SSA directly parallels independent subspace analysis (ISA) with the only difference that SSA maximizes slowness instead of sparsity. We find a large discrepancy between the filter shapes learned with SSA and ISA. We argue that SSA can be understood as a generalization of the Fourier transform where the power spectrum corresponds to the maximally slow subspace energies in SSA. Finally, we investigate the trade-off between slowness and sparseness when combined in one objective function.Following earlier studies which showed that a sparse coding principle may explain the receptive field properties of complex cells in primary visual cortex, it has been concluded that the same properties may be equally derived from a slowness principle. In contrast to this claim, we here show that slowness and sparsity drive the representations towards substantially different receptive field properties. To do so, we present complete sets of basis functions learned with  A key question in visual neuroscience is how neural representations achieve invariance against appearance changes of objects. In particular, the invariance of complex cell responses in primary visual cortex against small translations is commonly interpreted as a signature of an invariant coding strategy possibly originating from an unsupervised learning principle. Various models have been proposed to explain the response properties of complex cells using a sparsity or a slowness criterion and it has been concluded that physiologically plausible receptive field properties can be derived from either criterion. Here, we show that the effect of the two objectives on the resulting receptive field properties is in fact very different. We conclude that slowness alone cannot explain the filter shapes of complex cells and discuss what kind of experimental measurements could help us to better asses the role of slowness and sparsity for complex cell representations. The appearance of objects in an image can change dramatically depending on their pose, distance, and illumination. Learning representations that are invariant against such appearance changes can be viewed as an important preprocessing step which removes distracting variance from a data set in order to improve performance of downstream classifiers or regression estimators Complex cells in primary visual cortex are commonly seen as building blocks for such invariant image representations e.g. . While csparseness and slowness. While in the context of unsupervised representation learning the two objectives have been proposed to similarly explain the receptive field properties of complex cells, there are important differences between them that may help to identify the algorithms used in biological vision. Intuitively, the slowness objective can be seen as a measure of approximate invariance or “tolerance”, whereas sparseness is better interpreted as a measure of selectivity. Tolerance and selectivity—or slowness and sparseness, respectively—can be understood as complementary goals which both play an important role for solving the task of object recognition The two main objectives used for the optimization of models of neural representations are Thus, based on the strong association between complex cells and invariance, one would expect that slowness rather than sparseness should play a critical role for complex cell representations. In this study, we investigate the differences between slowness and sparseness for shaping the receptive field properties of complex cells.complete sets of filters learned with slow subspace analysis (SSA) independent subspace analysis (ISA) While for natural signals it may be impossible to find perfectly invariant representations, slowness seeks to find features that at least change as slowly as possible under the appearance transformations exhibited in the data For sparseness it is common to show complete sets of filters, but this is not so in case of slowness. Based on the analysis of a small subset of filters, it has been argued that SSA may generally yield similar results to ISA Complete representations optimizing slowness have previously been studied only for mixed objective functions that combined slowness with sparseness The central result of this paper is the observation that the effect of the slowness objective on complex cell learning is substantially different from that of sparseness. Most likely this has gone unnoticed to date because previous work either did not derive complete representations from slowness or combined the slowness objective with a sparsity constraint which masked the genuine effect of slowness. Therefore, we here put a large effort into characterizing the effect of slow subspace learning on the complete set of filter shapes under various conditions. We first study a number of analytically defined transformations such as translations, rotations, and scalings before we turn to natural movies and the comparison between slowness and sparseness.The general design common to SSA and ISA is illustrated in In the first part of our study, the input sequence consisted of translations. As time-varying process for the translations, we implemented a two-dimensional random walk of an smaller the area under the curve the better the average slowness.In fact, one can think of SSA as learning a generalized power spectrum based on a slowness criterion. While the power spectrum is known to be invariant against translations with periodic boundary conditions, perfect invariance—or infinite slowness—is not achieved for the translations with open boundary conditions studied here see . The sloCompared to random subspaces, the decrease in In Given that the SSA representation learned for translations is very similar to the Fourier basis and since the Fourier basis achieves perfect invariance for cyclic shifts we proceeded to investigate whether the Fourier basis is optimal even for non-cyclic translations as well. We created three different data sets, with random translations as in the first study, but the maximal shift amplitude of the 2D random walk was 1, 2, and 3 pixels, respectively. As initial condition, we used the Fourier basis , ‘’ instDuring optimization, the basis slightly departs from the initial condition but remains very localized in the Fourier domain , ‘’. The set cf. . The sloEven though we expect changes in natural movies to be dominated by local translations, it is instructive to study other global affine transforms as well. Therefore, we applied SSA to 3 additional data sets: The first data set contains After characterizing the result of slow subspace learning for analytically defined transformations we now turn to natural movies and the comparison between slowness and sparseness. Specifically, we compare slow subspace analysis (SSA) to independent subspace analysis (ISA) in order to show how the slowness and the sparsity objective have different effects on the receptive field shapes learned. To this end, we combine the two objectives to obtain a weighted mixture of them for which we can gradually tune the trade-off between the slowness and the sparseness objective. In this way, we obtain a 1-parametric family of objective functionsindependent slow subspace analysis (ISSA). If a representation is optimized for For tion see ,11. RemaThe trade-off in performance with respect to slowness and sparsity for natural movies, translation, rotation, and scaling is summarized in Unsupervised learning algorithms are a widespread approach to study candidate computational principles that may underly the formation of neural representations in sensory systems. Slowness and sparsity both have been suggested as objectives driving the formation of complex cell representations. More specifically, it has been claimed that the filter properties obtained from slow subspace analysis would resemble those obtained with independent subspace analysis underestimation of the fraction of small bandwidth filters. Principal component analysis will always select for low spatial frequency components and thus reduce the fraction of small bandwidth filters because it is the high spatial frequency components which have the smallest bandwidth.The different implications of slowness and sparseness are most notable in filters containing high spatial frequencies. For low spatial frequency filters the number of cycles is small simply because it is constrained to be smaller than the product of spatial frequency and simulation window size. Since previous studies have inspected only low spatial frequency filters the different effect of sparseness and slowness has gone unnoticed or at least not been sufficiently appreciated While it is difficult to make rigorous statements that are model-independent, there are general arguments why the lack of localization is likely a generic consequence of slowness rather than a spurious property that was specific to SSA only: By definition a neuron cannot be driven by stimuli outside of its receptive field (RF). Therefore, whenever a stimulus is presented that drives the neuron inside its RF, the neuron must stop firing when the stimulus is shifted outside the RF. This suggests very generally, that in the presence of motion the objective of slowness or invariance necessarily requires large RFs. Sparsity, in contrast, encourages neurons to respond as selectively as possible. One obvious way to achieve this is to become selective for location which directly translates into small RF sizes.In addition, analytical considerations suggest that slowness is likely to generate global filters with small bandwidth. For small image patches it is reasonable to assume that the spatio-temporal statistics are dominated by translational motion. Thus, it is not surprising that the filter properties of SSA found for natural movies resemble those for translations. In computer vision, there is a large number of studies which derive features that are invariant under specific types of transformations such as translations, scalings and rotations. An analytical approach to invariance is provided by steerable filter theory The receptive fields of complex cells determined from physiological experiments rarely exhibit multiple cycles as predicted by SSA. This indicates that complex cells in the brain are not fully optimized for slowness. It may still be possible though that slowness plays some role in the formation of complex cells. The trade-off analysis with the mixed objective has shown that giving up some sparsity allows one to achieve both relatively large sparsity and slowness at the same time with localized receptive fields.decreases throughout development, we are not aware of the equivalent evaluation of any change in neuronal slowness.Having established how exactly sparseness and slowness differ in their implied receptive fields also helps to address the roles of sparseness and slowness experimentally. Li & DiCarlo Independent of what happens during development, the comparison of slowness and sparseness raises questions about how we should view the role of complex cells with respect to the tolerance-selectivity trade-off. Given that large receptive fields are advantageous for invariance or slowness, the small receptive field size of complex cells suggests that complex cells do not aim at achieving maximal tolerance but rather lean towards preserving a high degree of selectivity. For both ISA and SSA some degree of invariance is already built into the architecture which resembles the energy model of complex cells and will always find two-dimensional invariant subspaces. Instead of prescribing the invariant subspace dimensionality we wanted to know what happens if the subspace dimensionality is learned as well. This can be done by learning complex cells with SFA on the full quadratic feature space and then investigating the spectrum of the resulting quadratic forms. Comparing the number of subspaces employed by SFA to maximize slowness to empirical measurements in V1 The deeper principle underlying both sparsity and slowness is the idea of generative modeling The algorithm of slow subspace analysis (SSA) has previously been described by Kayser et al et al.The objective function of SSA has been called “temporal smoothness” objective by Kayser In order to compute the gradient of the objective function we have to compute the temporal derivative of the output signal http://bethgelab.org/software/natter/.The optimization is initialized with a random orthonormal matrix http://research.ics.aalto.fi/ica/imageica/.Independent subspace analysis (ISA) has originally been proposed by Hyvärinen and Hoyer The time-varying input signal The extracted Figure S1Model complex cells derived with SFA fail to reproduce the small numbers of significant eigenvalues found empirically with STC analysis. We computed SFA filters on the quadratic feature space of the 100 lowest Fourier components of (PDF)Click here for additional data file.


