Embryonic pattern of cartilaginous head development in the European toad, Bufo bufo.

The craniofacial skeleton of vertebrates is a major innovation of the whole clade. Its development and composition requires a precisely orchestrated sequence of chondrification events which lead to a fully functional skeleton. Sequential information on the precise timing and sequence of embryonic cartilaginous head development are available for a growing number of vertebrates. This enables a more and more comprehensive comparison of the evolutionary trends within and among different vertebrate clades. This comparison of sequential patterns of cartilage formation enables insights into the evolution of development of the cartilaginous head skeleton. The cartilaginous sequence of head formation of three basal anurans (Xenopus laevis, Bombina orientalis, Discoglossus scovazzi) was investigated so far. This study investigates the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen until the premetamorphic larvae in the neobatrachian species Bufo bufo. Clearing and staining, histology, and 3D reconstruction enabled the tracking of 75 cartilaginous structures and the illustration of the sequential changes of the skull as well as the identification of evolutionary trends of sequential cartilage formation in the anuran head. The anuran viscerocranium does not chondrify in the ancestral anterior to posterior direction and the neurocranial elements do not chondrify in posterior to anterior direction. Instead, the viscerocranial and neurocranial development is mosaic-like and differs greatly from the gnathostome sequence. Strict ancestral anterior to posterior developmental sequences can be observed within the branchial basket. Thus, this data is the basis for further comparative developmental studies of anuran skeletal development.

Sequence of chondrocranial development in basal anurans-Let's make a cranium.

BACKGROUND: The craniofacial skeleton is an evolutionary innovation of vertebrates. Due to its complexity and importance to protect the brain and aid in essential functions (e.g., feeding), its development requires a precisely tuned sequence of chondrification and/or ossification events. The comparison of sequential patterns of cartilage formation bears important insights into the evolution of development. Discoglossus scovazzi is a basal anuran species. The comparison of its chondrocranium (cartilaginous neuro- & viscerocranium) development with other basal anurans (Xenopus laevis, Bombina orientalis) will help establishing the ancestral pattern of chondrification sequences in anurans and will serve as basis for further studies to reconstruct ancestral conditions in amphibians, tetrapods, and vertebrates. Furthermore, evolutionary patterns in anurans can be studied in the light of adaptations once the ancestral sequence is established. RESULTS: We present a comprehensive overview on the chondrocranium development of D. scovazzi. With clearing and staining, histology and 3D reconstructions we tracked the chondrification of 44 elements from the first mesenchymal Anlagen to the premetamorphic cartilaginous head skeleton and illustrate the sequential changes of the skull. We identified several anuran and discoglossoid traits of cartilage development. In D. scovazzi the mandibular, hyoid, and first branchial arch Anlagen develop first followed by stepwise addition of the branchial arches II, III, and IV. Nonetheless, there is no strict anterior to posterior chondrification pattern within the viscerocranium of D. scovazzi. Single hyoid arch elements chondrify after elements of the branchial arch and mandibular arch elements chondrify after elements of the branchial arch I. CONCLUSIONS: In Osteichthyes, neurocranial elements develop in anterior to posterior direction. In the anurans investigated so far, as well as in D. scovazzi, the posterior parts of the neurocranium extend anteriorly, while the anterior parts of the neurocranium, extend posteriorly until both parts meet and fuse. Anuran cartilaginous development differs in at least two crucial traits from other gnathostomes which further supports the urgent need for more developmental investigations among this clade to understand the evolution of cartilage development in vertebrates.

The biogenetic law and the Gastraea theory: From Ernst Haeckel's discoveries to contemporary views.

