Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles for experimentally observed plasticity in HCN channels accompanying synaptic plasticity in hippocampal neurons, and uncover potential links between HCN-channel plasticity and calcium influx, dynamic gain control and stable synaptic learning. Theoretical and computational frameworks for synaptic plasticity have a long and cherished history, with proven utilities ranging from understanding the underlying biophysical and biochemical mechanisms to solving complex engineering problems h current, have been postulated as a prominent mechanism that can mediate activity homeostasis in hippocampal neurons h conductance. In doing this, we first posed the question on what changes in the h conductance would be required for it to maintain activity homeostasis, when perturbed by a calcium-dependent bidirectional synaptic plasticity mechanism. We employed the answer to this question to arrive at a calcium-dependent plasticity rule (CDPR) for the h conductance such that firing rate homeostasis was maintained when h-channel plasticity accompanied synaptic plasticity. Finally employing calcium-dependent plasticity rules for synaptic strength and h conductance magnitude, we demonstrate that the synergy between these two plasticity rules accomplish much more than firing rate homeostasis. Specifically, we show that the co-occurrence of the two forms of plasticity enabled retention of synaptic weights within a useful dynamic range, introduced metaplasticity in the synaptic learning rule so that the positive feedback introduced by repeated synaptic potentiation was nullified, and facilitated reliable rate-based information transfer across the neuron when faced with positive feedback introduced by repeated synaptic potentiation.The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, that mediate the hyperpolarization-activated h conductance are required for it to maintain firing rate homeostasis, when it is perturbed by bidirectional synaptic plasticity? What should be the relationship between HCN channel plasticity and synaptic plasticity for the former to counteract the perturbation in the input-output relationship that was imposed by the latter? To answer these questions, we employed a conductance-based model of a hippocampal pyramidal neuron with ion channel kinetics derived from experimental measurements and inserted a synapse made of colocalized AMPAR-NMDAR in the model 2+ influx through NMDARs fi, when plasticity was induced through 900 synaptic stimuli  for h-conductance plasticity based on a gradient descent algorithm to minimize the mean-squared error (MSE) between the target FF and post-LTP FF for all considered SFs a, and shift parameter b) and empirically arriving at the dependence of the sigmoidal parameters on the h conductance, odelsand Methods for the derivation of the IPR). Consequently, after the induction of LTP, we implemented the IPR over several iterations (k), involving an update of odelsand Methods for the derivation based on the Lyapunov stability criterion; When long-term potentiation (LTP) was induced in the model synapse through 900 stimuli at 25 Hz , the FF-  as in   was set h conductance of the model after the synapse underwent LTP  would be analogous to the corresponding dependence of synaptic plasticity on [Ca]i. Specifically, we postulated that moderate levels of [Ca]i, higher than a certain depression threshold, would result in h-conductance depression and even higher levels of [Ca]i would result in h-conductance potentiation.The analysis employing the IPR was geared towards the purpose of validating the postulate on a homeostatic role for the asticity  togetherh-channel and synaptic plasticity (h-conductance plasticity (see Modelsand Methods for the derivation of the CDPR). In deriving this rule, we noted that the slope of the linear relationship between h-conductance and synaptic plasticity was variable and depended on the values of baseline HCN channels conductance and values of permeability of the AMPAR. We accounted for this variability and derived a generalized rule that worked under various baseline values (see Modelsand Methods for more details). The CDPR that we used for the normalized h conductance wh was as in Our results with the IPR provided us a quantitative foundation for testing this postulate. We employed the linear relationship between asticity  in conjurameters .fi. To do this, we updated both the synaptic weight and the h conductance simultaneously through respective Ca2+-dependent mechanisms. Consistent with the linear relationship that was the basis for the CDPR, we found that h conductance evolved in a manner , incorporating these into a morphological realistic model would constitute an enormous increase in computational cost given the increase in the number of neuronal compartments. Second, bidirectional plasticity in the mR = 28 kΩ cm2 and Cm = 1 µF/cm2. Fast Na+, delayed rectifier K+ (KDR), A-type K+ (KA) and HCN channels were introduced, with kinetics adopted from experimental measurements of these channels in hippocampal pyramidal neurons 2) were set to qualitatively match (see vs. current (f-I) curves of hippocampal pyramidal neurons h, Na+ and K+ channels, respectively were (in mV), Eh = –30, EK = –90, ENa = 55. All simulations were performed at –65 mV in the NEURON simulation environment The passive parameters associated with the model were: atch see  the firiA synapse was modeled as a co-localized combination of NMDA and AMPA receptor currents. A default value of NMDAR:AMPAR ratio was set at 1.5 and synaptic plasticity was induced in the model synapse by presenting 900 pulses at a various induction frequencies −6, Current through the AMPA receptor was modeled as the sum of currents carried by these sodium and potassium ions:w , an experimentally well-established plasticity protocol e.g.,Using this framework, the direction and strength of plasticity were analyzed by presenting stimuli made up of 900 pulses at various induction frequencies  changed as a consequence of this altered synaptic weight  as the output variable-SF plot . In ordeht e.g., , then, tTo minimize this function using the standard gradient descent algorithm, we needed a differentiable, parameterized form for the FF-SF curve From (16), this reduces to:Rewriting (17),h conductance h current, we used conductance as a means to do this, although the procedure outlined above is generalizable to the half-maximal activation voltage of the h conductance.Under what constraints will the algorithm derived in the previous section converge? Is there a limit on the learning rate parameter function  to achiefunction  as the LLyapunov theory states that if there is a function For a given SF Substituting the value of Substituting Simplifying this, we got,To satisfy the Lyapunov stability criterion, this gradient, From this, we derived the constraint for the system to be globally asymptotically stable and ensuring convergence in terms of the learning rate parameter Thus, the Lyapunov stability criterion would be satisfied, ensuring convergence of IPR, if the learning rate parameter satisfied the inequality provided in (40). To ensure this, in implementing the IPR, we always set The linear interaction between intrinsic and synaptic plasticity forms the basis for the derivation of the calcium dependent ing form :(44)withgiven as :(45)wherh-conductance, h-conductance value vsThe rationale behind using  the IPR , it was rameters . Therefo ), and   would ex, and  ).Furthermore, as we wanted to retain w and the baseline value If the linear relationship between e value  –11). Wheh-channel plasticity to underlying parameters where error   was lessMutual information between the output spikes and the input stimulus was employed as a measure of encoding capabilities of a neuron, and was computed as the difference between the total response entropy and the noise entropy ion for  . This prion for  . The resputed as :Noise entropy was then computed as:We computed mutual information by inserting the computed values of response and noise entropies into h-channel plasticity on the FF-SF curve, without altering driving forces and/or the voltage-dependent conductance of all the channels present in the model h conductance, the two parameters that underwent plasticity. This was to ensure the sigmoidal parametric form for the FF-SF curve, which would be lost if firing entered the high frequency regime where it would be limited by the refractory period or depolarization-induced inactivation of the fast Na+ channels.The synaptic drive to the model neuron was modeled as Poisson-distributed pre-synaptic action potentials arriving at various stimulus frequencies (SF). We presented the model with 100 trials of inputs at each SF and measured the corresponding firing frequencies FF;  to conste.g.,Firing frequencies in response to non-synaptic pulse current injections e.g., was deteFigure S1Sensitivity analysis for the iterative plasticity rule. (A) Plot depicting the amount of change required in h-conductance to achieve firing rate homeostasis at various permeability values and baseline h-conductance values for the maximum synaptic perturbation (25 Hz/900 pulses LTP). (B–D) FF-SF plot for the parameter values indicated by the arrow B (B), C (C) and D (D) in panel (A). Black: Baseline; Red: after LTP inducation; Green: after LTP induction and IPR. At lower values of baseline h-conductance to compensate for the change (B). At intermediate values of baseline h-conductance was large because the baseline activity levels are higher . At larger values of baseline h conductance required (D). (E) The offset correction factor h conductance. (F) To quantify this variability depicted in (A–D), we employed the term h conductance quantified the variability shown in (A–D) across different values of baseline h conductance.(TIF)Click here for additional data file.Figure S2Sensitivity analysis for the calcium-dependent plasticity rule (CDPR), also demonstrating changes in excitability obtained after CDPR. (A) Plot depicting the effectiveness of CDPR in achieving firing rate homeostasis after synaptic plasticity induced with 900 pulses of different induction frequencies. As CDPR runs in parallel to synaptic plasticity, homeostatic gain control runs in parallel, reducing the root mean squared error (RMSE) between the achieved FF-SF curve and the target FF-SF curve below 1 Hz across all induction frequencies. (B) 3-D plot showing the updated h-conductance for various AMPAR permeability and baseline h-conductance values obtained by implementing CDPR for h channel plasticity in parallel with synaptic plasticity. Note that the sensitivity analysis presented here for CDPR is over a range smaller than the one presented for IPR. This was because the FF-SF lost its sigmoidal characteristic at larger AMPAR permeabilities and/or lower baseline C) Traces showing neuronal firing for a non-synaptic pulse current injection, before (black) and after LTP and CDPR (green). (D) Firing frequencies for various amplitudes of non-synaptic pulse current injections (of 500 ms) shown for cases before (black) and after LTP and CDPR (green). (E–F) Same as (C)–(D), but for LTD, when implemented in parallel with CDPR.(TIF)Click here for additional data file.Figure S3Sensitivity analysis for the information transfer across the neuron. (A) Impact of repeated LTP  on AMPAR permeability depicted as a function of the number of LTP inductions, plotted for cases where synaptic plasticity was accompanied (green) and not accompanied (black) by CDPR. 2. (B) Mutual information plotted as a function of number of successive LTP runs, under cases where CDPR accompanied (green) or did not accompany (black) synaptic plasticity for parametric values as in (A). (C) Impact of repeated LTP  on AMPAR permeability depicted as a function of the number of LTP inductions, plotted for cases where synaptic plasticity was accompanied (green) and not accompanied (black) by CDPR. 2. (D) Mutual information plotted as a function of number of successive LTP runs, under cases where CDPR accompanied (green) or did not accompany (black) synaptic plasticity for parametric values as in (C). (E) Impact of repeated LTP  on AMPAR permeability depicted as a function of the number of LTP inductions, plotted for cases where synaptic plasticity was accompanied (green) and not accompanied (black) by CDPR. 2. (F) Mutual information plotted as a function of number of successive LTP runs, under cases where CDPR accompanied (green) or did not accompany (black) synaptic plasticity for parametric values as in (E). The frequencies employed for induction are the sliding threshold frequencies for the corresponding baseline parameters of (TIF)Click here for additional data file.


