It is shown that the addition of graphene pins the grain growth of the ceramic matrix grains, leading to a change of the microstructure at low filler concentrations. As a consequence, the composites exhibit two percolation thresholds and their dielectric properties are not only determined by the dielectric properties of the constituents and their relative fractions but also the microstructure of the composite must be considered. Additionally, a giant increase of the dielectric constant has been found around the percolation thresholds in barium titanate–graphene composites. In particular, values of the dielectric constant up to 45,000 and 15,000 were found at 1 kHz in composites containing 0.4 and 0.6 wt. % graphene, respectively.Al It is increasingly being recognized that new applications for materials require functions and properties that are not achievable with monolithic materials. In this sense, composites formed by a ceramic matrix and a conductive second phase are very interesting materials since their physical properties, such as optical, electrical, and magnetic properties as well as tribological, corrosion resistance, and wear properties can be tailored, making them attractive for many new applications –4. In pa2O3/graphene composites with graphene concentrations between 0.56 and 1.24 % weight and BaTiO3/graphene composites with graphene concentrations between 0.1 and 0.6 % weight are studied in the frequency range of 10−1–106 Hz.The dielectric properties of insulator–conductor materials, such as conductivity and permittivity, show a critical behavior when the fraction of the conductive phase reaches the percolation threshold , 7, whic3 and KMnO4. Concentrated H2SO4 (360 mL) was added to a mixture of synthetic graphite (7.5 g) and NaNO3 (7.5 g), and the mixture was cooled down to 0 °C using an ice bath. Afterwards, KMnO4 (45 g) was slowly introduced in small doses to keep the reaction temperature below 20 °C. The solution was heated to 35 °C and stirred for 3 h. At that point, a hydrogen peroxide (H2O2) 3 % solution (1.5 L) was slowly poured, giving rise to a pronounced exothermal effect up to 98 °C. The reaction mixture was stirred for 30 min and centrifuged (3700 rpm for 30 min) to discard the supernatant. The remaining solid material was then washed with 600 mL of water and centrifuged again; this process being repeated until the pH was neutral.Graphene oxide was synthesized from synthetic graphite by the modified Hummers method . This me−1) was prepared in 1-L batches and kept under ultrasound for 10 h. Afterwards, the suspension was centrifuged (3700 rpm for 30 min) to discard the filtered supernatant. To prepare the suspensions for organic solvents, water was evaporated in a rotary evaporator and 10 mL of each solvent was dispersed into 1 mg of solid to then sonicate for 30 min. Details on the characteristics of graphene oxide are given elsewhere [A colloidal suspension of individual graphene oxide sheets in purified water  and BaTiO3  powders for ceramic matrices with an average particle size of around 150 and 700 nm, respectively, were dispersed in distilled water then mixed with the graphene oxide dispersion and finally ball milled for 1 h. The homogeneous mixtures were spray dried and uniaxially pressed (30 MPa) prior to spark plasma sintering in an FCT-HP D25/1 apparatus with a heating rate of 50 °C·min−1 under an applied pressure of 80 MPa, a holding time of 1 min and in vacuum (10−1 mbar). The final sintering temperature was 1500 °C in the case of Al2O3/graphene composites and 1100 °C for BaTiO3/graphene. Densities close to 100 % were found in all composites.Al−1. Microstructure was characterized by field emission scanning electron microscopy (FESEM) on a QUANTA FEG 650 and by transmission electron microscopy (TEM) on a JEOL-JEM 2100F. The dielectric properties of the samples were studied by standard low-frequency impedance measurements (PSM1735-NumetriQ).Powder X-ray diffraction analysis  was used to determine possible high-temperature chemical reactions between components in sintered samples. Raman spectroscopy was performed on a Renishaw 2000 Confocal Raman Microprobe  using a 514.5-nm argon ion laser. Spectra were recorded from 1100 to 3500 cm2O3/graphene composites  and G (1583 cm−1) are narrow and present similar intensities (ID/IG = 0.92). A shoulder corresponding to the D’ band (1620 cm−1) is observed next to the G band and a well-defined structure is also present in the range 2500–3500 cm−1. A single and narrow 2D peak is centered around 2700 cm−1; therefore, according to Ferrari et al. [D and G bands together with the shape of the 2D band, indicates that the state of the graphene in these composites is intermediate between those treated at 1000 and 2000 °C in that work, which agrees with the sintering temperature of 1500 °C.X-ray diffraction analysis of Altes Fig. a only shtes Fig. b. Bands i et al. , the prei et al. , consideElectron-dispersive X-ray spectroscopy performed along the yellow line in Fig. 2O3 grain sizes as a function of the graphene concentration in the samples. While monolithic alumina sintered by SPS under the same conditions presents 10-μm grain sizes [2O3 grain growth. This result is analogous to the one reported in ref. [Field emission scanning electron microscopy images Fig. a–d show in sizes , the gra in ref.  for alum2O3/graphene composites at a frequency of 1 kHz. It should be noted that at low frequencies, the conductivity reaches a plateau regime, as it corresponds to a d.c. regime. In the case of monolithic alumina, its relative dielectric constant and conductivity are respectively ε′ = 10 and σ = 10−11 S·cm−1 in agreement with previously reported values in the literature [Figure terature , 26. Forterature . In thisterature . The losThe existence of a double percolation threshold in ceramic matrix–graphene composites can be very interesting from a technological point of view as it can be used to obtain a giant increase of the dielectric constant in a high permittivity matrix by the addition of very small amounts of graphene as will be shown next.3. Again, no reaction phases are noticeable in the diffractogram corresponding to the composite with the largest amount of graphene. In this case, this is a very relevant result because some impurities, such as TiC, or different barium oxides could be expected in dense compacts if they were sintered by conventional methods. The use of SPS has allowed obtaining dense composites  by sintering them at low temperatures and for very short times. In order to study the structure of the graphene after sintering, Raman spectra were taken and are shown in Fig. D (1355 cm−1) and G (1583 cm−1) are narrow and present similar intensities, with band D being even more intense than band G (ID/IG = 1.06). A qualitative comparison with the results by Botas et al. [D, G, and 2D bands, indicates that the state of the graphene in these composites is very similar to graphene treated at 1000 °C in the reference given above, which agrees with the sintering temperature of 1100 °C. The 2D peak is centered around 2690 cm−1 and presents a single component; therefore, according to Ferrari et al. [The X-ray diffraction analysis Fig. a of the s et al. , considei et al. , the pre3/graphene composites  and the dielectric losses of the composites (~4–5 %) are due to a reduction of the Ti4+ to Ti3+ originated during the sintering process in vacuum. With increasing graphene content, the conductivity increases until it reaches a maximum value (~2.5·10−5 S·cm−1) at 0.4 % graphene. Then, conductivity abruptly decreases for the 0.45 % graphene composite, displaying again a conductivity behavior similar to that of BaTiO3. In a similar way as it happens in the Al2O3/graphene composites shown above, the pinning effect on the matrix grains by graphene sheets induces a local decrease (at the grain boundaries) of the effective graphene concentration and, therefore, a decrease in the composite conductivity. Finally, the conductivity of the composites increases again for concentrations over 0.45 %.The dependence of the microstructure versus graphene content is expected to have a deep influence on the dielectric properties of the composites. Fig. 3/graphene composites at a frequency of 1 kHz. In particular, the 0.4 % composite exhibits a permittivity value of ε′ ≈ 45,000 and the 0.6 % composite of ε′ ≈ 15,000 compared to the value of 2900 for the monolithic BaTiO3 ceramic. However, these large values of the dielectric constant are accompanied by large increases of the loss tangent  of the dielectric constant has been found in BaTiO3/graphene composites by introducing very small amounts of graphene. In particular, a permittivity value around 45,000 was obtained for the composite of 0.4 wt. % of graphene and around 15,000 for the 0.6 % graphene composite. This opens a route to develop giant dielectric constant composites with very low concentrations of conductive phases.In summary, it has been shown that low concentrations of the filler produce a strong change in the microstructure of the ceramic matrix composites, particularly on the average grain size of the ceramic matrix. When the aspect ratio of the conductive filler is large enough, the pinning effect of graphene on ceramic matrix modifies the spatial distribution of the conductive phase in such a manner that the percolation threshold is reached twice as the result of the change of local graphene concentration. The dielectric properties of Al


