Only 1-3% of ischemic stroke patients receive thrombolytic therapy. Provider barriers to adhering with guidelines recommending tPA delivery in acute stroke are not well known. The main objective of this study was to describe barriers to thrombolytic use in acute stroke care.Twenty-four hospitals were randomly selected and matched into 12 pairs. Barrier assessment occurred at intervention sites only, and utilized focus groups and structured interviews. A pre-specified taxonomy was employed to characterize barriers. Two investigators independently assigned themes to transcribed responses. Seven facilitators  conducted focus groups and interviews of emergency physicians (65), nurses (62), neurologists (15), radiologists (12), hospital administrators (12), and three others .The following themes represented the most important external barriers: environmental and patient factors. Important barriers internal to the clinician included familiarity with and motivation to adhere to the guidelines, lack of self-efficacy and outcome expectancy. The following themes were not substantial barriers: lack of awareness of the existence of acute stroke guidelines, presence of conflicting guidelines, and lack of agreement with the guidelines.Healthcare providers perceive environmental and patient-related factors as the primary barriers to adherence with acute stroke treatment guidelines. Interventions focused on increasing physician familiarity with and motivation to follow guidelines may be of highest yield in improving adherence. Improving self-efficacy in performing guideline concordant care may also be useful.NCT00349479ClinicalTrials.gov identifier:  Ischemic stroke is a devastating disease, affecting approximately 600,000 adults in the U.S. every year, leaving many survivors with significant functional limitations. IntraveA large proportion of patients are excluded from treatment due to factors outside of physician control, such as delayed presentation to the hospital. In spite of this, provider-specific barriers remain a significant determinant of low treatment rates,6. PreviThe INcreasing Stroke Treatment through INterventional behavioral Change Tactics (INSTINCT) trial is a cluster randomized, controlled trial aimed at increasing appropriate tPA use in ischemic stroke by first determining hospital-specific barriers and then providing targeted, professional educational interventions. Barriers were determined using a partial grounded theory method, whereby qualitative data obtained through focus group discussions is coded into themes using a previously-described taxonomy. QualitaThe protocol was approved by the University of Michigan Institutional Review Board (IRBMED) and all relevant site IRBs. Written informed consent was obtained from all participants in focus groups and interviews.The INSTINCT trial is evaluating the hypothesis that initial barrier assessment focused on tPA use in stroke followed by targeted, interactive educational interventions will increase appropriate tPA use. These eTwenty-four hospitals were randomly selected from the population of Michigan acute care hospitals and matched into 12 pairs based on emergency department volume and number of stroke patients - Stroke. While GThe qualitative data collection and analysis methods have been described in detail previously and are summarized below. During There were 30 participants in the six initial focus groups (phase 1): 10 EPs, 15 nurses, 3 neurologists, 1 hospitalist, and 1 pharmacist. Focus group composition during phase 1 was mixed by site and occupation and the groups ran concurrently. In phase 2, two focus groups were conducted at each of the 12 intervention sites, one of EPs and one of primarily emergency department nursing staff. A total of 55 EPs and 48 nurses participated in phase 2 focus groups. Additionally, one-on-one structured interviews were conducted with a neurologist, an administrator, and a radiologist at each intervention site. Focus group participants were recruited by the local principal investigator from each site. Participants with disparate opinions and past experience were sought to enhance the diversity of responses. The demographics of these participants were not collected to protect anonymity.The focus group discussion guide was developed with a professional focus group consultant. It is included in Additional file A pre-specified taxonomy was employed to characterize major barriers to clinical guideline adherence. BarrierResponses from participants were coded into nine major themes. The three external barriers were environmental factors , patient factors , and guideline factors . The six internal barriers were the lack of familiarity, agreement, awareness, motivation, outcome expectancy, or self-efficacy. Each paragraph (the coding unit) was coded for all themes found; thus each paragraph could be assigned zero to nine themes. See Table Major themes were derived in advance of data collection. After completion of phase 1, the two coders independently used the phase 1 data to inductively derive minor themes, including the various aspects of acute stroke presentation and treatment, conceptual models of acute stroke presentation, and the overall process of stroke onset to outcome. These minor themes were then coded for both phase 1 and 2 data for the development of the site-specific educational interventions. Barriers were also related to the various phases of acute stroke presentation and treatment. External barriers were related to the conceptual models of the acute stroke presentation. Barriers were related to the points in the overall process from stroke onset to outcome.Phase 1 of the barrier assessments occurred at the initial site investigators' meeting on 3/26/2007. Phase 2 of the barrier assessments was conducted at each of the intervention hospitals from 6/12/2007 to 10/05/2007. The thematic analysis occurred from July to October 2007 and was used to design and prioritize educational interventions for the trial. The short lead time from barrier assessment to intervention was the rationale for the semi-quantitative approach (relative barrier proportions) that was utilized to determine the most discussed barriers from each site.Since the external barriers of environmental and patient factors comprised most of the cited barriers, sub-categories were inductively derived from these two major themes to better inform the sites during the educational intervention. The derived subcategory themes of barriers external to the EP are described in Table Examples of responses which are illustrative of important internal barriers are provided in Table The external barriers of environmental factors and patient factors dominated the barriers discussed for every hospital Figure  and for Within the internal barriers Figure , most paWe investigated barriers to the provision of thrombolysis for stroke in a randomly selected group of community hospitals, focusing on the beliefs and experiences of health care providers most likely to be involved in providing acute stroke care. In general, almost half of the discussed barriers were considered external to the provider . The barriers internal to the provider were prioritized according to a model of physician behavior change.The hospital barrier assessment process was important because although the top barriers were similar across sites, there was still inter-site variability. This stresses the need for interventions targeted to individual hospital and provider barriers. The optimal and efficient design of interventions to improve health processes requires a firm understanding of the knowledge and attitudes of the group targeted for the intervention. This isPast work on barriers to thrombolysis has focused on patient- or hospital-level characteristics and not provider-level barriers,6. Our fThe finding that EPs frequently cited lack of motivation to adhere to, and lack of familiarity with, the content of guidelines for stroke thrombolysis is intuitive. An example of this is the observation that physicians will repeatedly examine patients looking for improvement to justify withholding treatment. Prior and current ongoing methods of examining thrombolytic utilization and eligibility have not captured this. Designing interventions that recognize treatment should occur promptly to combat this hesitancy is important, particularly since providers may delay even further with the recent publication of data that potentially expands the time window for thrombolysis,19. The The relative minimal importance ascribed to a lack of agreement with the guidelines is surprising, especially in light of the past controversy that stroke thrombolysis has generated within the field of emergency medicine,22. ThisOur separate interviews with nurses and EPs provided unique findings. The repeated re-examination phenomenon was described by emergency department nurses. This specific example typifies the perceived barrier that was cited as most important by many nurses: lack of motivation. The picture that is painted is that of the clinician who is uncomfortable and unsure when faced with the potential of having to administer a thrombolytic agent. It is doubtful if this barrier would have been articulated as clearly without interviews restricted to individual provider types.This work has several important limitations. We did not generally seek "saturation" by performing repeat focus groups with the intent of further delving more deeply into specific themes. We used an existing taxonomy to classify responses, which might have missed barriers that did not fit well into any of the categories. The integration of these results with quantitative methods and overall response to the targeted educational interventions (as evidenced by change in tPA treatment rates), is not possible at this point in the overall trial. We focused only on 12 hospitals within Michigan, and while these hospitals came from diverse geographic and socioeconomic areas, these findings may not be widely generalizable. There is a potential that participants in the focus groups and interviews were generally more positive towards stroke thrombolysis, although it is also plausible that participants with strong negative opinions would also be extremely motivated to participate. Overall it appears that a range of opinions were represented by our participants. This contributes to the richness of the findings of the current investigation.In summary, healthcare providers responsible for acute stroke treatment perceive environmental and patient factors as the most important barriers to adherence with the AHA acute stroke guidelines. With respect to internal barriers, nurses perceived lack of guideline familiarity as the biggest barrier whereas physicians (both EPs and neurologists) perceived physician motivation as the primary barrier. Overall, the minimal discussion of lack of physician agreement as a barrier is interesting in light of ongoing controversy over the use of tPA for stroke in the field of emergency medicine. Greater knowledge of the barriers which impede the widespread adoption of acute stroke thrombolysis is crucial to designing effective educational interventions to improve guideline adherence and may be informative in other areas where difficult risk/reward decisions are made on an emergent basis.The authors declare that they have no competing interests.PAS conceived, obtained funding, and supervised this study. WJM developed the analysis and data collection methods. WJM, SAF AMS, and PAS all participated in data collection. WJM and JJM performed the data analysis. WJM wrote the first draft of the paper; all authors have read and edited the paper for content and approve of this manuscript. WJM and JJM have full access to all of the data in this study and take responsibility for the integrity of the data and the accuracy of the data analysis.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-227X/11/5/prepubThis is the final focus group script that was used for emergency physician or nurse focus groups.Click here for fileThis is the coding guide developed by the investigators with conventions used in assigning themes.Click here for file