The vertebrate peripheral nervous system contains sensory neurons that arise from ectodermal placodes. Placodal cells ingress to move inside the head to form sensory neurons of the cranial ganglia. To date, however, the process of placodal cell ingression and underlying cellular behavior are poorly understood as studies have relied upon static analyses on fixed tissues. Visualizing placodal cell behavior requires an ability to distinguish the surface ectoderm from the underlying mesenchyme. This necessitates high resolution imaging along the z-plane which is difficult to accomplish in whole embryos. To address this issue, we have developed an imaging system using cranial slices that allows direct visualization of placode formation.We demonstrate an effective imaging assay for capturing placode development at single cell resolution using chick embryonic tissue ex vivo. This provides the first time-lapse imaging of mitoses in the trigeminal placodal ectoderm, ingression, and intercellular contacts of placodal cells. Cell divisions with varied orientations were found in the placodal ectoderm all along the apical-basal axis. Placodal cells initially have short cytoplasmic processes during ingression as young neurons and mature over time to elaborate long axonal processes in the mesenchyme. Interestingly, the time-lapse imaging data reveal that these delaminating placodal neurons begin ingression early on from within the ectoderm, where they start to move and continue on to exit as individual or strings of neurons through common openings on the basal side of the epithelium. Furthermore, dynamic intercellular contacts are abundant among the delaminating placodal neurons, between these and the already delaminated cells, as well as among cells in the forming ganglion.This new imaging assay provides a powerful method to analyze directly development of placode-derived sensory neurons and subsequent ganglia formation for the first time in amniotes. Viewing placode development in a head cross-section provides a vantage point from which it is possible to study comprehensive events in placode formation, from differentiation, cell ingression to ganglion assembly. Understanding how placodal neurons form may reveal a new mechanism of neurogenesis distinct from that in the central nervous system and provide new insight into how cells acquire motility from a stationary epithelial cell type. Most studies on generation of neurons in vertebrates have focused on the central nervous system, which is composed of the brain and the spinal cord. The peripheral nervous system contains sensory neurons whose neurogenesis has been much less explored. In the head, ectodermal placodes contribute to a majority of the peripheral neurons, including sensory neurons in the cranial ganglia of the trigeminal V, facial VII, glossopharyngeal IX, and vagal X nerves responsible for somatosensation, general visceral sensation, and gustation . TheyEctodermal placodes together with another embryonic cell population, the neural crest, are responsible for formation of the entire peripheral nervous system in vertebrates ,3. CompaPlacode formation involves induction of placode-specific fates and often acquisition of thickened columnar morphology followed by two key steps: first, the birth of neuronal cells in the ectoderm and the second, their detachment and migration to the site of ganglion assembly. The first step likely involves intricately regulated patterns of mitoses to generate the proper number and position of placodal neurons in the ectoderm. Previous studies show that placodal cells differentiate as neurons in the ectoderm prior to their delamination, as evidenced by co-labeling of fluorescently tagged ingressing placodal cells by GFP or vital dye with pan-neuronal markers Islet-1, neuronal beta-III tubulin (TuJ1), and neurofilament ,9, and oThe second step requires that placodal neurons make a transition from an epithelial to a mesenchymal like state in order to detach from the ectoderm. This process is often referred to as either ingression or delamination. However, placodal cells appear to undergo a different process of delamination than that of neural crest that undergo an epithelial-to-mesenchymal transition (EMT). For example, placode cells do not express typical EMT genes  and To date, in vivo imaging of placodal cell ingression in whole intact embryos remains optically difficult, largely due to the insufficient z-axis resolution in three-dimensional fluorescence microscopy. In particular, poor cellular resolution along the z-axis prevents clear delineation of cells in the surface epithelium from those that have migrated deep inside the head. To circumvent these issues, we have developed an imaging assay using cranial slice culture to monitor placodal cell behavior in real time at a single cell resolution. This novel imaging assay is based on adaptation of a long-term embryo slice culture imaging system previously established for chick spinal cord . SimilarTo image directly placodal cell delamination from the epithelial ectoderm and subsequent cell migration into the underlying mesenchyme, we sought to develop a method to visualize these tissues in a section of the embryonic head using three-dimensional fluorescence microscopy. Using 3-D wide-field deconvolution microscopy on embryonic slices, Wilcock et al. achieved the first time-lapse imaging of chick neurogenesis in the developing spinal cord at single cell resolution for >36 hours of imaging. Cell survival and cell cycle times in such cultures were similar to those in the intact embryo, demonstrating that the slice culture technique maintained the tissue and can be used to capture normal cell behavior. The general scheme of this imaging assay involves four main steps: first, introduction of fluorescent reporter constructs in the chick embryo by in ovo electroporation, second, surgical explant of a slice section from the embryo head, third, embedding slice onto a glass-bottom dish (see Methods) to image sample on an inverted microscope, and fourth, time-lapse imaging.As chick embryos can be easily transfected by in ovo electroporation with a fluorescent reporter construct in a tissue-specific manner , this meElectroporated embryos were screened in ovo for strong fluorescent reporter expression, and selected embryos were subsequently dissected out. To prepare embryos for slice explants, they were arranged and pinned around their extraembryonic tissues on clear sylgard dishes (see Methods). A surgical microknife was used to make two parallel sections across the head as shown in the Figure 2 using the same collagen based culture conditions in neurobasal media as used for spinal cord slices [Cranial slices were maintained for ~ 12 hours at 38°C incubation with 5% CO2/95% air mix , which has precise x-y stage control for automated imaging of multiple samples and z-stage control for collecting z-stacks Figure . For eacx Figure . Using wThis imaging assay was applied to visualize mitosis in the living trigeminal ophthalmic placodal tissue, which gives rise to the first placodes that later form the ophthalmic lobe of the trigeminal ganglion. Imaging was carried out at stages 13-14 when abundant placodal ingression has begun. Target tissue was double-labelled with a membrane localizing GFP reporter to visualize cell morphology and fine processes and a histone H2B-RFP reporter to mark cell nuclei and follow the progression of mitosis. These constructs were introduced into the trigeminal ectoderm by in ovo electroporation, which transfects ectodermal cells that constitute a heterogeneous population of non-placode and placode cells. Placode cells form in an apparently salt-and-pepper manner in the trigeminal ectoderm based on expression of placodal markers in discrete spots, such as Brn-3a , NeurogeUsing this cranial slice imaging assay many dividing cells were apparent in the placodal ectoderm and some were also observed in the mesenchyme. This prevalent detection of mitoses provides confidence in the viability and integrity of the cranial slices imaged. We found that the trigeminal placodal ectoderm contains mitotic cells all along its apical-basal axis without an apparent restriction to either side of the epithelium Figure . This isIn the time course of these time-lapses, we were generally unable to capture delamination following observation of mitosis. This is likely due to the long temporal separation of these two events and also in some cases the fact that the placodal cell nucleus in the ectoderm went out of focus in the z-axis. However, a few instances in which delamination followed soon after mitosis were observed , although the peak period is during stages 14-16 . As it iThe generation of new placodal cells from the ectoderm and incorporation into the ganglionic anlage is a continuous process that occurs over about two days of normal chick development. Imaging short time windows of about 3 to 8 hours using this cranial slice imaging assay was sufficient to capture dynamic changes in placodal cell morphology during ingression, migration, and ganglion assembly as well as to monitor mitoses. Interestingly, the time-lapse imaging data revealed that trigeminal placodal cells begin their ingression early on from within the ectoderm. This extends previous observations made in fixed tissues, which identified spurs or thickenings on the basal epithelial surface and concluded that ingression commenced at these points. We found that ingressing cells do not leave the ectoderm through individual exit points, but instead move into the mesenchyme via exit points used by placodal cells that ingressed before them. The data suggest that there are discrete common openings in the ectoderm from which placodal cells leave. The imaging data provide clear evidence of constant and abundant contacts between placodal processes throughout gangliogenesis. They show that mitoses in the trigeminal placodal ectoderm are not restricted to either apical or basal sides and have different cleavage orientations. The data also suggest that many, but not all, ingressed trigeminal placodal cells are post-mitotic. Taken together, the data shed new light on the early migration of placodal cells from within the ectoderm and their highly interactive processes throughout their development, which were not apparent in previous static analyses.In summary, we describe a chick cranial slice imaging assay which is highly effective for visualizing and analyzing sensory placode development. Combination of this live placodal imaging assay with molecular perturbations involving gain and loss of gene function will allow study of the spatiotemporal dynamics of the mechanisms that drive formation of the placode-derived cranial sensory ganglia, which until now have remained limited to static analyses on fixed whole mount and sectioned embryos. Study of the regulation of how these cells delaminate may also help to determine whether this is similar to the classical epithelial-to-mesenchymal transition and would further address the long-standing question of how cells acquire motility from a stationary epithelial cell type. Detailed investigation into how placodal neurons form would contribute significantly to our current understanding of neurogenesis. As inter-kinetic nuclear migration is lacking, the way in which placodal cells become neurons may reveal a new mechanism distinct from that