t-test. Sediment carbon concentration (g C cmtion see, the dep


In normal or nonmalignant cells, TGF-β inhibits cellular proliferation through activation of the SMAD-dependent canonical signaling pathway. Recent findings demonstrate that the protein TMEPAI1 can block the cytostatic effects of the canonical TGF-β signaling pathway, while activating cellular proliferation through the noncanonical, SMAD-independent TGF-β signaling pathway. As TMEPAI1 shows increased expression in the poor prognosis basal and HER2 intrinsic subtypes of breast cancer, these findings point to a new avenue of targeted therapy with considerable therapeutic potential. TGF-β is a pleiotropic cytokine that has been found to modulate the function of virtually every cell type. While important developmental and normal homeostatic processes are dependent upon TGF-β, it has been primarily studied for its role in cancer development and progression, where it has been found to function both as a tumor suppressor and a tumor promoter . In normet al. [In 2010, a pair of publications defined key mechanisms by which the protein TMEPAI1  could act a potent modulator of the TGF-β signaling pathway , 7. TMEPet al.  extends et al. [Singha et al.  employedet al. . Using sThese studies suggest that inhibition of TMEPAI may be a useful therapeutic approach for TNBC patients, although methods to accomplish this are unclear. It may be that identification and development of novel reagents to disrupt the interaction of TMEPAI with SMADs could be used in combination with PI3K- or Akt-targeting therapeutics currently in clinical trials to reactivate the cytostatic capability of TGF-β. It may be that other breast cancer subtypes could benefit from this approach as well. Interrogating a large meta-analysis of published breast cancer microarray datasets  in which


C. elegans. We have exploited this model of Aβ toxicity by carrying out an RNAi screen to identify genes whose reduced expression modifies the severity of this locomotor phenotype. Our initial finding was that none of the human orthologues of these worm genes is identical with the genome-wide significant GWAS genes reported to date (the “white zone”); moreover there was no identity between worm screen hits and the longer list of GWAS genes which included those with borderline levels of significance (the “grey zone”). This indicates that Aβ toxicity should not be considered as equivalent to sporadic AD. To increase the sensitivity of our analysis, we then considered the physical interactors (+1 interactome) of the products of the genes in both the worm and the white+grey zone lists. When we consider these worm and GWAS gene lists we find that 4 of the 60 worm genes have a +1 interactome overlap that is larger than expected by chance. Two of these genes form a chaperonin complex, the third is closely associated with this complex and the fourth gene codes for actin, the major substrate of the same chaperonin.The human Aβ peptide causes progressive paralysis when expressed in the muscles of the nematode worm,  APP) APP by β-secretase and then γ-secretase results in the generation of a spectrum of peptides, known as amyloid β (Aβ), that are the major component of senile plaques in the brains of patients with AD. The γ-secretase is of particular interest because its cleavage of APP determines which amino acids constitute the C-terminus of Aβ. Normally, Aβ peptides are predominantly 40 amino acids in length (Aβ40), while 5–10% have a further 2 amino acids at the C-terminus (Aβ42). The bulk of cases of FAD can be accounted for by mutations that either increase the total amount of Aβ APP gene, or the genes PSEN1 or PSEN2 encoding catalytic subunits of γ-secretase Once age is taken into account, the most powerful determinants of risk for Alzheimer’s disease (AD) are genetic; indeed, the heritability of the disorder is estimated to be 58–79% −7 as an empirical cut off for genome-wide significance (the GWAS “white zone”), the first generation of multicentre AD GWAS apoE, clusterin, PICALM, and CR1) associated with disease risk. With the publication of the second generation of GWAS findings, the total number of risk genes is now twenty one The advent of genome-wide association studies (GWAS) has made the investigation of multigenic disorders, such as sporadic AD, experimentally tractable. Using p<10APP, PSEN1, or PSEN2 from the list of candidates identified by GWAS is that the studies were lacking sufficient power to detect small or rare contributions to risk −5. This larger list of genes will include false positives, that can safely be ignored, but equally there will be genes that carry risk and are involved in the pathogenesis of sporadic AD. Our