More than 150 years ago, in 1866, Ernst Haeckel published a book in two volumes called Generelle Morphologie der Organismen (General Morphology of Organisms) in the first volume of which he formulated his biogenetic law, famously stating that ontogeny recapitulates phylogeny. Here, we describe Haeckel's original idea as first formulated in the Generelle Morphologie der Organismen and later further developed in other publications until the present situation in which molecular data are used to test the "hourglass model," which can be seen as a modern version of the biogenetic law. We also tell the story about his discovery, while traveling in Norway, of an unknown organism, Magosphaera planula, that was important in that it helped to precipitate his ideas into what was to become the Gastraea theory. We also follow further development and reformulations of the Gastraea theory by other scientists, notably the Russian school. Elias Metchnikoff developed the Phagocytella hypothesis for the origin of metazoans based on studies of a colonial flagellate. Alexey Zakhvatin focused on deducing the ancestral life cycle and the cell types of the last common ancestor of all metazoans, and Kirill V. Mikhailov recently pursued this line of research further.

Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis.

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I-IV, components of the branchial basket, follow this strict anterior-posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.

A novel approach for removing micro-stimulation artifacts and reconstruction of broad-band neuronal signals.

BACKGROUND: Electrical stimulation is a widely used method in the neurosciences with a variety of application fields. However, stimulation frequently induces large and long-lasting artifacts, which superimpose on the actual neuronal signal. Existing methods were developed for analyzing fast events such as spikes, but are not well suited for the restoration of LFP signals. NEW METHOD: We developed a method that extracts artifact components while also leaving the LFP components of the neuronal signal intact. We based it on an exponential fit of the average artifact shape, which is subsequently adapted to the individual artifacts amplitude and then subtracted. Importantly, we used for fitting of the individual artifact only a short initial time window, in which the artifact is dominating the superimposition with the neuronal signal. Using this short period ensures that LFP components are not part of the fit, which leaves them unaffected by the subsequent artifact removal. RESULTS: By using the method presented here, we could diminish the substantial distortions of neuronal signals caused by electrical stimulation to levels that were statistically indistinguishable from the original data. Furthermore, the effect of stimulation on the phases of γ- and ß- oscillations was reduced by 85 and 75 %, respectively. COMPARISON WITH EXISTING METHODS: This approach avoids signal loss as caused by methods cutting out artifacts and minimizes the distortion of the signal's temporal structure as compared to other approaches. CONCLUSION: The method presented here allows for a successful reconstruction of broad-band signals.

Bapx1 is required for jaw joint development in amphibians.

The acquisition of a movable jaw and a jaw joint are key events in gnathostome evolution. Jaws are derived from the neural crest derived pharyngeal skeleton and the transition from jawless to jawed vertebrates consists of major morphological changes, which must have a genetic foundation. Recent studies on the effects of bapx1 knockdown in fish and chicken indicate that bapx1 has acquired such a role in primary jaw joint development during vertebrate evolution, but evidence from amphibians is missing so far. In the present study, we use Ambystoma mexicanum, Bombina orientalis, and Xenopus laevis to investigate the effects of bapx1 knockdown on the development of these three different amphibians. Using morpholinos we downregulated the expression of bapx1 and obtain morphants with altered mandibular arch morphology. In the absence of bapx1 Meckels cartilage and the palatoquadrate jaw joint initially develop separately but during further development the joint cavity between both fills with chondrocytes. This results in the fusion of both cartilages and the loss of the jaw joint. Despite this the jaw itself remains usable for feeding and breathing. We show that bapx1 plays a role in jaw joint maintenance during development and that the morphants morphology possibly mirrors the morphology of the jawless ancestors of the gnathostomes.

Bapx1 upregulation is associated with ectopic mandibular cartilage development in amphibians.