Ciona intestinalis, an invertebrate chordate belonging to the sister clade of vertebrates, in which the prototypical chordate body plan is established through very derived morphogenetic processes. Ciona embryos are constituted of few cells and develop according to a fixed lineage; elongation of the tail occurs largely by rearrangement of postmitotic cells; mesoderm segmentation and somitogenesis are absent. We show that in the Ciona embryo, the antagonism of the RA and FGF/MAPK signals is required to control the anteroposterior patterning of the tail epidermis. We also demonstrate that the RA, FGF/MAPK and canonical Wnt pathways control the anteroposterior patterning of the tail peripheral nervous system, and reveal the existence of distinct subpopulations of caudal epidermal neurons with different responsiveness to the RA, FGF/MAPK and canonical Wnt signals. Our data provide the first demonstration that the use of the antagonism between the RA and FGF signals to pattern the main body axis predates the emergence of vertebrates and highlight the evolutionary plasticity of this patterning strategy, showing that in different chordates it can be used to pattern different tissues within the same homologous body region.Vertebrate embryos exploit the mutual inhibition between the RA and FGF signalling pathways to coordinate the proliferative elongation of the main body axis with the progressive patterning and differentiation of its neuroectodermal and paraxial mesodermal structures. The evolutionary history of this patterning system is still poorly understood. Here, we investigate the role played by the RA and FGF/MAPK signals during the development of the tail structures in the tunicate  Xenopus, RA induces the expression of MKP3, a phosphatase that inhibits the MAPK branch of the FGF signalling pathway, while FGF controls the expression of Cyp26, a P450-family cytochrome that hydrolyses RA Antagonism between the Retinoic Acid (RA) and FGF signalling pathways controls the development of the posterior vertebrate body The strategy relying on opposing FGF/WNT and RA gradients to coordinate embryo elongation and patterning has been identified only in vertebrates, and it is unclear whether it is exploited by other chordates. Some support for the existence of a two opposing gradient system in the last common chordate ancestor comes from amphioxus, currently seen as the most basal extant chordate Ascidians belong to the subphylum Tunicata (or Urochordata), the sister group to vertebrates If the last common chordate ancestor already exploited opposing FGF/WNT and RA signals to coordinate AP elongation and patterning, then such a mechanism is likely to have undergone profound modifications in tunicates: it may have been altogether lost for lack of selective constraints, simplified, adapted to different constraints such as a determinative mode of development or recycled to fulfil novel functions. In any case, a better understanding of the molecular and functional interactions between FGF, WNT and RA pathways in the development of the posterior structures of ascidian larvae will not only contribute to clarifying the existence of a common minimum blueprint for generating the chordate body plan, but also provide insights into how the evolution of gene regulatory networks and other complex patterning mechanisms accompanies and/or underlies morphological and morphogenetic simplification in an extremely derived organism that retained a prototypical chordate body plan Ciona intestinalis, the RA pathway is specifically active within the anterior tail epidermis, while the activities of the FGF/MAPK and WNT canonical pathways are restricted to the tailtip epidermis, a situation reminiscent of the two opposing anterior RA and posterior FGF/WNT gradients described in vertebrates. The RA and FGF/MAPK signals counteract each other, and this antagonism is required for the proper AP patterning of the tail epidermis and PNS. Thus, our data provide the first functional evidence that a strategy relying on the mutual antagonism between RA and FGF acts to pattern the posterior body of an invertebrate chordate, although in the very derived Ciona this may be used only to control the patterning of epidermal structures. This could reflect either a shared ancestral chordate usage of this system to control the anteroposterior patterning of all posterior body structures, followed by taxon-specific losses, or a case of repeated cooption of a particularly efficient developmental regulatory network during the course of chordate evolution.Here we show that in the elongating embryonic tail of Ciona embryonic posterior structures, we first defined the territories where these pathways are active during the processes of tail patterning and elongation.To address the potential role of the RA, FGF/MAPK and WNT signals in the patterning of Ciona embryo tail, immunoreactivity against diphosphorylated Erk, a hallmark of the activation of the RTK-dependent MAPK pathway, is detected in the epidermal cells surrounding the tailtip from neurula Ciona FGF receptor, Ci-FGFR  of epidermal cells at the tip of the extending tail from the gastrula stage onwards . Although WNT5 and WNT11 have often been considered as members of a “non-canonical” signalling subgroup, it has been recently shown that whether the canonical or the non-canonical pathways are activated depends more on the receptor/co-receptor context than on strictly defined WNT functional classes Ci-LRP5/6, the Ciona homologue of LRP5/6, the WNT co-receptor required for canonical pathway activation, is expressed in the ventral midline epidermis  constituting the posterior larval peripheral nervous system (PNS) are distributed with some periodicity along the dorsal and ventral midlines of the tail  are consistently born earlier than the anterior ones (Ci-Vesicular Glutamate Transporter (Ci-VeGluT) promoter We next searched for differentiated the tail  and, togior ones . We nextCiona CENs and support the existence of distinct anterior and posterior subpopulations of vCENs. Consistent with this hypothesis, posterior and anterior vCENs derive from different epidermis precursor blastomeres at the onset of gastrula and thus have a partially distinct lineage history Halocynthia roretziThus, our data reveal unexpected morphological differences among Ci-etr-positive vCENs at late tailbud stage  use different segmentation and patterning mechanisms Although Ciona embryos, the details of interaction between the RA and FGF signals differ from the model described in vertebrates. Accordingly, the mutual inhibition between RA and FGF in Ciona seems to take place only at the level of signal transduction or degradation rather than synthesis, as is the case in mouse or chicken Hox genes Ciona, the unique Cdx factor Ci-cdx is surprisingly expressed throughout the tail epidermis with the exception of the posteriormost cells, where FGF/MAPK signalling is active Ciona and vertebrates: as in vertebrates, RA promotes the expression of the anterior gene Ci-hox1 and represses the posterior Ci-hox12, while FGF has the opposite effect Ciona the loss of temporal collinearity has allowed for  the disintegration of Hox clustering, the maintenance of spatial collinearity is accompanied by the conservation of upstream regulatory factors. To what extent this holds true in other metazoans with various degrees of Hox gene clustering and temporal/spatial collinearity Our data show that not only small cell-autonomous kernels based on interactions among transcription factors Ciona and the vertebrates may be related to the different dynamics of the antagonistic RA and FGF activities: rather than generating a moving determination front that progressively shifts posteriorly during elongation, in Ciona embryos RA and FGF define two stable determination fields in the epidermis before the onset of the tail extension process or at its very beginning.Our work thus provides a further addition to a growing list of cases where the precise relationships among regulatory network components vary from one metazoan species to the next, but the ultimate outcome of the network is comparable Ciona the opposition between these two signals is required to pattern the posterior epidermis. This raises the possibility that in ancestral chordates, the RA/FGF antagonistic coupling was exploited to regulate the patterning of a broad range of embryonic derivatives, including mesoderm, neurectoderm and non neural epidermis, and that in the course of evolution, different chordate taxa have undergone germ layer-specific losses of this patterning mechanism. Such a situation could reflect taxon-specific differences in the degree of patterning complexity of each germ layer, exemplified by the complex patterning of epidermis and the absence of paraxial mesoderm patterning in Ciona. Alternatively, it is possible that in some cases the requirement for the antagonism between RA and FGF has been overlooked. As an example, it has been shown in amphioxus that RA affects the AP patterning of the epidermis and the distribution of the epidermal neurones In vertebrates the antagonism between RA and FGF has been shown to control the patterning of the paraxial mesoderm and spinal cord Ciona life cycle, which is still unknown. Whatever its function is, it is tempting to speculate that in Ciona, the presence of a strongly regionalized tail PNS