Parapenaeus longirostris 6%). This is partly attributed to existing environmental policy instruments (e.g. Habitats Directive and Mediterranean Regulation EC 1967/2006) aiming at minimising impacts on coastal priority habitats such as seagrass, coralligenous and maerl beds. The new knowledge on the distribution and persistence of demersal nurseries provided in this study can support the application of spatial conservation measures, such as the designation of no-take Marine Protected Areas in EU Mediterranean waters and their inclusion in a conservation network. The establishment of no-take zones will be consistent with the objectives of the Common Fisheries Policy applying the ecosystem approach to fisheries management and with the requirements of the Marine Strategy Framework Directive to maintain or achieve seafloor integrity and good environmental status.The identification of nursery grounds and other essential fish habitats of exploited stocks is a key requirement for the development of spatial conservation planning aimed at reducing the adverse impact of fishing on the exploited populations and ecosystems. The reduction in juvenile mortality is particularly relevant in the Mediterranean and is considered as one of the main prerequisites for the future sustainability of trawl fisheries. The distribution of nursery areas of 11 important commercial species of demersal fish and shellfish was analysed in the European Union Mediterranean waters using time series of bottom trawl survey data with the aim of identifying the most persistent recruitment areas. A high interspecific spatial overlap between nursery areas was mainly found along the shelf break of many different sectors of the Northern Mediterranean indicating a high potential for the implementation of conservation measures. Overlap of the nursery grounds with existing spatial fisheries management measures and trawl fisheries restricted areas was also investigated. Spatial analyses revealed considerable variation depending on species and associated habitat/depth preferences with increased protection seen in coastal nurseries and minimal protection seen for deeper nurseries (e.g.  Understanding the patterns of spatial distribution of wild populations is a critical consideration in the development of effective management and conservation strategies . For marMerluccius merluccius) and the deep-water rose shrimp (Parapenaeus longirostris). Due to poor enforcement and reduced trawl selectivity, some sectors show up to 70% of the catch of specimens below the MCRS . Ththe MCRS .Cystoseira forests , Cy forests , Peyssonlia beds  and crinlia beds .The Council Regulation EC 1967/2006 provides some guidelines for the implementation of habitat conservation in the Mediterranean Sea, with particular attention devoted to the protection of nursery areas. This regulation has the potential to yield important conservation benefits and it is primarily based on two assumptions: (1) juvenile fish are particularly vulnerable to a fine mesh trawl fishery , especiaIn the last 20 years, a line of research has focused on identifying the most important nursery habitats based on their contribution to adult populations , 15 intehttp://mareaproject.net/). Annual nurseries were identified by modelling the spatial distribution of recruits with the purpose of reconstructing the distribution of “density hot-spots” (i.e. areas of higher density), regarded as more productive areas. As a second step, each annual density hot-spot was ranked according to its temporal persistence throughout the study period. Our main assumption was that the average contribution to the adult population could be expected to be higher from nurseries with higher densities of recruits and higher spatio-temporal stability. In fact, the maintenance of a population mostly depends on the success of the displacement of young fish into nursery areas and their recruitment from nursery areas to the parental population . H. H65]. HIn GSA 18, the Southern Adriatic vortex produces an upwelling system, which enhances the productivity of the basin, while two main currents, the south-western, formed by colder and low salinity waters from the Po river and the south-eastern, carrying warmer and saltier waters from the eastern basin  contribuIn the Northern Adriatic Sea the nursery areas observed in the western coastal area are linked with the same north-west—south-east currents , while tBakun’s ocean triad hypothesis cannot, however, be applied to elasmobranchs, such as the blackmouth catshark and thornback ray, producing benthic eggs. Nursery grounds of these species correspond to the areas where gravid females concentrate for hatching. From a conservation perspective the observed temporal stability of the identified nurseries of these two species implies a certain level of philopatry for their natal nursery grounds. This behaviour, furthermore, makes them even more susceptible to regional overfishing and localised stock depletion and extinction .Our analyses of recruit distribution have demonstrated that the only nurseries consistently protected in European Mediterranean waters are those of coastal species, such as red mullet, common Pandora and common sole with 66.8%, 54.1% and 46.1% respectively of persistent nursery areas under protection. This is mostly due to the trawling ban within 3 nautical miles of the shoreline or 50 m depth, applied through current management measures as defined by Article 13 of EU Council Regulation 1967/2006. The proportion of nursery areas protected by ongoing conservation measures is much less consistent for species with offshore recruitment areas such as European hake and the deep-water rose shrimp, whose recruitment occurs on the shelf-break and upper slope below 150 m depth. Finally, there was minimal overlap between TFRA and recruitment areas for offshore species distributed over the continental slope deeper than 200 m . It is also worth noting that the overlaps seen here between spatial measures and persistent nurseries could represent an underestimate as the temporal extent of fishing closure can vary considerably in the Mediterranean. As a consequence the duration of closures might not always be sufficient, or at the appropriate time to coincide with recruitment periods.Traditionally, Mediterranean fisheries have been oriented towards a multispecies complex, and the application of a mix of management tools based only on technical measures and fishing capacity control have mostly failed to ensure the long-term sustainability of fisheries, or the conservation of important habitats , 72]. W. W72]. WA combination of poorly selective gears (i.e. fine mesh sizes) and lack of spatial conservation measures currently leads to fisheries with high catches and discards of specimens below the MCRS, established for Mediterranean commercial stocks by the EC reg. 1967/2006 .The new knowledge provided in this study on the distribution and persistence of demersal nurseries in EU Mediterranean waters can support the designation of no-take MPAs and their inclusion in a conservation network , 76–77]–7776–77]S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Memories compete for retrieval when they are related to a common retrieval cue. Previous research has shown that retrieval of a target memory may lead to subsequent retrieval-induced forgetting (RIF) of currently irrelevant competing memories. In the present study, we investigated the time course of competitive semantic retrieval and examined the neurocognitive mechanisms underlying RIF. We contrasted two theoretical accounts of RIF by examining a critical aspect of this memory phenomenon, namely the extent to which it depends on successful retrieval of the target memory. Participants first studied category-exemplar word-pairs (e.g. Fruit—Apple). Next, we recorded electrophysiological measures of brain activity while the participants performed a competitive semantic cued-recall task. In this task, the participants were provided with the studied categories but they were instructed to retrieve other unstudied exemplars (e.g. Fruit—Ma__?). We investigated the event-related potential (ERP) correlates of retrieval success by comparing ERPs from successful and failed retrieval trials. To isolate the ERP correlates of continuous retrieval attempts from the ERP correlates of retrieval success, we included an impossible retrieval condition, with incompletable word-stem cues (Drinks—Wy__) and compared it with a non-retrieval presentation baseline condition (Occupation—Dentist). The participants’ memory for all the studied exemplars was tested in the final phase of the experiment. Taken together, the behavioural results suggest that RIF is independent of target retrieval. Beyond investigating the mechanisms underlying RIF, the present study also elucidates the temporal dynamics of semantic cued-recall by isolating the ERP correlates of retrieval attempt and retrieval success. The ERP results revealed that retrieval attempt is reflected in a late posterior negativity, possibly indicating construction of candidates for completing the word-stem cue and retrieval monitoring whereas retrieval success was reflected in an anterior positive slow wave. Memories that are associated with a common retrieval cue are reactivated and compete for retrieval when the shared cue is presented. An everyday-example of such competitive cued recall is when someone asks you about your friend’s address. In this situation, the memory of her current address, the target-memory, and memories