Caretta caretta) is an endangered marine reptile for whom assessing population health requires knowledge of demographic parameters such as individual growth rate. In Cape Verde, as within several populations, adult female loggerhead sea turtles show a size-related behavioral and trophic dichotomy. While smaller females are associated with oceanic habitats, larger females tend to feed in neritic habitats, which is reflected in their physiological condition and in their offspring. The ratio of RNA/DNA provides a measure of cellular protein synthesis capacity, which varies depending on changes in environmental conditions such as temperature and food availability. The purpose of this study was to evaluate the combined use of morphometric data and biochemical indices as predictors of the physiological condition of the females of distinct sizes and hatchlings during their nesting season and how temperature may influence the physiological condition on the offspring. Here we employed biochemical indices based on nucleic acid derived indices  in skin tissue as a potential predictor of recent growth rate in nesting females and hatchling loggerhead turtles. Our major findings were that the physiological condition of all nesting females (sRD) decreased during the nesting season, but that females associated with neritic habitats had a higher physiological condition than females associated with oceanic habitats. In addition, the amount of time required for a hatchling to right itself was negatively correlated with its physiological condition (sRD) and shaded nests produced hatchlings with lower sRD. Overall, our results showed that nucleic acid concentrations and ratios of RNA to DNA are an important tool as potential biomarkers of recent growth in marine turtles. Hence, as biochemical indices of instantaneous growth are likely temperature-, size- and age-dependent, the utility and validation of these indices on marine turtles stocks deserves further study.The loggerhead turtle ( The impact of anthropogenic activity on biodiversity has now been demonstrated throughout terrestrial and aquatic ecosystems The usefulness of sRD as a measure of physiological condition is related to the fact that the concentration of cellular DNA is relatively constant in the somatic cells regardless of any changes in the organism's environment, while the RNA content of a cell increases as the cellular demand for protein synthesis and growth increases Despite its widespread use in marine fishes and invertebrates, sRD has mainly been validated for application to studies with small marine organisms. The application of this method in studies with large migratory organisms, such as marine vertebrates, should offer important insights, particularly in view of the conservation concern for most marine vertebrate taxa Caretta caretta, is an endangered species with a circumglobal distribution. Human exploitation of this species over the last few centuries has led to dramatic population declines Of the marine megavertebrates, marine turtles are one of the best studied taxa, with all seven species having now been tracked worldwide Satellite tracking has now demonstrated that many populations of marine turtles exhibit plasticity with multiple foraging strategies seen in populations of adult turtles. For example, in loggerhead turtles in some areas, larger adult turtles may be more likely to forage in shallow neritic habitats, whereas smaller adult turtles appear to be more likely to forage in oceanic habitats e.g., standardized RNA/DNA ratio-sRD, RNA concentration and DNA concentration) as predictors of the physiological condition of the females and hatchlings during their nesting season, and to assess how temperature may influence the physiological condition of offspring. We suggest this may help towards development of minimally invasive growth and viability measurement strategies, which are essential in planning future conservation strategies.Establishing techniques with which to supplement mark-recapture and length-frequency analyses for estimating recent growth rates of turtles upon first capture would also substantially improve our ability to evaluate the status of loggerhead turtle populations on a shorter timescale. In addition, loggerhead turtles expend considerable energy migrating from foraging areas to breeding and nesting areas. Loggerhead turtles nest on average every 12 to 17 days during the breeding season and return to breed every 2 to 4 years, presumably because the energetic cost of doing so prohibits breeding more frequently In this study, we aimed to investigate the following hypotheses: regarding adult turtles, (i) Does the physiological condition of females decrease during the nesting season (A1)?; (ii) Is the physiological condition of “large” adult sea turtles associated with neritic habitats different to the physiological condition of “small” adult sea turtles associated with oceanic habitats (A2)? Regarding hatchling sea turtles, (i) Are the hatching and emergence successes of “small” adult sea turtles lower than in “large” adult sea turtles (O1)?; (ii) Are hatchlings produced by “large” females larger than the hatchlings produced by “small” females (O2)?; (iii) Is hatchling vigour correlated with physiological condition (O3)?; and (iv) Does incubation temperature influence the physiological condition of the hatchlings (O4)?The Cape Verde Islands are located in the eastern North Atlantic between 14° 45′ and 17° 18′ N and 22° 38′ and 25° 22′ W, 500 km off the coast of Senegal . The locReserva Natural da Tartaruga, which is included in the national protected area network of Cabo Verde (Decreto-Regulamentar n° 14/2013 de 9 de Maio), operated by the “Direcção Geral do Ambiente”, an agency of the Cape Verdian Ministry of the Environment. The work was ethically approved by the “Direcção Geral do Ambiente” and loggerhead sea turtle tissues were imported to Portugal (University of Algarve) under a CITES permit.The present study was carried out on Ervatão beach, within the min) using a fiberglass tape measure (±0.1 cm) and turtles classified as “small”  and “large”  was established on Benguinho Beach, a natural nesting beach area adjacent to Praia Ervatão.We collected a total of 57 tissue samples from the right front flipper from randomly selected nesting females during the beginning (7th to 12th of July) and end (19th of September to 10th of October) of the 2012 nesting season from one of the main nesting beaches, Praia Ervatão. The skin tissue samples (<2 mg) were taken using a 5-mm biopsy punch, while the female was covering her completed nest with sand. We stored samples in a RNAlater solution at 4°C. Nesting females were measured  over all nests 45 days after egg-laying to collect hatchlings after emergence. Nests were checked throughout the night and again at daybreak to check for hatchling emergence. Within two hours of emergence, we measured hatchling straight carapace length (SCL) with a digital caliper (±0.1 mm), assessed hatchling vigour and collected tissue samples from 3 randomly selected hatchlings from each nest. To assess hatchling vigour we placed hatchlings (n = 10 per nest) on their dorsal side and the time taken for hatchlings to ‘right’ themselves was recorded 2) with one front flipper held flat against the board and isopropyl alcohol applied to disinfect the biopsy site. A single sample was then taken using a 2-mm biopsy punch (with plunger) from the trailing edge of the front flipper proximal to the body out to midway along the flipper In order to investigate if the temperature might influence hatchling physiological condition (as measured using sRD) we manipulated nest temperature as follows: We marked and randomly assigned the 20 nests to one of two experimental treatments: either shading them under a shade cloth made of plastic mesh over the sand surface above the nests ; or leaving them uncovered  . We assuet al. (2001) et al. (2000) E. coli RNA (4 ug uLl–1) (Roche), in the appropriate range of values. The average ratio of DNA and RNA slopes (mean ± SE) was 2.84±0.20 SE. RNA/DNA ratios were standardized (sRD) using this information and the reference slope ratio of 2.4, according to Caldarone et al. (2006) Nutritional condition was assessed using the following nucleic acid acid-derived indices: sRD (standardized RNA/DNA ratios), DNA/mg and RNA/mg per tissue. Nucleic acid analysis was carried out for ecophysiological condition of breeding females in the beginning and at the end of the breeding season and for hatchling condition at the shaded/unshaded treatments. The procedures used to quantify nucleic acids in individual females and hatchlings are outlined in Caldarone www.r-project.org).All graphics and statistical analyses were performed using the open source software R version 3.0.0  and independent variables (time to righting) on dependent variables  were analyzed. We also performed two regression analyses to investigate the relationship among the ecophysiological condition of hatchlings and the hatching success of their nests and their righting time. Significance was assigned at p<0.05.1,45 = 23.4, p<0.001; 1,55 = 25.95, p<0.001; F1,55 = 7.70, p = 0.008, respectively) such that females sampled at the beginning of the season  had more than the double the RNA concentration than the females sampled at the end .Ecophysiological condition of nesting females was higher at the beginning of the season (sRD = 2.9±0.95) compared to the end of the season (sRD = 1.9±0.98) (Fmin≤85 cm and >85 cm respectively) , the analysis of sRD for the two distinct size groups reveal significant differences for the biochemical index   .1,52 = 3.932,p<0,0001; The ecophysiological condition of the hatchlings (sRD) was negatively correlated with righting time . On average, hatchlings from unshaded nests had a higher sRD (1.39±0.75) than the hatchlings from shaded nests (0.9±0.5)  than those from unshaded nests (2.76±2.75) and hatchlings from unshaded nests had a higher average RNA concentration (3.19±2.77) than hatchlings from shaded nests (3.03±2.86), they were not statistically significantly different . In fact, the purpose of this study was to evaluate the ecophysiological condition of loggerhead turtles using novel and cost effective biochemical behavioral indices to infer life history traits. We were able to support the utility of this approach using a population for which a strong dichotomy in foraging ecology is known to exist (Hypothesis A2). Validation of assays for this method with substantial predictive power for estimating growth rates would provide a less invasive alternative to mark and recapture programs for marine vertebrates and would feed into monitoring programs