Enhancement of heat transfers in counterflow plate heat exchanger due to presence of an intermediate auxiliary fluid flow is investigated. The intermediate auxiliary channel is supported by transverse conducting pins. The momentum and energy equations for the primary fluids are solved numerically and validated against a derived approximate analytical solution. A parametric study including the effect of the various plate heat exchanger, and auxiliary channel dimensionless parameters is conducted. Different enhancement performance indicators are computed. The various trends of parameters that can better enhance heat transfer rates above those for the conventional plate heat exchanger are identified. Large enhancement factors are obtained under fully developed flow conditions. The maximum enhancement factors can be increased by above 8.0- and 5.0-fold for the step and exponential distributions of the pins, respectively. Finally, counterflow plate heat exchangers with auxiliary fluid flows are recommended over the typical ones if these flows can be provided with the least cost. Counterflow plate heat exchangers are widely used in various engineering applications especially preheat, chemical, pharmaceutical, and food processing applications . This isThe most recent literature reviews on passive heat transfer enhancements in heat exchangers , 9 show The proposed plate heat exchanger is composed of hot and cold fluid channels separated by an auxiliary fluid channel. This auxiliary channel may contain as many passive enhancement tools as possible. Accordingly, both the velocity profile and the composition of the hot and cold fluids are preserved. The heat transfer enhancement in the proposed system is due to the following combined effects: (1) convection of the auxiliary fluid and (2) passive enhancement mechanisms in the auxiliary channel. In the present work, transverse pins connecting the facing boundaries of both hot and cold fluid channels are considered as one of passive enhancement mechanisms , 17. MorIn the present work, heat transfer inside plate heat exchanger with auxiliary fluid channel separating the hot and cold fluid channels is modeled and analyzed. Both hot and cold fluid flows are considered to be laminar under hydrodynamically fully developed condition. The energy equations of the hot and cold fluids are coupled with the energy equations of the auxiliary fluid boundary layers. The solution of the momentum and energy equations within the boundary layers is well established –20. AccoL and width W. The first channel confines the hot fluid flow while the second one contains the cold fluid flow. The solid boundaries of these channels facing each other are perfectly connected together via cylindrical pins of diameter df and length Lf as shown in T∞ and convection heat transfer coefficient hf. The convection heat transfer coefficient between the auxiliary fluid stream and the channel boundaries facing that stream is hu. Accordingly, heat transfers between the channels and the auxiliary fluid are due to convection over the unfinned surfaces and conduction via the connecting pins. In contrast, the outermost solid boundaries of the heat exchanger are considered to be adiabatic so that heat transfer rates in the heat exchanger are maximized.Consider two parallel channels of length Uh and Uc are the dimensionless axial velocity fields for the hot and cold fluids, respectively. θh and θc are the hot and cold fluid dimensionless temperatures, respectively. Xh and Xc are the dimensionless axial positions of the hot and cold fluids, respectively. Yh and Yc are the dimensionless transverse positions of the hot and cold fluids, respectively. The channels aspect ratios ah,c as well as the hot and cold flow Péclet numbers Peh and Pec are given byHh and Hc are the heights of the hot and cold fluid channels, respectively. , , and  are the density, specific heat, and thermal conductivity pairs of the hot and cold fluids, respectively. umh and umc are the mean axial velocities of the hot and cold fluids, respectively.The dimensionless momentum and energy equations of the hot and cold fluids are (1)d2Uh,xh and yh are the axial and transverse positions of the hot fluid, respectively. xc and yc are the corresponding positions of the cold fluid, respectively. xh and xc start from zero at the fluid inlets while yh and yc start from zero at the adiabatic boundaries. uh and uc are the axial velocity fields for the hot and cold fluids, respectively. Th and Tc are the hot and cold fluid temperatures, respectively. Th1 and Tc1 are the inlet temperatures of the hot and cold fluids, respectively.The dimensionless variables given in  and 2)  2) are dqfh′′ and qfc′′ are the conduction heat fluxes through the pin bases at the hot and cold surfaces, respectively. However, quh′′ and quc′′ are the convection heat fluxes at the unfinned portions of the hot and cold surfaces, respectively. The local pins base area concentration denoted by ϕ can be calculated from the following expression:dNf/dx is the local axial gradient of the number of pins (Nf).Equation  is basedtions of  are giveXf, the dimensionless pin local temperature, θf, and the dimensionless pin thermal length, m*, are given byf = hfdf/kf is the pin Biot number and af = df/Lf is the pin aspect ratio. The boundary conditions of  and θc,W = θc are the dimensionless temperatures of the hot and cold boundaries, respectively. S is the dimensionless cold excess temperature. It is equal toS ≤ 1 as auxiliary fluids are usually hotter than the cold reservoir. The solution of , where ThW and TcW are the temperatures at the hot and cold boundaries facing the auxiliary fluid, respectively. As such, (9)d2θfdXqh′) and that to the cold fluid (qc′) can be calculated fromTmh2 and Tmc2 are the mean bulk temperatures at the hot and cold fluid exit ports, respectively. θmh2 and θmc2 are the dimensionless values of Tmh2 and Tmc2, respectively. In terms of the dimensionless parameters, θmh and θmc are given byThe heat transfer rate per unit width from the hot fluid (HhTh1−Tmh=kh(Th1−TLo/L) ≤ (Lm/L) = 1 − (ϕo/ϕm). Lm is the maximum value of Lo that produce 99% of the upper limit of pins base area concentration (ϕm). ϕm when pins have parallel arrangement is equal to ϕm = 0.99(π/4). The quantity ϕo is the average pins base area concentration. To have the pins concentrated uniformly near channels midsection (see Lm/L) = (ϕo/ϕm) ≤ (Lc/L) ≤ 1.Different distributions for pins will be analyzed in this work. These distributions allow having concentrated pin distribution either far from the middle section of the channels or about this section. Two families of distributions are considered. They are the step function and exponential distributions. The step function distribution that has concentrated pins far from the channels midsection see  has the tion see , 19) ch ch(19)ϕϕB| < Bc. Bc is the upper limit value that makes ϕ = ϕm. It can be accurately correlated to ϕo through the following correlation:B > 0. However, they are more concentrated around the channels midsection when B < 0. These trends are seen in The exponential distribution of the pins has the following functional form:hh and hc, respectively, is defined ash and Nuc are equal toThe convection heat transfer coefficient for hot and cold fluid flows qhMax′ and that to the cold fluid qcMax′ are obtainable when Th2 = Tc1 and Tc2 = Th1. Using 7d) changλh, λc) are defined as the ratios of the heat transfer rate from the hot fluid and that to the cold fluid to the corresponding quantities when ϕ = ϕo, respectively. Mathematically, they are equal toThe last set of performance indicators for the present heat exchanger denoted by , , 15b), aThW−T∞=B1.Using .Using , εh andh*, Nuc*} can be obtained when assuming that the pins are very long such that the heat transfer rates in both channels are uncoupled. For this case, (h)eff and (Bic)eff are equal toh)eff, (Bic)eff} ≤ 100. It can be noticed form (h)eff, (Bic)eff} values as the maximum variation (relative) in the Nusselt numbers is less than 10%.Approximate Nusselt numbers {Nuis case,  and  =  and Bih → 0. Accordingly, their dimensionless quantities areZh = (kf/kh)(Hh/df) and Zc = (kf/kc)(Hc/df).The maximum heat fluxes due to only pin conduction are obtained when  =  and kf → 0. They are equal toc = Bih(kh/kc)(Hc/Hh).The dimensionless maximum convection heat fluxes in absence of pins can be obtained when   acPec and kf are very large, while ahPeh is very small. For these conditions, the minimum average pin base area concentration denoted by ϕhMin,cMin, respectively, can be shown to be equal toLet the auxiliary stream be laminar flow along the direction of the channels width axis. Therefore, the convection heat transfer coefficients for the fin and unfinned surfaces can be computed using the following correlations :(43)NudYh,c directions. Furthermore, two-point backward differencing quotients were used in the discretization of the first derivatives with respect to Xh,c directions. For  and  represent the location of the discretized points in the numerical grids of the hot and cold fluid domains, respectively. M is the total number of either i or I sections. Nh and Nc are the total number of the discretized points per i and I sections, respectively. M, Nh, and Nc were taken to be equal to M = 1001 and Nh = Nc = 201.Equation  is couplons. For  and θcW)iassumed.The dimensionless cold boundary temperatures were assumed ((2)Nh algebraic equations for i = 2,…, M were solved.The (3)Nc algebraic equations for I = 2,…, M were solved.The (4)θcW)icorrected were found using the finite difference equation of (The corrected cold boundary temperatures (ation of .(5)θcW)iassumed with the corrected (θcW)icorrected until the following condition is satisfied:Steps (2)–(4) were repeated by replacing , respectively. These excess temperatures increase as S decreases. Accordingly, the heat transfer rate from the hot fluid increases which causes γh to increase as both Reh and (1 − S) increase as seen in γh decreases as Rec increases. This indicates that the increase in the reference case heat transfer rate (qo′) is larger than that for the present system (qh′). Also, it is noticed from γh can be larger than one at both smaller S and Rec values and larger Reh values. Similarly, γc can be larger than one for smaller (1 − S) and Reh values and larger Rec values.As Reh decreases the hot fluid effectiveness εh as shown in S is noticed to cause an increase in εh due to the associated increase in ThW − T∞. Figures c causes an increase in εh. This is because both T∞ − TcW, ThW − TcW increase as Rec increases; thus, the conduction through the pins is enhanced. The latter enhancement cannot be clearly identified from af = 0.05. As indicated earlier, when af → 0 the heat transfer rate in both channels will be uncoupled. In a similar manner, it can be concluded that εc decreases as Rec increases and as (1 − S) decreases and it increases as Reh increases as noticed in The increase in ReLf increases (af decreases), the pin surface area increases causing the fully developed maximum fluxes Θfh and Θfc to increase until they reach their asymptotic values as shown in εh to increase as af decreases when S = 0.25 as shown in S = 0.5, Θfc ≫ Θfh; thus, TcW sharply increases causing sharp reduction in ThW − TcW. Therefore, εh decreases as af decreases when S = 0.5. The cases considered in Figures ahPeh ≫ acPec > 1. Thus, the hot fluid flow is dominated by the thermal entry region. For this condition, ThW is less sensitive to Lf, while TcW increases apparently as Lf increases. As a result, T∞ − TcW,  and ThW − TcW decrease as Lf increases. Since pins conduction is linearly dependent on T∞ − TcW, and ThW − TcW while it is less sensitive to Lf, εc decreases as af decreases as seen in γh and γc increase as af increases except when S = 0.25 where γh is noticed in af increases. Also, it is shown from this figure that γh > 1 when S = 0.25 and Rec = 10 as well as γc > 1 when S = 0.5 and Rec = 20. This demonstrates the superiority of the present heat exchanger over the conventional counterflow plate heat exchanger.As ϕo → 0 and (2) pure conduction between the channels when ϕo → ϕm. As seen in uh, Θuc) >  when af = 0.2 which represents the condition for Figures εh, εc) pair is expected to decrease as ϕo increases. This is shown in εh plot when S = 0.25. For this case, ThW − T∞, and ThW − TcW are very close to their upper limit (Th1 − Tc1) while this limit is reduced to (Th1 − T∞) for the pure convection condition as ϕo → 0. And since the heat flux is linearly proportional ThW − T∞, and ThW − TcW as can be seen in  pair decreases as ϕo increases except when S = 0.25 where γh is noticed in ϕo increases. Furthermore, it is shown from this figure that γh > 1 when S = 0.25 and Rec = 10 while γc > 1 when S = 0.5. This demonstrates the superiority of the present heat exchanger over the conventional counterflow plate heat exchanger.Two limiting cases can be encountered in the present heat exchanger. They are as follows: (1) pure convection between the channels and the auxiliary stream when  seen in  and . These excess temperatures become closer to that limit near the hot fluid inlet. Far from this region, they tend to apparently decrease as ϕ decreases since Θuh > Θfh. Consequently, the uniform distribution of pins reveals the maximum λh in which λh = 1 as seen from these figures. On the other hand, T∞ − TcW turns out to be much smaller than (T∞ − Tc1) particularly near the cold fluid exit and as ϕ decreases because Θuc > Θfc. But the pin conduction from hot fluid to the cold fluid increases as ϕ decreases near the cold/hot fluid exit/inlet as explained earlier. Accordingly, λc is maximized for specific ϕ distribution distinguished by having critical values of Lo/L, Lc/L and B as shown in Figures ahPeh, and acPec reduce Tmh, and Tmc, respectively. As a result, ThW − T∞, and T∞ − TcW increase causing {(λh)max, (λc)max} to increase as ahPeh, and acPec increase, respectively. These trends are seen clearly in Figures h*, Nuc*} approximations. Some critical parameters that produce the maximum quantities {(λh)max, (λc)max} are listed in λh)max, (λc)max} as seen from Figures Since The heat transfer rate from/to the hot/cold fluid of the present system can be higher than that for the conventional counterflow plate heat exchanger under the following conditions: (1) large hot/cold flow Reynolds number, (2) small cold/hot flow Reynolds number, and (3) large hot/cold fluid excess temperature.Increasing either the number pins or pins length may increase the hot/cold heat transfer rate above that for the conventional counterflow plate heat exchanger under the following conditions: (1) effective hot/cold fluid Biot number due to only pins conduction being larger than that due to only convection with the unfinned surface and (2) large hot/cold fluid excess temperature as when having large hot/cold flow Reynolds number.Uniform distribution of the pins leads to maximum heat transfer enhancement ratios when thermal entry regions are significant.Larger heat transfer enhancement ratios are obtained when both hot and cold fluid flows are fully developed.The maximum enhancement factors can be increased by above 8.0- and 5.0-fold for the step and exponential distributions of the pins, respectively.Heat transfer inside counter-flow plate heat exchanger subject to internal convections with an auxiliary fluid was investigated in this work. The auxiliary fluid passage is surrounded by the hot and cold fluid channels and it is supported by highly conductive pins connected to both channels. Good agreement was noticed between the numerical solution and an approximate analytical solution based on fully developed flow and very long pin conditions. The results of the current study can be summarized by the following concluding remarks.Finally, the counterflow plate heat exchanger with auxiliary intermediate channel can be recommended over the conventional one, if the auxiliary fluid flow is provided with negligible cost.


Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI. Myosin VI is an ATPase motor protein responsible for many cellular functions including endocytosis, protein secretion, and the maintenance of both the Golgi morphology and stereocilia In this study, to clarify how myosin VI generates its backward steps, we analyzed ATP-dependent stepping rate changes and step-type ratio changes using FIONA To clarify how the ATP hydrolysis cycle relates to myosin VI forward and backward steps, we analyzed stepping rates by single molecule imaging. Myosin VI labeled with Qdot585 had their processive movement visualized by total internal reflection fluorescence microscopy , and thefk+bk, where fk and bk describe the actual forward and backward stepping rates, respectively. The apparent ATP-dependent stepping rates of all three step types were measured at 10–500 µM ATP  L_distantr): small (s_distantr): backward (B_distantr)  =  (1−s)fk: fsk: bk.Because we have previously reported that the type of forward steps taken from the distant binding state is determined by the structural state of the leading head's lever arm (i.e. the pre-power or post-power stroke state) when the leading is bound and the trailing head is detached fsk+bk, was added to s_distantr to calculate the probability of the total number of small forward steps, resulting in sr = 2fsk+bk, where sr equals the number of total small forward steps. The probabilities of large steps (Lr) and backward steps (Br) equaled L_distantr and B_distantr, respectively, since these steps can occur only from the distant binding state. Therefore, Lr: sr: Br can be rewritten as (1−s)f:k (2fsk+bk): bk, which means s can be described as:From the adjacent binding state myosin VI will always take a small forward step s was found independent of ATP concentration and equaled 0.14±0.021 (14±0.021 .fsk+bk)/2, but at the same time equally overcount the number of steps following the adjacent binding state made by the other head, (fsk+bk)/2, meaning which head takes the ensuing step from the adjacent binding state is unlikely to compromise our results.When myosin VI is in the adjacent binding state, the terms ‘leading head’ and ‘trailing head’ no longer apply, as we have shown that either head has an equal probability of taking the next step Lr: Br  =  (1−s)fk: bk means fk: bk  =  Lr/(1−s): Br. Thus, the actual forward and backward stepping rates can be estimated as follows.That ATPk[ATP] and an ADP-release waiting time of 1/ADPkfk was well fitted to the function 1/fk  = 1/ATPk[ATP]+1/ADPk with a ATPk of 0.035 µM•s−1 and ADPk of 8 s−1 , where 0,ATPk = 0.05 µM•s−1 and δ = 4 nm ATPk = 0.02 µM•s−1, a value in complete disagreement with biochemical results, which suggests instead that ATP-binding does not couple with myosin-VI backward steps.Therefore, according to of 8 s−1 , values 0.62 s−1 . Should −1 has been reported previously −1 is consistent with myosin V, indicating that myosin VI too generates backward steps by spontaneous detachment of the leading head.A spontaneous detachment of the myosin V leading head at about 1 sHere we show that ATP-binding is coupled only to myosin VI forward steps. The two prevailing models for myosin V steps, however, assume that all steps are done in an ATP-dependent manner In the strain sensor model, intramolecular backward strain to the detached trailing head promotes phosphate release from the motor domain, resulting in preferentially binding to the forward binding site because of the transition from a weak to strong actomyosin binding state et al. proposed that ATP-binding coupled with trailing head detachment induces a conformational change in the lever arm from the post-power stroke to pre-power stroke state, causing preferential binding to the forward binding site because of the geometry between the actin filament and the myosin head In the case of the toe up-down model, Shiroguchi −1 spontaneous detachment rate of the leading head. Therefore, at no load, the low frequency of backward steps should ensure myosin VI functions as a transporter for endocytosis At cellular ATP concentrations  and no load, myosin VI performs unidirectional movement as a transporter and mainly takes forward steps. This is due to a higher ATP binding rate  to the tUnder high backward load, however, the forward stepping rate of the trailing head decreases At the same time, under backward load and in the adjacent binding state, the forward small stepping rate is sufficient for myosin VI to take the distant binding state. The result is a loop of small forward steps and backward steps, causing myosin VI to alternate between the adjacent binding and distant binding states. We recently reported that the tail domain of myosin VI, which is where myosin VI attaches to membrane structures via myosin VI binding proteins k. In the case the rate constant of an event does not change, the waiting time for the next event follows a single exponential decay function. Thus, the apparent distributions of the durations of competing reactions are described below with the rate constant i is described as ik, while the probability of that event is described as ik/kHere, the actual reaction rate of event To create dimeric myosin VI constructs, human myosin VI cDNA was truncated at Ala1021. This fragment included the motor domain, neck domain, and coiled-coil domain. To ensure myosin VI dimerization, the coiled-coil domain of the chicken gizzard smooth muscle myosin was appended at the Ala1021. For biotin labeling, a HaloTag (Promega) fragment was attached at the N- terminals. The sample contained His-tag at the C-terminus for protein purification. Protein purification was performed as previously described Qdot585 streptavidin conjugates and biotinylated myosin VI were incubated for single label imaging using FIONA 2 and 1 mM EGTA) was adsorbed onto the glass surface, followed by a 3 min incubation, a 20 µl AB wash, and finally an injection of 2 µg/ml non-fluorescent phalloidin labeled actin filament solution in AB into the chamber. After another 3 min incubation and 20 µl AB wash, 5 mg/ml α-casein in AB was injected into the chamber. After a third 3 min incubation and 20 µl AB wash, MB  mixed with Qdot labeled myosin VI was flowed into the chamber and the chamber was sealed with nail polish. Qdot conjugated myosin VI movement was imaged using total internal reflection fluorescence microscopy All experiments were performed according to previously reported methods Figure S1Myosin VI stepping traces at various ATP concentrations: 10 µM (A), 20 µM (B), 30 µM (C), 50 µM (D), 100 µM (E) and 500 µM (F).(TIF)Click here for additional data file.Figure S2Myosin VI dwell time distributions and step size distribution: (A) Histograms of dwell times just before forward large (60–150 nm) and (B) small (0–60 nm) steps. The histograms were best fit by a convolution of two exponentials (tk2 exp (-kt)). The value of k for large and small steps was 3.6 s−1 and 4.5 s−1 at 200 µM ATP, respectively. (C) Histogram of dwell times just before backward steps. The histogram was best fit by a single exponential function with a rate constant of 4.7 s−1 at 200 µM ATP. .(TIF)Click here for additional data file.Figure S3The binding state of myosin VI at the start and end of processive movement. At the start of processive movement (left), myosin VI takes the distant binding (61%) or adjacent binding states (39%) (n = 54). After myosin VI moves processively along an actin filament, it detaches to terminate processive moment (right). At this stage, the distant binding (61%) or adjacent binding states (39%) occur at the same frequency as the start of processive movement (n = 61). Here, we defined the distant binding state as when the inter-head distance is over 30 nm and the adjacent binding state as when it is under 15 nm. The inter-head distance was directly measured at the single molecule level using SHREC (TIF)Click here for additional data file.


