The Barents Sea Ice Sheet was a marine-based ice sheet, i.e., it rested on the Barents Sea floor during the Last Glacial Maximum (21 ky BP). The Bjørnøyrenna Ice Stream was the largest ice stream draining the Barents Sea Ice Sheet and is regarded as an analogue for contemporary ice streams in West Antarctica. Here, the retreat of the Bjørnøyrenna Ice Stream is simulated by means of two numerical ice sheet models and results assessed against geological data. We investigate the sensitivity of the ice stream to changes in ocean temperature and the impact of grounding-line physics on ice stream retreat. Our results suggest that the role played by sub-shelf melting depends on how the grounding-line physics is represented in the models. When an analytic constraint on the ice flux across the grounding line is applied, the retreat of Bjørnøyrenna Ice Stream is primarily driven by internal ice dynamics rather than by oceanic forcing. This suggests that implementations of grounding-line physics need to be carefully assessed when evaluating and predicting the response of contemporary marine-based ice sheets and individual ice streams to ongoing and future ocean warming. In particular, the Barents Sea Ice Sheet was largely marine based, as its largest portion rested hundreds of meters below sea level on the Barents Sea floor. The Bjørnøyrenna Ice Stream was the largest ice stream draining the Barents Sea Ice Sheet2. At the LGM the ice stream extended up to the continental shelf edge in the western Barents Sea3  comprised three large ice sheets: the British-Irish Ice Sheet, the Fennoscandian Ice Sheet and the Barents Sea Ice Sheet ky BP co7. Therefore, it is interesting to identify the dynamic processes driving the retreat of the Bjørnøyrenna Ice Stream over the western Barents Sea continental shelf during the last deglaciation. Here, we focus on the role exerted by the sub-shelf melting and by grounding-line dynamics as triggers of the ice stream retreat. Several recent studies show that ice shelves and marine-terminating glaciers in the WAIS are thinning8 and retreating9 primarily due to sub-shelf melting occurring where relatively warm Circumpolar Deep Water reaches the ice-shelf cavities11. Analytical and numerical studies suggest that an initial retreat of the grounding-line position can lead to abrupt changes in volume and extent of marine-based ice sheets in regions where the bedrock is sloping towards the interior of the ice sheet 14. However, these studies analyzed the instability of marine-based ice sheets on retrograde slopes under conditions of one dimensional horizontal (1DH) flow14. When two dimensional horizontal (2DH) flow is considered, specific numerical examples of stable grounding lines on retrograde slopes have been simulated with various numerical Ice Sheet Models (ISMs)15. Nevertheless, several studies expanded and incorporated the 1DH boundary layer analytical solution proposed by Schoof13 for the flux across modelled grounding lines in large-scale three dimensional ISMs17. Therefore, the question of whether the WAIS will be susceptible to marine-ice sheet instability in the future remains unanswered, although geological evidence shows that the WAIS retreated frequently over the past 5 million years18.Due to similar morphological and glaciological features, the Bjørnøyrenna Ice Stream is acknowledged as an analogue for several present-day ice streams in the West Antarctic Ice Sheet (WAIS)19 and the Stockholm Branch of Penn State University glacial model (PSU)20, in order to investigate the role played by sub-shelf melting and grounding-line dynamics as triggers of the retreat of the Bjørnøyrenna Ice Stream after the LGM. The timing of the simulated retreat from Sentraldjupet is compared with deglacial chronologies derived from marine geological data6 Material and Methods) GBvol and PBvol yield an Eurasian Ice Sheet grounded-ice area at the LGM of 6.5 and 6.1 106 km2, respectively . In our climate forcing, negative July and annual mean near-surface air temperatures occur over these regions at the LGM , while proxy data suggest higher air temperatures21.GRISLI and PSU best fit simulations , whereas PBvol slightly overestimates ice loss from the British-Irish Ice Sheet . This leads to to a massive ice transport towards the relatively warm, low-latitude southern margins of both ice sheets , where the ice is melted via surface ablation. In the case of the high-latitude marine-based Barents Sea Ice Sheet, the combined effect of sea-level rise  which appears to be overly sensitive to atmospheric warming. Due to its high latitude location, the simulated Barents Sea Ice Sheet is less sensitive to atmospheric warming and its retreat is more reduced to that of the Fennoscandian Ice Sheet  than in the PBvol simulation (0.32 106 km2).Between 15 and 14 ky BP, the rate of ice loss accelerates in both GBvol and PBvol simulations 23 and is mostly in agreement with the DATED-1 reconstruction, except over Severnaya Zemlya, where the ice sheet is more expanded. In Patton et al.23 the evolution of the Fennoscandian and British-Irish ice sheets throughout the deglaciation is adequately simulated compared to the DATED-1 reconstruction, in spite of an overestimation of the area treated with fast flow physics in the southern Fennoscandian Ice Sheet, similarly to our GBvol simulation. Until 16 ky BP, the evolution of the Barents Sea Ice Sheet in Patton et al.23 is similar to the DATED-1 reconstruction. After this time, Patton et al.23 simulate a larger northern and eastern Barents Sea Ice Sheet than observed, although the discrepancy with the DATED-1 reconstruction is lower than that observed in GBvol and PBvol simulations. The better agreement obtained by Patton et al.23 with the DATED-1 reconstruction can be explained by the different approach used to force the ISMs in terms of atmospheric temperature, precipitation and ocean temperature. Although both studies use the same positive degree-day scheme to determine the surface mass balance over the ice sheet, Patton et al.23 tuned the regional reference climatologies and the associated climate forcings for the Fennoscandian, Barents Sea and British-Irish ice sheets in order to match a suite of empirical data23. Furthermore, in contrast to what is done in the present study, Patton et al.23 do not explicitly compute the sub-shelf melting. The retreat of the marine-terminating margins of the ice sheet is determined by an empirical function which relates calving to ice thickness and water depth23. Similarly as for the regional climate indexes, the sensitivity of the Fennoscandian, Barents Sea and British-Irish ice sheets to calving is independently tuned throughout the deglaciation23. In our study, climatic and oceanic forcings are derived from a transient climate simulation22  and are not modified. The two studies are therefore not directly comparable.Recently, Patton It emerges from our simulations that the retreat of the Barents Sea Ice Sheet is mostly driven by changes in ocean temperatures and sea level in the GBvol simulation. In contrast, in the PBvol simulation the ice sheet appears to be less sensitive to variations in ocean conditions and the major role in triggering the ice sheet retreat is played by grounding-line dynamics. In order to better establish the interplay between ocean warming, sea level and grounding-line physics in triggering the retreat of the Bjørnøyrenna Ice Stream, we focus on the evolution of its southern branch between 21–10 ky BP . Therefore, when the simulated grounding-line is particularly thick, the outgoing ice flux computed with the solution by Schoof13 is very large compared to sub-shelf melting and ablation , thus dampening the effect of ocean warming on grounding-line retreat as observed in the GBvol simulation. As a result, the final deglaciation of Bjørnøyrenna Ice Stream in PBvol simulation is delayed with respect to the oceanic warming observed between 14.5 and 13 ky BP. Nevertheless, in both simulations the deglaciation timings are in agreement with the timing suggested by the geological data from the area6.By 13 ky BP, the 400 m deep Sentraldjupet . Without the grounding-line flux correction, the combined effect of sea-level rise and high sub-shelf melting (around 1.0 m/yr) at 14.5 ky BP does trigger a grounding-line retreat of 50 km in PBvolNS simulation  the ice stream/shelf geometries are similar to that simulated in GBvol, with a 100-km long and 400-m thick ice shelf  and floating ice (ice shelves). As opposed to the GRISLI ISM, the 1DH solution from13 is imposed as a condition on the ice flux across the grounding line in the PSU ISM. The ISMs’ dynamics is fully described in19 and20, whereas the boundary conditions and a list of model parameter values are included in the Supplementary Material. All the GRISLI/PSU simulations were performed using a horizontal resolution of 20 km/18.5 km, respectively. With both ISMs, transient spin-up simulations were run between 122 ky BP (MIS5) and the LGM, in order to initialize the thermodynamical state of the Eurasian Ice Sheet. At the beginning of each spin-up simulation, a masked ice-free present-day surface topography based on the IBCAO chart27 and the PI annual mean air temperature and precipitation simulated with the IPSL-CM5A-LR28 AOGCM were prescribed . During the spin-up simulations, the climatology was progressed from PI to the LGM by means of a climate index based on the NGRIP δ 18 O record29. At the LGM, the annual mean air temperature and precipitation simulated by the IPSL-CM5A-LR28 model were prescribed . The PI climate fields were downscaled on the ISM grids by means of the present-day surface topography based on the IBCAO chart27. The LGM surface topography based on the ICE-5G30 glacio-isostatic reconstruction was used to downscale the LGM climate fields. A low constant sub-shelf melting rate was prescribed during all spin-up simulations in order to allow the Barents Sea Ice Sheet to spread over the Barents and Kara Seas . In the transient simulations of the last deglaciation the downscaled LGM and PI annual mean air temperature and precipitation simulated with the IPSL-CM5A-LR28 model were prescribed as initial and final climate snapshots, respectively, with both ISMs. During the simulations, the climatology was progressed from the LGM to PI by means of three different climate indexes for annual mean air temperature and precipitation derived from TraCE-21ka, a non-accelerated transient climate simulation of the last 21 ka22. The three different indexes for annual mean air temperature and precipitation were taken as representative of Fennoscandia, Svalbard/Barents Sea and Siberia/Kara Sea macro-regions . During the transient simulations of the last deglaciation, the sea level was progressed from −130 to 0 m following an index based on the NGRIP δ 18 O record29 . With the GRISLI ISM a Latin Hypercube Sampling (LHS) of five model parameters was constructed  and a statistical ensemble of 101 transient simulations of the last deglaciation was generated. The Eurasian Ice Sheet Complex ice volume evolution during the deglaciation simulated with the GRISLI ISM was compared with those simulated with the global glacio-isostasy model ICE-5G30, and a best fit simulation (referred to as GBvol) from the statistical ensemble was identified . Due to large computational costs, it was not possible to pursue the same statistical approach with the PSU ISM. Therefore, the optimal parameter values identified with the GRISLI ISM were used to run a corresponding PSU best fit transient simulation . In order to investigate the impact of the grounding-line flux correction13 implemented in the PSU ISM on the retreat of the Bjørnøyrenna Ice Stream, the simulation PBvol was repeated inhibiting the grounding-line correction (referred to as PBvolNS).The numerical ISMs employed are the Stockholm Branch of PSUd29 Fig. . In bothSupplementary Material