The proliferation and expansion of the malignant PC producing a monoclonal immunoglobulin eventually results in BM failure with anemia, skeletal involvement with osteolytic lesions and hypercalcemia, and renal failure due to the excessive production of paraprotein.2 However, most patients will still eventually suffer a fatal relapse after an initial effective therapy. This is due to the persistence of chemotherapy-resistant precursor cells3 in the BM even after destruction of the bulk of tumor cells4 and, accordingly, the disease will become more and more refractory to chemotherapy after each additional line of treatment.There have been significant therapeutic advances over the past decade and the median survival of myeloma patients has increased to ~6 years.5 have shown significant clinical efficacy in different solid tumors7 and also hematological malignancies.8 PD-L1 is expressed on different antigen-presenting cells and it has been suggested that PD-L1 is absent from normal PCs10 while it is expressed on myeloma cell lines9 and primary myeloma tumor cells from patients with MM.10 However, so far it has remained controversial whether normal PCs from myeloma patients or healthy donors are also PD-L1 positive and it is unclear whether quiescent myeloma-propagating cells express PD-L1 as a therapeutic target.Immunotherapy could play an important role in eradicating chemotherapy-resistant myeloma cells from the BM of the patients and one approach in particular has been shown to be very promising. Monoclonal antibodies targeting PD-L1 or its receptor PD-1, which is expressed on the surface of exhausted T and B cells and is involved in the maintenance of peripheral self-tolerance,We recently performed a flow cytometry-based analysis of BM samples from 11 healthy donors, 15 patients with monoclonal gammopathy of undetermined significance, 6 patients with smoldering MM and 51 patients with MM. All the patients and healthy subjects had identifiable CD38+/CD138+ PCs in their BM but, as expected, the number of PCs was the highest in our patients with active MM . When wen=22) as defined by the absence or presence of CD19/CD56 and CD45/CD28, respectively. We did not detect any difference in the expression of PD-L1 among the patients' PC subtype as defined by positivity for CD19 and CD56, in particular we did not observe an abnormal expression on those PCs showing the typical CD19−/CD56+ aberrant phenotype  in a contact-dependent manner. These Tregs demonstrated a suppressive phenotype and an increased PD1 expression compared with naturally occurring Tregs.12 Furthermore, It has recently been shown that the PD-1/PD-L1 pathway not only promotes progression of myeloma indirectly by causing failure of immune control but that BM stromal cells induce expression of PD-L1 on myeloma cells, which results in increased proliferation of the tumor cells and dampened susceptibility to anti-myeloma chemotherapy. Accordingly, clinical progression was observed in patients whose myeloma cells expressed high levels of PD-L1.13In conclusion, we have shown here in a large collection of patients with PC dyscrasias including MM that the immunomodulatory molecule PD-L1 is commonly and broadly expressed on the patients' PCs. This and our observation that PD-L1 shows an increased expression on those myeloma cells with an aberrant phenotype suggests an important function of this molecule in suppressing local immunity in the tumor microenvironment of MM. Indeed, it has been shown that myeloma cells overexpressing PD-L1 inhibit the generation of cytotoxic T cells 14 would support the initiation of clinical trials investigating the efficacy of anti-PD-1 or -PD-L1 antibody treatment in MM either alone or in combination, for example, with lenalidomide.15 In addition to indirectly targeting MM by reversing local immunosuppression through anti-PD-1/PD-L1 antibody treatment one could also imagine to directly target PD-L1 expressed on the myeloma cells, for example, by the adoptive transfer of PD-L1-specific chimeric antigen receptor-transduced T cells. As we have shown here, PD-L1 is also expressed on myeloma-propagating pre-PCs and, accordingly, immunotherapeutic approaches directly targeting this surface molecule would have the potential to eradicate chemotherapy-resistant disease. It remains to be determined whether the expression of PD-L1 on normal PCs, as demonstrated in our current study, represents an obstacle to approaches like this.These combined data and the observation made by others that the expression of the PD-L1 receptor PD-1 is upregulated on T cells isolated from patients with MM,SY performed the research, performed the statistical analysis, analyzed and interpreted the data, and wrote the manuscript; JM performed the research; MS collected the data; AL collected the data; TK collected the data; MB performed the research; NK collected the data and contributed vital new reagents or analytical tools; TL wrote the manuscript, DA designed the research, performed the statistical analysis, analyzed and interpreted the data, and wrote the manuscript.


There are errors in the ninth and tenth sentences of the Abstract. These sentences should read: Also there was a decrease in the risk of progression (RR of PFS: 0.82 IC 0.73–0.92) compared to placebo. Conclusions: We observed significant differences in physiologic and clinically relevant outcomes such as reduction in all-cause mortality, IPF related mortality, worsening of IPF and improvement of PFS. So pirfenidone treatment should be considered not only for its benefits in pulmonary function tests but also by its clinically relevant outcomes.There are multiple errors in the Results described below.The third and fourth sentences of the “Progression-free Survival (PFS)” section should read: The meta-analysis includes 786 patients in intervention group and 728 in placebo group . PirfeniThe third sentence of the “Worsening of IPF” section should read: The meta-analysis includes 858 patients in intervention group and 763 in placebo group . Pirfenidone improves worsening of IPF with a RR of 0.64  compared to placebo.The second sentence of the “Adverse events” section should read: The meta-analysis includes 859 patients in intervention group and 763 in placebo group .There is an error in the third sentence of the fourth paragraph of the Discussion. It should read: We also observed differences in clinically relevant outcomes such as reduction in all-cause mortality, IPF related mortality, worsening of IPF and risk of progression; but no benefit on acute exacerbation of IPF.There are errors in the fourth and fifth columns of


The moist cell and the dry cell on the antenna of the male honeybee were exposed to humidities slowly rising and falling at rates between –1.5%/s and +1.5%/s and at varying amplitudes in the 10 to 90% humidity range. The two cells respond to these slow humidity oscillations with oscillations in impulse frequency which depend not only on instantaneous humidity but also on the rate with which humidity changes. The impulse frequency of each cell was plotted as a function of these two parameters and regression planes were fitted to the data points of single oscillation periods. The regression slopes, which estimate sensitivity, rose with the amplitude of humidity oscillations. During large-amplitude oscillations, moist and dry cell sensitivity for instantaneous humidity and its rate of change was high. During small-amplitude oscillations, their sensitivity for both parameters was low, less exactly reflecting humidity fluctuations. Nothing is known about the spatial and temporal humidity variations a honeybee may encounter when flying through natural environments. Microclimatic parameters  were measured from an automobile traveling through different landscapes of Lower Austria. Landscape type affected extremes and mean values of humidity. Differences between peaks and troughs of humidity fluctuations were generally smaller in open grassy fields or deciduous forests than in edge habitats or forest openings. Overall, fluctuation amplitudes were small. In this part of the stimulus range, hygroreceptor sensitivity is not optimal for encoding instantaneous humidity and the rate of humidity change. It seems that honeybee's hygroreceptors are specialized for detecting large-amplitude fluctuations that are relevant for a specific behavior, namely, maintaining a sufficiently stable state of water balance. The results suggest that optimal sensitivity of both hygroreceptors is shaped not only by humidity oscillation amplitudes but also according to their impact on behavior. Hygroreceptors provide an insect with background information such as the direction and rate with which atmospheric humidity is changing. This information contributes significantly to the insect's ability to maintain a stable water balance by avoiding dangerous and preferring favorable humidities. Electrophysiological identification of hygroreceptors has been successfully accomplished in only few species In earlier studies, humidity changes were produced by switching rapidly from one conditioning air stream at a constant humidity to a second at a different constant humidity Understanding the function of hygroreceptors requires knowledge of the adequate stimulus. The relative humidity is obviously the most familiar and practical way of measuring atmospheric humidity and defining the content of water of hygroscopic materials. The water content of a human or horse hair under tension changes its length due to changes in the relative humidity of the air. Accordingly, it was assumed that the sensillum wall acts as a hygro-mechanical transducer which changes its dimension due to the uptake (adsorption) and loss (desorption) of water, much like a hair hygrometer depending on changes in relative humidity Regardless of how the process of humidity transduction is best described, the most important function of insect hygroreceptors is to acquire information about changes in atmospheric humidity. Hygroreceptors represent two input parameters (instantaneous humidity and its rate of change) with a single output parameter (impulse frequency) We chose to study the dynamic sensitivity of the moist and dry cells in the male honeybee. The moist and dry cells of the honeybee offer advantages. They occur together in peg-shaped sensilla located in heavily walled pits which are readily found on worker bee and drone antennae  and someOur knowledge of the properties of humidity stimuli occurring in the insect's natural environment is limited to stationary measurements. Nothing is known about the amplitudes and rates of humidity changes a flying insect may encounter in its natural environment. Plausible ranges of humidity fluctuations could be measured from an automobile traveling with constant speed through different landscapes. Such measurements would indicate what temporal humidity profiles may hit the hygroreceptors of a flying insect. Normal worker bee flight speed is about 7.1 to 8.2 m/s  for unobstructed humidity stimulation. The hygro-thermoreceptive sensilla  was calculated from running averages of three consecutive 0.5-s intervals.Impulse frequency , water vapor density was measured at a rate of 100 Hz. The voltage outputs of the hygrometer and the thermistor were passed through the AD-converter , fed into the PC and recorded online. Based on the digitized