approach has been to probe this larger group of GWAS white+grey zone genes using the results of a genome-scale screen in a model system that reports exclusively on the toxicity of the Aβ peptide. We postulate that if Aβ toxicity were distinct from sporadic AD then we would expect to see an overlap between the worm and human genes lists that is consistent with chance. However were the GWAS data reporting a role for Aβ in clinical AD we would expect to see a significantly increased overlap in the network of genes identified in the human and worm screens.One explanation for the absence of genes such as C.elegans and we used a subset of the Ahringer C. elegans RNAi library We identified 7970 human protein-encoding genes that have orthologues in −11 , indicating that the hits in the worm screen are much more likely to have human orthologues than one would expect by chance.In our initial analysis, we generated a list of the human orthologues of the worm modifier genes using EnsemblCompara Using worm ontology enrichment analysis (clueGO plugin for Cytoscape) we found that a number of labels were over-represented in our screen hits, implicating genes involved in ATP synthesis, purine metabolism, protein binding and chaperone activity, and components of the translational machinery .When we compared this list of 61 human orthologues of the worm modifier genes with the 63 genes in the GWAS white+grey zone  we foundIn order to determine which, if any, of the worm screen hits interacted with the GWAS list to a statistically significant degree we supplemented the candidate gene lists with genes encoding the first-degree physical interactors (+1 interactome) of the protein products of the primary members using the BioGrid database (v. 3.2.96). We then asked, for each member of the worm-screen hit list, whether their +1 interactome overlap with GWAS was greater, or less, that one would expect by chance . Of the It was immediately apparent from the relative sizes of the +1 interactomes  & 2 thatIn order to determine whether the observed bias towards highly interconnected genes in the worm modifier list was statistically significant, we used a computational approach to compare our observed population of genes to similar, randomly generated, lists. Firstly we ranked the population of human genes in the Biogrid database (13940 genes) according to their number of interactors. This ranked list was then divided into 10 bins such that each bin contained the same number of human orthologues of worm genes (that is 797 in each bin). For example, this resulted in the most highly interconnected 952 human genes in the database being put into the first bin; the bin boundaries for the whole database were: 1–952, 953–2006, 2007–3175, 3176–4403, 4404–5735, 5736–7134, 7135–8624, 8624–10200, 10201–12104 and 12105–13928. Using a Monte-Carlo approach As expected the genes in the random lists segregated with equal frequency into the various bins  of the overlap between the +1 interactome of each worm screen hit and the GWAS +1 interactome was achieved by generating 1000 random worm +1 interactomes of equivalent size for each experimental worm gene. We then counted how often the overlap of the random worm +1 interactomes with the GWAS +1 interactome was either> = or< = to the experimentally observed overlap. To quantify the statistical significance of our findings, we repeated this process for 100 random lists of 60 worm genes.Overall, we found that, of the 100 random lists of 60 worm genes, 3.14  genes had +1 interactome overlaps with GWAS that differed significantly from expected at a level of p<0.05. As such, this number is consistent with the random nature of the simulated gene lists. However when we examined the experimental worm screen list, we found that 7 genes had a non-random +1 interactome overlap with the GWAS network. When we reviewed our simulations we found that out of 100 runs only 5 resulted in 7 or more genes that had a +1 interactome overlap that differed from chance, yielding p = 0.05 for our observation.When we considered the overlap between the 7 non-random worm genes we found that 3  and all were significantly different from the expected zero value.One possible explanation for the highly interconnected nature of the worm screen hits could be summarised as “sociological bias” Another explanation is that genetic modifiers of worm phenotypes really do have more highly interconnected protein products as compared to the average gene For each gene in the worm-screen hit list we estimated whether its interactome overlapped with the GWAS interactome to a greater or lesser extent than would be expected by chance. We employed a Monte Carlo simulation approach where