At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca2+]i in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca2+ peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca2+]i. Furthermore, ATP-induced Ca2+-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca2+]i similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca2+]i response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca2+ responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca2+ appeared to contribute to the generation of ATP-induced Ca2+ response in ASDN cells. Importantly, flow- and hypotonic-induced Ca2+ elevations are markedly blunted in P2Y2 −/− mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca2+]i elevation during purinergic stimulation. Consistent with this, ATP-induced Ca2+ plateau are dramatically attenuated in TRV4 −/− mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca2+ plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.Mechanical forces are known to induce increases of [Ca Abnormal regulation and/or dysfunction of the transporting systems in the ASDN are linked to a number of pathological states associated with changes in the circulating plasma volume and electrolyte imbalance It is recognized that the distal part of renal nephron , which includes the connecting tubule (CNT) and the cortical collecting duct (CCD), is responsible for the final regulation of electrolyte  Dynamic changes in renal tubular flow and fluid composition can be sensed by the cells of the ASDN 2+ entry from the extracellular medium, thus, possibly contributing to the elevation of [Ca2+]i in response to mechanical stimuli +-secretion in the CCD has been recently proposed Transient receptor potential (TRP) channels are known to participate in cellular adaptations to a variety of environmental stimuli, including thermo-sensation, chemo-sensation, mechanical forces etc (reviewed in + reabsorption + pressure-natriuresis ATP is constitutively released from the ASDN cells q-coupled P2Y receptors to regulate electrolyte and water transport in the ASDN (reviewed in 2+]i via PLC-IP3-mediated Ca2+ release from the endoplasmic reticulum (ER) 2+-permeable TRPC3 channel to drive Ca2+ flux from tubular lumen in IMCD-3 cell line ATP release in the ASDN is markedly augmented by changes in tubular flow rate as well as cell volume 2+]i and to test if purinergic signaling is involved in mechano-sensitivity of mammalian distal nephron. We found that ATP uniformly increases [Ca2+]i in a PLC-sensitive manner in ASDN cells. Both extracellular and intracellular Ca2+ sources contribute to these Ca2+ elevations. Disruption of purinergic signaling in P2Y2 −/− mice markedly reduces cellular responses to mechanical stimulations. Importantly, ATP activates Ca2+-permeable TRPV4 and TRPC channels to elicit a sustained raise of [Ca2+]i with TRPV4 having a major role. We conclude that activation of purinergic signaling by mechanical stimuli reciprocally contributes to the mechano-sensitivity by activating Ca2+-permeable TRPV4 channel.In this study, we used freshly-isolated split-opened ASDNs to define the molecular mechanism of ATP-induced elevation of i) were selected and the baseline values were subtracted for generation of average time course for multiple cells. Typically, 3–5 individual ASDNs from an average of 3 mice were used for each experimental set.Experimental traces from individual cells were inspected visually prior to acceptance. For analysis, traces with initial F2 camera (Photometrics) interfaced to a PC running NIS elements software.Split-opened tubules were fixed with 4% paraformaldehyde in PBS (pH = 7.4) for 20 min at RT. After fixation the samples were permeabilized by addition of 0.1% Triton in PBS for 5 min and washed in PBS 3 times for 5 min. Nonspecific staining was blocked with 10% normal goat serum  in PBS for 30 min at RT. After washing with PBS (3 times for 5 min) the samples were incubated for 1.5 hr at RT in dark with anti-aquaporin 2 labeled with ATTO-550  in 1% serum+0.1% Triton in PBS. After washing with PBS (3 times for 5 min) the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI)  to visualize nuclei. Subsequently the samples were dehydrated, and mounted with permanent mounting media . Labeled tubules were examined with an inverted Nikon Eclipse Ti fluorescent microscope using a 40× Plan-Fluor oil-immersion (1.3 NA) objective. Samples were excited with 405 and 561 nm laser diodes and emission captured with a 16-bit Cool SNAP HQ2, 2 MgCl2, 5 glucose and 10 HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca2+]i, the rate of perfusion was instantly increased from 1.5 ml/min (∼15 mm H2O) to 15 ml/min (∼80 mmH2O). The linear velocity of flow increased from ∼0.13 cm/sec to 1.3 cm/sec under these conditions. For experiments testing the effect of hypotonic cell swelling, the isotonic solution contained (in mM) 100 NaCl, 5.4 KCl, 0.5 MgCl2, 0.4 MgSO4, 3.3 NaHCO3, 1.0 CaCl2, 10 HEPES, 5.5 glucose, 90 mannitol, pH 7.4, with osmolarity of 305 mOsm/L. The hypotonic solution contained (in mM): 100 NaCl, 5.4 KCl, 0.5 MgCl2, 0.4 MgSO4, 3.3 NaHCO3, 1.0 CaCl2, 10 HEPES, 5.5 glucose, pH 7.4, with osmolarity of 220 mOsm/L.Typical bath solution was (in mM): 150 NaCl, 5 mM KCl, 1 CaClt-test. Data from different experiments were compared with a Student's (two-tailed) t-test or an One-Way ANOVA as appropriate. P≤0.05 was considered significant.All summarized data are reported as mean ± SEM. Data from before and after treatment within the same experiment were compared using the paired 2+]i in perfused CCD of rabbits and mice 2+-imaging in freshly isolated split-opened murine ASNDs with multiple regions of interest (ROI) representing fluorescent signals from individual cells within a split-opened area. Previous studies suggested that luminal application of ATP caused a prominent elevation of [Ca2+-responses in ASDN cells. 2+]i in individual cells in response to 10 min application of 10 µM ATP. Purinergic stimulation caused a rapid (<20 sec rise time) Ca2+-spike followed by a sustained plateau with no obvious decline after 10 min of ATP application. [Ca2+]i returned to basal levels upon ATP removal. Quantitatively, ATP increases [Ca2+]i to 197±4 nM in the peak followed by 165±4 nM during the sustained plateau from the baseline of 123±3 nM (n = 37). We next tested if repetitive 2 min ATP stimulations elicit reproducible rises of [Ca2+]i in ASDN cells. As is clear from the average time course of changes in F340/F380 ratio in 2+ response were not attenuated during the second application.We first aimed to quantify the ATP-induced Ca2+ responses. We first generated a histogram of the peak magnitude  of ATP-induced [Ca2+]i elevations in ASDN cells  and intercalated cells (IC). Several studies also point out that cells of CCD and CNT might posses different expression/activity of electrolyte transporting systems DN cells . We expe2+ responses in CNT and CCD cells. For analysis, we reasonably assumed that cells located upstream to the tubule bifurcation belong to the connecting tubule and cells located downstream to the merging point belong to the cortical collecting duct (see 2+]i in response to ATP for CCD (n = 135) and CNT (n = 66) cells. As can be seen, ATP-induced Ca2+-responses are not different between CNT and CCD cells.We further address this by testing if ATP elicits similar Caduct see . Figure 2+]i from individual cells and then stained ASDNs with the specific marker of principal cells, AQP-2, for discrimination between cell types. As demonstrated by a representative micrograph (2+ peak (202±6 nM and 203±9 nM) and the plateau (170±4 nM and 173±4 nM) for identified PC (n = 34) and IC (n = 12), respectively.We next directly probed a role of purinergic signaling in principal and intercalated cells. For this, we quantified ATP-induced elevations of i in response to increases in the linear velocity of flow from 0.13 cm/sec to 1.3 cm/sec in wild type and P2Y2 −/− mice (2+]i and genetic deletion of P2Y2 receptors significantly blunts this response. The mean ΔF340/F380 was 0.038±0.003 (n = 17) and 0.019±0.002 (n = 27) for wild type and P2Y2 −/− animals, respectively.We next quantified the relative elevations in i. Therefore, we next aimed to determine the molecular determinants responsible for ATP-induced elevations of [Ca2+]i.Of note, flow-induced Ca2+-response in ASDN cells. Stimulation of P2Y2 receptors typically leads to PLC activation and elevation of [Ca2+]i via IP3-mediated mechanism. Thus, we next tested a role of PLC in generation of ATP-induced Ca2+-response. 2+]i during ATP application alone and in the presence of a PLC inhibitor, U73122 (10 µM). As summarized in 2+ transients . Moreover, treatment with U73122 decreases basal Ca2+ levels  signaling in innermedullary collecting duct cells 2+-response in the ASDN (2+ responses: ΔF340/F380 = 0.045±0.002 in the control and ΔF340/F380 = 0.035±0.002 after AACOCF3 pre-treatment (n = 52). Therefore, the results in 2+]i in a PLC-dependent manner and ATP-induced stimulation of PLA2 contributes to this response.Elevations of [Cathe ASDN . Inhibit2+ release from the ER. 2+]i in response to ATP in the control and after 10 min pre-treatment with 2 µM Thapsigargin (TG) to inhibit the Ca2+-pump SERCA of the ER. As summarized in 2+ responses were markedly attenuated after TG treatment. The amplitude of relative Ca2+ elevations was ΔF340/F380 = 0.035±0.002 in the control and ΔF340/F380 = 0.010±0.003 after TG (n = 21).Stimulation of PLC in response to purinergic activation can lead to the Ca2+ response after depletion of intracellular Ca2+ stores. We hypothesized that activation of Ca2+-permeable ion channels on the plasma membrane during purinergic stimulus may contribute to the elevated [Ca2+]i. Therefore, we next quantified ATP-induced Ca2+-responses in Ca2+-free extracellular media (buffered with 5 mM EGTA). Ca2+ removal markedly decreased basal [Ca2+]i which was returned to a control value after perfusing with standard solution (2+ spike: ΔF340/F380 = 0.044±0.004 and ΔF340/F380 = 0.028±0.03 (n = 52) in the control and in Ca2+-free media, respectively  in response to ATP in the control, after application of BTP2, RuR, and both inhibitors together (BTP2+RuR). As can be seen, inhibition of both TRPC and TRPV channels attenuated the sustained elevation of Ca2+ in response to ATP application with RuR having the greater effect.Several Ca2+-elevations suggests a major role of TRPV4 channels in generation of the plateau of ATP-induced Ca2+ responses. To further test this, we next assessed the actions of ATP on [Ca2+]i in mice lacking TRPV4 channels (TRPV4 −/− mice). 