signals of the hygrometer and the thermistor, the water vapor pressure (Pw) and the relative humidity (rH) were monitored offline. The saturation water vapor (Ps) and the saturation deficit (SD) were determined offline by running a self-written script.Air from a pressure-regulated source was cleaned, dried and split into two streams. Their flow rates were equalized by matching the rates in mass flow meters. The first stream was bubbled out through many openings in a polyethylene tube firmly anchored in a tank containing ion-exchange purified water at constant depth and at 42°C. The second stream was conducted through the tank in a spiral tube and remained dry as it was warmed to 42°C. The temperature of the two streams was then set at different temperature levels by driving them through a further self-made, thermostatically controlled heat exchanger. After emerging from the heat exchanger, the two air streams passed through electrical proportional valves  and then were combined to a single stream. The water vapor pressure of this stream was sinusoidally modulated by mixing the two streams in a ratio determined by the proportional valves. To hold the flow rate of the mixed air constant at 2.5 m/s, the control voltages  of the proportional valves were phase shifted by 180°. The mixed air was divided into two streams. For stimulation, the first stream was directed towards the antenna by way of a Plexiglas tube 7 mm in diameter. The hygroreceptive sensillum was 5 mm away from the outlet of the tube. The temperature within the air stream was measured within ±0.03°C by a small thermistor  3 mm downstream from the sensillum. By passing the second air stream through a 1-cmAir humidity was measured by an UV krypton absorption hygrometer at a rate of 100 Hz , ambient temperature in real time by a fine-wire thermocouple , and wind speed in real time by a hot wire anemometer. These sensors were installed at the front end of a 4-m rod that was mounted with its back end on the leading edge of the roof rack of an automobile so that it juts out on the right side. In this way, data were collected at a height of 1.5 m above the ground and more than 3 m within the roadside vegetation. The safety distance of more than 5 m minimized the influence of the exhaust fumes. The voltage outputs of the sensors were displayed on an oscilloscope inside the automobile and recorded on a DAT-recorder. In the laboratory, the data were passed through a CED 1401-micro  interface, stored on a hard disc of a PC and analyzed using the commercial software Spike2.Measurement trips lasting 1.5 to 2 h were made in the countryside of Lower Austria  on calm days in June and July during late morning and early afternoon. The sky was generally clear, with some scattered clouds. The automobile travelled at a mean speed of 25 km/h (7 m/s) along vegetated roadsides, open farm fields, grasslands with clusters of trees shadowing the ground, and edge habitats. Deciduous forests with low canopy cover were also crossed. The trips were continuous with no stops or road intersections. No specific permissions were required for the measurement trips in these landscapes. The field studies did not involve endangered or protected species.The basic measures chosen to characterize the temporal profiles of temperature, humidity and wind speed were the amplitudes and positions of the peaks and troughs, and the values of the rising and falling slopes . Peaks aIn both worker bees and drones, the hygroreceptive sensilla are located at the mid region of most antennal segments. Their external structure is shaped like a mushroom with a slightly dilated cap. They are positioned in the central opening of shallow cuticular depressions so that only the cap is visible from outside A. An elThe same antagonistic responses of the two cell types to an increase or decrease in the relative humidity can also be elicited by changing the air temperature. For example, a sudden increase by 4.5°C from 26.4 to 30.9°C F at a crH between 20 and 70% rH was covered and the rates of humidity change were between –2% rH/s and +2% rH/s. In the moist cell, impulse frequency varied between values of 5 and 30 imp/s , and in the dry cell between 22 and 50 imp/s . Impulse frequency of the moist cell tended to be higher at the higher values of instantaneous humidity and lower at the lower values . Conversely, impulse frequency of the dry cell tended to be higher at the lower values of instantaneous humidity and lower at the higher values . Frequency values during humidity oscillations may simply be interpreted as the response to instantaneous humidity, i.e., the succession of humidities at particular instants in time. Impulse frequency, however, is not in step with instantaneous humidity but ahead of it. A second stimulus parameter that is also in advance of instantaneous humidity must influence their responses. The rate of humidity change was the obvious candidate. To estimate the double dependence on instantaneous humidity and its rate of humidity change, the impulse frequency of the moist and dry cells was plotted in Ba and Bb as a function of both parameters.Three kinds of experiments were performed involving slowly changing humidity. In the first kind a humidity amplitude of roughly 50% 30 imp/s Aa,g, a50 imp/s Ab,g. Ir values Aa,c. Cr values Ab,c. FF = a+b drH/dt+c rH; where F is the impulse frequency and a the height of the regression plane) were calculated to determine the simultaneous effects of the rate of humidity change (b slope) and the instantaneous relative humidity (c slope) on the response frequency of both cell types . The slopes demonstrate the two properties that characterize each cell: the sign of the slopes is positive for the moist cell and negative for the dry cell; that is, an increase in both instantaneous humidity and its rate of change raises the impulse frequency of the moist cell and lowers that of the dry cell; and the slopes are similar for the moist and the dry cells, sign ignored. Accordingly, given changes in instantaneous humidity or in the rate of humidity change have similar effects on the frequency of the moist and the dry cell with due consideration of the sign.Multiple regressions  show a strong relationship between impulse frequency, instantaneous humidity and rate of humidity change. For the moist cell, 2R indicates that in a series of oscillating humidity changes, an average of 97% of the variation in impulse frequency can be explained by a multiple regression; for the cold cell, the value is 96%. The orderly relationships of impulse frequency to the instantaneous humidity and its rate of change in both cells provide an opportunity to determine their differential sensitivity. The differential-sensitivity values are the expression of the mean change in impulse frequency for each unit change in stimulus magnitude. On average, differential sensitivity to instantaneous humidity was +0.3 imp/s per % rH for the moist cell and – 0.4 imp/s per % rH for the dry cell; differential sensitivity to the rate of humidity change was +5.2 imp/s per % rH/s for the former and –5.4 imp/s per % rH/s for the latter. Thus impulse frequency of both cells can be influenced more by changing the rate of humidity change by 1% rH/s than by changing instantaneous humidity by one additional percent. Actual measurements show that an increase of 1 imp/s in the moist cell can be elicited either by a +3.3% rH increase in instantaneous humidity (at constant rate of change), or by a +0.19% rH/s rate of humidity change. In the dry cell, it takes a decrease of –2.5% rH in instantaneous humidity – or a rate of change of –0.18% rH/s – to increase frequency by 1 imp/s. Both cells display similar response properties, with due consideration of sign: both are more sensitive to the rate of humidity change than to the humidity at which the change takes place.The mean values of the instantaneous relative humidity and the rate of humidity change from 12 moist and dry cells are listed in rH between 15 and 45% rH were produced. The rate of humidity change lay between –1.5% rH/s and +1.5% rH/s , that of the dry-cell between 5 and 30 imp/s . The time course of impulse frequency of both cell types displayed small irregularities. In the moist cell, the higher impulse frequency values were associated with higher humidity , and in the dry cell with the lower humidity . In order to determine the extent to which the impulse frequencies are governed by instantaneous humidity and the rate of humidity change, the frequency values were plotted as functions of both humidity parameters . The emerging coefficients of determination (2R) indicate that, in a series of oscillating humidity changes, an average of 76% of the variation in the moist-cell' impulse frequency can be explained by a multiple regression; in the dry cell the value is 88%. Thus the mean b slope of +0.5 imp/s per % rH/s for the moist cell and – 0.8 imp/s per % rH/s for the dry cell drawn from all multiple regression provides an estimate for their sensitivity to the rate of humidity change. The mean c slope of +0.2 imp/s per % rH for the moist cell and –0.2 imp/s per % rH for the dry cell estimates their sensitivity to instantaneous humidity. To produce an average increase of 1 imp/s in the moist cell, instantaneous humidity must increase by +5% rH or the rate of change by +2% rH/s. The corresponding dry cell values are either a decrease in instantaneous humidity of –5% rH or a decrease in the rate of change of –1.2% rH/s. Thus the dry cell is more sensitive to both humidity parameters.In the second experiment, slow humidity changes covering a range of roughly 30% .5% rH/s B. Moist.5% rH/s Aa,g, t30 imp/s Ab,g. Thumidity Aa,g, ahumidity Ab,g. Irameters Ba,b. TrH between 10 and 20% rH. The rate of humidity change was between –1.5% rH/s and +1.5% rH/s , in the dry cell from 5 to 15 imp/s . Plotting the impulse frequency of both cells as a function of instantaneous humidity and its rate of change yielded flat regression planes . For the moist cell, 2R indicates that, in a series of oscillating humidity changes, an average of only 12% of the variation in impulse frequency can be explained by a multiple regression; for the dry cell, the value was 14%. Thus, both cells are only slightly affected by humidity changes, if at all.In the third experiment, the humidity changes covered roughly 10% .5% rH/s C. In th.5% rH/s Aa,g, i15 imp/s Ab,g. Pn planes