represents the selective force requiring the maintenance of a complex signalling system responsible for controlling the AP pattern of the posterior body. In this respect, it is worth noting that in the larvacean Oikopleura dioica, which lacks a tail PNS analogous to that of Ciona , most of the RA signalling machinery is missing Our work reveals the existence of subpopulations of Ciona CENs with distinct morphologies and different susceptibility to the RA, FGF and Wnt signals. The morphological heterogeneity of the tail PNS neurons is most likely imposed by their function during the Ciona intestinalis exploit a very similar mechanism, based on the antagonism between the RA and FGF/MAPK signals, to pattern the posterior body of their embryos and larvae. Thus this mechanism, until now described only in vertebrates, likely represents an ancestral trait common to all olfactores. Remarkably, while in the vertebrates the opposition between RA and FGF/MAPK coordinates the patterning of the posterior neural tube and paraxial mesoderm, in Ciona it controls the AP patterning of the posterior epidermis and the associated PNS, suggesting a high degree of functional plasticity throughout evolution. We also propose that the presence of a relatively complex posterior PNS might have been the selective constraint responsible for retaining the RA vs FGF/MAPK antagonism in Ciona. Altogether, our work supports the existence of a common minimum blueprint for generating the chordate body plan but also shows how this has been modified and restricted or redeployed to only a subset of tissues in an extremely derived organism that retained a prototypical chordate body plan.We show that, regardless of the different morphogenetic strategies they adopt to build the chordate body plan, the vertebrates and the tunicate C. intestinalis adults were collected by the Biological Sample Collection Service of the Station Biologique de Roscoff, France. Fertilization and embryo culture were performed as previously described Ciona Fucosyl Transferase control region that drives expression in the b-line from late gastrula stage .Embryo electroporation was performed as described in In situ hybridization and X-gal staining were performed as described in 3VO4 to inhibit endogenous phosphatases. After EtOH dehydration, embryos were re-hydrated in PBTri (PBS+ Triton 0.1%), treated for 10′ with H2O2 0.6% in PBTri, blocked in PBTri, 1% Roche Blocking Reagent, 10% goat serum, then incubated overnight with an anti-dpErk antibody . After extensive washing in PbTri, embryos were incubated overnight with a HRP-conjugated anti-mouse antibody , followed by revelation with the TSA Plus Fluorescein Kit (Perkin Elmer). After the revelation, the embryos were fixed for 30′ in 4% paraformaldehyde, extensively washed in PBTri, then blocked and incubated overnight with an AP-conjugated anti-fluorescein antibody (Roche), followed by NBT/BCIP staining.Detection of diphosphorylated Erk was performed with a modified version of the protocol described in http://chirouble.univ-lyon2.fr/~ricco/tanagra/en/tanagra.html).vCENs were stained by ISH against Ci-etr, and their number was counted in control and treated embryos. To quantify their distribution along the tail, embryos were photographed and analysed with the ImageJ software. The following parameters were analysed: total embryo length ; total tail length ; anteriormost vCEN position (distance between the trunk/tail junction and the anteriormost Ci-etr+ cell); posteriormost vCEN position (distance between the tailtip and the posteriormost Ci-etr+ cell). All the statistical analyses on neuron number and position were performed using ANOVA with Tukey post hoc test in R environment. To quantify the morphological differences between anterior and posterior vCENs, fertilized eggs were electroporated with the pVGluT::EGFP construct, then let develop until hatching larva stage in controls condition or in the presence of RA or bFGF from late gastrula stage. The larvae were processed for anti-GFP immunofluorescence as described above, then individual vCENs were photographed and analysed with the ImageJ software. The following parameters were measured: cell body surface, axonal length and distance of the cell body from the trunk-tail junction. Cluster analysis was performed with the TANAGRA software : treatment with recombinant bFGF leads to the ectopic diphosphorylation of Erk, while treatment with U0126 results in a loss of the dpErk signal throughout the embryo, distinct from the tailtip specific loss due to RA treatment shown in B): Ci-fgf9/16/20 is expressed by a few muscle posterior muscle cells close to the tail tip (image kindly provided by C. Hudson and H. Yasuo).((TIF)Click here for additional data file.Figure S2A): Ci-LRP5/6, the Ciona homologue of LRP5/6, the WNT co-receptor required for canonical pathway activation, is expressed in the ventral midline epidermis. Left panel, side view of an early tailbud stage embryo, anterior is to the left. Right panel, ventral view of the same embryo, anterior is to the top. (B): X-Gal staining of embryos electroporated with low amounts of the Wnt canonical pathway reporter construct p12xTCF::LacZ. Following mosaic inheritance of the electroporated plasmid, the activity is detected in the posterior ventral midline tail epidermis, but not in the endodermal strand. The epidermal activity is independent from the endodermal activity and is first detected at neurula stage in the precursors of the posterior ventral midline epidermis. (C): Co-expression of an epidermally-targeted dominant-active form of β-catenin leads to ectopic canonical Wnt activity. Black arrows point to the posterior ventral tail midline.((TIF)Click here for additional data file.Figure S3A): treatment with DEAB blocks the activity of the pCi-Hox1(intron2)::lacZ reporter construct, while treatment with RA leads to its ectopic activation throughout the embryo. (B): treatment with RA at late gastrula stage rescues the U0126-induced loss of Ci-cyp26 expression in the anterior tail epidermis. (C): bFGF treatment of pCi-Hox1(intron2)::lacZ electroporated embryos results in a decrease in the number of embryos showing epidermal activity.((TIF)Click here for additional data file.


The recent findings brought the necessity of testing the mutational status of a series of genes which had been already identified as responsible for melanomas development and progression, such as BRAF, CKIT and PTEN: the consequent results are, in fact, essential to guide the assessment of the novel treatment protocols based on tailored targeted therapies. We present here the case of a 66 year-old male patient, diagnosed with an advanced melanoma in June 2011, and treated with Dabrafenib for double mutant metastatic disease. The patient was referred to our attention for a large exophytic malignant melanoma on the left shoulder. After complete surgical excision and elective lymph node dissection for presence of metastatic sentinel lymph node, the patient has started high-dose interferon alfa-2b injections as adjuvant therapy for a complete negative staging. The treatment was interrupted in August 2011 due to the appearance of metastatic lymph nodes. Tumor burden was rapidly growing reaching in few months the size of a tennis ball for the tumor mass located in the shoulder. Mutational study of the tumor revealed a double BRAF mutation on V-600E and V600M. This finding incited us to enroll the patient in compassionate Dabrafenib clinical trial. The therapy was started on may 2012 at 150 mg bid dosage. Almost surprisingly for the rapidity of the effect, one week later the lesion on the shoulder has reduced its size by 60% and one month later it has completely disappeared from sight. CT scan of June 2012 documented the astonishing clinical response. The last year has finally experienced a real breakthrough in advanced melanoma therapy, that had probably been awaited for decades: molecular targeted therapies have been added to old and often impotent treatments in the battle against its metastatic disease.It is nowadays well known that prognosis is dramatically influenced by primary tumor stage and chanches to be cured are very low in presence of non-operable metastatic disease. In fact, metastatic melanoma faces a poor outcome, with a predicted 5-year survival rate less than 10%, according to the metastases site and serum LDH levels [The recent findings brought the necessity of testing the mutational status of a series of genes which had been already identified as responsible for melanoma development and progression, such as BRAF, CKIT and PTEN (MEK and mTOR pathways): the consequent results are, in fact, essential to guide the assessment of the novel treatment protocols based on tailored targeted therapies. Among them, Vemurafenib  and Dabrafenib GSK2118436), are the selective inhibitors of BRAF kinase activity that competitively inhibit ATP [6, are thAt this regard we present here the case of a 66 year-old male patient, diagnosed with an advanced melanoma in June 2011, and treated with Dabrafenib for double mutant metastatic disease.The patient was referred to our attention for a 5 cm large exophytic skin lesion on the left shoulder, that revealed to be a malignant melanoma Figure a. After Mutational study of the tumor revealed a double BRAF mutation on V-600E and V600M. Preliminary evidences showed an antiprolifetive activity of Dabrafenib also on rare BRAF somatic mutations different from the V600E ,5. This Obviously, the therapeutic benefit improved patient quality of life and mood. Based on previous experiences in the use of anti-BRAF molecules we cannot be optimistic and a relapse of the disease is expected at any time, so the patient is nowadays in constant monitoring. While we eagerly keep monitoring the evolution of our patient’s disease, we are awaiting the new advances in melanoma targeted therapy. Molecular targeted therapies (e.g. B-RAF inhibitors) have reached high response rates but unfortunately rather short response duration , while Immunotherapy showed slower but more durable results . MoreoveIt is necessary to understand and overcome the limitations of these strategies, especially those concerning resistance mechanisms, in order to transform ephemeral remissions in long-lasting healing.Written informed consent was obtained from the patient for publication of this case report and accompanying images.The authors declare that they have no competing interests.GP was responsible of the clinical management of the patient, acquisition of data, drafting the manuscript; AT was supervisor of management and interpretation of data; GP was responsible of the scientific revision, discussion and editing of the manuscript. All authors read and approved the final manuscript.


