As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality. A large body of evidence indicates that both acute exercise and habitual physical activity affect platelet function. This is of special interest as the inflammatory and immunomodulatory consequences of platelet activation are increasingly recognized and platelets therefore seem to be of central importance not only to the final stages of cardiovascular disease (CVD), but also to the development of these diseases. Therefore, a modulation of platelet function by acute exercise and/or habitual physical activity might represent a mechanistic link between physical exertion and its observed effects on CVD. This would be especially interesting as a remarkable strong correlation exists between CVD-related mortality and physical activity, as we will discuss later in this review.In order to give a structured overview based on currently available literature, the first part of this review will deal with the influence of acute (mostly strenuous) exercise on platelet function. To introduce the reader to different aspects of platelet activation as well as platelet function tests , this will be done from a platelet-centered view where different aspects of platelet activation are treated separately. Subsequently, the impact of exercise intensity and the subjects' cardiorespiratory fitness on the effects of exercise on platelet function is summarized and discussed, including the modulating effects of cardiorespiratory fitness/physical activity on platelet function in the resting state.Platelets represent the smallest formed elements of blood. They are anucleate cells with a life-span of 7–10 days and contain a variety of intracellular organelles, including different types of secretory granules.Activation of platelets, finally resulting in platelet degranulation and aggregation, is essential for hemostasis and can be triggered by several specific platelet-stimulating mediators  as well as by shear and oxidative stress.As blood platelets are of central importance to the process of (primary) hemostasis and coagulation, abnormalities in platelet function (resulting in thrombosis or bleeding) result in severe and potentially lethal consequences. In principle, activation of platelets immediately results in platelet aggregation and subsequent thrombus formation—however, these consequences of platelet activation can to some extent be antagonized by functional endothelium, pointing to a crucial role of endothelium-derived mediators that counteract platelet activation  in this process. However, as activated platelets compromise endothelial function, the situation is complex.The importance of platelets to the development of atherosclerotic disease is apparent from the fact that platelet activation is associated with accelerated atherosclerosis and correlates with severity of this disease in humans. An injection of activated platelets exacerbates the formation of native atherosclerotic lesions and an increase in systemic platelet activation has been described for a variety of atherosclerotic diseases including coronary artery disease , 2 and cWhile the in vitro assessment of platelet reactivity in different settings (including acute exercise and training) might include the final consequences of platelet activation , measurement of in vivo platelet activation depends on more subtle parameters.As different pathways of platelet activation might result in distinct patterns of “platelet activation markers,” such studies require the consideration of several aspects of platelet activation for a final interpretation of obtained results.Several studies show that acute exercise results in a transient increase in platelet count. This increase is caused by hemoconcentration and by platelet release from the liver, lungs, and, importantly, the spleen –6. The lαIIbβ3) is constitutively expressed on the surface of resting platelets and represents the main fibrin(ogen) receptor on these cells. Upon platelet activation, this receptor undergoes a conformational change resulting in a drastically increased affinity towards fibrin(ogen), enabling the firm binding of platelets to fibrin(ogen) and thus facilitating platelet-platelet interaction, that is, platelet aggregation and the formation of stable thrombi. Only a limited number of studies found an increase in GPIIb/IIIa activation after exercise [Glycoprotein IIb/IIIa  is carried out by the Platelet Function Analyzer (PFA-100), where platelets in whole blood are activated by the combined action of shear stress and agonists, resulting in closure of an aperture. After exercise, shorter closure times have been reported , 29, 53;Various platelet adhesion assays have been used in the literature to test for an effect of exercise on platelet function. These studies have yielded inconsistent results; whereas one study showed decreased platelet adhesion to fibrinogen  , other gβ-thromboglobulin  and platelet factor 4 , can be used as a measure of (in vivo) platelet degranulation/activation.Degranulation represents an integral consequence of platelet activation that results in the concomitant release of a variety of soluble mediators and the expression of distinct proteins on the platelet surface due to membrane fusion of the granules with the platelet membrane. Consequently, de novo protein expression, for example, of P-selectin (CD62P), on the platelet plasma membrane as well as plasma levels of soluble mediators, for example, β-thromboglobulin [β-thromboglobulin and platelet factor 4 represent very sensitive marker of platelet activation, these findings represent strong evidence that exercise activates platelets, although a small number of studies reported unchanged [β-thromboglobulin after exercise.After exercise, several studies found increased plasma levels of globulin , 60, 61 globulin , 60, 62.nchanged , 63 or dnchanged  levels oAlthough the (activation-dependent) expression of P-selectin is not restricted to platelets, the fact that degranulation initially results in the expression of membrane-bound P-selectin (that is later on cleaved off to form soluble P-selectin) makes this glycoprotein an invaluable marker of platelet activation. P-Selectin represents a pivotal adhesion molecule mediating cell-cell contact of platelets with leukocytes, endothelial cells, and other platelets. Binding of platelet P-selectin to its receptor PSGL-1 on leukocytes results in aggregate formation of platelets with leukocytes (see below), promoting the stimulation of leukocytes—a process involved in many inflammatory conditions.The influence of acute exercise on the expression of P-selectin on platelets has been addressed by several studies and yielded contradictory results. Basal P-selectin expression  has been found to be increased , 64–68 aConsistent with results of studies showing increased platelet P-selectin expression after exercise, formation of aggregates with (various subsets of) leukocytes  has been detected in response to acute exercise.Overall PLAs were found to be increased , 69 and Within these studies, both basal –71, 74 aμm and 1.0 μm) membrane vesicles that represent an important link between platelet activation and plasmatic coagulation, as PDMPs provide a large surface of phosphatidylserine necessary for tenase and prothrombinase complex formation. After exercise, the levels of PDMPs in plasma have been reported to be increased [A further possible consequence of platelet activation is the release of platelet-derived microparticles (PDMPs) that constitute small , and CD39/CD73 represent the physiologic effectors of the three major pathways that are relevant to the inhibition of platelet function (for a detailed review see [Nitric oxide (NO), prostacyclin (PGIview see ).2 inhibit platelet function via an increase of cyclic nucleotides (NO mainly via cGMP and prostacyclin via cAMP), which subsequently activate their respective protein kinases , which in turn phosphorylate key proteins ultimately leading to platelet inhibition. Notably, since platelet activation is associated with an increase in cytosolic calcium concentration, NO generation within platelets represents also an early consequence of platelet activation, thereby playing an important autoregulatory role that limits excessive aggregation, adhesion, and thrombus growth [2), or also a result of platelet activation. Consequently, direct and indirect quantification of NO and PGI2 may provide information concerning the production of these compounds but do not necessarily indicate inhibition of platelets as the net effect of activating versus inhibitory stimuli remains obscure without a direct assessment of platelet function.(Increased) production and release of both NO and prostacyclin is mediated by an increase in cytosolic calcium concentration, which can be triggered by shear stress (what is obviously relevant to exercise) and a multitude of other mediators. Whereas NO is produced by platelets as well as other cells including endothelial cells and red blood cells, prostacyclin synthesis occurs in endothelial cells and smooth muscle cells but is absent in platelets. Redundancy between these two systems has been reported, where one system takes over when the other one is compromised, for example, increased NO generation when prostacyclin production is impaired . Both NOs growth , 84. Thu2 are commonly quantified by their degradation products nitrite and nitrate (in the case of NO) and 6-keto-PGFα1 (in the case of prostacyclin).Due to their short in vivo half-life, both NO and PGIAvailable studies indicate increased prostacyclin generation in response to acute exercise , 85–88 aSimilarly, increased plasma levels of NO degradation products nitrite and nitrate have been observed together with platelet activation , 80, 85 2-receptors and a subsequent increase in cAMP. After exercise, basal as well as ADP-induced CD39 expression on platelets has been reported to be decreased while ADP-stimulation increased CD39-expression before as well as after exercise [CD39/CD73 constitutes a further pathway that interferes with platelet function. CD39 is expressed on a number of vascular cells, including endothelial cells and platelets , 91. CD3exercise . This isShort Summary. Taken together, despite some inconsistency and contradictory results, the majority of available studies indicate that acute exercise exerts a significant influence on virtually all aspects of platelet activation. Although  different protocols might partly account for contradictory results found in the literature, results from several studies indicate that the applied exercise intensity might represent a critical determinant for the platelet-activating effects of acute exercise.To test for the hypothesis that exercise intensity affects platelet function, one has to consider that there are several ways to define exercise intensity—each with certain advantages and drawbacks. Although it is beyond the scope of this review to discuss this topic in detail , one cr2max). Whereas the 30-minute submaximal trial resulted in decreased platelet adhesion and aggregation (β-TG and PF4 remained unchanged), all of these parameters were increased after the incremental test until exhaustion. These results were basically confirmed by a study with similar study design [2+ levels were measured. Intraplatelet Ca2+—basal as well as after ADP stimulation—was shown to be increased only after the incremental test. Similarly, Chicharro et al. [2max and even decreased in response to low concentrations of platelet agonist [2max) with respect to their effects on platelet function. These authors did not observe any effects on platelet aggregation but found reduced closure time measured with the PFA-100 in response to epinephrine and ADP. Notably, these effects were more pronounced at the higher intensity. Comparable intensities  were applied in a study where intensities were defined by means of 90% or 130% of the first ventilatory threshold . In this study, hemostatometry readings in terms of increased thrombus formation were only affected by the higher intensity [Nevertheless, a few studies directly compared different exercise intensities with regard to their effects on platelets. These studies provide substantial evidence that exercise intensity indeed represents a critical determinant of platelet activation. Wang et al.  comparedy design , where ao et al.  reported agonist . Cadroy  agonist  applied ntensity .2max compared to rest, but less PLAs (compared to rest) in response to an exercise intensity of 40% VO2max. Notably, there were no stimulatory effects of exercise on PLAs in the absence of agonists. Also Hilberg et al. analyzed P-selectin expression as well as different subsets of PLAs in a study where they compared effects of an incremental step test until exhaustion with those of 45 minutes at 90% of the IAT  [More recent studies also addressed P-selectin expression and platelet-leukocyte formation. Wang et al.  observedse test) . Whereas2 was increased after an incremental exercise test until exhaustion but not after 15 minutes of continuous exercise at 75% of maximal heart rate, platelet aggregation increased in response to both exercise protocols, but this increase was more marked after the incremental test.Experiments performed with an ergometer for the upper limbs  indicateBesides exercise intensity, also cardiorespiratory fitness, that is, the adaptation to long-term exercise training, might represent a critical determinant for alterations of platelet function in response to acute exercise.