Misplaced screw during the internal fixation of acetabular fractures may penetrate the hip joint which might cause chondrolysis and traumatic osteoarthritis in the future. This study aims to acquire the safe path for screw insertion along inferior border of the arcuate line fixation route at acetabular area.Computed tomography (CT) scans of 98 patients without pelvic trauma were rebuilt for three-dimensional models of pelvis. After depicting the fixation route curve, five cross-sections perpendicularly to the curve were established from the anterior of pelvis to the posterior along inferior border of the arcuate line. The safe screw lengths for section 1 and 5 were measured from the computer models. In section 2, 3 and 4, a line from the screw entry point tangent to the inferior edge of the acetabulum was depicted and the measurements of minimum safe direction of screw insertion were performed then marked with angle θ.n = 98). The minimum safe angles of screw insertion for the middle three sections 2, 3, and 4 were 65.38 ± 10.23°, 74.20 ± 10.20°, and 57.88 ± 11.11°(n = 98), respectively. The results for the male group (n = 98) indicated smaller minimum safe angles in these three sections compared with the female (n = 98).The safe screw lengths for section 1 and 5 were 22.29 ± 4.41 mm and 32.64 ± 4.70 mm (Compared to male, the minimum safe angles of screw placement at acetabular area for female should be more away from inferior edge of acetabulum and tilt to the bottom of pelvis along inferior border fixation route in surgical management of acetabular fractures. Rigid and accurate internal fixation is required for acetabular fractures with displacement to achieve anatomical reduction and good prognosis , 2. HoweThe Stoppa and the illoinguinal approaches, classical anterior approaches, are both commonly employed for treating acetabular fractures . PreviouBased on 3D reconstructions and measurements of pelvic CT scans, a retrospective observational study was performed. The objective of the present study is to acquire safe path for screw insertion along the inferior border fixation route at acetabular area.Ninety-eight patients including 60 males and 38 females without pelvic trauma or deformity, diagnosed with varicose veins in lower extremity, were recruited in Shanghai General Hospital from December 2009 to November 2010. The mean age of these available samples was 60.1 years  scan. To obtain clearly pelvic images, the needed thickness and scanning time for each slice was 0.75 mm and 0.2 s, respectively. Stored with DICOM format, all raw imaging data of each patient were then processed by Mimics 13.0 where three-dimensional (3-D) pelvic model was reconstructed. After that, thresholding segmentation, region growing and surface smoothing were performed to the 3-D pelvic model and saved with Stereo Lithography (STL) format. Then, the Geomagics 10.0 software was used for further smoothing and noise reduction.In the beginning, via Geomagics 10.0 software, three fundamental planes defined as the sagittal, coronal and horizontal plane were set up, respectively. The sagittal plane was recognized as the plane through from the pubic symphysis to the midpoints of the whole sacrum and the coccygeal tip. In coronal plane, the anterior superior iliac spine and pubic tubercle were positioned at the same level to comply with anatomical position during the process of the study. Perpendicular to both above-mentioned planes, the horizontal plane was established. A curve was delineated by collecting some points, 5 mm lateral and inferior to the pelvic rim, along the inferior border fixation route from pubic tubercle, pubic ramus inner side, arcuate line, to sacroiliac joint. The site with distance of 5 mm at this area is frequently used as the entry point of screw. Saved with STL format, all these objects were imported to the Imageware 23.0 where an optimal ball fitting the acetabular fossa was established. And after being saved with IMW format, the whole model was imported to the Unigraphic NX 7.0.Passing through the center of the ball, a section perpendicular to the space curve was built as the reference plane. Parallel to this section, two planes with interval of the quarter of diameter forwardly and backwardly along the space curve were built, respectively. These five planes segmented the space curve at acetabular area into four equal parts. Thus, five intersection points between each plane and the curve were acquired as the screw entry points Fig. . ThroughThrough the screw entry point of each section and perpendicular to the cortical surface, another reference plane was built. The section and reference plane intersected at one point which was recorded as the intersection point. In section 1 and 5, located in the outermost of acetabular area, screws can be placed perpendicularly to the bone surface directly and there is no need to worry about screw penetration into the hip joint. In section 2, 3, and 4 with the shape similarity, the same measurement methods were performed. With several points selected along the edge of acetabulum then expended by 5.0 mm, an arc was delineated to represent the subchondral bone thickness. A line through the screw entry point and tangent to the inferior edge of the arc was defined as the screw-placement direction. Formed by the tangent line and the normal plane, the angle was marked as θ. The distance, defined as the screw length d from the screw entry point to the intersection point, was measured in section 1 and 5. Due to the similar shaping, the measurements for section 2 and section 3 were represented with the same model .Data were reported as mean ± standard deviation. Normality tests were used for analysis of data distribution and n = 98), 72.66 ± 9.35° (n = 98), and 57.88 ± 11.11° (n = 98), respectively. The results for the male group (n = 98) indicated smaller minimum safe angles in these three sections compared with the female (n = 98). Significance of differences between male and female groups for θ in section 2, 3 and 4 were indicated . The safe screw lengths for section 1 and 5 were 21.94 ± 3.07 mm (n = 98) and 33.01 ± 4.47 mm (n = 98). The differences of screw lengths between male and female groups in section 1 and 5 were not statistically significant  (Table The experimental results showed the differences on minimum safe angles and safe screw lengths between the male and female in different sections. In section 2, 3, and 4, the minimum safe angles were 67.40 ± 7.11° (The special anatomical morphology of acetabular area is more complex and irregular compared with the long bone which increases the difficulties for surgical treatment. Although considerable achievements have been obtained for management of acetabular fractures in recent years, such fractures remain challenging to orthopedic surgeons . During As two main approaches, the Stoppa approach and the ilioinguinal approach are widely employed in the fixation of acetabular fractures . Our preThe safe screw insertion angles can be found from the comparisons between male and female. The male group showed smaller minimum safe angles obviously in the middle three sections. Compared with male, angles for the female should be more away from inferior edge of acetabulum. Thus, it can be interpreted that during the operation, more tilt to the bottom of pelvis should be required for the female to avoid intra-articular screw insertion. And proper length screws used in middle three sections should be selected in accordance with the actual depth detecting during the surgery procedure. Unlike other human bones, the pelvic shape appears markedly different between the male and female. For the general pelvis, there are four different shapes termed gynecoid, android, platypelloid and anthropoid. That means although these samples were divided into two main groups, male and female, because of the overlap of pelvic shapes in these two groups, the results inevitable appear overlapped to some extent. The finding concluded by us provides a general guidance for the surgeons. However, before the surgery, a 3D geometrical reconstruction should also be performed to the patient so as to acquire the accuracy. Thus, a combination of the results of the current study and the preoperative 3-D geometrical reconstruction would provide a relatively accurate guidance in the planning of the surgery.. However, it didn’t set up an operative reference plane, thus the relative position between surgeon and entry point might be changed. In our study, perpendicular to the entry point of the cortical surface, the reference plane was built in each section which could be convenient for the surgeons to get the accurate screw insertion during the operation. Ebraheim et al. [Focusing on safe path of screw insertion, previous researchers have reported several conclusions with different methods. Xianquan Wang  sectionem et al.  performem et al.  reportedTo conclude, results of the present study provided the safe angles for screw insertion along the inferior border fixation route at acetabular area. The minimum safe angles for female should be more away from inferior edge of acetabulum and tilt to the bottom of pelvis along inferior border fixation route in surgical management of acetabular fractures. The definition of safe angles for screw insertion with Stoppa approach made it more practical to guide the surgery. On the basis of accurate 3D reconstructions and digital measurements, a reliable reference on safe path of screw insertion for acetabular surgery was offered by this study.