of previous addresses, competitors, will compete for retrieval. Memory research has suggested that the ability to retrieve the currently relevant target-memory comes at a cost, namely forgetting of the competing memories . The pheThe experimental paradigm used to investigate RIF  typically involves three phases . In the dependent upon successful target retrieval in the intermediate retrieval-practice phase. This theory holds that target retrieval strengthens the association between the cue and the target (e.g. Fruit and Mango). When the category-cue (Fruit) is later represented in the final recall test, it will activate the strengthened exemplars (Mango) and block retrieval of the non-strengthened (non-practiced) memories associated with the same cue (e.g. Kiwi). In contrast, the inhibitory control account assumes that RIF is independent of target retrieval success in the retrieval-practice phase. This theory postulates that presentation of the category-plus-word-stem cue (Fruit–Ma__?) reactivates studied exemplars belonging to the category (e.g. Mango and Kiwi) and that cognitive control mechanisms are recruited to inhibit the currently irrelevant competitors (e.g. Kiwi) to facilitate retrieval of the target memory (Mango). The inhibition of the competitors causes lowered accessibility and forgetting of these exemplars in the ensuing final recall test.The associative blocking account (e.g. ) posits Previous research has lent support to the notion that RIF is independent of target retrieval. In fact, in two studies, RIF has even been observed in a task where retrieval-practice is impossible and there can be no target memory strengthening ,7. PartiA semantic version of the retrieval-practice task was used in the present study. In this task, participants were given the same category-plus-word-stem cue as in the standard episodic retrieval-practice task, but they were asked to retrieve an exemplar from semantic long-term memory instead of an exemplar that had been presented in the encoding phase (none of the studied exemplars matched the word stem cues). This semantic competitive-retrieval task has been shown to induce RIF in previous studies –8, and wThe three conditions in the present experiment differed only in the competitive semantic-retrieval phase, where participants were provided with either a completable retrieval cue , an incompletable retrieval cue , or an intact category-exemplar word-pair . The associated tasks were to retrieve an exemplar from semantic memory that completed the word-stem cue when a word-stem was given  and to read the exemplar when an intact exemplar was presented (presentation baseline condition), i.e. only the former two tasks involved memory retrieval.We examined the relationship between target retrieval and RIF with behavioural measures in two ways: a) if unsuccessful retrieval attempt causes forgetting in the impossible retrieval condition, and b) if target retrieval in the possible retrieval-practice condition predicted RIF in the ensuing test.Importantly, the inhibitory control account and the associative blocking account have opposite predictions regarding the outcome of these two tests. According to the blocking account, RIF is dependent on target retrieval and this theory hence predicts that there should be no RIF in the impossible retrieval condition. This theory also predicts that there should be a positive correlation between target retrieval success in the competitive semantic retrieval phase and RIF in the episodic final test. On the contrary, the inhibitory control account holds that RIF is independent of target retrieval and predicts that a mere retrieval attempt is sufficient to induce forgetting without the prerequisite of retrieval success. Only one research group so far has reported RIF in an impossible retrieval-practice task , 7]. We. We7]. WIn addition to these behavioural tests of the role of target retrieval in RIF (i.e. strength dependence), we examined the relationship between RIF and the ERP correlates of retrieval attempt and retrieval success in competitive semantic retrieval. The logic of the ERP analyses was that ERP correlates of retrieval attempt should be evident in contrasts between failed retrieval trials  and the presentation baseline condition, whereas ERP correlates of retrieval success should be evident in comparisons between successful retrieval trials and failed retrieval trials within the possible retrieval condition. The two accounts of RIF have opposite predictions regarding the relationship between RIF and these ERP correlates. The associative blocking account assumes that RIF is dependent upon target retrieval and hence predicts a positive correlation between RIF and the ERP correlates of retrieval success, but no such correlation between RIF and ERP correlates of retrieval attempt. The inhibitory control account, on the other hand, assumes that RIF is independent of target retrieval and predicts no correlation between RIF and ERP correlates of retrieval success. Inhibitory control is theorized to be recruited to handle interference during competitive retrieval attempts even if retrieval is unsuccessful. The inhibitory control account accordingly suggests that potential ERP correlates of inhibitory control should be evident in the retrieval attempt ERP contrast and that an index of inhibitory control will be positively related to RIF. Previous research on inhibitory control during memory retrieval has suggested that inhibition is reflected in enhanced N2 amplitude e.g. –11). Thi. Thi11])semantic cued recall task. Previous ERP studies of cued recall have predominantly focused on episodic rather than on semantic memory . ). semantry . Thi. Thisemary . The LPN ERP effect has been related to a) the continuous reconstruction and evaluation of attribute conjunctions (e.g. item-source or item-context associations) when such conjunctions are not readily recovered by the test probe, and to b) error monitoring mechanisms that are engaged during memory retrieval (for review of the LPN see , th, th7], tThere was no correlation between target retrieval in the competitive semantic retrieval phase and RIF. This finding does not support the predictions from the associative blocking account, which predicts that RIF is dependent on target retrieval. On the other hand, our measure of memory performance is limited to overt target retrieval and it is still possible that covert retrieval of task irrelevant memories  may correlate with RIF as would have been predicted by the associative blocking account.Unexpectedly, we did not observe any correlations between RIF and the ERP correlates of retrieval attempt or retrieval success in the present study, making the the ERP results inconclusive regarding the role of target retrieval in RIF.The question of whether RIF is dependent of strengthening of the association between the target and the retrieval cue has been examined with a different approach in a recent meta study . In lineTo summarize, the behavioural results in the present study suggest that RIF is independent of target retrieval. This result supports the inhibitory control account, but does not necessarily rule out the associative blocking account, since covert retrieval of task irrelevant memories may cause RIF in the impossible condition. The ERP results are inconclusive regarding the role of target retrieval in RIF given the absence of correlations between the ERP effects and RIF. In addition to investigating the role of target retrieval in RIF, the present study allowed new insight into the ERP correlates of retrieval attempt and retrieval success in competitive semantic cued-recall.We included the impossible retrieval condition for two reasons. First, it enabled us to isolate retrieval attempt from retrieval success, since target retrieval was impossible and erroneous responses that did not match the cue were excluded in this condition . Second, as a consequence of excluding incorrect responses, the impossible retrieval condition made it possible to investigate continuous retrieval attempts throughout the recording epoch, whereas the retrieval attempts were discontinued in the possible retrieval condition if the participant retrieved the target  or retrieved an erroneous exemplar .The earliest retrieval-attempt-related effect was a positive deflection in all of the retrieval conditions  compared with the presentation baseline condition that peaked approximately 300 ms after the presentation of the word-stem cue (900 ms into the recording epoch). Such P300-like effects have previously been related to the allocation of attentional resources and may consequently indicate the allocation of attention to the relatively more difficult semantic cued-recall task compared with the reading task in the presentation baseline condition .The P300 effect was followed by an N2-like effect. Similar N2 effects have previously been suggested to reflect inhibitory control during memory retrieval . ). 