for populations of endangered species. This study represents the first application of tissue nucleic acid content analysis to evaluate the physiological condition or potential growth of loggerhead sea turtles.et al. (2014) Loggerhead turtles gather the energy necessary for reproduction over several years while in their foraging areas, before they migrate to mate and nest. Loggerhead turtles return to beaches in the region in which they hatched (termed ‘philopatry’), typically after intervals of two to four years , thought to depend in part on the quality and quantity of food available in foraging areas et al. (2012) Meyer Larger nesting females exhibited a higher biochemical index (sRD) than “small” nesting females, which may mean that large females have a higher growth potential, due to differences in their diets. Adult female loggerhead sea turtles show a size-related behavioural and trophic dichotomy within several populations: oceanic planktivory by small females and neritic benthivory by large females et al. 2012 Although “large” nesting females exhibited a higher growth potential than smaller nesting females, there were no significant differences in hatching or emergence success (Hypothesis O1). This may suggest that egg quality was similar between the two foraging groups et al. (2012) Buskirk & Crowder (1994) et al. (2004) The results of the present study also indicate that shaded nests produce hatchlings of lower ecophysiological condition (Hypothesis O4). Other studies have suggested that temperature is one of the main factors with the potential to influence reproductive fitness in marine turtles, as well as a number of phenotypic traits, including hatchling sex Nucleic acid concentrations and ratios have become an important tool as biomarkers of recent growth in fish


The concentration of Li ion vacancy generated by the inclusion of aliovalent dopants such as Al3+ plays a key role in stabilizing the cubic LLZO. However, it is found that the site preference of Al in 24d position hinders the three dimensionally connected Li ion movement when heavily doped according to the structural refinement and the DFT calculations. In this report, we demonstrate that the multi-doping using additional Ta dopants into the Al-doped LLZO shifts the most energetically favorable sites of Al in the crystal structure from 24d to 96 h Li site, thereby providing more open space for Li ion transport. As a result of these synergistic effects, the multi-doped LLZO shows about three times higher ionic conductivity of 6.14 × 10−4 S cm−1 than that of the singly-doped LLZO with a much less efforts in stabilizing cubic phases in the synthetic condition.Here, we investigate the doping effects on the lithium ion transport behavior in garnet Li Due to their high ionic conductivities reported, it was proposed that they could be potential alternatives to conventional organic electrolytes. However, the practical issues arising from the instability and handling difficulties related to the unavoidable degradation of sulfide solid electrolytes in contact with oxygen or humid in general atmospheric condition, have significantly impeded the utilization of the sulfide solid electrolytex3Lax(2/3)−□x(1/3)−2TiO3 x1+AlxTix2−(PO4) (LATP)−4 to 10−3 S cm−1 at RT. Nevertheless, there are still serious intrinsic problems that the unwanted decomposition of electrolytes can occur upon the contact with Li metal anode, where Li ions are intercalated into the electrolyte causing the reduction of Ti4+ to Ti3+ at the potential below 1.7–1.8 V vs. Li+/LiOnes of the representative solid electrolytes that have been widely studied include the sulfide systems such as glass-ceramic Li7La3Zr2O12 (LLZO) has been extensively studied owing to several advantages since it was firstly introduced by Murugan et al. in 2007−4 S cm−1 at RT) is about two orders of magnitude higher than that of the latter (~10−6 S cm−1 at RT)2122232425262728293031212223242526272Zr2O7 or other second phases, thereby reducing the ionic conductivity22242526272829303132et al. have revealed that Al contamination from alumina crucible plays a major role in stabilizing the cubic LLZO during thermal reaction283024252627282931322430The Li garnet-type LiIn this study, we introduce a novel route to prepare the cubic LLZO phase with an enhanced Li ionic conductivity by exploiting multi-doping strategy. While multiple steps of thermal reaction are generally required for preparing the cubic LLZO even utilizing doping elements, it is demonstrated that the multi-doping of Al and Ta can stabilize the cubic LLZO more easily with a one-step calcination. Moreover, the ionic conductivity of the multi-doped LLZO increases by about three times compared to the singly-doped LLZO. Our density functional theory (DFT) calculation reveals that the stabilization of the cubic phase is attributed to the more Li vacancy offered by multi-doping of aliovalent dopants, whose disordering nature favors more symmetric cubic phase. More interestingly, the addition of Ta dopants alters the energetically favorable sites of Al dopants from 24d to 96 h Li site in the crystal structure, thereby providing more open space for Li ion transport.1/acd (No. 142) and cubic structure with space group Ia-3d (No. 230). Both structures have unit cell (Li56La24Zr16O96) containing 8 formula units (Li7La3Zr2O12), which La and Zr ions are located in the center of LaO8 dodecahedrons and ZrO6 octahedrons, respectively  the cubic phase began to appear with a noticeable amount of tetragonal phase still present. In contrast, doping with Al  was capable of stabilizing the pure cubic phase, which was in agreement with previous literatures35x−y7−3AlxLa3Zry2−TayO12; x = 0.2, y = 0.2) the cubic phase LLZO was formed more quickly, reducing thermal reaction time to a half (2 h) at 1000 °C. For a comparison, the increased amount of a single dopant up to the level of the multi-doping were also attempted to verify the effect of the absolute amount of dopants in stabilizing the cubic phase  between cubic and tetragonal phases linearly decreases with increasing Li vacancy concentration in good agreement with other previous literatures39233637ic phase , howeverx−y7−3AlxLa3Zry2−TayO12; x = 0.2, y = 0) and multi-doped  LLZO were investigated. The optimized sintering process with the LLZO powder is necessary to obtain a sintered body with an adequate intrinsic ionic path (12 h at 1200 ˚C). However, the doped LLZO has begun to decompose with prolonged hours of sintering leading to the formation of second phases such as La2Zr2O7 and LaAlO3, which might be due to the significant loss of volatile Li during long thermal reaction  than that of the single Al-doped LLZO (2.54 × 10−4 S cm−1) at the same experimental condition. The temperature dependence of total ionic conductivity for both single Al-doped and multi-doped LLZO sintered for 12 h, was plotted according to the Arrhenius equation  was lower than that of single Al-doped LLZO (0.36 eV). The experimental results including the Arrhenius plots of the single Al-doped and multi-doped LLZO sintered for different time, are also summarized for a comparison in where As shown in 313142For this reason, we focused on the preferred sites of Al dopants in both single Al-doped and multi-doped LLZO cubic phases. In −4 S cm−1 and activation energy of 0.29 eV, which are comparable or even superior to the results in literatures wherein the multiple steps and long thermal reaction time are required2122232425262728293031In summary, we have demonstrated that the fast stabilization of cubic phase is feasible for the multi-doped LLZO compared to single Al-doped LLZO, through Li vacancies generated by the inclusion of aliovalent dopants. In spite of the stabilizing effect of Al, the site preference of Al substituting a 24 d Li sites can block the Li ion conductive pathway in the garnet framework. However, the additional Ta doping moves a majority of Al from the 24 d to 96 h Li sites, providing more open space for Li ion transport as well as the increased amount of Li vacancy. Owing to these synergistic effects, the multi-doped cubic LLZO yields a total ionic conductivity of 6.14 × 102CO3 (99.9%), La2O3 (99.9%), ZrO2 (99.9%), Al2O3 nanopowder (<50 nm particle size), Ta2O5 (99.9%) were purchased from Sigma-Aldrich and used as received. Garnet-structured cubic LLZO solid electrolytes with the desired amounts of doping elements  was prepared through a solid-state reaction. The whole precursor materials were weighed according to the stoichiometry and mixed by planetary ball-milling in isopropyl alcohol at 200 rpm for 10 h. No excess Li was added into the mixture. After drying the mixture, the collected powder was calcined at 1000 ˚C for 2–4 h to obtain the cubic LLZO powder. The calcined LLZO powder was reground and pressed into a pellet at 50 MPa. Finally, the pellet was sintered at 1200 ˚C from 1 to 24 h to ensure the formation of well-dense body. In the sintering step, the pellet was covered with the mother LLZO power to minimize the Li loss at high temperature. During the synthesis of cubic LLZO powder and the sintering of pellet, Al2O3 crucible with boron nitride (BN) coating was used to prevent the unintentional Al doping.Li−1  to 105 Hz) in the temperature range of −20 to 100 ˚C. Prior to measurement, the ~6 μm thick Cu electrode was evaporated on both sides of LLZO pellet to form the current collector.The structural analysis of LLZO power and the sintered pellet was performed by X-ray diffraction (XRD) pattern using an X-ray diffractometer  equipped with a Cu target and accumulative detector. The cross-section microstructure of solid electrolyte was analyzed with a scanning electron microscopy . The bulk density of the sintered LLZO was calculated from the weight and physical dimensions. By dividing the bulk density by theoretical density, the relative density was determined. The ionic conductivity of LLZO pellet was measured by the AC impedance method using a frequency response analyzer , with Perdew-Burke-Ernzerhof (PBE) spin-polarized generalized-gradient approximation (GGA) functional4556La24Zr16O96) contain 8 formula units (Li7La3Zr2O12). It is noted that the obtained cubic structure has excess Li sites because of partially occupied Li sites . With the assigned oxidation states, excess Li atoms were removed by highest electrostatic energy first in sequence.Initial structures of tetragonal and cubic LLZO were obtained from previous literatures33Li sites . To deteFor the calculation in How to cite this article: Shin, D. O. et al. Synergistic multi-doping effects on the Li7La3Zr2O12 solid electrolyte for fast lithium ion conduction. Sci. Rep.5, 18053; doi: 10.1038/srep18053 (2015).