operating in the CNS.Fertilized chicken  eggs were obtained from Winter Egg Farm  and incubated at 38°C to the desired stages. Reporter constructs were transfected into the trigeminal ectoderm by in ovo electroporation at stages 9-10 (7-11 somites stages). Plasmids were injected overlying the presumptive trigeminal placodal ectoderm at the approximate axial level between the posterior forebrain and anterior hindbrain. To electroporate, platinum electrodes were placed vertically across the chick embryo delivering current pulses of 5 - 8 V in 50 ms at 100 ms intervals as described [2 level for at least one half hour prior to imaging.Slices were prepared as described in Results and Discussion. Transfected embryos that were selected for strong fluorescence expression were collected at the desired developmental stages between 12-18 for imaging. Transverse sections were surgically excised using a microknife with a 15 degrees blade slant  from the midbrain level of the head ectoderm, targeting the trigeminal ophthalmic region in the dorsal half Figure . One to Prior to imaging, generally at least one day in advance of experiment, the cover glass base of the imaging chamber was treated and coated with poly-L-lysine . Poly-L-lysine was incubated on glass for 15 minutes or longer up to one hour at room temperature, and was removed followed by 4-6 quick rinses with double-distilled or sterilized water. Excess leftover polymer can be toxic to the tissue. The cover glass was left to dry at room temperature over several days, in the 38°C incubator overnight, or for a quick dry by low power microwave heating at 2 minutes interval. A completely dried cover glass is necessary for setting collagen on the glass for slice preparation.Cranial slices were imaged on a DeltaVision Core widefield fluorescence system  as described in Results and Discussion. The microscope system uses an Olympus inverted microscope with a xenon lamp as the light excitation source and a high-speed cooled CCD camera (Photometrics CoolSnap HQ2). Slices were maintained at 38°C within a Solent environmental chamber. The DeltaVision acquisition software Resolve3D was used to control image acquisition.We used SoftWoRx (Applied Precision) for analyzing and projecting 3D stacks into 2D images. To measure speed and velocity of placodal cell migration in 3D volume, we performed 4D tracking with Imaris (Bitplane) by modeling moving placodal nuclei with spot objects using the ImarisTrack module with manual corrections and Imaris MeasurementPro for calculations of the tracks. The orientation of cleavage planes with respect to apical surface was measured by the angle made between a line bisecting two sets of chromosomes separating at anaphase . For each z-stack, a bright-field image was taken at the middle slice as control to monitor integrity of tissue in parallel with the fluorescence imaging. Images were taken at 3-minute intervals at 20×, and shown at 6.5 fps. ecto, ectoderm; mes, mesenchyme; nt, neural tube.Click here for fileTime-lapse movie of a dividing cell labeled by H2B-RFP undergoing a parallel cleavage in the basal side of the placodal ectoderm. Metaphase plate rotates prior to division (arrow). 72 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.Click here for fileTime-lapse movie of a dividing cell labeled by H2B-RFP undergoing a perpendicular cleavage in the apical side of the placodal ectoderm. 60 minutes in real time taken at 3-minute intervals; 24 um z-stack; 20×; 6.5 fps.Click here for fileTime-lapse imaging of cell divisions (dotted lines around cells) in the trigeminal placodal ectoderm as labeled by nuclear H2B-RFP and membrane GFP. Dotted white line demarcate basal edge of ectoderm. Yellow arrow points to a placodal cell that divides prior to ingression and a different placodal cell (blue arrow) undergoes division after it is already in the mesenchyme. Video covers 336 minutes of development in real time captured at 3-minute intervals; 45 um z-stack; 20×; 7.5 fps.Click here for fileSame as Movie 4 but showing only the H2B-RFP channel for clearer visualization of only nuclei, 6.5 fps.Click here for fileTime-lapse imaging of the cranial slice in Figure 3C showing only nuclear H2B-RFP labeled ectoderm and ectoderm-derived cells to follow movements of placodal cell bodies within the ectoderm and during delamination. Two examples of placodal cells that move from within the ectoderm are each marked by a different colored dot (red and green) starting at a time point when the cell can initially be tracked. Tracking of the green cell begins after it has already begun migration. Some cells move in different directions before appearing to follow a path to the site of exit on the basal side of the epithelium (see red cell). 210 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 10 fps.Click here for fileTime-lapse imaging of actively ingressing cells labeled by membrane GFP and nuclear H2B-RFP from the placodal ectoderm. Movie shows highly interacting placodal cells as they stream out of a discrete exit point on the basal side of the epithelium, forming contacts among themselves and with already ingressed ganglionic placodal neurons. Imaging shows 336 minutes of development captured at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.Click here for fileSame as Movie 6 but showing only the membrane GFP channel for clearer visualization of the cell morphology.Click here for fileTime-lapse imaging of placodal neurons labeled by membrane GFP and nuclear H2B-RFP in the ganglionic anlage show intimate contacts by placodal processes. Movie shows highly dynamic interactions at sites of axon-axon contacts which eventually make a connection at time of growth cone collapse. 336 minutes in real time taken at 3-minute intervals; 45 um z-stack; 20×; 6.5 fps.Click here for file


The cause of tako tsubo cardiomyopathy remains unclear. We used a multidisciplinary approach to investigate if a common pathophysiological denominator could be outlined.Within 3 days following symptom presentation and again after 3 months we investigated all patients coming to our institution and diagnosed with tako-tsubo cardiomyopathy. Patients underwent extensive biochemical screening. Left ventricular function was evaluated by echocardiography and contrast-enhanced cardiac magnetic resonance imaging. Cardiac autonomic function was studied by heart rate variability and signal-averaged electrocardiogram and posttraumatic stress and depression were investigated by questionnaires .During 2 years, 13 consecutive patients were included. Markers of myocardial damage and heart failure were slightly to moderately elevated and ejection fraction (echocardiography and MRi) was moderately reduced at hospitalization and improved to normal values in all patients. Signal averaged ECG demonstrated a statistically significant shorter duration of the filtered QRS complex in the acute phase as compared to follow-up. In heart rate variability analysis, SDNN and SDANN were shorter acutely compared to follow-up. Two patients fulfilled criteria for posttraumatic stress syndrome while 7 patients were in the borderline zone. There was a statistically significant inverse correlation between PTSS-10 score and QRS duration in the signal-averaged ECG .Patients with tako tsubo cardiomyopathy have altered cardiac autonomic function and a high incidence rate of borderline or definite posttraumatic stress syndrome acutely. This is in line with findings in patients with myocardial infarction and does not allow conclusions on cause and effect. Tako-tsubo cardiomyopathy is a rapidly resolving condition of unknown etiology. The typical patient is female, postmenopausal and present with symptoms, electrocardiographic (ECG) and clinical findings suggestive of ST-elevation myocardial infarction. However, coronary arteriography is normal or findings are unrelated to the often severe transient heart failure involving hypokinesia of the left ventricular mid segments and apical ballooning .2+-handling proteins [Tako tsubo cardiomyopathy has been associated with acute emotional stress  but thisproteins .The purpose of this study was to offer a comprehensive multidisciplinary approach to all patients presenting with tako tsubo cardiomyopathy in our institution during a 2-year time period in order to better understand and treat the disease. We hypothesized a relation between scores of emotional stress and depression on one side and cardiological, physiological and biochemical measures of disease severity, on the other. In order to address our hypothesis we used an array of conventional cardiac examinations, scores of posttraumatic stress and heart rate variability and signal-averaged ECGs to assess ventricular late potentials at baseline and after 3 months.All patients suspected of having ST-elevation myocardial infarction and referred to our institution for acute for coronary arteriography between April 2008 and March 2010 were screened for the study. Criteria for inclusion were in line with the recently published Mayo clinic criteria for tako tsubo cardiomyopathy : hypokinIn the acute phase and at the 3-months follow-up HRV was recorded using a battery operated solid-state recorder  for 24-hour continuous recording of ambulatory ECG data and analyzed using an Aspect Holter system . The following time-domain variables were calculated: average R-R interval value calculated from accepted beats (mean RR), standard deviation of all the NN  R-R intervals (SDNN), and the baseline width of the minimum square difference triangular interpolation of the highest peak of the histogram of NN intervals (TINN). The long-term HRV was quantified by the standard deviation of all 5-min NN interval mean values (SDANN). NN50 count, that is the number of interval differences of successive normal-to-normal intervals greater than 50 ms as well as SDSD, that is the mean of standard deviations of normal RR intervals for all 5-minute segments, were also calculated. Finally, the beat-to-beat HRV was estimated by the square root of the mean squared differences of successive NN intervals (RMSSD), and the ratio of NN interval differences of successive NN intervals greater than 50 ms to the total number of NN intervals (pNN50) .Power spectral analysis was performed on the RR-interval data by means of fast Fourier transformation. The following components were measured: very low frequency (VLF), low frequency (LF), high frequency (HF) and the total power.The SAECGs were obtained using a Megacart ECG recorder . In total, 300 beats were analysed using a noise reduction of 0.3 μV and a correlation of 0.98. The bipolar X, Y and Z lead system was used. The duration of the filtered QRS-complex (fQRS), the root mean square voltage, that is the RMS-amplitude during the last 40 ms of the filtered QRS-complex (RMS40) and the low amplitude signal, that is the time during which the amplitude of the filtered QRS-complex last part remains below 40 μV (LAS40) [A Vivid 7 ultrasound machine  equipped with a multi-frequency phased array transducer  was used for the echocardiographic examinations and measurements were made after the examination using stored images on an EchoPac workstation (GE Vingmed). The subjects were examined in the left lateral recumbent position. Left ventricular ejection fraction (LVEF) was measured using the biplane Simpson's methodMRi was done with a Philips Achieva 1.5-T scanner. Standard