Phaseolus vulgaris L.) productivity is severely affected by drought stress due to low technology cultivation systems. Our purpose was to identify differentially expressed genes in roots of a genotype tolerant to water deficit (BAT 477) when submitted to an interruption of irrigation during its development. A SSH library was constructed taking as “driver” the genotype Carioca 80SH (susceptible to drought). After clustering and data mining, 1572 valid reads were obtained, resulting in 1120 ESTs (expressed sequence tags). We found sequences for transcription factors, carbohydrates metabolism, proline-rich proteins, aquaporins, chaperones and ubiquitins, all of them organized according to their biological processes. Our suppressive subtractive hybridization (SSH) library was validated through RT-qPCR experiment by assessing the expression patterns of 10 selected genes in both genotypes under stressed and control conditions. Finally, the expression patterns of 31 ESTs, putatively related to drought responses, were analyzed in a time-course experiment. Our results confirmed that such genes are more expressed in the tolerant genotype during stress; however, they are not exclusive, since different levels of these transcripts were also detected in the susceptible genotype. In addition, we observed a fluctuation in gene regulation over time for both the genotypes, which seem to adopt and adapt different strategies in order to develop tolerance against this stress.In Brazil, common bean ( Plants are frequently exposed to stress-inducing environmental conditions, such as drought or floods, intense heat or cold, excessive soil salinity, insufficient availability of nutrients, changes in lightness, and the presence of heavy metals, that affect their growth, development and productivity . The tolP. vulgaris L.) is the second most important legume crop in the world , + in the cytoplasm that was identified is the kidnapping of this ion by the cell vacuole. In plants with heavy accumulation of Na+, Na+/H+ vacuolar transporters (antiport system) are activated by a differential on chemical potential through the tonoplast by protein pumps called V-H(+)-ATPases , and promote such capture of Na+ excess.Another system to prevent the accumulation of NaRicinus communis, other (GenBank ID: |75748631|) similar to PIP2 of G. max and the third (GenBank ID: |75749342|) was a putative aquaporin identified previously in P. vulgaris.When under water deficit, the osmotic balance of cells may also be regulated by the presence of transmembrane protein channels known as aquaporins that make both plasma and vacuolar membranes more permeable to water . The levet al. (2003)  × 100.A parallel study was also conducted with the same genotypes, Carioca 80SH and BAT477, with the goal of obtaining the RWC (%) of leaves induced through the application of a 10% PEG solution during 6 h. Leaves were rapidly weighed after harvesting in order to obtain the “fresh weight” (g), the supernatant was transferred to a new tube. The extract was centrifuged again for 2 min for additional leaf tissue removal and the supernatant was stored in ultrafreezer (−80 °C).Bulks composed of leaves from five plants collected from the experiment were used for total protein extraction. Extraction buffer  and 1% PVPP (polyvinylpolypirrolidone) were added to 50 mg of sample. After centrifugation  following manufacturer’s recommendations. The RNA samples were quantified by spectrophotometry at 260 nm and 280 nm wavelengths using NanoDropTM 2000c, and RNA quality was verified by electrophoresis on a 1.6% agarose gel. mRNA of each sample were purified using the Dynabeads® mRNA Purification Kit  according to the manufacturer’s instructions.Before RNA extraction, biological replicates for each treatment were ground in liquid Nitrogen and mixed together in order to obtain four bulks. Total RNA was extracted with Trizol . BAT 477 cDNA library was used as the “tester,” and Carioca 80SH was used as the “driver”.From the mRNA samples, we constructed two cDNA libraries, one for each genotype under water deficit, using the PCR cDNA Synthesis Kit SMARTer™ and Advantage®—T Easy Vector  and cloned in Escherichia coli (strain DH5α) for sequencing. A total of 1632 clones were sequenced at the Center for Molecular Biology and Genetic Engineering of Unicamp , using the BigDye Terminator v3.1 Kit  and an automated DNA capillary sequencer .PCR products obtained at the end of the SSH library construction were loaded in agarose gel Low-melting-point 1.6% in 1× TSB buffer, and the smear pattern observed was excised and purified using the GFX™ PCR DNA and Gel Band Purification Kit . The fragments obtained were bonded to the pGEMad hoc pattern-matching written in Pearl. For data interpretation, it was applied Parsers software built in the Pearl language [Phred tool  was usedlanguage .Resulting reads were grouped by phrap tool  with argE-value ≤ 1 × 10−5 were annotated; in addition, other parameters such as greater significant alignment (“best hit”) measured by the score and personal criteria based on related published data of genes described as involved on plant drought-stress response were also used.Functional annotation of the ESTs was performed based on similarity with common bean sequences and other related species, available in public databases , using tFor functional classification of the transcripts the Combined Scheme (CS) model developed by  was elecArabidopsis thaliana has one of the most comprehensively annotated genomes, and in many cases is supported by extensive experimental data, after annotation and functional classification, 31 individual bean ESTs obtained in our library were selected for their abundance, as well by classification in important metabolic pathways related to stress signaling and response, and then were aligned (tBlastX) with Arabidopsis transcript sequences using the TAIR10 database [E-value ≤ 1 × 10−5 were annotated.As database ; for thiArabidopsis gene orthologs (AGI numbers) obtained were used to identify probes designed for the ATH1: 22 k array Arabidopsis platform and then accessed under different stress conditions by using Genevestigator clustering analysis [The putative analysis .Total RNA extraction procedure was conducted in the aforementioned manner by using the root samples of BAT 477 and Carioca 80SH in both their drought-stressed and control conditions.Primers were designed using Primer3 program  and theiRT-qPCR was carried out in two steps. cDNA synthesis was obtained with Maxima™ First Strand cDNA Synthesis Kit  in the following reaction: 2 μL of 5× Reaction Mix, 1 μL of Max Enzyme; 1 μL of RNA (100 ng/μL) and 6 μL of DEPC-water, which was incubated at 25 °C for 10 min, 50 °C for 15 min and 85 °C for 5 min. Quantitative PCR reactions were carried out using the Maxima SYBR Green qPCR Master Mix Kit  in the StepOnePlus™ RealTime PCR System  equipment. The reactions contained 1 μL of cDNA, 5 μL of SYBR Green 2X, 1 μL of forward and reverse primers (2.5 pmol each) and 2 μL of DEPC-water. Amplification conditions were as follows: 10 min at 95 °C; 40 cycles of 15 s at 95 °C, 20 s at 59 °C and 20 s at 72 °C (with fluorescence data collection); and 95 °C for 15 s, 59 °C for 1 min and 95 °C for 15 s with data collection at each 0.7 °C increase in temperature to obtain melting curve data.E) and starting concentration (N0) values for each transcript. After normalization to the reference genes, relative expression ratios were calculated between contrasting samples.All samples were amplified in triplicate in order to quantify transcript levels of the target genes and two reference genes (act and skip2) . After qP. vulgaris, the AGI numbers (Table S2) that were obtained as described in the previous section were used. These were then used as “access codes” to map our ESTs to the identifiers in the Affimetrix Ath_AFFY_ATH1_TAIR9_Jan2010 platform according to their cellular functions and then to visualize expression data in PageMan tull (inside MapMan 3.5.1R2).From the Relative Expression data obtained for the time scale experiment, HeatMaps were constructed using the software MapMan 3.5.1R2 . As the A new set of ESTs from the root system of a drought-tolerant common bean (BAT 477) was obtained. Analyzed together, they revealed the potential metabolic pathways activated by these plants in order to acquire tolerance. Moreover, temporal gene expression analyses showed that, despite some transcripts having also been detected in the susceptible genotype (Carioca 80 SH), their regulation differs substantially between genotypes, thereby indicating molecular mechanisms as a target for breeding programs or for genetic engineering to create new cultivars with improved drought tolerance.