Ba,b. Fi) open farm fields and areas of short grass with clusters of trees shadowing the ground (ii) through deciduous forests with low canopy cover (iii) through fragmented forests with transitions (edges) to grasslands with ruderals and other herbs . Habitat affected the extremes of humidity peaks and troughs as well as the mean values. Specifically, the humidity transitions were more round and smooth in open grassy fields  than in deciduous forests (a.b.c), but they were abrupt in edge habitats . In open grassy fields, light winds were interspersed with brief periods during which wind speed fluctuated or remained broadly stable at a new level (d). Air temperature was relatively constant (e). In fragmented deciduous forests, however, rapid changes in wind speed were evident, and the rate at which peaks occurred ranged considerably (d). Temperature tended to fluctuate constantly rather than peak (e). Crossing the edges of a fragmented habitat yielded rapid changes in wind speed with multiple peaks (d). In the same situation, temperature changed quickly and fluctuated around a new value (e). A comparison of the temporal profiles of the parameters measured in different landscapes revealed that the absolute humidity  affects the relative humidity (a) and the saturation deficit (––Cb) more than it does the temperature (e) or wind speed (d).These experiments center on the questions of what kind of humidity stimuli a honey bee may encounter during long-distance flights. We made 25 measurement trips using an automobile that traveled along vegetated roadsides, passing  py cover B, and . Moreover, when the absolute humidity is high, the peaks and the troughs are also high, and when the absolute humidity is low, the peaks and troughs are also low. The almost parallel slopes of the regression lines used to approximate the dependences of the peaks and troughs of the relative humidity and the saturation deficit on the absolute humidity indicate that the fluctuation amplitudes are almost the same at different absolute humidities. By contrast to the absolute humidity, the peak and trough of the relative humidity were affected neither by temperature  nor wind speed . Similarly, the peak values of the saturation deficit display no dependence on temperature or wind speed. However, the trough values of the saturation deficit slightly decrease with increasing temperature.The temporal profiles in .8% rH/s . The ratperature Ab,Bb nnd speed Ac,Bc. In his study of the cockroach's hygroreceptors, Yokohari (1978) suggested that hygroreceptors function as mechanical hygrometers. He provided a descriptive model based on the assumption that the cuticular wall of the sensillum acts as a hygro-mechanical transducer that changes its dimension due to the uptake (adsorption) and loss (desorption) of water like a hair hygrometer. This water uptake detector functions because a human or horse hair under tension becomes longer when the relative humidity rises and shorter when it falls. It became accepted wisdom that relative humidity is the adequate stimulus. However, the relative humidity is not a direct measure of the water vapor in the air. Identical relative humidity values do not indicate identical atmospheric moisture conditions unless the temperature is also the same. A 50% relative humidity at low temperatures contains much less water vapor than a 50% relative humidity at high temperatures. The reason is that the higher the temperature, the more thermal energy is in the air and the more evaporative work can be done in that air parcel. Evaporation will therefore occur more rapidly from a moist surface (under the same conditions) into an air with a relative humidity of 50% at high than at low temperature. The relative humidity of the air alone does not indicate the moisture conditions of the air. An air parcel with a 50% relative humidity is dry when the temperature is high but wet when the temperature is low: 50% relative humidity indicates dryness at high temperatures or wetness at low temperatures. Relative humidity is a useful measure of evaporation provided that moisture conditions are being assessed at a single constant temperature. Thus if an insect defends its water content by keeping evaporation constant, it must maintain a constant vapor pressure deficit and search for regions of similar saturation deficit. To maintain the same saturation deficit at high temperatures, the relative humidity must be increased and vice versa. If observations are to be extended over a range of temperatures, however, the saturation deficit is the exact measure. For this reason we recorded the water vapor pressure, the saturation deficit as well as the relative humidity. However, nothing is known about the amplitudes and rates of both humidity and temperature changes encountered by a running or flying insect in its natural environment. In this study we addressed these issues by mobile measurements of the fluctuating strength of humidity and temperature in different landscapes.Mobile humidity measurements from an automobile have the disadvantage of being confined to passable landscapes. Nonetheless, the measurements can be made representative very easily by repeating a trip so that humidity values at different times or weather conditions can be quantified for given landscapes; the common evaluation method facilitates data interpretation. Importantly, this study did not attempt to characterize the microclimatic conditions of various landscapes. The potential effects of landscape structures such as topography, slope and aspect, or composition, density and heights of vegetation were not considered. Moreover, the measurements were done on calm and clear summer days, which are no doubt the most suitable for bee flight activity.The important finding was that the dominant form of changes in the absolute humidity was slow, regardless of landscape type. Accordingly, the rates of changes in relative humidity and the saturation deficit were low. The landscapes, however, differ in the range and amplitude of humidity changes. In contrast, the temperature changes were small in the different landscapes; similarly small was their effect on the humidity peak and trough values, regardless of how humidity was expressed. Wind speed generally changed more pronouncedly than temperature but without affecting the humidity peaks or troughs.rH) and then stimulated with higher humidity levels up to saturated air. Conversely, the dry cell was adapted to extremely moist air (100% rH) and then stimulated with lower humidity levels down to dry air. In the moist cell, impulse frequency was higher the higher the humidity level used for stimulation, and conversely, the dry-cell's impulse frequency was higher the lower the stimulating humidity level. Nonetheless, the technique used to stimulate the worker-bee's hygroreceptors requires comment The first recordings from a single sensillum containing a hygroreceptor were reported by Lacher (1964) 50 years ago. He identified a moist cell on the antenna of the worker bee. Waldow (1970) demonstrated electrophysiologically on the antenna of the locust a dry cell together with a moist cell in the same sensillum. Yokohari et al. (1983) reinvestigated the worker-bee's hygroreceptive sensilla and found a moist cell and a dry cell combined in a single sensillum with a cold cell. Stimulation consisted of rapid, step-like changes in relative humidity. To this end, the moist cell was adapted to extremely dry air (0% rH), a 1 imp/s increase in the moist-cell's discharge can be elicited by increasing instantaneous humidity by +3.3%. In the same situation, it takes a –2.5% decrease to increase frequency of the dry cell by 1 imp/s. When the oscillation amplitude is reduced to a medium value (∼30% rH), it takes in the moist cell a +5% increase in instantaneous humidity for a 1 imp/s increase; in the dry cell the value is –5%. A further reduction of the oscillation amplitude to a small value (∼10% rH) requires even larger humidity changes to affect the discharge rates of the two cells by the same amount.The range over which humidity oscillated determined the response magnitude of the moist and dry cells as well as their sensitivity. This magnitude decreased with diminishing oscillation amplitude. In effect, they responded well to large and poorly to small amplitude oscillations. In accordance with the responsiveness, sensitivity decreased with diminishing oscillation amplitude. Sensitivity is usually described by the reciprocals of the slope values of the input-output functions, i.e., the increment in stimulus strength required to change impulse frequency by 1 imp/s. Insects are believed to regulate their water content by avoiding areas with extremes and by moving along the humidity gradient into a more suitable environment. While avoidance or search behavior is certainly very efficient in dealing with the insect's water balance, it has the disadvantage that the insect's entire activity is devoted to improving its humidity environment. It therefore competes with other forms of behavior and its presence at a given time means that humidity is at that time the most important motivation for the insect. In trying to understand the contribution of hygroreceptors to specific behaviors, the responses of the moist and dry cells were plotted as functions of the instantaneous humidity and its rate of change. The range of impulse frequency spanned by the regression planes and their slopes indicate hygroreceptor sensitivity. High values are much more likely to lead to behavioral responses than low ones. Reduction in sensitivity suggests, but does not strictly demonstrate, a decrease in information content.In the honeybee's hygroreceptors, high sensitivity is devoted to large-amplitude humidity oscillations, which may seriously affect body water content. Small-amplitude oscillations are associated with low sensitivity, probably because they are less harmful. Accordingly, sensitivity is related not only to the hygroreceptors' inputs but also to the outputs relevant for the specific behavior of improving water balance. Low-amplitude oscillations will be relevant too if they occur in the range of extreme humidities but they may be of little use for orientation as the stimulus field has no definite directions. However, some insect tolerate extremes of their water content and finally carry out escape movements intended to improve the humidity environment.


