Cell line development (CLD) is a critical step in the generation of biotherapeutics, but it is still hindered by several pain points, including the lengthy and labor-intensive workflow needed to isolate desirable clones, lack of reproducibility, as well as potential protein quality issues. Over the last decade, antibody titers in mammalian cell culture systems in excess of 3 g/L have been achieved through the use of novel media and feeds. However, it is still a challenge to consistently and rapidly create a stable cell line and a cell culture process capable of supporting both high antibody yield and acceptable post-translational modifications while managing the effort required for execution of the workflow. The goal of the study was to develop a robust and reproducible stable cell line workflow to generate scalable high-producing clones in less than 6 months, with industry-standard titers and desirable product quality using minimal effort.Using CHO-S™ as the host cell line, we first evaluated if a single medium could be used for the entire CLD workflow, therefore avoiding the issues and complications of changing media during this process. We investigated if a formulation previously shown to increase titer as a production medium could in fact be used for all CLD steps, from transfection to stable pool isolation all the way through to clone productivity, without compromising titers or performance. The same rich production medium was used in limiting dilution cloning and compared to a lean cloning medium prototype. Furthermore, robustness of the workflow was verified by testing multiple molecules. We also explored reducing effort by streamlining all the steps of the workflow. Finally, we assessed top clone scalability and expressed product quality. We tested whether clones chosen only by titers responded well to scale-up and process development in a model bioreactor setting. In addition, product glycosylation from these clones was compared to the same molecule produced in CHO DG44 cells, a well-characterized production platform.TM Medium or CD FortiCHOTM Medium gave the most desirable outcomes in terms of recovery time and productivity. We also found that CD FortiCHOTM Medium can be used for every step of the CLD workflow, including limiting dilution cloning. When we performed a limiting dilution cloning using the same pool seeded in either a lean cloning medium prototype or in CD FortiCHOTM Medium, we found that the top 10 clones isolated from each medium showed similar average titer trends (Figure TM cells was indistinguishable.Our results show that cell growth during selection and productivity assessment were affected by both the basal medium and nutrient feed strategy. Stable pools generated in either CD OptiCHOTM clones was comparable to historical data from CHO DG44-derived clones expressing the same molecule. In addition, the establishment of clone stability and acceptable glycosylation patterns are key attributes required for regulatory approval of biotherapeutic production. Together, these results demonstrate the capabilities of the FreedomTM CHO-STM Kit as an efficient and robust stable CLD platform, which can be accessed without the burden of milestone or royalty payments.The use of a single medium for the entire workflow is revolutionary, and has the distinct advantage of avoiding any need for media adaptation at any point in the workflow, whether it be preceding or following cloning, or for productivity assessment. This in turn avoids any undesirable genetic selection that may occur during such adaptation steps, and facilitates streamlining the workflow for ease of use and efficiency. Clones are easily scaled from shake flask to bioreactor and produce 2-3 g/L with minimal process development. Product glycosylation in top CHO-S


The recovery of functional movements following injury to the central nervous system (CNS) is multifaceted and is accompanied by processes occurring in the injured and non-injured hemispheres of the brain or above/below a spinal cord lesion. The changes in the CNS are the consequence of functional and structural processes collectively termed neuroplasticity and these may occur spontaneously and/or be induced by movement practice. The neurophysiological mechanisms underlying such brain plasticity may take different forms in different types of injury, for example stroke vs. spinal cord injury (SCI). Recovery of movement can be enhanced by intensive, repetitive, variable, and rewarding motor practice. To this end, robots that enable or facilitate repetitive movements have been developed to assist recovery and rehabilitation. Here, we suggest that some elements of robot-mediated training such as assistance and perturbation may have the potential to enhance neuroplasticity. Together the elemental components for developing integrated robot-mediated training protocols may form part of a neurorehabilitation framework alongside those methods already employed by therapists. Robots could thus open up a wider choice of options for delivering movement rehabilitation grounded on the principles underpinning neuroplasticity in the human CNS. Stroke or spinal cord injury (SCI) often leaves an individual with persistent functional movement deficits that impact on independent living and quality of life, whilst putting an enormous healthcare and macro-economic burden on societies . Such suSeveral potential therapies may assist or guide this spontaneous recovery and include constrained induced movement therapy (CIMT), robot-mediated therapy, pharmacological treatments , brain-machine interfaces (BMIs), goal oriented physiotherapy, epidural spinal stimulation, non-invasive cortical stimulation, electromechanical-mediated therapy, and combinations thereof –13. TakiUnderlying many existing motor therapies is the central tenet that repetitive, progressive, and engaging practice using the affected limb induces plastic changes in neural networks subserving motor control and learning. The changes could be both functional and structural-anatomical and the neurophysiological processes by which these changes might occur, have been collectively termed as neuroplasticity , 14, 15.in vitro and in vivo animal studies and which contribute to changes in neurophysiology such as increased or decreased evoked post-synaptic potentials (EPSPs) that can persist for long periods Can these re-organizations of brain function and structure be “tuned” or enhanced by motor training? There is strikingly little information available to answer this especially in the early stages of recovery . Longituassistance is rather scarce. However, it has been suggested that robot assistance may promote motivation, because motor performance during training can be better than without assistance  or ankle (lower limb).One common paradigm incorporates adaptation to robot-induced force fields which physically perturb ongoing arm movement . ChangesPerturbation stimulates the healthy motor system to adapt, that is, to counteract the external force and can involve adaptation of predictive “feedforward anticipatory” movement or force production  and adaptation of reactive “feedback” adjustments of limb movement in response to the perturbation. Adaptation is a fast process that can be distinguished from learning by repeating the same unperturbed movement over and over again . WhereasIn healthy subjects, cortical excitability of a brain region can be modulated by applying unilateral anodal tDCS during motor adaptation Figure A. Cerebein vitro motor cortex brain slices has suggested that tDCS interacts with coincident low frequency stimulation  to increase BDNF secretion and TrkB activation  has been combined with a robotic training device in gait training after stroke and can significantly augment gait improvements more than robot therapy alone . The impUnfortunately a significant number of stroke survivors often see little progress in their training because improvement is slow and post-stroke depression may devalue reward. Several rewarding features can be built into training robots, such as those providing immediate feedback about movement errors as well as delayed rewards, for example by collecting points or virtual money. VR can provide an excellent framework for reward presentation. Reward regions in the brain have been proposed to contribute to motor skill learning in an animal model via dopaminergic pathways . Recent The idea of using a combination of a BMI and robots for rehabilitation has been explored in several recent studies and is justified by the absence of rehabilitation therapies for paralyzed and severely impaired stroke patients. These patients cannot benefit from existing therapy since residual movement ability is generally necessary. Rehabilitation robotic devices hold the potential to bridge the gap between the intention to move  and actual movement of an orthosis or robot device without the need of a limb –91. In tStroke patients can acquire control over a hand orthosis (opening/closing of the hand) by volitionally modulating sensorimotor frequency-dependent rhythms in the lesioned hemisphere. While most of the patients were able to learn controlling the orthosis via the BMI, clinical scales used to measure hand function showed no improvement after training . In anotIn summary, this rapidly expanding field is yielding significant methodological steps forward in the design of upper and lower limb rehabilitation using BMI with robots or other hybrid approaches such as acquired self-control of brain activity , 101. ThThe use of robot-mediated therapy for augmenting recovery from neurological injury is now becoming more attractive as evidence for cost effectiveness of robot-mediated therapy is becoming stronger , 102. AsHowever, critically assessing the evidence for neuroplasticity induced by robot use has highlighted several large gaps in our knowledge and some paradoxes that should be addressed Table .assisted therapy is the most frequently robot-based intervention for neurological injury, we know little about the neural correlates of this type of movement (per se (The first paradox is that whilst robot-movement , 42 and  (per se , 48, 49. (per se .The second paradox is that whilst we know a significant amount about the neural correlates of motor learning and neuroplasticity induced by robot-mediated perturbations in healthy subjects, this type of robot usage is rarely used in neurological therapy . The neuThe third paradox is that whilst cortico-striatal neural activation is modulated during and after robot perturbing motor adaptation in healthy subjects  and rewaThe fourth paradox involves the interaction of brain and robot without the need for an actuating human limb. Using brain signals to drive a robot device directly to undertake everyday tasks and to induce motor rehabilitation is feasible following severe stroke , 111. ThThere are several large gaps in our knowledge on the neural correlates of effective robot-mediated therapy Table . The rapRobots are more than aids for or simply replacements of therapists to deliver movement therapy. From a technical perspective, robots can be excellent research tools, because they provide ways to standardize rehabilitative training, to precisely monitor recovery of motor function in patients [Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Climate plays an important role in determining the geographic ranges of species. With rapid climate change expected in the coming decades, ecologists have predicted that species ranges will shift large distances in elevation and latitude. However, most range shift assessments are based on coarse-scale climate models that ignore fine-scale heterogeneity and could fail to capture important range shift dynamics. Moreover, if climate varies dramatically over short distances, some populations of certain species may only need to migrate tens of meters between microhabitats to track their climate as opposed to hundreds of meters upward or hundreds of kilometers poleward. To address these issues, we measured climate variables that are likely important determinants of plant species distributions and abundances (snow disappearance date and soil temperature) at coarse and fine scales at Mount Rainier National Park in Washington State, USA. Coarse-scale differences across the landscape such as large changes in elevation had expected effects on climatic variables, with later snow disappearance dates and lower temperatures at higher elevations. However, locations separated by small distances (∼20 m), but differing by vegetation structure or topographic position, often experienced differences in snow