BACKGROUND: The emergence of novel structures during evolution is crucial for creating variation among organisms, but the underlying processes which lead to the emergence of evolutionary novelties are poorly understood. The gnathostome jaw joint is such a novelty, and the incorporation of bapx1 expression into the intermediate first pharyngeal arch may have played a major role in the evolution of this joint. Knockdown experiments revealed that loss of bapx1 function leads to the loss of the jaw joint, because Meckel's cartilage and the palatoquadrate fuse during development. We used Xenopus laevis and Ambystoma mexicanum to further investigate the function of bapx1 in amphibians. Bapx1 expression levels were upregulated through the use of Ly-294,002 and we investigated the potential consequences of the enhanced bapx1 expression in amphibians to test the hypothesized joint inducing function of bapx1. RESULTS: We show that Ly-294,002 upregulates bapx1 expression in vivo. Additionally, ectopic mandibular arch derived cartilages develop after Ly-294,002 treatment. These ectopic cartilages are dorsoventrally oriented rods situated lateral to the palatoquadrate. The development of these additional cartilages did not change the muscular arrangement of mandibular arch-derived muscles. CONCLUSIONS: Development of additional mandibular cartilages is not unusual in larval anurans. Therefore, changes in the bapx1 expression during evolution may have been the reason for the development of several additional cartilages in the larval anuran jaw. Furthermore, our observations imply a joint-promoting function of bapx1, which further substantiates its hypothetical role in the evolution of the gnathostome jaw joint.

Sequence and timing of early cranial skeletal development in Xenopus laevis.

Xenopus laevis is widely used as a model organism in biological research. Morphological descriptions of the larval cartilaginous skeleton are more than half a century old and comprehensive studies of early cartilage differentiation and development are missing. A proper understanding of early cranial skeletal development in X. laevis requires a detailed description that can function as a baseline for experimental studies. This basis makes it possible to evaluate skeletal defects produced by experiments on gene interactions, such as gain- or loss-of function experiments. In this study, we provide a detailed description of the pattern and timing of early cartilage differentiation and development in the larval head of X. laevis. Methods used include antibody staining, confocal laser scanning microscopy and 3D-reconstruction. Results were than compared to earlier studies based on classical histological approaches and clearing-and-staining. The first cartilage to chondrify is, in contrast to other vertebrates investigated so far, the ceratohyal. The components of the branchial basket chondrify in anterior-to-posterior direction as reported for other amphibians. Chondrification of different cartilages begins at different stages and the majority of cartilages are fully developed at Ziermann and Olsson stage 17. Our baseline data on the pattern and timing of early cartilaginous development in X. laevis is useful for evaluation of experiments which alter head skeletal development as well as for identifying heterochronic shifts in head development in other amphibians.

Depressive symptoms as a novel risk factor for recurrent venous thromboembolism: a longitudinal observational study in patients referred for thrombophilia investigation.

BACKGROUND: Increasing evidence suggests that psychosocial factors, including depression predict incident venous thromboembolism (VTE) against a background of genetic and acquired risk factors. The role of psychosocial factors for the risk of recurrent VTE has not previously been examined. We hypothesized that depressive symptoms in patients with prior VTE are associated with an increased risk of recurrent VTE. METHODS: In this longitudinal observational study, we investigated 271 consecutive patients, aged 18 years or older, referred for thrombophilia investigation with an objectively diagnosed episode of VTE. Patients completed the depression subscale of the Hospital Anxiety and Depression Scale (HADS-D). During the observation period, they were contacted by phone and information on recurrent VTE, anticoagulation therapy, and thromboprophylaxis in risk situations was collected. RESULTS: Clinically relevant depressive symptoms (HADS-D score ≥ 8) were present in 10% of patients. During a median observation period of 13 months (range 5-48), 27 (10%) patients experienced recurrent VTE. After controlling for sociodemographic and clinical factors, a 3-point increase on the HADS-D score was associated with a 44% greater risk of recurrent VTE (OR 1.44, 95% CI 1.02, 2.06). Compared to patients with lower levels of depressive symptoms (HADS-D score: range 0-2), those with higher levels (HADS-D score: range 3-16) had a 4.1-times greater risk of recurrent VTE (OR 4.07, 95% CI 1.55, 10.66). CONCLUSIONS: The findings suggest that depressive symptoms might contribute to an increased risk of recurrent VTE independent of other prognostic factors. An increased risk might already be present at subclinical levels of depressive symptoms.

Depressive symptoms, perceived social support, and prothrombotic measures in patients with venous thromboembolism.