Atmospheric iodine causes tropospheric ozone depletion and aerosol formation, both of which have significant climate impacts, and is an essential dietary element for humans. However, the evolution of atmospheric iodine levels at decadal and centennial scales is unknown. Here, we report iodine concentrations in the RECAP ice-core  to investigate how atmospheric iodine levels in the North Atlantic have evolved over the past 260 years (1750–2011), this being the longest record of atmospheric iodine in the Northern Hemisphere. The levels of iodine tripled from 1950 to 2010. Our results suggest that this increase is driven by anthropogenic ozone pollution and enhanced sub-ice phytoplankton production associated with the recent thinning of Arctic sea ice. Increasing atmospheric iodine has accelerated ozone loss and has considerably enhanced iodine transport and deposition to the Northern Hemisphere continents. Future climate and anthropogenic forcing may continue to amplify oceanic iodine emissions with potentially significant health and environmental impacts at global scale. Despite its chemical importance, the evolution of atmospheric iodine concentrations over time is unknown. Here, the authors show that North Atlantic atmospheric iodine levels have tripled since 1950, and propose ozone pollution and enhanced biological production Arctic sea ice thinning as a primary driver. Iodine is also a key trace element in the endocrine system of mammals, and is essential for the production of hormones in the thyroid gland. In humans, iodine deficiency causes neurological damage and developmental delays in children, and represents the most common cause of preventable mental retardation5. Iodine intake by mammals thus represents the last reservoir in the biogeochemical cycle of iodine. Atmospheric iodine is transported from its dominant oceanic source to the continents, where it is adsorbed onto soil and vegetation. Between ocean emission, soil deposition, and ultimate ingestion by animals and humans, iodine participates in a complex variety of physical and chemical atmospheric processes1. Laboratory studies have demonstrated the oceanic emission of hypoiodous acid (HOI) and molecular iodine (I2) following the deposition of tropospheric ozone (O3) to the surface and the subsequent reaction with iodide (I−) ions7. This ocean release of inorganic iodine is estimated to account for 75% of the total source of atmospheric iodine, with the remainder coming from organic iodine 8. Consequently, an increase in global ocean iodine emissions has been hypothesized in response to the human-driven increase in tropospheric ozone levels during the industrial period9. The lack of long-term measurements of atmospheric iodine has so far prevented the study of its evolution in the atmosphere.The biogeochemical cycle of iodine involves ocean emission, atmospheric transformation, new particle formation, uptake on aerosols, heterogeneous recycling, and deposition on land, where iodine enters terrestrial ecosystemsIn this work iodine and sodium were measured in the upper 130 m of the RECAP (REnland ice Cap Project) ice-core spanning the Industrial Period , providing an opportunity to understand changes in atmospheric iodine levels in the North Atlantic region and their responses to natural and anthropogenic forcings. We find that although the iodine concentrations do not vary significantly from 1750 to 1950, the abrupt increase observed from 1950 to 2010 is linked to enhanced tropospheric ozone pollution and Arctic sub-ice biological activity.−1 (σ = 0.009) and 10 µg m−2 year−1 (σ = 4), respectively  to 1940 C.E., followed by a drop to values of 0.01 ng g−1 during the 1940s . Iodine fluxes have doubled from 1750 to 2010, and tripled from 1950 to 2010.The Renland ice cap is located on a high elevation plateau on the eastern coast of Greenland  and was the site of a 584-m ice-core drilled to bedrock in 2015. The Renland iodine record displays mean concentrations and deposition fluxes of ~0.023 ng g10. The weak iodine–sodium correlation since 1950 C.E. suggests that the recent iodine variability since 1950 C.E. cannot be attributed to natural processes, such as the atmospheric transport of sea salts. In addition, the iodine–sodium ratio in the Renland ice-core is two orders of magnitude higher than that in seawater11  show a 33% increase in ocean iodine emissions over the North Atlantic since 1950 in response to a 30% ozone increase during this period ). The estimated decreases in ozone precursor emissions over North America and Europe during the 1940s16 suggest a similar link during the concurrent observed ice-core iodine decrease. Overall, this suggests that ozone-driven ocean emissions of iodine may have controlled the variability of atmospheric iodine levels during the 1950–1989 period. Note that the increase in atmospheric iodine levels followed by deposition to ice covered areas would in turn make the local heterogeneous processing and recycling of iodine in ice/snow more efficient, as recently measured in the Arctic18. Interestingly, during the period from 1990 to 2011, iodine concentrations increase despite stable atmospheric ozone concentrations  and Arctic sub-ice biological productivity  . Thicker Arctic sea ice for the period 1958–197623 . The abrupt decrease in the Arctic sea ice thickness below 1.5 m since 1990  to a 130-m ice-core depth (1750 C.E.). The RECAP ice-core chronology is based on annual layer counting using the StratiCounter algorithm36, which accounts for the annual signal in a large array of chemical species37—including black carbon, calcium, conductivity, ammonium, sodium, dust, water isotope , as well as electrical conductivity records ; in total 17 data series. The timescale was constrained by five prominent volcanic eruption markers within the considered time interval . Given the volcanic constraints, the uncertainty in the age scale is estimated to be ±2 years through the period discussed here. The density profile was obtained by fitting a three-stage exponential densification model (χ2-fit prob = 7.7%) to density measurements  obtained every 55 cm. The annual accumulation rates were calculated from the annual layer thickness, the density profile and a linear thinning function, which was inferred from a Dansgaard and Johnsen38-type ice flow model constrained to well-dated horizons. The average accumulation in the period from 1750 C.E.–present is 447 kg m−2 year−1 (1σ = 65 kg m−2 year−1), which is consistent with the Holocene value of 50 cm ice equivalent (i.e. 459 kg m−2 year−1) reported in a previous work39. The ice-core samples were collected in October–December 2015 using a continuous flow analysis (CFA) system at the University of Copenhagen40. One discrete sample was collected for each 55-cm rod of melted ice. Meltwater was collected in pre-cleaned polyethylene tubes, subsequently refrozen and stored shielded from light until analysis. Samples were sent to the IDPA-CNR, University Ca’ Foscari of Venice for sodium and iodine determination.The RECAP ice-core was drilled at 71.30° N, 26.72° W (2315 m asl), on the Renland ice cap in Scoresbysund Fjord (Eastern Greenland) in 2015 using the Danish Hans Tausen intermediate drill system. An ice-core 10 cm in diameter was drilled to bedrock, 584 m below the snow surface. The depth range of the Renland ice-core reported here spans the Industrial Period from a 5.5-m ice-core depth (2011 41. Iodine (127I) and sodium (23Na) isotopes were determined, respectively, at a low and medium mass resolution with plasma stability evaluated by the continuous monitoring of 129Xe. The sample line was thoroughly cleaned using 2% nitric acid and ultrapure water  between each analysis. Instrumental blanks (UPW samples and UPW ice samples from the CFA campaign) were measured throughout the analytical phases. Detection limits of sodium and iodine were respectively 0.8  ppb and 5 ppt—calculated as three times the root mean square of the blank samples. Instrumental-associated errors for iodine and sodium concentration measurements are 10%. Air-to-snow iodine annual fluxes (JI) were calculated by the following: JI = CIA, where CI is the iodine concentration in the ice and A is the annual accumulation rate.Measurements were carried out at the Environmental Analytical Chemistry laboratory of the IDPA-CNR, University Ca’Foscari of Venice. Samples were measured by collision reaction cell-inductively coupled plasma mass spectroscopy  using a Scott spray chamber fitted with a Microflow PFA nebulizer . The operational methodology was carried out as described in a previous work42, which is included in the CESM framework (Community Earth System Model). The model setup is based on the CCMI-REFC1 experiment described in a previous work43, but incorporates an updated halogen chemistry scheme for halogens 45. This model configuration includes an explicit state-of-the-art scheme of iodine emissions (both organic and inorganic) and photochemistry (both gas and particle phase), which explicitly account for chemical transformation during transport from the ocean source to deposition in the Renland region. The iodine chemical scheme includes in addition to a complete gas phase chemical mechanism considering independent photolysis rate coefficients, bi-molecular and termolecular reactions15, and a parameterized representation of heterogeneous reactions occurring both on sea-salt aerosols and tropospheric ice crystal14, as well as independent representation of dry and wet deposition (including below-cloud wash-out and ice-uptake) for each inorganic iodine species15. These processes change the chemical partitioning of each of the 14 individual inorganic halogen species by calling the chemical solver coupled with the transport module at each time step.The model employed in this work is the global 3-D chemistry-climate model CAM-Chem (version 4)Br = CHBr3, CH2Br2, CH2BrCl, CHBrCl2, CHBr2Cl) and four iodocarbons , which are naturally emitted from the ocean to the atmosphere following its production by phytoplankton and photochemical processes occurring at ocean surface46. Additionally, abiotic oceanic sources of HOI and I2 have been included in the lowest layer of the model9, based on recent laboratory studies of the oxidation of aqueous iodide by atmospheric ozone deposited on the ocean surface7. Therefore, the model includes both organic and inorganic global iodine emission sources.Halogen sources include the photochemical breakdown of five very short-lived bromocarbons  based on the A1 halogen scenario from WMO47 are considered, while the surface concentrations of CO2, CH4, H2, and N2O are specified following a previous work48. The model was run in free-running mode42 considering prescribed sea surface temperatures , as in previous studies49. While the model shows very good skill for the recent past, there is a tendency for the model simulation to overestimate ozone prior to the 1970s. This would, in turn, reduce the rate of ozone increase over that period, and would therefore underestimate the iodine response to an ozone change. The effectiveness and importance of each reaction and process included in the chemical mechanism has been evaluated in the global atmosphere both during the day and night51, while all the sources and tropospheric mixing ratios of organic and inorganic iodine species have been validated46.The model has been extensively compared with available measurements, especially long-term trends of ozone at the surface and in the mid-troposphere13. The differences are most likely because there is difficulty in accurately modeling ozone emissions in the past, when measurements were scarce. Furthermore, the model does not implement online the biological activity underneath the sea ice in the Arctic region, and the sea surface production of CH2I2, CHClI2, and CHI3 from the reaction of HOI and I2 with marine dissolved organic matter53, although their contribution to the global atmospheric iodine budget is likely to be smaller than that from HOI + I2. Therefore, the model is only representative of the ozone-induced inorganic iodine emissions (HOI and I2) from the North Atlantic Ocean. Hence, these values must be considered as lower limits, because the modeled trend is lower than that registered in the ice-core. This first difference has been already reported in a previous work13, suggesting that in order to reproduce the long-term ozone trends, chemistry-climate models should be improved by introducing additional processes that might have climatic impacts.The modeled iodine values display a more continuous and less increasing trend than the observed values in the ice-core. The same can be said for the modeled ozone, in comparison to observations from previous worksThe ice-core and model data that support the findings of this study are available upon request.Supplementary Information