2+ responses for wild type and TRPV4 −/− mice. As is clear, genetic deletion of TRPV4 dramatically decreases the amplitude of the plateau. Importantly, the magnitude of this inhibition closely resembles the effect of RuR on ATP-induced [Ca2+]i (2+ response (340/F380 ratio during the second ATP response (after application of a respective inhibitor or during genetic deletion of TRPV4) in The stronger RuR inhibition of ATP-induced Ca [Ca2+]i . For betresponse . For thi2+ spike and the sustained plateau (data not shown). This may suggest that amiloride, in addition to inhibiting TRPP2 channels, possesses non-specific inhibitory actions on P2Y receptors. This remains to be tested further in future studies.Interestingly, we have also attempted to investigate a role for TRPP2 channel which is known to utilize TRPV4 to form a heteromeric mechano-sensitive molecular sensor in the cilium 2+]i signalization in response to mechanical stress. Mechanical forces lead to activation of a mechano-sensitive TRPV4 channel. At the same time, ATP is released from the cells where Cx30 hemi-channels most likely mediate this process. ATP targets P2Y2 receptors and stimulates PLC in the same (autocrine action) or neighboring cells (paracrine action). Activation of PLC triggers Ca2+ release from the ER and also stimulates Ca2+-permeable mechano-sensitive TRPV4 and, to a lesser extent, TRPCs channels to further potentiate mechano-sensitive elevations in [Ca2+]i in ASDN cells.Overall, 2+ permeable channels, such as TRPV4, which, in turn, augments cellular responses to mechanical stimuli. ATP failed to evoke a sustained raise of [Ca2+]i in mice lacking the TRPV4 channel  from whole perfused murine CCDs was also demonstrated In this study we performed careful characterization of a role for purinergic signaling in regulation of [Ca plateau  and thattization . In cont2+ responses in CNT and CCD cells . A recent study suggested that luminal ATP can stimulate TRPC3 in cultured IMCD-3 cells and that this channel is critical for sustained elevation of [Ca2+]i in response to ATP 2+ response in native ASDN , while participates in this process, is mainly involved in the development of PKD.An important aspect of mechano-sensitivity of the ASDN is how cells sense the mechanical forces. Strong experimental evidence argues for a critical role of central cilium as a reporter of velocity of tubular fluid flow. For instance, cellular responses to flow are blunted in 2+-permeable TRP channels, such as TRPV4, which underlie the mechano-sensitive properties of the ASDN. Disruption of purinergic signaling in P2Y2 −/− mice does not lead to PKD development but does impair mechano-sensitivity in the distal nephron. Interestingly, growing experimental evidence suggests that cysts formation also leads to a dysfunction of purinergic signaling In summary, we defined a role of purinergic signaling in regulation of mechano-sensitivity in the distal nephron. In contrast to the TAL where purinergic signaling accounts for the mechano-sensitive response


The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations. DSB are a major threat for genomic integrity. However, some pathways for DSB repair can mis-rejoin DNA ends from different break sites, thus creating chromosome aberrations. The influence of initial distance between DSB sites on the probability of mis-rejoining is under debate, as well as the possibility of mechanisms bringing DSB sites into closer vicinity during damage processing232(Δt)>MSD(Δt) = <r reflect movement of the nucleus rather than that of the foci within the nucleus.Experimental data on the mobility of break sites in mammalian chromosomes are inconclusive. Mostly, they rely on labelling the chromatin region containing the DSB by visualizing specific chromatin modifications known to surround the breaks site, such as γ-H2AX, or proteins known to bind chromatin in the vicinity of break sites, such as MDC1. The labelled chromatin regions microscopically appear as ionizing radiation-induced foci (IRIF). Analyzing fixed cells, inferences on mobility are made by investigating the development of foci number and size over time, or their position with regard to the sites of damage induction6789101113D can be determined15αD being a generalized diffusion coefficient and α being the anomality parameter quantifying the deviation from normal diffusion. In the case of subdiffusion, α < 1, while in normal diffusion, α = 1. Γ represents the Gamma function and d is the dimension of the analyzed space.A plot of MSD versus observation time allows determining if the particle observed moves via normal diffusion, in which case the MSD is linearly related to time and a diffusion coefficient 2(Δl)σ. While measuring distance changes has been introduced by others182(Δl)σ is even largely invariant in the case of additional changes of nucleus size.To characterize the mobility of damaged chromatin regions, we chose to investigate distance changes between two IRIF over time rather than the MSD, using the standard deviation of the distance changes 12C) ions per point. The distances il of adjacent MDC1 foci pairs numbered by i were monitored for several hours post-irradiation (i(Δt)Δl during different time intervals Δt (i.e. i(Δt)Δl = i(tj + Δt) − li(tj)l for all time points jt of the time series) were determined.U2OS cells expressing GFP-tagged repair protein MDC1 were irradiated in a matrix pattern with single carbon (adiation . The dis2(Δl(Δt))σ of the changes in foci distances i(Δt)Δl between neighbouring MDC1 foci as a measure for the underlying random walk process (see SI). Then the time-ensemble average of the “neighbouring” 2(Δt)σ from all analyzed carbon irradiated cells was calculated for each Δt and plotted versus Δt in a double-logarithmic plot (2σ) with log(Δt) is evident, which demonstrates a power-law dependence of 2σ on Δt (cf. α = 0.49 ± 0.05 (SEM) and D0.49 = (3.7 ± 0.5) × 10−4 μm2/s0.49 for the next neighbour foci (l ≈ 5 μm) of the carbon irradiated cells . The value obtained for α significantly differs from α = 1 demonstrating that the mobility of the IRIFs follows a subdiffusion process4 s) observed. In addition, the best least-square fits of constrained diffusion2 = 0.91, reduced χ2 = 5.0) or a combination of constrained diffusion and normal diffusion of the confinement region  did not fit the experimental data . As shown in 2σ analyzed from both data sets are very similar for Δt < 180 s, but increase faster in the case of second next neighbours at Δt > 180 s, hinting at non-corrected additional relative foci movements which we attribute to gross morphological deformations of cell nuclei. We determined α = 0.58 ± 0.05 (SEM) and D0.58 = (3.1 ± 0.5) × 10−4 μm2/s0.58.To examine the influence of the initial distances on the determined dynamics, the cells were additionally analyzed using the distances between second next foci neighbours (Comparison between time-series recorded early (3–10 min) and late (80–100 min) after irradiation shows that the subdiffusion of the MDC1 foci is time-invariant, justifying the used method of ensemble- and time-averaging as it can be done for any ergodic process .iΔl for a single time interval Δt obtained for the carbon-irradiated samples  of the i(Δt)Δl values for initial distances much larger than the diffusional lengthα = 0.5 (see Δt = 60 s) and applying a two dimensional random walk situation for distance analysis and thus substituting x → Δl and αK → α2dD: The anomality of the diffusion of MDC1 foci can also be recognized in the histogram of distance changes  samples . Normal Δx = Δy = 6 μm with 32 protons  per point. The number of protons was chosen to create on average 3 DSB per matrix point so that within the depth of view each detected IRIF contained on average only one DSB, while after carbon irradiation several DSB are expected to occur in each IRIF. As in the case of carbon-irradiated cells, a linear increase of log(2σ) with log(Δt) is evident  and D0.50 = (8.1 ± 1.8) × 10−4 μm2/s0.50 for the proton-irradiated cells, showing that the time dependence of foci after carbon and proton irradiation is very similar. As the anomality parameter α for proton and carbon irradiation  is the same within the errors, the number of induced double-strand breaks within one focus does not seem to influence the degree of anomality of the foci diffusion. The absolute 2σ values, however, differ by a factor 2.2. This difference may be attributed to the fact that the dynamics observed in proton irradiated cells is mainly that of an isolated, single DSB (number of DSB per IRIF pN ~ 1), whereas due to clustered DSB induction and poor axial microscopic resolution a number of CN > 1 foci contribute to each detectable IRIF after carbon irradiation. Thus, the diffusional behaviour observed is that of the centre of mass of all DSB in this IRIF, where contributions of individual DSB can neutralize each other. In a pure statistical approach we expect 2p/σ2C = NC/Npσ    and the  evident  that theΔt ≤ 104 s, with α ≈ 0.5. Subdiffusive behaviour of chromatin regions has been described in yeastE. coli chromosomes2325In the present work, we demonstrate that radiation-induced MDC1 foci, which are assumed to reflect the chromatin region surrounding one DSB or several clustered DSB, exhibit a subdiffusive mobility in the time range 5 s ≤ E. coli is a consequence of both viscoelasticity of the nucleoplasm and the restrictions on monomer mobility caused by neighbouring monomers in a polymer29The underlying mechanisms leading to subdiffusion have mainly been investigated in the context of anomalous mobility of macromolecules such as proteins and microinjected beads in nucleoplasm and cytoplasm e.g.28 where 15appD from a variety of data on mobility measurements of chromosomal loci available in the literature (app with observation time were performed with single particle tracking (Δt). In contrast, distance tracking as used in our work, intrinsically corrects for translational and rotational movements and, when using the standard deviation of distance changes as it is used here, even largely