Measuring the impact of alcohol consumption on morbidity and mortality depends on the accurate measurement of alcohol exposure, risk relationships, and outcomes. A variety of complicating factors make it difficult to measure these elements. This article reviews these factors and provides an overview of the articles that make up this special issue on current research examining alcohol’s role in the burden of disease. These topics include estimating alcohol consumption as well as alcohol-related morbidity and mortality in various demographic groups, and the burden of alcohol use disorders. Alcohol Research: Current Reviews examines the public health impact of alcohol consumption beyond the role of alcohol use disorders alone  causing the burden b;The measurement of the risk relations  a; andThe measurement of outcomes.This issue of rs alone —that is,rs alone :The meaAlcohol Research: Current Reviews.These different measurements are central to this issue of Numerous challenges exist when measuring the extent and predictors of alcohol-related mortality and morbidity. Those challenges also affect our ability to evaluate the effectiveness of interventions to reduce alcohol-related morbidity and mortality. Different challenges exist for acute alcohol-related mortality and chronic disease mortality and morbidity, although some of the same challenges confront measurement in both areas. The following list of challenges is illustrative but not exhaustive.Postmortem alcohol test data are not consistently available for many types of acute injury or poisoning deaths. The best available U.S. estimates indicate alcohol-attributable acute deaths outnumber chronic disease deaths 44,000 to 35,000 . TrafficIn addition, by examining the characteristics of crashes and drivers in fatal crashes where alcohol is present, the National Highway Traffic Safety Administration (NHTSA)  has deveUsing this approach, researchers have been able to determine annual State and national estimates of alcohol involvement in fatal traffic crashes since 1982. Furthermore, having those accurate direct-test results and imputed results has permitted researchers to make epidemiologic estimates of the increased odds of fatal crashes and other crash involvement at various BALs . DriversBecause these data are available monthly on a national, State, and community level, researchers also have been able to monitor trends in fatal crashes involving alcohol relative to fatal crashes where alcohol is not involved over time. In addition, researchers are able to use quasi-experimental and other research designs to evaluate whether State-level traffic safety legislation and community-level education and law enforcement and treatment programs are effective in reducing alcohol-related traffic deaths .Such studies have guided policy-makers to select and implement effective programs and policies. Since the early 1980s, alcohol-related traffic death rates per 100,000 have been reduced more than 50 percent versus the decline in traffic crash death rates where alcohol is not involved . It has Unfortunately, unlike traffic deaths, postmortem alcohol testing is not nearly as complete for other types of unintentional or intentional poisoning and injury deaths. In 18 States , a violeSecond, research is emerging indicating that alcohol may interact with and pharmacologically potentiate the effects of other drugs, thereby increasing risks of motor-vehicle crashes  and poisThird, it is important to calculate the secondhand harm alcohol misuse poses. Just as awareness of the secondhand negative consequences of passive smoke inhalation has heightened the public health resolve to curb smoking, learning about the secondhand effects of alcohol misuse may heighten the resolve to study and implement effective interventions to reduce alcohol misuse. For example, 40 percent of people who die in traffic crashes involving drinking drivers in the United States are not driving. Half of the deaths in crashes involving drinking drivers under the age of 25 are those other than the driver. This has incited citizen activists and policymakers to pass more than 2,000 laws at the State and Federal levels to reduce alcohol-impaired driving .Fourth, many prevention activities are implemented at the community level, and community-level data are needed to stimulate the planning and evaluation of those interventions (Hingson and White 2012). Yet most surveillance data-monitoring systems measure behavior and consequences at the State and Federal levels. Strategies are needed to either facilitate more community-level data collection or to offer technical assistance to concerned communities and researchers so that they can collect their local data using standardized questions and sampling procedures for comparison with other communities, their State, and the Nation.When examining either acute-disease and chronic-disease mortality and morbidity, a variety of measurement challenges may produce underreporting. First, drinking levels reported in surveys account for only 40 to 60 percent of alcohol sales . UnderreSecond, especially with chronic disease, in etiology studies the time proximity of drinking data collection to disease outcome must be carefully evaluated. Although some chronic diseases may take years to develop, cessation or reduction of drinking may stop the process and reduce morbidity or mortality consequences almost immediately. This can be seen in the immediate gains in mortality and life expectancy in Russia following the Gorbachev reforms that led to a reduction of drinking . HoweverThird, maintaining high response rates in surveys and longitudinal studies has become increasingly difficult over time, particularly using telephone methods, as the percentage of the population who uses mobile phones increases. If nonresponse becomes high and disproportionately involves people with characteristics and behaviors , that may cloud our understanding of alcohol’s role in the development and progression of disease. It also can limit the ability of researchers to monitor disease and death-rate trends over time.Fourth, both in estimates of acute and chronic conditions, attributable fractions from meta-analyses of epidemiologic studies are used to estimate alcohol’s contribution to mortality and disability. Yet, these attributable fractions may change over time. For example, the percentage of factual traffic-crash deaths that involve alcohol have dropped from 60 percent to just under 40 percent in the past 30 years . If the Fifth, when chronic disease morbidity and mortality attributions are made, the range of diseases considered may vary. Current U.S. estimates may not fully consider alcohol’s role in chronic diseases such as HIV.This issue of ARCR examines the methodology involved in measuring the burden of alcohol use in greater detail. Dr. Cheryl Cherpitel focuses more in depth on one of these data sources for the community in her examination of hospital emergency departments . She desThe next two chapters deal with two other outcomes of alcohol use. No overview on alcohol use and consequences would be complete without mentioning the efforts to enumerate alcohol-attributable economic costs. The sidebar focuses on the last attempt to estimate such costs for the United States, focusing on heavy drinking .The first part of the volume is complimented by three sidebars. In the second part of the volume, the focus is on alcohol use and its consequences over the lifespan. It starts chronologically with use by children and adolescents . AnotherFor children, adolescents, and college students, despite the problems with establishing causality, a number of alcohol-attributable consequences have been identified. Some of them may be further in the future, as there are links between age of onset of alcohol use and alcohol dependence or other consequences in later years in the United States . The mosOther groups of concern highlighted in this issue include women  and ethnAlcohol use disorders (AUDs) are one of the most important consequences of alcohol use. Diagnostic and Statistical Manual of Mental Disorders–IV (Grant 2013). Notes from a special NIAAA expert panel on alcohol and chronic diseases (The issue concludes with contributions on the new diseases  also are


How does the organization of phenotypes relate to their propensity to vary? How do evolutionary changes in this organization affect large-scale phenotypic evolution? Over the last decade, studies of morphological integration and modularity have renewed our understanding of the organizational and variational properties of complex phenotypes. Much effort has been made to unravel the connections among the genetic, developmental, and functional contexts leading to differential integration among morphological traits and individuation of variational modules. Yet, their macroevolutionary consequences on the dynamics of morphological disparity–the large-scale variety of organismal designs–are still largely unknown. Here, I investigate the relationship between morphological integration and morphological disparity throughout the entire evolutionary history of crinoids (echinoderms). Quantitative analyses of interspecific patterns of variation and covariation among characters describing the stem, cup, arm, and tegmen of the crinoid body do not show any significant concordance between the temporal trajectories of disparity and overall integration. Nevertheless, the results reveal marked differences in the patterns of integration for Palaeozoic and post-Palaeozoic crinoids. Post-Palaeozoic crinoids have a higher degree of integration and occupy a different region of the space of integration patterns, corresponding to more heterogeneously structured matrices of correlation among traits. Particularly, increased covariation is observed between subsets of characters from the dorsal cup and from the arms. These analyses show that morphological disparity is not dependent on the overall degree of evolutionary integration but rather on the way integration is distributed among traits. Hence, temporal changes in disparity dynamics are likely constrained by reorganizations of the modularity of the crinoid morphology and not by changes in the variability of individual traits. The differences in integration patterns explain the more stereotyped morphologies of post-Palaeozoic crinoids and, from a broader macroevolutionary perspective, call for a greater attention to the distributional heterogeneities of constraints in morphospace. Heterogeneous patterning of morphospaces  has been frequently documented in clade-wide temporal studies, and is now widely acknowledged as a prominent feature of phenotypic macroevolution ), disparity arguably also has an underlying, non-trivial structure. This structure potentially reflects aspects of the hierarchical organization of phenotypes into quasi-independent units of evolutionary transformation, i.e., evolutionary modules Although morphological disparity analyses have been undertaken primarily as a means to globally characterize patterns of stability and change of realized morphospace during the long-term history of clades  over the Phanerozoic. The evolutionary history of crinoids is marked by two distinct radiations, occurring firstly in the early Palaeozoic  and secondly in the Triassic-Early Jurassic, as part of the recovery from the end-Permian mass extinction (∼251 Myr ago). Both radiations are characterized by rapid morphological diversifications at relatively low taxonomic diversity. Nevertheless, Foote itself, reflecting the highest levels of the hierarchical embedding of evolutionary modules within the crinoid body plan.Given the temporal scale, the taxonomic level and the degree of morphological resolution, the temporal changes in the overall degree of integration do not focus on patterns comparable to those that are generally described at low taxonomic levels and concerned with small-scale aspects of organismal organization and variation. Rather, the evolutionary dynamics of integration quantified here is more closely related to the dimensionality of the crinoid morphospace The morphological dataset used in the present study has been compiled and regularly augmented by Foote I followed two complementary approaches in order to allow the use of different measures of morphological disparity and integration. The first approach treats discrete characters directly and is hereafter referred to as the discrete character space approach; the second approach consists in extracting a dissimilarity matrix from the discrete character space using the mean character difference as the measure of morphological dissimilarity between two species i and j is defined as the total number of characters compatible with both i and jWith these two approaches, morphological disparity is measured as the mean pairwise dissimilarity and as the sum of univariate variances respectively, which are both standard indices of disparity relatively insensitive to sample size For the continuous variables obtained via PCoA, I used the relative standard deviation of the eigenvalues of the correlation matrix proposed by Pavlicev et al n, large p” problem makes the sample correlation matrix an unreliable estimator of the population correlation matrix. Indeed, when the number of individuals becomes too small compared to the number of variables, the sample correlation matrix loses its full-rank and positive definiteness, thereby biasing the distribution of its eigenvalues and, in the present context, the measures of morphological integration. In addition, it has been shown analytically that the lower bound of the range of the standard deviation of eigenvalues for finite sample correlation matrices varies as (1/n)1/2R package corpcorp/n ratio of 2.73 with a maximum of 6 .The temporal partitioning of the morphospace into successive time intervals often leads to the extraction of matrices with more variables than individuals from the total morphological dataset. This “small Comparison between morphological disparity and integration cannot be made directly because of the potential effect of trends and serial correlation inherent to most time series. To circumvent these effects, I used the generalized differencing approach Finally, to trace the evolution of patterns of integration in greater details, I also used the metric recently proposed by Mitteroecker and Bookstein Assessing patterns of correlation and compatibility among characters can be hindered by the fact that species are not independent entities but parts of a hierarchically structured phylogeny resulting from branching evolution Even though characters provide ecological and functional information about crinoid morphology, some also enable taxonomic distinctions