We also used Δδ15N (δ15N plant - δ15N soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances.Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the δ13C in hay and δ15N in both soil and hay between management types, but showed that δ13C abundances were significantly lower in soil of organic compared to conventional grasslands. Δδ15N values implied that management types did not substantially differ in nitrogen cycling. Only δ13C in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice.Isotopic analyses revealed no significant differences in δ However, in nature δ15N abundances in plants are affected by a multiplicity of factors such as type and degree of mycorrhization, the chemical type of N-compounds taken up or further soil characteristics, which can be barely separated from each other Distinguishing organic and conventional products is a major issue of food security and authenticity and much research on method development has been conducted to tackle this issue 13C in plant and soil are also of broad ecological interest 13C abundances in biomass are first of all affected by water availability and drought stress, but show also significant interactions with nutrient availability and fertilization 13C values are related to a different contribution of CO2 from soil respiration to plant photosynthesis y Similarly, δ15N values in soil and vegetation Although grasslands play a central role in the production of organic meat and dairy products 13C and δ15N of soil and hay (plant biomass). Furthermore, Δδ15N values (δ15N plant - δ15N soil) were calculated to estimate differences in nitrogen dynamics. We also assessed the time since organic certification to test for temporal trends. In detail, we analyzed whether (a) differences in isotopic abundances among organic and conventional grasslands exist and whether there are (b) significant trends in isotopic composition with time since conversion to organic management. Additionally (c), we used discriminant analysis to deduce the respective management type from the isotopic composition of hay and/or soil samples.Here, we studied 21 organic and 34 conventional grasslands in two German regions and analyzed δField work permits were given by the responsible state environmental offices of Baden-Württemberg, Thüringen, and Brandenburg (according to § 72 BbgNatSchG).Biodiversity Exploratories project Hainich-Dün in Thuringia in central Germany situated in and around the National Park Hainich and (II) the Biosphere Reserve Schwäbische Alb in Baden-Württemberg in south-west Germany. In grasslands of both regions Cambisols occur, while in the Schwäbsiche Alb Leptosols and in Hainich-Dün Stagnosols and Vertisols can also be found. Grassland types could be categorized as pastures, meadows and mown pastures −1) from 2006 to 2010 −1*a−1 . Accordingly, study plots can be distinguished in two sub-sets: organic plots which are managed according to an official organic farming certificate We studied agriculturally used permanent grasslands in two regions in Germany which belong to the 2 from mid-May to mid-June 2011 in both regions simultaneously. Temporary fences ensured that no mowing or grazing took place before yield was sampled. Plant material was dried for 48 h at 80°C and ground to fine powder for lab analyses. Both isotopic abundances of biomass samples and δ15N of soil samples were determined by mass spectrometry . Isotope ratios are given in per mille (‰), whereby δ13C is relative to the international reference standard v-PDB using NBS19 and δ15N relative to AIR-N2Soil sampling was conducted in early May 2011. On each plot mixed samples of 14 soil cores from 0 to 10 cm depth were collected using a split tube sampler with a diameter of 5 cm. Cores were taken along two 20 m transects at each plot. Roots were removed from the samples in the field and soil samples were air-dried, sieved to <2 mm and ground. For δ13C analyses, soil samples were weighted into tin capsules and treated with sulphurous acid inside the capsules to remove carbonates. Samples were dried again at 70°C before combustion in an oxygen stream using an elemental analyser . Evolved CO2 was analysed using an isotope ratio mass spectrometer . For biomass sampling, we harvested aboveground community biomass in four quadrates of 0.25 mHainich-Dün, located in Schönstedt (193 m A.S.L.), revealed that the precipitation prior to sampling (April and May 2011) was 70% lower than the mean of the same month from 2003–2010 (33.8 mm instead of 116.2 mm) Schwäbische Alb, the next climate station is situated in Münsingen-Rietheim (732 m A.S.L). Compared to the same month in the period 2005–2010, precipitation in April and May 2011 was 20% lower (153.8 mm instead of 191.5 mm) The nearby climate station in n = 7), soil type (n = 4), study region (n = 2) and grasslands type  including all two-way interactions with management type. Therefore, the lm and step functions in R for stepwise reduction of explanatory variables were used. Obtaining model results by using the anova output assured that the order of variables entering the model had no effect on significance gained for the respective variable, because all other variables were taken into account previously as co-variables. Model assumptions were checked using diagnostic plots function. Similarly, also applying the lm function analysis of co-variance (ANCOVA) models were calculated to relate the isotopic composition to the time since certification in all organic plots including farm, soil type and study region. Soil texture  was also incorporated in lm models but removed for final analysis as they did not add to explained variance. Additionally, we employed the qda function from the MASS package 15N and 13C isotopic values in soil and biomass samples. To ensure normal distribution of variables log10 transformation was applied prior to all analyses where necessary. All statistical tests were performed with R Multiple analysis of variance (ANOVA) models were used to examine differences among management types  while also accounting for farm or land owner  kg N a−1. However, both management systems show wide variation in fertilization intensity at the plot level. Organic plots received only organic fertilizers, as prescribed by organic management guidelines, whereas conventional plots received mostly mineral fertilizers .Farmer's questionnaires revealed that organic grasslands received average fertilizer applications of 21.6 (±37.0) kg N a13C and 15N isotopic abundances in soil and hay  15N isotopic composition of grassland soils and vegetation 15N vales in soil and vegetation probably due to increased microbial activity and subsequent losses of 15N depleted N from the system 15N in organic and conventional grasslands. This is supported by findings from Wrage et al. 15N in soil and vegetation due to spatially heterogeneous conditions and short-term changes in stocking densities in pastures. Moreover, as only a certain proportion of the N stored in biomass and soil originates from fertilization, biologically fixed N (e.g. through legumes) have additionally diluted isotopic signals of fertilizer applications Organic grasslands received only organic fertilizers and were characterized by significantly lower fertilization intensity, in line with Klaus et al. 15N between organic and conventional plots suggest that the studied grasslands do not substantially differ in nitrogen cycling 15N indicating that nitrogen cycling is strongly driven by individual decisions and practice at the farm level. Because plant species richness was shown decease Δδ15N in plant and soil assumedly via nitrogen partitioning 15N did not respond to organic grassland management. Contrary, in our study organic grasslands had higher mean plant species richness compared to conventional ones , apparently not leading to significant differences in Δδ15N.Missing differences in Δδ15N isotopic abundances revealed no significant indication for fewer losses of N from the studied organic grasslands.Haas et al. 13C abundances between organic and conventional soils and trends in decreasing δ13C abundances with time since certification in both organic soil and hay samples. Missing statistical significances of soil types in the analyses suggested that this is not only caused by differences in water availability among study plots. However, clear differences in δ13C in hay might only get apparent in dry years, because δ13C isotopic patterns can differ considerably among seasons 13C abundances in soils are probably a more reliable indicator for water supply for plants, because they integrate δ13C signals over a longer period of time, while δ13C in hay strongly depends on the photosynthetic activity and stomatal conductance influenced by current weather conditions 13C in hay can get more pronounced than in the studied year as prior to sampling the weather was particularly dry.We found significant differences in δ13C decrease with plant diversity in grassland experiments due to facilitation and/or complementarity among plant species 13C with time since certification cannot be traced back to changes in plant diversity, because diversity was stable over time (data not shown).It was shown that vegetation δ13C. They suggest higher soil respiration rates in organic soils due to higher microbial activity might cause a lowering of the δ13C value of the soil CO2 pool available to plants. As soil density fractions can have different 13C signals There are several other mechanisms, which could have also occurred, but cannot be easily separated from the whole complex of factors influencing isotopic abundances. Georgi et al. 13C values in organic grasslands could be direct or indirect effects of (former) fertilization. As organic farms use less often corn (a C4 plant) to feed their livestock, conventional slurry is thus enriched in 13C and may have led to higher δ13C values in soil organic matter in conventional grasslands 13C in soil with time since certification. Similarly, it was shown that high N availability can lead to higher δ13C in plant biomass mostly due to structural changes in plant tissue further tightening drought conditions 13C values of vegetables observed by Georgi et al. 13C abundances.Other reasons for lower δ15N abundances. These were significantly higher in organic fruits used in the study by Rapisarda et al. 15N and δ13C abundances. This is especially true, if we would also include uncertified but unfertilized grasslands, which occur frequently Although Rapisarda et al.


However, reliably predicting the spatial distribution of δDh and δ18Oh requires confirmation from animal tissues of known origin and a detailed understanding of the isotopic routing of dietary nutrients into felid hair.Several felids are endangered and threatened by the illegal wildlife trade. Establishing geographic origin of tissues of endangered species is thus crucial for wildlife crime investigations and effective conservation strategies. As shown in other species, stable isotope analysis of hydrogen and oxygen in hair (δDh and δ18Oh measurements from the North American bobcat (Lynx rufus) and puma (Puma concolor) with precipitation-based assignment isoscapes to test the feasibility of isotopic geo-location of felidae. Hairs of felid and rabbit museum specimens from 75 sites across the United States and Canada were analyzed. Bobcat and puma lacked a significant correlation between H/O isotopes in hair and local waters, and also exhibited an isotopic decoupling of δ18Oh and δDh. Conversely, strong δD and δ18O coupling was found for key prey, eastern cottontail rabbit  and white-tailed deer .We used coupled δD18O and δD isoscapes for use in forensic investigations. The effective application of isotopes to trace the provenance of feline carnivores is likely compromised by major controls of their diet, physiology and metabolism on hair δ18O and δD related to body water budgets. Controlled feeding experiments, combined with single amino acid isotope analysis of diets and hair, are needed to reveal mechanisms and physiological traits explaining why felid hair does not follow isotopic patterns demonstrated in many other taxa.Puma and bobcat hairs do not adhere to expected pattern of H and O isotopic variation predicted by precipitation isoscapes for North America. Thus, using bulk hair, felids cannot be placed on δ Many carnivore species are currently threatened and are the