bTFE  cine-images were acquired as single 8 mm slice in long-axis, 3-chamber and 4-chamber view. Full ventricular coverage was achieved with 15-20 short-axis slices of 5 mm thickness. A delayed enhancement protocol was used 15 minutes after intravenous injection of Gadiodiamid (0.15 mmol/kg) with breath-hold inversion-recovery TFE using 10 slices of 10 mm thickness in long-axis, 3-chamber and 4-chamber view. About 15 slices were acquired in short-axis to get full ventricular coverage.Patients answered questionnaires on posttraumatic stress  and depression . The PTSS-10 is a self-administered questionnaire that measures the current presence of specific posttraumatic stress symptoms. Patients are asked to rate the presence of each symptom during the past 7 days and its severity on a scale from 1 (never) to 7 . A total score > 35 is associated with a high probability that the diagnostic criteria for post-traumatic stress disorder are fulfilled and a score between 27 and 35 is borderline . The MADAll blood sampling was done in the morning after admission to the hospital, echocardiography within 24 hours and all other testing within 72 hours after admission. Most tests were repeated at a 3 months clinical control. The study protocol was approved by the Regional Ethical Committee in Uppsala, Sweden.Statistical analysis was performed using SigmaStat 3.5 software . Most variables were not normally distributed and we therefore used the Wilcoxon signed rank test for pairwise comparisons and Spearman's rank correlation coefficient for non-parametric assessment of statistical dependence between two variables. Differences were considered statistically significant when P < 0.05.In the 2-year study period, between April 2008 and March 2010, 15 patients, all women, fulfilled criteria for inclusion. Two patients declined participation and 13 patients were included in the study  and the standard deviation of sequential 5-min heart period mean values (SDANN) which were both shorter acutely as compared to three months follow-up table . No otheOnly one patient had a history of mental illness. This patient had been diagnosed with manio-depressive psychosis years before being admitted with tako tsubo. Her manio-depressive disease was considered stable at admittance and she scored 31 and 33 in the PTSS-10 scale (borderline) while the score was 13 on MADRS-S scale (no depression). At admission, 2 patients fulfilled criteria for posttraumatic stress syndrome while 7 patients were in the borderline zone. At 3 months follow-up one patient still fulfilled criteria for posttraumatic stress syndrome while 4 had borderline scores Figure .The main findings of this study on consecutive patients with tako tsubo cardiomyopathy were that in addition to clinical and laboratory indices of heart failure, cardiac autonomic function was altered and we found a relation between emotional stress and QRS duration.Although the signal-averaged ECG values were within the limits of previously published normal values  we did fHRV measured shortly after coming to the hospital with acute symptoms and again after recovery at 3 months demonstrated lower values of the time domain parameters SDNN and SDANN acutely. A German group recently made similar findings on HRV parameters  and the Posttraumatic stress is not uncommon in patients following myocardial infarction and was reported in 22% of patients in one study . Thus, wBiochemical markers of myocardial damage were slightly to moderately elevated in the present study and this corresponds with previous findings in patients with tako tsubo cardiomyopathy ,27. In cAlthough the bulk of literature on tako tsubo cardiomyopathy is confined to case reports this study is limited by its size and by the fact that data come from a single center. Only a subset of patients was examined by MRi and had plasma catecholamine investigations but complete data on both parameters are unlikely to have changed our overall conclusion. Nevertheless, to our knowledge, this study is one of the most comprehensive and detailed investigations of patients with tako tsubo cardiomyopathy and the novel combined assessment of scores of emotional stress and HRV parameters seems clinically relevant and might inspire future studies of this disease.We conclude that patients with tako tsubo cardiomyopathy, in addition to clinical and laboratory indices of heart failure, have altered cardiac autonomic function and a high prevalence of borderline or definite posttraumatic stress syndrome. This is in line with findings in patients with myocardial infarction and does not allow conclusions on cause and effect in tako tsubo cardiomyopathy. In our opinion terms such as "stress-induced cardiomyopathy" or "broken heart syndrome" for this condition should be avoided.The authors declare that they have no competing interests.MW performed echocardiography, data management and analysis. MS participated in the design of the study and patient inclusion. AK designed the MRI investigations and did the MRI analyses. KE participated in study design, echocardiography and helped to draft the manuscript. OF designed the study, performed coronary angiographies, statistical analyses and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/11/14/prepub


Understanding the molecular mechanisms that underlie plant responses to drought stress is challenging due to the complex interplay of numerous different genes. Here, we used network-based gene clustering to uncover the relationships between drought-responsive genes from large microarray datasets. We identified 2,607 rice genes that showed significant changes in gene expression under drought stress; 1,392 genes were highly intercorrelated to form 15 gene modules. These drought-responsive gene modules are biologically plausible, with enrichments for genes in common functional categories, stress response changes, tissue-specific expression and transcription factor binding sites. We observed that a gene module (referred to as module 4) consisting of 134 genes was significantly associated with drought response in both drought-tolerant and drought-sensitive rice varieties. This module is enriched for genes involved in controlling the response of the plant to water and embryonic development, including a heat shock transcription factor as the key regulator in the expression of ABRE-containing genes. These results suggest that module 4 is highly conserved in the ABA-mediated drought response pathway in different rice varieties. Moreover, our study showed that many hub genes clustered in rice chromosomes had significant associations with QTLs for drought stress tolerance. The relationship between hub gene clusters and drought tolerance QTLs may provide a key to understand the genetic basis of drought tolerance in rice. Drought is a major environmental stress factor that affects the growth and development of plants. Plant drought stress response is one of the most complex biological processes, and it involves numerous changes at the physiological, cellular, and molecular levels. The increasing molecular information has shown complex gene networks that consist of numerous different genes are involved in plant responses to drought stress The development of high-throughput data-collection techniques, such as microarrays and deep sequencing, enables gene function to be uncovered on a global scale. Microarray analyses can simultaneously measure the expression levels of a large number of genes, but may not provide much information on gene-gene interrelationships. Due to rapid advances in network biology, network-based analysis of large-scale datasets has offered a novel view of biology In recent years, global co-expression networks have been constructed for rice Oryza sativa), one of the most important cereal crops, has been used as a model plant for the grass family. A better understanding of complex interactions among drought-stress related genes is of great importance to improve the stress resistance of rice and other cereals. In this study, we used network-based gene clustering to identify drought-responsive modules to provide a better understanding of the underlying molecular mechanisms of drought response in rice. This method can help in uncovering gene interrelationships for studies into the complex response to drought stress in rice, as well as provide a reference for other crop plants.Rice  were calculated for each pair of the 2,607 drought-responsive genes. To determine a biologically relevant correlation coefficient for gene co-expression, we examined the changes in the actual number of edges in the co-expression network and all possible edges in the control network consisting of these non-singleton nodes as a function of r cutoff values. As the cutoff increased, both the actual number of edges and all possible edges decreased; however, as the cutoff reached a relatively high value, the decreasing rate of the actual number of edges became slower than all possible edges  algorithm To determine whether these gene modules in our drought-responsive gene network were robust, another gene correlation network was generated using the WGCNA package One of the aims of network analysis is to identify sets of functionally related genes based on the high gene connectivity in expression. To determine the drought-responsive modules comprised of functionally similar genes, we carried out gene ontology (GO) enrichment analyses for all modules −11). There are a total of 119 genes whose functions are associated with responses to environmental stimuli, which represent approximately half of the annotated genes in the module. Most genes in the module are downregulated by drought stress, while only 16 genes are induced by dehydration, which implies that the module may serve as a negative regulator in rice response to drought stress.Module 1 consists of 303 gene nodes and is the largest module in the drought-responsive gene co-expression network. It is not surprising that the module is enriched with stimulus response genes  and participate in photosynthesis .Another important negative player is module 2, which contains 213 drought-responsive genes; 189 genes are downregulated by drought stress. Functional enrichment shows that the module genes are enriched with products that target the thylakoid . The module contains five genes involved in this process, which accounted for 41.6% of the annotated water responsive genes in the background datasets. Functional enrichment revealed that the module was also enriched for genes involved in embryonic development , including six annotated late embryogenesis abundant LEA genes. It is of interest that almost all genes within the module are significantly induced by drought stress.Module 4 is enriched for genes known to control the response of plants to water , representing approximately 38% of all of the 37 annotated genes in the module. These data suggest that modules 4 and 8 may serve as key positive players in rice response to drought stress.Module 8 is another module that is upregulated by drought stress, which is principally involved in the response to abiotic stimuli. The gene module contains 14 abiotic stimulus responsive genes . There are 39 annotated genes that encode protein kinases, which represent 29% of the annotated genes in the two modules. This implies that these two gene modules may play important roles in the signal transduction networks that