disappearance date and soil temperature as great as locations separated by large distances (>1 km). Tree canopy gaps and topographic depressions experienced later snow disappearance dates than corresponding locations under intact canopy and on ridges. Additionally, locations under vegetation and on topographic ridges experienced lower maximum and higher minimum soil temperatures. The large differences in climate we observed over small distances will likely lead to complex range shift dynamics and could buffer species from the negative effects of climate change. Biologists have long recognized the fundamental role climate plays in determining the geographic distributions of species and biomes However, these projections of climate change-induced range shifts (and subsequent extinction risks) are sensitive to the spatial scale at which the analyses are conducted The implication of significant fine-scale climatic heterogeneity that is not captured by coarse-scale climate maps is that projections based on these maps could fail to capture important range shift dynamics. For example, cool microhabitats (such as north-facing slopes in the Northern Hemisphere) near the contracting edge or core of a species’ distribution may allow populations of that species to persist if individuals can disperse to them from warmer microhabitats (such as south-facing slopes in the Northern Hemisphere), even if most of the surrounding landscape becomes unsuitably warm  quantify snow disappearance date and soil temperature as a function of coarse-scale differences in elevation and exposure to storm tracks (i.e. being on the windward or leeward side of the mountain) and fine-scale differences in vegetation structure or topography, 2) compare fine-scale differences in climatic variables (that would be missed by climate models) to coarse-scale differences (that would be captured by climate models), and 3) determine whether fine-scale patterns in climatic variables related to topography (but not vegetation structure) are correlated with fine-scale patterns in vegetation characteristics. We focus on snow disappearance date and growing season soil temperature because snow disappearance date influences the length of the growing season  while soil temperature strongly influences plant growth rates and other physiological processes It has long been known that climate can vary dramatically at fine spatial scales .Mount Rainier National Park encompasses 95,354 ha of land in the western Cascade Mountains in Washington State, USA . The regThe large climatic gradients create three major biomes in the Park. The forest biome extends from the lowest elevations of the Park up to about 1600–2000 m and is dominated by coniferous trees. The subalpine biome typically extends about 300 m above the upper limit of the forest and is a mosaic of conifer tree patches and subalpine meadows. The alpine biome occupies the highest elevations, stretching from 1900–2300 m to the summit of Mount Rainier, and consists of large patches of mostly continuous alpine meadows  near the lower limit of the biome, with exposed rock, glaciers, bare soil, and cryptogams  predominating above.From September 2009 through October 2010, we deployed 284 soil temperature sensors (HOBO Pendants made by the Onset Computer Corporation and iButtons made by Maxim Integrated Products) across Mount Rainier at elevations ranging from 638 m to 2164 m as part of two different plant ecology studies where microclimate was measured as an explanatory variable. The first study took place in the forest biome and spanned the elevational range of forests on the south side of Mount Rainier. The second study took place in the subalpine and alpine biomes, with study sites set up at the lower limit of the subalpine biome and the upper limit of alpine meadows on three sides of the mountain . MicroclFor each sensor, we calculated the values of four climatic variables: snow disappearance date, and average daily mean, maximum and minimum soil temperature. We could assess snow cover from soil temperature measurements because snow insulates soil from fluctuations in air temperature so that temperatures beneath the snowpack in this region remain constant around 0°C. Thus, the soil temperature data allowed us to determine whether snow was covering the sensor for each day the sensor was deployed using an algorithm that considers daily temperature ranges and maxima 2 on average. Within each gap or non-gap location, we placed one sensor in a 5.5×1.5 m area where all understory vegetation up to 2 m tall had been experimentally removed since early Summer 2009 (the “removed” plot) and one sensor at an adjacent location 2 m away where the vegetation had been left undisturbed (the “control” plot)  and in locations under intact canopy within 20 m of one of the gaps (“non-gaps”). Each study site contained five of these gap/non-gap pairs. Gaps were ∼130 ml” plot) , Table 1For the subalpine/alpine study, we quantified microclimate at study sites on three sides of the mountain  which have different exposures to storm tracks and experience different precipitation regimes. On each side, we established two study sites, one close to the lower limit of the subalpine biome and one close to the upper limit of continuous alpine meadows, . These sites were about 200–300 m apart in elevation on each side of the mountain. Within each site, we established six linear transects that ran from a depression in the landscape up to a ridge  and were about 20 m in length. Within each transect, two sensors were located in the depression and two sensors were located on the ridge , Table 1At each of the sensors in the subalpine/alpine biomes , we measured vegetation characteristics in order to compare patterns in microclimate to ecological patterns. At the study sites near the lower limit of the subalpine biome, where closed-canopy forests transition to open meadows with increasing elevation, we measured percent cover by tree canopy above each sensor using a spherical densiometer , allowing us to assess the density of trees . At the study sites near the upper limit of alpine meadows, where meadows transition to bare ground with increasing elevation, we estimated the percent of ground covered by vegetation at each sensor using a square-shaped PVC frame (1×1 m) placed on the ground adjacent to the sensor. String tied to the PVC frame created 100 evenly spaced grid points, allowing us to count the number of grid points overlaying vegetation in the area within the frame.We used linear mixed effects models (LMMs) to characterize the relationships between the potential drivers of climate and each of the four climatic response variables For each model, we used Akaike’s information criterion (AIC) to select the most parsimonious combination of fixed and random effects to derive the “best-fit” model. Specifically, we used a three-step process following t-tests (when no random effects were included in the best-fit model). We then used the explanatory variable coefficients of the best-fit models to calculate the magnitude of differences in microclimate response variables relative to differences in the explanatory variables. For example, if the coefficients related to topographic position in the model of snow disappearance date at the subalpine/alpine sites indicated that the difference between ridges and depressions was 20 days, controlling for differences in other variables, then the effect of topographic position on snow disappearance date would be equal to 20 days. In order to compare the effects of elevation  to the effects of other explanatory variables, we calculated the difference in snow disappearance date or temperature between two points 100 m apart in elevation for each model (controlling for differences in other variables). Like differences in climate amongst different sides of the mountain, differences in climate caused by a 100 m difference in elevation can typically be captured by coarse-scale climate models (e.g. PRISM), while differences caused by vegetation structure and fine-scale topography cannot. If one of the explanatory variables was not included in the best-fit model, we included the main effect of that variable in the final model for comparative purposes. This happened for one explanatory variable in one model (the understory structure variable in the snow disappearance date model in the forest study).We assessed the significance of the model coefficients using Markov chain Monte Carlo sampling implemented with the MCMCglmm package in R t-tests to assess the significance of the microclimate variable coefficient in the best-fit model in order to assess the significance of that microclimate variable. When modeling percent tree canopy cover at the lower elevation sites as a function of average daily minimum temperature and side of the mountain, neither explanatory variable nor their interaction was included in the best-fit model (i.e. the best-fit model was the null model with only an intercept). For this situation, we performed a t-test on the minimum temperature coefficient in a model that included both minimum temperature and side of the mountain, but not their interaction, as explanatory variables  in order to assess the significance of minimum temperature.For sites in the subalpine/alpine biomes, we also fit linear models (LMs) to characterize the relationships between each of the four microclimate variables and percent cover by tree canopy at the lower elevation sites, and the relationships between each of the four microclimate variables and percent cover by ground vegetation at the higher elevation sites, for a total of eight LMs. In these models, the response variable was the vegetation characteristic while the explanatory variables were the microclimate variable, side of the mountain (included as a covariate) and their interaction. Using LMMs with sensor transect designated as a random effect did not improve model fit for any of the relationships, so we used the simpler LMs for all of these analyses. Next, we used AIC to select the best-fit LM. With one exception, this best-fit model included both the microclimate variable and side of the mountain, but not their interaction, as explanatory variables. In these cases, we used r2) for all models, following We calculated the proportion of variance in the response variable explained by variance in the fixed effect explanatory variables  had higher mean and maximum temperatures, but lower minimum temperatures, than lower elevations . Compared to ridges, depressions had higher mean and maximum temperatures but lower minimum temperatures . We founp<0.0001). At study sites near the upper limit of alpine meadows, percent cover by ground vegetation was lower where snow disappearance date was later and average daily minimum soil temperature was lower (p<0.0001).Several microclimate variables were significantly correlated with vegetation characteristics . At studOur study suggests that climatic heterogeneity at the fine spatial scales most organisms experience their environment is substantial, implying that projections based on coarse-scale climate models will not capture the full complexity of range shifts in response to climate change. Specifically, we found large differences in snow disappearance date and growing season soil temperatures over small distances , 4, diffIn the forest biome, a complex interplay between elevation and vegetation structure is likely responsible for the heterogeneous patterns in snow disappearance date and soil temperature we observed. For example, locations under tree canopy gaps likely experienced later snow disappearance dates than locations under an intact canopy  because Similarly, in the subalpine/alpine biomes we found that both coarse- and fine-scale features had large effects on climate. For example, snow disappeared substantially later from depressions in the landscape than from ridges only ∼20 m away, likely because snow typically collects in these depressions while it is blown off of ridges and because shading from surrounding slopes can reduce ablation rates A striking pattern to emerge from our data was that mean and maximum soil temperatures were greater at higher elevations within the subalpine/alpine biomes (though minimum soil temperatures were lower). Feedbacks between climate and vegetation likely play important roles in producing this temperature pattern. First, tree cover declines with increasing elevation, leading to less shading and potentially higher daytime soil temperatures, especially during the sunny growing season when our data were collected. This explanation is supported by the