we generated 1000 random interactomes that were the same size as the experimental interactome  and deteThree of these genes  have +1 interactome overlaps with GWAS white+grey zone genes that are smaller than one would expect by chance, suggesting that they are artifactual modifiers of the worm phenotype and unlikely to be involved in the human disorder. Consequently, these data should dissuade us from investigating the ribosomal The details of the role for TRiC/CCT in AD are unknown; however the finding that RNAi knockdown in the worm protects against Aβ-induced paralysis indicates that the chaperone in some way mediates Aβ toxicity. TRiC has been shown to remodel oligomeric protein aggregates in a worm model of polyQ toxicity, with a concomitant reduction in toxicity C. elegans indicates that AD cannot simply be considered synonymous with Aβ toxicity. However using a +1 interactome network approach we have shown that the experimental pattern of overlap between worm and GWAS gene products was significantly non-random. Of those worm genes that showed increased overlap with GWAS lists, three are part of an abundant cytoplasmic complex with important chaperone activity; the fourth gene coded for β-actin that is an important substrate of the same chaperone. The interaction that the TRiC/CCT chaperone has with tubulin, STK24 and the MAPK pathway may explain how Aβ promotes tau-linked cytoplasmic pathology.In summary, the lack of a direct overlap between genes implicated by the GWAS studies and the hits from our modifier screen in C. elegans strain CL4176 [smg-1(cc546ts) dvIs27(pAF29 Pmyo-3::Aβ42+pRF4)], expressing Aβ in body-wall muscle, and the non-Aβ expressing control, CL802 [smg-1(cc546ts) rol-6(su1006)] have been described previously The E. coli carrying RNAi expression plasmids from the whole-genome C. elegans feeding library  were cultured separately, in 500 µl LB medium containing 50 µg/ml ampicillin, overnight at 37°C. A control culture, carrying empty plasmids without an RNAi insert, was grown simultaneously under identical conditions. Bacteria were seeded drop-wise onto solid NGM plates, supplemented with 25 µg/ml carbencillin and 1 mM IPTG, and incubated overnight at room temperature. Worm embryos were harvested from synchronised worms as described previously Clones of HT115 unc-22 that normally results in uncoordinated body wall twitching. As a quality control, any plate in which the unc-22 RNAi-fed worms failed to twitch was discarded and the experiment was repeated.The interaction of each RNAi-expressing clone with the Aβ paralysis phenotype in the worm was assessed in triplicate and compared with triplicate wells of worms fed with control bacteria carrying an empty vector. Each plate also had a well containing bacteria expressing RNAi to Locomotor behaviour was assessed at two time-points following the increase in ambient temperature. The first assay was performed 22 h post-induction, at which time Aβ-expressing worms fed control bacterial cultures were all moving normally. When all worms in each of the triplicate wells for a particular RNAi clone at this time point were seen to be paralysed, then the clone was defined as an enhancer of Aβ toxicity. This enhancement was specific to Aβ-expressing worms as no similar paralysis was seen in the corresponding treatment of non-Aβ-expressing CL802 worms. Clones that non-specifically paralyse worms were not considered. The second time-point, at 32 hr, permitted the detection of suppressors of the paralysis phenotype. At this time-point, Aβ-expressing worms under control conditions were all paralysed and so any RNAi-expressing clone that protected the locomotor function of all the worms, in triplicate wells, was defined as a suppressor of Aβ toxicity.The secondary screen consisted of a further three experiments, each performed in triplicate wells, for each hit from the primary screen. Paralysis curves were generated by hourly observation of locomotor behaviour and were analysed using Kaplan-Meier plots with statistical significance being estimated using the Mantel-Cox Log Rank test ; p<0.05 was considered significant.C.elegans that have one or more human orthologues. We used the EnsemblCompara phylogenetically based orthology prediction method (http://www.ensembl.org/) http://orthology.phylomedb.org/) According to our pre-planned study design, we considered only those genes in http://www.ncbi.nlm.nih.gov/projects/gapplusprev/sgap_plus.htm, p<10−5, Disease = “Alzheimer Disease”, access date June 2013).Using this method, human orthologues for 61 out of 78 worm genes were found. The human orthologues of this series of worm genes was then compared with the GWAS white+grey zone genes .1 as