are activated in the drought response in rice.Modules 6 and 10 are other two important gene groups that are preferentially involved in the processes of protein amino acid phosphorylation , which are shown in cis-regulatory motifs in their promoters. To gain an overall view on motif organization of drought response in rice, the putative binding site motifs enriched in drought-responsive gene modules were investigated by MEME tool cis-motifs were found in 15 drought-responsive modules with MEME E-value less than 1. The Pearson correlation coefficient between each motif pair was calculated based on the corresponding position weight matrices and similar motifs were clustered using a correlation coefficient cutoff of 0.65. After merging motifs within each cluster, 10 nonredundant motifs were identified to be involved in the response to drought stress  and GCC-box, were known to be involved in drought stress response GCCACGTGKC sequence was found to be enriched in six modules, including 4, 8, and 14, which were significantly induced by drought stress. Among those revealed elements, most motifs were described to be involved in known functions, while only one motif with the AGCTAGCTAG sequence had an unknown function. We found many of discovered motifs were enriched in more than one drought-responsive gene module, especially the three global motifs that included the REGION1OSOSEM (CSGCGGCGGC), the SBOXATRBCS (CCCCCTCC) and the GAGA8HVBKN3 (GGGAGGGAG) element which occurred in nine or more modules. Interestingly, the RY/Sph box with the conserved CATGCATGCA sequence and the AGCTAGCTAG element were identified to be uniquely enriched in module 1. Although the role of the two motifs in drought stress response remains unclear, the root-specific expression of associated genes suggests that both of them are likely to be involved in transcriptional repression in rice root in response to drought stress.Genes in the drought-responsive modules are likely to possess the same t stress . A compa2-fold expression changes of module genes to measure module changes in the response to drought stress. The significances of module changes greater than the network average were assessed using t-tests in the drought-tolerant  and drought-sensitive (IR64) rice cultivars based on the independent datasets. The data showed there were one to five modules whose expression changes were significantly greater than the network average in each rice variety  and abscisic acid (ABA); furthermore, their expression patterns exhibited obvious coherence in IRAT109 seedlings under PEG and ABA treatments  induced by drought stress had the highest intramodular connectivity value in the drought-responsive TFs. The was 0.90 . Many HSeatments . These rThe highly connected hub genes within the networks are thought to play important roles in organizing the functional modules. Here, we identified hub genes as the top 20% highly connected nodes in each module, and 262 hub genes were found in fifteen drought-responsive modules. Functional enrichment revealed that these drought-responsive hub genes were preferentially involved in the response to stimulus , which wAlthough some data have shown that co-regulated stress response genes tend to cluster in rice chromosomes http://qtaro.abr.affrc.go.jp/) p<0.005) (Compared to the mapping of quantitative trait loci (QTLs) for drought tolerance on rice chromosomes  . For insMost of the existing studies used the Pearson-based correlation method to construct co-expression networks, but value-based methods are significantly limited by a homogeneous correlation threshold for all genes in the network OsbZip23, OsbZip72 and AP37 which have been validated as important factors affecting drought tolerance traits for rice The major objective for this study was to use the network-based approach to identify drought-responsive gene modules, providing new insights into the organization of functional modules in the response to drought stress. From publically available microarray data, we identified 2,607 rice genes that showed significant changes in gene expression under drought stress and more than half of the drought-responsive genes were highly intercorrelated to form 15 functional modules. These correlated transcriptional modules are biologically plausible, with enrichments for genes in common functional categories, stress response changes, tissue-specific expression and TF binding sites. Functional annotations show that some modules are enriched for genes involved in the response to stress, including Among these identified drought-responsive modules, module 4 consisting of 134 genes was significantly associated with drought response in both drought-tolerant and drought-sensitive rice varieties. The involvement of many of these genes in drought response was not previously known, but it was understood that they were involved in controlling the response of the plant to water and embryonic development. Motif enrichment analysis revealed approximately 70% genes in the module contained the ABRE element in their promoter regions, and most of them are induced by drought stress Gene organization in rice chromosomes is non-random with respect to the complex traits of drought tolerance. The distribution of drought-responsive hub genes that was detected varied both among the rice chromosomes and within regions of each chromosome. Many hub genes in the drought-responsive modules were clustered in rice chromosomes. These hub gene clusters overlapped with QTLs for drought tolerance, which suggests that hub gene clusters are likely to be major components of drought tolerance QTLs. This evidence should facilitate the identification and cloning of drought-related genes at target QTLs. The interesting association between hub gene clusters and drought tolerance QTLs might be the key to understanding the complex genetic basis of drought tolerance in rice.The dataset of 852 Affymetrix rice arrays was downloaded from the NCBI Gene Expression Omnibus  and the EBI ArrayExpress Archive. Thirty-seven slides were removed from the dataset due to genomic DNA hybridization. A total of 815 slides remained and these slides were normalized using the RMAExpress software for network construction The Affymetrix rice genome array was designed based on the early annotation of rice genome, and not all probe sets had one-to-one mapping to rice genes. Some probe sets matched more than one gene, and some were redundant with multiple probe sets mapping to a single gene. To avoid such ambiguous information, we mapped the probe sets to gene models by searching the newly released genome data (version 6.1) of the MSU Rice Genome Annotation Project p<0.05 and at least two-fold changes (either up- or downregulation). To reduce the noise within the gene co-expression network, the data subsets were restricted to genes that were differentially expressed in at least two out of the five experimental sets.Identification of the drought-responsive genes was based on the normalized expression profiling data of rice seedlings (NCBI GEO: GSE6901), and the Azucena and Bala rice varieties (NCBI GEO: GSE24048), as well as N22 and IR64 (EBI ArrayExpress: E-MEXP-2401) under drought stress conditions. Average signal intensities of biological replicates for each sample were used to calculate the fold change of gene expression. The differentially expressed genes were identified using significance analysis by t-tests with The correlations between genes were determined by the Pearson correlation coefficient, and it was calculated using Perl script based on the following formula:The density of a gene co-expression network D was defined as a ratio of the actual number of links to all possible links of the non-singleton nodes. It uses the following formula:r cutoff, we apply the approach as described by Aoki et al. to examine the changes in the gene correlation network density as a function of r cutoff values To determine a biologically relevant The value-based co-expression network was created for the drought-responsive genes. An edge in the network represented two genes with a correlation coefficient greater than 0.71. We then limited the maximum link number for each node to reduce the connections between genes in different modules. The edges connecting two genes in each other's top correlation were kept and other links were removed in the network. The resulting co-expression network was drawn using Cytoscape Gene co-expression networks with thousands of nodes are difficult to visualize and analyze. A useful strategy for analyzing such a network is to partition it into sub-networks as modules that share regulatory mechanisms and functional relationships. Many graph-clustering algorithms have been developed for partitioning graphs into node clusters based on structure topology p-value was less than 0.01 in comparing with the dataset of all MSU rice loci.The drought-responsive modules were tested for GO annotation enrichment using the agriGO program cis-motifs of between six and ten nucleotides The sequences of the promoters (1 kb upstream sequences from transcription start site) were extracted from the MSU rice genome database. The upstream sequence groups of co-expressed module genes were analyzed by the MEME program to find For transcript level analysis of the inferred co-expression genes under stress, IRAT109 plants were planted in plastic trays walled with sandy soil and placed in the greenhouse. Four-leaf-old seedlings were prepared for PEG and ABA treatments. Seedling roots were submerged in 20% PEG solution for drought stress, and seedling leaves were sampled at 0, 3, 6, 12 and 24 h after the treatment, as well as rewatering after 24 h. For ABA treatment, seedling leaves were sprayed with 0.1 mM ABA solution and sampled at 0, 30 min, 3, 6, 12 and 24 h after treatment as well as rewatering after 24 h.Actin1 gene  was used as the internal control with primers.Total RNAs of the collected samples were extracted using TRIzol reagent (Invitrogen). Then, 2 µl RNA sample was reverse-transcribed to first-strand cDNA with AMV Reverse Transcription System  using oligo(dT) primers. Real-time RT-PCR using SYBR Green I technology on ABI PRISM 7000 Sequence Detection System  was performed. A PCR master mixture of 20 µl contained 1×SYBR® Green PCR Master Mix, 1 µl cDNA, and 0.5 µl 10 µM sense and anti-sense primers see . The expp-value was thus generated equal to n/1000.Permutation tests were used to determine whether the overlap between hub gene clusters and drought tolerance QTLs was higher than expected by chance. For the test, we randomly shuffled gene connectivity and chromosome position, and then counted the number (n) of times that the number of random hub gene clusters  overlapped with the drought tolerance QTLs was higher than the actual overlaps. We repeated this procedure 1,000 times, and the Table S1Microarray samples used in the network construction.(XLS)Click here for additional data file.Table S2Gene modules detected by MCL in the drought responsive network.(XLS)Click here for additional data file.Table S3Functional enrichment of drought-responsive module genes.(XLS)Click here for additional data file.Table S4Real-time PCR primers specific for HSF and its neighborhood genes.(XLS)Click here for additional data file.Dataset S1Cytoscape session file of the drought-responsive network with the mapping modules.(RAR)Click here for additional data file.