β-TG and PF4 tended to be more pronounced in the sedentary group. Coppola et al. [Some studies directly compared the effects of acute, strenuous exercise on platelet function between two groups differing in cardiorespiratory fitness and habitual physical activity, respectively. By and large, results obtained from these studies suggest that low cardiorespiratory fitness results in greater platelet activation after acute exercise. Kestin et al.  compareda et al.  also com2max and their platelets were exposed to a higher shear stress. This is especially relevant in light of the fact that shear stress is able to activate platelets and therefore should be considered when interpreting the results of this study.Another recently published study assessed the effect of acute (moderate) exercise on the occurrence and procoagulant activity of platelet-derived microparticles. In untrained as well as highly trained individuals, 90 minutes of cycling at 80% of IAT caused a significant increase in both the number of PDMPs and their coagulant activity. However, after two hours the number of PDMPs remained significantly elevated only in untrained subjects . Such a In addition, also longitudinal studies have been carried out, where acute effects of exercise on platelet function were assessed before and after a long-term exercise training program. In an early study including only 6 healthy volunteers, 20 minutes of cycling with 70–80% of maximal heart rate was shown to cause a significantly increased slope of ADP-induced platelet aggregation, while 12 weeks of regular exercise training abrogated this response to acute exercise. In line with these findings, Wang et al. , 48 obseIn a population predestined to be particularly unfit, namely, (untrained) patients with spinal cord injury, El-Sayed et al.  observedTaken together, these studies show that regular exercise training mitigates the activating effect of acute, strenuous exercise on platelet function. In addition, a number of studies indicate that long-term exercise training also affects platelet function at rest.2max, 5x/week for 45–60 min) for 12 weeks led to a reduction of secondary platelet aggregation in blood that was sampled at physical rest [An early study performed in overweight, hypertensive men showed that low intensity training , decreased both platelet adhesion and aggregation in response to several stimuli, including oxidized LDL and shear stress [2max, 3x/week for 30 min) increased plasma and intraplatelet nitrite/nitrate levels and decreased platelet aggregation in response to collagen and ADP [2max 3x/week for 30 min) on platelet aggregation at rest [Importantly, effects of exercise training on basal platelet function have also been shown in healthy volunteers. Exercise training performed on a bicycle ergometer 5x/week for 30 min over a period of ~8 weeks with healthy, but  sedentary, men or women, respectively  was sufficient to decrease platelet adhesion to fibrinogen (while showing no effect on platelet aggregation) . In healIn any way, physical activity habits and cardiorespiratory fitness appear to have major impact on platelet reactivity, but it should be kept in mind that platelet function observed in physically active subjects obviously represents the physiologic state; therefore, the conclusion that a sedentary lifestyle and low cardiorespiratory fitness affects platelet function in a prothrombotic, proinflammatory manner would probably describe this issue in an accurate way.2max) was used in the cited study reporting a decrease in platelet adhesion in response to exercise [β-TG plasma levels were measured after exhaustive exercise, unchanged or decreased levels were only observed after 20–30 min exercise protocols with an intensity not higher than 70% of VO2max or maximal heart rate, respectively [Considering exercise intensity as well as physical activity and fitness, inconsistencies between different studies cited in the first, platelet function centered part of this review can be partly explained. For example, in those studies reporting inhibition of platelet aggregation , 27, 28 exercise . Moreoveectively , 26.Available literature that has been summarized in this review indicates that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness prevent platelet activation in response to acute exercise, at least to a certain degree, and (iii) although a remarkable low number of studies have been performed on this topic in healthy populations, habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest.Therefore, physical activity habits and cardiorespiratory fitness appear to have major impact on platelet reactivity, but it should be kept in mind that platelet function observed in physically active subjects obviously represents the physiologic state, since endurance exercise—especially running—played a key role in the evolution of the human species . TherefoObviously, platelet function is directly and indirectly affected by physical (in)activity. In this regard, several mechanisms and cell/tissue types are supposed to contribute to the observed effects. Acute exercise results in increased levels of catecholamines as well as increased shear and oxidative stress, all of which are known to activate platelets. This is especially relevant as artery blood flow and shear rate increase in parallel to exercise intensity . While eAs both NO production and antioxidant defense are increased as a consequence of training, also bioavailability of NO is affected by regular exercise and this would help to explain several effects of physical (in)activity on platelet function even more as low shear rates have been shown to stimulate endothelial ROS generation .Notably, while short-term exercise training enhances production and bioavailability of NO, long-term adaptations taking place in specific vascular regions induce structural changes to the vessel resulting in an increase in lumen diameter, thereby decreasing shear rates while normalizing endothelial NO production , 108. InWhile the contribution of platelets to the final stages of CVD is obvious, the central importance of these cells and their activation state to the development of cardiovascular disease is increasingly appreciated as chronic platelet activation and platelet hypersensitivity result in increased liberation of growth factors and proinflammatory mediators. Therefore, it is of special interest that the effects of (both acute and chronic) exercise on platelet function show obvious similarities to outcomes from epidemiologic studies on the impact of exercise on the development of cardiovascular events where it has been shown that vigorous exercise transiently increases the risk for myocardial infarction –112. AppThis obvious parallelism poses the intriguing question if the assessment of platelet function in response to acute and habitual exercise might help to predict the beneficial effects of physical activity/fitness on (the development of) CVD. This would offer the possibility of acquiring information from platelet-related studies that is not available from long-term studies with cardiovascular events defined as the primary endpoint. Consequently, this would also allow directly studying and comparing the potential effectivity of different training programs and exercise intensities for primary and secondary prevention of CVD. Although this is speculative at the moment, clearly more research is required on this highly relevant topic as well as on the mechanisms that are involved in the modulation of platelet function by habitual exercise.


The ability to imitate complex sounds is rare, and among birds has been found only in parrots, songbirds, and hummingbirds. Parrots exhibit the most advanced vocal mimicry among non-human animals. A few studies have noted differences in connectivity, brain position and shape in the vocal learning systems of parrots relative to songbirds and hummingbirds. However, only one parrot species, the budgerigar, has been examined and no differences in the presence of song system structures were found with other avian vocal learners. Motivated by questions of whether there are important differences in the vocal systems of parrots relative to other vocal learners, we used specialized constitutive gene expression, singing-driven gene expression, and neural connectivity tracing experiments to further characterize the song system of budgerigars and/or other parrots. We found that the parrot brain uniquely contains a song system within a song system. The parrot “core” song system is similar to the song systems of songbirds and hummingbirds, whereas the “shell” song system is unique to parrots. The core with only rudimentary shell regions were found in the New Zealand kea, representing one of the only living species at a basal divergence with all other parrots, implying that parrots evolved vocal learning systems at least 29 million years ago. Relative size differences in the core and shell regions occur among species, which we suggest could be related to species differences in vocal and cognitive abilities. Vocal learning, a critical behavior for spoken language, is the ability to modify acoustic and/or syntactic features of sounds produced, including improvisation and imitation. Complex vocal learning has so far been found in five distantly related groups of mammals  and three distantly related groups of birds  –5. VocalPsittacus erithacus), and corvid songbirds, such as crows and the Greater Indian Hill Mynahs (Gracula religiosa intermedia), and humans [Tursiops truncatus), killer whales (Orcinus orca), harbor seals (Phoca vitulina), and elephants [Interestingly, not all vocal learners display vocal mimicry, defined as the ability to imitate heterospecific vocalizations –13. Specd humans –16. Somed humans ,15,17,18lephants –23, but Melopsittacus undulatus) [The neural pathways for vocal learning have been characterized in multiple songbird and hummingbird species, but in only one parrot species, the budgerigar (dulatus) ,24–29. Idulatus) ,30–34). dulatus) ,30 best dulatus) .Thus far, differences found between vocal learning bird species include that the posterior vocal motor pathway is also anatomically adjacent to the auditory pathway in songbirds, but more distant to it in hummingbirds and parrots , blue 2,27. The PVALB), glutamate receptors, FOXP1, and SLIT1 [To better understand the organization of the song system in parrots, its evolution, and specialized molecular properties relative to other vocal learning avian species, we performed analyses of brain gene expression profiles in nine different parrot species representing all the three superfamilies, Strigopoidea, Cacatuoidea, and Psittacoidea, for genes with known specialized expression in vocal learning nuclei, and compared those findings with vocalizing-driven gene expression and neural tracing experiments. The set of genes that show specialized expression, meaning higher or lower expression in the song nuclei compared to the surrounding regions, include among many others Parvalbumin  the inner core song nuclei; (2) the outer shell song nuclei that surround the cores; and (3) the outer non-vocal