within and among higher taxa P<0.01 in both cases with a Mann-Whitney U test).r = −0.449, P = 0.042; I further investigate the relationships between degree of correlation among traits and level of morphological variety by calculating the correlation between the generalized differences of integration and disparity estimates . Only thP<0.001; Mann-Whitney U test) despite the roughly equivalent duration separating successive intervals. This suggests greater magnitudes of transition between successive patterns of integration in post-Palaeozoic crinoids. Similar results are obtained when the space of correlation matrices is built from the set of taxonomically non-significant characters or from a drastic culling of data preserving only characters with less than five percent of missing data.Finally, I computed the pairwise distances among the trait correlation matrices associated with each geologic time interval so as to ordinate and visualize patterns of integration within the space of correlation matrices . The temThe present work examined large-scale patterns of evolutionary integration among morphological traits in crinoids and tested if and how changes in these patterns were associated with concomitant changes in the level of morphological disparity expressed by the clade. The analyses reveal relatively stable measures of the overall degree of integration despite marked temporal variations in taxonomic diversity and morphological disparity. Correlation analyses accounting for and limiting the effect of phylogenetic autocorrelation did not detect a significant one-to-one relationship between integration and disparity. Nevertheless, significant differences in the degree and pattern of integration are observed between Palaeozoic and post-Palaeozoic crinoids. Post-Palaeozoic crinoids have a higher overall degree of integration and occupy a different region of the space of correlation matrices. Their location indicates heterogeneously structured correlation matrices with larger blocks of correlated traits, which could explain the less disparate and more stereotyped post-Palaeozoic morphologies reported previously Hence, if the amount of morphological disparity does not appear to be conditional upon any given degree of overall integration, the results suggest that disparity is related to the modular nature of the correlation matrix, that is, to its pattern of organization into evolutionarily quasi-independent blocks of integrated traits. With regards to the two competing hypotheses presented in the introduction, the temporal trajectory of morphological disparity in crinoids would then be tied to changes in the pattern of correlation among traits rather than to changes in their individual variability.In a study comparing the disparity levels of ecological and non-ecological  characters before and after mass extinctions, Ciampaglio In summary, morphological disparity should be seen as more than a mere summary statistic of the amount of morphospace occupied. On the one hand, disparity reflects the building-up of the genealogical hierarchy over long timescales, with for instance the changing taxonomic composition of clades and the signature of mass and background extinctions. On the other hand, the behaviour of morphological disparity in face of these macroevolutionary phenomena is tied to the dimensionality of phenotypic variation constrained by the apportionment of variability among units of evolutionary transformation. Hence, the distribution and dynamics of taxa in morphospace should provide insights into the architecture of phenotypes and the constraints on their evolvability. This challenges the frequent conceptualization of morphospace as a homogeneous state-space. Such an interpretation of morphospace is unlikely to hold at the level of macroevolutionary phenotypic variation where development imposes a strong structure on the evolutionary accessibility of phenotypes e.g. . To diffAppendix S1Crinoid data (Foote 1999). The file includes details of the stratigraphic intervals used, a description of the 90 discrete morphological characters, and the coding of these characters for the crinoid species retained in the analyses. See M. Foote, 1999. Paleobiology Memoir 1∶1–115  for additional details (doi:10.1666/0094-8373(1999)25[1:MDITER]2.0.CO;2)(TXT)Click here for additional data file.Appendix S2R functions. The file includes the R functions for running disparity and integration analyses as described in the main text. Crinoid data are available in R can be downloaded from http://www.r-project.org/. The shrinkage estimators of correlation matrices were obtained using the R package corpcor (http://strimmerlab.org/software/corpcor/). For additional details or questions: s.gerber@bath.ac.uk.(DOCX)Click here for additional data file.Appendix S3Time series for morphological disparity and integration. The file provides the numerical values of the temporal trajectories of disparity and integration throughout the Phanerozoic as displayed in (XLS)Click here for additional data file.


Salmonella enterica are significant causes of human food poisoning. Fowl carrying these bacteria often show no clinical disease, with detection only established post-mortem. Increased resistance to the carrier state in commercial poultry could be a way to improve food safety by reducing the spread of these bacteria in poultry flocks. Previous studies identified QTLs for both resistance to carrier state and resistance to Salmonella colonization in the same White Leghorn inbred lines. Until now, none of the QTLs identified was common to the two types of resistance. All these analyses were performed using the F2 inbred or backcross option of the QTLExpress software based on linear regression. In the present study, QTL analysis was achieved using Maximum Likelihood with QTLMap software, in order to test the effect of the QTL analysis method on QTL detection. We analyzed the same phenotypic and genotypic data as those used in previous studies, which were collected on 378 animals genotyped with 480 genome-wide SNP markers. To enrich these data, we added eleven SNP markers located within QTLs controlling resistance to colonization and we looked for potential candidate genes co-localizing with QTLs.The serovars Enteritidis and Typhimurium of the Gram-negative bacterium In our case the QTL analysis method had an important impact on QTL detection. We were able to identify new genomic regions controlling resistance to carrier-state, in particular by testing the existence of two segregating QTLs. But some of the previously identified QTLs were not confirmed. Interestingly, two QTLs were detected on chromosomes 2 and 3, close to the locations of the major QTLs controlling resistance to colonization and to candidate genes involved in the immune response identified in other, independent studies.Salmonella colonization and carrier state. These observations provide evidence of common genes controlling S. Typhimurium colonization and S. Enteritidis carrier-state in chickens.Due to the lack of stability of the QTLs detected, we suggest that interesting regions for further studies are those that were identified in several independent studies, which is the case of the QTL regions on chromosomes 2 and 3, involved in resistance to both  Salmonella enterica species are responsible for diseases in several animal species and in humans. Salmonella enterica serotypes Enteritidis and Typhimurium are two of the main sources of human food poisoning. One of the most important factors is that poultry can carry these bacteria without any clinical symptoms, and thus the carriage of disease goes unnoticed prior to culling. Selection and breeding of chickens more resistant to Salmonella carrier state, i.e. able to rapidly clear Salmonella, could reduce the spread of these bacteria in poultry stocks and hence improve food safety. Selection for resistance to carrier-state requires the use of genetic markers in poultry breeding programs. Identification of such markers by several authors has been done using different approaches. These include candidate gene analyses, genome scans with single marker analyses, quantitative trait loci (QTLs) detection studies by interval mapping with microsatellites and more recently SNP markers (reviewed in , whe, whe11],1 lines are highly but not totally inbred, possibly accounting for the reason why more genome-wide QTLs were found in this study than previously observed  and andSLC11er-state . This reer-state ‐[4]. It Salmonella carrier-state using maximum likelihood. The role of several genomic regions in the control of Salmonella carrier-state was confirmed, consistent with previous findings, while testing the existence of two segregating QTLs allowed the identification of novel regions. The observed differences with QTLExpress were most probably due to the different hypotheses concerning allele segregation, to the strong dominance effect of some of the QTL and to the weakness of some QTL effects. In this context, the most interesting QTLs are probably those that were identified several times in independent studies, even more so when they also co-localize with candidate positional or functional genes. This is why the QTL regions identified on chromosomes 2 and 3 are of particular interest for further research and potentially for marker assisted selection. In addition, their involvement in the resistance to both S. Typhimurium colonization and S. Enteritidis carrier-state is particularly interesting for selection purposes.We observed numerous QTLs for resistance to The authors declare that they have no competing interest.Salmonella challenges and contributed to the candidate gene analysis. AV obtained the original set of SNP data. ELD contributed to the QTLMap analyses. All authors contributed to the revision of the manuscript and approved the final manuscript.ST performed QTL analyses and drafted the manuscript. FC and CB contributed to data analyses and to manuscript drafting. MF, NS and PK contributed to obtaining the marker data. PV performed the


Aspergillus ssp.Suppurative fungal infection of the thyroid is an extremely rare condition even more so in an immunocompetent patient. Fungal infections of the thyroid usually occur in immunocompromised patients with hematological malignancies, recipients of bone marrow and solid organ allografts on immunosuppression, and patients with AIDS. A 65-year-old male presented with swelling in the front of the neck for 2 years. Examination revealed a 4 × 4 cm non-tender, firm swelling of the right lobe of the thyroid. The patient was taken up for a subtotal thyroidectomy. Intra-operatively, an abscess cavity with pus was found in the right lobe of the thyroid. Histopathology revealed features of fungal abscess and staining demonstrated fungal hyphae characteristic of  Staphylococcus and Streptococcus species) remain the most common causes, there has been a marked decrease in the number of cases caused by mycobacteria, Salmonella species, and anaerobes when compared with the early part of the 20th century [Aspergillus spp. in a patient who presented with a lump in the front of his neck with a competent immune system. Thyroid abscess was a common condition in the era before antibiotics. In the current medical environment, however, it is a clinical entity that is seldom encountered. Review of the published causes of thyroid abscess since 1980 demonstrated that although Gram-positive bacteria ( century , thyroid century , 5. Of 4Aspergillus ssp. (Figures A 65-years-old male diabetic patient presented with swelling in the front of the neck for 2 years . There wAspergillus spp. are the predominant causative fungus for thyroiditis and asymptomatic thyroid infiltration [Aspergillus spp., with thyroid infiltration found postmortem in 10% to 35% of aspergillosis cases [P. jiroveci is the most common cause of fungal thyroiditis in patients with AIDS, reflecting the high incidence of pneumocystosis in these patients [P. jiroveci. Whether this pattern reflects the degree of glandular dysregulation by the aforementioned fungi or results from differences in the timing of diagnosis remains unknown. Patients with acute severe fungal infections may develop euthyroid sick syndrome, characterized by decreased levels of triiodothyronine and increased levels of normal thyroxine and thyrotropin-releasing hormone, as described in cases of paracoccidioidomycosis [Fungal infection of the thyroid is extremely uncommon. The ability of the thyroid gland to resist infection is well known and infection in the thyroid gland is rare, particularly so with the advent of widespread usage of antibiotics. The remarkable resistance of the thyroid gland to infection is attributed to many factors. A prosperous lymphatic and vascular supply, well-developed capsule, and high iodine content of the gland are various mechanisms suggested to account for this relative resistance to infection . The patltration . The litpatients . Hyperthomycosis .Thyroid fungal involvement can manifest as subacute thyroiditis with local and systemic symptoms. Neck swelling and tenderness are common because of thyroid enlargement and dysphagia from esophageal compression; in severe cases, fatal respiratory failure from tracheal obstruction has occurred . ClinicaDiagnosis of fungal thyroiditis is made by direct microscopy and culture of a fine needle aspirate and/or biopsy in most cases . Fungal Reported modes of management of thyroid abscess vary, but drainage remains an integral component of therapy for resolution of the infection . If a diFungal thyroiditis is a rare condition but should be kept in mind in cases of immunocompromised conditions and disseminated disease. It is not uncommon for fungal thyroid infections to present as abscess which cannot be clinically distinguished from other benign lesions of the thyroid. Our case underlines the need for all cases of infective thyroiditis to be sent for both bacterial and fungal cultures, especially in the wake of pandemics of immunocompromised states like AIDS. Treatment of fungal thyroid abscesses usually involves aggressive surgical management, in addition to systemic antifungal therapy.62 cases of fungal thyroiditis were reported in the last 110 years.Fungal thyroiditis has been reported in patients with immunocompromised states or in the setting of disseminated disease.Our patient developed a fungal thyroid abscess despite a healthy immunity which is an extremely rare presentationFungal thyroiditis has been reported to present as an abscess in the thyroid, though it is difficult to clinically determine this.In the wake of pandemics of immunocompromised diseases like AIDS, all cases of infective thyroiditis should be sent for both bacterial and fungal cultures.