negative correlation we observed between percent canopy cover (a measure of tree density) and mean/maximum soil temperature in the subalpine/alpine biomes . Second,Since snow disappearance date and growing season temperature vary dramatically over short distances, species whose distributions are primarily constrained by these climate variables may not need to migrate long distances to remain in suitable habitat even when there are large changes in climate. For example, in the subalpine/alpine biomes we found that the average difference in snow disappearance date between depressions and ridges separated by only ∼20 m was often one month or more. This is an especially large difference considering the ground is typically only free of snow for 3–5 months out of the year at these elevations. Snow manipulation experiments have shown that differences of this magnitude have large impacts on the phenology, species composition, diversity and productivity of plant communities The importance of topographic heterogeneity for creating climatic heterogeneity shown in this study and others Different kinds of cool or snowy microhabitats will likely differ in their effectiveness as refugia in a warming world. First, the abundance of microhabitat types will strongly influence how effective they can be as refugia. For example, depressions in the landscape in the subalpine/alpine biomes may have a high likelihood of serving as refugia because they are a common topographic feature. Second, the longevity of microhabitat types will affect their ability to act as refugia. For example, canopy gaps may disappear relatively quickly as trees establish in them, forcing species that might use gaps as refugia to migrate amongst gaps, which may not be possible for some species (though others may be adapted to this migration). In contrast, depressions in the landscape could provide more long-term refugia. Third, the temporal climatic heterogeneity experienced in microhabitat types may affect how well they can serve as refugia. For example, gaps had lower daily minimum and higher daily maximum temperatures compared to non-gaps, showing that these microhabitats experience a wide variety of temperatures. This heterogeneity may favor some species but not others. A final complicating factor influencing how and whether microhabitat types can serve as climatic refugia are the non-climatic conditions associated with them. For example, depressions may differ from other topographic positions in soil characteristics, which could prevent some species from using them as snowy microrefugia.An important caveat to these findings is that they are based on one year of data. Spatial patterns in climate can change from year to year due to differences in prevailing synoptic weather patterns To best protect biodiversity in a period of rapid climate change, conservation biologists and resource managers will require realistic assessments of future species distributions In addition to more realistically forecasting range shifts, knowledge of fine-scale climatic heterogeneity may also allow managers to increase species and ecosystem resilience to climate change. For example, protecting microhabitats with cooler temperatures or later snow disappearance dates could become increasingly important as climate change occurs because these microhabitats may provide critical refugia for species. Additionally, our results suggest that planting seedlings and sowing seeds at a variety of microhabitats when restoring degraded sites is an important bet-hedging strategy because it could increase the probability that species establish in microsites that remain suitable as climate change progresses, even if those microsites are only marginally suitable now. Thus, information on fine-scale climate heterogeneity has the potential to be useful for natural area protection and restoration when taken together with other important factors such as edaphic constraints, biotic interactions, genetic diversity and financial costs We have shown that snow disappearance date and growing season soil temperature vary dramatically over small distances due to differences in vegetation structure and topography. In fact, fine-scale features such as gaps in the forest canopy or small depressions in the landscape can produce differences in snow disappearance date and soil temperature as large as those produced by shifting hundreds of meters up a mountain slope. This large degree of fine-scale spatial heterogeneity may provide an important buffer against the negative effects of rapid climate change, as many species may only need to migrate tens of meters from one microhabitat to another in order to track suitable climate, as opposed to shifting hundreds of meters upward in elevation or hundreds of kilometers poleward. Climate change will undoubtedly pose serious threats to biodiversity, but knowledge of fine-scale climatic heterogeneity may allow managers to better assess and potentially alleviate some of these threats.Appendix S1Model fitting and selection procedure.(PDF)Click here for additional data file.Appendix S2Soil characteristics in the subalpine and alpine biomes.(PDF)Click here for additional data file.Appendix S3Climatological context of the study.(PDF)Click here for additional data file.


Dear Editor,We read with keen interest the recently published paper entitled "Ribavirin impairs salivary gland function during combination treatment with pegylated interferon alfa-2a in hepatitis C patients" by Aghemo et al in your Even at the beginning of the third millennium, hepatitis C virus (HCV) infection remains a major global public health problem, and to date, combination therapy seems to be the most effective treatment option. Nonetheless, this treatment might result in some side effects such as lichen planus, skin rash, psychological disturbances, poor appetite, anemia, and leucopenia .Derived results of the study by Aghemo et al might be affected by some confounding factors. First, although the authors had mentioned that a limitation of their study was using patients who had hepatitis B virus (HBV) infection as the control subjects, it cannot iron out all the attributed results of choosing an unsuitable control group. Previous studies have shown that HCV infection in the epithelial cells of the salivary glands is a factor associated with the development of sialadenitis and Sjogren's syndrome . HoweverAn advantage of their study was that they used stimulated saliva for evaluating sialadenitis. Previous studies have confirmed that stimulated whole saliva is especially useful in measuring salivary gland function in patients with common autoimmune disorders. The main advantage of gustatory stimulation is reduction in the variability of salivary flow rates among study subjects . SeveralFurther studies are required to assess the variations in the levels of oral fluid secretion in patients with viral hepatitis. If such changes can be proven, it might help identify why the rate of dental caries is significantly higher in patients with HCV infection than in the general population .We suggest that randomized controlled trials and experimental observations, including salivary gland function monitoring before and after treatment, to be conducted in HCV-infected patients. Evaluating the presence of HCV in salivary gland tissues could also yield accurate results.


Medial entorhinal grid cells and hippocampal place cells provide neural correlates of spatial representation in the brain. A place cell typically fires whenever an animal is present in one or more spatial regions, or places, of an environment. A grid cell typically fires in multiple spatial regions that form a regular hexagonal grid structure extending throughout the environment. Different grid and place cells prefer spatially offset regions, with their firing fields increasing in size along the dorsoventral axes of the medial entorhinal cortex and hippocampus. The spacing between neighboring fields for a grid cell also increases along the dorsoventral axis. This article presents a neural model whose spiking neurons operate in a hierarchy of self-organizing maps, each obeying the same laws. This spiking GridPlaceMap model simulates how grid cells and place cells may develop. It responds to realistic rat navigational trajectories by learning grid cells with hexagonal grid firing fields of multiple spatial scales and place cells with one or more firing fields that match neurophysiological data about these cells and their development in juvenile rats. The place cells represent much larger spaces than the grid cells, which enable them to support navigational behaviors. Both self-organizing maps amplify and learn to categorize the most frequent and energetic co-occurrences of their inputs. The current results build upon a previous rate-based model of grid and place cell learning, and thus illustrate a general method for converting rate-based adaptive neural models, without the loss of any of their analog properties, into models whose cells obey spiking dynamics. New properties of the spiking GridPlaceMap model include the appearance of theta band modulation. The spiking model also opens a path for implementation in brain-emulating nanochips comprised of networks of noisy spiking neurons with multiple-level adaptive weights for controlling autonomous adaptive robots capable of spatial navigation. How our brains acquire stable cognitive maps of the spatial environments that we explore is not only an outstanding scientific question, but also one with immense potential for technological applications. For example, this knowledge can be applied in designing autonomous agents that are capable of spatial cognition and navigation in a GPS signal-impoverished environment without the need for human teleoperation.Lesion and pharmacological studies have revealed that hippocampus (HC) and medial entorhinal cortex (MEC) are critical brain areas for spatial learning, memory, and behavior Since the time of the proposal of Sensory context includes properties of the following kind: The primary determinants of grid cell firing are, however, path integration-based inputs Along similar lines, Discussion section reviews how hexagonal grid structures can be learned in the brain even when stripe cells of multiple spacings converge initially on entorhinal cells Why do grid cells learn to fire at hexagonally-located positions as an animal navigates in an open field? Most computational models focused on learning of either hippocampal place cells The original GridPlaceMap model uses neurons that interact using rate coding; that is, they interact via signals based on spiking frequency, rather than in terms of their individual spike trains. The goals of the current model are threefold; namely, to test whether the insights gained from the rate-based GridPlaceMap model can be applied and extended to simulating and explaining the development of spiking grid and place cells, as an instantiation of a general method for converting rate-based adaptive neural models, without the loss of any of their analog properties, into models whose cells obey spiking dynamics; to develop a neural system that makes it possible to address, for the first time, known temporal coding properties of hippocampal place cells and medial entorhinal layer II grid cells, such as theta band modulation Additional extensions of the GridPlaceMap and sGridPlaceMap models will be needed to achieve a general-purpose neural architecture for spatial navigation. It has, for example, been proposed how top-down attentive matching processes from hippocampal to entorhinal cortex may facilitate fast learning and dynamic self-stabilization of learned spatial memories, provide a pathway whereby environmental cues may modulate properties of grid and place cells that arise through path integration, and may help to explain a wide range of additional data about modular grid orientations, grid realignment, place remapping, and gamma and beta oscillations e.g., .A receptors, and whose synaptic plasticity is governed by a spike timing-dependent variant of the competitive instar learning law The spiking GridPlaceMap model, called sGridPlaceMap see , employs+ channels and delayed rectifier K+ channels that underlie the generation of stereotypical spike waveforms in membrane potential dynamics, synaptic transmission delays, axonal conduction latencies, and refractory periods are not considered. GABAA-gated channel conductances are approximated by single exponentially decaying traces because their rise times are typically negligible  via voltage-dependent Ca2+ channels, and an EPSP mediated