Phaeocystis pouchetii, caused near depletion of the surface nitrate inventory and a decline in dissolved inorganic carbon by 16 ± 6 g C m−2. Ocean circulation characteristics in the area indicated that the bloom developed in situ despite the snow-covered sea ice. Leads in the dynamic ice cover provided added sunlight necessary to initiate and sustain the bloom. Phytoplankton blooms beneath snow-covered ice might become more common and widespread in the future Arctic Ocean with frequent lead formation due to thinner and more dynamic sea ice despite projected increases in high-Arctic snowfall. This could alter productivity, marine food webs and carbon sequestration in the Arctic Ocean.The Arctic icescape is rapidly transforming from a thicker multiyear ice cover to a thinner and largely seasonal first-year ice cover with significant consequences for Arctic primary production. One critical challenge is to understand how productivity will change within the next decades. Recent studies have reported extensive phytoplankton blooms beneath ponded sea ice during summer, indicating that satellite-based Arctic annual primary production estimates may be significantly underestimated. Here we present a unique time-series of a phytoplankton spring bloom observed beneath snow-covered Arctic pack ice. The bloom, dominated by the haptophyte algae  Annual phytoplankton net primary production in the Arctic Ocean has increased by 30% since the late 1990’s mainly due to the declining sea ice extent and an increasing phytoplankton growth season2Phaeocystis pouchetii was actively growing beneath snow-covered pack ice at higher latitudes and earlier in the season than previously observed.Phytoplankton production beneath the ice-covered Arctic Ocean is assumed negligible because of the strong light attenuation properties of snow and sea ice, despite sporadic reports of phytoplankton growth beneath Arctic sea ice over the past decades45679101191011a) concentrations in the water column were low (<0.5 μg L−1) until 25 May when we first drifted into an under-ice phytoplankton bloom over the Yermak Plateau (YP) 80 km north of the ice edge (a concentrations of 7.5 μg L−1 were observed on 2 June and 50 m depth-integrated Chl a and particulate organic carbon (POC) standing stocks ranged between 109–233 mg Chl a m−2 and 9–22 g C m−2. The under-ice bloom (10–80 km from open waters) nearly depleted the surface nitrate inventory (P. pouchetii (a m−2) throughout the drift indicating that contributions from the ice to water column stocks were negligible. A detailed list of protist plankton taxa observed during the bloom period can be found in the We studied the ice-associated ecosystem and the environmental factors shaping it in the Arctic Ocean north of Svalbard from 11 January to 24 June 2015 during the Norwegian young sea ICE (N-ICE2015) expeditionice edge  and remaice edge . The onsice edge  and reduice edge . This renventory  and redunventory . The depouchetii , which aouchetii  and as louchetii . FurtherPAR) to the underlying water column. On the other hand, EPAR transmittance for thin ice examined near camp in a refrozen lead was 20% on average, ranging from 6.3–42.2%. Leads in the ice pack  modal thickness of 1.8 m, with a secondary mode at 0.2 m, representing thin, lead ice . Local sice pack  were freice pack  prior toice pack  indicateice pack . Melt poht field  experienP. pouchetii was modelled based on 14C photosynthesis-irradiance (PE) relationships obtained from a P. pouchetii bloom in the Greenland SeaPhaeocystis is particularly well adapted to low light environments17In vivo photosynthetic parameters, obtained with the Pulse Amplitude Modulation (PAM) method to assess the photo-acclimation status of the bloom, corroborate this finding (a fluorescence of dark-acclimated cells) of 0.48–0.66 showed that the bloom-forming species were in good condition and actively growing. The maximum light utilization coefficient (α) of 0.188–0.295, obtained from Rapid Light Curves, also illustrates that the bloom exhibited high photosynthetic rates at low irradiances. Furthermore, the low POC/Chl a ratio of 31.4 in the upper 25 m of the under-ice water column suggests a relatively high investment in photosynthetic pigments, indicative of shade-acclimation. On the other hand, light saturation (Ek) values of 137–584 μmol photons m−2 s−1 suggests that the phytoplankton community was at the same time acclimated to relatively high irradiances. This apparent inconsistency can be explained by the plasticity in photosynthetic performance of P. pouchetii that seems to be a characteristic feature of this species91011−1  were weak. Time-mean current velocity components in PSW at 20–30 m depth for the bloom period were 2.2 cm sdue west . While tdue west  shows sidue west  at the sa concentrations of <0.1 μg L−1 in open waters across the shelf slope north of Svalbard on transit to floe 3. Thus, the weak re-circulation pattern over YP implies that the bloom grew in situ beneath the ice pack. The area that floes 3 and 4 drifted over towards the end of their respective drifts in June was open water in mid-April when the ice edge was at its northern-most position during the period April to June 2015  masses at greater depths . The oveune 2015 . Conside−2 in the DIC inventory and a nitrate uptake equivalent to 15 ± 5 g C m−2 for the bloom period agreed well with the build-up in POC standing stocks. The biogeochemical footprint of the bloom was comparable to a diatom bloom beneath ponded, more transparent sea ice at a lower latitude−2 d−1 during the bloom period. Inspection of 100 m depth sediment-trap material revealed that the bulk of vertical carbon export was mediated via P. pouchetii aggregates, while zooplankton faecal pellets accounted for <2%. Out of the 63 zooplankton taxa identified in 200 μm MultiNet samples taken during the bloom period, the three dominant Calanus species  accounted for 89 ± 8% of the total zooplankton biomass. The apparently low grazing impact on the P. pouchetii bloom by the dominant Calanus copepods is supported by the finding that P. pouchetii does not significantly contribute to Calanus dietP. pouchetii does not contribute much to deep carbon sequestration, which is generally mediated by diatoms25Phaeocystis, in benthic macrofaunaP. pouchetii biomass below 100 m has been reported previously28The mean integrated drawdown of 16 ± 6 g C mP. pouchetii, despite the thick snow cover and limited light transmission. High lead fractions in Fram Strait, the Barents Sea, and in other parts of the Arctic OceanP. pouchetii, a conjecture that is corroborated by a shift in dominance from diatoms towards P. pouchetii in Fram Strait since 20063334P. pouchetii blooms under snow-covered sea ice would constrain diatom blooms during the melt season, with far-reaching repercussions on bloom timing and composition, strength of the biological carbon pump and energy flow through Arctic marine food webs.Our observations extend the spatial and temporal domains of known under-ice blooms. High lead fraction provided sufficient light to initiate and sustain an under-ice spring bloom dominated by a samples were collected on 25-mm GF/F filters (Whatman), extracted in 100% methanol for 12 h at 5 °C on board the ship and measured fluorometrically with an AU10 Turner Fluorometer . Phaeopigments were measured by fluorescence after acidification with 5% HCl. Calibration of the Turner Fluorometer was carried out following the JGOFS protocola measurement uncertainty  was estimated from triplicate water samples taken from depths ranging between 5 and 100 m. Particulate organic carbon (POC) and particulate organic nitrogen (PON) samples were collected onto pre-combusted 25-mm GF/F filters (Whatman), dried at 60 °C and stored at room temperature in PALL filter slides until analysis with continuous-flow mass spectrometry (CF-IMRS) carried out with a Roboprep/tracermass mass spectrometer . All POC/N values were corrected for instrument drift and blanks. Water samples for inorganic nutrients were collected in 20 mL scintillation vials, fixed with 0.2 mL chloroform and stored refrigerated until sample analysis approximately 6 months later. Nitrite, nitrate, phosphate and silicic acid were measured spectrophotometrically at 540, 540, 810 and 810 nm, respectively, on a modified Scalar autoanalyser. The measurement uncertainty for nitrite is 0.06 μmol L−1 and 10% or less for nitrate, phosphate and silicic acid. Seawater for DIC analyses was sampled in 250 mL borosilicate bottles, preserved with 60 μL saturated mercuric chloride solution and stored dark and cool. DIC was determined using gas extraction followed by colourometric detectionChl a, POC, plankton taxonomy, and faecal pellets.Ice-tethered sediment traps (KC Denmark) were deployed four times at 5, 25, 50 and 100 m depth during the bloom period. Deployment time varied between 36 and 72 h, but was usually close to 48 h. Before deployment, each trap cylinder was filled with a saturated NaCl solution to reduce microbial activity and thus increase the retention of organic matter. The traps were carefully deployed and retrieved to avoid loss of trap material. Swimmers (copepods and other zooplankton) were removed before sub-sampling for Chl a fluorescence (ΦPSII-max), the light saturation parameter (Ek), the maximum light utilization coefficient (α) and the maximum relative electron transfer rate (rETRmax) were obtained using the Pulse Amplitude Modulated (PAM) fluorometry method with a Phyto-PAM  following established protocolsThe maximum quantum yield of charge separation in photosystem II Chl Phytoplankton samples were settled in 50 mL Utermöhl sedimentation chambers  for 48 h. Phytoplankton was identified and enumerated at 100–600× magnification using an inverted Nikon Ti-S light and epifluorescence microscope. The organisms were identified to the lowest taxonomic level possible under inverted light microscopy, ideally to species level, otherwise to genus level or grouped into size-classes. Microscopic counts of the dominant organisms at each depth were always well above the recommended number of 50 per sample. Further, the water column stocks presented in 2 opening and 200 μm mesh size at the following depth strata: 0–20, 20–50, 50–200 and 200 m-bottom. Zooplankton were preserved using 4% formaldehyde solution in seawater buffered with hexamethylentetramine and identified to species and stageMesozooplankton was sampled with a MultiNet  consisting of five nets with a 0.25 mλ) was measured simultaneously with two upward-looking Ramses spectral radiometers with cosine collectors . One measured the incident and the second the transmitted irradiance at the bottom of the ice. These measurements were integrated over the wavelength band of photosynthetically active radiation  and then used to estimate the transmittance (fraction of transmitted to incident radiation) of EPAR, photosynthetically active radiation  through the ice and snow. The measurements were conducted continuously during floes 3 and 4 at a site representative of the thick snow-covered icePAR for