Arctic amplification of global warming has led to increased summer sea ice retreat, which influences gas exchange between the Arctic Ocean and the atmosphere where sea ice previously acted as a physical barrier. Indeed, recently observed enhanced atmospheric methane concentrations in Arctic regions with fractional sea-ice cover point to unexpected feedbacks in cycling of methane. We report on methane excess in sea ice-influenced water masses in the interior Arctic Ocean and provide evidence that sea ice is a potential source. We show that methane release from sea ice into the ocean occurs via brine drainage during freezing and melting i.e. in winter and spring. In summer under a fractional sea ice cover, reduced turbulence restricts gas transfer, then seawater acts as buffer in which methane remains entrained. However, in autumn and winter surface convection initiates pronounced efflux of methane from the ice covered ocean to the atmosphere. Our results demonstrate that sea ice-sourced methane cycles seasonally between sea ice, sea-ice-influenced seawater and the atmosphere, while the deeper ocean remains decoupled. Freshening due to summer sea ice retreat will enhance this decoupling, which restricts the capacity of the deeper Arctic Ocean to act as a sink for this greenhouse gas. The Arctic is considered a region where noticeable and rapid global change is taking place and where the effects, particularly on sea ice, are dramatic, albeit that the area is relatively small globally seen12359101314Tracing this process the current study reveals a unique two stage scenario: methane release from sea ice into sea water and seasonal efflux to the atmosphere. We combine oceanographic and geochemical data and highlight new information on the impact of oceanographic processes i.e. convection and haline stratification on the geochemical behaviour of methane, especially considering the pronounced seasonality resulting in the increasing decline of summer sea ice. Our studies focus on unexpected feedbacks concerning the methane cycle in the Arctic e.g. the buffering capacity of the winter mixed layer in summer and the isolation of the deeper Arctic Ocean. The permanent isolation influences the sink capacity of the Arctic Ocean which becomes relevant for methane released by terrestrial sources in the Arctic. Hence aiming on the interaction between sea ice and sea water is fundamental and has up to now not yet been considered when it comes to understanding the balancing of trace gas fluxes in the ice covered ocean. Concerning the pronounced seasonality in trace gas fluxes our observations highlight the differences between trace gas exchanges in ice-covered Polar Regions relative to open water areas in lower latitudes.The current study is based on the analysis of concentrations, stable carbon isotope ratios and aerobic oxidation rates of methane in surface water and sea ice brine coupled with the hydrographic data along two transects in the central Arctic Ocean occupied by RV Polarstern between early August and late September 2011 (Sea ice influenced surface water encompasses the winter mixed layer (WML) and the melt water layer (MWL). The MWL comprises the upper 20 m and is formed by melting summer sea ice . The WML1314In the interior Arctic methane is super-saturated in the WML and MWL and under–saturated below the halocline in UHW (>60 m) . We sugg13C signature from the atmospheric background illustrate the strong evidence for the presence of additional sources of methane in surface water. We compare the measured data with calculated curves in order to establish whether the methane pool in seawater is modified by oxidation and mixing * ln f + (δ13CCH4)0 is used to calculate the δ13C signature of methane which results when methane is being microbially consumed  is the methane consumed by oxidation and (δ13CCH4)0 is the initial isotopic composition). RC-1 0 is −45‰ corresponding to the atmospheric δ13CCH4 value (−47‰)13C and deviates clearly from the RC-1 curve. These values are localized between RC-1 and a second Rayleigh curve 0 −75‰, i.e. the end member of methane detected in brines (blue squares). A Rayleigh curve describes ideal conditions, assuming that only one source and only one sink (oxidation) exists. The measured data have been compared with calculated curves in order to check if the methane pool in seawater is modified by oxidation. Deviations of the measured values from the calculated Rayleigh curves reflect the influence of additional processes, i.e. mixing with sources with different isotope signatures and sea-air flux. Since not only oxidation but a combination of several processes modifies the isotopic signature of methane in sea water we also displayed our data using a Keeling type plot.i.e., δconsumed .  is more depleted in 13C than methane in UHW and values scatter around an oxidation line with methane from brine as end member (Compared to the atmospheric backgroundype plot . Differed member  and 5.13C signature (−36‰ to −75‰) of methane in brine are indicative of extensive methane cycling within sea ice i.e. production and consumption, as both processes induce isotopic fractionation. Oxygen depletion in brine channels−1The large potential of sea ice to discharge methane is evident in methane super-saturation of up to 8000% in brine . FurtherMethane in sea ice may be present in two phases; dissolved in brine or most likely in small gas bubbles formed during freezing. Temperature and salinity changes lead to a sharp decrease in methane solubility, in which case a much larger amount of methane will accumulate as gas in the ice12121314In spring, methane in the ice may be released to the MWL, when basal melting starts but the top of the sea ice still remains impermeable, inhibiting direct methane release from sea ice to the atmosphere. Subsequently, sea water or melt water will flush the brine channels and gas bubbles included in the brines will re-dissolve. Several freezing and melting events may contribute to locally different levels of super saturation in the MWL  and 6.3H-CH4 tracer and with a median of 0.0009 nM/d with a 14C-CH4 tracer, respectively. Although both tracers increase ambient methane concentrations by 1.7 nM and 457 nM, respectively, an increase in uptake rate due to an increase in substrate was not observed over three days incubation. These pulse chase experiments illustrate that the abundance and activity of methanotrophic bacteria is low in the water investigated. Methanotrophs might be limited due to unfavourable growth conditions  ranges between 1.5 and 1.9 nM relative to the methane equilibrium concentration (see methods). By extrapolating the methane surplus in the top 10 m to the area of seasonal sea-ice melting  with a flame ionization detector (FID). For gas chromatographic separation we used a packed column (Porapac Q 80/100 mesh). The GC oven was operated isothermally (60 °C) and the FID was held at 250 °C. Two sets of standard gas mixtures were used for calibration. The standard deviation of duplicate analyses was 5%. This high overall error is almost exclusively due to the gas extraction procedure and not to GC precision, which had an error of only 1%. After GC analyses an aliquot of the extracted gas was transferred into pre-evacuated glass containers for analysis of the stable carbon isotopic signature on shore.We measured concentrations, stable carbon isotope ratios and aerobic oxidation rates of methane in surface water and sea ice brine along two hydrographic transects in the central Arctic Ocean occupied by RV Polarstern between early August and late September 2011. The first transect runs from 82.5°N, 50en Basin . Coverinδ13CCH4values were determined using a Delta XP plus Finnigan mass spectrometer. The extracted gas was purged and trapped with PreCon equipment (Finnigan) to pre-concentrate the sample. All isotopic ratios were given in a δ-notation relative to the Vienna Pee Dee Belemnite (VPDB) standard using conventional delta notation. The reproducibility derived from duplicates was 1–1.5‰.The 13C values (R2 = −0.84) is given for methane in UHW when y- intercept is −47‰. In sea ice-influenced water (i.e. WML and MWL) a correlation is missing when y- intercept is −47‰ (R2 = 0.0554) but given when the y- intercept is −55‰. This different correlation refers to deviating methane sources in both water masses.A significant correlation between 1/methane concentration vs. the δex situ tracer incubations in order to quantify the methane consumption. Methane oxidation rates were determined from ex situ incubations of water samples in 100 ml serum vials. Samples were incubated with 50 μl gas mixture comprised of 3H-labelled CH4 (200–300 Bq) and a second set was incubated with 10 μl of 14C-labelled CH4 (12000–15000 Bq). The samples were subsequently shaken to facilitate tracer dissolution and then incubated in the dark at 0 °C. Tracer consumption was measured after 48 h and 72 h. Incubations with 14C-CH4 were terminated by adding a 5 ml headspace and injecting 0.5 ml of 10 M NaOH so that the remaining 14C-CH4 accumulated in the headspace and the produced 14C-CO32- and 14C-biomass was trapped in the aqueous NaOH solution. Separation and activity measurement of 14C-CH4 and 14C-CO32− was carried out analogous to previous measurements of CH4 turnover in sediments14C-CH4 in the headspace was combusted to 14C-CO2, while 14C-CO32− was converted to 14C-CO2 through acidification with HCl. In either case, 14C-CO2 was then trapped and the radioactivity was measured by wet scintillation counting. Incubations with 3H-CH4: Total activity (3H-CH4 + 3H-H2O) was measured in 1 ml of sample aliquot by wet scintillation counting and activity of 3H-H2O was measured after sparging the sample for ≥30 min with nitrogen gas to remove remaining 3H-CH4. CH4 oxidation rates (rOX) were calculated assuming first order kinetics: rOX = k’ [CH4] where k’ is the effective first order rate constant calculated as the fraction of labelled CH4 oxidized per unit time and [CH4] is the ambient CH4 concentration. Killed controls resulted in methane oxidation rates near zero.Water was sampled directly from the ice floes through ice core holes for How to cite this article: Damm, E. et al. Methane excess in Arctic surface water-triggered by sea ice formation and melting. Sci. Rep.5, 16179; doi: 10.1038/srep16179 (2015).


The mechanism of droplet drying is a widely concerned fundamental issue since controlling the deposition morphology of droplet has significant influence on printing, biology pattern, self-assembling and other solution-based devices fabrication. Here we reveal a striking different kinetics-controlled deposition regime beyond the ubiquitous coffee-ring effect that suspended particles tend to kinetically accumulate at the air-liquid interface and deposit uniformly. As the interface shrinkage rate exceeds the particle average diffusion rate, particles in vertical evaporation flow will be captured by the descending surface, producing surface particle jam and forming viscous quasi-solid layer, which dramatically prevents the trapped particles from being transported to drop edge and results in uniform deposition. This simple, robust drying regime will provide a versatile strategy to control the droplet deposition morphology, and a novel direction of interface assembling for fabricating superlattices and high quality photonic crystal patterns. The evaporation of solvent brings about not only the concentration of solute, but also the spatial redistribution of the dispersed phase1234579108131415311678131842021In this work, we find that if the droplet dries rapid enough, particles tends to accumulate at the air-liquid interface rather than at the drop edge and directly deposit in the interior . Thus, u34ω) varying from 10−1 to 10−4 and droplet sizes (V) vary from 100 μL to 0.5 μL. Similar results are obtained for other particles with hydrophobic surface or polydispersed size —ranging from room temperature to near boiling point of water (90 °C) with constant humidity (50%)—to study the drying behavior at different evaporation rate. Water drops containing monodispersed hydrophilic polystyrene nanospheressed size .Profile of each deposition obtained from different drying conditions demonstrates the evolution of the gradually suppressed coffee-ring effect, growth of the center-region and consequent diminution of the ring-region. In order to quantify the behavior shown in 252627Why particles tend to deposit at center when the evaporation temperature increases? We initially deduced that the suppressed coffee-ring effect might be caused by the thermal Marangoni flow because of enlarged temperature difference between drop edge and drop apex on hot plate  However,3As the influence of thermal Marangoni flow shows to be tiny, we speculate that this suppressed coffee-ring phenomenon may stem from a surface accumulation process caused by rate-controlled interface capture effect313233Snapshots of region at drop apex  inset co4The colors evolution of this layer from red to green further confirms the ordered assembling of particles on surface. Photonic band gap (PBG) of periodic dielectric materials, known as photonic crystal, demonstrates typical structure color3637vi = h/tf grows faster than the particle average diffusion rate xp = 2(Dt/π)1/2 (per second) when rising the evaporation temperature. Here h is the original height of droplet, tf the final evaporation time; the diffusion constant D is estimated from the Stokes-Einstein relation D = kBT/6πηr, where kB is Boltzmann’s constant, T the temperature, η the viscosity of solvent, and r the hydrodynamic radius of the particles which approximates to spherical particle radius. When the surface descends faster than the particle diffusion, particles of random walk in vertical evaporation flow will be captured by the drop surface ) and buffer agent of ammonium bicarbonate (6.30 mmol) is dissolved previously. Keep the reaction mixture at 70 °C for 0.5 h. Following the addition of initiator for three times, aqueous solution of ammonium peroxodisulfate , keep stirring at 80 °C for 1.5 h, then increasing the temperature to 80 °C before the second addition of APS, and react for another 2 h. Finally, the polymerization carries out at 80 °C for 2 h with continuous stirring for the third addition of APS solution. The resulting latex spheres are used directly without purification. The dispersity of the latex spheres is about 0.005, which is determined by using a ZetaPALS BI-90plus (Brookhaven Instrument) and SEM image. The hydrophobic monodispersed (diameter 255 nm) polystyrene nanospheres are bought from Unisizetech Co. Ltd., China.Monodispersed hydrophilic polystyrene nanospheres are synthesized by one-step emulsion polymerization using our modified previous method24T = 90 °C) for every ten degrees with constant humidity (50%). We study mass fraction (ω) varying from 10−1 to 10−4 and droplet sizes (V) vary from 100 μL to 0.5 μL. Trace amount of surfactant  is added to investigate the fixation effect of surfactant molecular to colloidal particles on the air-liquid interface. Droplets containing other particles with hydrophobic surface or polydispersed size (diameter 170 nm and 360 nm) are measured at the same condition. The environment humidity when dying on hot plate is controlled with the help of a humidifier.Water drops containing monodispersed nanospheres (diameter 216 nm) on untreated clean silicon wafer with contact angle about 45° are evaporated in constant temperature & humidity chamber with precise temperature and humidity control—ranging from room temperature to near boiling point of water  to measure the profile of each deposition obtained from different drying conditions. The profile variation of each deposition formed at different drying conditions indicates the degree of the suppressed coffee-ring effect. The cross section area fraction of the ring-region and center-region in final deposition are measured by software program . The ordered assembling of the monodispersed nanospheres in deposition are confirmed by SEM image (JEOL S-4800).The drying process is recorded via stereomicroscope (Zeiss SteREO Discovery. V8 microscope integrated AxioCam MRc 5 CCD camera) on hot plate  customized for microscope. Two separate light sources from side view of droplet are used to obtain real three-dimensional visualization stereoscopic images, which could reveal detail information on drop surface. The surface assembling process is observed via optical microscope (Leica DM2500), focusing on region about 1mm at drop apex and tracing the descending surface to acquire snapshots of different drying stages. The air-liquid interface decreasing rate is directly recorded on an OCA20 machine  at different substrate temperature and measured by software program .Surface reflection spectra and its evolution are detected by Ocean Optics  HR 4000 fiber optic UV−vis spectrometer in reflection mode, with incident light normal to the drop apex. The spectrometer is integrated on a microscope (OLYMPUS BX51) to monitor microdomain reflection spectral on droplet, characterizing the formation of crystal domain shown in Real time infrared thermal image is acquired by using an IR camera (FLIR A655sc) to investigate the temperature distribution and its evolution on droplet surface. The image is obtained from vertically by drying droplets on silicon with different temperature controlled by hot plate .How to cite this article: Li, Y. et al. Rate-dependent interface capture beyond the coffee-ring effect. Sci. Rep.6, 24628; doi: 10.1038/srep24628 (2016).