by NMDA receptors, respectively The adaptive weights, These variables are initialized to zero at the start of each trial. The weights are only initialized once, at the start of the first trial, by sampling from a uniform distribution between 0 and 0.1. The learning law in A channel conductance gate that is opened by the spiking of the The membrane potential A channel conductance gate For this stage too, all gates are initialized to zero, and all membrane potentials are initialized to The adaptive weights, These variables are also initialized to zero at the start of each trial. The pre-learning weights are sampled from a uniform distribution between 0 and 0.03. The initial weights of projections from stripe cells to MEC cells have a higher individual mean to compensate for the relatively lower number of input cells; see below.The parameter values used in the simulations were The model was run with 100 HC map cells receiving adaptive inputs from three distinct populations of 100 MEC map cells, each of which was driven by adaptive inputs from stripe cells of one of three spacings. Stripe cells were activated in response to linear velocity estimates derived from realistic rat trajectories of ∼10 min in a 100 cm×100 cm environment  from its adaptively smoothed firing rate map. Distinct fields were indentified from circular templates around local peaks based on the criteria that the maximal rate within a field is at least more than 50% of the overall peak rate 16 points) whose amplitude response is squared, and normalized to the maximal value, to yield the power spectrum between 0 Hz and 250 Hz.Temporal modulation in the spiking responses of cells was assessed by computing the power spectra of the corresponding spike trains, with a temporal resolution of 2 ms, using a standard procedure This section shows that all the results of the rate-based GridPlaceMap model ly Data: . Both arer Data: . While tsquare box of 100 cm×100 cm , each human brain consumes just about 20 W of power. This power budget contrasts dramatically with that required  to run the most advanced supercomputer in the world; namely, the Blue Gene/Q at the Lawrence Livermore National Laboratory in Livermore, CA. Moreover, such advanced supercomputers occupy a lot of physical space, and need to be explicitly programmed for each specific task that they are supposed to perform. Aggressive efforts are currently underway across the world to develop a fundamentally new class of computer chips that closely mirror biological brains to herald the arrival of a transformative new technological field of natural intelligence. With respect to sGridPlaceMap model computations, the spiking competitive instar learning law described in This form reveals a single inhibitory term  currents mentioned above, and the m-current IhIh is known to generate subthreshold MPOs The spiking grid and place model developed in this article makes it feasible to ascertain more directly the relative contributions of and interactions among different synaptic currents in setting up the spatial scale topography of grid cells, and to probe them further computationally; namely, the AHP, leak K


Performance of timed motor sequences relies on the cerebellum and basal ganglia, which integrate proprioceptive information during the motor task and set internal timing mechanisms. Accordingly, these structures are also involved in other temporal processes, such as the discrimination of the different afferent information in the domain of time. In the present study we tested temporal processing of proprioceptive and tactile stimuli in 20 patients with neurodegenerative cerebellar ataxia and 20 age- and sex-matched healthy subjects. Tactile temporal discrimination threshold was defined as the value at which subjects recognized the two stimuli as asynchronous. Temporal discrimination movement threshold of the first dorsal interosseous and flexor carpi radialis was defined as the shortest interval between two paired electrical stimuli in which the subjects blindfolded perceived two separate index finger abductions and wrist flexions. Both tactile and movement temporal discrimination thresholds were higher in patients with cerebellar ataxia. No correlation was found with disease duration and severity. Our study demonstrates that temporal processing of tactile and proprioceptive stimuli is impaired in patients with cerebellar neurodegeneration and highlights the involvement of cerebellum in temporal processing of somatosensory stimuli of different type. The performance of timed motor sequences relies on the cerebellum and basal ganglia, which integrate proprioceptive information during the motor task and set internal timing mechanisms. Specifically, the lateral cerebellum seems to be implicated mainly in the process of synchronization, whereas the basal ganglia seem to be implicated in internally paced timing mechanisms Tactile temporal discrimination threshold (TDT) and proprioceptive temporal discrimination movement threshold (TDMT) are psychophysical measures of temporal processing of tactile and proprioceptive stimuli We recruited 20 patients  affected by neurodegenerative cerebellar ataxia (CA), from the outpatient clinic of the Neurology Institute “Carlo Besta” in Milan, Italy. All subjects gave their written informed consent before participation. The study was approved by the Department of Neurological, Psychological, Morphological and Motor Sciences, University of Verona. Inclusion criteria were the presence of a slowly progressive cerebellar syndrome without clinical evident involvement the motor  and the sensory systems . Eight patients had a positive family history compatible with an autosomal dominant disorder, and twelve cases were classified as sporadic cerebellar ataxia All participants were tested by a single neurologist expert in clinical neurophysiology (A.P.) in a single experimental session lasting about 2 hours. Each upper limb was tested separately, and the order of presentation including the stimuli procedure was counterbalanced across subjects. Verbal instructions about the experimental tasks were also provided before each testing session, as well as examples of single or double stimuli. To maintain subjects’ attention throughout the procedure and to disclose possible perseverative responses, catch trials (3 for each series) with inter-stimulus interval (ISI) of 0 ms were included during the ascending series for each procedure. Subjects were allowed to pause between each block.Tactile TDT testing was conducted according to previous standardized protocols The first dorsal interosseous (FDI) and flexor carpi radialis (FCR) muscles were selectively stimulated with a procedure extensively described in previous studies Two-sample t-test and Fisher’s exact test were used to explore between-groups differences in demographical variables. Separate repeated measures analyses of variance (R-ANOVAs) were employed to compare groups for each variable . R-ANOVA for PT and TDT had one between-subjects factor: “Group”  and one within-subjects factor: “Side” . R-ANOVA for PPT and TDMT had one between-subjects factor: “Group”  and two within-subjects factors: “Side”  and “Muscle” . Conditional on significant F-value, post-hoc comparisons were performed by means of two-samples t-tests with Bonferroni’s correction where necessary. Correlations between TDMT or TDT abnormalities with disease severity (by SARA) and duration were explored by Spearman rank correlation. Significance level was set at P<0.05. Unless otherwise stated, data are given as mean ± standard error (SE).p = 0.3, independent-sample t-test) and gender distribution . PT and PPT did not significantly differ between CA patients and HC for both sides , due to higher TDT in CA patients compared to control subjects. Side  and Side by Group interactions were not significant  , due to higher TDMT in CA than in HC , with higher TDMT in FCR than in FDI muscle in both groups. Side , Side by Group interaction  and Side by Muscle by Group interaction were not significant . Further statistical details are reported in the For TDT, R-ANOVA disclosed a main effect of Group p = 0.4) , panel Aan in HC , panel BIn CA patients, Spearman rank correlation coefficients disclosed no significant correlation between TDMT or TDT abnormalities and disease duration  or severity, as evaluated with SARA .Our study showed that temporal processing of tactile and proprioceptive stimuli, evaluated by a psychophysical discriminative paradigm, is impaired in patients with neurodegenerative cerebellar ataxia. Abnormal temporal processing did not correlate with disease duration or severity. This might be explained by the lack of patients with short disease duration, as most of the subjects had a long disease duration (≥10 years in 70% of them). Alternatively, together with the lack of correlation with disease severity, we can speculate that impaired temporal discrimination is an abnormality not related to motor disability but rather reflecting dysfunction of the cerebellar pathways.The novel finding of our study is the increase of TDMT in patients with cerebellar degeneration. TDMT is a psychophysical technique measuring the temporal aspects of kinaesthesia Kinaestesia is an aspect of proprioceptive sensation with refers to the ability to sense the position and movement of a body part Our previous studies demonstrated that TMDT – which explores the sense of movement in the domain of time – mainly involve activation of muscle afferent fibres In conclusion, increased TDMT in patients with cerebellar degeneration support the role of cerebellum in updating the somatosensory cortex with proprioceptive inputs coming from muscle afferents during performance of movements; the alteration of temporal proprioceptive discrimination in patients with cerebellar degeneration support the role of cerebellum in performing computations of time differences, for all sensory modalities There are few possible interpretations about the increase of TDT in the patients with cerebellar disease. First, subtle abnormalities of peripheral sensory nerves might have influenced our results; however, this is unlikely as nerve conduction studies and evoked potentials did not disclose any significant abnormality in the sensory system in none of the patients. Moreover, perceptual tactile and proprioceptive thresholds were similar among patients and controls. As second hypothesis, presence of extra-cerebellar pathology in the basal ganglia might have determined higher tactile TDT; again, none of subjects presented with dystonic or parkinsonian features, which are reported to be associated with increased TDT Finally, impaired temporal processing of tactile stimuli might be caused by cerebellar degeneration itself. This finding was unexpected, as discriminative threshold for tactile stimuli has been considered to engage mainly basal ganglia processing; indeed, increased tactile TDT has been consistently reported in PD Against the hypothesis of cerebellar involvement in time processing of tactile stimuli, a recent study found that continuous theta-burst transcranial magnetic stimulation over of the lateral cerebellum left TDT unchanged Overall, our data of increased tactile TDT in patients with cerebellar degeneration highlight the contribution of cerebellum in temporal discrimination of tactile stimuli and challenges the view that this psychophysical paradigm might represent a marker of basal ganglia impairment.Accurate performance of voluntary movements relies on correct integration of sensory inputs which provide information on velocity and position, as well on visual and auditory feedback.Sensory information necessary to provide adequate motor control is conveyed both in the basal ganglia and the cerebellum. Basal ganglia function is to select between competing options in order to produce either goal-directed or habitual behaviours, which in turn depend on segregated functional territories On the other side, the cerebellum receives massive sensory inputs as demonstrated by functional neuroimaging studies disclosing cerebellar activation during cutaneous, kinaesthetic, visual or auditory stimulation Our data of abnormal TDT and TDMT in cerebellar neurodegeneration are consistent with the role of the cerebellum in timing processing of stimuli of different type and specifically in timed event prediction. Impairment on perceptual tasks that require precise timing and poor acuity on a time discrimination task has been reported in patients with cerebellar lesions Similarly to PD patients and differently from subjects with dystonia , our datThere are few possible explanations for this finding: first, it should be also considered that the pattern of neurodegeneration in PD is not limited to the basal ganglia, but can functionally affect interconnected areas such as the cerebellum itself Finally, the alteration of TDT in patients with cerebellar degeneration lead us to reconsider the meaning of higher TDT in dystonia