pubhttp://thebiogrid.org, v. 3.2.96) The “GWAS +1 interactome” consists of all the GWAS white+grey zone genes plus all the genes encoding proteins with which their products physically interact. The “worm +1 interactome” was generated in a similar manner, using 61 human orthologues of the worm modifier screen “hits”. The list of interactions was retrieved from the “Biogrid” database .Steps 2–3 were repeated 1000 times, yielding a Monte Carlo set of 1000 overlap scores.right,g  to give the p-value for the overlap  induces Aβ expression and results in progressive paralysis in worms fed on E. coli containing empty vector . By contrast, a typical suppressor clone rescues this locomotor deficit (square symbols). The paralysed worms  are straighter and largely immobile whereas non-paralysed worms exhibit a marked “roller” phenotype , but otherwise move normally.(TIF)Click here for additional data file.Figure S2Computational pipeline for determining the significance of the overlap between the +1 interactomes of worm-screen hits and GWAS white+grey zone genes. The algorithm is described more fully in the “Monte Carlo simulations” section of the methods section.(TIF)Click here for additional data file.Figure S3Generation of equivalent random +1 interactomes. An illustration of how random +1 interactomes (right) were generated to compare with the +1 interactomes of each of the worm screen hits. Random +1 interactomes were generated containing the same number of genes . Human orthologues of worm-screen hits are labeled with “1” (left). Random human genes with a worm orthologue are labeled with “2” (right). Human +1 interactors are represented by small green dots.(TIF)Click here for additional data file.Figure S4Estimating the significance of highly overlapping/poorly overlapping +1 interactomes. The +1 interactome of the human orthologues of worm screen genes may overlap more than expected  or less than expected . Generation of 1000 random +1 interactomes allows the p value for the experimental +1 interactome to be derived. Further details can be found in the “Monte Carlo simulations” section of the methods section.(TIF)Click here for additional data file.Figure S5Venn diagram indicating the degree of the +1 interactome overlap for GWAS white+grey genes and each of the significant genes from the worm screen. Seven genes from the worm screen have human orthologues that have +1 interactomes that overlap more or less than one would expect by chance with the GWAS +1 interactome. Four overlap more (left panel), and three less (right panel), than expected. The area of each circle or overlap is proportional to the number of genes. Worm screen genes that have human orthologues that interact directly with GWAS white+grey zone gene products are marked with asterisks.(TIF)Click here for additional data file.Table S1The 63 human genes in the AD GWAS white+grey zones.(DOCX)Click here for additional data file.Table S2Worm RNAi modifiers for screens of various disease models. The genes that modified fly phenotypes when targeted with RNAi are listed for a) polyglutamine, b) tau and c) α-synuclein.(DOCX)Click here for additional data file.


While these individual phenomena have been investigated in detail, it remains unclear how they are related to each other and to what extent uranium f-electron valence fluctuations influence each one. Here we use ligand site substituted URu2Six2-Px to establish their evolution under electronic tuning. We find that while hidden order is monotonically suppressed and destroyed for x≤0.035, the superconducting strength evolves non-monotonically with a maximum near x≈0.01 and that superconductivity is destroyed near x≈0.028. This behavior reveals that hidden order depends strongly on tuning outside of the U f-electron shells. It also suggests that while hidden order provides an environment for superconductivity and anomalous metallic behavior, it's fluctuations may not be solely responsible for their progression.The heavy fermion intermetallic compound URu The heavy fermion compound URu2Si2 displays a hidden order phase and superconductivity at low temperatures. Here, the authors perform substitution studies—partially replacing silicon with phosphorus—and study the effects on hidden order and superconductivity. These studies follow high resolution X-ray diffraction2Si2 differs from most other unconventional superconductors, which are typically found near the zero temperature termination point of a line of phase transitions, a ‘quantum critical point,' where strong fluctuations are believed to be favourable for the superconducting pairing1819202Si2 is fully contained inside the ordered phase, as revealed by numerous tuning