motor regions that surround the song nuclei shells. We described our more in depth findings in budgerigars first, and then comparisons with other parrot species.PVALB gene, which generates a calcium-binding protein with a role in neuroprotection and critical period plasticity, preferentially in GABA-expressing interneurons [PVALB mRNA expression serially throughout the budgerigar forebrain. We found that PVALB expression revealed core (very high) and shell (moderate) regions of expression that included the posterior song nuclei NLC and AAC and surrounding motor areas relative to the surrounding nidopallium (N) and arcopallium (A), respectively  in the thalamus. There was also higher expression at the boundaries of the ventral mesopallium (MV) with the adjacent nidopallium and the dorsal mesopallium (MD) with the intercalated hyperpallium  and NAO  . However, we noted a uniformly moderate higher expression than the surrounding striatum (St) in sections that spanned the MMSt song nucleus rostro-caudally  Fig –3c. The ium) Fig . The NAOentation  as previPVALB-defined core and shell regions, and whether core and shell song nuclei regions were confined to the Nissl-defined boundaries we identified, we performed side-by-side comparisons of comparable serial brain sections that we had collected with vocalizing-driven (warble song) and non-singing motor-driven (hopping) DUSP1, EGR-1 , and C-FOS IEG expression [DUSP1 is an activity-dependent dual sensitivity phosphatase that shows motor-driven gene expression only in song nuclei of vocal learners and sensory-driven expression in the primary sensory neurons in all species [EGR-1 and C-FOS are activity-dependent transcription factors that show behaviorally-driven expression in many cell types, including in song nuclei and the surrounding motor and auditory regions [To determine the functional makeup of the pression ,35,44,45 regions ,35,44,46EGR-1 and C-FOS boundaries encompassed the entire PVALB-defined cores and part of the PVALB-defined shells of the NLC and AAC  around the NLC shell and the lateral intermediate arcopallium (LAI) around the AAC shell (EGR-1 or C-FOS (DUSP1 not shown) induction in the PVALB-defined song nuclei and surround  and shells (moderate to low expression) that are vocally active; and the remainder of the PVALB-defined shells (moderate expression) that are non-vocal motor active regions . The anterior song nuclei shell regions share non-specialized PVALB expression with the surrounding motor regions  and the Anna’s hummingbird , respectively.Core versus shell gene expression specializations have not been described in the ‘vocal-motor active’ brain regions of songbirds or hummingbirds. To validate whether the vocal-motor pattern we found is unique to parrots, we examined published and previously unpublished serial sections containing song nuclei that were hybridized for ingbirds ,38,44,45PVALB expression “within” the song nuclei. Instead, in support of previous conclusions [PVALB expression was uniformly high throughout each song nucleus  [SLIT1 downregulation corresponds to our PVALB-defined AAC core, and that SLIT1 expression in the PVALB-labeled AAC shell was not different relative to the surrounding arcopallium  expression in what we define here as both NLC, AAC, and NAO core and shells, and only the MO core (PVALB-defined NLC shell and the motor part around the NLC shell compared to the NLC core where TH fibers were sparse (C (AACd) ,39. We flium Fig  and 7b. ongbirds , was repongbirds ,54; we fund MMSt ,55. NR2Ation Fig  and 8b an in NLC   NLC core, MO core, and NAO core relative to the shell regions  gene expression profiles and Nissl-defined patterns, we found that targeted biocytin injections into the AAC core  with verions Fig ; Table 2ions Fig  consisteions Fig  with litions Fig  and 12b ions Fig ; Table 2ions Fig . These fPVALB-defined MO core  by Durand et al , labeledTaken together, these findings indicate that the core regions of NLC, AAC, MO, and NAO are primarily interconnected to each other whereas the shell regions are primarily interconnected to each other. There are sparser connections between cores and shells within and among each song nucleus.PVALB mRNA expression, we discovered the presence of similarly labeled song nuclei-like regions in eight other parrot species, namely the yellow lored Amazon (Amazona xantholora), yellow crowned Amazon , peach-faced lovebird (Agapornis roseicollis), African Grey parrot (Psittacus erithacus), the peach-fronted conure (Aratinga aurea), and the blue and gold macaw (Ara ararauna), all from the Psittacidae Family, the cockatiel (Nymphicus hollandicus) from the Cacatuidae Family, and the kea (Nestor notabilis) from the Nestoridae Family  and shell (moderate expression) regions  was barely detectable relative to the surrounding brain regions , with sOur study helps clear up contradictions between past studies, in that different investigators had been studying the core, shell, and/or both without consistent recognition of the differences between them . In the We did not find gene expression specializations for the two smaller parrot cortical song nuclei, LAN  in the nidopallium and LAM  in the mesopallium  , except Sayornis phoebe) has a rudimentary RA (AAC-like) song nucleus not found in other bird species, such as quails [Procnias contigas), shows evidence for vocal learning [One fundamental shared connection among the vocal learning birds is the parrot’s AAC core, songbird’s RA, and hummingbird’s VA specialized direct projection to the brainstem vocal lower motor neurons ; a diffes quails , and at learning  whereas learning . We alsoThe remarkable relative size differences between core and shell regions among parrot species could reflect functional and/or brain size differences. Parrot species generally considered to be more limited in their vocal imitation abilities, such as the budgerigar, peach-faced lovebirds, and cockatiels, have larger and conspicuous core nuclei relative to the shell nuclei, whereas those thought to have considerably more complex communication abilities such as the blue and gold macaws, peach-fronted conures, African Grey ,17, yellIn most species, the lowest core to shell area ratios (meaning shell is bigger) were in NLC. The NLC in budgerigars is required for production, but not memory of English words and natural vocalizations . NLC lesSLIT1 belongs to a class of proteins involved in axon guidance and cell migration, and in a recent study we also showed that its mRNA is downregulated in the human laryngeal motor cortex [ROBO1 receptor [FOXP2 transcription factor (involved in speech acquisition) more than the chimpanzee FOXP2 [ROBO1 cause speech sound disorders and dyslexia [SLIT1 is a candidate gene involved in the formation and maintenance of the specialized direct projection of the ACC core to brainstem vocal motor neurons [SLIT1 is mostly repulsive for forming connections, and its down-regulation could be a mechanism to allow a direct projection to form.Identification of core and shell functional differences is informed by the gene expression specializations. r cortex , the anareceptor , with a receptor , and is ee FOXP2 . Mutatiodyslexia . One pot neurons ; SLIT1 iSLIT1 downregulation in the AAC core, a subset of them expressed intermediate specialized levels of SLIT1 in the PVALB-defined shell that includes non-vocal motor areas. Together with the limited specializations of mENK and TH noted here in the adjacent motor regions of this or other parrot song nuclei suggest that the brain areas involved in movement control may also have some specialized connectivity and functions. Only vocal learning birds (parrots in particular) and mammals  show auditory-motor entrainment, synchronizing body movements to the rhythm of the downbeat in music (i.e. dance to music heard) [Although all nine parrot species examined showed c heard) ,80–82. Sc heard)  as notedFOXP1 in the NLC core and shell, and NR2A and GLUR1 genes in the core, could mean that the downstream consequences on the genes that they target (FOXP1’s direct regulation or NR2A’s and GLUR1’s indirect single transduction regulation of genes) and their ultimately influence on behavior would be different than in the surrounding regions. The higher levels of gene products with functions in Ca2+ buffering, neuroprotection and cell metabolism in all the cores , suggest that the cores could be more active than the surrounds [The differential regulation of urrounds , as in sDUSP1 in MO and NAO cores, there is no noticeable difference in the relative degree of vocalizing-driven EGR1 or C-FOS mRNA induction between the core and shell region of any song nucleus in budgerigars. One explanation could be that both core and shell regions are equally required for production of warble song. Alternatively, IEG mapping techniques could be inadequate to parse out more finely tuned functions of the core and shell regions in vocal production. It is also possible that behavioral experiments that are designed for parrots to perform more specific vocal behaviors would be required to answer these questions. In either case, future IEG and other functional studies can now be guided by our discoveries to help determine potential functional differences between the core and shell regions.Interestingly, except for higher In conclusion, we believe our study presents a new and viable hypothesis concerning the nature of unique specializations that characterize the parrot song system, which distinguish it from those of other vocal learning species. This hypothesis could inform future studies aimed at understanding the mechanisms of mimicry of human speech and the neural substrates that govern motor imitation in parrots, and provide a basis for deepening our understanding of the neural underpinnings of vocal learning.Melopsittacus undulatus)  from the University of Copenhagen, Denmark colony, and from Duke University, USA ; peach-fronted conures (Aratinga aurea) , cockatiels (Nymphicus hollandicus) , and peach-faced lovebirds (Agapornis roseicollis)  from the University of Copenhagen; a male yellow lored Amazon parrot (Amazona xantholora), a male yellow crowned Amazon parrot , a female blue and gold macaw (Ara ararauna) and a female kea (Nestor notabilis) from the Denmark Zoo ; and a female African Grey parrot (Psittacus erithacus) from the Diagnostic Pathology Laboratory of the Dutch Research Institute for Avian and Exotic Animals . The birds in the University of Copenhagen colony were briefly anaesthetized with CO2, whereas the birds collected from the Copenhagen Zoo were anaesthetized with sevoflurane and pentobarbital IV, or with medetomidine and ketamine before decapitation. The remaining birds used for behaviorally regulated gene expression were not anaesthetized prior to decapitation, in order to not disrupt the behaviorally-regulated gene expression. All collected brains were dissected within minutes of decapitation, frozen immediately in a dry ice-ethanol bath, and stored at -80°C. Brains were kept frozen on dry ice during transportation. We embedded the frozen brain in Tissue-Tek OCT Compound  in a plastic block mold and quickly froze the Tissue Tek around the brain on a dry ice-ethanol bath. Brains were sectioned in sagittal and coronal planes at 16μm in 6–20 series on a cryostat and the sections mounted on plus charged slides . Only the coronal plane sections are shown, where it was easier to identify core and shell regions.We obtained brains from budgerigars .PVALB and SLIT1 genes we used radioactive 35S in situ hybridization following previously described procedures [PVALB riboprobe we used a clone from our zebra finch full-length cDNA collection [SLIT1 clone containing 1.8Kb of the coding region that we cloned [35S-UTP-labeled sense and antisense riboprobes by reverse transcription, and hybridized each slide containing brain sections with 1x106 cpm at 60°C . We exposed the slides to NTB emulsion  diluted 1:1 in distilled water for ~14–30 days at 4°C and processed the slides with D-19 developer (Kodak) and fixer (Kodak). We visualized the bound riboprobe as silver grains, and the cell bodies by counterstaining with cresyl-violet acetate solution . Tissue incubated with the sense probe showed no significant signal above background.To localize mRNA expression of the ocedures ,83. To mllection , 46  core and shell regions and the anterior song nuclei (MO and NAO) core regions, whereas only cresyl violet was used to visualize the shell regions of MO and NAO, as PVALB does not distinguish their shell boundaries. All statistical analyses were performed using IBM SPSS Statistics for Macintosh (Version 22.0).To perform quantification of area measurements, we captured digital images of brain sections of emulsion dipped (in darkfield) plus Nissl-stained (in brightfield) slides using an Olympus MVX10 microscope  connected to a DP71 camera (Olympus) and DP Controller software. We quantified the core and shell area boundaries from all available sections from each animal per species. However, to remove any size measurement biases due to the number of available sections, we selectively analyzed the core and shell regions in sections where the core region was the largest, in 3–4 adjacent sections per brain region in each animal. We measured the area sizes using the Freehand tool in the Fiji software from ImageJ with the scale bar set in millimeters for spatial calibration. To investigate connectivity of the core and shell regions of NLC, AAC, MO, and NAO, we imaged and analyzed slides (> 4000) containing serial brain sections of budgerigars (n = 29) from neural tracing studies previously conducted in the Brauth lab ,34 and cS1 Figa) Boundaries of the NLC and AAC song nuclei consisting of core and shell regions (delineated by dashed black lines); (b) Boundaries of the core region of NLC; (c) Boundaries of the core region of AAC; (d–f) Views (from low power to high power) of tightly packed clusters of cells arranged in a sphere like shape comprising the core region of NLC, in a black and white setting under brightfield; (g–i) Views of widely dispersed clusters of cells in the shell region; (j–l) Views of closely packed cells in the core region of AAC; (m–o) Views of widely dispersed cells in the shell region of AAC. The shell boundaries in these images are based on the PVALB expression, and thus include the non-vocal motor areas. Sections are in the coronal plane; medial is to the left, dorsal is top.((TIF)Click here for additional data file.S2 Figa) Boundaries of the MO song nucleus consisting of core and shell regions (delineated by dashed black lines); (b) Boundaries of the NAO song nucleus consisting of core and shell regions (delineated by dashed black lines); (c) Schematic diagram showing relative positions of MO and NAO song nuclei; (d–f) Views (from low power to high power) of large loosely distributed cells arranged like a sphere in the MO core region; (g–i) Views of the shell region of MO consisting of widely distributed cells; (j–l) Views of closely packed cells of the NAO core region; (m–o) Views of widely distributed cells in the shell region of NAO. Sections are in the coronal plane; medial is to the left, dorsal is top.((TIF)Click here for additional data file.


We combined sediment and faunal data to explore the role of the sedimentary regime in shaping the distribution of subtidal sandbank environments and the associated meiofaunal nematode communities at Broken Bank and Swarte Bank, in the southern North Sea. A variety of sediment transport processes occur in the area, differing in the frequency and magnitude of sediment mobility, and the continuum between erosion, translation and sediment accumulation. The seabed contained a variety of bedforms, including longitudinal furrows, and small to very large sandwaves. The bed sediments were dominated by fine and medium sands, with admixtures of silt and gravel. Based on sedimentary bedforms and grain size analysis, a total of 11 sedimentary facies were delineated, of which 8 were analysed in detail for their relationships with the meiofauna. The sedimentary facies fell clearly into groups of facies, respectively representing high, high-moderate and moderate, and episodic sediment mobility. For those sedimentary facies where daily movement of sediments and bedforms occurred (‘high’ sediment mobility), the resulting spatially homogeneous environments were dominated by an impoverished nematode community comprising small deposit feeders and large predators. Resistance to sediment movement and the ability to exploit alternative food sources were prominent functional features of the successful colonisers. Those facies characterised by relatively infrequent sediment mobility (‘episodic’ and ‘high-moderate and moderate’ sediment mobility) comprised a heterogeneous suite of benthic habitats, containing taxonomically and functionally diverse assemblages of nematodes of various sizes, feeding types and reproductive potential. Faunal distribution patterns here indicated trade-offs between the resistance to sediment movement, environmental tolerance and competitive abilities. Our focus on diverse assemblages of organisms with high turnover times, inhabiting highly dynamic sedimentary environments, has revealed new animal-sediment relationships of relevance to pure and applied science. Scientists, socio-economists, environmental managers, policy-makers and the public all have interests in marine environmental studies and their outcomes. A common motivation of marine studies is the implicit or explicit search for indicators of natural and anthropogenic change, with an aim of identifying and assessing human impacts and the effects of management actions. As an example policy driver, the EU Habitats Directive for the protection of species and habitats Effective conservation of marine habitats in European seas requires specific conservation objectives. These need to be achieved by closely linked management measures that regulate the activities of those marine industries which may threaten the integrity of the habitat, or the viability of the associated populations Reviewing the literature on animal-sediment relationships, A general appreciation of the highly heterogeneous nature of continental shelf sediments has progressively emerged in recent decades. Multi-beam bathymetric surveys have revealed numerous types of sedimentary and geomorphological features on the sea floor that occur at a variety of spatial scales, ranging from a few meters to several kilometres There are obvious possible dependencies of meiofauna upon the distribution and mobility of sediments A detailed description of the benthic fauna associated with two linear sandbanks off the North Norfolk coast in the southern North Sea by All data reported in this article were collected by the Centre for Environment, Fisheries and Aquaculture Science (Cefas), as part of a government-funded research project. As an executive agency of the UK Government, Cefas has an exemption from The Crown Estate (i.e. land owner of our study site) to collect sediment and other scientific material (except vertebrates) in UK waters. The activities involved in the survey of the seabed and the collection of benthic invertebrates were not subject to specific regulation and did not involve endangered or protected species. Throughout the survey, scientists and ship crew acted in a responsible and environmentally friendly manner.We analysed the bathymetry, sedimentary bedforms, sediments and meiofaunal nematodes at and near Broken Bank (BB) and Swarte Bank (SB), in the southern North Sea, to examine the association of faunal assemblages and their sedimentary habitat. The sampling strategy was not designed to fully characterise nematode-sediment relationships at the two sandbanks, but rather to enable us, with minimal cost and effort, to make comparisons between nematode communities inhabiting a diverse range of sedimentary environments.−1−1 have been recorded −1 and 1.0 m s−1 on spring and neap tides, respectively Broken Bank and Swarte Bank are the outermost of the Norfolk Banks in the southern North Sea. Broken Bank lies 67 km offshore, and has dimensions typical of the tidally-controlled open-shelf linear sandbanks In April 2006, the southern portions of Broken Bank and Swarte Bank were surveyed using RV Cefas Endeavour to run a Kongsberg EM3000D multi-beam echo-sounder to provide high-resolution bathymetric data, and a Benthos 1500 digital side-scan sonar to measure seabed acoustic reflectance, which can indicate substrate hardness 2 Day grab (2 surface area) to a depth of 5 cm, one for the analysis of grain size distribution and one for the study of meiofaunal nematodes. The location of sampling stations was largely dictated by the seabed topography because sufficient draught was necessary under the vessel to allow the collection of sediment samples.Sediments from both Broken Bank and Swarte Bank were sampled by means of a 1 mDay grab ; Table 1Sub-samples for grain size analysis were combined by station and frozen at −20°C prior to analysis. After thawing, the 22 sediment samples were wet sieved through a 500 µm sieve, and the fraction> 500 µm was oven dried at 90°C for 24 h. This fraction was then dry sieved at 0.5 phi intervals, down to 1 phi (500 µm). The fraction <500 µm was freeze dried and analysed on a Malvern Mastersizer S laser particle sizer. The sieve and laser sizer data were merged to form one uniform distribution spanning the size range 0.49 µm −22 mm. It is recognised that freezing samples can alter the grain size distribution, especially of clay-sized grains, but microscope and size analysis both indicated that no clay-sized grains were present in any samples, consistent with previous findings Grain size data were grouped using the computer programme ENTROPY Distribution patterns of the various sedimentary bedforms and the grain size data were used together to delineate a series of sedimentary facies for the study area. In the geological literature, there are a number of ways that the term facies has been used All faunal samples were fixed in 5% formaldehyde in filtered (63 µm) sea water. After washing the meiofauna samples onto a 63 µm sieve, the fauna from all replicate samples were extracted by floatation in Ludox 40 Nematodes were identified to genus or species level, using taxonomic keys based on a phylogenetic classification scheme 1 and N2Species and trait diversity and dominance in each sample were expressed as Hill numbers Na priori sedimentary facies and distinguished between them. The dissimilarity between faunal distribution patterns recorded in the sedimentary facies was computed and an average calculated. We then calculated the contribution of each species and trait to the average similarity within each sedimentary facies and the average dissimilarity between facies. All statistical analyses were performed using the software packages Statgraphics Plus version 3.3 and Primer version 6.1.5 Non-metric multi-dimensional scaling (MDS) ordination using the Bray-Curtis similarity measure was applied to relative abundance data to compare spatial patterns in the taxonomic (i.e. species) and functional (i.e. traits) composition of nematode communities amongst the putative sedimentary facies. One-way analysis of similarities (ANOSIM), the multivariate analogue of analysis of variance, was used to test for significant taxonomic and functional differences between sedimentary facies. Similarity percentages (SIMPER) were calculated to identify the species and traits which both characterised A variety of publications have used the term disturbance to cover a wide range of anthropogenic and natural sediment transport and mixing processes including dredging, dredge material emplacement, trawling and natural sediment mobility The banks' gross morphologies include measured lee slopes of nearly 5° . SuperpoThe bedforms (numbered i – viii) superposed on and between the sandbanks vary across the survey area in a relatively ordered fashion . On the The key physical features are therefore that ; Table 4For the positive relief features of Broken Bank and Swarte Bank, bedform transport is relatively active, with transport of sand to the NW across the stoss slope and along the crest, and to the SE along the lee slope, i.e. in an apparent clockwise circulation  which indicate episodically fast flows. This is in contrast to the upper stoss where the sediment is finer and where the mostly smoother seabed is inferred to be more continuously mobile.The seabed sediments are mostly fine and medium sands, with a modal grain size of 180 – 250 µm, and with a minor gravel component in some