The nanoscale friction between an atomic force microscopy tip and graphene is investigated using friction force microscopy (FFM). During the tip movement, friction forces are observed to increase and then saturate in a highly anisotropic manner. As a result, the friction forces in graphene are highly dependent on the scanning direction: under some conditions, the energy dissipated along the armchair direction can be 80% higher than along the zigzag direction. In comparison, for highly-oriented pyrolitic graphite (HOPG), the friction anisotropy between armchair and zigzag directions is only 15%. This giant friction anisotropy in graphene results from anisotropies in the amplitudes of flexural deformations of the graphene sheet driven by the tip movement, not present in HOPG. The effect can be seen as a novel manifestation of the classical phenomenon of Euler buckling at the nanoscale, which provides the non-linear ingredients that amplify friction anisotropy. Simulations based on a novel version of the 2D Tomlinson model , as well as fully atomistic molecular dynamics simulations and first-principles density-functional theory (DFT) calculations, are able to reproduce and explain the experimental observations. The mechanical behavior of materials at the nanoscale may present completely novel and unexpected features as compared to their bulk counterparts. Particularly in the process of understanding and controlling friction at the nanoscale one faces new challenges and paradigm shifts24et al.The advent of single layer graphene opened up new possibilities for studies on the mechanisms of nanoscale friction. Major efforts have been addressed, for instance, to understand why friction increases as the number of atomic layers is reduced789One is then led to the question of friction anisotropy in graphene, which has been observed in exfoliated monolayer graphene, but with a 180° period angular pattern, and not the 60° period expected from the sixfold rotational symmetry of the graphene lattice11121314In this work, we demonstrate that friction anisotropy in graphene is tunable by the normal force and can reach values as high as 80% , which represent a giant enhancement with respect to graphite. This behavior is quite unexpected, since the linear elastic properties of graphene  are isotropic, as expected from symmetry considerations. Using a combination of simulations based on the Prandtl-Tomlinson model16Graphene flakes were prepared in ambient condition by micromechanical cleavage of bulk graphite. A typical flake used in our measurements is shown in L as illustrated in the inset of −1) and armchair (2.3 nm−1) lattice periodicities, respectively (we have considered the graphene lattice constant to be a = 0.246 nm). When the tip scans along the zigzag crystallographic direction (30°), a single prominent peak at 4.0 nm−1 is clearly observed in the FT spectrum. On the other hand, when the tip scans along any other direction, at least two prominent peaks are observed in the FT spectra. These two features have spatial frequencies of 1/λn and 1/λn+1, and the largest separation between them occurs for scanning along the armchair direction (±30°). λn and λn+1 as a function of the scanning angle (with respect to the zigzag direction): full circles and triangles are experimental data, empty squares are results from Prandtl-Tomlinson simulations, and the solid lines represent an analytical approximation , and armchair (c) directions. All profiles present short-period stick and slip jumps superimposed with slow build-up of friction forces, eventually reaching saturation after the tip completes approximately 10 stick-slip cycles. The orientation dependence of the stick-slip patternOnce we have determined the crystallographic orientation of the graphene sheet, friction forces can be measured along specific directions. N). Surprisingly, the energy dissipated along the armchair direction is about 80% larger than the energy dissipated along the zigzag direction, for large normal forces. Interestingly, this giant friction anisotropy can be tuned by the applied normal force: friction enhancement along armchair with respect to zigzag increases from roughly 15% to reaching saturation at ~80% as the normal force increases from 50 nN to 150 nN as shown in Strengthening of tip-surface interaction induced by the tip motion certainly increases energy dissipation, by converting kinetic energy of the tip into lattice vibration energy. The energy dissipated in each scanning cycle is obtained by integrating the friction force over the lateral displacement in the forward and backwards scan loops, as illustrated in 3N4 tip scanning over a single graphene layer. The anisotropy in HOPG observed in our experiments has atomic-scale origin and it is discussed in detail in the Friction anisotropy has been previously observed in bulk graphite, and was associated with the incommensurability of an “effective tip” generated by adhesion of a small flake of graphite to the tip apexAs it is evident from the comparison to the HOPG case, the giant and tunable anisotropy observed in graphene must be related to flexural deformations of the monolayer, which are likely enhanced when the tip moves along the armchair direction, with respect to the zigzag. This is surprising, because linear elastic constants are isotropic in graphene, so one should not expect to be any easier to buckle a graphene sheet by applying forces along any specific direction. Besides, assuming that the graphene sheet is flat when the motion begins, lateral forces exerted by the tip on the sheet are only 15% larger along armchair (the friction anisotropy of HOPG), therefore there must be some sort of nonlinear ingredient responsible to amplify the anisotropy of flexural deformations as the tip moves.m and position rt, interacting with the surface via a potential energy V(rt). For HOPG, we useWe are able to model and understand the giant friction anisotropy in a graphene using a modified version of the well-stablished Prandtl-Tomlinson model16a = 0.246 nm. This potential energy corresponds to placing the zigzag direction along x. The base of the microscope is located at position rM, and the elastic energy of the strained tip is modeled by an isotropic harmonic potential: with Ft of the elastic force along the base trajectory is integrated along the path to give the dissipated energy. Technical details of the simulations are presented in the The stick-and-slip motion of the tip apex gives rise to friction and the component X to describe flexural displacements, which evolves according to the following equation of motion:We then introduce flexural degrees of freedom of graphene by adding an effective dynamical variable M is an effective mass that describes the inert, interacting with the surface via a potential energyrtia of the flexural mode and W(X) is the restoring potential of the flexural mode, in which we include a non-harmonic cubic component on the potential C > 0. The cubic term is important to make the system stable after we introduce the feedback mechanism by which the increase in flexural amplitude enhances the tip-surface interaction, to be discussed below. Damping is introduced by the ΓFL is the lateral force due to the tip, which drives surface buckling only if it becomes larger than a critical force Fc. This is the well-known condition for Euler buckling which occurs not only for macroscopic rods and membranes1921where V0 on the flexural amplitude X: V0→V0 + αX. This is illustrated schematically in 2, it should have no effect on friction anisotropy. The anisotropy amplification effect can be easily understood by considering an extreme situation in which the lateral forces along armchair, initially only 15% larger than zigzag ones when the sheet is planar (as for the HOPG case), become larger than the critical buckling force as the tip moves, whereas zigzag forces do not. In this special case, armchair scanning causes buckling and zigzag scanning does not, leading to giant anisotropy amplification. The effect can be tuned by increasing the normal forces, which in turn increase the lateral friction forces.A feedback mechanism by which the increase in flexural amplitude enhances the tip-surface interaction is introduced simply by adding a linear dependence of the tip-surface potential energy amplitude σvm known as the von Mises stress24σij as:Finally, in order to provide an atomistic visualization of the mechanisms leading to these phenomena, we perform classical molecular dynamics (MD) simulations. Our simulation model is composed of an elastic tip (the AFM tip) and a single layer of graphene, which interacts via van der Waals forces with a horizontal substrate which is held fixed below it. The AFM tip is pushed against the surface using a constant force (~0.3 pN) perpendicular to the graphene plane and forced to move along the horizontal direction, parallel to the membrane, as illustrated in In summary, we demonstrate the existence of a giant and tunable anisotropy in nanoscale friction between an AFM tip and graphene. The microscope tip was observed to move in a discontinuous stick and slip mode allowing the identification of the graphene crystallographic directions. By increasing the normal force, the anisotropy increases from 15% (same as HOPG) to roughly 80% larger for scanning along the armchair direction, with respect to the zigzag one. The nonlinear mechanism responsible for amplifying the anisotropy is related to Euler buckling in membranes, which leads to flexural deformations of the graphene sheet, causing puckering of graphene around the tip and enhancing tip-surface interactions, in a feedback mechanism. The proposed mechanism is supported by theoretical calculations in three levels: density functional theory, molecular dynamics simulations and Tomlinson model. This novel behavior is an important step in understanding and controlling friction in single-atom-thick membrane at the nanoscale.2 layer on top of a Si substrateGraphene flakes were prepared in ambient conditions by micromechanical cleavage of bulk graphite onto a 300 nm SiOAll nanoscale friction measurements showing atomic-scale lattice resolution were acquired using silicon nitride V-shape cantilevers, with tip radii of about 20 nm and calibrated normal and lateral spring constants of 0.40 ± 0.01 N/m and 85.4 ± 4.2 N/m respectively. During quantitative FFM measurements, the scanning direction was kept perpendicular to the cantilever main axis in such a way that the friction force experienced by the tip was measured by the lateral torsion of the cantilever. The microscope was calibrated for each tip used, and the lateral force was obtained from the product between the cantilever’s lateral spring constant and the lateral tangential displacement of the tip measured in the position sensitivity photodetectorHow to cite this article: Almeida, C. M. et al. Giant and Tunable Anisotropy of Nanoscale Friction in Graphene. Sci. Rep.6, 31569; doi: 10.1038/srep31569 (2016).