focus of intense conservation concern t) with the isotopic composition of the amount-weighted mean annual or mean-growing season precipitation (δDp). The latter correlates inversely with latitude and elevation across the continents, especially in North America 18O measurements of the organic or inorganic fractions of animal tissues despite the strong covariance between these isotopes in environmental waters  of animals include abiotic  and biotic (diet and physiology) factors Measurements of the stable isotopes of hydrogen (δD) and oxygen (δ18O) of animal keratinous tissues have been used to track the geographic origin and migratory patterns in a wide variety of animals e.g. . To dateNeotoma cinerea and Neotoma stephensi; Rattus norvegicus; Coturnix japonica; Passer domesticus; Homo sapiens; Sus scrofa domesticus; Artemia franciscana; Chironomus dilutus; 18O reflect both biological  and environmental signals  and puma (Puma concolor). Both species were ideally suited to test the strength of the isotope approach in assigning geographic origins of felidae. The availability of skins from museum collections, high-resolution precipitation δ18O and δD isoscapes for North America and ecological differences between these study animals  allowed us to assess the application and efficacy of H/O isotope fingerprinting for forensic spatial assignment in feline carnivores.Here, we provided the first large-scale δD and δ18Oh and δDh. The controlling factors and possibilities to quantify these will be discussed.Our study was designed to determine whether puma and bobcat hairs varied predictably in their isotopic composition among isotopically distinct geographic locations and reflected the spatial pattern of isotopic variation in precipitation. Furthermore, we examined if species- or sex-specific effects existed, and whether these could be explained by differences in diet, body size and foraging ecology. Our results demonstrated that the application of water isotopes for provenance determination of feline carnivores was compromised by major controls of their diet, physiology and metabolism on δAll CITES permits (MA 125284-0) for the export and use of museum materials from puma and bobcat were issued by the U.S. Fish and Wildlife Service.Lynx rufus, n = 45) and puma , as well as the eastern cottontail rabbit , the latter representing the preferred prey species of the bobcat, were obtained from the Smithsonian National Museum of Natural History in Washington D.C. and the Utah Museum of Natural History, Utah. Published isotope data of bone-phosphate (δ18Obp) and bone collagen (δ18Obc) from white tailed deer (Odocoileus virginianus), constituting the major prey of the puma, were included for comparative analysis 18Oriv  =  −17.5‰ to −0.1‰; δDriv  =  −132.7‰ to 0.6‰).Eighty-eight hair samples from two North American felid species bobcat  standard scale Sample preparation and H/O isotope analysis were conducted at Environment Canada. All keratin samples were physically cleaned of adhering debris and washed twice in a 2∶1 mixture of chloroform and methanol to remove lipids from the keratin surface. After cleaning, all samples were air-dried for 24 h. Hair samples were then cut into 0.5 cm increments  and weighed into pre-combusted silver foil capsules for H and O isotope ratio analysis. For δD, in order to account for exchangeable hydrogen in hair proteins, we used comparative equilibration with in-house keratin working standards, BWB (−108‰), CFS (−147.7‰), CHS (−187‰), for which the δD value of non-exchangeable H had been previously established 18O values for precipitation were determined using the Online Isotopes in Precipitation Calculator (OIPC) version 2.2 (http://www.waterisotopes.org). The OIPC provided a model estimation of long-term annually or monthly averaged precipitation isotope ratios at specified locations through spatial modelling of a large database of precipitation isotopic data covering the time period 1960–2004 18O data of the OIPC model were compared to those measured for local river waters riv and δ18Oriv and δDp and δ18Op for relatively small- to medium-sized drainage catchments  2, 2, 18O values of ingested prey-derived drinking water. Therefore we compared the hair δDh and δ18Oh data with the river water data.The H and O isotopic composition of water ingested by both felid species indirectly from their prey were inferred from modeled isoscape values p and δ18Op values, because the formation and isotopic incorporation of cat hair is limited to a rather short time period. For instance hair growth in domestic cats is not continuous h and δ18Oh not only to annual average δDp and δ18Op values but also to seasonal spring and summer precipitation to test if a better relation with water isotope values of the likely main hair growing season was obtained  and summer  precipitation δDobtained .18Oriv, δDriv and δ18Oh, δDh of puma, bobcat and rabbit hairs were investigated using linear regressions  or spring  OIPC modeled precipitation values did not significantly change the results . Relative humidity did show a significant modest effect on δ18Oh of bobcats  but no effect on δ18Oh of puma . Relative humidity, however, did not affect δDh of bobcats  and puma     but not in puma   and a moderate δ18Oh–δ18Oriv  positive relationship  (All hair δD results . The δ18 n = 30) . The iso in puma  and 3. N n = 30) . Likewis n = 30) . No effe n = 30) . There w n = 30) . Resultstionship  and 3. T n = 13) .18Oh and δDh. This finding contrasted strongly with results from numerous previously published studies on keratin tissues of omnivores and herbivores. Hence, tracing the provenance of feline carnivores such as puma and bobcat based on δ18Oh and δDh isoscapes does not appear to be possible, as individuals could not be reliably placed on δ18Op and δDp maps. Potential explanations for this lack of correlation between hair and ambient water isotope compositions are discussed below.Both puma and bobcat lacked the expected correlation between water isotopes in local water and hair, and also exhibited a complete decoupling between δ18Oh of bobcats  but not on puma  , most likely because they prey upon rabbits, whose δ18Obp compositions are humidity dependent , probably because they feed on white-tailed deer, whose δ18Obp is affected by both relative humidity and δ18Oph values of both feline carnivores were not influenced by relative humidity . Similar observations were made for δDbc (bone collagen) of white-tailed deer by Cormie et al. 18Ot of predators (e.g. bobcats) that feed on drought -tolerant herbivore species like rabbits. However, relative humidity did not explain the lack of a correlation between δDh-δ18Oh observed in both felids we studied.In our study, relative humidity showed a significant modest effect on δ= 0.818) . Previounts e.g. . For exah is not well correlated with δDp, if (i) ingested food or water sources  are not isotopically related to local meteoric water and/or (ii) migration between isotopically distinct habitats takes place. We tested whether the ingested food sources (i.e. key prey species) of bobcat and puma were in disequilibrium with δDp, and so caused the lack of a correlation between H/O isotopes in precipitation and those in felid hair. In North America, the preferred prey species of puma is the white-tailed deer (Odocoileus virginianus) 18O of bone phosphate (δ18Obp) bc) 18Op and δDp, respectively  water at the place of residence Falco amurensis; Accipiter cooperii; p was weaker Measurements of δD in feathers have been successfully applied in many bird species to estimate the origins of migrating and wintering individuals The natural diet of wild felids contains a high proportion of the energy as protein, a variable percentage as fat and a very low percentage as carbohydrate h. This may also affect the relative contributions of all sources to protein synthesis and hair formation. Recent findings from Pecquerie et al. Currently we lack a testable explanation for our observed and confounding isotopic patterns, but considering the unique felid physiology, we hypothesized that the food metabolism of strict carnivores may exert a vital effect particularly on δD18Obp with δ18Op18Obw and δ18Oh values than animals that sweat because water vapour lost in panting is more depleted in 18O bw and δDh.The water metabolism in feline carnivores also differs from herbivores and omnivores. Cats drink to a limited extent h and δ18Oh and meteoric water δ18Op and δDp (c and δDp was observed in a recently published study of migrating pumas in the USA c and δDwater (R2 = 0.6) compared to hair (R2 = 0.9) from the same individuals In contrast to the weak correlation between feline carnivore hairs δD and δDp  and 3, aThe isotopic values of keratins are generally defined by the isotopic composition of their constituent amino acids h and δ18Oh) correlation between hair and meteoric waters  data from bobcat and puma hairs from a range of locations across North America revealed that feline carnivores cannot be placed on δpes e.g. . With thTable S1Sample list.(XLS)Click here for additional data file.Table S2Statistical analysis.(DOC)Click here for additional data file.


Most individuals  were classified as residents (i.e. fledged within our study area) and estimated minimum dispersal distances of immigrants were between 40 and 240 km. Even when considering maximum dispersal distances, the likely origin of most first-year breeders was<200 km from our study area. Our method identified recruitment into our population from large geographic areas with relatively few samples whereas previous mark-recapture based methods have required orders of magnitude more individuals to describe dispersal at such geographic scales. Natal dispersal movements revealed here suggest the spatial scale over which many population processes are taking place and we suggest that conservation plans aiming to maintain populations of Ovenbirds and ecologically-similar species should consider management units within 100 or at most 200 km of target breeding populations.Adult birds tend to show high fidelity to their breeding territory or disperse over relatively short distances. Gene flow among avian populations is thus expected to occur primarily through natal dispersal. Although natal dispersal is a critical demographic process reflecting the area over which population dynamics take place, low recapture rates of birds breeding for the first time have limited our ability to reliably estimate dispersal rates and distances. Stable isotope approaches can elucidate origins of unmarked birds and so we generated year- and age-specific δ Seiurus aurocapilla) and American Redstart (Setophaga ruticilla) in western Canada based on the number of first-year birds that were “isotopic outliers” relative to expected baseline isotopic values for six study sites. Studds et al. Catharus bicknelli) and assignment tests to determine the area of likely origin of first-year breeders . That study provided minimum natal dispersal distances and evidence for spatio-temporal discrepancy in demographic connectivity. Although single isotope approaches provide important insights into natal dispersal, it is generally recognized that multiple isotopes or combinations of techniques improves the spatial resolution for assigning individuals to their natal area (e.g. 34S) may be particularly useful in distinguishing individuals growing feathers in coastal regions vs. inland since marine-derived sulfates are generally more enriched in 34S than terrestrial sources and volatilized sulfates from ocean spray can be deposited inland over considerable distances directly or through precipitation Using δrea e.g. . Stable 2Hf and δ34Sf to obtain a finer-scale assignment than using δ2Hf alone. We anticipated significant longitudinal structure in δ34Sf based on distance from the coast and prevailing weather. We first created species-, age-, and year-specific feather isoscapes a priori expectations regarding the spatial extent of natal dispersal movements by Ovenbirds as they have never been quantified. Most information available on distance of natal dispersal has been provided by mark-recapture studies. While mark-recapture studies are biased against detection of longer-distance dispersers, the isotope approach is biased against short-distance dispersers We investigated the use of two stable isotope tracers to estimate natal dispersal