Despite evidence implicating dietary sodium in the pathogenesis of cardiovascular disease (CVD) in chronic kidney disease (CKD), quality intervention trials in CKD patients are lacking. This study aims to investigate the effect of reducing sodium intake on blood pressure, risk factors for progression of CKD and other cardiovascular risk factors in CKD.The LowSALT CKD study is a six week randomized-crossover trial assessing the effect of a moderate (180 mmol/day) compared with a low (60 mmol/day) sodium intake on cardiovascular risk factors and risk factors for kidney function decline in mild-moderate CKD (stage III-IV). The primary outcome of interest is 24-hour ambulatory blood pressure, with secondary outcomes including arterial stiffness (pulse wave velocity), proteinuria and fluid status. The randomized crossover trial (Phase 1) is supported by an ancillary trial (Phase 2) of longitudinal-observational design to assess the longer term effectiveness of sodium restriction. Phase 2 will continue measurement of outcomes as per Phase 1, with the addition of patient-centered outcomes, such as dietary adherence to sodium restriction (degree of adherence and barriers/enablers), quality of life and taste assessment.The LowSALT CKD study is an investigator-initiated study specifically designed to assess the proof-of-concept and efficacy of sodium restriction in patients with established CKD. Phase 2 will assess the longer term effectiveness of sodium restriction in the same participants, enhancing the translation of Phase 1 results into practice. This trial will provide much-needed insight into sodium restriction as a treatment option to reduce risk of CVD and CKD progression in CKD patients.Universal Trial Number: U1111-1125-2149. Australian New Zealand Clinical Trials Registry Number: ACTRN12611001097932 Cardiovascular disease (CVD) is the leading cause of mortality in the chronic kidney disease (CKD) population, with CKD patients being 5–10 times more likely to die of CVD than to progress to end-stage kidney disease (ESKD) . DietarySeveral meta-analyses of dietary sodium intake and blood pressure (BP) have been published, consistently demonstrating that a reduction in dietary sodium intake leads to reduced BP, particularly in hypertensive populations -14. UnfoAn issue with translation of research in sodium restriction into practice is poor dietary adherence. This has been reported in several research trials -21. FurtDietary sodium shows great promise as a modifiable risk factor for reduction of cardiovascular risk and risk of renal function decline in CKD, but high quality evidence is lacking. There is a need for a research trial showing the efficacy of sodium restriction for reducing the risks of CVD and CKD progression in CKD. Further, the effectiveness of recommendations to restrict sodium should be assessed to ensure the results are translatable to practice.The aim of the LowSALT CKD study is to examine the impact of reducing dietary sodium intake in decreasing cardiovascular risk in CKD patients. Our primary hypothesis is that a low sodium intake (LSI) (60 mmol/day) in patients with stage III or IV CKD will be independently associated with decreased ambulatory BP compared to a moderate sodium intake (MSI) (180 mmol/day). Our secondary hypothesis is that LSI (60 mmol/day) will result in the following outcomes compared with MSI (180 mmol/day): decreased arterial stiffness; decreased proteinuria and albuminuria; decreased extracellular fluid and markers of volume overload and thirst.The LowSALT CKD study is a single-centre double-blind randomized cross-over trial (Phase 1) with a prospective observational follow-up Phase 2). Phase 1 entails a 7-day run-in period followed by two, 2-week interventions  non-dialyzed, non-transplanted patients under the care of a nephrologist at the Princess Alexandra Hospital; aged ≥ 18 years with systolic BP 130–169 mmHg and diastolic BP ≥ 70 mmHg; non salt-wasting CKD (as diagnosed by nephrologist); prescribed ≤ 20 mmol sodium per day via medications; not pregnant or breastfeeding; life expectancy > 6 months; no current involvement in other intervention study and mental capacity to adhere to the study protocol .The target population for the trial will be stage III and IV non-dialyzed CKD patients. Eligibility criteria will include hypertensive, CKD stage III or IV . To confirm that a potential participant’s blood pressure is within range, BP will be measured once in seated position when the participant attends clinic and previous BPs will be collected from the medical notes. Once a potential trial participant has been screened as meeting the eligibility criteria, recruitment by informed consent will be sought either at the time of the clinic visit or at a later date . Once consented, patients will undergo 24-hour ambulatory blood pressure (ABP) measurement at baseline to confirm eligibility. Patients with 24-hour BP ≤ 130/70 will be discontinued from the study due to the potential risk of hypotension with sodium reduction. Patients and the treating doctors will be asked to keep the anti-hypertensive medications unchanged for the duration of the trial within bounds of patient safety.All patients will be counseled to follow a low sodium diet for the entire trial duration. Initial dietary education will occur face-to-face during run-in, with ongoing education during follow up visits and phone calls Table . Topics An external statistical consultant will undertake the computer-generated randomization for the order of the intervention in blocks of 8, blinded to the investigators. To ensure all investigators and participants are blinded to the treatment, study medications are packaged off-site by an external pharmacy with a generic label as medication 1 and 2 for the first and second intervention.As per Figure 24-hour ABP was selected as the primary outcome as it is the most accurate reflection of BP, with measurements over an entire 24-hour period indicating diurnal variation, while avoiding error due to white-coat or masked hypertension -35. DataABP will be measured using the TM-2430 Ambulatory Blood Pressure Monitor . This device is a non-invasive lightweight portable device that is validated in accordance with British Hypertension Society protocol, with A/A rating . ABP meaTM CPV  which employs applanation tonometry to measure the shape and velocity of the pulse wave, with good repeatability and reproducibility [Arterial stiffness will be assessed by carotid-femoral Pulse Wave Velocity (PWV), which is the current gold standard for non-invasive measurement of aortic arterial stiffness , and by cibility .To ensure accuracy and reproducibility, measurements will be taken in the morning after fasting blood samples. Participants will be provided with a standardized breakfast  and will be asked to abstain from tobacco and caffeine at least 4 hours before the measurement. PWV will be assessed by measuring carotid and femoral artery wave-forms sequentially over the common carotid and femoral arteries. Radial PWA will be used to estimate central pulse pressure and augmentation index. Both PWV and PWA measurements will be measured in duplicate. Validity of the measurements will be assessed as per the SphygmoCor manual , with an24-hour urine samples   will be Fluid status, a predictor of cardiac alterations such as left ventricular hypertrophy , will beThirst will be measured by the Xerostomia Index (XI) -49. The Serum blood samples will be stored for analysis of markers of inflammation (CRP), adiponectin , markers of renin-angiotensin-aldosterone system stimulation  and a marker of fluid overload (NT-proBNP) in addition to the standard tests taken in practice (e.g. full blood count and urea and electrolytes). Blood samples will be collected after overnight fast and drawn from a peripheral vein while participants are seated. Blood will be centrifuged within 30 minutes after blood collection. After separation, plasma will be transferred using a graduated plastic disposable pipette into color coded storage vials and placed in freezer storage boxes in a −80°C freezer. Additional serum will be stored for the analysis of novel risk factors in the future. Participants and investigators will be blinded to the serum outcomes.A multi-method approach to measuring dietary sodium intake was chosen to employ both objective and self-reported methods, optimizing accuracy of dietary intake estimation, while minimizing participant burden . The curIn addition to objective measures, a diet history and food frequency questionnaire (FFQ) will be used to measure dietary intake. In the diet history method, information about what is consumed over an extended period of time is collected using open-ended questions . This isThe final method for measuring intake will be a daily checklist, used throughout the entire 6 weeks of Phase 1. This is designed to capture intake of foods likely to contribute to a high sodium intake  Study, Isbel N et al.), as well as daily record of study medication. The information on this checklist will be verified for completeness and accuracy. Adherence to the study medication will also be measured by pill count at trial end. Medication changes will be recorded, with all efforts made to avoid change of anti-hypertensive medications during the six week trial.Phase 2 of the trial  aims to assess long-term changes in outcomes and adherence to a sodium-restricted diet, as well as establishing common barriers and enablers to achieving dietary sodium restriction in this group. In this phase, barriers and enablers to adherence will be measured via four self-administered questionnaires. Self-efficacy will be measured using the health-related general self-efficacy scale  and healAnother outcome specific to Phase 2 is taste assessment of the five basic taste senses: salty, sweet, bitter, sour and umami. Evidence suggests that hedonic liking and taste sensitivity of salty taste can be modified with adherence to a low sodium diet , howeverCKD patients have been found to have significantly decreased quality of life (QoL) with QoL decreasing as GFR declines . HyperteOverall, Phase 2 aims to translate the promising indications from basic science into clinical practice demonstrating the effect of dietary sodium on blood pressure, risk factors for CKD progression and cardiovascular disease.The primary outcome will be change in 24-hour SBP from matched pairs of study subjects (by cross-over design) from MSI to LSI. Prior data indicate that the mean response of matched pairs is a 5 mm Hg change in BP with a standard deviation of 7 mm Hg . If theAnalyses will be undertaken by analysis of covariance (ANCOVA) adjusting for baseline values using STATA (Version 12 ). To tesThis trial will provide evidence on the effects of reducing dietary sodium on cardiovascular risk factors and risk factors for kidney function decline in participants with mild to moderate CKD. We anticipate this intervention, a low sodium intake (60 mmol/day) , will result in significant reductions in 24-hour BP, arterial stiffness, excess fluid and inflammation, all of which have been shown to be predictors of cardiovascular abnormalities and/or morbidity and mortality in CKD patients ,66,67. TA number of potential confounding factors will be considered in this trial. Potassium intake reduces BP; therefore changes in