A significant challenge in Prehistory is to understand the mechanisms involved in the behavioural evolution of human groups. The degree of technological and cultural development of prehistoric groups is assessed mainly through stone tools. However, other elements can provide valuable information as well. This paper presents two bone retouchers dated to the Middle Pleistocene MIS 9 used for the shaping of lithic artefacts. Originating from Bolomor Cave (Spain) and Qesem Cave (Israel), these two bone retouchers are among the earliest of the Old World. Although the emergence of such tools might be found in the latest phases of the Acheulean, their widespread use seems to coincide with independently emergent post-Acheulean cultural complexes at both ends of the Mediterranean Sea: the post-Acheulean/pre-Mousterian of Western Europe and the Acheulo Yabrudian Cultural Complex of the Levant. Both entities seem to reflect convergent processes that may be viewed in a wider cultural context as reflecting new technology-related behavioural patterns as well as new perceptions in stone tool manufacturing. HomoMeat consumption is deeply rooted in human existence and seems to have been essential as early as the appearance of the genus Cervus elaphus, Equus ferus and Oryctolagus cuniculus, and 4 remains belonging to Canis cf. lupus. The proportion of long bone fragments  is higher than flat bones . Among the long bones, mid-shaft fragments show a significant representation . Anthropogenic evidence caused during bone breakage has been documented on 117 ungulate remains (6.8%) in form of percussion notches, impact flakes and peeling Bolomor Cave is located in Valencia (Spain) at 100 m a.s.l. . Its strDama cf. mesopotamica and in a lesser extent, of Cervus elaphus, Equus ferus, Bos primigenius, Sus scrofa and Testudo cf. graeca. Cone (percussion) fractures were recognized on 19% of bones from Unit III H. erectus but rather similar to later modern populations  of this region, with some Neanderthal affinities as well. These cultural and biological transformations might indicate the emergence of a new hominin lineage in the Middle Pleistocene Levant Qesem Cave is situated 12 km east of the Mediterranean coast in a hilly limestone terrain 90 m.a.s.l. . The cavCervus elaphus)  of a red deer  because the striations are well-defined, isolated and not associated with a loss of cortical tissue. In contrast, the bone retoucher from Qesem Cave displays a slight increase of pits, scores, and exfoliation in the very well-defined active area. This type of modification seems to be consistent with the employment of a semi-fresh bone. Another difference between the two retouchers is the presence of scraping-marks. All flesh had to be removed from bone blanks, and several authors have proposed that for hammer and retoucher efficiency, it was also necessary to remove the periosteum The two bone retouchers presented herein originating at these two sites respectively are of the few earliest examples of such tools known to date and although early they both possess typical morphological and functional characteristics of such tools. Despite the distance between sites, the use of bone as retoucher emerged in a similar chronology (MIS 9). Simultaneously, other European sites show similar objects, such as sublevel TD10-1 of Gran Dolina, Spain Interconnections between the Spanish and Near Eastern post-Acheulean cultural complexes are not feasible, thus this similar technological  advancement indicates possible convergent developments. Both of these disparate cultural complexes were innovative at their time, consisting of a series of newly introduced behaviours and qualities unknown from earlier Acheulean sites, such as the habitual use of fire, the presence of large amounts of burnt bones (roasting), and lithic recycling. In the case of bone retouchers, their appearance and generalized use seem to have marked a new manner of bone use possibly reflecting a novel view of discarded bone recycling and therefore, an innovative human behaviour. The employment of bone tools in achieving an end hitherto achieved by use of other raw materials is not merely a technological innovation. The introduction of bones that originated in hunted, defleshed, and consumed animals into the sphere of lithic production brings together the two basic elements of prehistoric life - stone tool making and animal hunting and consumption. Such an early integration of these two primordial defining elements of human Palaeolithic existence suggests cultural convergence. In the case of Bolomor and Qesem Cave it evokes thoughts on the reason why two such different and geographically remote entities, and probably also two different hominins, show such similar and simultaneous innovation.The Qesem Cave project has a permit by the Antiquities Authority of Israel following the law of Antiquities of Israel issued annually since the year 2001. The field-work/research at Bolomor Cave  is carried out in strict accordance with the Cultural Heritage law of Valencia. Fossil remains from Bolomor Cave are stored in the Prehistory Museum of Valencia under the authority of the Provincial Council of Valencia, Spain. Fossil remains from Qesem Cave are put in storage in the Department of Archaeology of the Tel-Aviv University, Israel. No permits were required for the described study, which complied with all relevant regulations.Surface alterations were treated at both macroscopic and microscopic level. For microscopic study an Olympus Europe SZ11 (magnification up to 110) and ESEM  were used. Percussion marks were identified according to criteria described by Spencer de Gruchy and Roberts


Myelin is the multi-layered lipid sheet periodically wrapped around neuronal axons. It is most frequently found in vertebrates. Myelin allows for saltatory action potential (AP) conduction along axons. During this form of conduction, the AP travels passively along the myelin-covered part of the axon, and is recharged at the intermittent nodes of Ranvier. Thus, myelin can reduce the energy load needed and/or increase the speed of AP conduction. Myelin first evolved during the Ordovician period. We hypothesize that myelin's first role was mainly energy conservation. During the later “Mesozoic marine revolution,” marine ecosystems changed toward an increase in marine predation pressure. We hypothesize that the main purpose of myelin changed from energy conservation to conduction speed increase during this Mesozoic marine revolution. To test this hypothesis, we optimized models of myelinated axons for a combination of AP conduction velocity and energy efficiency. We demonstrate that there is a trade-off between these objectives. We then compared the simulation results to empirical data and conclude that while the data are consistent with the theory, additional measurements are necessary for a complete evaluation of the proposed hypothesis. This process is called saltatory conduction  into account simultaneously. The best performing models displayed one of many optimal combinations of high conduction velocity and high energy-efficiency. The frontier of such models with optimal performance combinations is the Pareto-front.Energetic cost was defined as the total charge and thus related to the total metabolic cost in one node: more charge offsets the ionic gradient more and thus the pumps needs to work more to achieve balance again .The data acquired for each species should ideally contain information at least about conduction speed, axonal thickness, myelin thickness, inter-node distance, node length and sodium and potassium concentration at the node. The data should also be available for comparable axonal tracts, across species, from the central and PNSs. Measurements should include classic electron microscopy (for measuring lengths and diameters) and immuno-gold electron microscopy for measuring ion channel densities  and g_k (0.036 mS/cm2).The optimization algorithm we used yielded axonal models with parameter sets . The axons optimized for fast AP propagation had the highest number of nodes, while the slower had fewer nodes but the exact number varies 10–40). A positive correlation exists between the number of nodes and the AP propagation velocity  among the Chondrichthyes and the modern bony fishes (teleostei), among the Actinopterygii, two groups which emerged during the Mesozoic Long, .Due to several factors involving the difficulty of obtaining specimen and of modifying existing methods for animals of a lesser-used species, modern neurobiology is mostly concentrating on relatively few species (“model organisms”). Thus, the desired data are available from fewer species than is desirable, with a glaring lack of data from aquatic vertebrates. There is some published information along these lines for common model organisms such as rats and cats. We compiled a table Table  with allWe performed a literature study that is summarized in Table The available data is too sparse at this point to convincingly test the hypothesis that the role of myelin changed from energy conservation to propagation speed maximization. The empirical data lacks both complete information  and the central nervous system (CNS) could operate along different speed/efficiency set-points. A number of time-dependent processes, such as the distinct types of oscillations seen in the CNS, could exert unique evolutionary pressures on conduction speed. On the other hand, the longer distances transversed by some PNS axons could exert pressure toward evolving higher conduction speeds as well. Better knowledge of the ontological changes in myelin and axonal conductance changes among the species included in the study would also help refine our understanding. In most cases, this kind of data is non-existent and the preponderance of the data included herein is from adult tissues.An additional, extremely useful, piece of empirical information would be knowledge of the molecular biology of the ontogeny of myelin across multiple species. A comparison of the proteins involved in ensheathing axons with myelin, such as the oligodendrocyte myelin glycoprotein (Vourc'h and Andres, Furthermore, an evolutionary divergence of myelin during the Mesozoic marine revolution would have significant consequences for the use of different animal species as models for demyelination diseases. If the model species has myelin located at an optimum different from the human myelin-parameter optimum, its use as model species would be limited.We hope that this hypothesis-paper serves as a motivation for our fellow neuroscientists to conduct measurements in less frequently investigated vertebrate species. The outcome of the testing of this hypothesis will have significant consequences for our understanding of vertebrate brain evolution. It will tell us if the Mesozoic marine revolution had macro-evolutionary consequences for vertebrate brains.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.


Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress.The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain a balance between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence indicates that several pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential loading of ribosomes onto mRNAs. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold yvc1 gene that is induced by ER stress in a UPR-dependent manner.ER stress was induced by treating the fungus with dithiothreitol, tunicamycin, or a thermal up-shift. The mRNAs were then fractionated on the basis of ribosome occupancy into an under-translated pool (U) and a well-translated pool (W). The mRNAs were used to interrogate microarrays and the ratio of the hybridization signal (W/U) was used as an indicator of the relative translational efficiency of a mRNA under each condition. The largest category of translationally upregulated mRNAs during ER stress encoded proteins involved in translation. Components of the ergosterol and GPI anchor biosynthetic pathways also showed increased polysome association, suggesting an important role for translational regulation in membrane and cell wall homeostasis. ER stress induced limited remodeling of the secretory pathway translatome. However, a select group of transcription factors was translationally upregulated, providing a link to subsequent modification of the transcriptome. Finally, we provide evidence that one component of the ER stress translatome is a novel mRNA isoform from the A. fumigatus and reveal a remarkable breadth of functions that are needed to resolve ER stress in this organism.Together, these findings define a core set of mRNAs subject to translational control during the adaptive response to acute ER stress in  Aspergillus fumigatus causes life-threatening pulmonary infections that have the potential to progress into invasive aspergillosis, a disseminated disease with a very high rate of mortality , as described in Methods.Click here for fileList of over-represented KEGG pathways in the dataset of translationally regulated mRNA following a shift to 37°C.Click here for fileList of mRNAs with increased polysome association during each of the three forms of ER stress: treatment with DTT, TM and thermal stress. Values represent log2[translational efficiency ratio], as described in Methods. #mRNAs subject to translational upregulation in the thermal stress dataset at 60 min.Click here for file


Reconstruction of large mandiblular defects following ablative oncologic surgery could be done by using vascularized bone transfer or, more often, primarily with simultaneous or delayed bone grafting, using load bearing reconstruction plates. Bending of these reconstruction plates is typically directed along the outer contour of the original mandible. Simultaneously or in a second operation vascularized or non-vascularized bone is fixed to the reconstruction plate. However, the prosthodontic-driven backward planning to ease bony reconstruction of the mandible in terms of dental rehabilitation using implant-retained overdentures might be an eligible solution. The purpose of this work was to develop, establish and clinically evaluate a novel 3D planning procedure for mandibular reconstruction.Three patients with tumors involving the mandible, which included squamous cell carcinoma in the floor of the mouth and keratocystic odontogenic tumor, were treated surgically by hemimandibulectomy.In primary alloplastic mandible reconstruction, shape and size of the reconstruction plate could be predefined and prebent prior to surgery.This study provides modern treatment strategies for mandibular reconstruction. Indications for partial and total mandibulectomy include malignancies especially squamous cell carcinomas (SCC), benign tumors such as ameloblastomas and sequelae of radiotherapy such as osteoradionecrosis. Defect size in ablative surgery mainly depends on dignity and dimensions of the pathology. Following mandibulectomy, reconstruction of the mandible is mainly done with vascularized bone grafts or alloplastic materials (reconstruction plates). However, irrespective of the type of primary hard tissue reconstruction is simultaneous adequacy of the soft tissue reconstruction equally important: only a well vascularized soft tissue envelope allows for a successful hard tissue reconstruction. Even large series of mandibular reconstruction after ablative surgery or major trauma in literature show that oral rehabilitation of patients with large segmental defects is still a challenge -4. BesidPre-operative planning is highly-developed -17 and iThree patients with tumors involving the mandible, which included squamous cell carcinoma in the floor of the mouth and keratocystic odontogenic tumor, were treated surgically by hemimandibulectomy. Written informed consent was obtained from the patient. The patient's preoperative demographic data are summarized in Table ®, Feldkirchen, Germany). This platform allows for alignment of the DICOM data set, viewing and analysis and provides 3D tools for the planning procedure such as mirroring tools, autosegmentation with atlas-function and a three-dimensional modification tool named SmartShaper. iPlan enables import of different imaging modalities and allows to automatically image-fuse these data. That facilitates not only the pre-operative analysis of the data but also the post-operative quality control. Furthermore this platform allows for intra-operative navigation of the virtual plan which is important for midface and skullbase procedures, however, this was not used in the presented cases.The imaging and planning platform used in this study was iPlan 3.0  imaging data with a maximum slice thickness of 1 mm. The data were transferred on the computer and reconstructed into 3D images. Different planning modalities include mirroring tools (case 1), autosegmentation tools (case 2) and the backward planning tool (case 3). The backward planning is mainly based on size-variable ideal dental arch models with adjustable and truly designable virtual hollow cylinders. These hollow cylinders define not only the implant position but also the needed peri-implant bone volume. Thus desired bone position as a required subvolume within the later bone grafts for later insertion of dental implants could be visualized and already considered during the primary alloplastic reconstruction of the mandible.®, Umkirch, Germany) was bent to bridge the prospective mandible defect.Outcome of the virtual planning is in each case a virtual template of the mandible (STL-file format) for production of a corporeal stereolithographic model. After prototyping of the model  a conventional 2.4 Compact UniLOCK reconstruction plate , secondly using the autosegmentation to virtual reconstruct the mandible (case 2) and finally presenting the prosthodontic driven "backward planning" for optimized bone graft position (case 3).The patient was a 53-year-old man with a large keratocystic odontogenic tumor confirmed by previous biopsy. Due to the huge extent of the lesion, there was an indication for hemimandibulectomy of the right side. After importing the data into the imaging and planning platform (iPlan 3.0) a virtual model was built by using mirroring tools Figure . The unaAfter ten months patient underwent secondary reconstruction with bone graft from the iliac crest. The operation was straightforward and quick, with no intra-operative complications. In the 2D-analysis of the panorex X-ray . The extent of the primary tumor and the nodal involvement was evaluated clinically and radiologically (CT scan). The expected size of mandible resection was defined virtually. Because of arrosion of the mandible and the large extent of the resection (complete corpus mandibulae), mirroring is no option for virtual planning. Brainlab iPlan 3.0 comes with an autosegmentation mode. Starting the procedure of autosegmentation, the so generated neomandible combines the existing bony structures with averaged segments of the autosegmentation. Following 3D analysis of the neomandible, a stereolithographic model was built and, as above mentioned, prebending performed .Similar to case 2, virtual mirroring as a planning tool was not feasible due to mandible destruction and atrophy. For the autosegmentation mode, atrophy and destruction has only minor effects on template building. After autosegmentation of the mandible with iPlan, backward planning by using virtual ideal upper and lower dental arches and virtual hollow cylinder as placeholders was performed Figure . The holThe result of placing these hollow cylinders illustrated the target (neomandible) which should be addressed with the bone graft in secondary reconstruction. Using the SmartShaper the virtual mandible is adjusted to the desired outline of the planned neomandible and exported as STL-file Figure .In primary alloplastic mandible reconstruction, shape and size of the reconstruction plate could be predefined and prebent prior to surgery. This could improve accuracy of the contour of the plate. In limited mandible defects, good results could be achieved with this simple planning method (case 1). In larger defects (case 2) simple mirroring tools are not feasible. The recent iPlan-Software allows by using the autosegmentation mode a virtual reconstruction of the mandible and following prebending of plates.The base of planning is up to now mostly the outer contour of the mandible. But in large reconstructions, this leads to an inappropriate positioning of bone grafts in secondary reconstruction due to the limited dimensions of bone grafts. In case 3 we could demonstrate the significant discrepancy between original mandible contour and desired neomandible contour regarding later oral rehabilitation.Adequate reconstruction should offer replacement of dentition and provide improved deglutition, mastication, and speech  and therSoft tissue lining has to be addressed at least twice: in mandibular reconstruction to allow a sufficient blood supply for the transplanted bone with its bradytrophic character and before or after implant insertion to allow a non-irritant interface between mucosa and implant. It is reported that in accompanying soft tissue deficiencies mandibular reconstruction is preferably treated with vascularized bone grafts. Buchbinder et al. demonstrate that osseocutaneous flaps offer the most rapid rehabilitation . In contProblems of bony reconstruction include undercorrection of the lower facial contour, unsatisfying midline match and unfavourable sagittal maxillary-mandibular relationship  due to lThere are many reports about virtual planning of mandible reconstruction. Virtual planning appears to have positive impact on the reconstruction of major mandibular defects . There aThe recent software version iPlan 3.0 allows for the described workflow in backward-planning of an ideal neomandible. A substantial limitation in commercially available planning platforms, at the moment, is the lack of an automated collision detection to find an ideal occlusal position with maximum intercuspidation. In edentulous mandible this is of subordinate importance and simplification by virtually rotation the mandible in the desired position seems to be admissible. Further software releases should address these limitations.Bending of reconstruction plates using 3-dimensional stereolithographic models is reported. These stereolithographic models are based on virtual templates which were standardized exported in STL-file format and produced by specialized companies ,21. The This study provides modern treatment strategies for mandibular reconstruction.The authors declare that they have no competing interests.HE, MR, HK, CS, MRU, FT and NCG conceived of the study and participated in its design and coordination. HE and MR made substantial contributions to data acquisation and conception of manuscript. HE and MR drafted and designed the manuscript and contributed equally to this work. NCG and MRU were involved in revising the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The article processing charges are funded by the Deutsche Forschungsgemeinschaft (DFG), "Open Acess Publizieren".