Diet has a great impact on the risk of developing features of metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and cardiovascular diseases (CVD). We evaluated whether a long-term healthy Nordic diet (ND) can modify the expression of inflammation and lipid metabolism-related genes in peripheral blood mononuclear cells (PBMCs) during a 2-h oral glucose tolerance test (OGTT) in individuals with MetS.n = 213) with MetS, randomized to a ND group or a control diet (CD) group applying an isocaloric study protocol. In this sub-study, we included subjects (n = 89) from three Nordic centers: Kuopio (n = 26), Lund (n = 30), and Oulu (n = 33) with a maximum weight change of ±4 kg, high-sensitivity C-reactive protein concentration ≤10 mg L−1, and baseline body mass index <39 kg m−2. PBMCs were isolated, and the mRNA gene expression analysis was measured by quantitative real-time polymerase chain reaction (qPCR). We analyzed the mRNA expression changes of 44 genes before and after a 2hOGTT at the beginning and the end of the intervention.A Nordic multicenter randomized dietary study included subjects (TLR4), interleukin 18 (IL18), and thrombospondin receptor (CD36) mRNA transcripts and significantly up-regulated the expression of peroxisome proliferator-activated receptor delta (PPARD) mRNA transcript after the 2hOGTT compared to the CD.The healthy ND significantly down-regulated the expression of toll-like receptor 4  contains supplementary material, which is available to authorized users. The metabolic syndrome (MetS) is a cluster of risk factors increasing the risk of type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD)  has been shown to improve lipid profile among hyper-cholesterolemic subjects  include monocytes and lymphocytes, which are cells central in inflammation. These cells circulate in the body and are exposed to nutrients, bioactive food components, and metabolic tissues. Alterations in gene expression levels in these cells may therefore reflect systemic health  is such a stress response, monitors the ability of the body to respond to glucose intake, and is primarily used for addressing the degree of glucose tolerance and insulin resistance  healthy ND could modify the expression of inflammation and lipid metabolism-related genes in PBMCs during 2hOGTT in individuals with MetS.P = 0.330, P = 0.845, P = 0.196, and P = 0.681, respectively)  to 2h (after OGTT) was observed in the whole study population (q < 0.0001) ) , q < 0.000 (Additioq < 0.000 (AdditioTLR4) , interleukin 18 (IL18) , and CD36  compared to the CD after the OGTT (Table PPARD)  compared to the CD after the OGTT  activity  and lipid metabolism related genes  were up-regulated, after the OGTT in the whole study population, confirming the use of PBMC gene expression analysis as a model system to detect metabolic responses after an OGTT  should be employed to extend our biological and clinical understanding of the data presented here.The study design and participants have been described in detail elsewhere  criteria for MetS  out of the 166 subjects who completed the SYSDIET study , Uppsala (n = 9), and Reykjavik (n = 15), because the Aarhus study center did not collect PBMC samples, and the number of PBMC samples was limited from Uppsala (n = 9) and Reykjavik (n = 5). So, by excluding these two centers, we reduced variance. We also excluded subjects with high-sensitivity C-reactive protein (hs-CRP) concentration higher than 10 mg L−1 at baseline or after the intervention (n = 6), baseline BMI above 39 kg m−2 (n = 3), and body weight change more than 4 kg during the intervention (n = 8). Since five subjects were excluded from the analysis due to low quantity of RNA (n = 3) or problems with the TaqMan Array Micro Fluidic Cards (n = 2), we analyzed data from 89 subjects (n = 49 in ND and n = 40 in CD)  was performed after an overnight fast at baseline and at the end of the intervention. Blood samples for PBMC isolation were taken at the time points 0 and 120 min.After blood collection, PBMCs were isolated at baseline and at the end of the intervention at time points 0 and 120 min by using the BD Vacutainer Cell Preparation tubes according to the manufacturer’s instructions  and stored as pellets at −80 °C for further analysis. Total RNA isolation was performed centrally at the Karolinska Institute . The total RNA was isolated using the RNeasy Mini Kit according to the manufacturer’s instructions . RNA quantity and quality measurements were performed using a Nanodrop ND-1000 Spectrophotometer  and Agilent 2100 Bioanalyzer , respectively. RNA from all samples was reverse transcribed by a high-capacity cDNA reverse transcription kit . Quantitative real-time polymerase chain reaction (qPCR) was performed on an ABI PRISM 7900HT (Applied Biosystems) using TaqMan Array Micro Fluidic Cards (Applied Biosystems). The target genes are shown in the Additional file TBP) was used as reference gene for normalization. Briefly, the Ct values of each target gene were normalized to the Ct values of the TBP (=ΔCt). The fold change in mRNA gene expression from TBP was calculated at fasting (0h) and after (2h) OGTT (2−ΔCt) at baseline and at the end of the intervention. The fold change in mRNA gene expression from fasting was calculated as 2−∆ΔCt at baseline and at the end of the intervention, as 2−ΔCt2hOGTT was divided by 2−ΔCt0hOGTT.The relative mRNA level for each transcript was calculated by the ∆Δ cycle threshold (Ct) method  were carried out on serum cholesterol, fasting glucose, and insulin . Data are given as the median (25–75th percentiles). Linear multivariable regression analyses were also carried out to test the independent effect of the study groups on the dependent variable fold change at the end of the intervention. The analyses were adjusted for the independent variables fold change at baseline and study center. In the presentation, β denotes the regression coefficient of the treatment group. Fold changes at baseline and at the end of the intervention (2−∆ΔCt) were log2 transformed before the analyses to improve normality. To account for multiple testing, we applied false discovery rate (FDR) analysis and q < 0.05 (FDR < 5 %) was considered significant. Calculations were performed using IBM SPSS Statistics version 20  and R version 3.2.0.Linear multivariable regression analyses were used to test the independent effect of the study groups on the change in glucose, insulin, triglycerides, and free fatty acids from 0h (fasting) to 2h (after OGTT) at the end of the intervention as well as on the dependent dietary intake variables at the end of the intervention. The effect of the independent variable study group was adjusted for the corresponding baseline variable, study centers, gender, log10-transformed age, and body weight at the end of the intervention. Changes in gene expression from 0h to 2hOGTT within the whole study population were tested with Wilcoxon signed rank test (2


Drosophila Genetic Reference Panel to map polymorphic variants affecting natural variation in mushroom body morphology. We identify 139 genes and 39 transcription factors and confirm effects on development and adult plasticity. We show correlations between morphology and aggression, sleep and lifespan. We propose that natural variation in adult brain size is controlled by interaction of the environment with gene networks controlling development and plasticity.Genetic variation in brain size may provide the basis for the evolution of the brain and complex behaviours. The genetic substrate and the selective pressures acting on brain size are poorly understood. Here we use the  The mushroom bodies (MBs) in an insect brain integrate and process sensory information. Using fully sequenced/inbred lines of the Drosophila Genetic Reference Panel, this study performs genome wide association analyses and identifies candidate genes affecting MB size, and uses RNAi to functionally validate the identified loci. The brain plays a central role in controlling the social interactions of animals and their interactions with the environment. Visual, auditory, olfactory, tactile and gustatory sensory stimuli are integrated by the brain and result in a behavioural response appropriate for the context. Key higher order brain centres for integration and processing of sensory information are the mammalian cerebral cortex and the insect mushroom bodies (MBs). The size of the cerebral cortex and the MBs has been regarded as a proxy for cognitive ability and behavioural plasticity. For instance, the social brain hypothesis states that the evolution of the large brain size of primates is driven by the requirement to live in large groups2Drosophila MBs are derived from a total of eight MB neuroblasts, while Apis MBs are produced by four neuroblast clusters each consisting of 500 neuroblasts2611Drosophila, MB fibre number has been related to age, sex and experience13Variation in brain size in the wild has been studied using two complementary approaches: interspecific comparative studies of the relationship between brain size and behavioural and environmental factors, and analyses of adaptive phenotypic plasticity6Drosophila MBs consist of ∼2,500 Kenyon cells projecting their axons rostroventrally through the peduncle, where at the MB heel they form distinct lobesThe Drosophila Genetic Reference Panel (DGRP)15In the current study, we use the inbred, sequenced lines of the We assessed morphology of the MB α and β lobes in 40 DGRP lines. These lobes are straightforward to visualize and we have previously optimized their morphometric