potassium intake between the high and low sodium periods may confound the trial results . As suchThere are several attributes to the study design that we believe will ensure robustness of the results. By employing the use of sodium chloride tablets versus placebo while blinding both the participants and the investigators to the treatment we will be avoiding a confounding effect of changes in other dietary nutrients, while also reducing risk of bias. The use of gold standard measurements such as 24-hour BP further strengthens the study. Conducting this trial in stage III-IV CKD allows us to investigate a strategy that may potentially delay or prevent adverse outcomes (such as CVD or ESKD) early in the disease. Furthermore, we believe the provision of dietary counseling by an accredited practicing dietitian with an individualized approach will assist dietary adherence. In addition, the multi-method approach to measuring dietary intake will ensure that dietary adherence will be measured as accurately as possible. Finally, the long-term follow up will enable us to assess long-term effects of sodium restriction, whilst also observing barriers and enablers to dietary adherence that may be targeted to aid sodium restriction.The outcomes of this research will provide valuable proof-of-concept data for a future study of dietary management of CVD in CKD, with the hypothesis that therapeutic control of sodium intake will reduce CV disease progression and events in CKD patients.The authors declare that they have no competing interests.EM participated in the trial design, developed the statistical plan and drafted the manuscript. KC conceived of the trial, and participated in its design, developed the statistical plan and helped to draft the manuscript. JB participated in the trial design, coordination and statistical plan and helped to draft the manuscript. CH, NI, MS, DJ participated in trial design. RH participated in trial design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/13/137/prepub


Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density.Our analyses show that (i) in addition to myogenic progenitors, myofibers also harbor non-myogenic progenitors of a distinct, yet close, lineage; (ii) myofiber-associated non-myogenic and myogenic cells share the same muscle-bound primordial stem cells of a lineage distinct from bone marrow MSCs; (iii) these muscle-bound primordial stem-cells first part to individual muscles and then differentiate into myogenic and non-myogenic stem cells. Skeletal muscles and axial skeleton share the same embryonic origin, the mesoderm In the adult, MSCs were first identified as a stromal population (distinct from hematopoietic stem cells) in the bone marrow and were then identified in virtually all adult organs. MSCs were shown to be capable of forming bone, cartilage, adipose, and to a much lesser extent muscle In the adult, satellite cells contribute myogenic progeny that account for postnatal growth, maintenance and regeneration of skeletal muscles To date the lineal relations between myofiber-associated myogenic (MA-M) and non-myogenic (MA-NM) progenitors is unknown, and addressing this question is the main aim of this study. To achieve this, we opted to determine the lineage relationships between myogenic and non-myogenic progenitors from several muscles of different embryonic origins  and the Masseter . These clones were compared to bone marrow derived MSCs.We applied a method of cell lineage tree reconstruction developed in our laboratory Myofibers and bone marrow were extracted and cells were seeded at clonal density. The DNA from these clones was amplified and served the basis for reconstruction of the lineage trees based on the fact that the genome of a clone reflects, on average, the genome of its founder It is noteworthy that cell lineage reconstruction can be applied to cells or clones from the same organism only. Cells taken from different organisms are generally incommensurable and would give rise to distinct cell lineage trees, the root of each would be the presumed zygote of that organism. Nevertheless, cell lineage trees of different organisms may have common features, e.g. similar topology and similar depth (number of cell divisions since the zygote) for cells of the same type or with the same biological markers. In addition, cell lineage trees may share common trends affected by age or disease progression, e.g. increase in the depth of certain cell types. Cell lineage analysis can and should be repeated to different organisms in order to explore such common features or common trends.Several scenarios can be hypothesized regarding the lineal relations between MA-M, MA-NM and bone marrow MSCs, each resulting in a different type of cell lineage tree, as depicted in There are three independent stem cell lineages: mesenchymal, MA-M and MA-NM .MA-NM progenitors belong to and are indistinguishable from the MSC lineage  and are Satellite cells have a bi-potent potential, retaining mesenchymal plasticity, and may stochastically become myogenic or non-myogenic .Primordial stem cells first differentiate into myogenic and non-myogenic stem cells, which migrate independently to individual muscles .Primordial muscle-bound stem cells first migrate to individual muscles and then differentiate into myogenic and non-myogenic stem cells within each muscle .Our data show that (i) The lineage of myofiber-derived non-myogenic clones is significantly closer to that of myogenic clones than to the lineage of bone marrow MSCs; (ii) Myofiber-associated myogenic and non-myogenic progenitors have common ancestors, which we term primordial stem cells, of a lineage different from bone marrow MSCs; (iii) muscle-bound primordial stem cells first part into the different muscles and then differentiate into myogenic and non-myogenic progenitors.Two mlh1−/− mice, M1 (330 days-old) and M2 (44 days-old) served for reconstructing the lineage trees. From these mice we extracted MSCs from the bone marrow, myofiber associated cells were extracted from individual myofibers from the left and right Gast and right Masseter aiming to elucidate the lineage relationships between cells that are tightly associated with an individual myofiber. Each individual myofiber contains, in addition to a few hundreds of post-mitotic nuclei, a few tens of satellite cells (depending on muscle type and age) and possibly some adherent cells adding to a total of much less than 100 cells per myofiber. Here we were interested in deciphering the lineal relationships between myogenic and non-myogenic ancestor cells that were derived from the same myofiber. Using a FACS method for isolating quiescent satellite cells was not applicable in this study due to the small amount of cells per myofiber. Even if the founders of the non-myogenic clones are adherent cells the total number of cells per myofiber  would have been several tens, a number that is far from the minimum number of cells needed for FACS analysis. In view of this limitation, cells were separated from their parent myofiber using enzymatic and physical means and were then cultured at clonal density see . These rWe isolated 116 myofiber-associated progenitors and 29 MSCs from M1. Myofiber- associated progenitors were extracted from the Gast and Masseter muscles and cloned. These cells gave rise to both myogenic (MA-M) and non-myogenic (MA-NM) clones and were characterized as shown in These data suggest that there is a common ancestor for the founders of the myogenic and non-myogenic clones in the same muscle, termed muscle-bound stem cells. These ancestors are distinct from the ancestors of bone marrow MSCs, postulating that bone marrow MSCs are not the founders of the developing non-myogenic clones.To elucidate the development and migration paths of myofiber-associated progenitors harbored in distinct muscles we performed lineage analyses comparing clones extracted from the Masseter and Gast muscles. Our data revealed that cells are clustered in a hierarchical manner, first according to the muscle from which they originated and then according to their myogenic or non-myogenic type . For exaWe next analyzed the reconstructed lineage trees of the myogenic and non-myogenic clones extracted from all tested muscles. For this we compared 6 sets of myofiber-associated cells: left Gast myogenic and non-myogenic, right Gast myogenic and non-myogenic, and right Masseter myogenic and non-myogenic. Data revealed that (i) the myogenic clones cluster separately from non-myogenic clones within the same muscle  and (ii)As detailed above, our data show that in addition to myogenic progenitors, myofibers also harbor non-myogenic progenitors of a distinct, yet close, lineage. To shed light on the question whether the non-myogenic progenitors are cells that adhere to the outer surface of myofibers' basal lamina  we performed an experiment that is detailed in the Supplemented Materials see . In thisIn the present study we analyzed the lineal relations between cells associated with myofibers and MSCs. MSCs were cloned from the bone marrow and myogenic and non-myogenic cells were cloned from individual myofibers. Our analyses suggest, as depicted in Although muscles are known to contain MSCs as part of their interstitium, and determining their lineage relations with myofiber-associated progenitors would have been informative, doing so in our current experimental setting was not practical, as isolating these MSCs requires digesting many muscles Satellite cells  as well as several other types of cells, such as MSCs or white fat cells were suggested to account for such non-myogenic cells Potential candidate cells for a shared ancestor of the myogenic and non-myogenic founders maybe the mesoangioblasts/pericytes. Pericytes are cells that surround endothelial cells in capillaries and microvessels and are thought to include progenitors of different cell types including skeletal muscle cells in-vivo approach was taken to determine the fate of satellite cells during limb regeneration after amputation. Data showed that some of the re-introduced labeled satellite-cell derived clones adopted non-myogenic fates. Alternatively, if the myofiber-associated myogenic progenitors represent the satellite cells pool, then satellite cells are homogenous with regards to their differentiation breadth and are only in close, but not same, lineal relations with the ancestors of non-myogenic cells. This accords with evidence implying that satellite cells are uni-potent cells, giving rise to myogenic cells only Whether the primordial stem cells or the myogenic progenitors represent the satellite cells pool cannot be conclusively established based only on the current lineage analyses. If the primordial stem cells represent the satellite cell population, then upon their population of target muscles they give rise to myogenic and non-myogenic cells. The notion that satellite cells can give rise to non-myogenic cell types accords with studies with rat and mice myofibers Regardless of their origin, the finding that muscle fibers consistently harbor a distinct lineage of non-myogenic stem cells immediately raises the question on the biological function of such cells in health and disease. One may speculate that the presence of non-myogenic progenitors within the muscle maybe of advantage. For example, there