samples. Based on their entire size distributions, the samples fall into four broad grain size groups  were included in our statistical analyses . Facies The facies names highlight the major features which define each facies and which distinguish them from adjacent deposits ; Fig. 6.At all stations, nematodes consistently comprised> 81% of the total meiofauna abundance. A total of 9171 nematodes was identified, with abundances per sample ranging from 47 in washed-out megaripples at the stoss off Broken Bank (station BB-D) to 434 in active megaripples at Swarte Bank (station SB-F). Nematode assemblages in the study area were taxonomically and functionally diverse. A total of 118 species was recorded which fell into 77 trait groups.1(species): F = 6.71, P <0.01; N1(traits): F = 4.66, P <0.01) and dominance (N2(species): F = 6.96, P <0.01; N2(traits): F = 3.72, P = 0.02). Taxonomic and trait diversity (N1) were significantly lower in facies 1 and 8  than in most of the others. High numbers of some dominant species and traits in facies 1 resulted in a low N2 value, whereas the low N2 value observed in facies 8 was a consequence of the distribution of nematodes across a limited number of species and traits  were also highly influential in determining taxonomic and functional distribution patterns across sedimentary facies.Functionally, nematode assemblages were dominated by epistrate-feeding species that combined fast turnover times and an ornamented, relatively small, slender body . A totalMetadesmolaimus pandus and large predators, respectively. The trait groups differed not only with regard to feeding habit, but also in cuticle pattern (striated versus smooth) and body size (<1 mm versus> 2 mm). The relative abundance of non-selective deposit feeders was significantly higher  in this facies and that of epistrate feeders significantly lower  compared to other sedimentary environments  and the dominant multi-species trait group  were abundant in facies 8,  as was a limited range of other trait groups which combined a deposit- or epistrate-feeding habit with a striated, medium-sized (1 – 2 mm), slender body. Nematode assemblages in this facies exhibited a relatively high similarity with those recorded in facies 6 and 7  differed little with regard to their taxonomic and functional structure. Small and medium-sized deposit- or epistrate-feeding species dominated nematode assemblages.These key features tend to indicate a range of faunal similarities between sets of facies, which led to readily interpretable patterns in the MDS ordination . DistincInterstitial habitats such as those occupied by meiofauna are broadly distributed across the planet, and the complexity of the associated physical sedimentary environments has been present throughout the evolutionary history of meiofauna e.g. . Hence, A variety of sediment transport processes in our survey area, operating at different magnitudes and frequencies, created a wide range of sedimentary environments for meiofaunal nematodes . The preDelineating and using sedimentary facies can help to better understand the way in which the local complexity of the seabed might determine the number and type of available niches for meiofaunal nematode communities. In our context, sedimentary facies inherently include the key features of the bedforms and sediments themselves e.g. , thus inWith an emphasis on the sandy facies appropriate to our study area, the general relationships between the groups of sedimentary facies representing high, high-moderate and moderate, and episodic sediment mobility can be illustrated by using three key parameters: the frequency of sediment mobility, its magnitude, and the continuum between erosion, translation (i.e. sediment throughput) and sediment accumulation . Our facMetadesmolaimus pandus who, together with a contrasting trait group comprising large predatory nematodes (including Viscosia), dominated colonist communities. These disturbance-resistant nematodes A common assumption of ecological theories addressing the causes of faunal variability in space and time is that species or functional groups with different morphology, physiology or behaviour dominate under different environmental regimes Larger sedimentary bedforms comprising small sandwaves and washed-out megaripples are located along the crests of both banks (facies 2 – 4) and indicate relatively high magnitudes and frequencies of bed sediment transport. However, in contrast to facies 1, the bedforms may persist for longer (i.e. weeks to years as opposed to hours to days) and be more spatially varied. Accordingly, we recorded a range of coexisting epistrate feeders and sub-dominant deposit feeders. As has been shown for other dynamic sedimentary environments Solitary sandwaves on the lower stoss and apron of Swarte Bank (facies 5) were characterised by moderate spatial gradients in the benthic habitat across and along the bedform  and 6. CThe relatively low elevation seabed between Broken Bank and Swarte Bank comprised sandwave fields of varying topography and extent (e.g. facies 6 and 7). In common with facies 8, at the steep lee slope of Swarte Bank, these areas of seabed probably undergo repeated and complex ecological transitions, associated with the irregular timing and nature of seabed mobilisation (e.g. as a result of storms in the study area). The initiation and episodic regeneration of bedforms  by strong storm-associated flows is related to very high rates of sediment transport, involving largely passive transport of meiofauna, and at these times, much of the study area would constitute a solely physically driven benthic system sensu . HoweverSuch an episodically influenced change in seabed character probably also extends to those facies located North and West of the furrowed areas, away from the SE tips of the banks. It is likely that, especially during periods of decreasing sediment transport associated with the decaying phase of storms, a phase of relatively high net sediment accumulation occurs here over a few hours, and perhaps up to a day or so, depending upon the particular storm's characteristics. This phase of accumulation would be followed by an extended period where colonisation and assemblage development will take place, associated with the ambient, strongly tidal, oceanographic regime. Resident nematode communities appear to reflect such sedimentary complexity. In our particular case, the dominance of small- to medium-sized deposit- and epistrate-feeders of high reproductive potential most likely represents communities at a relatively early post-mobility stage.Taxonomically and functionally disparate communities inhabiting the sedimentary facies defined in our survey area can be considered a complex product of the various sedimentary contrasts inherent within different sedimentary facies and the general sedimentary nature of the area being considered. Regarding variation within facies, the sedimentary environments experienced by meiofauna will be distinctly different across the variety of sub-environments across a sandwave-dominated facies, where troughs, stoss slopes, crests and lee slopes are dynamically and texturally different. There will also be great differences in those facies where longitudinal furrows occur with differing levels of infill. Further, sandwaves are much larger physical features than megaripples, and therefore their rates of horizontal migration and morphological change tend to be much slower. In our study area, sandwaves also tend to be much more patchy in their distribution than megaripples. Therefore, we might expect that 1) a similar number of sediment grabs in sandwave facies is more likely to include a greater variety of seabed types (and meiofauna) than in megarippled facies and that 2) the more frequent and regular mobility of megaripples tends to favour the presence of fewer meiofauna species and functional groups.The long-term  tendencies for seabed erosion, sediment throughput or sediment accumulation are important because they exert key controls upon habitat type and sediment mobility . For exaTwo main points emerge from our data. Firstly, a broad-scale characterisation of sedimentary environments is unable to account for the microhabitat heterogeneity to which the associated meiofauna will tend to respond. A key aim of future studies should be to incorporate fine-scale spatial information on sedimentary environments into the analysis of broader-scale animal-sediment relationships. For deeper insights, such studies should build on a good conceptual understanding of sedimentary processes operating at a range of spatial and temporal scales, including the movement of sediment across the full range of marine sedimentary bedforms Secondly, small-scale faunal distribution patterns do not always reflect only those marine processes occurring concurrently with sample collection. Whilst this is not a new finding, our results emphasise how important past events can be in affecting animal distributions. Caution must be exercised when attempts are made to explain spatial distributions of animals from physical or biological conditions measured at a single point in time. For example, analyses conducted of pre-storm samples from our facies 8  would inevitably generate different faunal results, and hence differing expectations regarding state, than analyses of samples collected shortly after major storms. Further, for those areas where longitudinal furrows occur, there would be relatively high variation in sediment types after storm-associated currents which activate the furrows, but the degree of variation would change through time. Within the furrows themselves, sediments would accumulate relatively rapidly and become finer through time, favouring mud-dwelling meiofauna whereas immediately outside the furrows, the sediments would change in completely different fashion in response to the re-development of ripples and megaripples, with associated different meiofaunal responses.The variety of sedimentary regimes at and around Broken Bank and Swarte Bank clearly shapes the meiofaunal nematode communities, and the interaction of local hydrodynamics and sediment transport is clearly a key driver of the processes involved. Their influence occurs in a variety of different ways, and, importantly, in ways that would not have been predictable from using 1) existing generalised theories of seabed disturbance based on frequency or intensity alone Our focus on diverse assemblages of organisms with high turnover times , inhabiting highly dynamic sedimentary environments , has revealed interesting animal-sediment relationships worthy of investigation by future studies of these and a broader range of marine environments. Results from such studies should facilitate a new generation of hypotheses concerning the relative importance of different environmental drivers in structuring benthic communities, and movement towards their rigorous testing. This will provide better indications of the stability of the sediments that benthic communities inhabit and allow better insights into how such communities may respond to natural sediment mobility and to anthropogenic disturbance. Taking account of the past evolution and modern processes of the sedimentary environment is paramount. Contemporary faunal distribution patterns reflect the integrated response of species and functional groups to a wide suite of sedimentary processes, so that effective descriptors of those sedimentary processes will help reveal meaningful animal-sediment relationships.As noted in the introduction, the EU Habitats Directive requires that “management plans be created in order to detect human-induced change”; such words are easy to write but are likely to be very difficult to achieve. In the case of the UK's candidate Special Areas of Conservation (SAC), which include subtidal mobile sandbanks, there is much to be learnt before such plans can realistically be formed. Our scoping study has indicated some physically and ecologically meaningful measures of seabed physical texture that will help improve seabed characterisation and associations with benthic assemblages.Table S1Nematode species abundance by sampling station.(XLS)Click here for additional data file.Table S2Relative weight of sediment [%] by grain size [μm].(XLS)Click here for additional data file.