34]). As expected, retrieval attempts gave rise to an LPN effect. This effect has previously been related to retrieval of attribute conjunctions, such as item-source and item-context associations, in episodic memory paradigms . In the precede semantic retrieval independently of whether the retrieved item was correct or not, whereas the error-detection interpretation predicts that the LPN should only be present after retrieval of exemplars that do not match the cue. We instructed the participants to withhold their response until the question mark appeared on the screen (after the ERP recording epoch had ended) to reduce the amount of muscle artefacts in the recording epoch. The participants did hence not respond within the ERP recording epoch in the present study, so we can not examine the relative contribution of the two processes to the LPN in the present study.Both retrieval of attribute conjunctions and retrieval monitoring are expected to be iterative and their ERP correlates in averaged EEG should hence be tonic in line with the LPN effect in the present study. An important difference between the two interpretations of the LPN effect is that the retrieval of attribute conjunctions interpretation predicts that the LPN should Both retrieval of attribute conjunctions and the error-detection interpretations can be regarded as essential parts of memory search processes. The finding that long-term memory search is related to posterior activity is in line with the attention to memory model . AccordiBoth semantic and episodic retrieval success were reflected in a PSW effect. This effect started approximately 300 ms earlier for semantic (500 ms post word-stem cue presentation) compared with episodic retrieval . The difference in onset-latency may be due to differences in cue-presentation procedures between tasks . In The anterior topography of the semantic PSW effect in the present study is consistent with the observations in studies of episodic cued recall and with the semantic word-fragment completion task in Rass et al (2010). Surprisingly, the topography of the PSW was however more anterior in the present study compared with our previous study, using the same task . AlthougThe results in the present study suggests that overlapping neurocognitive processes are recruited during retrieval from the semantic and the episodic memory system. Successful retrieval was reflected in a PSW effect with similar timing and topography in both semantic and episodic retrieval. We primarily designed the present study to investigate the role of target retrieval in RIF and it should be noted that a side effect of optimizing the RIF effect was that the semantic and episodic retrieval tasks differed in procedures and difficulty. First, while the category and word-stem cues were presented sequentially in the competitive semantic retrieval phase they were presented simultaneously in the test phase . The reaBesides the PSW the present study also provides novel data suggesting that the LPN is involved in semantic retrieval. The LPN has previously been reported in episodic cued recall and recognition memory paradigms. The present study extends current literature by reporting an LPN in a semantic retrieval task, and suggests that the LPN reflects processes that are involved in both episodic and semantic retrieval.To further investigate similarities and differences between episodic and semantic cued recall future studies should compare the ERP correlates of retrieval attempt and retrieval success in both memory systems within the same experiment using the same cueing procedure, similarly to how episodic and semantic retrieval tasks have been compared in recognition memory by contrasting recognition tasks and semantic judgement tasks Click here for additional data file.


The energetics of cerebral activity critically relies on the functional and metabolic interactions between neurons and astrocytes. Important open questions include the relation between neuronal versus astrocytic energy demand, glucose uptake and intercellular lactate transfer, as well as their dependence on the level of activity.Vcyc) explains part of the uncoupling between glucose and oxygen utilization at increasing Vcyc levels. Thus, we are able to characterize different activation states in terms of the tissue oxygen-glucose index (OGI). Calculations show that glucose is taken up and metabolized according to cellular energy requirements, and that partitioning of the sugar between different cell types is not significantly affected by Vcyc. Furthermore, both the direction and magnitude of the lactate shuttle between neurons and astrocytes turn out to depend on the relative cell glucose uptake while being roughly independent of Vcyc.We have developed a large-scale, constraint-based network model of the metabolic partnership between astrocytes and glutamatergic neurons that allows for a quantitative appraisal of the extent to which stoichiometry alone drives the energetics of the system. We find that the velocity of the glutamate-glutamine cycle (ad hoc activity-related constraints on neuronal and astrocytic metabolism, the glutamate-glutamine cycle does not control the relative energy demand of neurons and astrocytes, and hence their glucose uptake and lactate exchange.These findings suggest that, in absence of  In parres ..in vivo,The difficult interpretation and integration of the experimental findings produced a substantial theoretical effort aimed at characterizing intra- and inter-cellular metabolic fluxes -12. So fOn the other hand, the known regulations of enzyme-catalyzed reactions implemented in dynamical models have so far proved insufficient to justify a fundamental energetic role for the CCLS . In partThe aim of this study is to examine the activity-dependent metabolic cooperation of glutamatergic neurons and astrocytes from a network-based perspective. Specifically, two issues lie at the core of our work: (i) the correlation between partitioning of glucose and lactate shuttling; and (ii) their functional modulation across varying levels of glutamate-glutamine cycle. We have employed a constraint-based setting where an extensive and controlled sampling of the solution space is possible ,22 on a (a) the OGI is able to distinguish states characterized by different levels of neurotransmission, as flux configurations with larger OGIs typically carry smaller values for the velocity of the glutamate-glutamine cycle;(b) the partitioning of glucose between neurons and astrocytes is roughly independent of the level of activity;(c) the magnitude and direction of the CCLS depend strongly on glucose partitioning while being roughly independent of the level of neurotransmission.In other terms, within a purely stoichiometric model, the system’s energetics is determined to a significant extent by the sharing of glucose. These results support the idea that neurotransmission does not impose significant constraints on glucose partitioning or CCLS. In addition, we show that (d) the overall degree of correlation among metabolic reaction fluxes between and within cells changes drastically in the presence of neurotransmission, pointing to an extended metabolic and possibly functional partnership between neurons and astrocytes.The reaction network we considered  . Both ce+ ions and counter-transport of one K+ ion. Glutamate is amidated to glutamine by astrocytic glutamine synthetase (GS) with the concurrent hydrolysis of one molecule of ATP. Glutamine is then exported to neurons where it is eventually converted back to glutamate and loaded into synaptic vesicles again, which costs another ATP. Astrocytic uptake of glutamate and release of glutamine, together with neuronal uptake of glutamine and release of glutamate configure the so-called glutamate-glutamine cycle. In this way, the clearance of neuronally released glutamate from the extracellular space is mostly accomplished by astrocytes . Th. Th11]. Third, simulations showed that glutamate-glutamine cycle is correlated with overall tissue glucose utilization and lactate production, but not with specific patterns of cellular glucose uptake and lactate shuttle Figure . These rWe found that intercellular shuttling of lactate increases only when glucose partitioning between neurons and astrocytes is significantly uneven Figure . TherefoInterestingly, if the functional partnership between neurons and astrocytes is suppressed via the zeroing of neurotransmission, the anticorrelation between glycolysis in the two cellular compartments further increases (i.e. correlation becomes more negative), a feature shared with many other fluxes related to energetics, including TCA cycle and oxidative phosphorylation. So, even when there is no functional relationship between neurons and astrocytes, their energy (primarily glycolytic) metabolism is anticorrelated. The reason for this behavior is that the two cell types share the same environment, thereby the same glucose availability. Glucose availability thus represents the primary drive on glucose partitioning. If the two cells are functionally independent, oxidative metabolism follows glycolysis, as also evidenced by the negative correlation between neuronal and astrocytic oxidative metabolism. As soon as neurons and astrocytes become coupled by neurotransmission, the network shifts to correlated patterns of activity, both within the same compartment and between different compartments. Accordingly, glycolysis in one cell type becomes slightly positively correlated with oxidative metabolism in the other. However, glycolysis in either neurons or astrocytes, which essentially reflect partitioning of tissue glucose, remains poorly correlated with the glutamate-glutamine cycle. Overall, glutamate-glutamine cycle positively correlates with cell TCA cycle and oxidative phosphorylation much more than it does with glycolysis, which is especially significant for the astrocytic compartment.ad hoc constraints on neurotransmitter cycling and ionic fluxes in the hope to capture the non-stoichiometric side of the energetics of brain activity.In conclusion, we developed a