corrects for a change of the nucleus size. It is important to note that reduced apparent mobility at longer observation periods is not a consequence of altered conditions with increasing post-irradiation time  since the mobility at short observation intervals is similar whether investigated within the first minutes after irradiation or after one hour , “subdiffusive molecules take longer than expected to reach distant targets but they are also more likely to retrace local regions of space than a freely diffusing particle”71213appD smaller than 10−5 μm2/s at Δt = 3 h (d = 3) which is less than 0.5% of the total volume of the cell nucleus of about 700 μm3.In any case, we conclude that any model invoking movements of foci over several μm within a few hours cannot be explained by typical mobility of chromosomal loci. Since the average distance of travel is small, the probability of joining wrong ends from DSB that initially were separated by several micrometers is low. For example, with typical values for Δt = 3 h , the aveR smaller than the so-called crossover radius cR, thus facilitating cellular processes such as protein complex formation with high probability−3 Pa·s at 37°C and an anomalous diffusion coefficient α = 0.5 lead to a search time-dependent crossover radius cR [μm] ≈ 0.5 × searcht1/4 (with searcht in [s]) of more than 5 μm for search times of 1–3 hours  can be estimated to be larger than 90% for subdiffusion (using the model ofR ~ 100 nm with maximum 2% probability (P(R) = a/ROur study investigated the mobility of IRIF surrounding break sites rather than the mobility of individual DNA ends. Little is known on the extent of tethering of both ends of a DSB site, resulting either from the original chromatin structure or from fast DSB detection and response mechanismsWhen there are more than one DSB within the initial separation distance R of the two ends of a single DSB, the probability to connect two ends would be the same for all DNA ends, thus resulting in a large fraction of translocations. Note that the ratio of correctly rejoined ends and translocations would be the same under subdiffusion or normal diffusion random walk, but many more single ends would be left in case of a normal diffusion. In the case that two DSB are created at a distance larger than R, however, the ratio of correctly rejoined ends to translocations increases more for subdiffusion than for normal diffusion.In conclusion, we propose that the subdiffusional behaviour of DSB sites guarantees a high probability of rejoining the ends of an individual DSB while reducing the probability of chromosomal aberrations produced by rejoining the ends of different DSB or of leaving a large fraction of DNA ends unrepaired.2 (72 hours before irradiation).Generation of a stable transfectant (clone F1) of human osteosarcoma cells (U2OS) with pEGFP-MDC12 matrix, with an accuracy of 0.58 μm in x and 0.86 μm in yThe irradiation was performed at the microbeam facility SNAKE36total  were recorded. Motion analysis was performed with the open source image analysis software ImageJ (http://rsb.info.nih.gov/ij) using the SpotTracker plugin (http://bigwww.ep.ch/sage/soft/spottracker/), developed by Daniel Sage et ali was determined for different time intervals Δt for analysis of the foci dynamics. For more details see Live cell observation started approximately 200–400 s after irradiation and time series of different interval length Δt  and total length tS.G., V.H. and G.D. designed research; S.G., V.H., C.G., G.A.D. and C.S. performed research; S.G., V.H., M.H. and G.D. analyzed data; and S.G., V.H., G.D. and A.A.F. wrote the paper.Supplementary Information


Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca2+]i. However, the relationship and interaction between salinity stress-induced [Ca2+]i increases and ROS-induced [Ca2+]i increases remain poorly understood. Using an aequorin-based Ca2+ imaging assay we have analyzed [Ca2+]i changes in response to NaCl and H2O2 treatments in Arabidopsis thaliana. We found that NaCl and H2O2 together induced larger increases in [Ca2+]i in Arabidopsis seedlings than either NaCl or H2O2 alone, suggesting an additive effect on [Ca2+]i increases. Following a pre-treatment with either NaCl or H2O2, the subsequent elevation of [Ca2+]i in response to a second treatment with either NaCl or H2O2 was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca2+]i seen with a second NaCl treatment more than that seen with a second treatment of H2O2. A similar response was seen when the initial treatment was with H2O2; subsequent addition of H2O2 led to less of an increase in [Ca2+]i than did addition of NaCl. These results imply that NaCl-gated Ca2+ channels and H2O2-gated Ca2+ channels may differ, and also suggest that NaCl- and H2O2-evoked [Ca2+]i may reduce the potency of both NaCl and H2O2 in triggering [Ca2+]i increases, highlighting a feedback mechanism. Alternatively, NaCl and H2O2 may activate the same Ca2+ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca The presence of high salinity affects almost every aspect of plant growth and development, and causes enormous losses in agricultural production worldwide. It is estimated that about 10 million hectares of agricultural land is abandoned every year because of high salinity, and salt stress affects as much as a quarter to a third of global agricultural land, particularly land which has been irrigated . Given tArabidopsis thaliana [2+ concentration ([Ca2+]i) that lasts about 2 min i are detected by cytosolic Ca2+ sensors. More than 250 Ca2+-binding EF-hand proteins have been identified in Arabidopsis i signatures, allowing the plant to tightly bring about the appropriate adaptation to its ever-changing environment.In plants, Cat stress -13. Thesembranes , and thebidopsis , includi2+]i leads to the activation of SOS3/CBL4, which functions as the primary Ca2+ sensor of [Ca2+]i changes under salt stress [+/H+ antiporter SOS1 that transports sodium ions out of the cell [2+]i increase is an essential component for bringing about the plant response to salt tress.The salinity stress-induced increase in [Ca2O2) [2+]i and ROS are about 3 sec and 400 sec, respectively, as estimated from previous studies [2+]i occurs earlier than the ROS elevation after salt stress treatment. Considering ROS have also been shown to trigger increases in [Ca2+]i [2+]i increases might serve as a feed forward mechanism in the salt stress signal transduction pathway. However, less is known about the relationship and interaction between the salt stress-induced [Ca2+]i increases and the [Ca2+]i increases evoked by ROS, which are produced in response to either salt stress specifically or other stresses in general i -26, it i general .2+]i increases and the ROS-induced [Ca2+]i increases in Arabidopsis. We found the increases in [Ca2+]i induced by both stimuli were higher than these induced by either single stress, suggesting that NaCl and H2O2 have an additive effect on [Ca2+]i. We have also found that NaCl-induced [Ca2+]i increases might inhibit both NaCl- and H2O2-gated channels by a feedback mechanism, but more NaCl-gated channels; a similar response was seen when the H2O2-induced [Ca2+]i increases were analyzed. These data suggest responses seen involve both feedback inhibitory mechanisms, as well as an interaction between two stimuli-mediated Ca2+ signaling pathways.In this study, we have systematically analyzed the relationship and interaction between salt stress-induced [Ca2+]i induced by NaCl and H2O2 interact each other in Arabidopsis, we attempted initially to identify optimum concentrations of NaCl and H2O2, which ideally could be applied to generate about half of the maximum amplitude of [Ca2+]i for potential up- and down-regulation. In addition, we attempted to establish the kinetics of NaCl- and H2O2-induced increases in [Ca2+]i for administrating these stresses in different sequential combinations. First, to analyze NaCl-induced increases in [Ca2+]i, we treated Arabidopsis seedlings expressing aequorin with solutions containing 0 to 600 mM NaCl. Aequorin bioluminescence images were taken every 10 sec for 500 sec, and the peak [Ca2+]i was calculated and analyzed, as NaCl induces a transient increase in [Ca2+]i [2+]i of 80 ± 21 nM  might be desensitized or adapted by unknown signaling elements upstream of NaC activation. To test whether the NaC is desensitized or adapted, we waited for 3 hr and were able to detect a normal (~1 µM) [Ca2+]i increase in response to NaCl, suggesting that the NaC is most likely desensitized (data not shown).To further characterize the potential interaction between the two stimuli-triggered i induced by H2O2 was clearly greater than that induced by 200 mM NaCl (P < 0.001). After 450 sec, the [Ca2+]i decreased to a new basal level under 200 µM  subsequently inhibited NaC more than HpC , inhibited HpC more than NaC  and calcineurin B-like proteins (CBLs), leading to the activation of downstream events [2+]i is maintained at a concentration about 10,000-fold below the extracellular Ca2+ concentration i [2+]i have long been proposed as being involved in the process perceiving the salt signal although the properties of the salt-activated Ca2+ permeable channel are poorly understood and its molecular nature remains to be identified i ,32. Saltentified .2+ permeable channels in the plasma membrane, which in turn lead to Ca2+ influx into the cell and thus increases in [Ca2+]i [2+]i increases precede the production of H2O2 signaling molecule i ,24,38. Nmolecule . Neverth2+]i induced by both NaCl and H2O2 seen in this study  constitutively expressing intracellular aequorin  under the control of the cauliflower mosaic virus 35S promoter was used i measurements i were calculated used the equation described previously i increases in response to NaCl treatment.H(A) Arabidopsis seedlings were treated with water (Control), the NADPH oxidase inhibitor DPI (15 µM), and ROS scavenger ascorbic acid (5 mM) and glutathione (5 mM) two hours prior to the NaCl treatment. The seedlings were then subjected to a 200 mM NaCl treatment, and aequorin luminescence was recorded continuously through the treatments in the dark. (B) Quantification of peak [Ca2+]i increases from experiments as in (A). Data for three independent experiments are shown .(PDF)Click here for additional data file.