Schizophrenia (SZ) is a chronic and severe mental illness for which currently there is no cure. At present, the exact molecular mechanism involved in the underlying pathogenesis of SZ is unknown. The disease is thought to be caused by a combination of genetic, biological, psychological, and environmental factors. Recent studies have shown that epigenetic regulation is involved in SZ pathology. Specifically, DNA methylation, one of the earliest found epigenetic modifications, has been extensively linked to modulation of neuronal function, leading to psychiatric disorders such as SZ. However, increasing evidence indicates that glial cells, especially dysfunctional oligodendrocytes undergo DNA methylation changes that contribute to the pathogenesis of SZ. This review primarily focuses on DNA methylation involved in glial dysfunctions in SZ. Clarifying this mechanism may lead to the development of new therapeutic interventional strategies for the treatment of SZ and other illnesses by correcting abnormal methylation in glial cells. Schizophrenia (SZ) is a chronic and severe mental illness that affects approximately 1% of the population , glutamate, as well as 5–hydroxytryptamine (5-HT) have been implicated as primary etiologies of SZ. In general, most studies have focused on the dysfunction of neurons in different regions of gray matter that include the ventral tegmental portion, the limbic system and the prefrontal cortex  are differentiated from the neural stem cells (NSCs). After specification, glial progenitor cells/oligodendrocytes precursor cells (OPCs) can further differentiate into oligodendroglial lineage cells or astrocytes, and the processes are controlled by specific transcriptional programs . In addition, the term glial cells also extend to Schwann cells and satellite cells in the peripheral nervous system (PNS). Besides basic functions like nutritional support or neuronal protection, glial cells have other integral roles including governing myelination, synapse formation and immunological reactions  in the gliogenic phase of gestation. After specification, OPCs continued to differentiate into immature oligodendrocytes and finally the mature oligodendrocytes  has also been cited as a strong candidate target gene for SZ. Rationale involving its implication in SZ revolves around the ability of DISC1 to negatively regulate the differentiation of oligodendrocytes , OPRM , the serotonin-2A receptor gene (HTR2A), brain derived neurotrophic factor (BDNF) and arachidonate 5-lipoxygenase  or azacytidine, Gfap is demethylated and this induces astrocytic differentiation from NSCs  is a childhood white matter disorder caused by mutations in MeCP2. It is an X-linked neuro-developmental disorder featured by autism and severe mental retardation and neurological dysfunction in females. Although RTT is generally attributed to be a primary neuronal dysfunction, it has recently been shown that astrocytes, oligodendrocytes and microglia also contribute to RTT pathophysiology , SOX9, glutamate-ammonia ligase (GLUL), the sodium/phosphate cotransporters NaPi-IIc (SCL34A3), gap junction alpha 1 (GJA1) and gap junction beta 6 (GJB6), brain-enriched guanylate kinase-associated protein (BEGAIN) and glutamate receptor ionotropic kainate 2 (GRIK2). These markers can serve as functional candidates involved in the clinical screening of patients for susceptibility to SZ  channel which is essential for the development of the CNS. As such, decreased Kir4.1 expression is associated with Dnmt1-mediated DNA hyper-methylation in medical conditions such as ischemic injury, epilepsy, and Alzheimer’s disease , as part of the DNA-demethylation pathway component, whose expression was increased by nearly threefold in SZ (Matrisciano et al., Since glial abnormalities are thought to be key players in SZ pathology, new strategies that target the glial cells may be beneficial in the treatment of SZ (Takahashi and Sakurai, 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.


Psoriasis is a chronic inflammatory disease, predominantly affecting the skin, being included in the group of Immune Mediated Inflammatory Diseases. Growing evidence from the last 10 years suggests that several systemic conditions like metabolic syndrome, cardiovascular disease, diabetes, psychological disorders or inflammatory bowel disease are prevalent in psoriasis patients. The linker might be the chronic secretion of pro-inflammatory cytokines. In this current review, the scientific evidence that explains the relationship between psoriasis and the metabolic syndrome in particular will be addressed, as the metabolic syndrome comprises a group of risk factors for cardiovascular disease, thus offering an overall picture of the systemic involvement in psoriasis. An integrated approach, with an early detection and treatment of the components of the metabolic syndrome, are important steps in psoriasis management. Attention should be paid on influence of psoriasis treatment upon comorbidities and vice-versa. In the last years, an important progress has been made towards the identification of the inflammatory pathways involved in psoriasis pathogenesis. A series of inflammatory molecules are produced in psoriasis skin lesions , interferon-γ, etc.) [2] and it seems that these molecules are released in the systemic circulation based on the severity and extension of the skin lesions . Moreover, experimental and epidemiological studies have linked certain interleukins, cytokines and hormones (adipokines) with cardiovascular disease (CVD), metabolic syndrome (MetS), obesity and diabetes, making psoriasis a risk factor in developing systemic comorbidities . Beside inflammation, other factors have to be considered to explain these associations, like shared risk factors , treatment  or shared genetic risk loci.Psoriasis is a chronic inflammatory skin disorder, immunologically mediated . Its prevalence ranges from 0.91 in the US to 8.5 in Norway , states that MetS diagnosis is based on the presence of at least three of the following five conditions, as defined by 1) fasting glucose 100 mg/dL or greater (or receiving drug therapy for hyperglycemia); 2) blood pressure 130/85 mmHg or higher (or receiving drug therapy for hypertension); 3) triglycerides 150 mg/dL or higher (or receiving drug therapy for hypertriglyceridemia); 4) HDL cholesterol (high density lipoprotein cholesterol) less than 40 mg/dL in men or less than 50 mg/dL in women (or receiving drug therapy for reduced HDL-C); and 5) waist circumference 102 cm (40 inches) or greater in men or 88 cm (35 inches) or greater in women; in Asian American, 90 cm (35 inches) or greater in men or 80 cm (32 inches) or greater in women. The main difference between NCEP ATP III definition and International Diabetes Federation (IDF) definition [9] is that the latter uses central obesity as a mandatory component, plus any two of the other four components described above. It is well known that MetS increases the risk of CVD, diabetes and cancer; however, most of the published reports indicate that the syndrome does not predict cardiovascular events or disease progression any better than the sum of its components [8].There are a number of definitions and criteria used to identify MetS. The National Cholesterol Education Program’s Adult Treatment Panel III (NCEP ATP III) revised criteria from 2005  found that psoriasis patients have higher prevalence of MetS compared with the general population, and patients with more severe psoriasis have greater odds of MetS than those with milder psoriasis. Armstrong et al. [10] found that most of the studies reported a high prevalence of MetS in psoriasis patients, ranging from 14 to 40%, suggesting that MetS is a common problem for psoriasis patients.A recent meta-analysis [11] found that psoriasis is associated with MetS in a ‘‘dose–response’’ manner, with a 22% increase in the odds of developing the MetS in those with mild psoriasis, 56% increase in those with moderate disease, and a 98% increase in those with severe psoriasis. Next, the scientific evidence so far, concerning interrelationships between inflammation/psoriasis and each of the components of MetS, will be briefly summarized.In a large population-based study from UK, Langan et al. [Insulin resistance and diabetes mellitus8]. It seems that inflammatory mediators may be involved in insulin resistance, such as TNF-α, Il-6, leptin, adiponectin , mediators that are also disturbed in psoriasis (further detailed below).Insulin resistance is a condition in which normal amounts of insulin are inadequate to produce normal insulin response from fat, muscles and liver cells; it is a state that precedes type 2 diabetes mellitus. Until now, insulin resistance has explained most if not all of the components of MetS [14] and insulin resistance was correlated with psoriasis index severity area [15]. Several studies have found an increased risk of diabetes in psoriasis patients . Thus, it seems that the risk of diabetes in psoriasis is linked to insulin resistance [18].Insulin resistance was found in non-obese patients with psoriasis . Several studies, including a recent meta-analysis, have shown an increased prevalence of hypertension among psoriasis patients .Psoriasis patients have an alteration of the renin-angiotensin-aldosterone system (RAAS), with elevated plasma renin activity and elevated angiotensin-converting enzyme activity [23]. All these evidence provide an explanation of the relationship between hypertension and obesity in the MetS.Adipose tissue is an important source of angiotensinogen, the precursor of angiotensin, which plays a major role in blood pressure control. Angiotensinogen microRNA expression is increased in visceral fat. Moreover, it is known that weight loss leads to blood pressure reduction. Beside angiotensinogen, resistin and leptin secretion from adipose tissue have also been implicated in hypertension from MetS [24], a pro-inflammatory cytokine, and reduce adiponectin expression [25], an anti-inflammatory cytokine. These cytokines are shown to be abnormal when expressed in psoriasis: increased osteopontin and decreased adiponectin levels, significantly associated with the severity of psoriasis and MetS [26].The RAAS may induce osteopontin expression [Dyslipidemia27] found that psoriasis patients have a higher incidence of dyslipidemia than the general population.Dyslipidemia is a widely accepted term that comprises any plasma lipid abnormality, usually defined as increased low-density lipoprotein, very low-density lipoprotein, and triglyceride levels and decreased high-density lipoprotein levels. A recent large systematic review [28]. Moreover, these interleukins increase lipid levels [29]. Psoriasis is associated with increased oxidative stress , as most human diseases are, and oxidized-LDL is found to be elevated in severe psoriasis [32].In vitro evidence shows a common inflammatory background between psoriasis and dyslipidemia. Il-1, Il-6 and TNF-α