Heart failure represents an end-stage phenotype of a number of cardiovascular diseases and is generally associated with a poor prognosis. A number of organized battles fought over the last two to three decades have resulted in considerable advances in treatment including the use of drugs that interfere with neurohormonal activation and device-based therapies such as implantable cardioverter defibrillators and cardiac resynchronization therapy. Despite this, the prevalence of heart failure continues to rise related to both the aging population and better survival in patients with cardiovascular disease. Registries have identified treatment gaps and variation in the application of evidenced-based practice, including the use of echocardiography and prescribing of disease-modifying drugs. Quality initiatives often coupled with multidisciplinary, heart failure disease management promote self-care and minimize variation in the application of evidenced-based practice leading to better long-term clinical outcomes. However, to address the rising prevalence of heart failure and win the war, we must also turn our attention to disease prevention. A combined approach is required that includes public health measures applied at a population level and screening strategies to identify individuals at high risk of developing heart failure in the future. Heart failure is a clinical syndrome that represents the end-stage phenotype of a number of different cardiovascular diseases. However, regardless of the underlying cause, the prognosis is generally poor once a patient develops symptoms or signs of heart failure. There has been considerable progress in management including the use of drugs that interfere with neurohormonal activation, device therapy in selected patients, and multidisciplinary disease management. This at least partly explains the reductions in heart failure mortality seen in population-based studies over the last two decades; however, the overall epidemiological impact has been modest. This paper will provide an overview of the epidemiology of heart failure and outline our continued efforts to fight the battle focusing on management strategies that have been shown to modify disease progression. I will then consider whether or not we are winning the war and suggest that efforts to minimize variation in care should continue coupled with increasing emphasis on heart failure prevention.Heart failure is one of the few cardiovascular diseases whose prevalence continues to rise, largely related to the aging population accompanied by improved survival in patients with heart failure and following myocardial infarction , 2. InciThe prognosis of patients with heart failure is generally poor with frequent admissions to hospital throughout the disease trajectory and median survivals of 3–5 years , 23–28. Despite heart failure representing an end-stage phenotype of different cardiovascular diseases, numerous large-scale, randomized, and placebo-controlled studies have extended our mechanistic understanding of this syndrome. Most of these studies have been conducted in patients with moderate to severe LVSD regardless of the underlying cause. Broadly speaking, the therapeutic approaches can be divided into pharmacological therapies, device-based therapies, and coordinated disease management –51.Treatments primarily focused on managing congestion and hemodynamic derangements play an important role in treating decompensated heart failure with loop diuretics remaining the most effective pharmacological approach to manage congestion. However, these approaches have a limited effect on long-term clinical outcomes. Over the last three decades, a number of multicenter studies conducted in patients with symptomatic and asymptomatic LVSD have highlighted the role of neurohormonal activation on disease progression see  21, 52––52–65. Angiotensin converting enzyme (ACE) inhibitors improve symptoms, reduce hospitalization, and prolong survival in patients with symptomatic heart failure associated with moderate to severe LVSD both early following myocardial infarction and in the setting of chronic heart failure regardless of the underlying etiology , 58, 65.However, despite the well-established benefit from blockade of the renin-angiotensin system, the long-term cumulative mortality rates remained high reflecting ongoing disease progression, often associated with progressive adverse LV remodeling. Subsequent large, placebo-controlled studies emphasized the need for a combined approach to neurohormonal blockade. The addition of a beta blocker reduced mortality by 34-35% and decreased heart failure hospitalization in chronic heart failure patients with underlying moderate to severe LVSD on top of standard therapy which included an ACE inhibitor and diuretic in the majority of patients –61.http://www.fda.gov/drugs/drugsafety/ucm300889.htm). Furthermore, a population-based study conducted in Canada reported increased hospitalizations and deaths related to hyperkalemia, associated with increased rates of prescribing spironolactone to heart failure patients treated with ACE inhibitors following the publication of the Randomized Aldactone Evaluation Study (RALES) , 7978, 7Approximately 20–30% of patients with heart failure due to LVSD have abnormal intraventricular conduction manifested by a prolonged QRS duration on the 12-lead electrocardiogram . Short-tThe management of chronic heart failure includes lifestyle adjustment, initiation and uptitration of medical therapy, and selective consideration of device therapy. These cases are often diagnosed following an acute episode of decompensation with 25–40% of patients admitted to hospital with acute decompensated heart failure having no prior documented history of heart failure –98. The A large body of evidence supports the use of coordinated, multidisciplinary models of care usually led by heart failure nurses to support the transition of care from the acute hospital setting to community health providers. These services have been shown to prolong survival, reduce hospitalizations, and improve quality of life –105. FurWhilst there is a rich body of evidence supporting the safety and clinical efficacy of pharmacological and device-based therapies in heart failure, the key question is whether this translates to clinical effectiveness in a real-world population. Recent registries and population-based cohorts of patients admitted to hospital with acute decompensated heart failure have highlighted treatment gaps and considerable variation in the quality of care provided in different hospitals , 109–115Whilst the rates of prescribing disease modifying agents in patients admitted to hospital with heart failure have improved over the last decade, recent registries conducted in Europe, the Asia-Pacific region, and the USA still report discharge prescription rates of 63–80% for ACE inhibitors or angiotensin receptor blockers, 41–74% for beta blockers, and 20–48% for aldosterone antagonists , 122. FuReasons for the gaps in treatment provided in real-world practice compared to clinical trials may include the limitation of using administrative datasets to code heart failure patients, the different populations being treated, the limitation of assessing treatment in hospital following an acute decompensation, and the level of care provided.Administrative datasets rely on appropriate documentation by the treating clinicians and accurate coding by administrative staff. A number of studies have evaluated the accuracy of administrative coding demonstrating high positive predictive accuracy compared with independent chart abstraction or objective scoring systems such as the Boston score criteria –143. WhiPatients treated in clinical trials are on average over a decade younger than the patients we see in our clinical practice. Furthermore, the clinical trials which guide our practice either exclusively or largely enrolled patients with moderate to severe LVSD; yet up to half the patients enrolled in heart failure registries and population-based cohorts have preserved LVEF , 144–147http://www.jointcommission.org/specifications_manual_for_national_hospital_inpatient_quality_measures.aspx). However, few patients will have new therapies commenced in the first few months following hospital discharge [Hospital-based registries generally report in-hospital metrics to measure quality performance. Whilst performance of echocardiography and discharge prescription of ACE inhibitors or angiotensin receptor blockers are well-accepted inpatient quality measures of healthcare, discharge prescription of beta blockers was not included in the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) heart failure performance indicators . Our local experience matches that seen in most studies that report prescribing patterns in clinical practice see  124–137137, 155,The role of supplementary monitoring strategies to guide therapy and support disease management including telemonitoring and biomarkers remains the active area of investigation. Noninvasive telemonitoring has shown promise , 160; hoFinally, most of the therapeutic gains made with drugs and device therapy have been in heart failure due to LVSD. Studies conducted in patients with preserved LVEF have been disappointing, which probably at least partly relates to the difficulty in clearly defining this population –178. FurWhilst efforts to improve the quality of care and clinical outcomes of heart failure patients should continue, this will have a limited epidemiological impact on the human and economic burden imposed by heart failure in our society. Unless we also focus on early disease detection and prevention, we are in effect chipping away at the tip of the iceberg as incident cases of heart failure continue to emerge. The American College of Cardiology Foundation/American Heart Association (ACCF/AHA) staging classification emphasizes the need for disease prevention akin to the approach applied in cancer prevention . This clTo address the presymptomatic phase of heart failure, a combined approach is required that encompasses both public health measures applied at a population level and identification and management of individuals at high risk of developing heart failure in the future. Public health measures should focus on environmental contributors with the ultimate aim being to encourage and facilitate healthy lifestyles through education, legislation, and urban planning . SmokingThe best example of this is asymptomatic LVSD which is associated with a five-fold increased risk of developing heart failure with over half dying (most from cardiovascular causes) before they present with clinical heart failure . This reIf we can accurately detect LVSD, it would seem reasonable to link this with the evidence from treatment efficacy studies conducted in subjects with asymptomatic LVSD to infer the clinical effectiveness of a screening strategy, provided the subjects detected through screening represent the same spectrum of disease as the patients enrolled in the clinical trials see  140, 18. HoweverRather than limiting a screening strategy to identify asymptomatic LVSD, another approach is to screen for a number of structural cardiovascular derangements that are associated with an increased risk of developing heart failure including left atrial dilatation, LV diastolic dysfunction, LV hypertrophy, valvular heart disease, and aortic stiffness , 214–218Despite considerable advances in the treatment of heart failure over the last two to three decades, its prevalence continues to rise. Registries and population-based cohorts have revealed substantial gaps and variation in care. Multidisciplinary disease management brings us closer to the models of care that were applied in the randomized, controlled trials that guide our practice. More importantly, these programs improve quality of care, decrease mortality, and are cost effective in reducing hospitalizations in high-risk patients. However, to win the war, we also need to focus on disease prevention to reduce the human and economic burden imposed by heart failure. These strategies include addressing cardiovascular risk factors at a population level and screening for structural cardiovascular derangements such as asymptomatic LVSD.