studies2223242526272829303132Materials that defy straightforward description in terms of either localized or itinerant electron behavior are a longstanding challenge to understanding novel electronic matter1232Six2−Px, where ligand site substitution is a ‘gentle' way to tune the electronic state. Recent advances developing a molten metal flux growth technique to produce high quality single crystal specimens of URu2Si2 enabled these experiments, which were previously inaccessible due to metallurgical challenges associated with the high-vapor pressure of phosphorusx≤0.035, the superconducting strength evolves non-monotonically with a maximum near x≈0.01 and that superconductivity disappears near x≈0.028. While the rapid suppression of hidden order indicates the importance of itinerant electrons, the unexpected maximum in superconducting strength may suggest the presence of a critical point that is defined by the termination of a phase boundary other than that of the hidden order. Owing to the relatively small chemical difference between silicon and phosphorus, we propose that in this series the dominant tuning effect is simply to change the chemical potential. Extrinsic factors such as disorder, as well as some intrinsic factors including changes in the unit cell volume or bond angles, spin orbit coupling and ligand hybridization strength play a minor role. Phosphorus substitution further has the advantage that it does not directly affect either the local electron count or the balance of the spin–orbit and Coulomb interactions of the d- or f-electrons on the Ru and U sites, making it an ideal and long desired tool for unraveling the physics of URu2Si2.To explore the mechanisms of hidden order, anomalous metallic behavior and superconductivity in this material we synthesized high purity single crystal specimens of chemically substituted URuR and heat capacity C versus temperature T for several phosphorus concentrations x (see χ(T)). The Kondo lattice behavior of the electrical resistance  for T≥T0 is unaffected by phosphorus substitution for x≤0.035  where correlated electron antiferromagnetism appears (See x URu2Six2-Px substitution series will be reported separately). Unlike for other tuning strategies2223242526272829303132In  x≤0.035 , suggesteasing x . There iears See   for the specimens reported here is RRR=10  (and the transition width), which evolve though a maximum (and a minimum) between 0.006 and 0.01, respectively  ,  evolves non-monotonically through a maximum value of 1.2 at x=0.01  , which gng x see . Further/γTc(x),  which, ft x=0.01 , where tHc2 (x and follows θ is measured from the c-axis), suggesting that the upper critical field is Pauli limitedx dependence in gc(x), the a-axis g-factor ga(x) significantly decreases before the superconductivity is destroyed near x≈0.028 (g-factor in the a-direction may differ significantly from the fitted ga(x) because of the increased importance of diamagnetic effects as the field rotates into the ab-plane. It remains to be seen whether these trends are consistent with recent theoretical proposals such as ref. Magnetoresistance data  show quaHc2  is highl x≈0.028 . We notex of the superconductivity and the underlying metallic state. H<9 T applied parallel to the c-axis at all dopingsx<0.028 the normalized magnetoresistance is described by a distinct fx(h) ), which itself evolves with doping. Notably, the function fx(h) evolves non-monotonically with x with a maximum near x=0.006 (fx(h) nearly coincides with the maximum in the thermodynamic signatures of the superconductivity inside the hidden order phase.Magnetoresistance measurements further highlight the non-monotonic evolution with  x=0.006 . The max2Si2 has focused on the uranium electronic structure and the symmetry of the hidden-order phase, a more fundamental question is the degree to which the f-electrons can be treated as being localized and the role of valence fluctuations. The continuity of experimental information extracted from well-developed applied pressure (P) and chemical substitution (x) series has proven essential to disentangle such effects in other correlated systems including high temperature superconducting cuprates, pnictides and heavy fermion compounds. To some extent, URu2Si2 has also benefited from such studies. For example, pressure drives a first order phase transition from hidden order into antiferromagnetism near Pc=0.5 GPa, with a simultaneous evolution of the Fermi surface222324T−x phase diagrams that closely resemble the T−P phase diagram2627293031322Si2 in clean single crystals at ambient pressure. In this context, ligand site substitution in URu2Si2 is an obvious target for investigation.Although much of the recent excitement