that mediate psoriasis may alter the hepatocyte function and the arterial smooth muscle cells, which will finally lead to arterial plaque development [Obesity33]. Central obesity plays a central role in MetS development and seems to precede the appearance of all the other components of MetS [34].Obesity is associated with a chronic low level of inflammation [35-37]. Most of the current available data refer to leptin and adiponectin [33]. Leptin circulating levels correlate with fat mass and leptin exerts important roles in inflammation  [33], stimulate keratinocyte proliferation and angiogenesis [38]. Psoriasis patients have high levels of leptin [39] and these high levels may derive both from the adipose tissue in obese patients and from inflammation. Adiponectin is inversely correlated with obesity, it is an anti-inflammatory molecule, with anti-atherogenic properties [40] and psoriasis patients have decreased levels of adiponectin [26].Activated macrophages in the adipose tissue stimulate adipocytes to secrete adipocytokines (adipokines), such as TNF-α, IL-6, leptin, and visfatin, which may also play a role in the pathogenesis of psoriasis [41]. Increased levels of VEGF are found in hyperinsulinemic states, such as MetS, deriving from adipocytes [42]. Among all adipose tissues in the body, omentum expresses the highest level of VEGF [43]. A recent meta-analysis that comprises 16 observational studies found that psoriasis patients have a higher prevalence and incidence of obesity, compared with the general population [21].VEGF promotes angiogenesis and endothelial cells activation and is increased in psoriatic skin and correlates with the severity of the disease [Therapeutic implications35]. A recent controlled study showed that weight loss improved the therapeutic response to a low dose of cyclosporine in obese patients with moderate-to severe chronic plaque psoriasis [44]. Patients on cyclosporine should monitor their blood pressure.The current therapies for psoriasis include topical agents, phototherapies, systemic therapy (immunosuppressive drugs) and biotherapies (biologics). Some of the drugs used for the psoriasis treatment have been shown to modify risk components of MetS while other exacerbate them. Obesity is a risk factor for MetS and weight loss may improve psoriasis , reduce lipid peroxidation and increase high-density lipoprotein levels [46], thus having a beneficial systemic effect. Dermatologists should be aware that patients with psoriasis both on anti-TNF therapies and insulin for diabetes may experience hypoglycemia [47].Some of the anti-TNF biologics are shown to reduce C-reactive protein levels . For statins, other authors suggest beneficial effects in psoriasis [50], that might derive from anti-TNF properties of statins.Other drugs used for psoriasis have negative effects like the alteration of lipids profile in the case of retinoids and cyclosporine, while some drugs used for MetS like fibrates or even statins may exacerbate psoriasis [2]. These pieces of evidence may explain the increased prevalence of MetS in psoriasis patients.Psoriasis is currently considered a “T-cell mediated disease”, and shares a common immune profile with MetS, both of them having increased Th type 1 proinflammatory cytokines, with a wide range of actions: on insulin signaling, lipid metabolism, adipogenesis [Information about the degree of psoriasis severity and duration still lacks, being necessary to increase MetS risk. However, the current recommendation is that all psoriasis patients should undergo a complete evaluation and treatment for all the components of MetS and physicians should be aware of the potential increased risk of psoriasis, in particular moderate to severe forms, for MetS and thus CVD and increased mortality risk.Sources of funding: NoneDisclosures: None


Iron accumulates in human atherosclerotic lesions but whether it is a cause or simply a downstream consequence of the atheroma formation has been an open question for decades. According to the so called “iron hypothesis,” iron is believed to be detrimental for the cardiovascular system, thus promoting atherosclerosis development and progression. Iron, in its catalytically active form, can participate in the generation of reactive oxygen species and induce lipid-peroxidation, triggering endothelial activation, smooth muscle cell proliferation and macrophage activation; all of these processes are considered to be proatherogenic. On the other hand, the observation that hemochromatotic patients, affected by life-long iron overload, do not show any increased incidence of atherosclerosis is perceived as the most convincing evidence against the “iron hypothesis.” Epidemiological studies and data from animal models provided conflicting evidences about the role of iron in atherogenesis. Therefore, more careful studies are needed in which issues like the source and the compartmentalization of iron will be addressed. This review article summarizes what we have learnt about iron and atherosclerosis from epidemiological studies, animal models and cellular systems and highlights the rather contributory than innocent role of iron in atherogenesis. The correlation between iron and heart disease was initially proposed by Sullivan in 1981. According to his “iron hypothesis,” iron overload promotes cardiovascular disease, while on the contrary, sustained iron depletion/deficiency exerts a primary protective effect against ischemic heart disease. This theory, continually debated for more than 30 years, was based on the observation that male gender is associated with higher risk of cardiovascular disease, but that the protective effect in women is diminished after menopause , that is released upon intravascular hemolysis or intraplaque hemorrhage  is a genetic disorder associated with progressive iron overload, resulting in oxidative stress and organ failure. HH is more common among individuals of Northern European descent and is caused by inherited mutations in proteins implicated in iron transport and regulation, such as the upstream regulators of hepcidin, the human hemochromatosis protein (HFE), hemojuvelin, transferrin receptor (TfR)-2, as well as hepcidin and ferroportin (FPN)   is an acute-phase plasma protein with the primary function to capture cell-free Hb and chaperon it to macrophages for degradation  can form three genotypes: Hp1-1, Hp2-1, and Hp2-2 with heterogeneous protein structure and functional differences  mouse, spontaneously deficient in macrophage-colony stimulating factor (M-CSF), displayed a reduction of 86% in plaque volume, demonstrating the essential role of macrophages in atherogenesis  into the left ventricles of anesthetized animals. HO-1 overexpression inhibits lesion formation and reduces iron overload in apoE deficient mice  that acts as a Fe3+ reducing agent, but requires neither H2O2 nor production of hydroxyl radical (OH•) by the Fenton reaction  and activation of NF-kB in mice , and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells stimulated by tumor necrosis factor alpha   proved that heme largely contributes to endothelial activation and dysfunction and cardiovascular alterations , thus promoting the recruitment of monocytes and initiating the inflammatory response. The enhanced cytokine and chemokine release by TLR4 activation could stimulate EC and VSMC migration and proliferation, thus accelerating plaque progression  and divalent metal transporter 1 (DMT1) expressed within phagolysosomal membranes. Iron is either stored coupled to ferritin or exported as ferrous iron via FPN, the only known iron exporter  and the alternative activation (M2) macrophages Figure . M1 macrIn atherosclerosis M1 macrophages were detected in both human and mouse lesions, in the lipid core of the plaque. M1 macrophages were the prevalent macrophage subtype in advanced lesions  with oxidized phospholipids reproduced differentiation toward this macrophage subtype that is distinct from both M1 and M2 subtypes. Considering the pro-oxidant action of iron, increased iron levels would be expected to enhance lipid oxidation, thus promoting Mox polarization. Whether this occurs in conditions of body iron overload has not been demonstrated.Atherosclerotic lesions are characterized by the accumulation of oxidized phospholipids that also play a role in macrophage polarization. A novel macrophage phenotype denominated Mox macrophages was identified in the lesions of mice deficient for the LDL receptor  pathways. HA-mac macrophages are distinct from the macrophages found in the lipid core and seem to play an atheroprotective role. In vitro stimulation of monocytes with Hb-Hp complexes showed a differentiation toward an HA-mac phenotype, suggesting that Hb released upon hemorrhage might model monocytes recruited to the lesion toward a specific HA-mac subtype  significantly inhibited VSMC proliferation, a hallmark of the synthetic phenotype in vitro -mediated osteoblastic transition and subsequent mineralization of VSMCs in vitro and in vivo studies are essential to clarify the particular role of iron in differentiation of VSMC into synthetic, inflammatory, osteochondrogenic, or macrophage-like phenotypes.Although increasing evidence suggests the critical role of VSMC phenotype switch in atherogenesis, the role of iron in these mechanisms still remains to be elucidated. Further Over the last 30 years, several studies in animals and humans assessed the effect of increased body iron levels on atherosclerosis, yielding conflicting results. In the last decade, our understanding of Hb and iron biology underwent a radical revision. This significantly helped in understanding the atherogenic effects of iron and iron-containing molecules. Numerous experiments support the idea that oxidized Hb, Heme, and iron—by interacting with plaque lipids, promoting endothelial dysfunction, dictating macrophage polarization, modulating VSMC phenotype and proliferation—may affect the atherogenic process. Complex systems have evolved to control and dispose cell-free Hb, heme, and iron but these systems may be eventually overwhelmed upon excessive hemorrhage or hemolysis and upon pathological iron overload. However, to date, the impact of iron on atherosclerosis is still debated. Future studies are required to clearly address whether iron overload is a risk factor for atherosclerosis and what iron source - systemic, tissue or macrophage iron—mainly affects the atherosclerotic process. Comprehensive understanding the role of iron on atherogenesis may lead to the development of improved diagnostics and therapeutics meant to interrupt the pathologic actions of excess iron.All authors contributed to the conception and design of this review. Viktória Jeney, Francesca Vinchi, and Milene Costa Da Silva wrote the manuscript, designed and made the figures. The manuscript was critically revised by Martina U. Muckenthaler, József Balla, and György Balla.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.