Familial Mediterranean fever (FMF) is an autosomal recessive hereditary disease which is characterized by recurrent attacks of fever and peritonitis, pleuritis, arthritis, or erysipelas-like skin disease. As such, FMF is a prototype of autoinflammatory diseases where genetic changes lead to acute inflammatory episodes. Systemic inflammation – in general - may increase procoagulant factors, and decrease natural anticoagulants and fibrinolytic activity. Therefore, it is anticipated to see more thrombotic events among FMF patients compared with healthy subjects. However, reviewing the current available literature and based upon our personal experience, thrombotic events related purely to FMF are very rare. Possible explanation for this discrepancy is that along with the procoagulant activity during FMF acute attacks, anticoagulant and fibrinolytic changes are also taking place. Colchicine which is the treatment of choice in FMF may also play a role in reducing inflammation thereby decreasing hypercoagulability. Familial Mediterranean fever (FMF) is an autosomal recessive hereditary disease which is characterized by recurrent attacks of fever and peritonitis, pleuritis, arthritis, or erysipelas-like skin disease.in vivo activation of clotting, fibrinolysis, endothelial cells and platelets are identified in vitro by specialized clotting techniques and by specific antibodies directed at biomarkers of clotting activation and damaged vasculature.Hypercoagulability is a laboratory phenotype whereby In general, systemic inflammation may increase procoagulant factors and decrease natural anticoagulants and fibrinolytic activity.11We review the available literature about coagulability in FMF and try to understand why thrombotic events are rare in this disease despite its being a typical inflammatory disease.Fibrinogen contributes to thrombosis risk in different ways and is important in regulating inflammatory response.Thrombin, formed by activation of the coagulation cascade, is essential to promote hemostasis but also stimulates several cell functions, including chemotaxis and mitogenesis. Thrombin not only converts fibrinogen to fibrin, it also acts as a proinflammatory agent, resulting in a positive feedback loop of the inflammocoagulatory response.D-dimer is formed as a result of the plasmin-mediated, proteolytic degradation of cross-linked fibrin.Plasminogen activator inhibitor 1 (PAI-1) is produced by endothelial cells and inhibits fibrinolysis. PAI-1 is also considered to be one of the markers of endothelial dysfunction. An increase in PAI-1 causes hypofibrinolysis and results in a hypercoagulable state.Tissue plasminogen activator (tPA) is the trigger of the fibrinolytic activity, and fibrinolytic pathways must be activated along with the coagulation activation.P-selectin is a marker of thrombocyte activation. Serum levels of P-selectin did not differ significantly between FMF patients during the attack or attack-free periods. However, these levels were significantly lower in both patient groups compared with controls. Again, the low levels of P-selectin might reflect a response mechanism to counteract the hypercoagulable state in FMF patients. Nevertheless, since the blood sampling was late (in the FMF attacks), one should be cautious in drawing firm conclusions.13Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factors Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection.20Protein C activity decreased in FMF patients during attack-free periods compared with healthy controls. This finding may indicate the problem in suppressing inflammatory response through the protein C pathway in the acute attack.Fibronectin (FN) and thrombospondin (TSP) are extracellular matrix proteins. Fibronectin, apart from being an adhesive solid phase element, has many different roles in wound healing and cellular adhesion. More recently, FN has emerged as a player in platelet thrombus formation and diseases associated with thrombosis, including vascular remodeling, atherosclerosis and cardiac repair following myocardial infarction.21Thrombospondin is a glycoprotein that interacts with a wide variety of molecules, including heparin, fibrin, fibrinogen, platelet cell membrane receptors, collagen, and fibronectin. It also plays a role in platelet aggregation, tumor metastasis, vascular smooth muscle growth and tissue repair in skeletal muscle following crush injury. As such, it is another extracellular matrix product which is primarily found in the granules of thrombocytes. However, it can also be synthesized and secreted by many inflammatory cells, including neutrophils. Both fibronectin and thrombospodin may play a role in promoting hypercoagulability in addition to their role in inflammation. Plasma FN and TSP levels during acute FMF attacks were significantly higher than after resolution of the acute attacks. In addition, significant correlations were observed between both FN and TSP levels and CRP and WBC counts in two different studies.Von Willebrand factor (vWF) is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium, megakaryocytes and subendothelial connective tissue.Although thrombocytes can be used as a marker for acute phase reactants, and MPV levels may be the first to increase in response to inflammation, no differences in thrombocyte count and MPV were found between FMF patients and controls.13Increased levels of prothrombin fragments F1+2 were found in attack-free periods in FMF patients compared with healthy controls. No differences were found in levels of activated protein C resistance (APCR), antithrombin or anti cardiolipin antibodies in FMF patients and controls.14Many studies have shown that in FMF inflammation exists not only during the acute attacks, but also between attacks. Increased CRP and serum amyloid A (SAA) and elevated cytokines levels between attacks reflect an ongoing sub-clinical inflammation during these periods.The above-mentioned studies which dealt with coagulability changes in FMF patients shed some light on the association between coagulation and inflammation during the acute attacks and in between them. Most of these investigations show that in FMF - especially during the acute attacks - some parameters of coagulation are abnormal. This theoretically may increase the risk for thrombotic events in FMF patients. Nevertheless, reviewing the English literature for thrombotic events in these patients disclosed very few case reports. A single case report presented a child with FMF who experienced a stroke. An extensive thrombophilia work-up revealed that the patient had concomitant multiple inherited and acquired risk factors for thrombosis.a priori prone to develop thrombosis.Following the identification and isolation of the MEFV gene, several studies searched for a possible role of MEFV mutations in hypercoagulability. In fact a study by Rabinovitch et al. found that in patients with Behcet’s disease who carried the MEFV mutation M694V, there were more vascular and thrombotic events compared with BD patients who did not carry this mutation.The above cases show that FMF alone is not a direct cause for thrombosis and, in fact, in all the above cases there was an additional procoagulant background which led to the thrombotic complication.This observation raises the question regarding the explanation for the scarce reports on thrombotic events in patients with FMF only.in vivo. The higher than normal tPA levels during attack-free periods in FMF patients may indicate the importance of such persistence of the fibrinolytic activity for the balance of ongoing inflammation and coagulation and may be the key for the prevention of future thrombosis.13One possible answer could be that along with the ongoing inflammation in FMF leading to the procoagulant activity during the acute attacks and in between them, anticoagulant and fibrinolytic changes are also taking place. Thus, there may be a balance between coagulation and fibrinolysis An additional possible explanation is that all the above factors which are associated with hypercoagulability in fact also serve as inflammatory components. It may well be that during the acute attack of FMF they are consumed or used for the purpose of inflammation so that nothing is left for their role in the coagulation pathway. It seems that during an acute FMF attack the inflammatory process overcomes the coagulation process.Another question which might be raised is whether colchicine – which is taken continuously by FMF patients - has any role in the coagulation pathway.The observation that the highest levels of D-dimer were found during attacks in FMF patients who did not use colchicine suggests that colchicine decreases fibrinolytic activity by suppressing inflammation. Colchicine treatment is known to reduce the frequency and intensity of attacks in FMF, and it apparently also lowers the level of circulating fibrin, thereby reducing coagulation and later fibrinolysis.28In summary, the studies reviewed herein have just started revealing the procoagulant activity in FMF and its relationship to the acute attacks. Although it seems that coagulability is increased and enhanced during these attacks and in between them, the patients do not experience increased thrombotic events.Yet the current studies are few and in some cases the results are conflicting. Larger studies are needed to confirm the observations about the changes in coagulation in FMF patients. Furthermore, large case age matched control prospective studies are require to disclose the potential association of coagulation parameters with MEFV mutations.