analysis1718Surprisingly, we found gross morphological defects in 31 of the lines (77.5%), including thin lobes, short lobes, missing lobes, abnormal lobes  and fusion of β lobes. Some of the phenotypes were highly penetrant in certain lines, while others were seen more sporadically . We attr1718r) for each MB parameter and aggressive behaviour, copulation latency, fitness, startle response, ethanol resistance, starvation resistance, lifespan, chill coma recovery and sleep traits2021r=−0.410, P=0.009), β lobe width and fitness , β lobe length and night time sleep , α lobe width with the number of sleep bouts during the day  and β lobe length with the number of sleep bouts during the night  associated with MB morphologyWe performed single marker GWA analyses for the four genetically variable traits (length and width of the α and β lobes). At a reporting stan), frizzled (fz), Protostome-specific GEF (PsGEF), Protein tyrosine phosphatase 10D (Ptp10D), misshapen (msn), vein (vn)), four act in pathways that modulate MB development but have themselves never been linked to MB development (APP-like protein interacting protein 1 (Aplip1), Ecdysone-induced protein 63E (Eip63E), Ecdysone-induced protein 75B (Eip75B), JIL-1) and two are involved in adult MB function (slowpoke (slo), pumilio (pum))282930313233343536We mapped 39 candidate genes associated with α lobe length, 37 with α lobe width, 35 with β lobe length and 54 with β lobe width. These comprised 139 unique genes, as 18 genes were associated with two MB traits and four were associated with three mushroom body traits . Six of lola), tailless (tll) and ftz transcription factor 1 (ftz-f1) have been previously shown to be involved in MB development or function1741(dsf) and lethal of scute (l(1)sc) are expressed in the MBs43Abd-B) and hunchback (hb) interact with pathways with a previously described function in MB development or function45To assess whether variants in non-coding regions that are associated with MB size could be in regulatory regions, we used the Multiple EM for Motif Elicitation tool to identify recurring motifs for these variants for each of the genetically variable traitsIn summary, we identified 139 candidate genes and 39 candidate transcription factors associated with morphological variation in MB lobes. Although many of these genes are involved in development, morphogenesis and metamorphosis, only eight genes and four transcription factors have been previously shown to act in the MBs.eyeless gene). In total, we found a total of 20 unique genes which showed expression in the MBs. We included pum as a candidate gene for functional validation because it is known to be expressed in the MB and play a role in synaptic plasticity in the adult brain, although a FlyLight line was not availableacj6). In addition, we determined that all enhancer sequences in the FlyLight lines corresponded to the reported gene by means of PCR  affected all four traits; lola affected α lobe length and width and β lobe length, and slamdance (sda) affected α lobe width and β lobe length. crooked legs (crol), pou domain motif 3 (pdm3) and bric a brac 1 (bab1) specifically affected α lobe length and tailup (tup), jim, lethal (3) neo8 (l(3)neo8) and trachealess (trh) specifically affected α lobe width  affect plasticity in α lobe length and width and β lobe width and jim lovell (lov) affects plasticity of α and β lobe width. The other genes have specific effects on plasticity: slamdance (sda) and doublesex-Mab related 99B (dmrt99B) for α lobe length; bubblegum (bgm) for β lobe length; and spire (spir) and schlank for β lobe width.In many insect species, the MB is a dynamic structure in the adult brain, able to change in an experience-dependent manner6913asticity . pum andAnalysis of the genomic architecture of the DGRP panel highlighted extensive natural variation in the genomes of these flies204951lola which was linked to all four MB traits, providing a proof of concept for our approachOur GWA analyses identified many variants that are associated with variation in MB morphology, allowing us to identify genes and putative transcription factors involved in MB development. Of the 139 unique candidate genes, only eight have been previously reported to be involved in MB morphology or function28293032333435pum, which is known for its role in behavioural and synaptic plasticity and is expressed in the MBs, had large effectspum is also required for adult MB structural plasticity.In addition to our analysis of the developmental roles of the candidate genes in the MBs, we performed the first systematic study of the genetic basis of variation in adult brain morphometry. We showed that our approach is valid to identify genes involved in structural plasticity in the MB. We investigated 22 genes, of which eight showed significant effects on adult MB morphology. Interestingly, RhoGAPp190 Drosophila Resource Center .Flies were reared on standard cornmeal-agar-molasses Adult brains from male flies were dissected and processed for immunohistochemistry. Mouse monoclonal anti-fasciclin 2 antibody  was used to visualize MB α and β lobes171819Length and width of the α and β lobes of the MBs were measured by using the analySIS FIVE software and expressed as values relative to the distance between the α lobe heels1718L+H+L × H+ɛ, where L denotes DGRP line, H is hemisphere and ɛ the within line variance. We computed the variance components (σ2) for each of these terms using restricted maximum likelihood and estimated broad sense heritabilities (H2) as H2=(σL2+σL × H2)/(σL2+σL × H2+σɛ2).We partitioned the variation in the length and width of the α and β MB lobes using random effects factorial analysis of variance models of form Y=Y=μ+SNP+ ɛ to evaluate each segregating siteY is the phenotype, μ is the overall mean, SNP is the genotype and ɛ is the variance among line means within each genotype class. We used a nominal P value<10−5 as the reporting threshold for nominating candidate genes for functional validation. We annotated the site class of each significant variant as genic  or non-coding We associated the line mean of each of the four MB traits with all segregating sites in the DGRP. We used the analysis of variance model P<0.05) with an occurrence of zero or one per sequence. We then used the TOMTOM Motif Comparison Tool to compare the significant motifs with a database of known Drosophila motifs and to predict putative transcription factors (P<0.05) binding to these motifsWe inferred possible regulatory functions of variants associated with MB size located in non-coding regions. We analysed 40 basepairs up- and down-stream of each of these variants as this size is likely to span possible transcription factor-binding sitesWe used the FlyLight databaseOK107-Gal4 .Percentage of gross MB phenotypes observed in the 40 DGRP linesANOVAs of mushroom body traits.Correlation between the analyzed MB traits in the 40 DGRP lines (Pearson's correlation coefficient)Correlation between the analyzed MB traits and different behaviors in the 40 DGRP lines (Pearson's correlation coefficient)GWA analyses of mushroom body morphological variationGenes with variants associated with multiple MB traitsEnriched gene ontology categories among genes with variants associated with MB morphology traitsCluster analyses of gene ontology termsSelected transcription factorsused FlyLight, TRIP and VDRC linesPercentage of gross MB phenotypes observed upon RNAi mediated knock down of the investigated genesPrimers used for the verification of used FlyLight lines


Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies.Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02 ± 0.008, 0.03 ± 0.017 and 0.08 ± 0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary.VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility. We read with great interest a recent review in Journal of Ovarian Research published by Silvestris et al.  on ovariBut we have shown the co-existence of two distinct populations of stem cells in OSE including the ovary germ stem cells (OGSCs) which are the same as OSCs and a small sub-population of very small embryonic-like stem cells (VSELs) , 4. The The protocol discussed by Silvestris group picked up only OSCs similar to earlier published data from Tilly’s group. But flow cytometry on enzymatically isolated mouse ovarian cells and taking care to spin at 1000G , we have reported the presence of LIN-/CD45-/SCA-1+ VSELs in normal (0.02 ± 0.008 %) and chemoablated (0.03 ± 0.017 %) adult mouse ovary [Silvestris et al.  also disIt is very true that protocols for the use of cryopreserved cortical tissue for fertility preservation are not yet perfect and a risk of re-introducing malignant cells exists. A total of 37 babies have been born by transplanting cortical tissue in cancer survivors  and DemeFor treating infertility–the million dollar question is whether we need to treat and administer more stem cells in the non-functional ovary or improve the microenvironment for stem cells (that may be existing) to function properly! Various groups have suggested that decline of ovarian function with age is because the niche gets compromised and is unable to support stem cells differentiation into oocytes. On transplanting stem cells from aged ovary into a young ovary resulted in normal oocytes formation . We founTo conclude, adult mammalian ovary harbors VSELs and OSCs just like VSELs and SSCs in the testis. Endogenous VSELs can be targeted to regenerate non-functional gonads and thus offer an interesting alternative strategy to cure infertility.