thin ice (<0.25 m) in a refrozen lead. In addition, incident irradiance and irradiance under thick and thin ice was measured with Satlantic HyperOCR hyperspectral radiometers with cosine collectors, at the surface and mounted to a remotely operated vehicle (ROV) respectively. From these measurements, transmittance of EPAR was calculated as with the Ramses data.Solar spectral planar irradiance  mounted on a sledgeHow to cite this article: Assmy, P. et al. Leads in Arctic pack ice enable early phytoplankton blooms below snow-covered sea ice. Sci. Rep.7, 40850; doi: 10.1038/srep40850 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Most of the patients who receive myeloablative therapy prior to stem cell transplantation develop oral mucositis (OM). This adverse reaction manifests as oral mucosal erythema and ulcerations and may require high doses of morphine for pain alleviation. OM may also interfere with food intake and result in weight loss, a need for parenteral nutrition, and impaired quality of life. To date, there have been very few studies of evidence-based interventions for the prevention of OM. Cryotherapy, using ice chips, has been shown to reduce in an efficient manner the severity and extent of OM, although clinical applications are still limited due to several shortcomings, such as adverse tooth sensations, problems with infectious organisms in the water, nausea, and uneven cooling of the oral mucosa. The present proof-of-concept study was conducted to compare the tolerability, temperature reduction, and cooling distribution profiles of an intra-oral cooling device and ice chips in healthy volunteers who did not receive myeloablative treatment, and therefore, did not experience the symptoms of OM.Twenty healthy volunteers used the cooling device and ice chips for a maximum of 60 min each, using a cross-over design. The baseline and final temperatures were measured at eight intra-oral locations using an infra-red thermographic camera. The thermographic images were analysed using two digital software packages. A questionnaire was used to assess the tolerability levels of the two interventions.p = 0.0118). The two interventions were equally effective regarding temperature reduction and cooling distribution.The intra-oral cooling device was significantly better tolerated than the ice-chips  contains supplementary material, which is available to authorized users. A majority of the patients who undergo treatment for cancer that involves radiation and/or chemotherapy are at risk of side-effects, such as nausea/vomiting , diarrhoA long-standing concern for patients with cancer of the blood or bone marrow  who are scheduled to receive high doses of chemotherapy is the establishment of an effective and well-tolerated preventive treatment strategy to alleviate OM in conjunction with hematopoietic stem cell transplantation (HSCT). Current recommendations regarding the prevention of OM involve the use of recombinant human Keratinocyte Growth Factor-1 , low-level laser therapy (LLLT), and cryotherapy (CT) .In the field of haematology/oncology, CT using ice chips before, during, and after chemotherapeutic drug infusion has proven to be an effective treatment modality for alleviating OM –15. The Despite the fact that CT efficiently reduces OM in conjunction with HSCT, this cooling method can have adverse events, such as chills, headache, numbness/taste disturbance, and teeth sensations . In addiThe main objectives of the present study were to compare in a randomised cross-over trial with healthy subjects, the tolerability, temperature reduction, and cooling distribution profiles of the intra-oral cooling device and ice chips.https://www.randomizer.org/) was used to assign randomly the subjects to the order in which the two procedures were to be commenced. Half of the subjects started with the cooling device and crossed over to ice chips, while the other half of the subjects undertook the two procedures in the reverse order.This was a prospective randomised cross-over trial to compare a new cooling device and ice chips as cryotherapies. The Research Randomizer software , who were recruited between April 2016 and May 2016 from the Institute of Odontology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. All the participants were healthy and had no medical conditions or used any drugs with substantial impact on the cardiovascular system. None of the participants had mucosal lesions, were smokers or users of oral tobacco products. The characteristics of the participants are listed in Table All the procedures performed in the studies involving human participants were approved by the local Ethical Review Board and in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.The study was approved by the local board of the Department of Oral Medicine and Pathology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. The regional ethical review board in Gothenburg did not consider an ethical application necessary. Informed written consent was obtained by from all the participants.The cooling device . In this manner, the questionnaire was face-validated to ensure that the questions were interpreted as intended.A questionnaire was applied that was specially designed by the authors for this study and that consisted of 15 questions, which were primarily aimed at evaluating the tolerability of the cooling methods for the subjects. The questionnaire also included questions regarding: the reasons why the cooling procedure was not completed or discontinued; and any adverse events experiences for each of the two cooling methods. There was also space to share other comments in running text. Prior to the study, all the questions and response alternatives were tested and discussed with an independent group of participants (http://www.biopix.se) was used to assess the cooling distribution by automatically quantifying the percentage of an image that corresponded to a specific temperature. The Omron M3 Comfort digital monitor  was used to measure blood pressure and heart rate.The FLIR E60(bx)  is an accurate thermographic camera with a level of resolution (320 × 240 pixels) that allows the detection of temperature differences of <0.05 °C  located at the Department of Oral Medicine and Pathology Institute of Odontology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Prior to inclusion, each individual was provided with detailed information and instructions regarding the usage of the two cooling methods, and written informed consent was obtained. The length (in centimetres) and weight (in kilograms) were measured and the BMI was calculated for each subject. Subjects were asked to complete a form to gather information about their medical history. The participants used the cooling device and ice chips for a maximum of 60 min in two separate sessions at least 24 h apart.The cooling device, which was available only in one size, was self-inserted under surveillance and adjusted by the subject until it felt comfortable. A staff member verified a good adaptation to the oral mucosa before the cooling was started.Subjects inserted an ounce of ice chips and were asked to move the ice chips around in the mouth so as to cool as large a part of the oral mucosa as possible. When a melted ice slurry was obtained, the subject gargled for a few seconds before swallowing or spitting out the slurry. If the ice had melted completely another table spoon of ice was inserted immediately.Prior to and immediately after cryotherapy, the temperature level at eight intra-oral locations  were measured using the FLIR E60(bx) system. Blood pressure (systolic and diastolic) and heart rate were measured in the left arm with the subjects in a sitting position using the Omron M3 Comfort digital monitor. Following each cooling session, the subjects answered the questions regarding completed cooling in the questionnaire.In total, 700 thermographic images were captured using FlIR E60(bx). The images were analysed by a blinded observer using the FLIR tools and BioPix software for assessments of temperature reduction and cooling distribution, respectively.The maximum, minimum, and mean temperatures were recorded for each of the eight intra-oral locations before and after the cooling session, using the FLIR tools software. In addition to the temperature data, the software assigned a colour to each temperature, which was subsequently used in BioPix to assess cooling distribution. The analysis with BioPix was, however, only performed on the thermographic images captured after cooling, by calculating the percentage of an image that was covered by a specific colour, equivalent to or less than the mean temperature achieved from the previous temperature analysis at each location.For the statistical analysis, the average value, taking into account the mean for each intra-oral location, was calculated and used both for temperature reduction and cooling distribution results.The primary end-point regarding tolerability was the extent to which an individual expressed a preference for one of the two methods by responding to the question: “Which of the two cooling methods did you tolerate better?”.The secondary end-points were: mean temperature reduction; and mean cooling distribution.t-test, with corresponding confidence intervals (95%). Systemic variables were analysed in the same manner. The quantitative variables were also analysed by ANOVA, including factors for treatment, sequence, period, and subject nested with sequence. The standard Pearson correlation was used for analysing associations between systemic variables and the secondary end-points, by cooling method. The associations between BMI and secondary end-points were analysed in the same manner. A significance level of 5% was used. The analysis of the primary variable was done using the SPSS ver. 23 statistical analysis software package .With 20 participants, the power of the study for detecting a difference in preference of at least 80% (at least 80% of the subjects favour one of the two treatments) was 80%, given a significance level of 5%, using a two-sided sign test. The primary end-point, expressed by “Which of the two cooling methods did you tolerate better?”, was analysed using a two-sided sign test (McNemar’s test) and an exact 95% confidence interval. The secondary end-points of mean temperature reduction and mean cooling distribution were analysed using a paired samples p = 0.0118; 95% CI 0.563 to 0.943). Among the subjects who started with ice chips followed by the cooling device, nine out of ten preferred the cooling device, and for the subjects following the opposite procedure, seven out of ten preferred the cooling device, (p = 0.291).The cooling device was preferred in comparison to ice chips as 16 out of 20 participants (80%) favoured this cooling method  were conducted during the study and thirty-six sessions were completed. Thirty-four of the total cooling sessions, which accounted for 17/20 individuals, completed the entire time span of 60 min with both cooling methods. Two of the remaining three individuals completed one of the two methods each and two sessions were interrupted (one cooling device and one ice chips) because of discomfort. One of the three individuals interrupted two of the four sessions with both the cooling device and ice chips due to hypersalivation and nasal congestion. All of the subjects completed the questionnaires related to tolerability.n = 12) and numbness (n = 11) were the most common adverse events reported for ice chips followed by teeth sensations (n = 8) and pain (n = 5). On the contrary, difficulties with swallowing (n = 15), rubbing discomfort (n = 12) and poor fit (n = 7) were the most common adverse events reported for the cooling device.The adverse events for each of the cooling methods are presented in Table p = 0.795; 95% CI −1.18 to 1.52). The mean cooling distribution was 48.44% for ice chips and 47.33% for the cooling device, i.e., a mean difference of 1.11% . Furthermore, we