surrounding URu2Si2 the U-5f electrons are treated as having mostly fixed valence in a particular atomic crystal field state6f valence electrons allows for fluctuations between different configurations. However, measurements of the pure compound so far give no insight into the role of these fluctuations in favouring hidden order and superconductivity. The rapid changes in the hidden order and superconductivity in our measurements confirm the importance of the itinerant electron states. This is further supported by the weak evolution of Kondo lattice physicsf- and conduction electrons) and strong evolution in the g-factor anisotropy . Together these results point towards this series as a platform for unraveling the relationship between local and itinerant behavior in URu2Si2.While in many theoretical scenarios for hidden order in URu2Six2-Px (monotonic versus non-monotonic) further suggests that hidden order, although necessary, may not be directly responsible for the superconducting pairing. Instead, the observation of superconductivity completely contained inside the hidden order region and non-monotonic evolution of thermodynamic and electrical transport properties may indicate the presence of an independent collapsing phase boundary within the hidden order state, as is ubiquitous in other unconventional superconductors. This scenario is reinforced by the observed non-monotonic evolution of the normal state electrical transport, which is also common in correlated electron systems181920Ag2)Bg2)Bg1)Eg)2Si2.The stark contrast in the evolution of the hidden order and superconductivity in URuf-electron physics with no guidance regarding the specificity of the transition metal ion. It is especially puzzling that hidden order and superconductivity are only observed in the U–Ru duo. Examination of silicon site substituted transition metal analogues , which can now be synthesized using molten metal flux growth  using the same flux growth recipe.), may be particularly illuminating in addressing the universality of hidden order and superconductivity in this fascinating uranium compound.Finally, the existing theoretical landscape focuses on 2Six2−Px were grown from elements with purities >99.9% in a molten in flux, as previously reported3 tantalum crucible in the ratio 1(U):2(Ru):2(Si):22(In). The crucible was then loaded into an alumina tube spanning the bore of a high-temperature horizontal tube furnace. Argon gas was passed through the tube and a zirconium getter was placed in a pot before the tantalum crucible in order to purify the argon at high temperatures. The crucible was heated to 500 °C at 50 °C/h, dwelled for 5 h, heated to 600 °C at 50 °C/h, dwelled for 5 h and heated to 1,450 °C at 70 °C/h. The dwells at intermediate temperature are intended to allow the phosphorus to completely dissolve into the indium flux without producing a dangerous high-vapor pressure. The crucible was then cycled between 1,450 and 1,400 °C at 100 °C/h 10 times. Finally, the furnace was turned off and quickly cooled to room temperature. The indium flux was subsequently removed using hydrochloric acid, to which the URu2Six2-Px crystals are insensitive. This technique produced single-crystal platelets similar to the ones previously reported.Single crystals of URuT<20 K. Magnetization M measurements were carried out for mosaics of single crystals for temperatures T=1.8–350 K under an applied magnetic field of H=5 kOe applied parallel to the c-axis using a Quantum Design Magnetic Property Measurement System. Magnetic susceptibility χ is defined as the ratio M/H. Zero magnetic field electrical resistance R was measured using the He3 option in Quantum Design Physical Properties Measurement System for temperatures 400 mK<T<300 K. Several individual crystals were measured for each concentration, which revealed a high degree of batch uniformity. The angular dependence of the superconducting upper critical field was measured using the superconducting magnet (SCM-1) dilution refrigerator system at the National High Magnetic Field Laboratory for H<18 T and T=20 mK. Additional magnetoresistance measurements were performed at the National High Magnetic Field Laboratory, Tallahassee, up to magnetic fields of 35 T and at T=50 mK.Heat capacity measurements were performed for mosaics of single crystals using the He3 option in a Quantum Design Physical Properties Measurement System for temperatures 400 mK<How to cite this article: Gallagher, A. et al. Unfolding the physics of URu2Si2 through silicon to phosphorus substitution. Nat. Commun. 7:10712 doi: 10.1038/ncomms10712 (2016).Supplementary Figures 1-6, Supplementary Table 1, Supplementary Discussion and Supplementary Reference