distances in Ovenbird. Given that our study area was located within 160 km of the Gulf of St Lawrence, we jointly used δ2 managed forest district owned by J.D. Irving Ltd. The landscape is a mosaic of spruce plantations and deciduous, mixedwood, and coniferous stands at the interface of the northern hardwoods and Acadian forest regions Since 2006, an individually-marked subpopulation of male Ovenbird has been monitored in northwestern New Brunswick, Canada  individuals following the criteria of Donovan and Stanley 2  and returning ASY males from Black Brook (2010–2012), and for all ASY males from the 22 additional sampling locations (2010–2012). Values of δ34Sf were also measured for all SY males (2010) from Black Brook, but only for returning ASY males from 2010 and 2012, owing to logistical constraints. For the same reason, only three individuals per additional sampling location were analyzed for δ34Sf (n = 66).In 2010, most territorial males in the 250-ha area monitored  captured in 2010 (feathers grown in 2009) as the benchmark values for the year-specific δ2Hf isoscape. We could not visit our entire study region in a single year to create a year-isoscape and so applied a correction factor for δ2Hf values from samples collected at locations surrounding Black Brook in 2011  and 2012  based on the yearly variation we saw in ASY males at Black Brook. Specifically, we compared mean δ2Hf values from returning ASY males (Black Brook) captured in 2010 vs. 2011 and 2010 vs. 2012. We then used these differences to adjust δ2Hf from ASY males captured at sampling locations outside Black Brook in 2011 and 2012. Only δ2Hf values from six sampling locations had to be calibrated as the ASY males from the remaining 16 locations were all captured in 2010. This approach assumes that year-to-year variation in δ2H from growing-season precipitation is similar across the total sampling area. Values of δ34Sf were not calibrated to account for annual variation as there was no a priori reason to expect annual variation in this isotope. However, we still tested for a year effect using a t-test.Stable-hydrogen isotope ratios from feathers collected in the same breeding area can vary among years 34Sf and δ2Hf from individuals captured at the 12 study sites in Black Brook were pooled in 4 locations (3 sites per location) covering the extent of the study area , Canadian Wildlife Service (Permit Numbers: SC2710 and SC2751), and U.S. Fish and Wildlife Service (Permit Number: MB11009A-0).34Sf in 2010–2012 and δ2Hf in 2010 and 2012) and the Colorado Plateau Stable Isotope Laboratory (δ2Hf in 2011). High-temperature (1350°C) flash pyrolysis generating an H2 pulse for each sample (350±20 μg) was used to obtain δ2Hf measurements by continuous-flow isotope-ratio mass spectrometry (CF-IRMS). To account for exchangeable hydrogen in keratins, comparative equilibration was done using in-house keratin working standards . All results are expressed as nonexchangeable deuterium (δ2Hf) and sulphur (δ34Sf) isotopic ratios in units of per mil (‰) and normalized to Vienna Standard Mean Ocean Water – Standard Light Antarctic Precipitation (VSMOW-SLAP) and Canyon Diablo Triolite, respectively. Based on within-run analyses of keratin standards, we assumed measurement error to be ca. ±2‰ for both δ2Hf and δ34Sf.Surface oils were removed from feathers using a 2∶1 chloroform:methanol solution. Samples were prepared according to 2Hf between juvenile  and ASY individuals growing feathers at the same site ranging between 6–32.6‰ 2Hf was on average ∼6‰ lower in first-year breeders/fledglings than in adult Bicknell’s Thrushes at the northern edge of their study area. This portion of the Bicknell’s Thrush range  corresponded to the extent of our isoscapes and this comparison based on values from first-year breeders/fledglings is more appropriate than values from nestling Ovenbirds obtained by Haché et al. 2Hf from growing (nestlings) versus inert (first-year breeders/fledglings) tissue provide appropriate age correction factors. Also, the Bicknell’s thrush is expected to be of the same guild as the Ovenbird and hence should make a reasonable surrogate species. Hobson et al. 2Hf. Although we believe that the most parsimonious approach was to use a range of age correction factors from no correction to adding 6‰ to all SY males 2Hf of each SY male prior to assigning their origin to the ASY feather isoscapes.Previous studies have reported differences in δj; 2 km×2 km cell size) within the feather isoscapes was a potential origin was estimated based on the following equation:iyf(xi|jμ) is the likelihood that an individual (i) with given δ2Hf (ix) and δ34Sf (iy) values originated from a raster cell j, while μ is the mean, σ is the standard deviation, and ω is the correlation between δ2Hf (x) and δ34Sf (y) predicted for each raster cell (j) within the feather isoscapes. Predicted mean isotopic composition for each raster cell (j) was generated from the calibrated isoscapes (δ2Hf and δ34Sf). The parameter μj is therefore a vector of means for each raster cell (j) within the feather isoscapes presented as:We generated spatially-explicit assignment tests using bivariate normal probability density functions BNPDF;  to deter2Hf and δ34Sf isoscapes and the following variance-covariance matrix:2δ2Hf and σ2δ34Sf are the expected variance for each isotope ratio and σδ2Hf, σδ34Sf is the covariance between the pair of isotope ratios. The variance-covariance matrix was validated to define the likelihood a cell within the extent of our isoscape corresponded to a potential origin. This was achieved by using a subset of 66 samples from ASY males . We assumed a spatial stationarity of variance-covariance across our study area 2Hf and δ34Sf, respectively, and the correlation coefficient from the covariance was 0.29. For all SY males (2010), we generated a binary raster layer to classify each cell as a likely (1) or unlikely (0) origin based on a 2∶1 odds ratio . Odds ratios between 2∶1 and 4∶1 provided the best depiction of the geographic distribution of assigned versus true origins for samples from multiple individuals  outside Black Brook in 2011 and 11.9‰ to those of 25 ASY males captured at the three other sampling locations in 2012. Mean δ34Sf values differed significantly between years , but this difference was negligible when considering the 2‰ measurement error. In 2010, we captured 35 SY males breeding in Black Brook  at each of the 22 sampling locations outside Black Brook, 6.5 were ASY . When added to the 23 ASY males captured in Black Brook in 2009 and subsequently recaptured in 2010 (5.8±2.2 ASY per sampling location), samples from a total of 165 and 89 ASY males were used to produce the δectively –2. In Bl2Hf isoscape was generated using ordinary kriging based upon a Gaussian semivariogram model (root-square-mean error = 4.59). The model-estimated parameters were a major range of 120.7 km, nugget of 0.81, and sill of 31.8. Data for δ34Sf were kriged to create an isoscape based upon a spherical semivariogram model (root-square-mean error = 1.03). The semivariogram model parameters for the δ34Sf isoscape included a major range of 120.7 km, nugget of 0, and sill of 11.8. The δ2Hf isoscape showed generally more enriched values in southeastern portion of the isoscape (∼50‰) with gradual depletion along a southeastern-northwestern gradient, whereas the δ34Sf isoscape corresponded to the anticipated coastal effect with more enriched values mostly along the coast (10–20‰) creating an east-west gradient  of SY males could not be estimated because in all cases, it reached the edge of the isoscape, suggesting that they could have originated from an area beyond our defined feather isoscape. However, the population-level assigned origin  indicated that likely origins of SY males only reached edges of the isoscapes in small areas in the southeast and northeast, suggesting that the majority of SY males tended to have a maximum natal dispersal distance<200 km from Black Brook . This ef34Sf and δ2Hf Ovenbird isoscapes and bivariate assignment tests along with the most parsimonious age correction factors , we showed that most (94%) SY males recruited in our coastal-breeding population likely originated locally . Given that stable isotope analyses have been shown to be better-suited to identify long-distance dispersal than mark-recapture studies, this finding was unexpected based on the low natal philopatry generally reported in songbirds By combining age- and year-specific δ2 (ca. 80 km× 25 km), suggesting that a high proportion of males recruited into the district’s Ovenbird population likely hatched “locally”. Based on δ2Hf outliers, Hobson et al. 2) and spatial scale must be considered when making such comparisons. Nonetheless, Paradis et al. Protonotaria citrea) and Tree Swallow (Tachycineta bicolor) also have different population sizes and breeding ranges Estimates of minimum natal dispersal distances suggest that most SY males can be considered as residents and, in the rare instances where individuals were classified as immigrants (n = 2 based on the most parsimonious age correction factors), immigrants would have originated within 240 km of our study area. The Black Brook district covers 2000 km34Sf and δ2Hf, respectively. Similar coastal conditions at the same latitude also do not occur elsewhere in the Ovenbird’s breeding range The assigned origin of all SY males reached the edge of our isoscape, limiting our ability to generate accurate estimates of maximum natal dispersal distances. This was especially the case when using a −6‰ age correction factor and assignment tests based on a 4∶1 odds ratio. However, when using the most parsimonious age correction factors, the likely origin only reached smaller areas at the edge of the isoscape  previously observed 34Sf across our isoscape as a function of distance to the ocean seems to have been altered by local processes that we do not fully understand  and −3‰ (B) δ2Hf age correction factors (2∶1 odds ratio). Maps represent the sum of all binary raster layers of each individual assignment. Blue points are the 26 sampling locations and in black is the Black Brook district.(TIF)Click here for additional data file.Figure S2Geographic distribution of the assigned origin of 35 SY male Ovenbirds known to have bred in the Black Brook district, New Brunswick. Assignments of SY males to the δ2Hf and δ34S isoscapes were based on using bivariate normal probability density functions and +3‰ (A) and +6‰ (B) δ2Hf age correction factors (2∶1 odds ratio). Maps represent the sum of all binary raster layers of each individual assignment. Blue points are the 26 sampling locations and in black is the Black Brook district.(TIF)Click here for additional data file.Figure S3Minimum detectable dispersal distance (in grey) for individuals captured at one of the four sampling locations (in blue) within the Black Brook district (in black). Predicted δ2Hf and δ34Sf from our isoscapes were used for the four sampling locations within the Black Brook district to generate fictional individuals for which their origin was considered “unknown”. The likely area of origin was assigned to the bivariate isoscape and we calculated the minimum detectable dispersal distance as the perpendicular distance from a sampling location to the closest area assigned as unlikely origin (0).(TIF)Click here for additional data file.Figure S4Geographic distribution of the assigned origin of 35 SY male Ovenbirds known to have bred in the Black Brook district, New Brunswick. Assignments of SY males to the δ2Hf and δ34S isoscapes were based on using bivariate normal probability density functions and –6‰ (A) and +6‰ (B) δ2Hf correction factors (4∶1 odds ratio). Maps represent the sum of all binary raster layers of each individual assignment. Blue points are the 26 sampling locations and in black is the Black Brook district.(TIF)Click here for additional data file.Table S1SY male Ovenbirds (n = 35) classified as resident (1) or immigrant (0) depending on whether or not the assigned area of origin overlapped the study area (Black Brook). Assignment tests were based on 5 different age correction factors. In parentheses is the minimum distance of dispersal for each immigrant. Unless indicated otherwise (4∶1), assignment tests were based on a 2∶1 odds ratio. ID represents individual birds.(DOCX)Click here for additional data file.