large-scale model for compartmentalized brain energy metabolism including the core carbon pathways of neurons and astrocytes, as well as further compartments . A constraint-based scheme was then employed in order to define feasible configurations of reaction fluxes. Our numerical analysis was based on a relaxation algorithm which allows to obtain a statistically controlled sampling of the solution space without any prior assumption on the behaviour of energy producing/consuming pathways. Results have shown that only a large imbalance of cell glucose uptake can explain the occurrence of a significant lactate shuttle between neurons and astrocytes, the latter being roughly independent of the rates of transmitter cycling. Our results therefore do not support a link between glutamate-glutamine cycle and CCLS as a mechanism for activation-dependent transfer of energy compounds within the brain. On the other hand, CCLS can be found by assuming that neurons have limited access to glucose and/or by bounding their glucose uptake flux. The lack of correlation we observe stems from the fact that the distribution of activity-related energy stress between neurons and astrocytes cannot be estimated by the stoichiometry of the metabolic network and should be a primary target of current experimental research. Future developments will focus on introducing minimal a Because of finite numerical precision, for our putposes a flux is null when it is smaller than 10-6.The authors declare that they have no competing interests.ADM, MDN, FG and EM designed research; all authors contributed data and/or analysis methods; FAM performed research, with contributions from all authors; all authors analyzed results; all authors drafted the manuscript. All authors read and approved the final manuscript.Supporting text.Click here for file


European and national administrative legislation require objective evaluation systems for organ lesions at pig slaughter. These results can be used as basis for herd health improvement programs by farmers and their consulting veterinarians. Various studies have shown that the current evaluation and recording of lesions by authorized meat inspectors are not reliable and produce significant inter-rater disagreement especially for lung lesions in pigs. The objectives of this study were to increase the usability of official meat inspection data by a developed and validated scheme and to analyze potential improvements in the reliability of the proposed system under industrialized slaughter conditions.A simplified evaluation scheme for surface-related lung lesions was developed based on morphometric evaluations of unaffected lungs with quantitative relationships of each lobe to the whole lung (“Rule of Tens”). Furthermore, a theoretical as well as a hands-on training program for meat inspectors was developed and applied. Based on 5,183 lungs, the authors established a baseline of the inter-rater reliability of current routine assessments of lung lesions as documented by meat inspectors compared with the assessments of an independent veterinarian using the developed simplified evaluation scheme. Most frequent inter-rater disagreements greater than 75% were found for moderate pneumonia. Sources of the deviations most frequently included misinterpretations of technical artifacts, which were erroneously assessed by the meat inspectors as pneumonic lung lesions. Results of the post-training investigation based on 4,646 lungs showed a significantly improved reliability of lung lesion evaluation and the inter-rater agreement increased in all respects. Especially the disagreement of recording moderate cases of pneumonia decreased in total to 15% deviations from reference.The presented simplified lung evaluation scheme showed its capability to standardize the evaluation of lung lesions according to administrative legislation under industrialial slaughter conditions. The reliability of official meat inspections can be significantly increased with the help of the presented scheme to assess surface-related lung lesions of slaughter pigs. Continuous standardization and optimization can be achieved by personalized training programs in the framework of quality assurance systems for meat inspectors. Official controls on products of animal origin intended for human consumption are important for protecting public health as well as animal health and animal welfare. The European Commission regulation calls for continuous improvement of all mentioned aspects, which have to be based on the most recent and relevant information available. This information should constantly adjust current meat inspection practice . SlaughtAccording to a national administrative regulation in Germany, an objective evaluation system for organ lesions has to be used for official meat inspections in abattoirs . OutsideThe score developed by Blaha  assesses2. Due to the anatomical conditions of the cranial lobes of the lung on both sides, lobes were subdivided along a simulated line with 45° angle based on a horizontal line drawn at the bifurcatio trachealis  were compared with the assessment results of an independent observer using the surface-related evaluation scheme. Between July and August 2011, the status-quo investigation of the actual evaluation of lung lesion according to the regulation of the German AVVLmH was performed in an abattoir in North Rhine Westphalia, Germany. The abattoir processed 550 pigs per hour. Pigs were stunned by an automatic electro perfusion, and scalding was performed in a scalding tank. The official slaughter check included scoring of the lung lesions according to the system of the German AVVLmH (2009)  and was In order to disclose the sources of the calculated inter-rater deviations within the status-quo inquiry, personal discussions between OV and AQAs specific evaluations were performed at the slaughter line. The most frequently analyzed sources of deviations were included in a specific training program for AQAs. The training was divided into two parts: (a) a theoretical training in two sessions lasting four hours each and (b) a practical on-site training of individual AQAs during operations at the slaughter line. The training took place during two weeks in August and September 2012. The core element of the training was the simplified surface-related lung lesion evaluation scheme as an easy-to-use and quick method to quantitatively evaluate pathologically-anatomically lung lesions. This was presented to the AQAs and discussed in detail. Each AQA received a laminated sheet with the scheme to be used at the slaughter line for the training period.In this second investigation, the results of post-training assessments of lung lesions by AQAs were compared with the assessment results of the OV, both using the simplified lung lesion evaluation scheme. During four slaughtering days in September 2012, lungs of 4,646 randomly selected pigs scored parallel and independently by 15 AQAs and by the OV as described for the status-quo investigation (see above). The results were compared and the inter-rater reliability was determined based on absolute and relative agreement per result key between AQAs and OV.Figure 2.A red square =1 mmIn Table Medians and arithmetic means of sample lobes differed not more than 0.79% per site. The greatest difference of 0.79% was observed for the accessory lobe , cultural differences and lack of understanding to fulfill an essential function in public health and animal welfare.The training program consisted of a theoretical and a practical part. The theoretical part of the training focused on the components:1. Describing the importance for standardized lung lesion evaluation by showing the aims of informing farmers of the frequencies of lung lesions and the positive effects on herd health caused by a standardized scoring system.2. Illustrating the anatomical, physiological, histological, immunological and pathological basis of the porcine lung as well as the differentiation of lung alterations caused by slaughter techniques and by diseases.3. Revising the legal requirements of lung evaluation ,13.4. Presenting the developed simplified surface-related lung lesion evaluation scheme to evaluate lung lesions according to its surface as a tool for homogeneous evaluations.5. Showing affected lungs via photographs and abstracting the relevant lesions by means of a scheme with colorization of the affected lung areas to describe the extent  a validated, simplified and standardized scheme to assess surface-related lung lesions of slaughter pigs (“The Rule of Tens”) and ii) an accompanying theoretical and practical training of authorized qualified assistants (AQA) in evaluating pathological lung lesions. A continuative standardization and optimization can be realized by repetitive and individual training programs based on controls within the framework of quality assurance.AQAs: Authorized qualified assistants = synonymous to official auxiliaries in Reg. (EC) No. 854/2004; OV: Observing veterinarian (Reference).The authors declare that they have no competing interests.TS participated in the study design and carried out the data collection, developed the simplified standard surface area scheme, conducted the training program and drafted the manuscript. TB and DM planned and designed the study, coordinated the research project and participated in drafting the manuscript. TS, TB and DM performed the statistical analysis and were involved in the interpretation of data. All authors read and approved the final manuscript.