found no statistically significant mean differences in blood pressure or heart rate change. The systolic BP increased 1.10 mmHg in average for ice chips compared to a decrease of 0.50 mmHg for cooling device, i.e., a mean difference of −1.60 mmHg . Corresponding figures for diastolic BP were average increase of 2.70 mmHg for ice chips, 0.55 mmHg for cooling device and hence a mean difference of 2.15 mmHg . The average heart rate decreased in both groups 8.55 vs. 5.95 meaning that the mean difference was 2.60 beats per minute . BMI did not have any statistically significant impact on temperature reduction or cooling distribution regardless of the method used.The cooling device showed equivalent properties as ice chips in terms of mean temperature reduction. As shown in Tables The ANOVA gave no statistically significant sequence effects. There were, however, two significant period effects for temperature reduction and change in systolic blood pressure Table . The sigThis study was conducted to compare the tolerability levels of an innovative cooling device and ice chips in healthy volunteers, as well as to investigate any adverse events. Overall, cooling was well tolerated, which is in accordance with a previous review article by Kadakia et al. . HoweverAdverse events related to cryotherapy have, to the best of our knowledge, not been given prominence in previous studies, and therefore, have not been carefully evaluated. In the present study, several adverse events were reported for each of the cooling methods, with coldness, numbness, and teeth sensations being more frequently perceived in the cooling sessions with ice chips. This is not surprising, since ice chips at −0.5 °C were used and they were in direct contact with the oral mucosa and the teeth. In contrast, the cooling device is an enclosed channel system with circulating water at 8 °C, so there is no direct contact between the cold liquid and the surrounding tissues. The reason for using a water temperature of 8 °C was to avoid the addition of an anti-freeze coolant. Difficulties related to swallowing, rubbing discomfort, and poor fit were the most frequently reported adverse events for the cooling device, and these complaints may reflect the fact that only one size of the cooling device was available for this study. However, a study using devices of different sizes needs to be conducted to test this hypothesis. Furthermore, poor fit and rubbing discomfort are of great importance as they may cause sensitivity and damage to the oral mucosa, which would potentially worsen the OM. Therefore, a pilot study needs to be conducted with various sizes of the device in patients who are receiving myeloablative treatment, together with an assessment of adverse events. This would ensure that the adverse events experienced in healthy volunteers will not result in damage to the oral mucosa and exacerbation of OM.Pain and nausea were also reported as adverse events for both methods, although these adverse events were reported more frequently in the sessions using ice chips. The nausea may be due to the volumes of water swallowed by the subjects as a consequence of the ice chips melting in the mouth, and the experienced pain is attributable to the low temperature of the ice. Nausea caused by the cooling device might be related to over-extension in some patients.Regarding the adverse events noted, there is a clear trend towards design-related problems with the cooling device, whereas cold sensations and numbness accounted for the majority of the problems experienced with the ice chips. One major advantage of the cooling device over ice chips is that there is the possibility to refine and improve the design to achieve even better comfort, which is not possible for the ice chips.This study also compared the effects of the cooling device and ice chips on mean temperature reduction, mean cooling distribution, and the association between oral cooling and systemic variables. In addition, we investigated the potential correlation between BMI and differences in temperature reduction and cooling distribution. However, analyses of the thermographic images did not show any statistically significant differences between the two methods.This outcome can be explained in two ways. First, the FLIR tools software and BioPix are not specifically designed for investigating intra-oral temperatures, leading to a risk of misinterpretation. Second, although the same conditions were used for both cooling methods, the temperature of the oral mucosa recovers quickly, which means that there is a narrow time-window to capture the images at all eight intra-oral locations. This could cause distortions and further complicate the image analysis.Ultimately, since the study was not dimensioned for these secondary end-points, there is a possibility of type II errors, i.e., the study does not have a sufficient sample size to be able to detect a difference between the methods. However, the possibility that ice chips are superior to the new cooling device and that the study failed to show this due to being underpowered seem unlikely, as the descriptive statistics actually point in the opposite direction, indicating superiority for the cooling device. These results, however, indicate that the same levels of temperature reduction and cooling distribution are achieved using water at 8 °C in the cooling device and ice chips at −0.5 °C.The ANOVA did show two significant period effects, although since both treatments were equally affected by the period effects, these did not affect the estimates of differences between the two methods.The ANOVA did not show any significant effect of sequence, which means that no significant implications of unequal carry-over effects were observed. Naturally, this may be a type II error due to lack of power to detect such a difference, given the limited sample size in the present study. However, we believe that there are good grounds to assume that there are no carry-over effects due to the nature of the present study. First, there are no changes in the underlying health conditions, since we are studying healthy volunteers. Second, none of the treatment effects, e.g., adverse events, such as teeth sensation or rubbing discomfort, are likely to be carried over to the second period.Little is known about oral cooling and its possible associations with systemic variables. Baydar et al. observed no local or systemic side-effects associated with the use of cryotherapy with ice chips . In contOral cooling did not show any statistically significant impact on any of the systemic variables, and BMI had no impact on oral cooling, which is not surprising since the majority of the subjects were within the normal range for BMI. These results are of importance for future clinical studies, as potential systemic effects can hamper medical rehabilitation after chemotherapy. However, further studies are needed to determine if there are genuine systemic effects following oral cryotherapy, particularly in patients who are undergoing myeloablative therapy. Furthermore, the same cooling capacity can be expected in subjects regardless of their BMI values.The prospective randomised cross-over design, which allows all the subjects to test and evaluate both cooling methods, combined with blinded analyses of the thermographic images, provides reliable data and strengthens the impact of the present study. Another advantage is that subjective and objective parameters, as well as systemic associations were evaluated.A limitation of the present study is that it was conducted on healthy dental students, which could have influenced the results, as they knew that the cooling device was a novel cooling method compared to ice chips. The cooling device might be tolerated differently by patients in clinical practice when used in cooling sessions that are longer than 60 min.This study demonstrates that the cooling device is superior to ice chips in terms of its tolerability for the subjects tested. Furthermore, this study shows that the levels of temperature reduction and cooling distribution achieved using these two cooling methods are equivalent. The next step in this research will be to evaluate the cooling device in patients who are receiving myeloablative therapy prior to stem cell transplantation.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 74 KB)Supplementary material 2 (JPG 41 KB)


Platelets are small anucleate blood cells involved in haemostasis. Platelet activation, caused by agonists such as thrombin or by contact with the extracellular matrix, leads to platelet adhesion, aggregation, and coagulation. Activated platelets undergo shape changes, adhere, and spread at the site of injury to form a blood clot. We investigated the morphology and morphological dynamics of human platelets after complete spreading using fast scanning ion conductance microscopy (SICM). In contrast to unstimulated platelets, thrombin-stimulated platelets showed increased morphological activity after spreading and exhibited dynamic morphological changes in the form of wave-like movements of the lamellipodium and dynamic protrusions on the platelet body. The increase in morphological activity was dependent on thrombin concentration. No increase in activity was observed following exposure to other activation agonists or during contact-induced activation. Inhibition of actin polymerization and inhibition of dynein significantly decreased the activity of thrombin-stimulated platelets. Our data suggest that these morphological dynamics after spreading are thrombin-specific and might play a role in coagulation and blood clot formation. Upon endothelial injury of the blood vessel wall, they become exposed to the subendothelial extracellular matrix, which leads to platelet activation and adhesion5. Activated platelets aggregate and form a blood clot to close the site of injury and prevent blood loss6. During activation, a major reorganization of the platelet cytoskeleton is induced10, leading to the transition from a discoid platelet shape to a more spherical shape11. The marginal band of microtubules, which maintains the discoid shape of the resting platelet13, expands after activation, driven by the microtubule motor dynein14. Activated platelets develop filopodia and lamellipodia15 promoting adhesion and spreading on a substrate18. During spreading, the actin network remodels and develops new actin fibers in order to maintain the shape of the spread platelet19. Microtubules in the spread platelet reorganize and redistribute in the cytoplasm20 to support the secretion of granules stored inside the platelet to the extracellular space21. Granula constituents propagate subsequent activation, adhesion, and spreading of platelets and ultimately the formation of a blood clot.Platelets are small cell fragments circulating down the vascular branch22, a non-contact scanning probe microscopy technique excellently suited for imaging the topography of living cells25. The contact-free imaging mechanism of SICM allowed us to investigate platelets without mechanical interference, thus avoiding an additional mechanical activation stimulus27. SICM has been used for imaging living platelets29, for investigating the shape30 and the spreading process31 of platelets, and for measuring their mechanical properties during activation32.In this study we investigated the influence of different activation agonists on the morphology of spread human platelets. We visualized and quantified morphological dynamics of spread platelets with high spatial and temporal resolution using scanning ion conductance microscopy (SICM)We found that thrombin-stimulated platelets exhibit highly dynamic changes in their morphology after completing the spreading process. These dynamics were dependent on thrombin concentration and did not occur in platelets stimulated with other agonists or in platelets activated by contact with various substrates. The dynamics in thrombin-stimulated