Evolutionary theory posits that resource availability and parental investment ability could signal offspring sex selection, in order to maximize reproductive returns. Non-human studies have provided evidence for this phenomenon, and maternal condition around the time of conception has been identified as most important factor that influence offspring sex selection. However, studies on humans have reported inconsistent results, mostly due to use of disparate measures as indicators of maternal condition. In the present study, the cross-cultural differences in human natal sex ratio were analyzed with respect to indirect measures of condition namely, life expectancy and mortality rate. Multiple regression modeling suggested that mortality rates have distinct predictive power independent of cross-cultural differences in fertility, wealth and latitude that were earlier shown to predict sex ratio at birth. These findings suggest that sex ratio variation in humans may relate to differences in parental and environmental conditions. This adjustment was strongly correlated with maternal condition around conception, such that mothers in good condition during this period produced more sons Average national sex ratio at birth (SRB) in humans is slightly male biased , with remarkable deviation for some countries Although, well corroborated by non-human studies, TWH still lacks support for human populations, even after identification of staggering number of factors. This is due to studies reporting conflicting results Physical health and environmental conditions are directly reflected in life expectancy, the duration an individual presumes they have left to live, with those living under economic constraints, diseased state and unstable environments expecting to die sooner Counts of the sex ratio at birth for the year 2009 were taken from the Central Intelligence Agency, World Factbook As a measure of mortality level for each nation, life expectancy at birth  and healthy life expectancy  were used Fertility and sex ratio at birth are negatively correlated in human populations The relationship between life expectancy and sex ratio at birth was studied using regression model. Sex ratio at birth was entered as dependent variable and life expectancy was entered as independent variable. Each regression model included latitude, fertility rate and wealth as control variables. For variables showing high collinearity (square root of variance inflation factor greater than 2.0), ridge regression model was employed r = 0.68 and 0.67, both p<0.001, 2 = 0.46, β = 0.69, p<0.001, HALE: adjusted R2 = 0.45, β = 0.67, p<0.001). This effect was linear: addition of polynomial function of these variables i.e. LE2 or HALE2  to the regression model did not change the R2 value.Globally, natal sex ratios were positively correlated with life expectancy and healthy life expectancy  or averaged mortality indices (life expectancy and HALE). The zero-order correlation between life expectancy and sex ratio was higher than that of any other variable for which there is a previously proposed correlation .r = 0.80, p<0.001), total fertility is reduced  and more sons are produced   . AlthougLife expectancy and mortality rates impose substantial influence on reproductive Table S1Descriptive statistics for countries included in this study.(DOCX)Click here for additional data file.Table S2Multiple regression analysis predicting sex ratio at birth by life expectancy at birth (1), healthy life expectancy (2), adult mortality rate (3), infant mortality rate (4), under 5 mortality rate (5) and maternal mortality ratio (6), after controlling for fertility, wealth and latitude (n = 167). Except (3), all are ridge regression models at λ = 0.1, see (DOCX)Click here for additional data file.


Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. The hematopoietic system can become dysfunctional via various distinct molecular pathologies. For example, the protein tyrosine kinase oncoproteins (PTKs) associated with a number of different leukaemias and preleukaemias have been identified as critical in the molecular pathology of these diseases. They can result from point mutations, reciprocal chromosomal translocations and internal tandem duplication; thus in Anaplastic Large Cell Lymphoma (ALCL) the t(2∶5) translocation leads to the formation of the NPM/ALK gene which encodes a lymphoma specific NPM/ALK protein with intrinsic and disregulated PTK activity A key feature of the leukaemias and lymphomas is their progressive nature. The potential reasons for this are numerous. Expression of oncogenic PTKs such as BCR/ABL have been associated with increased genomic instability Whilst targeted therapies such as the paradigmatic use of imatinib in treatment of CML has altered the landscape in oncology research, the treatment does not eradicate the disease, resistance can develop and the CML stem cell persists Methods of finding such aberrations using high throughput, objective screens are now available. In an attempt to identify common downstream targets we have previously described a systematic analysis of a range of leukaemogenic kinases on the whole cell proteome of Ba/F3 cells. Ba/F3 cells were chosen because of their widespread usage in studies on leukemogenesis. Leukaemia generally progresses as cells acquire further mutations within the nucleus due to aberrant signalling cascades. We have shown primitive hematopoietic and embryonic cells alter their proteome with no concomitant change in mRNA We have previously described the generation and characterisation of Ba/F3 cells expressing six distinct leukaemia/lymphoma related oncogenic protein tyrosine kinases namely BCR/ABL, TEL/PDGFRβ, Fip1L/PDGFRα, KIT D816V, NPM/ALK or FLT3ITD The quality of the quantitative data required for such a study was ensured by running the experiment outlined in Ten million cells yielded 620 µg +/−24 µg of protein (mean+/−sem) with an average nuclear to cytoplasmic ratio of 1∶4 and no significant difference in ratio or protein yield between any of the oncogene-transfected or control cells. In addition to protein yields, lamin and tubulin expression/distribution were used as both loading controls and as a measure of quality of the fractionation .The number of proteins detected and relatively quantified with high confidence in the 8 channels was 3619 with a false discovery rate (FDR) of 4.6%. All the data was normalised such that the median was 1.0 and the ratios checked to ensure they had approximately a normal distribution . The totWe next defined what constituted a change in protein expression. To be called as changing a protein must have a ratio outside the range in which 95% of protein ratios for the internal replicate are found and a p-value of 0.05 or less in the majority of experiments that a ratio is recorded and not changing in any internal replicate. This “significance interval” was determined for each experimental run and attempts to account for the technical and biological variation seen in each run see . ProteinThe lack of overall harmonisation in protein changes emanating from the 6 PTKs led us to look at potential differential layers of control in these systems. To determine whether transcription or post-translational mechanisms were responsible for these changes we compared the data on major protein changes we observed against mRNA expression data we have previously published Given these data we determined if the involvement of any specific transcription factor binding site on genes whose protein levels change gave a strong correlation between protein changes and mRNA changes. In other words do some transcription factors drive an mRNA-mediated change in proteome more readily than others? We analysed the transcription factor binding sites and the most common included NFκB, Fos, Jun, PPAR-γ and Glucocorticoid receptor (GR). One outstanding observation from the consideration of upstream transcription factor binding sites from major changing proteins was the data seen with proteins changing that have upstream glucocorticoid receptors (GRs) binding sites. In no instance did increased protein expression with these gene products correlate with increased mRNA expression . Where dNFκB1/2 binding sites were the most common transcription factor binding site found on major changing proteins. This is of interest because NFκB can modulate the GR response and vice versa. IκB levels are a key determinant of NFκB complex activation in cells and all the PTKs with the exception of BCR/ABL decreased IκB see . But a cApproximately 25% of the proteins identified as changing in response to five or six of the PTKs are linked to DNA repair mechanisms e.g. Mum1, Xpc and PMS2. Genomic stability plays a major role in the development and progression of leukaemia We identified a total of 1889 unique phosphoentities, giving approximately 1558 distinct sites of phosphorylation on 705 different proteins (FDR of 0.32%). The amalgamated list is shown in Phosphopeptides whose expression was altered as a consequence of oncogene expression are shown in . As with the nuclear proteome analysis there was very little similarity in PTK-induced changes. Two receptor tyrosine kinases (TEL/PDGFRβ and Kit D816V) did group together though the effect was tenuous. There was no significant pattern to the phosphorylated effectors downstream of the 6 PTKs. This diversity in PTK-induced changes is also illustrated in the Venn diagram shown in Phosphopeptide changes due to the PTKs were used to classify PTK effects using hierarchical clustering . As withProteins previously associated with the development of leukaemias, such as hnRNP1 Similarities in changes in the protein level are shown in NF-IL3 (E4bp4) is up-regulated by the action of 4 out of 6 PTKs  a targetAnother protein fundamentally linked to DNA repair and markedly decreased  was PMS2. We validated this observation by western blot analysis . This deDNA mismatch repair plays an important role in maintaining genomic stability. Given our proteomic findings that pathways that potentiate genomic stability are common targets mediated by 6 oncogenic PTKs we investigated whether such pathways were perturbed in the oncogene expressing cells. Cells were treated with the DNA mismatch repair inducing drug 6-thioguanine and viability assessed 24 hours later. As is clearly illustrated in The data on major protein changes and their interactions is shown in Our substantial analysis of the effects of 6 leukemogenic PTKs has revealed a great deal of disparity in their effects in the nucleus, a novel and important observation in itself. We then went onto show that modulation of mRNA levels and nuclear/cytosolic shuttling were not responsible for the major changes seen in the nuclear proteome instigated by the leukaemogenic PTKs. The very fact that translocation and mRNA modulation do not account for many of the changes observed underlines the need for organellar proteomic analyses to identify the targets downstream of oncogenic PTKs and also the need for further research on mRNA processing and translation regulation plus protein hydrolysis mediated by leukaemogenic PTKs.A key example of a changing protein is HMGA1. This protein has been shown to be relevant in the leukaemias showing increased expression in a major proportion of Acute Myeloid Leukaemia cases Other interactors of HMGA1may therefore also be important. HMGA1 can modulate the response to insulin and to interact with the key hematopoietic regulator C/EBPβ As described above, GR gene targets are potentiated by the 6 PTKs and the levels of this key receptor are decreased markedly by 6 out of 6 of the leukaemogenic PTKs. Phosphorylation on a GR site associated with activation A study on the occurrence of mutations in two proteins involved in DNA mismatch repair, MSH2 and MLH1, has previously implicated alterations in this pathway in AML pathogenesis Protein phosphorylation effectors of the oncogenic PTKs is likely in part explained by their differential subcellular localization as well as pathways activated in the cell. Choudhary et al http://www.scalpl.org/scope3/. Further