INTRODUCTION: Psychosocial factors have been associated with both a prothrombotic state and an increased risk of venous thromboembolism (VTE). We examined the relation of depressive symptoms and social support with D-dimer, an integrative measure of enhanced coagulation activity, and several additional prothrombotic measures in patients with VTE. METHODS: We studied 173 patients with a previous deep venous thrombosis and/or pulmonary embolism (mean age ± SD 45 ± 14 years, 55% men). Clinical and lab assessments took place ≥ 3 months after VTE and ≥ 1 month after discontinuation of oral anticoagulants. The patients rated depressive symptoms and social support by validated questionnaires. RESULTS: After adjusting for sociodemographic and medical covariates, interactions emerged between depressive symptoms and social support for D-dimer (p=0.012) and aPTT (p=0.002). As opposed to patients with high levels of social support, those with low levels of social support showed a direct association of depressive symptoms with D-dimer (r=0.19, p=0.014) and an inverse relationship with aPTT (r=-0.14, p<0.09). Depressive symptoms were associated with levels of thrombin-antithrombin complex (r=0.19, p=0.016). Greater social support was associated with prolonged aPTT (r=0.16, p=0.046). There were no significant associations of depressive symptoms and social support with D-dimer, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FX:C, INR, and thrombin time. CONCLUSIONS: Depressive symptoms are associated with enhanced coagulation activity in patients with VTE, particularly so in those who perceive low levels of social support. Conversely, high levels of social support may contribute directly and through buffering the effect of depressive symptoms to attenuated clotting activity in VTE patients.

Effects of social support on the clinical course of Crohn's disease.

BACKGROUND: Social support has been found to be protective from adverse health effects of psychological stress. We hypothesized that higher social support would predict a more favorable course of Crohn's disease (CD) directly (main effect hypothesis) and via moderating other prognostic factors (buffer hypothesis). METHODS: Within a multicenter cohort study we observed 597 adults with CD for 18 months. We assessed social support using the ENRICHD Social Support Inventory. Flares, nonresponse to therapy, complications, and extraintestinal manifestations were recorded as a combined endpoint indicating disease deterioration. We controlled for several demographic, psychosocial, and clinical variables of potential prognostic importance. We used multivariate binary logistic regression to estimate the overall effect of social support on the odds of disease deterioration and to explore main and moderator effects of social support by probing interactions with other predictors. RESULTS: The odds of disease deterioration decreased by 1.5 times (95% confidence interval [CI]: 1.2-1.9) for an increase of one standard deviation (SD) of social support. In case of low body mass index (BMI) (i.e., 1 SD below the mean or <19 kg/m(2) ), the odds decreased by 1.8 times for an increase of 1 SD of social support. In case of low social support, the odds increased by 2.1 times for a decrease of 1 SD of BMI. Low BMI was not predictive under high social support. CONCLUSIONS: The findings suggest that elevated social support may favorably affect the clinical course of CD, particularly in patients with low BMI.

Relationship between psychological distress and endogenous anticoagulants in patients with a previous venous thromboembolic event.

BACKGROUND: Psychological distress, particularly anxiety and depression, has been associated with a prothrombotic state. However, the relationship between psychosocial factors and endogenous anticoagulants protein S (PS) and protein C (PC) has not previously been investigated. We explored the association between psychological distress, PS, and PC in patients with an objectively diagnosed venous thromboembolic event (VTE). METHODS: We investigated 126 consecutively enrolled patients ≥3 months after VTE (ie, deep venous thrombosis and/or pulmonary embolism) and ≥1 month of discontinuation of oral anticoagulants. Approximately 10 days before blood collection for thrombophilia workup, anxiety and depression scores were assessed using the Hospital Anxiety and Depression scale (HADS). Protein C and S were determined by routine laboratory assays. RESULTS: After controlling for demographic and medical factors, PC, as measured by the PC-activated partial thromboplastin time (aPTT) method and the PC-chromatin substrate method, was positively associated with psychological distress (sum of anxiety plus depression symptoms; P ≤ .027), anxiety (P ≤ .055), and depression (P ≤ .031), explaining between 3% and 6% of the variance. Total PS antigen showed a direct relationship with psychological distress (P = .025) and depression (P = .005), explaining 5% and 7% of respective variances. Free PS showed a positive association with depression (P = .046), explaining 3% of the variance. Anxiety showed no independent association with either PS measure. CONCLUSIONS: Psychological distress is independently associated with enhanced endogenous anticoagulant potential. This might reflect a counterregulatory mechanism to outweigh the previously observed hypercoagulability in individuals under chronic stress and with elevated symptoms of anxiety and depression.