Although the relevance of understanding spinal kinematics during functional activities in patients with complex spinal deformities is undisputed among researchers and clinicians, evidence using skin marker-based motion capture systems is still limited to a handful of studies, mostly conducted on healthy subjects and using non-validated marker configurations. The current study therefore aimed to explore the validity of a previously developed enhanced trunk marker set for the static measurement of spinal curvature angles in patients with main thoracic adolescent idiopathic scoliosis. In addition, the impact of inaccurate marker placement on curvature angle calculation was investigated.Ten patients (Cobb angle: 44.4±17.7 degrees) were equipped with radio-opaque markers on selected spinous processes and underwent a standard biplanar radiographic examination. Subsequently, radio-opaque markers were replaced with retro-reflective markers and the patients were measured statically using a Vicon motion capture system. Thoracolumbar / lumbar and thoracic curvature angles in the sagittal and frontal planes were calculated based on the centers of area of the vertebral bodies and radio-opaque markers as well as the three-dimensional position of the retro-reflective markers. To investigate curvature angle estimation accuracy, linear regression analyses among the respective parameters were used. The impact of inaccurate marker placement was explored using linear regression analyses among the radio-opaque marker- and spinous process-derived curvature angles.The results demonstrate that curvatures angles in the sagittal plane can be measured with reasonable accuracy, whereas in the frontal plane, angles were systematically underestimated, mainly due to the positional and structural deformities of the scoliotic vertebrae. Inaccuracy of marker placement had a greater impact on thoracolumbar / lumbar than thoracic curvature angles. It is suggested that spinal curvature measurements are included in marker-based clinical gait analysis protocols in order to enable a deeper understanding of the biomechanical behavior of the healthy and pathological spine in dynamic situations as well as to comprehensively evaluate treatment effects. Upper-body kinematics during daily activities such as walking are known to be important for movement control . A comprFor these reasons, an enhanced trunk marker set (IfB marker set) was introduced and validated for the assessment of sagittal lumbar and thoracic curvature in healthy adults . AlthougAn important aspect to consider in AIS is that the spinal deformity is always three-dimensional with the basic components being intervertebral lordosis , lateral inclination  and axial rotation (transverse plane), i.e. the vertebral bodies rotate toward the convex side and the spinous processes toward the concave side , 18. In In addition to the malrotation of vertebral bodies, inaccurate marker placement might also contribute to an under- or overestimation of the spinal curvature. Studies showed that the rate of accurately locating the spinous processes by palpation was somewhere between 45% and 83% with a mean distance of inaccurate identification either above or below the targeted level of 19.3±18.6 mm –24. ThisUsing biplanar radiography and marker-based motion capture techniques, the primary aim of the current study was to explore the static validity of skin marker-based measurements of sagittal and frontal plane spinal curvature angles in a group of patients with AIS. In a secondary aim, the accuracy of spinous process identification by palpation and the impact of inaccurate marker placement on curvature angle measurements in the frontal plane were addressed.This study was approved by the local ethics committee , Ref.-No.: 33/13). All patients as well as their legal guardians provided written informed consent to participate in this study.A consecutive sample of ten patients with AIS participated in the current study . All parStandard biplanar radiographic examinations  of the entire spine while standing were performed, including spherical radio-opaque markers (diameter: 5 mm) that were attached to the skin in a configuration described below. Measurements in the motion analysis laboratory were carried out using standard retro-reflective markers (diameter: 9–14 mm) and a 12-camera motion capture system  at a sampling rate of 300 Hz.Selected markers from a previously developed trunk marker set (IfB marker set) , placed Spinous processes of interest were located and marked by an experienced physiotherapist using a skin-compatible pen and with the patient in an upright seated position. First, the spinous process of C7 was located using two different methods: palpation of the two most prominent cervical spinous processes and identification of C7 by 1) flexion and assisted extension of the cervical spine (spinous process that remained stationary) , 27, 28 Radiographic images were processed using the software ImageJ . Cobb-angles were determined as described in the literature . The cenCurvature angle calculation in the sagittal and frontal planes was based on the radiographic  and motion capture data (retro-reflective markers). In the sagittal plane, the lumbar curve was defined by the vertebral bodies T12-L5 and the markers placed on T11, L1-L5, while the thoracic curve was defined by the vertebral bodies T3-T12 and the markers placed on T3, T5, T7, T9 and T11. In the frontal plane, thoracolumbar / lumbar and thoracic curves were defined by the vertebral bodies and markers that corresponded to the Cobb-angle boundaries , with a The accuracy of marker placement by palpation was evaluated on the basis of the posterior-anterior radiographic images (spinous processes and radio-opaque markers). Due to the laterally deviated spine, the calculated CoA’s of the spinous processes and the markers were vertically aligned according to the tilt angle of the longitudinal axis of the respective spinous process . VerticaStatistical calculations were performed using the software package SPSS 21  and statistical significance was set at p = 0.05.Primary outcomes: To determine the validity of skin marker-based curvature angle measurements, linear regression analyses among the parameters vertebral body (VB)-, radio-opaque marker (RO)- and retro-reflective marker (RR)-derived curvature angles were carried out.Secondary outcomes: The accuracy of marker placement by palpation was analyzed descriptively and expressed as numbers and percentages. In addition, absolute displacement values of the markers that were not correctly identified were presented. To explore the impact of inaccurate marker placement on curvature angle measurements in the frontal plane, linear regression analyses among the parameters spinous process (SP)-, RO- and RR-derived curvature angles were carried out with higher correlations indicating a smaller impact and lower correlations a greater impact.Primary outcomes: Statistically significant moderate to strong correlations between the VB- and RO- as well as RR-derived curvature angles were found for the sagittal as well as frontal curves . ConsideSecondary outcomes: Six of the 110 radio-opaque markers were not visible on the radiographs and could therefore not be included in the evaluation. A total of 57.7% of the spinous processes were palpated correctly in the vertical and 38.4% in the horizontal direction . Mean diThe current study aimed at validating skin marker-based measurements of spinal curvature angles in the sagittal and frontal planes in patients with main thoracic AIS. In addition, the accuracy of marker placement by spinous process palpation and the impact of inaccurate placement on spinal curvature angle measurements were addressed.In the sagittal plane, lumbar and thoracic curvature angles could be estimated with a reasonable accuracy by both the RO- and RR-markers, whereas in the frontal plane, thoracolumbar / lumbar and thoracic curvature angles were systematically underestimated by the skin markers. About half of the spinous processes were not identified correctly by palpation in the vertical and another half in the horizontal direction, but with fairly low displacement values. Inaccurate placement of markers appeared to have a greater impact on curvature angle estimation in the thoracolumbar / lumbar than the thoracic spine. While the consequence of these results for assessing the kinematics of the spine during dynamic activities remains to be elucidated in further studies, these data suggest that it might be possible to perform a systematic correction of the marker data or to focus on movement patterns (relative angular differences) in order to provide clinical understanding to dynamic data.Only few studies