platelets ceased following inhibition of actin polymerization and following inhibition of dynein, while the integrity of microtubules seemed to play a minor role. Our data suggest that these dynamics in spread platelets are thrombin-specific and might affect coagulation when high thrombin concentrations occur during blood clot formation.We imaged the spreading process of platelets without and with thrombin stimulation prior to contact with the surface. Unstimulated platelets spread until they reached a roundish shape Fig. . The sprt = 60 s,d, pre-factor c, time t, and power-law exponent α 33, indicates a more directed type of motion for the lamellipodium waves and a more random walk-like type of motion for the protrusions. The breakdown observed for the MSD vs. time relationship for times above 60 s was probably caused by the restricted travelling space owing to the confinement by the platelet edge. Some features changed their direction at higher lifetimes (s. above).For quantification of their lateral velocity, lamellipodium waves, and protrusions were tracked  of 0.13 U/mL . An influence of the substrate or its coating on the activity of contact-activated platelets was not observed: platelets spread to glass substrate or poly-L-lysine-, collagen-, or fibrinogen-coated polystyrene substrates had low activity after spreading -adenine) resulted in a similar decrease of the activity  and adenosine deaminase, which are inhibited by EHNA at this concentration40, did not affect platelet activity , when microtubules were in a more fragmented state, redistributed in the cytoplasm 47. Despite the different lateral velocities, lamellipodium waves and protrusions were similarly affected by cytoskeleton inhibitors and had similar lifetimes. This suggests that waves and protrusions have the same underlying cytoskeletal mechanism. Protrusions on the platelet body might move more freely, as they are not confined by the platelet edge, thereby possibly explaining the different velocities and the more random walk-like motion.Lamellipodium waves moved with a higher lateral velocity (50 nm/s) and in a more directed type of motion than the protrusions 20 nm/s) , which typically induce complete platelet activation60 and aggregation61. In contrast, stimulation with ADP, adrenaline, or AA or contact of platelets with polystyrene, glass, collagen, or fibrinogen did not induce dynamics. Our data suggest that the dynamics after spreading are thrombin-specific and occur at high thrombin concentrations prevailing inside blood clots during coagulation62. In this stage, platelets generate contractile forces63, leading to stiffening of the blood clot64. The increased morphological activity, which occurs on a similar timescale as the contraction65 and softening32 of single platelets (about 20 minutes), might facilitate or speed up coagulation and the closure of the injured vessel wall.The dynamics occurred when platelets were stimulated with high thrombin concentrations  was gained from whole blood by centrifugation at 250 g for 20 min. Tyrode-HEPES buffer , 4 mM HEPES), pH 6.5, was added to the PRP at a ratio of 1:1. Remaining red and white blood cells were removed with a second centrifugation step (100 g for 20 min). The platelet containing supernatant was centrifuged at 900 g for 10 min. The resulting supernatant was then discarded and the platelet pellet was re-suspended in Tyrode-HEPES buffer, pH 7.4. For SICM measurements, about 108 platelets were added to a “TC”-treated (“tissue culture”) polystyrene culture dish  containing Tyrode-HEPES buffer and allowed to adhere and spread. For the experiments in Figs All procedures were approved by the institutional ethics committee (270/2011BO1) and comply with the declaration of Helsinki. Informed consent was obtained from all participants. Human platelets were isolated from freshly drawn blood of healthy volunteers mixed with acid citrate dextrose to prevent coagulation as described35 (erythro-9-(2-hydroxy-3-nonyl)-adenine; biomol, Hamburg, Germany) solved in DMSO for dynein inhibition at 1 mM final concentration or ciliobrevin D34  solved in DMSO for inhibition of dynein ATPase; ATA41  solved in ethanol for kinesin inhibition at 10 µM final concentration.The following cytoskeleton inhibitors were used at the given concentrations, unless stated otherwise: Cytochalasin D (Sigma Aldrich) solved in dimethyl sulfoxide (DMSO) for inhibition of actin polymerization at 10 µM final concentration; blebbistatin  solved in DMSO for myosin II inhibition at 100 µM final concentration; Y-27632 (Sigma Aldrich) solved in DMSO for inhibition of rho-associated protein kinase (ROCK) at 50 µM final concentration; nocodazole (Sigma Aldrich) for inhibition of microtubule polymerization at 33 µM final concentration; EHNAFor immunofluorescence measurements, “TC”-treated glass bottom culture dishes were used (Greiner Bio-One). Platelets were fixed in Tyrode-HEPES buffer containing 2% formaldehyde for 5 min during SICM imaging. Afterwards, the platelets were post-fixed for 5 min with ice-cold ethanol at −20 °C. The platelets were rehydrated in phosphate buffered saline (PBS) for 30 min and then blocked with 1% BSA in PBS for 10 min. The following antibodies and concentrations were used for staining cytoskeletal components with 0.1% BSA in PBS for 60 min at room temperature: Anti α-tubulin monoclonal mouse antibody, Alexa Fluor 594 conjugate ; anti dynein intermediate chain monoclonal mouse antibody, Alexa Fluor 647 conjugate ; phalloidin iFluor 488 conjugate . Confocal fluorescence images were recorded with a laser scanning confocal microscope  using a 100 × oil immersion objective and Nikon Elements AR software.67 and the average fluorescence intensity was measured for each individual platelet, indicating its degree of activation.To determine the degree of activation, the expression of extracellular P-selectin of unstimulated and thrombin-stimulated spread platelets was quantified by immunofluorescence. Two separate compartments containing either unstimulated or thrombin-stimulated platelets in one dish were created using two-well culture inserts . Platelets were fixed in PBS containing 2% formaldehyde for 10 min after removal of the culture insert with sterile tweezers. Fixed platelets were blocked with 1% BSA in PBS for 10 min and incubated for 60 min with PBS containing anti CD62P/P-selectin monoclonal mouse antibody, PE conjugate , dilution 1:200. Epifluorescence images of platelets in both compartments were recorded with the same exposure time. The images were then analyzed using the CellProfiler software68 with high temporal and spatial resolution. The setup consisted of a 200 µm xy-scanner  for lateral positioning of the sample, a 15 µm z-scanner  for fast vertical positioning of the nanopipet and a patch clamp amplifier  for ion current measurement. The setup was mounted on an inverted optical microscope  for optical access to the nanopipet and the sample. Borosilicate nanopipets with a typical inner diameter of 80 nm were fabricated using a CO2-laser-based micropipet puller . Topography images were recorded with an ion current trigger of 99.5% of the free ion current and a constant pipet approach and retract speed of 340 µm/s. A pixel resolution of 125 nm/pixel was chosen for all images to fully utilize the lateral imaging resolution of 1.5 times the inner diameter of the nanopipet70. Image sequences were recorded with an average duration of 4 seconds per image consisting of 32 × 32 pixels in a 4 × 4 µm² scan area or with an average duration of 17 seconds per image consisting of 80 × 80 pixels in a 10 × 10 µm² scan area. To increase the contrast of protrusions on the platelet surface, the large-scale curvature of the platelet surface was removed in some topography images . In the obtained activity maps, regions of the platelet with fast and frequent movements during that time interval show higher activity values than regions with slow movements. A global value for the activity of the whole platelet was then gained by averaging all local values within the platelet area. The global values of the momentary activity maps were used in the graphs displaying the time dependence of the activity. The global values of the averaged activity maps were used to compare the activity before and after the addition of substances. For activation and cytoskeleton inhibitor measurements, the activity was determined during 5 min before and 15–20 min (activators) or 10–15 min (inhibitors) after the addition of the respective substance.For tracking the position of moving features in the image sequences, images of the height difference between two consecutive images were calculated , unless stated otherwise. All results were tested using Tukey’s test. Results were considered significantly different for Supplementary Movie S1Supplementary Movie S2Supplementary Movie S3Supplementary Movie S4Supplementary Movie S5Supplementary Movie S6Supplementrary PDF File


Q203 blocks growth of Mycobacterium tuberculosis at nanomolar concentrations, however, it fails to actually kill the bacteria, which may limit the clinical applicability of this candidate drug. In this report we show that inhibition of cytochrome bd, a parallel branch of the mycobacterial respiratory chain, by aurachin D invoked bactericidal activity of Q203. In biochemical assays using inverted membrane vesicles from Mycobacterium tuberculosis and Mycobacterium smegmatis we found that inhibition of respiratory chain activity by Q203 was incomplete, but could be enhanced by inactivation of cytochrome bd, either by genetic knock-out or by inhibition with aurachin D. These results indicate that simultaneously targeting the cytochrome bcc and the cytochrome bd branch of the mycobacterial respiratory chain may turn out as effective strategy for combating M. tuberculosis.Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome  The global tuberculosis epidemic is larger than previously estimated. In 2015, 10.4 million people fell ill with TB, among them 480000 cases of multi-drug resistant TB, and 1.4 million TB patients died in this year1. The WHO ‘End TB’ strategy calls for a drastic reduction of both TB deaths and TB incident rates by 2030. In order to address this unmet medical need and to move forward towards the ‘End TB’ goal, the development of new TB drugs and the design of effective drug combinations is needed.Tuberculosis (TB) chemotherapy has averted 49 million deaths globally between 2000 and 2015, but important treatment gaps still persistMycobacterium tuberculosis as target for treatment of tuberculosis5. Small molecules inhibiting various components of this key energy metabolic pathway have recently been identified9. In oxidative phosphorylation, electrons flow along the enzymes of the respiratory chain and are finally used for reduction of molecular oxygen. Coupled to this electron transport, a proton motive force across the bacterial cytoplasmic membrane is established by the respiratory chain enzyme complexes. The energy of the proton motive force in turn is utilized by the ATP synthase enzyme for synthesis of ATP. In M. tuberculosis, the respiratory chain and ATP synthase are required for growth and for survival. Impairment of the respiratory chain functionality causes a rapid loss of cell viability11.New TB drugs have been discovered and currently are evaluated in clinical trials. The regulatory approval of the ATP synthase inhibitor bedaquiline (BDQ), the first within 40 years for a TB drug, validated the oxidative phosphorylation pathway in bcc complex12, a variant of the cytochrome bc1 complex (complex III) found in the respiratory chain of mycobacteria and other actinobacteria13. Q203 potently blocks growth of M. tuberculosis in vitro and in human macrophages at lower nanomolar concentrations and also displayed activity in a mouse TB infection model12. These features make Q203 a promising candidate TB drug and this compound currently is evaluated in phase 1 