Piwi-interacting RNAs (piRNAs) are a class of small RNAs; distinct types of piRNAs are expressed in the mammalian testis at different stages of development. The function of piRNAs expressed in the adult testis is not well established. We conducted a detailed characterization of piRNAs aligning at or near the 3’ UTRs of protein-coding genes in a deep dataset of small RNAs from adult mouse testis.We identified 2710 piRNA clusters associated with 3’ UTRs, including 1600 that overlapped genes not previously associated with piRNAs. 35 % of the clusters extend beyond the annotated transcript; we find that these clusters correspond to, and are likely derived from, novel polyadenylated mRNA isoforms that contain previously unannotated extended 3’UTRs. Extended 3’ UTRs, and small RNAs derived from them, are also present in somatic tissues; a subset of these somatic 3’UTR small RNA clusters are absent in mice lacking MIWI2, indicating a role for MIWI2 in the metabolism of somatic small RNAs.The finding that piRNAs are processed from extended 3’ UTRs suggests a role for piRNAs in the remodeling of 3’ UTRs. The presence of both clusters and extended 3’UTRs in somatic cells, with evidence for involvement of MIWI2, indicates that this pathway is more broadly distributed than currently appreciated.The online version of this article (doi:10.1186/s12864-015-1662-6) contains supplementary material, which is available to authorized users. Among the numerous types of small RNAs that have been investigated in recent years, piRNAs have been of interest primarily because they seem to be essential for maintenance of germ cells. They range in size from 24–32 nt, complex with members of the PIWI clade of Argonaute proteins, and are enriched for a 5’ uridine –6. piRNATransposon-derived piRNAs are produced via a ping-pong amplification loop in which primary piRNAs antisense to transposon mRNA sequences direct the cleavage of the transposon mRNA, leading to the production of secondary sense piRNAs which then direct the cleavage of the complementary sequence from the primary piRNA transcript. This results in sense/antisense piRNAs characterized by a 10-bp 5’ overlap, and secondary piRNAs with an A at position ten .Less is known about the functions of pachytene piRNAs. They are derived from single-stranded precursors, are not enriched for transposable element sequences, and do not show evidence of ping-pong amplification, suggesting that they are not involved in transposon silencing , 14. TheWe sequenced small RNAs from the adult mouse testis to a depth of approximately 139 million reads. Adapter stripping, removal of known ncRNAs  and collapsing of identical reads left approximately 13 million distinct small RNA sequences  contain antisense reads with the 10-bp overlap typical of ping-pong amplification  used for the analysis. However by using an RNA-Seq dataset from adult mouse testis  we were Pdpr) by Northern blotting of testis RNA . Finally, in many cases the xUTR was present but no small RNAs were detected (585 (62 %) liver and 573 (61 %) spleen, bottom right quadrants in Fig. Since we find evidence of xUTRs in somatic tissues, we asked if small RNAs aligning to xUTRs are present in somatic tissues. To address this question we sequenced small RNAs from mouse liver and spleen. After processing the sequence data and removing sequences matching known noncoding RNAs, we were left with approximately 328,000 and 410,000 unique small RNA sequences in liver and spleen, respectively  mRNA in liver and spleen , a pathway that stimulates the proliferation of stem cells and progenitor cells [Although our dataset overlapped with 3’ UTR piRNA clusters identified by reanalysis of previously published piRNA datasets , 17, dueor cells .The evidence we have found is consistent with the interpretation that piRNAs and smaller somatic RNAs are derived from extended 3’UTRs, implying that their production may be connected in some way to regulation of 3’UTR length. The 3’ UTR is critical for regulating mRNA stability, localization, and translation (reviewed in ). Part oMiwi2 (Piwil4) gene. The small RNAs aligning to extended 3’UTRs in somatic tissues differ from canonical piRNAs: they are shorter, lack the predominant 5’ uracil, and much less abundant than their testis counterparts. We assessed the role of MIWI2 in these clusters because we and others have found piRNAs in somatic tissues and cancer cells [Miwi2 mRNA in somatic tissues  cells [Miwi2−/− mice, but any consequences of this absence must be subtle: we find no apparent change in expression of the genes associated with these UTRs in Miwi2−/− mice, and no other effects have been reported. MIWI2 has been shown to associate with piRNAs in the prenatal testis, and to mediate transcriptional silencing and methylation of retrotransposons at this stage of development [Perhaps the most intriguing finding of this study is that small RNAs aligning to extended 3’UTRs are also found in somatic tissues, and that a subset of these somatic 3’UTR clusters is absent in mice lacking a functional er cells , 27, andues Fig. . No phen−/− mice , 27, 28,elopment . If it ielopment –12.Using a very deep dataset of piRNAs from mature mouse testis, we show that clusters of piRNAs mark extended 3’UTRs that have not been previously annotated. These clusters are present on many mRNAs expressed in the testis, but also in somatic cells. A subset of somatic 3’UTR-derived small RNA clusters are dependent on MIWI2. MIWI2 is a Piwi protein that has been shown to associate with piRNAs in the prenatal testis, and to mediate transcriptional silencing and methylation of retrotransposons at this stage of development. Its potential involvement in the generation or maintenance of small RNAs derived from 3’UTRs in somatic cells would imply some very different function. These findings extend the biological scope of MIWI2 function, and also indicate the existence of a class of extended 3’UTRs that are processed into small RNAs.For testis piRNAs, 10 μg of total RNA from adult mouse testes was electrophoresed on an 18 % denaturing polyacrylamide gel with NEB miRNA markers, the gel stained with SYBR Gold, and the readily visible band migrating at 29 nt (range ~27-31 nt) excised. RNA was eluted from the gel fragment and purified by standard methods. Illumina libraries were constructed from RNA specimens using the Illumina Small RNA Library kit, following the manufacturer’s protocol. Libraries were sequenced on an Illumina GAII apparatus to collect reads of 36 nucleotides. Raw data was processed with the Illumina pipeline v1.3.2. Data are deposited into the GEO database with accession number GSE26251.Miwi2−/− mice [For deep sequencing of somatic small RNAs, liver and spleen were harvested from WT and −/− mice  and flasAdapter sequences were removed from the 3’ ends of raw reads; up to 25 % mismatch was allowed between the adaptor and read sequences. Exact duplicate sequences were collapsed (only a single representative read was retained). Known contaminants and small RNAs were removed by alignment with Bowtie , allowinPublished small RNA sequences were downloaded from the NCBI Gene Expression Omnibus (GEO) , includiCleaned RNAs were aligned to the NCBI37/mm9 assembly of the mouse genome using Bowtie  allowingIn the testis dataset, we used the feature density estimator F-Seq  to identTo generate other piRNA clusters, we started with the F-Seq defined regions above. We then expanded these regions by 500 bp if there was 1 piRNA in a 500 bp window. As intergenic piRNA clusters are known to span regions with repetitive elements, which would not contain our uniquely aligned reads, we then merged the clusters with the annotated RepeatMasker track elements within 50 bp. We then expanded the clusters by 500 bp windows if they contained 1 piRNA and checked the clusters that had expanded by more than 20 % of their length. We separated clusters if two clusters with reads aligning to different strands had merged. We then shrunk the cluster boundaries to the locations of aligned piRNAs. Clusters were annotated based on the presence of 50 % or more of their contained sequences within a given annotation track . Clusters with fewer than 50 % of sequences aligning to a genic track were classified as intergenic.In the liver and spleen small RNA datasets, we identified 3’ UTR clusters by first identifying genes where the density of sense small RNAs in the 3’ UTR was at least two fold that of the density in the coding region. We then selected 3’ UTRs with at least 3 small RNAs and a density of over 7.5 reads per kilobase 3’ UTR per million reads aligned. We set the cluster boundaries as the region of the largest 3’ UTR isoform and extended the 3’ boundary if there was at least 1 sense small RNA per 2 kb.www.ingenuity.com). IPA core analysis was conducted using the default parameters. The Functional Analysis identified the biological functions that were most significant to the combined data set. A right-tailed Fisher’s exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone.Genes with 3’ UTR piRNA clusters identified in our testis dataset, Robine et al. , and GanWhen analyzing the spleen and liver datasets, enrichment of specific gene ontology terms for genes with clusters of small RNAs overlapping their 3’ UTRs was determined using the Functional Annotation tool of the DAVID Bioinformatics Resources , 49.To analyze antisense reads in 3’ UTR piRNA clusters, we first aligned the small RNA sequences to the genome allowing up to two mismatches and up to 25 alignments. Sites of 10-bp overlap were defined as regions within a 3’ UTR cluster where the first 10 bases of sequences aligned to the plus and minus strands overlapped each other. To determine the most likely genomic source of sequences aligning antisense within the 3’ UTR clusters, these sequences were extracted and re-aligned to the genome under strict conditions . These positions were then annotated based on their presence in an original 3’ UTR cluster, an intergenic piRNA cluster (see above), a different piRNA cluster, or any other location.3’ UTR clusters were selected that extended beyond the annotated 3’ UTR by more than 30 % of the length of the cluster. Clusters that completely overlapped an annotated gene, in the sense or antisense direction, were removed from this analysis. For the expression analysis, only the portion of the cluster that did not overlap the annotated 3’ UTR was considered.9. We then used BEDTools [To calculate the expression of the unannotated portion of the extended 3’ UTR cluster precursors, we used mouse testis RNA-Seq data  downloadBEDTools  to generExpression of the RefSeq mRNA for genes with extended 3’ UTR clusters was calculated as for the annotated portion of the clusters. To calculate expression of the unannotated portion of extended 3’ UTR piRNA precursors in somatic tissues, we first downloaded RNA-Seq data for spleen, lung, liver, kidney, heart, cortex, cerebellum and bone marrow from the ENCODE project  using a Tissue Tearor homogenizer (Biospec Products) and total RNA was purified according to the manufacturer’s instructions. mRNA was purified from total RNA using a polyA Spin mRNA Isolation Kit (New England Biolabs) according to manufacturer’s instructions. Northern blotting used the Ambion NorthernMax kit (Invitrogen) following the manufacturer’s instructions. 1 % agarose gels were run with 1 ug mRNA per well and transferred to Ambion BrightStar-Plus membrane (Invitrogen) using downward capillary transfer. RNA was then cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene) and stored at −20 °C. Cross-linked membranes were hybridized to radiolabeled single-stranded DNA probe overnight at 42 °C, washed, and exposed to a phosphorscreen for one week. Phosphorscreens were scanned using a Storm 840 phosphorimager (Molecular Dynamics), and images were analyzed using ImageQuant 5.0.Pdpr, using primers NblotEx-F and NblotEx-R for the exon-specific probe and NblotUTR-F and NblotUTR-R for the extended 3’ UTR-specific probe  according to the manufacturer’s instructions. Liver DNA was then used as a template for PCR amplification of the probe sequences for Primer extension was then used to make radiolabeled single-stranded DNA probes. Radiolabeled probes were purified using SigmaSpin™ Sequencing Reaction Clean-up, Post-reaction Clean-up Columns (Sigma-Aldrich) according to the manufacturer’s instructions.Total RNA was purified as in the Northern blot analysis, and contaminating genomic DNA was removed with the RNase-Free DNase Set (Qiagen) according to the manufacturer’s instructions. RNA was then cleaned with the RNeasy Mini Kit (Qiagen) RNA cleanup protocol. cDNA was synthesized using the ProtoScript™ M-MuLV Taq RT-PCR Kit (New England BioLabs) according to the manufacturer’s instructions and used as template in the RT-PCR reactions. See Additional file Since our somatic small RNA datasets were so much smaller than the testis dataset, we used a different approach for identifying 3’ UTRs associated with small RNAs. In the liver and spleen small RNA datasets, we identified 3’ UTR clusters by first identifying RefSeq genes where the density of sense small RNAs in the 3’ UTR was at least two fold that of the density in the coding region. We then selected 3’ UTRs with at least 3 small RNAs and a density of over 7.5 reads per kilobase 3’ UTR per million reads aligned. We set the cluster boundaries as the region of the largest 3’ UTR isoform and extended the 3’ boundary if there was at least 1 sense small RNA per 2 kb.Miwi2−/− mice as above. If a cluster region had small RNAs in both the WT and Miwi2−/− datasets, it was determined to be MIWI2 independent. Clusters without any small RNAs in the Miwi2−/− dataset, and in the bottom quartile of density in the WT dataset, were filtered because they were too small to determine MIWI2 dependence. The remaining clusters, in the top quartiles of small RNA expression in the WT dataset and lacking small RNA alignments from the Miwi2−/− dataset, were called as MIWI2 dependent clusters.To determine which somatic 3’ UTR smRNA clusters were MIWI2 dependent, we first calculated the density of small RNAs in each 3’ UTR cluster region using datasets from WT and For quantitative RT-PCR of somatic expression of Miwi2 and of mRNA derived from genes with somatic 3’ UTR small RNA clusters, cDNA was purified as for “RT-PCR of extended 3’ UTR clusters” above. PCR reactions were carried out using FastStart Universal SYBR Green Master (Roche) on an ABI 7900 Real-Time PCR machine according to the manufacturer instructions. All reactions were run in duplicate (quadruplicate for Miwi2 expression), and the mean CT was used for downstream analysis. Expression values were calculated using a standard ladder of pooled cDNA, and normalized for GAPDH expression in that cDNA prep. Values shown are the average and standard deviation obtained from two different mice.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26251) and GSE47093 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47093).The data sets supporting the results of this article are available in the NCBI GEO repository, with accession numbers GSE26251 (