evaluated the validity of skin marker-derived spinal curvature measurements , 32, 33.To investigate the validity of skin marker-based measurements for the quantification of spinal movement in the sagittal plane, Mörl and Blickhan  used MRIWhile the two MRI-based studies above only described spinal curvature measurements in the sagittal plane, Hashemirad et al.  validateThe current study further showed that VB-derived sagittal thoracic curvature angles could be better estimated by the RO than the RR markers. This might be explained by the fact that in the movement analysis laboratory, the standing position of some patients did not exactly match the posture during the radiographic examination. For the lateral view images, patients were required to hold on to a horizontal bar elevating their arms to the front, which might have caused slight positional differences especially in the upper thoracic spine. If possible, future validations should therefore aim to perform the radiographic and motion capturing measurements simultaneously.Concerning the differences between RO- and RR-derived measurements, the slightly increased underestimation of the frontal plane curvature angles by the RR compared to the RO markers might be due the fact that the RR markers were mounted on a plastic socket that allowed slight tilting towards the concave side when placed on the paravertebral muscles in the area of the curvature’s apex. In general, regardless of the different types of markers, the underestimation of the curvatures in the frontal plane could be explained mainly by the pathology related structural deformity of the vertebrae as well as a systematic marker placement error towards the concave side of the curvatures. The fact that the palpable parts of the spinous processes were in most cases approximately one level below the position of the respective vertebral body  might have led to an additional falsification of the actual frontal plane curvature.Considering the available evidence on the identification accuracy of selected spinous processes, the results of the current study seem to be in agreement for the lumbar but not for the cervical spine. Harlick et al.  evaluateSkin marker-based motion capture techniques can be used for the non-invasive assessment of spinal curvature angles in the sagittal and frontal planes in patients with AIS. However, while absolute values in the sagittal plane could be measured with reasonable accuracy, frontal plane angles were systematically underestimated, mainly due to the rotational deformities of the scoliotic vertebrae. Skin markers on the trunk should therefore be used for the assessment of movement and postural change  rather than for the measurement of absolute angles.Inaccuracy of marker placement by palpation had a greater impact on the determination of the thoracolumbar / lumbar than the thoracic curvature angles. In order to keep such inaccuracies minimal, only health care professionals with experience in palpation should place markers.Based on the current and previous findings, it is suggested that spinal curvature measurements are included in marker-based clinical gait analysis protocols. This would enable a deeper understanding of the behavior of the healthy and pathological spine in dynamic situations and would open up the possibility for a more comprehensive evaluation of treatment effects as movement seems to be detected reasonably well. In addition, the data can be used to drive complex spinal models in order to get an insight into the dynamic loading of the spine during movement.


Coronal fractures of the anterior teeth are common sequelae of dental trauma. In case of complex fractures, where the fractured segment is available and there is close approximation of the segment to the remaining tooth, root canal treatment followed by reattachment of the fractured segment with fiber post reinforcement is a feasible option. The procedure is simple and economic and needs less chair-side time as compared to many conventional methods. In addition, the procedure provides good and long-lasting esthetics, because the original morphology, color, and surface texture are maintained. This paper reports three cases of complex coronal tooth fracture successfully managed using tooth fragment reattachment. Complicated crown fractures involving the enamel, dentin, and pulp constitute a major share of all dental injuries and are most common in maxillary central incisors , 2. A frManagement of complicated crown fractures is a multifactorial process influenced by the extent and pattern of fracture , restorability of fractured tooth (associated root fracture), secondary injuries (soft tissue status), presence/absence of fractured tooth fragment and its condition for use (fit between fragment and the remaining tooth structure), occlusion, esthetics, finances, and prognosis . In caseThis paper reports three cases of complicated coronal tooth fracture successfully managed by tooth fragment reattachment.A 27-year-old male injured in a road traffic accident (RTA) was referred to the Department of Conservative Dentistry and Endodontics, Amrita School of Dentistry. Clinical and radiographic examination revealed a complicated oblique crown fracture on 11 that extended subgingivally in the mesiopalatal aspect. The fractured segment was held in place by the gingival attachment . Ellis cIt was planned to perform single visit root canal treatment (RCT) on 11 followed by reattachment with fiber post reinforcement. 22 was also planned for RCT and post-core restoration.+ Nanoceramic Restorative, Denstply, Konstanz, Germany) was provided for 2 weeks. The RCT for 22 and subsequently 21 was performed. The patient was kept on periodic review and it was observed that both endodontic and restorative treatments remained clinically acceptable through each visit. The clinical and radiographic pictures after 1 year revealed favorable healing  and the fractured segment in relation to 11 was atraumatically removed . It was  Figures .A similar case of a 28-year-old male presented with Ellis class III fracture of the right maxillary lateral incisor incurred in an RTA, with subgingival extension beyond junctional epithelium Figures . As the After RCT and post space preparation , an esthTo gain access to the gingival extent of the fracture line and to better evaluate its relation to the bone crest, buccal and palatal full thickness mucoperiosteal flaps were elevated using number 15 B-P blade. Hemostasis was achieved . The posThe third case is of a 35-year-old female who presented with pain and mobility of her fractured left maxillary central incisor. Hard tissue examination revealed an Ellis class III fracture of 21 and a class I fracture of 11 . RCT, fiConventional methods employed in the restoration of fractured teeth include partial and full coverage crowns, laminate veneers, and composite resins all of which are time-consuming, high priced, and not conservative . First dThe success of the reattachment depends on several factors: hydration of the fractured segment while outside oral cavity is one of them. This is necessary to maintain the vitality and original esthetic appearance of the tooth and also ensures adequate bond strength . In all When there is a substantial associated periodontal injury and/or invasion of the biological width, the restorative management of the coronal fracture should also consider the rehabilitation of those associated tissues . In caseReinforcement of the reattached fragments using posts has been widely reported in the literature. Although many techniques with various materials have been suggested, resin-based restorative materials with tooth-colored fiber post may be considered the best option because of several advantages such as a suitable elastic modulus, esthetics, good bonding between post and cement, lower chair time, and minimal tissue removal , 8. It iIn addition to the preparation of the post space, in all cases a vent was created in the coronal separated segment as a leeway for the excess cement to flow out without buildup of any hydrostatic pressure. A similar technique has been recommended by Tosun et al. in reattachment using Ribbond material .The 3 cases presented in this paper suggest that, with the materials available today along with appropriate clinical technique, reattachment of tooth fragment is a viable and conservative treatment option for fractured incisors. It is hoped that this report of 3 cases will add to the increasing volume of evidence which supports the viability of reattachment of the broken fragment of the anterior tooth reinforced by suitable restorations. Future reports may need to focus on reporting longer followup to bolster the evidence in favour of this treatment option.