In the course of decentralization, pastoral communities in Namibia have had to find new ways to share their most salient resource, water, and the costs involved in providing it. Using data from sixty communities, we examine (1) whether and to what extent different sharing rules emerge, (2) how variations can be explained, (3) how rules are perceived and influence success, and (4) what economic consequences they have. Our results reveal that either all members pay the same  or payment is according to usage . We find that although proportional equality provides more success, the rule can only pertain where the state maintains an active role. Simulations show that where it does not prevail, wealth inequality is likely to grow. These findings have political implications and suggest that, in the context of the widespread decentralization policies, the state should not withdraw if it aims to ensure the success of common-pool resource management and to fight poverty. As the impact of humans on communal resources grows, there is an increasing need to better understand how resources can be governed sustainably. Elinor Ostrom has identified eight principles that explain failure and success in shared communal resource management. While the significance of some of the original variables  has been explored to some extent during the last decades Ostrom , comparanumerical and proportional . With proportional equality, the payment received varies according to the time each individual spends actually working, whereas with numerical equality all those who work are paid the same amount. Logically, the latter is a special case of the former. The parable of the workers in the vineyard offers an illustration (Matthew 20:1–16). Here, the winegrower decided to pay all workers the same, independent of when they began working during the day. Thus, he established a relationship between the two variables at stake, ‘work’ and ‘pay’ which is not proportional but numerical in Aristotle’s sense. Not surprisingly, those who had been working since dawn complained.Sharing agreements are based on notions of equality. Equality is a functional relationship between two variables, for example, time worked and payment received. According to Aristotle, there are two kinds of equality: In the case of communal resource governance, cost- and benefit-sharing institutions must resolve the same fundamental issue , and she concluded that institutions are perceived to be fair if there is a ‘congruence between appropriation and provision rules’ Ostrom , p. 90, Currently, Namibia offers a unique opportunity to test these expectations in an investigation of the emergence, success, and consequences of sharing agreements on a large scale. In the course of decentralization, rural communities have had to find new ways to share their most salient resource, water, and the costs involved in providing it. In the arid environment of northwestern Namibia, pastoralism is the dominant livelihood strategy, and almost all households own cattle and small stock. Until some 50 years ago, most African pastoralists obtained water through natural springs, surface water, and hand-dug wells McCabe . Open waIn northwestern Namibia, between 1960 and 1990 the number of boreholes increased almost by a factor of ten Bollig . Until iStarting in the mid 1990s, the implementation of CBNRM has led to a drastic reconfiguration of the organizational and institutional landscape , longitudinal data, or simulations is appropriate. Such data were not available prior to the study reported here. We explore for the first time (1) whether and to what extent different sharing rules emerge, (2) how possible variations can be explained, (3) how different rules are perceived and hence influence success in community water management, and (4) what economic consequences distinct rules are likely to have.The arid Kunene region is sparsely populated. Small communities dot the vast landscape and, on average, they contain 13.1 (SD 3.5) households with 13 (SD 9.3) household members each. In general, dependency on natural resources is high. Pastoralism is the dominant subsistence strategy and the pastoral livelihood is constrained by the environment, most notably the low and unpredictable precipitation Bollig . Annual Livestock possession is unequally distributed in Kunene and other parts of Namibia  as part of a German Research Council (DFG)-funded research project LINGS . The two principle investigators, M. Schnegg and M. Bollig, have been conducting ethnographic fieldwork in the region since 1994 (M. Bollig) and 2002 (M. Schnegg) respectively, and are responsible for the overall design and comparative analysis of the data. In the first phase of the current fieldwork, three anthropologists  stayed for roughly 1 year between 2010 and 2011 in seven communities in the southern (Fransfontein), central (Otwani), and northern (Okangwati) parts of the research area to gain an in-depth understanding of processes entailed in negotiating and crafting new institutions through daily routines. During this time, all 80 households were interviewed about their social and economic livelihoods, including economic possessions and social networks.2 around Fransfontein, Otwani, and Okangwati, all communities were interviewed. We can thus treat the data as a complete sample for those three areas. We decided against a representative sample of the entire Kunene region due to its size, bad road infrastructure, logistical constraints, and the lack of a list of communities that could serve as a sampling frame. In addition, our approach allowed us to make use of the fact that fieldworkers were already known and trusted in the target areas.After an initial analysis of the ethnographic data collected during 2010 and 2011, we returned to the field in late 2012 to conduct the ‘upscaling’ research we had designed to study the distribution of some of the phenomena found in the community ethnographies. Since our study treats communities as cases, it is challenging to collect relatively large numbers of observations that permit meaningful comparisons. To facilitate this requirement, we designed an interview guide to elicit information at the community level. For geographical areas of approximately 250 kmThe research protocol contained three sections. First, we elicited the rules of water management and the composition of community-based organizational structures for water governance in the community. Each interview took place in public and included both female and male informants, some of whom were active in the water point committee. The second and third sections dealt with the success of the water management and the conflicts communities had experienced or observed. Since those questions are more confidential, interviews were done in private, aiming at a sample of males and females of different age groups and from different economic positions. In total, we researched sixty communities. Since information remains incomplete for some of them, the number of communities included in the analysis is sometimes lower than that.All group interviews were taped and two independent interview protocols were written by the main researcher and his/her assistant. Discrepancies were resolved thereafter. The data were entered in MySQL database, designed for the project. The coding was done by the principal investigators in collaboration with the researchers.The measurement of success derives directly from the questions posed and is coded on an ordinal three-point scale. To measure the involvement of the state, we use two indicators. During the interview, we collected two types of information. First, the frequency and purpose of visits by state officials in the community during the last 2 years. If the state officials had visited the community at least once during the last year for consultancies and activities other than urgent repairs of broken infrastructure, we took this as an indication of more than average state involvement. Since CBNRM and the decentralization strategy are highly standardized, we did not observe any variations in the type of rules recommended. Generally, state officials try to push communities in certain directions above all concerning the payment schemes. Second, our ethnographic observations have shown that if employees of the ministry or commissioned NGOs lived in the communities at issue, the impact of the state was significantly stronger, because those people typically wanted their communities to be flagship cases for the state’s mission and ideology (see below). For the analysis, we coded the state involvement to be above average, if either of the two indicators was given.‘‘Recommended is a rate per head of large or small stock, each member paying a certain rate per head of large or small [stock] accordingly, as to raise enough money to sustain the water point”  and/or NGOs contracted by the government  agreed that individuals paid fees according to the number of livestock they owned . Thus, the more water one uses, the more they pay. This fits the notion of proportional equality. In addition, seven communities (12.5 %) used an attenuated form in which the rich paid more, but not exactly in proportion to the number of their livestock.4.6 % agrBefore we offer an explanation why, we want to rule out two common alternative hypotheses: technology and a “majority cost/benefit vote” Ostrom . WhereasThe account we provide instead is formulated on the basis of our ethnographic work and includes four factors. Of those, three apply in all communities and favor a numerical rule. Only the last factor, the involvement of the state, makes a difference, as the statistical analysis reveals.To establish and maintain a proportional rule is more costly than a numerical rule. Most importantly, it requires counting animals. Counting livestock is difficult. Counting individual animals of small stock in large herds from different owners flowing toward a well is nearly impossible. In addition, there are cultural taboos against counting livestock, and people complain that counting livestock brings about bad luck Bollig . In geneNot surprising, wealthy herd owners oppose a proportional rule and opt for numerical equality . Thus, water is only one resource in a larger sharing arrangement and a moral model exists that short-term imbalances in one domain will equal out across all types of transfers and time , the involvement of the state can explain the institutional outcome to a significant degree. In contrast, in communities where the state is only weakly involved the first three dynamics analyzed above are dominant and numerical equality prevails.We have outlined when and why different equality regimes evolve. Since the groundwater in northwestern Namibia comes from aquifers fed in Angola, ecological success cannot be measured locally. We, therefore, base our analysis of the success of the water management regimes on social indicators developed in cooperation with communities. These include (1) satisfaction with the rules, (2) satisfaction with the work of the water point committee (committee), (3) general level of satisfaction expressed concerning cooperation in the community, and (4) the general satisfaction with the water management in the community.Table As we have indicated, livestock is unequally distributed. Thus, with numerical equality in a hierarchical setting, where all individuals pay the same, the institutional regime must have economic consequences that diverge from equality. To explore these in depth, we developed a simulation model. The model starts with the distribution of livestock that we found among 80 households for which we did detailed livestock counts. Small and large stock are aggregated into one measure, livestock units (LSU), for which a head of cattle equals one LSU and small stock  one sixth  has become a guiding principle of many development efforts—the idea that turning ownership and responsibility over to local communities will empower them and help eradicate poverty Ribot . Left al