Association of fatigue and psychological distress with quality of life in patients with a previous venous thromboembolic event.

Health-related quality of life (QoL) has been associated with several social and medical conditions in patients with deep vein thrombosis (DVT) and pulmonary embolism (PE). To the best of our knowledge, there is no study investigating the relationship of QoL with psychological variables in this patient population. We assumed as a hypothesis an association between heightened levels of fatigue and psychological distress, as well as decreased QoL in patients with an objectively diagnosed venous thromboembolic event. Study participants were 205 consecutively enrolled out-patients (47.4 years, 54.6% men) with DVT and/or PE. Approximately 10 days before blood collection for thrombophilia work-up, QoL, fatigue, and psychological distress were assessed using the Short Form Health Survey (SF-12), the Multidimensional Fatigue Symptom Inventory Short Form (MFSI-SF) as well as the Hospitality Anxiety and Depression scale (HADS). After controlling for demographic and medical factors, fatigue (p < 0.01) but not psychological distress (p>0.05) was negatively associated with physical QoL, explaining 11.0% of the variance. Fatigue (p < 0.001) and psychological distress (p < 0.001) were significant predictors of mental QoL, explaining an additional 36.2% and 3.6% of the variance. Further analyses revealed that all subscales of the HADS (e.g. anxiety and depression) and of the MFSI-SF (e.g. general fatigue, physical fatigue, emotional fatigue, mental fatigue and vigor) were significant predictors of mental QoL. MFSI-SF subscales also predicted physical QoL. The findings suggest that fatigue and psychological distress substantially predict QoL in patients with a previous venous thromboembolic event above and beyond demographic factors.

Independent association of sleep quality, fatigue, and vital exhaustion with platelet count in patients with a previous venous thromboembolic event.

Elevated platelet count might reflect increased inflammation as an etiological factor for venous thromboembolism (VTE). Poor sleep, fatigue, and exhaustion are all associated with inflammation and are also common sequelae of chronic psychological stress that previously predicted increased risk of VTE. We hypothesized that platelet count would be high in patients with VTE who sleep poorly and who are fatigued and exhausted. We investigated 205 patients scheduled for thrombophilia work-up > or =3 months after an objectively diagnosed venous thromboembolic event. They completed the Jenkins Sleep Questionnaire to rate subjective sleep quality and the short forms of the Multidimensional Fatigue Symptom Inventory and Maastricht Vital Exhaustion Questionnaire. Platelet count was determined by a mechanical Coulter counter. Analyses controlled for age, sex, body mass index, time since the index event, and medication. After taking into account these covariates, poorer sleep quality (p = 0.001; DeltaR(2)= 0.046), high fatigue (p = 0.008; DeltaR(2)= 0.032), and vital exhaustion (p = 0.050; DeltaR(2)= 0.017) were all associated with elevated platelet count. In addition, high level of fatigue mediated the relationship between poor sleep quality and elevated platelet count (p = 0.046). Poor sleep quality, high levels of fatigue, and vital exhaustion were identified as correlates of an elevated platelet count in patients with a previous episode of VTE. Given the emerging role of inflammatory processes in VTE, the findings suggest a mechanism through which behavioral and chronic psychological stressors might contribute to incident and recurrent venous thrombotic events.