To incorporate formal assessments of potential efficacy into feasibility and pilot studies of complex interventions, appropriate methods for sample size determination are required. In particular, the implications of clustered patient outcomes resulting from cluster randomisation or treatment provision must be addressed. In this paper we will show how small-sample methods used to design phase II drug trials can be extended to allow for this clustering. In particular, we will propose a single-arm single-stage design for continuous data clustered in a two-level hierarchical structure. A number of alternative methods for determining the choice of sample size will be described. We will report the results of a simulation study comparing these methods in terms of the resulting error rates, suggested sample size parameters, and computational burden. A sensitivity analysis will demonstrate the implications of nuisance parameter misspecification at the design stage. Particular attention will be given to the scenario where the number of cluster-level units is severely limited, as will often be the case in the early phases of complex intervention development and evaluation.


A major limitation to current cancer therapies is the development of therapy-related side-effects and dose limiting complications. Moreover, a better understanding of the biology of cancer cells and the mechanisms of resistance to therapy is rapidly developing. The translation of advanced knowledge and discoveries achieved at the molecular level must be supported by advanced diagnostic, therapeutic and delivery technologies to translate these discoveries into useful tools that are essential in achieving progress in the war against cancer. Nanotechnology can play an essential role in this aspect providing a transforming technology that can translate the basic and clinical findings into novel diagnostic, therapeutic and preventive tools useful in different types of cancer. Hematological malignancies represent a specific class of cancer, which attracts special attention in the applications of nanotechnology for cancer diagnosis and treatment. The aim of the present review is to elucidate the emerging applications of nanotechnology in cancer management and describe the potentials of nanotechnology in changing the key fundamental aspects of hematological malignancy diagnosis, treatment and follow-up. Nanotechnology is the management of material properties at nanometer levels. The concept of nanotechnology was first introduced by the American physicist Richard Feynman who gave a talk entitled 'There's Plenty of Room at the Bottom' at an American Physical Society meeting at Caltech in 1959. Feynman did not mention 'nanotechnology', yet considered the potential to manipulate individual atoms at the molecular level. This talk inspired the conceptual beginning for nanotechnology science through creating the concept of 'molecular manufacturing' .Nanotechnology is defined as the creation and use of structures, materials, devices and systems with completely novel properties and functions obtained from their size which ranges from 1 to 100 nm. It is applied in many fields including electronics, information technology, material developments and biomedicine . NanobioCancer nanotechnology is a striving field , currentin vivo. Multiple types of nanomaterials can be used to generate the nanoscale drug delivery systems including lipid-based nanocapsules or liposomes and natural or synthetic polymers. Liposomes are biocompatible vesicles formed of amphipathic lipid bilayer membranes. Polymers could be derived from biocompatible drug conjugates or micelles that self-assemble to form a hydrophilic outer corona and a hydrophobic inner core for drug encapsulation. The choices of different nanoscale systems are based on the components, applications, advantages and limitations of each preparation. These novel nanotherapeutics are rapidly progressing as they offer solutions to many limitations of conventional drug delivery systems such as nonspecific targeting, limited biodistribution, poor bioavailability and low therapeutic indices. Factors that determine nanomaterial design and characterization include size and shape, blood half-life, controlled drug release and active targeting of nanoparticles. Efficient cell uptakes were reported with nanoparticles with a size range of 40–50 nm  and cytogenetic or molecular analyses of diagnostic genetic mutations associated with prognosis . As antiet al  have choet al  in 1995 for treatment of metastatic ovarian cancer and AIDS-related Kaposi sarcoma, several other drugs with similar features have been developed and were either approved or in different phases of clinical trials . Nanotecet al  was the an et al  who loadet al  loaded with adriamycin and tetrandrine to interfere with MDR of K562/A02 cells. Measurement of several biological parameters has shown that adriamycin- and tetrandrine-loaded Fe3O4-MNPs can help in counteracting the effects of MDR in K562/A02 cells, due to the buildup of drugs caused by its polymerization with Fe3O4-MNPs. In another study, Chen et al  is a novel approach developed to increase the concentration of anticancer drugs in the tumor and decrease the amount of leakage into the surrounding healthy tissues . Several advantages of NDs favor their use in the field of medicine such as being inert, transparent, having high surface area and being biocompatible . NDs canet al  adjustedl  to differentiate into mature myeloid cells and ATRA may induce complete remission in APL patients  is a specific subtype of AML. The hallmark of APL is the presence of specific t chromosomal translocation. The translocation occurs between retinoic acid receptor-α (RARα) gene and the PML gene on chromosomes 17 and 15, respectively . APL celpatients . Howevers  are among the interesting materials for use in the field of nanotechnology. AuNPs have many optical properties that make them suitable for bioimaging applications . AuNPs hAuNPs have also proven to be biocompatible, have limited toxicity and low susceptibility to photobleaching making them suitable for use in studies with human cells . These ein-situ hybridization (FISH) as well as reverse transcriptase polymerase chain reaction analyses (RT-PCR). However, those methods are costly and time-consuming creating the need for developing methods that are sensitive and cost effective at the same time  is a clonal disorder characterized by accumulation of mature-appearing lymphocytes in the peripheral blood, bone marrow, lymph nodes as well as the spleen. CLL cells are frequently monoclonal B-lymphocytes that express CD19, CD5 and CD23, with weak or no expression of surface immunoglobulin (Ig), CD20, CD79b and FMC7 . CLL celet al  and express VEGF membrane receptors (VEGF-R1 and VEGF-R2). VEGF also plays a significant role in CLL cells resistance to apoptosis . These fet al  studied et al . The comet al .et al  is a CD30-directed antibody-drug conjugate that received approval from FDA for the treatment of patients with HL after relapse . CD30 haet al  together with the surface expression of CD30. Based on the presence or the absence of ALK translocation, the tumor is classified into either ALK-positive or -negative-ALCL. The presence of these unique and specific features can provide the base of targeted therapy for treatment of ALCL . For exaet al  have devet al . Accordiet al (Mantle cell lymphoma (MCL) is a high-risk subtype of NHL characterized by an aggressive clinical course and by having the worst prognosis among B-cell NHL . These cet al  designedin