The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. Genes containing introns can produce several transcripts and protein products via alternative use of exons and introns. Alternative splicing (AS) exploits these gene structures to increase transcriptome and proteome complexity and to serve regulatory purposes in higher eukaryotes.Splicing requires a complex molecular machinery, the spliceosome, which is directed by three important splice signals: the 5’ splice site, the branch point and the 3’ splice site with associated polypyrimidine tract. These signals are detected by components of the spliceosome and define the exon-intron boundaries. To enable AS in higher eukaryotes, many auxiliary factors are present such as SR proteins or hnRNPs . These cIn S. cerevisiae, about 4% of all genes contain introns and the organism is able to correctly splice those genes. While complete splicing is well studied in S. cerevisiae, the use of alternative splice sites is only described in individual examples, which we will review below. These examples have been reported in the literature and are not annotated in resources like the Saccharomyces genome database (SGD) or Ensembl. The simple gene structure and certain properties of the S. cerevisiae genome suggest that AS in S. cerevisiae is limited: its genome shows strong 5’ splice sites and a highly conserved branch point sequence (TACTAAC), which facilitate the splicing of introns . FurtherIndividual examples of AS have been described, especially in the context of environmental stress or certain states of the cell cycle. Intron retention and subsequent nonsense mediated decay (NMD) are reported for certain genes during mitosis  or by auMore complex AS has been confirmed on the transcript level, leading to multiple alternative mRNAs for a single gene locus . Intron Finally, changes in the amino acid sequence by AS have been observed. Intron retention generates two different isoforms in PTC7, which determine the protein localization. The intron does not disrupt the reading frame and introduces 93 bases into the mRNA. The resulting additional 31 amino acids contain a transmembrane domain and change the localization and cellular function of the protein . Usage oMoreover, Pelechano et al. have shown that S. cerevisiae is capable of producing a high diversity of transcript isoforms by using different transcription start and end sites. More than 26 transcript isoforms per protein-coding gene have been observed . These rIntron retention, a common mechanism in fungi and plants, is often observed in S. cerevisiae splicing. Most known introns will disrupt the reading frame, leading to a premature stop codon. S. cerevisiae is able to degrade the affected transcripts using the nonsense mediated mRNA decay pathway , and useIn this study we show that S. cerevisiae makes extensive use of its limited alternative splicing capabilities. We analyze a publicly available mRNA sequencing data set of S. cerevisiae and report novel introns, the pervasive use of alternative splice sites and evidence for unspliced transcripts that possibly occur due to intron retention. Results are validated in three different ways. Firstly by using multiple mapping programs, secondly in a complementary mRNA sequencing dataset and thirdly, via PCR, which we performed in independent S. cerevisiae samples. Advances in next generation sequencing technology provide a large number of sufficiently long reads (≥ 100 bp) that can be mapped confidently to unique positions of the genome, even if a read spans a splice junction and two ends of the read need to be mapped individually. We analyze one of the first of these available data sets by Nookaew et al. , observewww.ensembl.org). Read mapping was performed using Bowtie [Sequencing data was obtained from Nookaew et. al. . The S. g Bowtie  and TopHg Bowtie  with defg Bowtie  and Contg Bowtie . TopHat Predicted introns were validated in another, independent next generation sequencing dataset . The rawFinally, several cases of novel introns and AS events are validated via PCR. Details are described in For a given transcriptome sequencing data set, we define a potential intron as chromosomal subsequence which is spanned by a split read mapping, i.e. the first part of the read is mapped to the genome before the potential intron and the other part after the potential intron. Potential introns provide evidence of splicing events and, in case of overlapping potential introns, AS events. They may match or overlap known introns, according to the annotation of the Saccharomyces Genome Database, and they can also constitute novel introns.We focus on potential introns that show similar characteristics to those of annotated S. cerevisiae introns in terms of length and splice signal, and which are supported by sufficient evidence. Potential introns meeting those three criteria are called predicted introns (PIs) as outlined in n) bases upstream or downstream due to repetitive sequence patterns at the junction boundaries, the exact position of a split is unclear. We observe that known introns are spanned by clearly defined split reads, i.e. the intron can be assigned to an exact genomic location in almost all cases. Furthermore, the supporting split reads match well before and after the junction exhibiting a long “anchor” on either side. Combining these two observations into a single score results in a measure for “junction quality”, defined as the anchor length minus n. Hence for each predicted intron we have two junction quality values. For PIs we require similar values as we observe for annotated introns, i.e. the maximum of both values must exceed 85 and the minimum must exceed 25  and two within small nucleolar RNA. Mitochondrial introns are not considered in this study because they are spliced differently from chromosomal genes .With, in general, only one intron per gene, the only possible forms of AS are intron retention, alternative 3’ and alternative 5’ splice site selection. These are summarized in After read mapping we obtain 880,312 mapped split reads, which define 5,093 potential introns in the genome. On average there are 173 reads per intron, but reads are distributed in such a way that a few of the potential introns are supported by a large fraction of all reads: 1,532 potential introns (30%) are supported by only one read, 2,418 by two or less reads. The median read support is 3. An overview of read support is given in Out of 447 predicted introns 277 match known introns. The remaining 170 predicted introns either define an alternative splice site at known introns (71 cases) or constitute novel introns (99 cases). Out of 99 novel introns, 27 are predicted as “single intron” see  and 12 aFour additional novel introns with sufficient read support are disruptive to the reading frame  we also find evidence of the unspliced transcript. Mostly, inclusion of annotated introns will lead to either a disrupted reading frame and/or an introduced stop codon.In our data we find 56 introns located in the 5’ UTR. Among these introns, we identify 20 of 24 known introns and 36 novel introns. In Under the most conservative assumptions we identify a total of 63 annotated introns with variable splice sites. These divide into 44 cases of an alternative 3’ splice site, 16 cases of an alternative 5’ splice site and three cases with combined alternative 3’ & 5’ splice sites. There are 22 (3’) and 8 (5’) cases with 10 or more supporting split reads. Compared to 3’ and 5’ alternative splice sites, combined alternative splice sites exhibit the lowest read support see .Among the alternative 3’ splice sites the median distance to the canonical splice site is 28 bases. There are 11 cases that do not disrupt the reading frame and are able to encode an altered protein product. The AS events are able to remove or introduce between 1 and 53 amino acids compared to the reference sequence. The remaining 33 cases will disrupt the reading frame, if the intron is located in the protein coding region. Examining some of those cases in more detail, we find examples in which the AS event may trigger well known biological functions: Alternative splicing in YDR099W (BMH2) and YBR078W (ECM33) introduces a single additional codon, the splice site is of the NAGNAG form, providing two adjacent acceptor sites. The additional codon introduces a new amino acid into the resulting protein sequence, which has been reported as a mechanism to control protein localization  and as aThe remaining alternative 3’ splice sites are located downstream of the canonical splice site, extending the intron and removing amino acids from the protein product. Intron extension may be explained by a variation in the linear scanning mechanism proposed for detection of the splice acceptor site starting from the branch point .We find evidence of three cases of novel introns overlapping the genes 3’ end . SplicinIn the spliced isoform of YBR101C (FES1) the terminal YVL* is substituted with TFCKMEFIKKVRRGK*. In case the terminal intron in YMR147W is spliced, the alternative protein terminus extends into the reading frame of YMR148W (OSW5), leading to a chimeric protein fusion. This confirms a previously described observation [The alternative splicing events reported in this paper were validated in three different ways: (A) by using different mapping software, (B) in an independent sequencing dataset, and (C) via PCR for selected splicing events.(A) The first validation was performed using two other mapping programs (Star  and Cont(B) Predicted introns were cross checked against read data from another next generation sequencing dataset . We cons(C) We used S. cerevisiae samples to validate our identified splicing and alternative splicing events by PCR. Primers could be designed if the following conditions were met: First, the length difference of the expected PCR products had to be large enough, such that different products would become visible on the gel. Second, the gene and isoform under consideration had to be expressed in our available S. cerevisiae samples. Primers must be unique and close enough to the alternative event and we require them to lie within genomic regions that are transcribed in the used samples. We also designed primers for three additional cases in which we found striking evidence of alternative splicing or splicing in our own S. cerevisiae samples. These cases are also found in our original dataset , but witUnder the abovementioned constraints, we designed primer pairs for 10 predicted novel introns: Five pairs were designed for novel introns in the 5’ UTR, three pairs for novel introns in protein coding regions and two pairs for alternative 3’ splice sites at two known introns. Out of those 10, PCR confirmed alternative splicing in 7 cases: splicing was confirmed for the 5’ UTR introns at genes YLR467W (YRF1-5), YBL111C and YGL063W (PUS2) . Furthermore, AS could be confirmed for the novel introns in YBR101C (FES1), and in YMR147W. In both cases the annotated stop codon is observed as both, included and spliced out in the isoform. The third novel intron in the protein coding region of YGL136C (MRM2) is alternatively spliced according to the sequencing data, and PCR confirms the presence of an intron. Finally, PCR confirmed the alternative 3’ splice site in YBL026W (LSM2), but not in YOR293W (RPS10A). Pictures of the gels and sequences of all primers are listed in the supporting information .We analyzed alternative splicing (AS) in the yeast S. cerevisiae. High throughput mRNA sequencing data with sufficient read length enable us to confidently map split reads to unique positions on the genome. As a result we can identify an unprecedented amount of previously unknown introns and observe several cases of alternative intron usage in S. cerevisiae. Some of these AS events have been described earlier and are referenced in the introduction; in most of these cases the alternative isoform shows high read support in our analyzed data and may be detected more easily in general. In addition to the previously described AS events we find novel cases, indicating a more widespread use of AS in S. cerevisiae than currently known. Strikingly, we validate most of the reported alternative events in a different, independent NGS dataset. We then validate reproducible splicing events via PCR in 7 out of 10 tested cases in a dedicated experiment.In about 20% of annotated introns we find evidence of alternative 3’ or 5’ splice sites, with a strong bias toward 3’ variation. Compared to 5’ splice sites, S. cerevisiae exhibits weaker splice signals at 3’ splice sites, which facilitates alternative splicing [Reads supporting intron retention can be found in every intron containing gene, albeit with low read support in general. The mechanism is common in fungi and plants and serves regulatory purposes, but the retained introns could also be an artifact of the stochastic nature of the splicing process. As such they might be yet unspliced transcripts