may be an advantage in the immediate availability of fibroblasts in case of injury as such cells are needed to synthesize extracellular matrix proteins that take part in scar formation which is necessary for adequate myofiber-repair In any event, this current study clearly demonstrates that myofiber-associated non-myogenic progenitors are not the progeny of MSCs. We thus conclude that at least some of the inter-muscular adipocytes and/or fibroblasts are the progeny of myofiber-associated progenitors rather than MSCs. This does not necessarily contradict with recent findings that MSCs also contribute to adipogenesis and fibrogenesis in skeletal muscles In summary, the combination of computation and biological approaches allowed analyzing three different cell types at the same time, a commodity that is not available by any other means up to date. This allowed better understanding the differentiation dynamics of the pools of these stem cells and the lineal relationships between the subpopulations.Mlh1+/−C57Bl/6 (obtained from Michael Liskay) th day cultures were inspected every other day for up to 14 days, a stage by which myogenic clones typically develop myotubes. As previously described Myofiber association cells were isolated and cultured as described 6 cells/ml medium. This amount of cells was seeded per well in 24 well dished. After 2–3 day incubation, wells were carefully washed to remove non-adherent cells and then supplemented with fresh culture medium. These selective conditions allowed only adherent fibroblastic cells to develop. Initial colonies were evident within 7–10 days and were followed every other day. These colonies were addressed as single Colony forming unit-fibroblast (CFU-F). Cells were allowed to proliferate for 14 days, before harvest and DNA isolation.MSCs were isolated and cultured as described and extensively characterized by other and by us in a series of studies WGA was performed using the Illustra GenomiPhi V2 DNA Amplification kit  according to the manufacturer's instructions as described by G. Kumar et al Microsatellite length was analyzed based on the capillary signals. Capillary signals that displayed more than one allele per locus were removed from the analysis. Only cells in which more than 25 alleles were amplified were included in the analysis. Trees were reconstructed using the distance-based neighbor-joining algorithm Pairs of cells were sequentially merged according to a distance matrix of lineage distances. Each entry in the distance matrix is taken as the maximum likelihood estimate of the number of divisions separating the two cells. The mutation step model and the mutation rates were estimated from the ex-vivo trees. Depth was read off the trees as the branch lengths leading from the root to each terminal leaf. Root signature was taken as the allele size values of tail normal cells (which represent a wide variety of cell types).P-values for the difference in distributions were calculated using Kolmogorov-Smirnov method. Hypergeometric tests were carried out for each internal branch to assess whether sub tree leafs are enriched for a cell population. P-values declared as significant are corrected for multiple hypothesis testing using false discovery rate of 0.2. Whenever sub trees were embedded only those with the most significant p-value are retained.Figure S1PCR repeats, in a 330 (A) days old mouse and 44 (B) days old mouse. Light blue nodes (•) indicate PCR repeats pairs with close genetic distance. Axis represents depth in arbitrary units.(TIF)Click here for additional data file.Figure S2Lineage tree of 178 Myofiber-associated cells (blue), and 28 MSC (brown) of a 44 days old mouse. Each terminal node (blue • or red •) represents a single sampled cell. The vertical axis represents the number of divisions a cell underwent since the zygote, i.e., cell depth. Blue and brown lines indicate significant clustering of myofiber-associated cells and MSCs, in distinct subtrees with a p value<1e-21.(TIF)Click here for additional data file.Figure S3Clustering of MSC and myofiber-associated cells of 44 days old mouse. Cells from the left Gast muscle are depicted in green and from the right Gast in red; cells from the right Masseter are depicted in purple. MSCs are depicted in brown. Myofiber-associated cells were significantly clustered according to the muscle they were extracted from with the p values denoted in the figure.(TIF)Click here for additional data file.Figure S4A Gast myofiber and its myonuclei. Laminin, that is part of the basal lamina, is shown in red and nuclei, visualized based on DAPI  incorporation, in blue. All nuclei in this segment of the myofiber are situated beneath the basal lamina of the myofiber.(TIF)Click here for additional data file.Table S1Microsatellite panel. Different microsatellites loci and sequence used in this paper. Name = Loci name, Color =  fluorescent colors ABI dyes: B = FAM Blue; Y =  NED Yellow; R =  PET Red; G = VIC Green. LIZ Orange was used as a size standard. fwd/rev primer are sequences of the primers used. #repeats is number of repeats in the micro sattelite.(DOC)Click here for additional data file.Video S1Reconstruction of a three-dimensional (3D)- fluorescent model of the myofiber basal lamina (red) and its associated nuclei (blue).(WMV)Click here for additional data file.


We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from  P. falciparum malaria. Erythrocytes containing mature malaria parasites adhere to the endothelium via a range of endothelial receptors in order to escape removal by the spleen. Endothelial activation is an early feature of malaria, which is likely to favor sequestration of parasitized erythrocytes The endothelium plays a central role in the pathophysiology of Plasmodium falciparum malaria from whom platelet poor plasma was obtained under special conditions to prevent ex vivo platelet activation. A second aim was to correlate plasma levels of these angiogenic proteins with circulating platelet numbers and markers of platelet activation.Both Ang-1 and VEGF are both stored in high quantities in alpha granules from platelets This study received ethical clearance for the use of human subject from the Eijkman Institute Research Ethics Committee, Jakarta, Indonesia and all enrolled patients gave written informed consent.P. falciparum and P.vivax malaria transmission. Consecutive patients presenting to hospital with recent or current fever, clinical symptoms of malaria and a positive blood slide for P. falciparum were enrolled in this study following informed consent. All patients were assessed according to a predefined protocol which included a standardized history and physical examination performed by an experienced internist-infectious diseases specialist. All patients were treated with intravenous quinine and an antipyretic (paracetamol). Concurrent administration of antibiotics for severely ill patients was on the discretion of the treating physician. Venous blood for this study was collected at enrollment and on day two and five after enrollment. A group of 25 healthy young adults were recruited among local hospital staff as controls. All controls had no signs or symptoms of any illness and a negative malaria blood slide. Treatment for malaria in the past two months was an exclusion criteria for both malaria patients and controls.This study was conducted in the Rumah Sakit Karitas Hospital in Waitabula, West Sumba, East Nusa Tenggara Province, Indonesia, an area of hypo- to meso-endemic In vitro platelet activation is almost completely absent in whole blood anticoagulated with CTAD Five ml of venous blood was collected in EDTA tubes for a full blood count and malaria slides and in CTAD tubes  for measurement of angiogenic and platelet activation marker proteins. Data are presented as median followed by interquartile range in parentheses unless otherwise stated. Within the group of malaria patients, the Freidman test with post-tests was used to compare laboratory parameters on the three time points.Mann-Whitney U test was used for comparisons with the controls. Relationships between laboratory parameters were assessed using Spearman correlation coefficient. All analyses were performed with GraphPad version 5.0.P. falciparum malaria and 25 controls were included. Demographic and clinical characteristics are shown in A total number of 27 patients with The time course of Ang-1 and VEGF resembled the course of the platelet activation markers P-selectin and CXCL7. Correlation analysis, presented in in vitro platelet activation ex vivo platelet activation, CTAD tubes almost completely prevent in vitro platelet activation and several authors have already recommended using CTAD when platelet-stored angiogenesis proteins are being measured ex vivo platelet activation was responsible for the lower Ang-1 and VEGF concentrations in previous studies. Serum concentrations of platelet-stored angiogenic proteins may also have prognostic value. Generation of serum results in complete in vitro platelet activation and serum concentrations will largely reflect the total platelet content of angiogenic proteins. Possible drawbacks of using serum for this objective is that clotting processes may not release all platelet-stored angiogenic growth factors into the serum These findings are in contrast to previous studies which showed that patients with malaria had lower Ang-1 levels than healthy controls 9/L) in the remaining volunteer.We only used a single centrifugation step to generate platelet poor plasma in our study, while a double centrifugation step is often advised. We assumed that only under special circumstances single centrifugation does not result in near complete removal of platelets from the top layer of the plasma. We tested this assumption by centrifuging venous blood in CTAD tubes from five volunteers using the same procedures as in our study and we found that a singly centrifugation step resulted in no detectable platelets in the plasma in four volunteers and a negligible platelet number (2×10Several groups of researchers have investigated the utility of angiopoietins and VEGF as biomarkers to identify patients with severe malaria. Due to its limited sample size, our study did not allow to explore the role of these angiogenic proteins in malaria. In general, we strongly recommend that a similar protocol for measurement of platelet-stored angiogenic proteins is used in future biomarker studies, including use of CTAD plasma and consideration of circulating platelet numbers. These precautions do not apply to Ang-2, which is derived from endothelial cells and of which plasma levels are not affected by platelet activation.What are the implications of these findings for the pathophysiology of malaria? Increasing evidence supports a central role for platelets in protecting the vasculature during inflammation In conclusion, we show that platelet activation is an important determinant of circulating Ang-1 and VEGF levels in malaria. Optimal biomarkers for malaria severity would rely on inexpensive point-of-care devices using whole blood. The fact that assays measuring these platelet-stored angiogenic proteins require special precautions to avoid inadvertent platelet activation during blood processing and are prone to artifacts limits widespread use of these proteins as possible biomarkers.