Acute myeloid leukemia (AML) is a clonal disorder of the hematopoietic stem cell, typical of the elderly, with a median age of over 60 years at diagnosis. In AML, older age is one of the strongest independent adverse prognostic factor, associated with decreased complete response rate, worse disease-free and overall survival, with highest rates of treatment related mortality, resistant disease and relapse, compared to younger patients. Outcomes are compromised in older patients not only by increased comorbidities and susceptibility to toxicity from therapy, but it is now recognized that elderly AML has peculiar biologic characteristics with a negative impact on treatment response.In older individuals prolonged exposure to environmental carcinogens may be the basis for similarities to therapy-related myeloid malignancies (t-MN), which result from toxic effects of previous cytotoxic treatments on hematopoietic stem cells. Age is itself a risk factor for t-MN, which are more frequent in elderly patients, where also a shorter latency between treatment of primary tumor and t-MN has been reported. t-MN following chemotherapy with alkylating agents and elderly AML frequently present MDS-related cytogenetic abnormalities, including complex or monosomal karyotype, and a myelodysplastic phase preceding the diagnosis of overt leukemia. Similarly, t-MN and elderly-AML share common molecular abnormalities, such as reduced frequency of NPM1, FLT3 and CEBPA mutations and increased MDR1 expression.Given the unfavorable prognosis of elderly and t-MN and the similar clinical and molecular aspects, this is a promising field for implementation of new treatment protocols including alternative biological drugs. Acute myeloid leukemia (AML) is year and represents 90% of acute leukemia in adults, mainly a disease of older adults. Worldwide, AML while it is rare in children. The AML incidence rises affects approximately 3 to 4 individuals in 100,000 per rapidly after 50 years, with a median age at diagnosis of 67 years.1Therapy-related AML (t-AML) accounts for 10 to 20% of AML cases in adults. The incidence of t-AML is increasing due to longer life expectancy of cancer survivors. Therapy-related myeloid neoplasms (t-MN), including t-AML and therapy-related myelodisplastic syndromes, are now recognized as distinct nosographic entities according to the 2008 WHO classification of tumors of Hematopoietic and Lymphoid Tissues (2008).3P = 0.01),6Age is a risk factor also for t-AML, which occurs at a higher median age compared to de novo AML.de novo AML in younger patients, elderly patients and those with t-MN more often present significant comorbidities, reduced tolerance to treatment, higher incidence of treatment-related complications and are commonly excluded from treatment protocols.This review will focus on similarities between elderly and therapy-related AML, both from the clinical and biological point of view. These diseases are commonly associated to worse prognosis related both to tumor biology and host-related factors. Tumor biology is characterized by adverse prognostic signature affecting cytogenetics, molecular genetics, epigenetics, GEP, and multidrug resistance . SimilarIn this review, we will use the terms “AML in older patients” and “elderly AML” to indicate AML occurring in individual aged 60 years or more. Changes of leukemia features will be described according to patients’ age considering different age intervals. Nevertheless, aging is a continuum of changes affecting not only individuals, but also several aspects of diseases. In particular, leukemia biological and clinical features change gradually with aging without any sharp distinction or any universally accepted temporal watershed to define distinct disease subsets.P < .001).de novo AML .4Morphological/laboratory characteristics as well as clinical course present several similarities between t-AML and elderly AML. Cytological and cytochemical differences have been described between “young” and elderly AML, indicating a less differentiated phenotype for “elderly” blasts.P = 0.011), lower median WBC count  and worse median EFS  and OS .The WHO Classification of Myeloid Malignancies identifies AML with myelodysplasia-related changes (AML-MRC) as a distinct AML subclass when at least one of the following prerequisites is fulfilled: a history of secondary AML arising from a previous MDS or MDS/MPN, AML with MDS-related cytogenetic abnormalities, and/or AML with multilineage dysplasia (MLD).Two distinct t-AML subtypes have been described according to the previous cytotoxic therapy.3Cytogenetics has been indicated as the most powerful outcome predictor in AML, independent of age and other potential confounders.9Monosomal kayotype (MK), defined as 2 or more distinct monosomies or a single monosomy in the presence of other structural abnormalities, identifies a distinct subset of AML with an extremely poor prognosis and a 4-year OS of 3–4%, even worse than that of unfavorable cytogenetics.15P < .0001) and adverse cytogenetics . The frequency of favorable-risk abnormalities is similar in t-AML and de novo AML, while the intermediate risk category is less frequent in t-AML. Common poor risk cytogenetic abnormalities significantly enriched in t-AML group include t, -5/5q-, -7/7q-, abnormalities (17p), complex karyotype and monosomal karyotype.4In the same line, t-AML frequently present abnormal karyotype  were shown to be less common in elderly compared to younger AML patients (23% and 37%).P < .001). The prevalence of TET2 mutations gradually increased with age, from 7% in adults younger than 30 to 32% in patients aged 70 years or older (P < .001).P = 0.035) AML.18TET2 mutations occur in 23% of AML patients and are associated with older age , particularly in those with normal cytogenetics and NPM1 mutated, and have an unfavorable impact on outcome. Patients carrying IDH mutations present a higher median age at diagnosis compared to IDH wild-type patients.21CEBPA mutations occur in 6–15% of AML patients and are more common in patients with normal kayotype. Double CEBPA-mutant have a better prognosis compared to wild-type and single mutant. Frequency of double CEBPA mutation decrease by increasing age.28de novo AML in younger patients, involving multidrug resistance, telomere shortening, gene expression profiling and gene promoter methylation.Several adjunctive molecular features make elderly and therapy-related AML biologically different from de novo AML ,P-glycoprotein (Pgp) is a membrane transporter encoded by the multidrug resistance  gene, which traps hydrophobic drugs, including key chemotherapeutic drugs in the plasma membrane of cells and effluxes them using an ATP-dependent process. High Pgp expression is detected in about 50% of AML blast samples, is more common in older (71%), than in younger cohorts (35%) and is associated with poor response to chemotherapy.P<0.001), while age-adjusted telomere length was found to be significantly shorter in patients displaying an aberrant karyotype than in patients with a normal karyotype .35Telomeres are specialized structures composed of TTAGGG repeats and associated proteins located at the end of eukaryotic chromosomes. Telomere repeats are lost with each cell division, eventually leading to genetic instability and cellular senescence when telomeres become critically short. Age-adjusted telomere length in AML patients is significantly shorter than in matched healthy controls. Surprisingly, patients younger than 60 years had significantly shorter age-adjusted telomere length than patients older than 60 years  compared to younger AML patients. In a cohort of 525 adult AML, the transcriptome of the 175 oldest patients (median age 59.4 years) compared to that of the 175 youngest patients (median age 31.1 years) revealed 969 differentially expressed probe sets, of whom 477 were up-regulated and 492 down-regulated with increasing age. The Gene Ontology analysis of 145 “Up-with-Age” as well as 257 “Down-with-Age” probe sets revealed that genes involved in biologic processes, as regulation of I-κB kinase NF-κB cascade and immune response were up-regulated with increasing age. On the other hand, biologic processes as establishment or maintenance of chromatin architecture, regulation of cyclin-dependent protein kinase activity, and cell matrix and cell-substrate adhesion were found to be downregulated with increasing age. In particular, among differentially-expressed genes, p16INK4A, encoded by the tumor-suppressor gene CDKN2A, is down-regulated in elderly AML samples, while it is normally induced during the physiologic aging of hematopoietic stem cells.37According to GEP, two distinct t-AML subgroups have been identified: the first group associated with del5q/-5 shows a higher expression of genes involved in cell cycle control , checkpoints (BUB1), or growth (MYC), and loss of expression of the gene encoding IFN consensus sequence-binding protein (ICSBP). The second t-AML group is characterized by down-regulation of transcription factors involved in early hematopoiesis  and overexpression of proteins involved in signaling pathways in myeloid cells (FLT3) and cell survival (BCL2). Moreover CD34+ cells from patients with t-AML present a more immature profile than normal CD34+ cells.38de novo AML, while no significant differences were found according to patients’ age.de novo MDS or AML .de novo AML blasts. This aberrant methylation affected particular chromosomal regions, occurred more frequently in Alu-poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways.43Epigenetic changes, in particular DNA methylation, are recognized as significant contributors to leukemogenesis, and in particular in therapy-related diseases. By a single gene analysis of promoter hypermetylation of several tumor-suppressor genes, BRCA1 and DAPK1 were found more frequently hypermethylated in therapy-related than in de novo AML.The most significant similarity between elderly and t-AML is the poor prognosis, compared to younger and Older age is a major adverse prognostic factor for AML in general. Surveillance, Epidemiology, and End Results (SEER) statistics from 1996 to 2002 show 5-year relative survival rates of 34.4% for adults aged under 65 and 4.3% for those over 65 years.44In the last three decades significant improvement in survival has been mainly observed in younger AML patients. In a large Swedish population-based cohort study including 9729 AML patients diagnosed between 1973 and 2005, one-year survival increased across all age categories, but the improvement in 5- and 10-year AML survival was confined to patients younger than 80 years and was most prominent in the young (<60 years) patient population.45de novo AML, t-AML still has worse outcome. According to the analysis of 6 prospective multicenter treatment trials of the German-Austrian AMLStudy Group (AMLSG), 4-year relapse-free survival was 24.5% in t-AML and 39.5% in de novo AML and 4-year overall survival was 25.5% and 37.9% (age-stratified log-rank test P = .001 for both). In multivariate analysis, t-AML remained an adverse prognostic factor for death in complete remission, but not relapse, and overall survival in younger intensively treated patients. On the other hand it predicted relapse, but not death in complete remission, in older, less intensively treated patients. In more intensively-treated younger adults, treatment-related toxicity had a major negative impact on outcome, possibly reflecting cumulative toxicity of cancer treatment.4Similar to elderly AML, patients with t-AML are often precluded from intensive treatment due to frequent comorbidities and cumulative toxicities, related to previous cytotoxic treatments. Also when appropriately treated as de novo and therapy related Core Binding Factor (CFB) AML, carrying the recurrent chromosomal aberrations inv16 or t. An inferior treatment outcome was observed in patients with t-AML, with a median overall survival of 100 weeks compared to 376 weeks in de novo CBF AML (P = .001).de novo cases, indicating the dominant prognostic role of good karyotypes.4An analysis matched for age, ECOG PS and additional cytogenetic abnormalities, compared de novo AML, so that most patients have to interrupt treatment after induction chemotherapy due to complications. Notwithstanding, t-AML patients with good performance status, favorable karyotypes and without significant comorbidities should still take advantage from standard therapy, since response rates are similar to younger patients with de novo AML. All these aspects should accurately be taken into account during treatment decision-making.Besides biological factors associated to a more aggressive disease behavior, host related factors, including performance and functional status, cumulative toxicities and comorbity significantly impact on prognosis, treatment tolerability and complications. Both elderly and therapy-related AML are associated with reduced bone marrow hematopoietic cell reserve, due to physiological aging-related bone marrow impairment or to the effect of previous cytotoxic treatment, respectively. This hampers patients to overcome intensive treatment or makes recovery time longer than that of younger patients with Before starting treatment, elderly leukemic patients should undergo comprehensive geriatric assessment (CGA) to identify problems that may interfere with cancer treatment, as recommended by the NCCN guidelines.49Evaluation of physical function provides information independent of comorbidity in older patients with AML. Score systems, such as the Eastern Cooperative Oncology Group (ECOG) and the Karnofsky performance status scales and the Basic Activities of Daily Living (ADL) and Instrumental Activities of Daily Living (IADL) scales, can detect subclinical disability that might predict tolerability and response.13Although the optimal therapy for older patients with AML remains debated, there is some evidence to guide decision making for individual patients. Older adults with newly diagnosed AML who present with any of the following characteristics are more likely to experience toxicity and less likely to benefit from standard induction chemotherapy: an ECOG score >2, significant comorbidity , and poor risk cytogenetics. It would be reasonable to offer these patients supportive care alone, low-intensity therapy, or a clinical trial investigating novel agents. Alternatively, older adults with good functional status , minimal comorbidity, and good-risk cytogenetics are likely to benefit from standard curative therapies regardless of chronologic age. The optimal treatment for the large majority of older adults falling between these two extremes is unclear.13de novo APL. Patients with t-AML associated with other favorable karyotypes should receive standard induction chemotherapy followed by high-dose cytarabine consolidation. Patients with indermediate and unfavorable cytogenetics who have an HLA-matched donor should be considered for allogeneic HCT. Supportive care or low dose chemotherapy remain the gold standard for patients with poor performance status.51Similarly to elderly AML, patients with t-AML should be accurately evaluated for performace status, comorbidities, status of the primary disease and presence of complications from primary therapy, as well as for cytogenetic abnormalities. Patients with therapy-related acute promyelocytic leukemia (APL) should be treated as patients with New investigational treatment approaches are under consideration for t-AML and elderly AML patients, including hypomethylating agents which induce significant response rates with limited toxicity in patients with unfavorable biologic factors and not susceptible of intensive chemotherapy.51De novo AML in younger patients, especially those with “balanced” karyotypes, are thought to result from a limited number of mutational events restricting the diversity of leukemia subclones and leaving many cell functions relatively intact. In contrast, AML in the elderly may more often be the result of a string of mutational events leading to multiple leukemic subclones with the opportunity to develop multiple mechanisms of chemoresistance.52The different biology of elderly AML favors the hypothesis of the involvement of specific pathogenetic pathways in this disease, similar to therapy-related AML. Both elderly and t-AML take their origin by the physiological or treatment-induced aging process of bone marrow hematopoietic system and microenvironment, associated to reduced efficiency of DNA repair system, reduced hematopoietic reserve and immune surveillance, and prolonged exposure to carcinogens .New therapeutic development are needed, because actual therapy strategies are limited due both to host-related factors responsible of poor tolerance to standard chemotherapy, and adverse biological features associated to treatment resistance.


Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations  of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus  Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signal transduction pathway is important controlling immune responses Suppressor of cytokine signaling (SOCS) proteins constitute a class of inducible negative feedback regulators of the Jak/Stat signaling transduction pathway IFN are important cytokines for innate immunity and for host protection from viral infections through the Jak/Stat signaling transduction pathway Siniperca chuatsi) in 1998 Iridoviridae. The iridovirus has a broad host range, which includes insects, fish, amphibians, and reptiles. Most members of the iridovirus family have been associated with serious diseases in frogs and fishes Iridoviridae has been subdivided into five genera, including Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and MegalocytivirusMegalocytivirus, ISKNV infects mandarin fish (Siniperca chuatsi), zebrafish (Danio rerio), tetraodon (Tetraodon nigroviridis), largemouth bass , and more than 50 species of marine fish Infectious spleen and kidney necrosis virus (ISKNV) was identified and isolated from mandarin fish , rock bream iridovirus (RBIV), and large yellow croaker iridovirus (LYCIV) vsocs genes only exist in the genus Megalocytivirus of the Iridoviridae family and not in any other virus species/stains.PCR was performed using ISKNV genomic DNA and cDNA extracted from the spleens of ISKNV-infected mandarin fish as templates to confirm the presence of ISKNV ORF103R. cDNA fragments of ∼400 bp were obtained. The sequencing results showed that the DNA sequences of two fragments were identical and perfectly matched ISKNV ORF103R. ISKNV ORF103R had 402 bp and encoded a predicted protein of 133 amino acids. SMART analysis and BLASTp results showed that ISKNV ORF103R encoded a protein having a conserved SH2 domain and was highly homologous to vertebrate SOCS1 proteins. Thus, this protein encoded by ISKNV ORF103R was named as ISKNV-vSOCS. Interestingly, similar Megalocytivirus. Moreover, ISKNV-vSOCS showed higher similarity with SOCS1 proteins from fishes than those from mammals, with 65%, 62%, 61%, 60%, and 51% identities to SOCS1 of stickleback (Gasterosteus aculeatus), medaka (Oryzias latipes), tetraodon (Tetraodon nigroviridis), fuge (Takifugu rubripes), and zebra fish (Danio rerio), whereas 42% and 41% identities to SOCS1 of mouse and human, respectively.vSOCS proteins  and SOCS1 proteins from different organisms, such as human, mouse, zebrafish, tetraodon, stickleback, fuge, and medaka were then compared . vSOCS pProtein sequences of SOCS family members were sought and a phylogenetic analysis was performed to further investigate the possible evolutionary origin of vSOCS . The phyThe activities of IFN-stimulated response element (ISRE)-promoter (ISRE-luc) reporter genes in response to IFN-α were observed after transfection with ISKNV-vSOCSmyc plasmid to investigate the function of ISKNV-vSOCS. Firefly luciferase activity was normalized to Renilla luciferase activity. The relative luciferase activity (RLA) levels of the cells transfected with empty plasmid without stimulation (control sample) were arbitrarily set as 1. Other RLA levels were presented as fold-increases over that of the control sample. As negative controls, the cells were transfected with a control reporter gene (TA-luc). The RLA levels were very low (<0.4) with or without IFN-α stimulation . HoweverDose-dependent assays were performed to further confirm whether the activation of ISRE promoters by IFN-α was inhibited by ISKNV-vSOCS. Briefly, cells were transfected with increasing amounts of ISKNV-vSOCSmyc (0–200 ng) and stimulated with IFN-α (5000 U) for 8 h. The RLA levels were then detected. The results show that significantly lower RLA levels in cells transfected with higher amounts of ISKNV-vSOCSmyc . RLA levAn immunoprecipitation assay was performed using cell lysates at 36 h post-transfection to investigate the mechanisms by which ISKNV-vSOCS inhibits the Jak/Stat signaling pathway. Jak1 precipitation with anti-Jak1 antibody and Jak1 was detected by western blotting to confirm the presence of Jak1 in both the control cells (transfected with the empty plasmid) and the cells expressing Myc-tagged ISKNV-vSOCS. to test Jak1 tyrosine kinase activity. Jak1 in both the control and recombinant cell lysates was subjected to immunoprecipitation with anti-Jak1 antibody and was used for the in vitro assays. The resulting protein complexes were detected by anti-Jak1 and anti-Myc antibodies (The tyrosine kinase activity of Jak family proteins is triggered by IFN Phospho-STAT1 (p-STAT1) and phospho-STAT3 (p-STAT3) were detected using the TransAM™ Stat family transcription factor assay to determine whether the activation of Stat transcription factors was also inhibited by ISKNV-vSOCS .Limnander et al. Mx, irf7, socs1, and socs3 genes, which are the downstream effector genes of IFN-induced Jak/Stat signaling, were strongly activated in mandarin fish cells after the previously described poly(I:C) stimulation vsocs gene (ISKNV-ΔvSOCS) was constructed using homologous recombination, where the vsocs gene was replaced with green fluorescent protein (GFP) in the ISKNV-ΔvSOCS genome (unpublished data). Time-course expressions of mx and socs1 genes were detected after the cells were infected with wild-type ISKNV and ISKNV-ΔvSOCS using quantitative real-time PCR to assess the function of ISKNV-vSOCS in ISKNV-infected cells. The results show that expressions of mx gene remained low in the period of 1–24 h and slightly increased in the period of 48–120 h after infection with wild-type ISKNV. However, the expression of the mx gene significantly increased at 1 h, peaked at 16 h (about 4-folds), and remained at a relatively high level within 48–120 h after infection with ISKNV-ΔvSOCS virus compared with wild-type ISKNV virus , but not by the wild-type ISKNV virus, suggesting that vSOCS serves as a suppressor that inhibits IFN-induced Jak/Stat signal transduction pathway in infected cells.A vSOCS from the virus, with functions similar to vertebrate SOCS1 protein, was identified for the first time in this study. Over-expressed ISKNV-vSOCS in HepG2 cells interacted with Jak1 protein to inhibit its tyrosine kinase activity, and impaired the phosphorylation and transcription activity of Stat1 and Stat3 proteins. Moreover, the expressions of Megalocytivirus in the family iridoviridae. We previously reported that the OSGIV ORF 99R encoded a putative SOCS iridoviridaeMegalocytivirus contain ORF-encoding vSOCS proteins, including ISKNV ORF103R, OSGIV ORF 99R, RBIV ORF 95.5R, and the LYCIV undefined ORF. However, vSOCS only existed in the megalocytivirus and may be used as a typical gene to distinguish megalocytiviruses from other viruses.vSOCS was not only present in ISKNV, but was also found in other viral genomes of the genus Interestingly, megalocytivirus vSOCS shares a similar architecture with SOCS1, but lacks a SOCS-box domain . TherefoThe mammalian SOCS1 directly bound to the Jaks through its SH2 domain, and inhibited the Jaks kinase activity via the KIR domain paramyxoviridae family to block the Jak/Stat signaling pathway led to the impaired biological activity of IFNs. The hepatitis C virus encoded a suppressor that decreased the phosphorylation of Stat tyrosine and strongly inhibited the IFN-α-induced signal transduction flaviviruses family have also been confirmed to inhibit IFN signaling. The Japanese encephalitis virus blocked the phosphorylation of tyrosine residues on TYK2, Stat1, Stat2, and Stat3 Papillomaviridae family of dsDNA viruses, expressed an E6 oncoprotein that interacted with Tyk2, thereby impairing Jak/Stat activation The IFNs-induced Jak/Stat signaling cascade responded to the viral infection. Several viruses developed various strategies to inhibit the IFNs-induced Jak/Stat signal transduction pathway to escape the antiviral immunity of the host Megalocytivirus was more closely related to the SOCS1 proteins of fishes than that from frogs, chickens, platypuses, and mammals. The megalocytivirus possibly gained its vsocs genes from fish during evolution. Compared with the SOCS1 of fishes, the vSOCS lacks a SOCS box, which may be attributed to the following two reasons. First, the vSOCS genes of the genus Megalocytivirus may have originated from a “full-length” SOCS1 gene and lost its SOCS box during evolution. Second, the genus Megalocytivirus may have selectively obtained a SOCS1 gene from a host without a SOCS1 box.A phylogenetic tree was constructed with the vSOCS proteins and other SOCS family proteins from the vertebrate to further study