or defective splicing, which will be targeted by NMD. However, we find evidence of eight novel introns that are not disruptive to the reading frame. The relatively weak evidence for the spliced, novel isoforms could explain why these introns have not been identified earlier. Intron retention has been shown to cause alternative proteins in at least one S. cerevisiae gene (PTC7) ), where There are 10 annotated cases of multi intron genes, and we predict a second, novel intron in the gene YFR031C-A (RPL2A). The respective genes contain two introns each and allow the possibility of AS induced exon skipping or cassette exons. Exon skipping has been reported for the SUS1 gene , but in We conservatively analyzed a single dataset with the hypothesis of limited AS in S. cerevisiae. We used a small dataset that comprises two conditions and systematically screened for variation among annotated introns. Nevertheless, we detect novel alternative splicing events that are subsequently validated using independent data. In general the predicted novel introns are supported by less reads than annotated introns. We conclude that the respective transcripts are minor isoforms, possibly triggered by specific conditions. They may coexist with the major isoform and are therefore rather hard to detect. PCR of alternative events resulted in the successful validation of most of the tested isoforms. While isoforms with weak evidence should be considered with care or examined under different experimental conditions, we suggest that highly supported alternative isoforms are more likely functional. This observation is supported by recent work of Kawashima et al. , in partKawashima et al. also publish a list of 728 splicing events, of which 522 suggest alternative splicing. However, the authors do not discuss them in detail. The overlap between our study and Kawashima et al. is not expected to be high, since our analysis pipeline is different  and AS is likely to be context specific. Consequently, differences in yeast strain and growth conditions result in different AS. Furthermore, technical factors like NGS library preparation method and sequencing depth and length will influence the detection of isoforms in each study. The overlap between 170 potential introns in our study and the 522 AS events is 13. Refer to In conclusion, AS in S. cerevisiae is possible and observed on the transcript level. Using split reads in mRNA-sequencing data we identify several cases of alternative transcripts, which can be explained by an alternative splicing mechanism in S. cerevisiae. Systematic analysis of AS events will help to investigate the impact on the proteome and to identify the regulatory mechanisms leading to isoform production. AS in S. cerevisiae is triggered by conditions like elevated heat  or restrS1 TableList of all predicted novel introns, sorted by evidence (read support). For completeness we also report potential introns with a read support lower than three. Columns include Chromosome, Strand, Start, End, Length specifying the position of the novel predicted intron. Additional columns areRead Support: evidence for the predicted intron.Anchor—Slack: junction quality scores.Gene: affected gene.PCR: if PCR has been performed, capital letters identify the lane in (PDF)Click here for additional data file.S1 FigPCR gels of 10 tested novel introns and alternative splicing events. The expected position of PCR products confirming novel events are marked with arrows. Numbers describe the adjacent PCR product size (in bases) of the two marker bands. Chromosomal locations of the events are listed below.μg of total RNA was transcribed in a 25 μl reaction using the M-MLV reverse transcriptase  according to manufacturer instructions. 1 μl of the resulting first strand cDNA was used as template in 50 μl PCR reactions  applying splice variant specific primer pairs .The S. cerevisiae strain BY4741   was cultivated in YPD at 30°C. Exponentially growing S. cerevisiae cells at an OD595 of 0.8 were collected and used for preparation of RNA applying the SV Total RNA Isolation System . For first strand cDNA preparation 1 er pairs  and GoTaThe above experiment was repeated three times. The first time without any sequencing attempt. The second time sequencing failed. The third time sub-clonig and subsequent sequencing was done:For sub-cloning, 50 µl PCR reactions were completely separated on 2% agarose gels and the bands corresponding to the splice product were purified using the MiniElute PCR Purification Kit . The purified splice product was sub-cloned using the TOPO TA Cloning Kit for Sequencing  according to the manufacturer’s protocol. Positive clones were selected after DNA-preparation by control PCRs with the respective primer set and further analyzed by sequencing .In Experiment J the “expected novel fragment length” could be sequenced and is matching the expected sequence. This coincides with the best visible PCR band out of any of the “expected novel fragment length” bands.(PDF)Click here for additional data file.S2 TableLocation and sequences of the PCR primers used.(PDF)Click here for additional data file.S2 FigWe compare our results with the recently published results of Kawashima et al. Venn diagrams compare potential intron assignments at two different levels of confidence in our analysis to introns reported by Kawashima et al. In each plot the set “All” contains all introns fulfilling our length requirements and presenting a valid splice signal. The set “Only accepted” only contains introns that additionally fulfill our support and junction quality criteria.A) Overlap limited to annotated introns. We observe a large overlap of about 90% introns reported in Kawashima et al.  and abouB) Overlap without annotated introns. In these diagrams we compare true alternative splicing events reported here and by Kawashima et al. The overlap among proposed isoforms is not expected to be high, since our analysis pipeline is different  and AS is likely to be context specific. Some isoforms are exactly found in both studies, so those results are reproducible under different experimental and technical conditions.c) Out of the highly confident AS events reported in the tables in the main text, five are also reported by Kawashima et al. Strikingly, all of them are at the 3’ splice site, and four preserve the reading frame. The set “Reported” only contains the predicted introns that are listed in the tables in this manuscript excluding any novel introns (n = 20), because those are not considered by Kawashima et al.The potential introns in the sets “All” and “Only accepted” are derived following our workflow, depicted in (PDF)Click here for additional data file.S3 FigThe data published by Kawashima et al.  enables In summary, we confirm the findings of Kawashima et al. Additionally, we detect some unknown AS events that seem to be unaffected by NMD:Panel A shows how the split read support for annotated introns changes between WT and the three NMD defective strains . Note that annotated introns have a higher median and mean read support in WT, than in any knockout strain. This changes among the unannotated introns in Panel B.Panel B shows the read support for predicted, unannotated introns. By requiring read support in at least two knockout strains, we gain confidence for each predicted intron. The negative fold change from knockout to WT is a result of efficient NMD in WT. This confirms the findings of Kawashima et al.In panel C we show all the predicted introns that are supported by at least two reads in WT, and by at least one read in one of the knockout strains. As a result, we are confident about the predicted introns in WT and able to compare them to the knockout strains. The boxplot clearly shows that the lack of NMD (in the knockout strains) has little or no effect on isoforms that are confidently expressed in WT. Consequently, such isoforms are unlikely targets of NMD in WT and, therefore, more likely functional. Also, there are other nuclear RNA decay pathways than NMD that might target those transcripts.Scatter plot D shows the same data as box plot B (including outliers): for each predicted intron, the WT read support is plotted against the mean read support of the knockout strains. In particular, the predicted introns with high support in WT do not show a significant fold change, so those transcript isoforms are not depleted in wildtype S. cerevisiae and, therefore, unlikely NMD targets.Unfortunately, a large scale comparison of the predicted isoforms based on our original dataset is not feasible, because the overlap of isoforms between the different datasets is not sufficiently large see .(PDF)Click here for additional data file.S4 FigSome additional evidence for a predicted intron would be its validation in a different, independent dataset. We successfully validated the most confident intron predictions by detecting their split reads in data from Waern and Snyder . In thisρ = 0.71). Among the unknown splice junctions (red circle), the correlation drops (ρ = 0.43); finally, those with a non-canonical splice signal (blue x) tend toward lower read support in general and the read support appears not to be correlated (ρ = −0.01).Figure on top: Evidently, annotated introns show high read support in either dataset and their read support seems to be correlated among datasets (Pearson’s ρ = 0.16), qualifying them as possible read or mapping errors. However, there are some potential introns covering long stretches of the S. cerevisiae genome. Some of these can also be validated in the independent dataset (green crosses around the center of the plot). These read mappings can either be explained by genomic rearrangements in the used strains or point toward post-transcriptional mechanisms in S. cerevisiae. Since there is no known mechanism explaining such transgenic splicings, the presence of these transcriptional products needs to be validated by other means.In the figure at the bottom we additionally show potential introns that do not pass our length filter (green x). The majority of those long introns shows a low read support and no correlation among datasets ((PDF)Click here for additional data file.S1 FileSome predicted isoforms might be translated into functional protein isoforms. In this file we list an in silico translation of all S. cerevisiae genes for which we predict alternative isoforms. The sequences might aid further research on the respective genes. In each record, the first line reports the gene along with the genomic location. The next line reports the predicted intron, followed by all annotated introns of the gene. Next, the length difference of the annotated and the predicted alternative transcript is given in bases: negative values indicate deletion and positive values indicate insertion events. Finally, the transcript and translated protein sequence are reported for the gene as annotated (ORI) and for the predicted isoform (VAR).(TXT)Click here for additional data file.S5 Fig(A) Split reads defining an intron. Reads r1 through r4 are mapped to the genome in a spliced form; the potential intron is supported by 4 reads. Reads r1 and r2 represent the same fragment, because they have exactly the same sequence and hence the same start and end position on the genome. As a result, the potential intron is supported by three distinct fragments. (B) Intron retention. The total read coverage (black) drops in the area of the intron and the intronic area is spanned by split reads (green). Ungapped (red) reads containing the 3’ or 5’ splice site are evidence of intron retention. (C) Alternative 3’ splice site. The total read coverage is lower in the intron, but shows an increase towards the 3’ site. There are two different types of overlapping split reads (green and blue), using the same 5’ splice site, but different 3’ splice sites. (D) Alternative 5’ splice site. Similar to alternative 3’ splice sites, but mirrored. (E) 5’ UTR intron. The intron is located before the start codon and the coding sequence (yellow) starts downstream. (F) Novel terminal intron. The intron overlaps the annotated stop codon and splices it out, extending the coding sequence to the next downstream stop codon.Alternative transcript models together with their idealized read coverage in next generation sequencing data. The black line represents the total read coverage of all mapped reads. The green, red and blue lines represent characteristic reads for certain isoforms. (PDF)Click here for additional data file.