Synaptic plasticity within the spinal cord has great potential to facilitate recovery of function after spinal cord injury (SCI). Spinal plasticity can be induced in an activity-dependent manner even without input from the brain after complete SCI. A mechanistic basis for these effects is provided by research demonstrating that spinal synapses have many of the same plasticity mechanisms that are known to underlie learning and memory in the brain. In addition, the lumbar spinal cord can sustain several forms of learning and memory, including limb-position training. However, not all spinal plasticity promotes recovery of function. Central sensitization of nociceptive (pain) pathways in the spinal cord may emerge in response to various noxious inputs, demonstrating that plasticity within the spinal cord may contribute to maladaptive pain states. In this review we discuss interactions between adaptive and maladaptive forms of activity-dependent plasticity in the spinal cord below the level of SCI. The literature demonstrates that activity-dependent plasticity within the spinal cord must be carefully tuned to promote adaptive spinal training. Prior work from our group has shown that stimulation that is delivered in a limb position-dependent manner or on a fixed interval can induce adaptive plasticity that promotes future spinal cord learning and reduces nociceptive hyper-reactivity. On the other hand, stimulation that is delivered in an unsynchronized fashion, such as randomized electrical stimulation or peripheral skin injuries, can generate maladaptive spinal plasticity that undermines future spinal cord learning, reduces recovery of locomotor function, and promotes nociceptive hyper-reactivity after SCI. We review these basic phenomena, how these findings relate to the broader spinal plasticity literature, discuss the cellular and molecular mechanisms, and finally discuss implications of these and other findings for improved rehabilitative therapies after SCI. Research into spinal plasticity over the past 50 years has shown that neurons within the spinal cord gray matter have a remarkable degree of plasticity, and in recent years we have seen a surge of interest in this field  stimulation both on the ipsilateral and contralateral hind-paw Woolf, . This suIn many ways central sensitization involves changes in the spinal cord gray matter that mimic hippocampal-mediated activity-dependent plasticity. In the hippocampus, tetanic stimulation of afferent pathways can increase responsiveness of subsequent post-synaptic potentials, a phenomenon known as long-term potentiation (LTP)  has the capacity to increase post-synaptic responses in the superficial spinal lamina, a phenomenon known as “wind-up”  modulate spinal plasticity within pain pathways by altering the plasticity of the ionotropic NMDA and AMPA receptors , stimulus association (Pavlovian conditioning), and response-outcome  learning  a spinally mediated flexion response. This habituation was not due to adaptation at sensory receptors in the periphery or to a change at the neuromuscular junction, suggesting that the memory for stimulus training history resided in spinal interneuronal synapses  conditioning. For example, the isolated spinal cord was shown to be capable of associating weak thigh stimulation  with strong plantar stimulation of the foot . With repeated CS–US pairings the thigh stimulation came to modulate the flexion withdrawal reflex, a type of Pavlovian conditioning known as “pairing-specific enhanced sensitization”  training of the spinal cord, with the goal of uncovering the basic principles that dictate spinal cord learning. The translational goal of this work is to provide basic knowledge that can help improve training-based rehabilitative therapies after SCI. Our spinal training model is based on the Horridge paradigm originally developed in headless insects and then later adapted to spinalized mammals to study learning and memory within the spinal cord  show persistent deficits in future learning when tested with response-contingent stimulation. This spinal learning deficit appears to reflect a long-term form of maladaptive spinal plasticity that endures for at least 48 h in completely transected rats Figure . Thus, uThe training-dependent effects of stimulus controllability on future spinal learning can be interpreted as “plasticity of plasticity” or “metaplasticity,” that regulates the capacity for future learning in a bidirectional manner  is given response-contingent stimulation and the other rat (yoked) receives stimulation whenever the master does. Even though the two rats receive stimulation at the same time, only master rats show instrumental learning. But is it necessary that stimulation pulses be separated at all to induce the yoked learning failures? Is it the total amount of shock delivered that predicts the induction of a spinal learning deficit, or the discontinuous nature of shock administration? Delivering 80 ms stimulation at an average of 2 s intervals over 6 min produces a total of approximately 180 shocks, and a total stimulation time of approximately 15 s. Thus, to test whether these temporal gaps are necessary, we applied a continuous 1.5 mA AC (60 Hz) shock for 15 s to one group, while another group received a continuous AC shock for the entire 6-min session. A third group received the typical intermittent stimulation schedule, and a control group received no stimulation. All rats were then subsequently tested for instrumental learning. As expected, the rats that received no stimulation were able to learn, and those that received intermittent stimulation failed. Interestingly, both groups that received continuous stimulation were able to learn, suggesting that the long-term effect of shock stimulation depends upon whether the pulses are separated by a temporal gap. Moreover, presenting a continuous tailshock while rats received intermittent legshock negated the adverse effect of intermittent stimulation, suggesting that continuous stimulation induces an opponent process that counters the induction of the learning deficit  to a noxious thermal stimulus applied to the tail  manner? Baumbauer et al.  exploredIn summary, several studies have demonstrated that the patterning of peripheral stimulation after SCI can have an enormous effect on whether adaptive or maladaptive plasticity emerges in the lumbar spinal cord. Response-contingent (master), predictable nociceptive stimulation promotes future spinal cord learning whereas unpredictable, intermittent nociceptive stimulation undermines future spinal learning and generates central sensitization. Therefore, discovering the biological mechanisms for these stimulus-induced forms of spinal plasticity has implications for both pain modulation and recovery of function after SCI.The preceding sections have outlined the fundamental features of spinal cord learning, and highlighted the importance of stimulus patterning in tipping the balance between adaptive and maladaptive forms of spinal plasticity. In the present section we will discuss the cellular and molecular mechanisms dictating spinal learning potential. The purpose of this line of research has been to discover biological mechanisms that modulate spinal learning, with the goal of developing therapeutic approaches to promote rehabilitation and recovery of function after SCI  that are known to modulate plasticity in the CNS through down-stream effects on iGluRs, including NMDA and AMPARs , did not exhibit a learning deficit, replicating the earlier finding from Crown and Grau  and persistent nociceptive stimuli can impair spinal cord learning. In the companion paper in this issue Grau et al.  discuss Dr. Crown is currently employed by Abbott Laboratories. The current research is not affiliated with his current duties at Abbott Laboratories. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Few distinctions in biology are as clearly drawn as the one between mitosis and meiosis. The function of mitosis is to produce two identical daughter cells, while the purpose of the first division of meiosis is to ensure the segregation of homologous chromosomes, and the second division is to create haploid gametes. These meiotic segregations usually rely on meiosis-specific processes such as the induction of preprogramed double-strand breaks, homolog pairing, synaptonemal complex (SC) formation, synapsis, and recombination between homologs to form crossovers. While not all meiotic systems function identically, enough commonalities exist to allow us to draw clear distinctions between meiosis and mitosis. Mitotic cells simply do not build SC, and the process of full-length homolog pairing is almost always a prerogative of cells entering meiosis, not mitosis.PLOS Geneticsmitotic divisions.Vexingly for those of us who like our distinctions made clearly, all this has changed, at least in Drosophila, as a result of two papers in this issue of Meiotic chromosome pairing in most organisms is thought to involve the formation of double-strand breaks and a subsequent single-stranded DNA homolog search, causing homologs to associate. This association is facilitated by the formation of the SC, a proteinaceous structure between homologs that is required for proper chromosome segregation. More recently, noncoding RNAs have been discovered that assist in the homolog search process Drosophila melanogaster, homologs undergo an early pairing event near the end of syncytial blastoderm development However, the study of meiotic pairing in Drosophila has been challenging for several reasons. First, it has been difficult to cytologically observe chromosome pairing in fixed images, and no imaging techniques currently exist to adequately follow the pairing process live. Moreover, there are no known mutants that specifically affect the pairing process in flies. Another impediment lies in the fact that in dipterans, such as not paired in either GSCs or their progenitors. Furthermore, homologous chromosome pairing occurs progressively through the early, pre-meiotic divisions in the germline The existence of ubiquitous pairing has led to the (far too popular) hypothesis that the pairing established early in Drosophila embryogenesis is maintained in the germline, and that meiotic homolog pairing is therefore simply an extension of this somatic pairing Female Drosophila meiosis occurs in the germarium of the ovariole, which is subdivided into three regions based on cytological observations of cell morphology X heterochromatin—which borders the rDNA genes—is paired in 80% of GSCs X heterochromatin in virtually any cell type may well reflect the fact that X chromosomes carry the 18S and 28S rDNA clusters and thus share a common nucleolus. However, for the two autosomes tested, Joyce et al. Using a novel technique to mark specific chromosomes It is possible that germline progenitor cells and GSCs contain a block to the somatic pairing process that is alleviated as cystoblasts begin mitoses in region 1 , allowinDoes the observation of SC in mitotically dividing cells reflect the pre-loading of SC that will assemble into mature SC as meiosis initiates in region 2, or does it reflect the existence of mature, structurally normal SC? And perhaps more importantly, does this mitotic SC play a role in mediating homologous centromere pairing? The meiosis-specific protein components of the Drosophila SC are the lateral element proteins Ord Christophorou et al. used mutants of these SC components to show that Cona and C(3)G promote centromere association during mitosis in region 1 and necessary for efficient homologous centromere pairing in mitotic cells in region 1 further erodes the notion that meiotic pairing is simply an extension of somatic pairing.It is unclear how the SC or its components function to help pair homologous centromeres in region 1. While SC mutants are known to disrupt centromere clustering in early region 2 proocytes, it is intriguing that although centromere pairing in region 1 occurs more slowly in these mutants, it is not abolished is built between the two pairs of sisters, then the only way a cell could mitotically divide would be to disassemble the SC at every division. Why would a cell build centromeric SC, only to dismantle and rebuild it after every division? It is becoming increasingly apparent that pericentromeric SC differs from the SC along chromosome arms These results create a puzzling situation. How can a cell undergo mitosis with SC present near homologous centromeres? Moreover, as Christophorou et al.