Furthermore, this process also exhibits very high selectivity (>1000:1) with conventional hard masks such as silicon carbide, silicon dioxide and silicon nitride, enabling deep Si etching. In these initial studies, etch rates as high as 9.2 μm/min could be achieved at 1150 °C. Empirical estimation for the calculation of the etch rate as a function of the feature size and oxygen flow rate are presented and used as proof of concepts.Using a combination of low-pressure oxygen and high temperatures, isotropic and anisotropic silicon (Si) etch rates can be controlled up to ten micron per minute. By varying the process conditions, we show that the vertical-to-lateral etch rate ratio can be controlled from 1:1 isotropic etch to 1.8:1 anisotropic. This simple Si etching technique combines the main respective advantages of both wet and dry Si etching techniques such as fast Si etch rate, stiction-free, and high etch rate uniformity across a wafer. In addition, this alternative O There are two main types of chemical-based Si wafer micromachining; isotropic and anisotropic silicon etching122), sulfur hexafluoride (SF6) alone or in combination with fluorocarbons (CxFx) (SF6 + CxFx), do not suffer from the detrimental stiction effect and can be easily calibrated and automated2 is preferred over SF6 for isotropic etching of Si as XeF2 is a plasma-less process, whereas SF6 is a plasma process. A plasma-less process means that the etching gas is spontaneously reactive, whereas a plasma process requires external excitation to create reactive species, which requires additional costs to implement. By alternating CxFx with SF6, the etching turns from isotropic to anisotropic Si etching as at each cycle one alternates between sidewall passivation (with CxFx) and trench etching with SF6 6 + CxFx is currently preferred for anisotropic Si etching due to its ability to form extremely high aspect ratio trenches 6 is a potent greenhouse gasOn the other hand, common dry etchants such as xenon difluoride XeF, sulfur uoride SF alone or2) at high temperatures. This process meets many of the aforementioned properties as we will show in this paper.Ideally, the perfect Si etchant would be an etchant that does not cause additional damage to the wafer being processed, etch only in the pre-determined locations, has simple setup requirements, high etch rates and good uniformity over the entire wafer, high selectivity to common mask materials, uses easy-to-dispose chemicals, and does not create environmental-unfriendly/toxic by-products. With these objectives in mind, we present here an alternative Si micromachining method using only oxygen (O2)82: Si etching via reaction (1) 2Si + O2(g) → 2SiO(g), or Si oxidation via reaction (2) Si + O2(g) → SiO2(s). More details on this mechanism are available in a thorough study by Smith and Ghidini2 to form the volatile gas silicon monoxide (SiO) (reaction (1))2) is formed on the exposed Si surface instead2 formation rate reduces over time due to increasing difficulty for O2 to diffuse through the thick SiO2. Therefore we have optimised process parameters to favour reaction (1), enabling deep Si etching and therefore achieve controllable Si micro-machining.Most studies of the reaction of oxygen with Si are primarily focused on the oxidation of Si for the purpose of converting the Si surface  into silicon dioxide  and is safer than toxic halogen based dry etchants, and so does not require expensive waste management systems. The main advantages and disadvantages of common Si etching techniques compared to our O2 based Si etching technique are summarised in Supplementary Table S1125In this paper, O2, and silicon nitride (SiN) thin films. As proof of concepts, we will also demonstrate the fabrication of single crystal SiC-based diaphragms, of MEMS-like structures, and long Si stripes mimicking photonic waveguide structures. To the best of our knowledge, no such etching method (deep Si etching using only O2 at high temperatures for Si micro-machining) has been reported to date. Thanks to its many advantages, we believe that this alternative Si etching technique can become an attractive Si micro-machining process for a broad range of applications including MEMS and silicon photonics.Here, we report the initial results of using this alternative Si etching technique with three different hard masks: silicon carbide (SiC), SiO2 flow rate of 20 sccm, the etch rate increased by 63% (from 1.9 μm/min to 3.1 μm/min). Importantly, etching at 1150 °C gives a reasonable etch rate (2.7 μm/min) and implies that a custom-built O2 etching equipment can use electronic grade quartz (maximum temperature ~1160 °C) in its construction. Hence 1150 °C is the etch temperature used for subsequent studies.By increasing the etch temperature from 1100 °C to 1200 °C at an O2 etching, higher than the activation energy of Si(100) etching using XeF2 (0.26 eV above 410 K)2 etching is expected in this process since the O2 molecules are converted into reactive O species at high temperatures2 is highly reactive at much lower temperatures2 etching of Si is therefore consistent with the higher activation energy. The etching behaviours for subsequent sections are examined in terms of etch rate, anisotropy, uniformity, and mask selectivity.An Arrhenius plot displayed on rgy of Si0 etching2 flow rate on the Si etch rate. Due to the high aspect ratio of the test patterns, the vertical and lateral etch rates at various O2 flow rate were measured and compared using a combination of optical microscopy (OM) and scanning electron microscopy (SEM) imaging. Lateral and vertical etch rate results at various O2 flow rates are plotted on 2 at 1150 °C (ie. isotropic etching), but anisotropic outside 10 sccm O2, as shown in 2 flow rate and so the saturation in the vertical-to-lateral etch rate ratio is not due to a vertical etch rate saturation at high O2 flow rates (>50 sccm).Firstly, we examine the effect of O2 flow rates is currently under investigation but might be related to various competing mechanisms such as chemisorption, surface diffusion, transition from molecular to viscous flow, and changes to O2 sticking coefficients. A detailed study will be reported in another manuscript. Similar anisotropic behaviour has also been observed in XeF2 etching, where the degree of anisotropic was found to be dependent on exposed Si area, XeF2 charge pressure, and number of etch cycles2 operation (~4 Torr operation pressure) compared to this O2 etching (10−3 to 10−4 Torr operation pressure), the anisotropy mechanism cannot be identical. For example, etch rate anisotropy in XeF2 is shown to be primarily caused by a short mean-free-path of ~20 μm2 etching operation is in the centimetre to metre range.The anisotropic etching behaviours at different O2 flow rates leads to higher etch rates. The higher etch rate at higher O2 flow rates is expected as more O atoms are available for etching. The vertical etch rates achieved varied between ~0.7 to 9.2 μm/min for O2 flow rates from 5 to 100 sccm at 1150 °C, much faster than the typical reported wafer-level etch rates2 and is comparable with typical HNA and KOH2 etching at 100 sccm O2 is also comparable with high etch rate, microwave enhanced KOH etching of ~10 μm/min2 flow rate, i.e higher O2 pressure. This illustrates that O2 etching combines advantage of fast etch rates of HNA with the stiction-free etching of dry etchants. Furthermore, an important issue with XeF2 etching is that the etch rate varies over the etching time. One cause of this is due to self-heating of the substrate during etching, which result in decreasing etch rates as etch time increases for etch temperatures below 400 K42 to diffuse into a cavity as its depth increases2 etching, we compared two etching runs done at 1150 °C using 10 sccm O2 with etch times of 30 mins and 75 mins respectively and found that the etch rates were identical. This implies that O2 etching does not suffer from time-dependent etch rate changes as typically seen in XeF2 etching.Nevertheless, 2, and a,b, and c are fitting variables that depend on the O2 flow rate  (sccm) and etch tool design. The adjusted coefficient of determination R2 values are calculated to range between 0.89 and 0.99 for all power law function fits . Such area dependent etch rate has also been observed with XeF2 etch process, and is linked to the so-called microloading effect2 flow rates (<10 sccm) or for large features (>30 × 103 μm2) at any O2 flow rate. Similarly, Sugano et al.2 resulted in reduced etch rate variations with changes in feature size. Xu et al.2 etchingThe etch rate variation is more pronounced for smaller feature areas, especially at high O2 flow rates dependent microloading effects, we have also observed lateral etch rate anisotropy depending on the O2 flow rate. 2 flow rate. For O2 flow rates of 30 sccm and below the under-etch region appears as a multi-sided polygon, direct evidence of an anisotropic etching. At O2 flow rate of 40 sccm and above, the number of sides reduces such that it is mostly a square with rounded corner, which better mirrors the original square opening.In addition to the observation of O2 etching process, the void formation is controlled as the Si is only exposed in the mask openings and protected elsewhere by the mask. With the addition of O2, the Si void formation is accelerated through formation of volatile oxides (SiO) via reaction of O atoms with Si surface dangling bonds, in addition to the spontaneous void formation purely due to high temperatures, resulting in several orders of magnitudes difference in Si void formation, and hence higher Si etch rates.For a passivated Si surfaces, Si voids have been known to spontaneously form after annealing the Si at high temperatures2 at 1150 °C, with a mask opening of 10 μm wide. The shape of the etched Si depends on the O2 flow rate, consistent with observation of lateral etch rate anisotropy in 2 flow rate, as the specific profile only expands, and not change shape, with a longer etch time. The appearance of several intermediate high indices, fast etching planes prior to being eventually bounded by the slowest etching planes has previously been observed in KOH etching of Si2 flow rates, the mechanism for facet formation is likely related to the supply of O atoms and available Si bonds for etching, behaving in a similar manner to KOH etching. The detailed physical process involved in lateral etch anisotropy formation mechanism is currently under investigation and will be reported in a later manuscript.However, in applications such as structure release and through-Si etch applications, faceting is not an issue. The faceting is also negligible when comparing it with the large features (channels may be centimetres long) that are typically used for microfluidic applications.2 flow rate affects the etched Si surface roughness. Smooth etched Si surfaces appear as a bright, white colour, and appear featureless under OM  etched Si surface coupled with the elimination of halogens (improves safety) and eliminating chemical waste hazards achieved in this initial study shows great potential for its use in large scale manufacturing.A novel technique for deep etching of Si using pure O2 formed either by using LP-CVD from SiH4 and O2 at 440 °C or thermally grown in a furnace by dry oxidation at 1000 °C. These three hard masks were tested for compatibility for this Si etching process but only the SiC mask was used for these studies for convenience as we had many SiC coated Si wafers already fabricated. Photoresist was then spin-coated onto the mask layers and then patterned using standard UV photolithography techniques, followed by dry etching to transfer the features of various shapes and dimensions between 2 to 600 μm wide and up to 1 mm long into the hard mask. The two inch wafers had an exposed Si area of approximately 23% and the 150 mm wafers had between 15% and 40% exposed Si areas.2 inch and 150 mm Si(100) substrates were first coated on both sides with a thin film for etch mask purpose and consist of either ~300 nm thick single-crystal SiC using a customised low-pressure chemical vapour deposition (LP-CVD) toolSi etching was performed using the same LP-CVD system used for the SiC thin film deposition2 etched samples were measured using an Olympus MX50 optical microscope (OM), a Jeol JSM-6510LV scanning electron microscope (SEM), and a Dektak 150 profilometer. The etch depth was determined using OM, SEM or a Dektak profilometer. Although OM measurements correlated to Dektak measurements for shallow etch depths, it should be noted that Dektak measurements were found to be unreliable for the narrower opening widths due to the difficulty for the relatively large stylus to reach the bottom of trenches which can be up to 100 μm deep. Hence OM was the predominant technique used to measure the etch depth. The lateral etch front anisotropy is studied using the OM. The etched Si roughness was evaluated using the OM and SEM. The mask selectivity was determined by measuring the mask thickness change before and after O2 etching using a Nanometrics Nanospec AFT 210 reflectometer.The lateral and vertical etch rate of the OHow to cite this article: Chai, J. et al. Silicon etching using only Oxygen at high temperature: An alternative approach to Si micro-machining on 150 mm Si wafers. Sci. Rep.5, 17811; doi: 10.1038/srep17811 (2015).