the evolutionary origin of vSOCS. vSOCS from the genus In conclusion, we demonstrated that the vSOCS of the megalocytivirus suppressed Stat1/3 signaling, which led to impaired IFN-α response. The discovery of vSOCS and further research on its multiple functions will help identify antiviral targets and develop more efficient antiviral drugs.2, as described previously HepG2 (ATCC HB8065) cells were cultured in a complete Dulbecco’s modified Eagle medium  supplemented with 10% FBS  and maintained at 37°C. Mandarin fish fry (MFF-1) cells were cultured in DMEM supplemented with 10% FBS at 27°C under humidified atmosphere containing 5% COAnti-Jak1 antibody was obtained from Upstate Biotechnology , and anti-Myc tag antibody was purchased from Clontech . Anti-phospho-Stat1 (Tyr701) and anti-phospho-Stat3 (Tyr705) were purchased from Cell Signaling Technology . Anti-β-tubulin antibody was purchased from Epitomics, Inc. .5′-GGA ATT CGG TGC TCC CCG ACG TCA TCG-3′ (sense), which contains an EcoRI site, and 5′-CCG CTC GAG TTA CTG TGT TTG CAC CCG CTT C-3′ (antisense), which contains an XhoI site. DNA fragment was amplified by PCR, digested with EcoRI and XhoI , and then inserted into the pCMV-myc  vector. The resultant plasmids were named ISKNV-vSOCSmyc. The point-mutations of KIR (F18D) and SH2 domains  were prepared with ISKNV-vSOCSmyc as template according to the site-directed gene mutagenesis kit . All plasmids were confirmed by sequencing.Genomic DNA and cDNA were extracted from the spleens of ISKNV-infected mandarin fish. Based on the DNA sequence of ISKNV ORF103R, a pair of primers was designed as follows: http://smart.embl-heidelberg.de/) and basic local alignment tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST). Sequence alignments were performed using ClustalX v1.83, and edited using the GeneDoc v2.60 software. The phylogenetic tree was constructed using the Bootstrap N-J method of the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 The amino acid sequence of ISKNV-vSOCS was analyzed using the Simple Modular Architecture Research Tool (SMART) (cis-acting element that responds to a particular pathway was used as a negative control (pTA-luc). Cells were cultured at 5×104 cells per well in a 48-well plate and co-transfected with 0.2 µg of pISRE-luc reporter plasmid, 20 ng of pRL-TK reporter plasmid, and 0.2 µg of pCMV-myc/ISKNV-vSOCSmyc plasmids (1 ng to 200 ng plasmids were employed in the dose-dependent assays) using Lipofectamine™ 2000 reagent . Cells were treated with recombinant interferon-α  for 8 h at 24 h after transfection. Cell lysates were harvested and assayed for the luciferase activity using the dual-luciferase® reporter assay system .The pISRE-luc reporter plasmid  was designed to monitor the induction of the interferon-α-triggered signaling transduction pathway 2 flasks were lysed using a modified RIPA lysis buffer  containing protease inhibitor cocktail after 36 h of transfection. Pelleted cellular debris was collected by centrifugation. Supernatants were precleared with protein A/G plus agarose for 1 h at 4°C on a rocker platform. A 5 µL of anti-Jak1 antibody was added to the supernatants and incubated at 4°C on a rocker platform overnight. The mixture was immunoprecipitated by incubating with fresh protein A/G plus agarose beads for 2 h. The beads were then washed four times with RIPA lysis buffer and once with PBS. The bound proteins were eluted from the beads by boiling with 1×SDS sample loading buffer. Anti-myc antiserum was used as primary antibody to detect immunoprecipitation.Cells from 75 cm2, 25 mM MnCl2, 15 µM Na3VO4, 25 mM ATP, 12.5 mM dithiothreitol), 10 µL of protein tyrosine kinase substrate (10 µg/mL), 20 µL of ultrapure water, and 10 µL of 5×ATP/MgCl2 solution . The mixture was then incubated for 60 min at 37°C. The enzyme reactions were stopped with 120 mM EDTA, and then the 50 µL products were transferred to streptavidin-coated 96-well strip and subsequently incubated at 37°C for 30 min. The phosphorylated substrates were detected at an absorbance of 450 nm after incubation with mouse anti-PY20-HRP, TMB substrate, and Stop Solution.Jak1 proteins were immunoprecipitated by the A/G plus agarose beads as described above. The activities of Jak1 kinase were assessed using the protein tyrosine kinase (PTK) activity assay kit  according to the protocol of the manufacturer. Briefly, the PTK reactions were mixed with the beads in 10 µL of 5×tyrosine kinase assay buffer  and exposed to polyvinylidene diﬂuoride membranes by Hyperfilm .TransAM™ Stat family transcription factor assay  is an ELISA-based assay used to detect and quantify the transcription factor activation for Stat signaling pathways studies The viruses were propagated in MFF-1 cells, and the infected cells were then lysed and subjected to three freeze-thawed cycles. Cell debris were pelleted at 10,000 rpm for 30 min at 4°C. The supernatants were centrifuged at 30,000 rpm in a Beckman-type 70 Ti rotor for 60 min at 4°C. The virus pellets were then resuspended and layered onto 30%, 40%, 50%, and 60% (w/v) sucrose gradients. The viral band was formed between 40% and 50% sucrose layer after centrifugation at 100,000 g for 2 h in a Beckman SW41 Ti rotor. The viral band was subsequently collected. The virions were resuspended and centrifuged at 100,000 g at 4°C for 30 min. The virion pellets were then finally collected and stored at −80°C.6 cells per well overnight. Each cell was then infected with wild-type ISKNV and ISKNV-ΔvSOCS viruses, respectively. The cells were harvested for RNA preparation using Trizol reagents  at various times  post-infection. All RNAs were reverse transcribed to cDNAs as described previously Equal multiplicity of infections (MOI = 10) of wild-type ISKNV and ISKNV-ΔvSOCS viruses were prepared for immune challenged experiments. MFF-1 cells were cultured in 6-well plates at 2×10Mx, irf7, socs1, and socs3 genes were performed by real-time quantitative PCR on a LightCycler  using SYBR premix Ex Taq™ , as described previously t-test The expressions of All data were expressed as a mean ± standard deviation (SD). Statistical significance was accepted at p<0.05, whereas high significance was accepted at p<0.01.Figure S1The point-mutations of ISKNV-vSOCS interacted with Jak1 protein via immunoprecipitation assay. Cells were transfected with ISKNV-vSOCSmyc or its mutants. Cell lysates were immunoprecipitated with anti-Jak1 antibody, and then detected by Western blotting using anti-myc antibody at 36 h after transfection.(TIF)Click here for additional data file.Figure S2The point-mutations in ISKNV-vSOCS altered its inhibitory activity on ISRE-promoter luciferase reporter genes. Cells were transfected with ISKNV-vSOCSmyc or its mutants, and then treated with IFN-α (5000 U) for 8 h. The activities of the ISRE-promoter luciferase reporter genes were detected. Relative luciferase activity in the cells transfected with empty plasmid after IFN-α treatment was arbitrarily set as 100. Error bars represent the mean ± S.D. (n = 3). **P<0.01(TIF)Click here for additional data file.Figure S3Activities of the gamma-interferon-activation sites (GAS)-promoter luciferase reporter gene. (A) IFN-γ-responsive GAS-luc promoter activity. Cells were treated with recombinant IFN-γ (1∼100 ng/mL) for 8 h at 24 h after transfection. The gray columns represent the RLA levels in cells transfected with empty plasmid, whereas the black columns represent the RLA levels in cells transfected with ISKNV-vSOCSmyc plasmid. RLA level in cells transfected with TA-luc reporter gene instead of ISRE-luc reporter gene was used as negative control. RLA levels of cells transfected with empty plasmid without stimulation were arbitrarily set as 1. (B) Activities of reporter genes in cells transfected with increasing amounts of ISKNV-vSOCSmyc plasmid. Cells were transfected with different amounts of ISKNV-vSOCSmyc plasmid (1∼200 ng), treated with IFN-γ (50 ng/mL) for 8 h, and then GAS-luc activity was analyzed. RLA levels in cells transfected with empty plasmid after IFN-γ treatment were arbitrarily set as 1. Error bars represent the mean ± S.D. (n = 3). **P<0.01(TIF)Click here for additional data file.Figure S4The point-mutations in ISKNV-vSOCS altered its inhibitory activity on GAS-promoter luciferase reporter genes. Cells were transfected with ISKNV-vSOCSmyc or its mutants and then treated with IFN-γ (50 ng/mL) for 8 h. The activities of the GAS-promoter luciferase reporter gene were detected. Relative luciferase activity in cells transfected with empty plasmid after IFN-γtreatment was arbitrarily set as 100. Error bars represent the mean ± S.D. (n = 3). **P<0.01(TIF)Click here for additional data file.