Conservation attention on charismatic large vertebrates such as dolphins is often supported by the suggestion that these species represent surrogates for wider biodiversity, or act as indicators of ecosystem health. However, their capacity to act as indicators of patterns or trends in regional biodiversity has rarely been tested. An extensive new dataset of >300 last-sighting records for the Yangtze River dolphin or baiji and two formerly economically important fishes, the Yangtze paddlefish and Reeves’ shad, all of which are probably now extinct in the Yangtze, was collected during an interview survey of fishing communities across the middle-lower Yangtze drainage. Untransformed last-sighting date frequency distributions for these species show similar decline curves over time, and the linear gradients of transformed last-sighting date series are not significantly different from each other, demonstrating that these species experienced correlated population declines in both timing and rate of decline. Whereas species may be expected to respond differently at the population level even in highly degraded ecosystems, highly vulnerable (e.g. migratory) species can therefore display very similar responses to extrinsic threats, even if they represent otherwise very different taxonomic, biological and ecological groupings. Monitoring the status of river dolphins or other megafauna therefore has the potential to provide wider information on the status of other threatened components of sympatric freshwater biotas, and so represents a potentially important monitoring tool for conservation management. We also show that interview surveys can provide robust quantitative data on relative population dynamics of different species. Charismatic large vertebrates represent a very small component of global biodiversity, but have always received a disproportionate amount of conservation attention and funding due to high public and scientific interest in such species Platanista gangetica) may be resilient to changes in fish community structure, and their status may not provide an accurate indication of the depletion of specific fish resources However, the ability of large vertebrates to act as surrogates for wider biodiversity has been increasingly questioned. There is little quantitative evidence that these taxa are actually efficient umbrella, keystone or indicator species Psephurus gladius) is a diadromous migratory species that was restricted to the Yangtze drainage and neighbouring East China Sea by the late twentieth century, although it was also historically recorded from the Yellow River and Yellow Sea Tenualosa reevesii), constituted one of the most important commercial fisheries in the middle-lower Yangtze until the mid-1970s, but the shad fishery closed in 1987, and the species is also now probably extinct in the Yangtze Lipotes vexillifer) and the Yangtze finless porpoise . Both of these apex predators have also experienced serious declines during recent decades due to incidental by-catch in fishing gear, vessel strikes, and wider-scale habitat degradation The Yangtze River drainage, the longest river system in Asia, supports approximately 10% of the world’s human population Although some historical catch records or abundance estimates are available for each of these species Interviews were conducted in 2008 in 27 fishing communities along the middle-lower Yangtze channel (1767 km stretch from Yichang downstream to the Yangtze estuary) and around two major lake systems (Dongting and Poyang Lakes) connected to the Yangtze mainstem . Baiji, F = 0.94, p = 0.395), demonstrating that the three target species experienced statistically similar rates of population decline over time. The intercepts of the linear gradients of transformed last-sighting date series for all three species are also not significantly different . The non-significant result was also borne out by non-parametric Monte Carlo tests: variance test p = 0.643; minimum difference p = 0.144; middle difference p = 0.669; maximum difference p = 0.346.In total, 365 baiji records, 338 paddlefish records and 375 shad records  were collected for comparison of last-sighting dates. Untransformed last-sighting date frequency distributions for all three species show similar decline curves over time . The modOnly 7.5% of informants who provided information on finless porpoise sightings (n = 563) stated that they had not seen the species in the year that interviews were conducted and provided pre-2008 last-sighting dates, and only a further 2.5% suggested they had not seen any porpoises in that year but were uncertain about when they had last seen the species . It is nPopulation trends are typically analysed using longitudinal studies that collect progressive data on the status of target taxa over time, a technique that can permit reconstruction of relative or absolute population status depending on data quality In addition to providing extensive new information on the extinction chronologies of baiji, paddlefish and shad, our last-sighting data demonstrate that these three species experienced correlated declines in terms of both the rate and timing of decline. This provides the first quantitative evidence that the status of dolphin populations can act as a population trend indicator of some other sympatric species, and perhaps of wider aquatic biodiversity. In order to assess the extent to which apex predators such as river dolphins can represent meaningful population trend indicators across other ecological systems, it is necessary to consider the relative significance of biological, ecological, and anthropogenic factors in determining patterns of extinction risk in freshwater taxa.Historical data on trophic ecology and landscape-level habitat use of baiji, paddlefish and shad are limited If the entire Yangtze freshwater biota has experienced a similar pattern of decline due to this overwhelming suite of extrinsic extinction drivers, the correlated declines of river dolphins and various fishes would be unsurprising and of little interest. However, both our porpoise last-sighting data and available Yangtze fisheries data Cyprinus carpio) and southern catfish  have remained relatively stable and widely distributed over much of the Yangtze drainage, and have become increasingly important target species for regional fisheries as other species have declined One key factor likely to explain at least some of the increased extinction vulnerability shown by baiji, paddlefish and shad in contrast to finless porpoise and other species is migratory status. Migratory reproductive behaviour is associated with higher extinction risk in fishes Even in an ecosystem such as the Yangtze that is exposed to a potentially overwhelming suite of extrinsic threats, different species can therefore be seen to respond differently to anthropogenic processes. However, our data suggest that the most vulnerable set of species  can experience correlated responses to extrinsic threats and display spatially and temporally similar patterns of decline, even if they represent otherwise very different taxonomic, biological and ecological groupings. The ability of temporal baiji population trend data to provide a meaningful indication of simultaneous trends in paddlefish and shad populations therefore does not represent a causal relationship associated with the baiji’s status as apex predator or putative keystone species, but instead the shared status of these species as being similarly highly vulnerable to regional threat processes due to specific aspects of their ecology . Conversely, finless porpoise population trend data do not represent a good surrogate for either paddlefish or shad population trends, because this species was less vulnerable to anthropogenic disturbance as a result of these specific ecological characteristics.Tenualosa ilisha in the Ganges-Brahmaputra-Meghna drainage) is necessary to test the generality of these findings, to define patterns of ecological causality and correlation between these taxa, and to assess the wider usefulness of dolphins as indicators of the status of regional biodiversity and the ‘health’ of aquatic ecosystems.Monitoring the status of river dolphins or other charismatic and vulnerable freshwater megafauna has the potential to provide wider information on the status of other highly threatened components of sympatric freshwater biotas. However, whereas river dolphins may behave as indicators of the status of wider trends in biodiversity under such conditions, other species with a variety of different biological and ecological traits may thus also be expected to perform well, and may even be easier to monitor in practice Informants were located through targeted sampling Acipenser sinensis, Yangtze sturgeon A. dabryanus) also found in the middle-lower Yangtze. Any appropriate regional names for paddlefish  recognized by at least one member of each fishing community were used in conjunction with the standard Chinese name for the species (baixun), and informants were required to demonstrate their ability to differentiate between these sympatric species  in order for their accounts to be recorded. Other data collected during the wider interview survey have been published elsewhere As part of a wider series of interview questions , informaInterview survey methods followed the Zoological Society of London (ZSL)’s guidelines for ensuring appropriate ethical standards in projects involving data collection from humans for research purposes, and standard fieldwork protocols for our longer-term research programme into local ecological knowledge in Yangtze communities have been approved by ZSL’s Ethics Committee . All informants were told about the aims of the survey and ensured that data would be analyzed anonymously; interviews were only conducted following verbal consent and approval of participants, such that their co-operation in completing an interview questionnaire represents our record of their verbal consent.Although about half of all last-sighting dates  were reported as direct calendar years, many informants also reported last-sighting dates in alternative formats: paired consecutive calendar years (e.g. ‘1986/1987’), decadal or other ranges , or decades/half-decades before 2008 . More recent sighting dates were more likely to be reported as direct calendar years because informants had more accurate recall over shorter time intervals, but to avoid excluding large amounts of additional sighting data from further analysis, dates reported in alternative formats were randomized to direct calendar years as follows: paired calendar years were given an equal probability of being assigned to either consecutive year; decadal or other specific ranges were given an equal probability of being assigned to any calendar year within this range; and dates rounded to a given number of decades or half-decades before 2008 were given an equal probability of being assigned to any calendar year ±5 the given value .The number of reported last-sighting dates for a recently extinct species is expected to decrease over recent time as a result of the species’ population decline, but will also drop off further back in time into the period before informants experienced their final sighting of the species. A frequency distribution of last-sighting dates will therefore be approximately bell-shaped . Only thp values.As the residuals from the above model were slightly non-normal, and subsequent transformations did not correct this, they were also analyzed using a non-parametric Monte Carlo test of the differences between gradients. Several metrics of the real differences between gradients were compared with a set of values for each metric expected under the null hypothesis. These metrics (test statistics) comprised: variance among gradients; maximum value of the two-way difference between gradients in the three gradient pairs; and middle and minimum values of the difference between gradients in the three gradient pairs. The null values were generated as follows: for each year from 1983–2008, the values of the response for each species were randomly reassigned without replacement to one of the three species. Decline gradients were calculated in the same way as for the real data, for 99,999 randomizations. Test statistics for real species data were ranked within those for 99,999 randomly allocated data points to give Spatial variation in last-sighting dates across the survey area was not further investigated, because previous analysis of interview data has shown that baiji population decline was not associated with any major contraction in geographical range across the middle-lower Yangtze drainage Text S1Yangtze fishermen interview questionnaire (English-language version).(DOCX)Click here for additional data file.Table S1Summary of interview data collected from the middle-lower Yangtze region.(DOCX)Click here for additional data file.