Drug addiction is considered a major risk factor that can influence maternal functioning at multiple levels, leading to less optimal parental qualities and less positive interactive exchanges in mother-child dyads. Moreover, drug abusers often report negative or traumatic attachment representations regarding their own childhood. These representations might affect, to some extent, later relational and developmental outcomes of their children. This study explored whether the development of dyadic interactions in addicted women differed based on attachment status. The longitudinal ongoing of mother-child emotional exchanges was assessed among four mothers with four different attachment statuses . Attachment representations were assessed using the Adult Attachment Interview (George et al.,  Parenting could be defined as a complex function, determined by multiple aspects, which has to do with the ability to take care of someone else, to provide them nurturance and to interpret correctly their needs and accept their subjectivity under different conditions Belsky, , 2014. TFrom an attachment perspective, parenting could be conceptualized as an organized behavioral system, goal corrected and maintained through internal working models Bowlby, . Part ofThe link between adult attachment representations, parental sensitivity and attachment security resulted moderate anyway, suggesting a “gap” in the intergenerational transmission of attachment and the consequent need to take into account other relational features in order to better understand parental functioning and the nature of early adult-child relationships  and their children (two girls two boys) aged from 9 to 14 months  at the beginning of the research. The subjects where extracted from a larger group of women following a rehabilitative program in a venetian Therapeutic Community (TC)The study examined longitudinally four addicted women (SD = 0.479), whereas the intoxication period lasted on average 8.75 years (SD = 1.50). The onset of substance use was due partly to the escape of personal or familiar problems (3/4) and partly to the identification with the partner or the group of pairs (3/4). When the study took place all the women were subject to substitutive opioid treatment. Regarding their past life experiences only one subject reported familiarity for drug-abuse disorder. At admission only one woman still attended school, while the others had previously interrupted studies due to low socioeconomic status (1/4) or to substance abuse (2/4). At entrance all the mothers where unemployed. Some of them (2/4) experienced important losses or traumatic experiences (2/4) concerning physical, sexual or psychological maltreatment. Two of them were engaged in prostitution acts. With respect to pregnancy and motherhood, only one subject declared to have wished pregnancy. Two of them reported a continuative use of drugs during pregnancy. Regarding newborn's medical status at delivery, mean values were 38.75 weeks (SD = 1.258) for gestational age, 3.05 kilograms (SD = 0.385) for weight, 34 cm (SD = 1.155) for cranial circumference and 50.63 cm for length. Apgar scores at 1′ and 5′ ranged all between 9 and 10. Only one child showed signs of Neonatal Abstinence Syndrome (NAS) at delivery. As far as it concerns maternal medical history, three mothers presented Hepatitis C virus at admission.Regarding substance abuse history, all mothers during the 12 months period before entrance in community showed a pathological pattern of abuse or use of substances which lead to a significant impairment or distress: for all of them, the main substance of abuse was heroin (4/4), followed by cocaine (4/4), cannabis (4/4), chemical drugs (3/4), methadone (3/4), hallucinogens (3/4), and medicines (1/4). The beginning of the rehabilitative program was partly chosen freely (2/4) partly consequence of a Juvenile Court decree (2/4). In all the cases the reason for entrance in TC was drug-addictionMaternal attachment representations were assessed at entrance in TC with the Adult Attachment Interview . Mother-child interactions where coded using the fourth version of the Emotional Availability Scales , the lack of negative/over involving behaviors and the use of verbal and non-verbal channels.Each EA dimension is given a global score on a 7 point scale, where higher ratings stand for more optimal features. Values between 5 and 7 are representative of an emotionally available dyad and considered index of a healthy relationship. Scores around 4 indicate complicated emotional availability, that is behaviors that are appropriate in some ways but that are not fully healthy (for example a mother apparently sensitive or inconsistent in structuring). Scores around 3 indicate less optimal aspects  while the range between 1 and 2 concerns more problematic behaviors As previously said, the mothers were extracted from a larger group in accordance with their adult attachment status, in order to compare the four main AAI categories with respect to the evolution in time of mother child-interactions.Table Figure Figure Figure Figure Figure Finally, Figure This study aimed to assess parenting of drug addicted mothers longitudinally, from different perspectives. Two of the main components of parental functioning were considered: maternal representations with respect to early attachment experiences and actual parenting behaviors enacted during adult-child interactions. To assess adult attachment representations we adopted the AAI  with higher maternal sensitivity and (b) with more stable emotional exchanges if compared to the insecure or the unresolved ones. As expected, the results highlighted marked differences in interactive competencies in the secure vs. insecure/unresolved mothers. More specifically the dyad with the secure (F) mother presented interactions of better quality, with the mother being more sensitive and responsive toward her child's cues and the child showing more optimal and positive responsiveness and involvement of the adult; moreover, these results appeared predictable over time . On the other side, the insecure (Ds) (E) dyads and the one with the unresolved (U) mother presented more changeable patterns of interactions and more difficulties during the periods observed, with the mothers resulting inconsistent in the ability to adequately perceive and respond to their infants' signals.secure attachment representations in the mother resulted associated with higher maternal sensitivity during mother-child interactions while mothers with insecure and unresolved attachment representations presented lower degrees of maternal sensitivity.the dyad with the secure mother experienced better emotional exchanges, that appeared more stable in time  while the dyads with the insecure mothers or with the unresolved mother showed more difficulties and more “jagged” patterns of mother-child interactions.In this sense, with respect to our initial hypothesis we can conclude that:oscillations in parenting”), emotion dysregulation and general distress . Moreover, all the subjects presented double diagnosis with borderline personality disorder, a condition often associated with particularly detrimental interactive features on the maternal side, such as rapid changes from sensitive to punishing responses . This woman, in fact, showed persistent difficulties in modulating negative affectivity during the interactions with her child. Negative affectivity and hostility have been pointed out in the literature as particularly adverse factors with respect to child development and mother-child interactions. Some authors . The same could be said concerning the change in time of emotional availability  mother of our study experienced). Secondly, a generalization of these results is not possible given the small amount of subjects considered and the absence of a control group; on one hand, in fact, the absence of a comparison croup does not allow to control the eventuality that the attachment style and the condition of drug addiction interacted together creating a unique style of mother-infant interaction; moreover, the presence of gender differences in each dyad might have somehow influenced the relational style, so that absolute conclusions cannot be drawn from the comparison of the four attachment styles. Anyway, despite these limits, these data seem to be in line with a large amount of theoretical and empirical literature on attachment, parenting, and early mother-child interactions, confirming in part the differences expected between dyads with secure mothers and dyads with insecure or unresolved mothers (van Ijzendoorn and Bakermans-Kranenburg, AS prepared the study design, supervised the research team, and wrote the discussions section of the manuscript; NC organized the recruitment of the sample and supervised data collection; AP and FD wrote the introduction section and the references, prepared data set, performed statistical analyses and prepared tables and figures; All authors reviewed the manuscript.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.


For any of these applications, PSi/nanoPS can be used directly after synthesis from Si wafers, upon appropriate surface modification processes, or as a composite biomaterial. Unedited studies of fluorescently active PSi structures for cell culture are brought to evidence the margin for new developments.For over 20 years, nanostructured porous silicon (nanoPS) has found a vast number of applications in the broad fields of photonics and optoelectronics, triggered by the discovery of its photoluminescent behavior in 1990. Besides, its biocompatibility, biodegradability, and bioresorbability make porous silicon (PSi) an appealing biomaterial. These properties are largely a consequence of its particular susceptibility to oxidation, leading to the formation of silicon oxide, which is readily dissolved by body fluids. This paper reviews the evolution of the applications of PSi and nanoPS from photonics through biophotonics, to their use as cell scaffolds, whether as an implantable substitute biomaterial, mainly for bony and ophthalmological tissues, or as an  According to Williams , tissue 4 stimulates calcification and subsequent hydroxyapatite formation  and nanostructured porous silicon (nanoPS) have found increasing applications beyond traditional uses in the field of photonics to the field of tissue engineering as cell scaffold given that its morphology at the micro- and nano-scales can be used to regulate cell behavior   and infrared Canham, . The mosPorous silicon is nowadays almost exclusively fabricated by the electrochemical etching of silicon wafers in hydrofluoric acid-based electrolytes . The Si–F bonds cause a polarization effect allowing a second F− anion to attack and replace the remaining hydrogen bonds. Two hydrogen atoms can then combine, injecting an electron into the substrate (3). The polarization induced by the Si–F bonds reduces the electron density of the remaining Si–Si backbonds making them susceptible to further attack by HF in such a manner that the remaining silicon surface atoms are bonded to the hydrogen atoms, which suffer a second nucleophilic attack by a F− anion forming silicon tretrafluoride (SiF4) (4). The SiF4 molecule reacts with HF to form the highly stable Many different models have been proposed to explain pore formation in PSi. However, the most accepted model concerning the silicon dissolution/PSi formation is the series of electrochemical reactions schematized in Figure All the key properties of PSi/nanoPS including porosity, thickness, pore diameter, and microstructure, depend on the properties of the Si wafer and the synthesis parameters , an oligopeptide sequence found in cell adhesion structures (Steps 3 and 4). Using rat mesenchymal stem cells (MSC) for cell cultures on these modified PSi surfaces, the authors showed that cell attachment increased with increasing cRGD density electrografting 6(OH)2] phase has been suggested. This is primarily because HAP is the main inorganic extracellular matrix component of skeleton cells  ceramics in its hydroxyapatite [HAP, CaThe most common processes for PSi/nanoPS functionalization of CaP are the biomimetic growth using simulated body fluids (SBF), and electrodeposition techniques using CaP aqueous solutions    were morphologically different for hMSCs in the central seeding area and those migrating on the Si/PSi triangle columns , Silicon Kinetics (USA), and pSivida (USA) are private companies dedicated to manufacture and market sustained-release drug-delivery systems based on PSi. In that sense, further research on PSi-based cell scaffolds should be performed to promote the transfer to real applied devices.The future of Psi-based scaffolds for tissue engineering passes first by the fabrication of new PSi surface terminations. Freshly produced PSi is extremely hydrophobic, though its natural degradation in physiological environment turns it into hydrophilic in a few minutes (Hajj-Hassan et al., JH-M drafted the initial manuscript. AM-N, JG-R, VT-C, RJM-P, and MM-S made contributions and modifications according to their field of expertise. All authors reviewed and approved the manuscript.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.