We tested laboratory rabbits from 2 US vendors for antibodies against hepatitis E virus (HEV); Seroprevalences were 40% and 50%. Retrospective analysis of an ocular herpes simplex 1 experiment demonstrated that HEV seropositivity had no effect on experiment outcome. HEV probably is widespread in research rabbits, but effects on research remain unknown. Hepeviridae (Hepatitis E virus (HEV) is a single-stranded RNA virus in the family HEV has 4 genotypes, and genotypes 3 and 4 are found in humans and other animals  were obtained from supplier A at LSUHSC. Supplier A also provides rabbits to the meat production industry. A third shipment comprising 10 Dutch Belted SPF rabbits (nos. DB1–DB10) were obtained from supplier B in August 2012 at LSUHSC. Supplier B rabbits were SPF for http://www.aphis.usda.gov/animal_welfare/). The LSUHSC is fully accredited by American Association for the Assessment of Laboratory Animal Care, Int. The animals were singly housed in stainless steel rabbit cages . On the day the rabbits arrived, blood was collected from each rabbit and stored for HEV testing. Rabbits from vendor A were treated for P. cuniculi and L. gibbus with selamectin . After arrival, the rabbits were acclimated to the facility for 7 days. After this period, they were used for research protocols approved by the Institutional Animal Care and Use Committee.The rabbits’ care and use were approved by the LSUHSC Institutional Animal Care and Use Committee and were consistent with the Guide for the Care and Use of Laboratory Animals  were obtained retrospectively from an investigator who used the rabbits in an HSV-1 ocular keratitis study. Survival data and HEV antibody status were examined statistically to determine whether HEV status had any effect on the survival of the rabbits in the study. Briefly, 4 different strains of HSV-1 with different virulence levels were tested, including MCK D-gK, ICP0-delete, 17 syn + mutant, and McKrae. Rabbits were infected with these viruses, and keratitis score and survival rate were recorded.ELISA was performed by using the protocol from Cossaboom with a few modifications .To evaluate the possible effect of HEV serology status on the survival of rabbits used in the HSV-1 study, we used the Fisher exact test when HSV-1 survival status was cross-tabulated with the HEV serology status. Odds ratios are also reported ; α was set at 0.05.We found that the seroprevalence of HEV in the 70 rabbits from supplier A was 40%. The 10 rabbits from supplier B exhibited a 50% seroprevalence for HEV  and 2.HSV-1 outcome results revealed no significant association between positive serologic HEV status and frequency of HSV-1 signs. Prevalence of positive HEV status was 32% (6/19) , compared with 36% (4/11)  for frequency of HSV-1 signs.We sought to determine the seroprevalence of HEV in research rabbits within LSUHSC. We were surprised to identify HEV-seropositive rabbits from the SPF research rabbit supplier (supplier B) because of the strict health quality standards employed at this supplier. The difference in HEV seroprevalence between the 2 vendors may be a function of sample numbers. Testing a larger number of samples from supplier B would answer this question. HEV may be widespread in research facilities throughout the United States.Statistically, we could not demonstrate any effect of HEV serology status on the survival outcome or keratitis scores of HSV-1–infected rabbits. However, the interpretation of these results is limited because only tested for HEV only by serologic testing and not reverse transcription PCR. Controlled studies in which rabbits are experimentally infected with HEV and then later infected with another pathogen are needed to determine the true effects of HEV co-infection on the outcome of systemic infection experiments in rabbits. ,Rabbits are commonly used in research and they often housed in a conventional setting. Because HEV is classified as a Biosafety Level-2 pathogen, wearing personnel protective protection equipment, such as masks, laboratory coats, and gloves, when working with rabbits is warranted (


In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs  or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca This Ca2+-induced Ca2+ release occurs in local release units or couplons, i.e. clusters of LTCC facing clusters of RyR, which are preferentially located in the T-tubules (TT). The preferential location of couplons in TTs 2+ release. Indeed, the [Ca2+]i transient results from spatiotemporal summation of elementary release events, or Ca2+ sparks In ventricular cardiac myocytes, Ca2+ from the external sarcolemma to the center mediated by a combination of Ca2+ release and diffusion 2+ release, and depress and slow the Ca2+ transient 2+ release during excitation-contraction coupling is consequently less synchronized and areas of delayed Ca2+ release were shown to result from local absence of TTs TTs are poorly developed or absent in neonatal myocytes and in cells from the specialized conduction system such as Purkinje cells, as well as in atrial cells. These cells are small and show a radial spread of Ca2+ sparks, reflecting intrinsic activity and properties of RyR clusters, may provide more information. In ventricular myocytes these mainly occur at the junctional SR Ca2+ transient properties during normally triggered SR Ca2+ release In the present study, we build on our earlier data where we identified areas without TTs within pig ventricular myocytes based on the local CaEthische Commissie Dierproeven, Katholieke Universiteit Leuven), with permit numbers P02042, P06048 and P10139.Animals were housed and treated according to the Guide for the Care and Use of Laboratory Animals  and experimental protocols were approved by the in-house ethical committee (pigs MI = 7) and compared to healthy weight-matched controls (Npigs CTRL = 12). To evaluate the effect of sarcolemmal fluxes and culture, additional control pigs were used in the present study (Npigs = 12).The MI data in the current study are derived from the same pigs described earlier 2+ Tyrode , the tissue was perfused with 1.4 g/L, collagenase A  and 0.1 g/L,protease XIV, , followed by washout with low Ca2+ tyrode. After removal of the tissue wedge from the setup, the infarct area and border zone were discarded and cells were obtained by gentle trituration of the digested midmyocardial tissue adjacent to the MI. The obtained cell suspension was filtered and resuspended in normal Tyrode . CTRL cells were isolated from the same area of matched healthy pigs.Cardiac myocytes were isolated enzymatically as described previously pigs CTRL = 7).In a number of experiments, freshly isolated cardiomyocytes from CTRL pigs were put in primary culture for 48 h as described previously Myocytes were fixed with 2% paraformaldehyde and permeabilised with 0.5% Triton X-100 in PBS. They were washed 3 times and incubated overnight with IgG-anti-RyR primary antibody  at 4°C. The primary antibody was rinsed with PBS and the secondary antibody, FITC-conjugated anti-mouse IgG, was added to the fixed myocytes for 2 hours .5fluo-3 0.05; pH 7.2). Inclusion of fluo-3 salt in the pipette solution allowed simultaneous measurements of ionic currents and [Ca2+]i transients. Patch pipettes  had a resistance of 2.0–3.0 MΩ. All experiments were carried out at 37°C in normal Tyrode solution. Where indicated NiCl2 (5 mmol/L) or CdCl2 (50 µmol/L) were added to this solution and applied through a fast perfusion system. Experiments with Ni2+ were performed in the presence of 1 µmol/L forskolin.Transmembrane ionic currents were recorded using an Axon 200B amplifier using whole-cell voltage clamp in the ruptured patch configuration. Pipette solution contained  from −70 to +10 mV at 1 Hz. NCX current density was measured as the inward tail current 10 ms after repolarization. SR Ca2+ content was measured by integrating the inward Na+/Ca2+ exchange (NCX) current during fast caffeine application (10 mmol/L) and expressed in terms of accessible cell volume.[Ca2+]i transients and spontaneous Ca2+ sparks were studied using a Zeiss Axiovert 100 M inverted microscope with a ×40/1.3 oil-immersion objective in combination with a Zeiss LSM 510 confocal laser point-scanning system . Quiescent, rod-shaped myocytes with clear striations were randomly selected. The 488 nm line of a 25 mW argon laser was used for excitation of Fluo-3. Ca2+ release was visualized by repetitive scanning of a line selected longitudinally through the cell, avoiding the nuclei. X and Y resolution were 0.2 to 0.4 µm and 1.54 ms respectively. Stimulated Ca2+ transients were recorded after reaching a steady state. Stimulation was subsequently stopped and spontaneous Ca2+ sparks were recorded for 15 s.Spatiotemporal characteristics of [Ca2+]i transients and spontaneous Ca2+ sparks were analyzed using a custom-made program, modified after 2+]i level (F/F0). Half-maximal Ca2+ release (F50) of the averaged [Ca2+]i transient served as reference and threshold for categorizing local Ca2+ release areas into early and late release areas, respectively near-TTs and more remote. This correlation with the TT network was established earlier 50 within 20 ms after the start of the [Ca2+]i transient; delayed release areas are those that did not.Linescan images of i transient in the corresponding part of the scan line and thus sparks were categorized as occurring near TTs (early release areas) or more remote. Spark properties were subsequently analyzed per category, i.e. associated with early or delayed release sites; spark frequency was likewise normalized within each category, i.e. number of events per area length with early or delayed release sites. Spark width and duration are shown as Full-Width at Half-Maximum (FWHM) and Full-Duration at Half-Maximum (FDHM) respectively. Sparks were considered as extremely long when more than three standard deviations above the average spark duration in CTRL (47.6 ms).Spontaneous CaData are shown as mean ± SEM, averaged by animal. Comparisons were done using Student's t-test, or Mann Whitney test, as appropriate. P<0.05 was considered significant.2+ sparks were recorded after stimulation at 1 Hz to load the SR to steady state and assigned to early or delayed release areas according to their position on the scan line, as shown in Spontaneously occurring Ca2+, 5 mmol/L NiCl2) applied for the duration of the spark recording, as shown in the inset of 2+ in early, but not delayed release areas . ICaL inhibition indeed did not affect spark frequency in early or delayed release areas . We next examined spark parameters which in CTRL were different between early and delayed sites.Subsequently, we compared sparks in early and delayed release areas within the MI group. As reported before, due to the decrease in TT density In contrast to CTRL, no relation between spark frequency and the type of release area was found in MI . This waIn contrast to CTRL animals, spark duration was not different between early and delayed release areas in MI . This wa2+ removal by NCX from myocytes was reduced in MI but this could be related to the reduced surface area with loss of TTs; protein expression levels were unchanged 2+]i measured at the same time point was comparable . Analysis per region . Compared to CTRL cells, spark duration tended to increase selectively in early release areas  spark duration was not affected by NCX inhibition in rat ventricular myocytes 2+ removal on NCX in rat compared to larger mammals The mechanism of modulation of spark duration by NCX is at present not resolved. A straightforward interpretation is a direct modulation of local Ca2+ by NCX reduces the duration of RyR openings. Modulation of RyR gating by NCX has been studied mainly with regard to the probability of opening. Several studies support the idea that reverse mode NCX during depolarization provides an additional trigger promoting RyR opening An alternative explanation is that the local removal of Ca2+ with or without an additional indirect modulation of RyR gating.Taken together, NCX modulates spark duration near TTs most likely directly through increased removal of local Ca2+ released per spark with larger width and amplitude. These data are consistent with the increased SR Ca2+ content, a feature not observed in heart failure. The relation between an increase of SR Ca2+ content and increased spark size 2+ leak 2+ content, leak measured as increase in cytosolic [Ca2+] in normal rabbit myocytes, increased two-threefold Global spark mass and calculated spark-mediated leak were increased significantly in MI. Whereas at first this feature seems to compare to observations in non-ischemic heart failure models 2+ release was also described in the dog with pacing-induced heart failure 2+ content. The latter, the absence of increase in early release sites, could be related to changes in the dyadic environment, such as a reduction in the amount of L-type Ca2+ channels per couplon o, which may offset the effect of an increased SR Ca2+ content. This needs further investigation.Further subcellular analysis shows heterogeneity in the changes in local spark properties. Such heterogeneity in local Ca2+ removal is reduced. The reduced NCX current density is consistent with this hypothesis, which is further supported by analysis of the phases of Ca2+ removal during caffeine application  was significantly shorter in MI  and the frequency of wave activity was higher in MI cells . These results are in line with an increased tendency for wave initiation and propagation in MI cells. A reduced density of NCX may however mitigate the arrhythmogenic potential of such waves.Reduced removal of spontaneous Ca2+ loss from the cell and contribute to the increased SR Ca2+ content. The increased spark mass and SR content can increase the propensity for spontaneous Ca2+ wave generation but the lower density of NCX may mitigate the arrhythmogenic potential.In ventricular myocytes with a sparse TT network, RyR cluster properties related to their position relative to TTs lead to intracellular heterogeneity of sparks. Remodeling in the area adjacent to a moderate MI differentially affects RyR subpopulations. Loss of TTs leads to a smaller fraction of RyR near TTs. Redistribution of sparks away from the membrane together with reduced NCX in TTs may limit actual Ca