validation studies will identify potential targets for therapy in PTK-mediated malignant disease.Our results are summarised in Ba/F3 cells 7 cells were washed in ice cold Hanks buffer with protein phosphatase inhibitors and incubated in hypotonic buffer (750 µl) as per manufacturer’s instructions. The preparation was then incubated on ice for 20 minutes in 50 µl TEAB (0.5 M), 0.05% (w/v) SDS and 0.5 µl of protease inhibitor cocktail (Active Motif) and 2 µl/ml Benzonase . The preparations were mixed, centrifuged for 10minutes at 14,000 g and the supernatant (nuclear fraction) recovered. The quality of the preparations was confirmed using western blot analysis for nuclear (lamin A/C) and cytosolic (α tubulin) standard marker proteins.Nuclear proteins were enriched using a kit from Active Motif  with some modifications. Briefly, 1×10Whole cell lysates were prepared by lysing cells in RIPA buffer . Briefly cells were washed three times with Phosphate buffered saline before lysing in RIPA buffer with the following additions; Protease Inhibitor Cocktail , Phosphatase Inhibitor Cocktail 2 and 3  and sodium orthovanadate (1 mM). Western blot analysis was carried out by standard protocols. Antibodies used were actin (A5060), HMGA1 (SAB2501305) from Sigma ; α tubulin (sc-5286), NFκB (sc-372), IκB (sc-371) and Glucocorticoid receptor (sc-1004) from Santa Cruz ; Lamin A/C (# 2032) Phospho Ser211 Glucocorticoid Receptor (#4161) from Cell Signalling Technology ; HMGb1 (ab18256), HMGb2 (ab11973), HMG14 (ab5212) from Abcam ; GSK3 phospho Y216 (612312), PCNA (610664) and PMS2 (556415) from Becton Dickenson ; HSP90b (RB-118-PO) from Thermo Scientific ; GSKα/β (KAM-SToo2) from Enzo Life Sciences (Exeter UK).2 columns. The ensuing nanoflow liquid chromatography plus tandem mass spectrometry were performed as described previously The work flow is outlined in 2 columns  were equilibrated with 150 µl of lactate loading buffer then the samples loaded. The tip was washed twice with 60 µl of lactate loading buffer and four times with 60 µl of wash buffer (80% (v/v) acetonitrile, 5% (v/v) TFA). Bound peptides were then recovered by elution in 60 µl of elution buffer . Samples were then concentrated to a few µl in a SpeedVac prior to RP-LC-MS/MS as above.100 µg of nuclear iTRAQ labelled peptide mixtures were re-suspended in 150 µl of lactate loading buffer (240 mg/mL lactate in 80% (v/v) acetonitrile, 1% (v/v) Trifluoro-acetic acid (TFA). TiOPrior to reversed phase LC-MS/MS peptides were fractionated off line using a reversed phase chromatography column  at a high pH using an LC Packings Ultimate LC system. The gradient was run at 700 µl/min using initially 99.5% high pH buffer A  0.5% high pH buffer B . Over a 30 minute time period high pH buffer B was increased to 50%, followed by a 4 minute time period to increase high pH buffer B to 75%. The concentration of high pH buffer B (75%) was held for 4 minutes then reduced back down to 0.5%. Fifteen second fractions were collected for the duration of the gradient, the volume of each fraction was then decreased under vacuum.18 PepMap100  using an LC Packings  UltiMate™ pump and separated as described previously . Briefly, peptides were separated over a 120 min solvent gradient from 5.9% (v/v) acetonitrile/0.1% (v/v) formic acid to 41% (v/v) acetonitrile/0.1% (v/v) formic acid on-line to a QStar® XL mass spectrometer . Data was acquired using an information dependant acquisition (IDA) protocol where, for each cycle, the two most abundant multiply charged peptides (2+ to 4+) above a 20 count threshold in the MS scan with m/z between 400 and 2000 were selected for MS/MS. Each peptide was dynamically excluded (±50 mmu) for 1 minute.Peptides were identified by RP-LC-MS/MS on either a QStar® XL, QStar® Elite or Tof/Tof 5800 mass spectrometer . For the QStar® XL dried peptide fractions were re-suspended in 180 µl of 2% (v/v) acetonitrile/0.1% (v/v) formic acid. For each analysis one third of the peptide sample was loaded onto a 15 cm reversed phase C18 column (75 µm inner diameter) packed with C+ to 4+) above a 10 count threshold in the MS scan with m/z between 400 and 2000. Each peptide was dynamically excluded (±50 mmu) for 90 seconds.For the QStar® Elite dried peptide fractions were re-suspended in 15 µl of 3% (v/v) acetonitrile, 0.1% (v/v) formic acid and 20 mM citric acid. For each analysis, 5 µl of the peptide sample was loaded onto a nanoACQUITY UPLC Symmetry C18 Trap, 5 µm, 180 µm×20 mm and flow was set to 15 µl/min of 3% (v/v) acetonitrile, 0.1% (v/v) formic acid and 20 mM citric acid for 5 min. Analytical separation of the peptides was performed using nanoACQUITY UPLC BEH C18 Column, 1.7 µm, 75 µm×250 mm. Briefly, peptides were separated over a 91 min solvent gradient from 3% (v/v) acetonitrile,0.1% (v/v) formic acid 20 mM citric acid to 40% (v/v) acetonitrile,0.1% (v/v) formic acid, 20 mM citric acid on-line to a QStar® Elite mass spectrometer . Data was acquired using an information dependant acquisition (IDA) protocol where, for each cycle, the four most abundant multiply charged peptides (2For the Tof/Tof 5800 system dried peptide fractions were re-suspended in 180 µl of 2% (v/v) acetonitrile/0.1% (v/v) formic acid and a third of the sample loaded onto a trap column using a U3000 liquid chromatography system  and the peptides fractionated by a capillary RP C18 HPLC column  at a flow rate of 0.8 µl/min with a gradient of between 2–40% (v/v) acetonitrile, 0.1% (v/v) TFA over 80 minutes. The flow through was spotted onto a MALDI plate  in 15 second fractions using an online Probot  with α-cyano-4-hydroxycinnamic acid mixing with the eluant to a final concentration of 1.25 mg/ml. Mass spectrometry was carried out on an AB SCIEX TOF/TOF 5800  using 1000 shots for MS. MS/MS was carried out on the top 27 precursors with a S/N of higher then 8 using 4000 laser shots, a 2 Kv acceleration voltage and air as the collision gas.www.ensembl.org) mouse database (release 58) containing 115,658 protein entries using ProteinPilot 3 software  with default settings including the allowance of one missed or nonspecific cleavage , MMTS and 8 plex iTRAQ fixed modifications. For a list of all variable modifications see ProteinPilot 3.Data was processed by a ‘Thorough’ search against the ensembl . As the number of identifications for an entity is usually very low the p-value was calculated where possible but not used to determine a change.Phosphopeptides identified with a confidence of http://bioinformatics.picr.man.ac.uk/vice/Protocols.vice). All data were analysed as previously reported RNA was prepared using TRIzol (Invitrogen), DNase treated then cleaned using MinElute RNeasy Clean up Kit (Qiagen) per manufacturers instructions. Transcriptome analysis was undertaken using murine GeneChip® Mouse Exon 1.0 ST Arrays by the CR-UK Affymetrix microarray facility . Cell viability was assessed at 24, 48 and 72 hours by trypan blue exclusion.(PDF)Click here for additional data file.Table S1All proteins identified. Table showing all 3763 nuclear proteins identified across the three replicate experiments of Ba/F3 cells transfected with 6 oncogenic PTKs. The first four columns display protein and gene accessions, gene symbol and name. The next nine columns give information from the search results for the identification of the protein in each experiment (blanks if not identified). “Coverage” indicates what percentage of the protein sequence was identified. “Unique peptides” indicates the number of unique peptides discovered whilst “Peptides used” indicates the number of unique peptides used to derive the quantification. The remaining columns give, for each oncogene in turn, quantification ratios (against MSCV control) and associated p-values and error factors for each replicate.(XLSX)Click here for additional data file.Table S2Change significance interval. Table showing the ratios employed to define a change. These are the values that 95% of ratios for the internal replicate lie between. This “significance interval” is individually determined for each experimental run and attempts to account for the technical and biological variation seen in each run.(DOCX)Click here for additional data file.Table S3Proteins changing as a result of expression of each oncogene. Proteins defined as changing are shown for each oncogene separately. To be called as changing a protein must have a ratio outside the range in which 95% of protein ratios for the internal replicate are found and a p-value of 0.05 or less in the majority of experiments that a ratio is recorded and not changing in any internal replicate. The first four columns display protein and gene accessions, gene symbol and name. The “change” column gives a significance for the likelihood of all the ratios representing a change and whether this is an increase or decrease in protein expression. The next three columns indicate in how many replicates the observation was made and whether it was an increase or decrease. “Coverage” indicates the percentage of the protein sequence identified. “Unique peptides” indicates the number of unique peptides discovered whilst “Peptides used” indicates the number of unique peptides used to derive the quantification. The remaining columns give, for each oncogene in turn, quantification ratios (against MSCV control) and associated p-values and error factors for each replicate.(XLSX)Click here for additional data file.Table S4All phosphopeptides identified. Table showing all phospho peptides identified across the three replicate experiments. The first four columns give protein and gene accessions, gene symbol and name from which each peptide originates. The next two columns list the modification assigned to each peptide and the peptide sequence. The next six columns display a value for the confidence with which the peptide was matched (Confidence) and the number of matches used for the quantification (Observations Used) for each of the three replicate runs. The remaining columns give, for each oncogene in turn, quantification ratios (against MSCV control) and associated p-values and error factors for each replicate.(XLSX)Click here for additional data file.Table S5Phosphopeptide spectra. Table showing all phospho peptides identified across the three replicate experiments and their observed mass and charge. The first column shows which experiment the peptide was seen. The next two columns display a unique number identifying the spectrum and the peptide match to the spectrum. The next three provide the accession number, peptide sequence and cleavage information. The next columns display a value for the confidence with which the peptide was matched (Confidence). The next four columns provide information on the charge, precursor mass and whether the peptide was used in the quantification calculation and an explanation as to why it was excluded if not used in the quantification.(XLSX)Click here for additional data file.Table S6Phosphopeptides changing as a result of expression of each oncogene. Phosphopeptides defined as changing are shown for each oncogene. To be called as changing a peptide must have a ratio outside the range in which 95% of peptide ratios for the internal replicate are found in at least one experiments and not changing in any internal replicate. The first four columns give phosphopeptide sequence, protein accession, gene symbol and name from which each peptide originates. The “change” column gives a significance for the likelihood of all the ratios representing a change and whether this is an increase or decrease in peptide expression. The next three columns indicate in how many replicates the observation was made and whether it was an increase or decrease. The remaining columns then display for each replicate a value for the confidence with which the peptide was matched (Confidence), the number of matches used for the quantification (Spectra Used), a value for the confidence in the likelihood of all the ratios representing a change (Change) and quantification ratios (against MSCV control) and associated p-values and error factors.(XLSX)Click here for additional data file.