The present study provides data to decide on the most appropriate method for sampling of ground-dwelling arthropods measured in a moist-deciduous forest in the Western Ghats in South India. The abundance of ground-dwelling arthropods was compared among large numbers of samples obtained using pitfall trapping, Berlese and Winkler extraction methods. Highest abundance and frequency of most of the represented taxa indicated pitfall trapping as the ideal method for sampling of ground-dwelling arthropods. However, with possible bias towards surface-active taxa, pitfall-trapping data is inappropriate for quantitative studies, and Berlese extraction is the better alternative. Berlese extraction is the better method for quantitative measurements than the other two methods, whereas pitfall trapping would be appropriate for qualitative measurements. A comparison of the Berlese and Winkler extraction data shows that in a quantitative multigroup approach, Winkler extraction was inferior to Berlese extraction because the total number of arthropods caught was the lowest; and many of the taxa that were caught from an identical sample via Berlese extraction method were not caught. Significantly a greater frequency and higher abundance of arthropods belonging to Orthoptera, Blattaria, and Diptera occurred in pitfall-trapped samples and Psocoptera and Acariformes in Berlese-extracted samples than that were obtained in the other two methods, indicating that both methods are useful, one complementing the other, eliminating a chance for possible under-representation of taxa in quantitative studies. Inclusion of ground-dwelling arthropods in biodiversity inventories and environmental assessment surveys has increased in the recent past . Since mPitfall trapping, the simplest and cheapest method among the three, is efficient in capturing those arthropod taxa that are nocturnally active on the surface, but is inefficient in capturing either the bottom dwellers or those that disseminate by flying . PitfallIn summary, the effectiveness of these three widely used methods to extract arthropods from soil substrates is being debated. Only a few replicated field studies have attempted to evaluate critically and compare quantitatively the extraction efficiency of the three sampling methods. Most of the earlier efforts evaluated the sampling efficiency of either the Berlese or the Winkler extraction method against pitfall trapping by sampling of a few specific arthropod taxa  were compared and evaluated using a field trial done in the moist deciduous forests in the Western Ghats in South India. The objective was to determine whether the three methods are equally effective in the separation and to determine which method achieves the best overall population numbers of as many taxa as possible.The study was carried out in the moist deciduous forests of Sholayar  , situated close to the Athirapally—Vazhachal waterfalls, 60 km south of the town of Trichur  located in the South-Western Ghats of moist deciduous ecoregion . Annual Sampling was done in the first week of February 2006. Although a thorough population assessment of any group of invertebrates necessitates sampling at different times in the year , the surEach litter sample for Winkler and Berlese extraction was obtained by placing a 50 × 50 cm wooden frame on the forest floor and collecting the leaves, litter and loose humus from within the frame area into a large polythene bag . SamplesFauna was extracted with Berlese extraction apparatus  over five days in 70% alcohol. Litter samples for Winkler extraction of the fauna  were plaEach pitfall trap consisted of a black plastic bowl , buried to its rim in soil and partly filled with 30 mm of nontoxic, propylene glycol. Each trap was topped with a dark-plastic tray supported on iron bars to prevent either desiccation or flooding; such a system operated for 24 h continuously to avoid bias in catches arising from diurnal activity variation of fauna . TrappedThe fauna obtained from the 40 litter samples from each method were available for data analysis. Taxa with > 30% frequency of occurrence in any of one of the sampling method was categorized as major, and the others as minor taxa. The sampling method, which trapped > 40% frequency of occurrence of a particular taxon, even if that taxon was trapped with < 40% frequency of occurrence in the other two methods was deemed to be ‘reasonably effective’ in sampling that particular taxon.Significant differences in the frequency of collection among sampling methods  made comparisons using common parametric statistics inappropriate. In the data analysis, emphasis was placed on seeking differences in the frequency of occurrence of arthropod types and less on testing for differences in the mean number of arthropod types . Higher From the three methods tested, 12,257 individuals belonging to 25 arthropod taxa were collected , Table 1In the Winkler extraction method, 18 taxa were obtained. The proportionate distribution of dominant taxa in the collection was in the following order: Acariformes (85%) > insect larvae (75%) > Coleoptera (60%) > Araneae (48%) > Formicidae (40%) . For theIn the Berlese extraction method, 23 taxa were obtained , Table 1In the pitfall trapping method, except Pseudoscorpionida, the remaining 24 taxa were recorded , Table 1Proportionate occurrence of the major taxa was in the order of: Acariformes = Formicidae = Orthoptera = Hemiptera = Diptera = Araneae > larval forms of diverse insect orders (93%) > Collembola (90%) > Blattaria (90%) > Coleoptera (88%) > Chalcidae (78%) > Thysanoptera (53%) , Table 1In summary, the pitfall trapping method yielded highest frequency of occurrence for 13 out of the 14 major taxa, Berlese extraction method for 3 out of the 14 taxa and also for one minor taxon, and the Winkler extraction method for none of the major taxa . For theResults of this study lead to recommendations on the relative usefulness of pitfall trapping, and Berlese and Winkler extraction methods for sampling ground-dwelling arthropods in subtropical, moist deciduous forests. A pronounced difference was found in the capture of major ground-dwelling arthropod taxa among the three tested sampling methods. Pitfall trapping yielded the maximal capture (both frequency and numbers) of 87% of the major taxa, followed by the Berlese extraction method (27%). The Winkler method proved ineffective for any of the major taxa in the moist, subtropical South Indian forest conditions. These percentages indicated that pitfall trapping was a useful standard arthropod collection method for ecological studies of ground-surface-dwelling arthropods compared with the Berlese and Winkler extraction methods. Irrespective of the method tested, few individuals of the minor taxa were captured and such an effect could be attributed to the low population densities of these taxa in the moist forests of the Western Ghats . Non-sigHowever, a bias was apparent in the samples obtained via pitfall trapping in comparison with the other two methods. Pitfall trapping captured high numbers of taxa active at the ground level, viz., taxa belonging to Orthoptera, Blattaria, Diptera, Araneae, Formicidae, Collembola, Hemiptera, Coleoptera, and other Hymenoptera , with 95Unlike the Berlese or Winkler litter extraction methods, pitfall trapping requires two trips to interior forests: one to set the traps and the second to retrieve traps and samples. A second field trip to retrieve the traps leads to additional expenditure, loss of time and practical difficulties in protected forests with restricted access. Moreover, multiple chances of wildlife disturbing the field-placed traps and inclement weather affecting the sampling effort in subtropical forests also exist. This leaves the researcher apprehensive about the success of his collection efforts until the second trip and makes pitfall trapping more laborious and chance oriented than the other two methods.Recent studies have highlighted the Winkler extraction method as a less expensive, more convenient, and a more efficient alternative method for exhaustive extraction of soil macro-invertebrates . Hence, Group and trap specific differences noted in the present study supports the findings by Edwards  and StanThe relative abundance and frequency of occurrence of fauna was different with the three sampling methods. When cost and time constraints dictate the limiting of ground-dwelling arthropod sampling to one method, the Berlese extraction method is ideal for quantitative estimates, and the pitfall trapping method is ideal for qualitative estimates.Since the three taxa  with a low catch probability with Berlese extraction were caught efficiently with pitfall trapping, inclusion of pitfall trapping with appropriate adjustments would be the method for comprehensive quantitative surveys of ground-dwelling arthropods. Although pitfall trapping samples tend to include more ground-active species, its efficiency indicates that pitfall trapping is certainly the method of choice for an individual quantitative sampling method for most major taxa except the Psocoptera and insect larval forms, for which the Berlese extraction method is a better option.As a cost effective, individual quantitative sampling method, Winkler extraction is suitable for obtaining Coleoptera and Acariformes in addition to litter Formicidae, for which it is an established method , but not