The glacial-interglacial δ18O variations in the Dim Cave speleothem are best explained in terms of changes in the trajectories of winter westerly air masses. These are along a northerly (European) track  during the early last glaciation but are gradually depressed southward closer to the modern westerly track along the North African coast (more depleted) after c.50 kyr and remain in the southern track through the Last Glacial Maximum. The southward displacement of the westerly track reflects growth of the Fennoscandian ice sheet and its impact on westerly wind fields. Changes in δ13C are interpreted as reflecting soil organic matter composition and/or thickness. δ13C values are significantly more negative in interglacials reflecting active carbonic acid production in the soil and less negative in glacial times reflecting carbonate rock values. Several Heinrich events are recorded in the Dim record indicating intensification of westerly flow across this part of the EM.Speleothem-based stable isotope records are valuable in sub-humid and semi-arid settings where many other terrestrial climate proxies are fragmentary. The Eastern Mediterranean is one such region. Here we present an 80-kyr-long precisely-dated (by U-series) and high-resolution oxygen (δ To date, high resolution (up to 250 kyrs12356n Turkey . The mai−1) and only 1% (12 mm yr−1) occurring in the core winter months  and summer, respectively. Isotopic data on a monthly basis reveals average winter and summer rainfall δ18O values of −6.12‰ and −3.83‰ (VSMOW), respectively (Table S2a online). Most recent data from 2009 shows average δ18O value of ~ −7‰ (VSMOW) for rainfall from November to March and no precipitation for the summer months103-type vegetationThe Dim  Cave  is located on the western flank of Cebel Reis Mountain 2 was liberated from carbonate samples by reaction under vacuum with H3PO4 acid at 90 °C for 1000 s and calibrated against NBS-18 and NBS-19 international standards. The replicate analyses of internal carbonate standards indicate that the analytical standard deviation of δ18O and δ13C averages at ±0.1‰  and ±0.04‰  respectively.Three stalagmites from narrow passageways and two water samples from pools of Dim Cave were collected in 2009 and 2012 . Due to mainly financial and logistic constraints, we were unable to establish cave monitoring but cave pool waters were sampled. The stalagmites were halved using a circular saw and micro-sampled along their growth axes using hand drills equipped with diamond-coated drill bits (0.5 to 2 mm in length) for U-series dating , as well as for stable isotope analyses. Sampling interval for the stable isotope analyses was 0.5–1.0 mm depending on the growth rate of the stalagmite. This sampling interval corresponds to a growth period of 100–200 years for Dim-E3, and an average of ~100 years for Dim-E2 and Dim-E4, respectively. An additional 30 sub-samples  were microdrilled from each of the growth phases of Dim-E3 and from one of the growth phases of Dim-E2 and Dim-E4 for Hendy tests230Th contributions are generally very small , which provide a centennial-scale continuous record for the time period ~90–13 kyr. 138 additional high-resolution (Table S2) stable isotope analyses of Dim-E2 and Dim-E4 extend the time series to ~10 kyr for the stalagmite deposition in the Dim Cave. Overall δ18O values of the stalagmites range between −3.46 and −6.81‰ VPDB, with the lowest value from Dim-E2 at around 9.9 kyr and highest value from Dim-E3 at around 76.7 kyr  while at Marine Isotope Stage (MIS) 4 the change averages closer to 0.5 per mil between uncorrected and ice volume-corrected records18Owater record has a ~31 per mil (in VSMOW) negative shift, assuming that average temperature was 8 °C degrees lower than today82018O speleothem values are consistent with the modern precipitation oxygen isotope range  to −0.21‰ (Dim-E3) VPDB, pointing to a varying degrees of contributions buffered with vegetation/soil-CO2 (associated with Mediterranean C3-type plants) and dolomitic host limestone  for the seepage waters42418O values (−6.4 to −4.4‰ VPDB) have not varied by more than 0.5‰  and do not correlate (R2 = 0.2) with δ13C values (−11.3 to −5.08‰ VPDB) along the tested growth laminae (Table S4 online), which means that the deposition of stalagmites was essentially in or close to quasi-isotopic equilibrium, allowing interpretation of the isotopic variations mainly as a climatic indicator in the cave environment.A high-resolution stable isotope profile of stalagmite Dim-E3 is based on 442 δ76.7 kyr . These a18O and δD ratios , which fall within the range of mean winter rainfall δ18O and δD reported from the GNIP Antalya station . Here we summarise the main findings.Based on the calcite growth rates in Dim-E3 , we argue that the moisture balance was net positive above the Dim Cave throughout the last glaciation. Growth rates in Dim-E3 are highest during the interglacial segments and between 72–63 kyr (11.9 mm/kyr). Rates varied between 2.0 mm/kyr at 79–72 kyr through 1.3 mm/kyr at 63–51 kyr to a low of 0.8 mm/kyr between 40 and 18 kyr  but deposition continued, which is also inferred from dominant compact/open columnar calcite fabrics18O and δD values of the months Dec-Feb: −6.12 and −31.3‰ VSMOW respectively; Table S2) and cave pool waters in the Dim Cave  confirms that the Dim Cave receives mostly winter rain.The heavy winter rain along the Mediterranean coast of Turkey is connected to westerly flows derived from the North Atlantic Ocean but the moisture for the rainfall is mostly sourced from the EM. The close match of the present-day isotopic composition between monitored winter rainfall in Antalya (average δ18O and δD) of the rain water232318O: −5 to −9‰; δD: ~ −25 to −50‰ SMOW28Currently the EM region is influenced by four distinct air mass tracks in winter, each characterised by varying moisture content and temperature, depending on the source of the air mass and the extent of interaction with the Mediterranean Sea that modifies the isotopic composition  which are typical of other speleothem records from the Mediterranean region . The variation can be explained as a response to changes in the track of the air mass from a northerly European track across the Balkans that yields less depleted δ18O values during MIS 4 to a southerly Mediterranean track that is dominant in interglacials  differences between the North Pole and the tropics. With polar amplification of cooling in glacial times, westerly circulation will intensify during glacial times and especially at times of maximal cooling. In the early part of the glaciation (MIS 4) ice sheet development of both the Laurentide and Fennoscandian sheets is restricted and the polar jet may lie at a northerly position pre-conditioning air mass trajectories largely through the Balkans . As the glaciation continues, both the Laurentide and Fennoscandian ice-sheets gradually extended southwards (and westward in the case of the Fennoscandian). This increase in surface elevation and pressure forces the polar jet southwards and consequently westerly winds and surface fronts will gradually track further south32We note that the gradual shift towards isotopically lighter water during the last glaciation would largely disappear if we had not provided an ice volume correction to the δThe longer-term records appear to reflect multi-millennial scale changes in ice volume, but we also note that those Heinrich events present in the record are also marked by phases of isotopic enrichment. This may reflect either the influx of fresh water into the North Atlantic, changing the isotopic composition of the air mass at the source region and/or enhanced westerly flow during Heinrich events. The enhanced flow would occur because of increased temperature contrast to areas further south and would amplify the effect of the northerly  track. This process would be most effective when the continental ice sheets are at their smallest extent (i.e. the YD and MIS 4 events show up most strongly) as there is a northerly track available for the westerlies. During MIS 2, the westerlies may be enhanced but there is little chance for NW-SE flow to dominate because the track of the westerlies is depressed far to the south.13C changes (−12.45 to −0.12‰) in the Dim speleothems . This reflects thinner density of forest/shrubland cover in the area as a consequence of depressed temperatures and reduced global availability of CO2372 (δ13C: ~ −7‰142413C record shows large positive extensions and strong relationships with δ18O during slower growth stages (i.e. marked as slow (s) growth periods of Dim-E3; Fig. S2a). Accordingly we think that degassing (kinetic fractionation) might have played an important role during short and drier periods, but overall we believe that bedrock contribution has an equally important role in the longer term variation of the δ13C record.The large range of δleothems  can be eon ~ −25‰36 with r18O depletions during interglacial episodes are obvious in both the Israeli and Dim records  C4 vegetation at glacial times24-type vegetation present now4 vegetation  to occur during glacial times. The isotopically heavier δ13C signal is consequently a function of reduced vegetation cover and enhanced bedrock interactions, rather than summer rainfall. Recent research from the Levant region44Previous studies have inferred a role of temperature for lighter oxygen isotopes in speleothems during glacial periods and have argued for increased summer rainfall during the late Pleistocene in this region404218O of the oceanic moisture source which is governed by latitudinal shifts in the westerly wind track. Swings in carbon isotope profiles are deduced mostly to reflect changes in the vegetation cover and subsequent interactions with the host dolomitic limestone over glacial-interglacial timescales. Although interrupted by short-term fluctuations, the long-term patterns of the Dim record show synchronous responses to global climatic variations driven by the evolution of the northern hemisphere ice sheets.In this study, we reconstruct the paleoclimate of the EM region for the past ~80 kyr using a well-dated continuous speleothem record from S-SW Turkey. Our findings demonstrate that the Dim Cave has had a positive moisture balance from ~9–90 kyr. The changes in composite Dim oxygen isotope time series are interpreted as changes in δHow to cite this article: Ünal-İmer, E. et al. An 80 kyr-long continuous speleothem record from Dim Cave, SW Turkey with paleoclimatic implications for the Eastern Mediterranean. Sci. Rep.5, 13560; doi: 10.1038/srep13560 (2015).