clinical trials. However, it has been reported that disabled assembly of cytochrome bcc in M. tuberculosis or genetic knock-out of cytochrome bcc in Mycobacterium smegmatis did not completely abolish bacterial growth15. In these mutants, network adaptation in the respiratory chain can lead to induction of cytochrome bd14, which constitutes an alternative branch of the respiratory chain and has been implied in the bacterial defense against a variety of stresses22. In mycobacteria, cytochrome bd is involved in the defense against hypoxia23, cyanide23, hydrogen peroxide24, nitric oxide25, and a variety of antibacterials including BDQ28. Cytochrome bd also facilitates metabolic adaptation of certain M. tuberculosis laboratory strains, including the reference strain H37Rv, to imidazopyridine-type cytochrome bcc inhibitors29. These adapted strains displayed considerably elevated minimal inhibitory concentrations (MICs) for Q203, effectively evading growth inhibition by these drugs29. Upon knock-out of cytochrome bd the susceptibility for growth inhibition by Q203 was restored29. In in vitro time kill kinetics experiments Q203 acted bacteriostatic against M. tuberculosis H37Rv, even when applied at concentrations of 200–300 × MIC31. However, a recent report showed that Q203 exhibited bactericidal activity against an M. tuberculosis bd-KO strain in vitro and in a mouse infection model31. The adaptability of M. tuberculosis strains and the lack of bactericidal activity may significantly diminish the suitability of the cytochrome bcc complex as antibiotic target and restrict the clinical applicability of Q203 as TB drug. It has been proposed that simultaneously targeting both branches of the mycobacterial respiratory chain might be required to effectively disrupt respiration in M. tuberculosis31.Q203, the lead compound of the imidazopyridine amide class of drugs, targets the cytochrome bd can enhance the activity of a cytochrome bcc inhibitor, Q203, against M. tuberculosis.In this report, we explore if small-molecule inhibition of cytochrome 30, treatment of the M. tuberculosis H37Rv strain used in our laboratory with Q203 resulted in only a marginal decrease of colony forming units  >100 μM  (Table bd (Table In line with previously reported results10 units decrease of colony forming units (cfu) triggered by 25 μg/ml aurachin D  into bactericidal activity Fig. . The enhn D Fig. . These rbcc complex. For safety reasons these experiments were performed with the strongly attenuated M. tuberculosis strain mc2 602034. Q203 inhibited oxygen consumption activity of inverted membrane vesicles (IMVs) from M. tuberculosis strain 6020 in a dose-dependent manner, with an IC50 of ~20 nM . This enhanced effect found for the Q203/aurachin D combination in the biochemical assay may explain why genetic or chemical inactivation of cytochrome bd can augment inhibition of bacterial growth and trigger bacterial killing by Q203.Next, we evaluated the ability of Q203 to inhibit its target, the cytochrome  nM Fig. . However nM Fig. . These r nM Fig. . The com nM Fig. . InhibitM. smegmatis, a fast-growing mycobacterial strain that is not sensitive to growth inhibition by Q203 (MIC > 50 μM)12, was also efficiently blocked by Q203  was comparable to the affinity for M. tuberculosis cytochrome bcc in the employed assay system, although M. tuberculosis and M. smegmatis vastly differ in drug susceptibility. These results demonstrate that the lack of growth inhibition found for M. smegmatis is not caused by insufficient affinity of Q203 for the cytochrome bcc complex in M. smegmatis. As observed for M. tuberculosis, inhibition of respiratory chain activity of M. smegmatis IMVs by Q203 was incomplete . IMVs isolated from an M. smegmatis strain lacking the cytochrome bcc complex14 did not show significant inhibition of respiratory chain activity by Q203  suggests that in M. smegmatis next to cytochrome bd additional cellular components are involved in the defense against Q203.Consistent with these results, we found that genetic inactivation of cytochrome M. tuberculosis. For effective antibacterial compounds bactericidal instead of bacteriostatic activity is highly desirable. Absence of bactericidal activity can be regarded as an argument against further consideration of a drug candidate. As an example, the development of a new imidazopyridine sub-class, the imidazopyridine ethers, was not further pursued, in part based on lack of bactericidal activity of these compounds35. Q203 efficiently blocks growth of M. tuberculosis at nanomolar concentrations, however, this drug acts bacteriostatic on M. tuberculosis and lacks bactericidal activity. Upon chemical inhibition or genetically knockout of the cytochrome bcc branch, respiratory electron transport through the alternate cytochrome bd terminal oxidase alone may be sufficient to maintain mycobacterial viability. Targeting both branches of the respiratory chain may be required for effective shutdown of mycobacterial energy conversion and concomitantly for killing M. tuberculosis31. As proof-of-concept for this strategy, we here showed that inactivation of cytochrome bd by a small-molecule inhibitor or by genetic modification can turn the bacteriostatic activity of Q203 into bactericidal activity.Development of new anti-tuberculosis drugs is urgently needed in order to combat multi-drug resistant strains of bcc complex, but inhibition of respiratory chain activity in M. tuberculosis is incomplete. We regard it as likely that imid(azo)pyridines and structurally related compounds42 as well as structurally not related compounds such as lansoprazol43 that share the cytochrome bcc complex as target also share incomplete respiratory chain inhibition and lack of bactericidal activity against M. tuberculosis. One study published during the peer-review/revision process of our work revealed incomplete growth inhibition and bacteriostatic activity for the phenoxoyalkyl benzimidazoles, which are hypothesized to target the cytochrome bcc complex in M. tuberculosis44.Our results reveal high affinity of Q203 for the cytochrome bd can contribute to efficient killing of M. tuberculosis. Interestingly, the opposite approach, killing M. tuberculosis based on activation of cytochrome bd, has recently been suggested for the triple drug combination Q203/BDQ/clofazimine. The strong bactericidal activity of this combination was attributed to increased cytochrome bd-mediated respiratory electron flux upon inhibition of cytochrome bcc by Q203, thereby facilitating production of reactive oxygen species by clofazimine30. Dysregulation of cytochrome bd function represents an efficient strategy for weakening the defense of M. tuberculosis and apparently can be achieved either way, by inhibition or by activation of this survival factor.Inhibiting the catalytic activity of cytochrome 45 and was kindly provided by Dr. Jennifer Herrmann . All other chemicals were bought from Sigma unless indicated otherwise.Aurachin D was synthesized as described earlierM. tuberculosis strain lacking cytochrome bd was a gift from Dr. Michael Berney (Albert Einstein College of Medicine). M. smegmatis mc2 155 mutants strains lacking either cytochrome bcc14 or cytochrome bd23 were gifts from by Dr. Bavesh Kana (University of Witwatersrand) and Dr. Valerie Mizrahi (University of Cape Town). Replicating bacterial cultures were grown in Middlebrook 7H9 broth (Difco) supplied with 0.05% Tween-80 and 10% Middlebrook albumin dextrose catalase enrichment (BBL) at 37 °C with shaking. If applicable, 50 μg/mL kanamycin or 50 μg/mL hygromycin was added to the medium to select for mutant strains. The attenuated M. tuberculosis strain mc2 602034 was kindly provided by Dr. William R. Jacobs, Jr. (Albert Einstein College of Medicine). Bacterial culture were grown in 7H9 medium (Difco) supplemented with 10% (vol/vol) OADC enrichment , 0.05% Tween-80, 0.2% Casaminoacids, 0.24 mg/ml Pantothenate and 0.8 mg/ml L-lysine. Culture and handling of strains were done in a Biological Safety Level 3 laboratory for M. tuberculosis H37Rv and at Biological Safety Level 2 for M. tuberculosis mc2 6020 and the M. smegmatis strains.A The resazurin microtiter assay (REMA) plate method was performed in 7H9 medium containing 10% (vol/vol) OADC enrichment , 0.05% Tween-80. If applicable, 50 μg/mL hygromycin was added to the medium to select for mutant strains. Q203 and aurachin D solutions were thawed and diluted in the 7H9 medium. Serial two fold dilutions of each drug in 100 µl of 7H9 medium were prepared directly in 96-well plates. Growth controls containing no antibiotic and sterility controls without inoculation were also included. The inoculum was prepared from exponential growth mycobacterial culture adjusted to OD 0.3 then diluted 1:100, and 100 µl was used as an inoculum. The plates were covered, sealed in plastic bags, and incubated at 37 °C in the normal atmosphere. After 7 days of incubation, 40 µl of fresh prepared 0.1 mg/ml resazurin was added to each well, incubated 48 hours at 37 °C, and assessed for color development. A change from blue to pink indicates reduction of resazurin and therefore bacterial growth. The MIC was defined as the lowest drug concentration that prevented this color change.6. The tested concentrations of Q203 and aurachin D were added to inoculum culture and incubated at 37 °C without shaking. At indicated antibiotic exposure time, samples were collected, serially diluted  and subcultured onto 7H11 agar plates supplemented with 10% (vol/vol) OADC enrichment , 0.5% glycerol, 0.4% activated charcoal. Plates were sealed in plastic bags and incubated for 28 days at 37 °C to determine colony forming units (cfu) counts. The lower limit of detection was 10 cfu/mL. All experiments were performed in duplicate.Fast-growing (log phase) mycobacterial cultures were grown to OD 0.8 to 1. Inoculum culture was prepared by diluting original culture to OD 0.4 then dilute 1:150 to achieve colony forming units around 1*1047. Briefly, M. smegmatis and M. tuberculosis mc2 6020 were grown in a pre-culture to late-exponential phase. Cells were sedimented by centrifugation at 6000 × g for 20 minutes. The pellet was washed with phosphate buffered saline  and centrifuged at 6000 × g for 20 min. Each 5 g of cells (wet weight) was re-suspended in 10 mL of ice-cold lysis buffer  including protease inhibitors . Lysozyme (1.2 mg/mL), deoxyribonuclease I  and MgCl2 (12 mM) were added and cells were incubated with shaking for one hour at 37 °C. The lysates were passed three times through a One Shot Cell Disruptor  at 0.83 kb to break up the cells. Unbroken cells were removed by three centrifugation steps (6000 × g for 20 min at 4 °C). The membranes were pelleted by ultracentrifugation at 222,000 × g for one hour at 4 °C. The pellet was re-suspended in lysis buffer and snap-frozen until use. The protein concentration was measured using the BCA Protein Assay kit (Pierce) as described by the manufacturer.Inverted membrane vesicles (IMVs) from the bacterial strains were prepared as described previously2 (pH 6.5). NADH was added as electron donor to a final concentration of 500 μM and oxygen respiration was measured for 3 minutes. Data were normalized relative to solvent (DMSO) control for full activity and to a sample with 10 mM potassium cyanide for complete inhibition. Statistical analysis (t-test) to determine P values and fitting of experimental results with one-site binding curves was done with GraphPad Prism software.Oxygen respiration and the effect of inhibitors on oxygen respiration were measured by polarography using a Clark-type electrode. The electrode was fully aerated (212 μM O2 at 37 °C) and calibrated with sodium hydrosulfite. The inverted membrane vesicles were pre-incubated for three minutes with the inhibitors in a pre-warmed (37 °C) buffer containing 50 mM MES and 2 mM MgClAll data generated or analyzed during this study are included in this published article.Supplementary Figure 1