vivo applications. Possible changes in the reactions of the particles with cells of the body may occur and changes in the absorption and secretion properties of such particles should be well investigated. FDA has recommended that meticulous research should be conducted in areas related to these novel properties that may cause toxicity. FDA also recommended that further research for development of devices and methods that can be used for measurement of nanoscale materials should be developed. Another important issue that is related to nanotechnology, being an emerging technology, is the ethical concern. In many cases, as science leaps forward, ethics lag behind. Part of the funding should be directed towards ethical, legal and social issues related to nanotechnology. One of the most famous examples for this readiness was during the Human Genome Project when James Watson suggested that 3–5% of the budget be devoted for study of ethical, legal and social issues.Nanotechnology represents a promising technology that can be of great value in the war against cancer. However, attention should be put towards safety issues and ethical concerns. Since particles at the nanosize have unique physical and chemical properties, care must be taken when dealing with particles or products for The future prospects of nanotechnology and nanomedicine are very promising. Nanotechnology development for targeted therapy of cancer is one of the most interesting areas in research currently underway where drugs are delivered specifically to the diseased tissues or cells of interest without affecting nearby normal cells. Development of biomarkers for early detection and imaging of cancer cells is another important topic for research using nanotechnology. Scientists hope that progress in proteomics and bioinformatics can be used by nanotechnology to identify and kill malignant cells with the highest efficacy and the least possible side-effects, therefore bringing us closer to the goal of more effective cancer care.


Gammaproteobacteria is proposed, which is designated Cellvibrionales ord. nov. and comprises the five novel families Cellvibrionaceae fam. nov., Halieaceae fam. nov., Microbulbiferaceae fam. nov., Porticoccaceae fam. nov., and Spongiibacteraceae fam. nov.In recent years a large number of isolates were obtained from saline environments that are phylogenetically related to distinct clades of oligotrophic marine gammaproteobacteria, which were originally identified in seawater samples using cultivation independent methods and are characterized by high seasonal abundances in coastal environments. To date a sound taxonomic framework for the classification of these ecologically important isolates and related species in accordance with their evolutionary relationships is missing. In this study we demonstrate that a reliable allocation of members of the oligotrophic marine gammaproteobacteria (OMG) group and related species to higher taxonomic ranks is possible by phylogenetic analyses of whole proteomes but also of the RNA polymerase beta subunit, whereas phylogenetic reconstructions based on 16S rRNA genes alone resulted in unstable tree topologies with only insignificant bootstrap support. The identified clades could be correlated with distinct phenotypic traits illustrating an adaptation to common environmental factors in their evolutionary history. Genome wide gene-content analyses revealed the existence of two distinct ecological guilds within the analyzed lineage of marine gammaproteobacteria which can be distinguished by their trophic strategies. Based on our results a novel order within the class  For several years an increasing effort in the characterization of bacteria from marine environments led to the isolation of a large number of gammaproteobacteria that belong to phylogenetic groups originally defined by 16S rRNA gene sequences retrieved from environmental samples. Many of these novel isolates can be allocated to the oligotrophic marine gammaproteobacteria (OMG) group as defined by Cho and Giovannoni . The fivGammaproteobacteria as shown for example in recent trees  distributed by the all-species living tree project . This leads to a confusing classification scheme of environmental important marine gammaproteobacteria that is not in line with the presumable evolutionary relationships. Part of the confusion may have been caused by a close relationship of recently isolated strains affiliated to the OMG group with species regarded as non-oligotrophic and belonging to established genera like Cellvibrio and Microbulbifer, which have been originally affiliated to the families Pseudomonadaceae and Alteromonadaceae, respectively. Consequently, unclassified members of the OMG group are intermingled with species that have been affiliated with several different orders of Gammaproteobacteria leading to a classification scheme comprising various paraphyletic taxa.Currently, most of the novel gammaproteobacterial strains that were obtained by high-throughput cultivation approaches and have been affiliated to the OMG group were not described as novel species and thus remain unclassified  or the correlation of group-specific oligonucleotide probes used for in situ hybridization experiments with the phenotypic characteristics associated with well-defined microbial taxa. For this reason the aim of this study was to define a taxonomic framework for major clades of the OMG group and related described species that is based on the unveiled evolutionary relationships among these bacteria and thus less arbitrary than the current classification.However, the definition of meaningful higher taxonomic units comprising members of environmental important clades of the OMG group and related species could be of great value for the cultivation-independent analyses of microbial populations in marine environments  and LTPs 115 (http://www.arb-silva.de/projects/living-tree/) databases a range of relevant type strains with a recognizable relationship to major clades of the OMG group were determined. Then, additional outgroup sequences were selected that represent type genera of all major families and orders within the class Gammaproteobacteria. Whenever possible only 16S rRNA gene sequences of type strains of type species with validly published names as given in the List of Prokaryotic Names with Standing in Nomenclature  with the GTRGAMMA model for DNA and PROTCATLGF for the RpoB data, which was determined as optimal empirical model using RAxML. In each case the robustness of the tree topologies was evaluated by performing 1000 rounds of bootstrap replicates.Phylogenetic trees based on datasets of single sequences were reconstructed using programs implemented in the ARB software package  were obtained from the IMG website (https://img.jgi.doe.gov/cgi-bin/er/main.cgi) with the exception of the proteomes of the strains MOLA455 and Congregibacter litoralis KT71T which were obtained from the NCBI website (http://www.ncbi.nlm.nih.gov/genome). The accession numbers of all used genome sequences are given in Table The selection of species used for phylogenetic analyses using genome-scale data was identical to the data set used for the RpoB protein trees. Total protein sequences of 59 strains belonging to the class e-value threshold of 10−5 and an MCL inflation parameter of 2.0. The clusters of homologs were converted to a gene-content matrix in analogy to the ortholog-content matrix.The OrthoMCL clusters were also converted to an ortholog-content matrix representing the presence or absence of a gene within a certain genome and clusters of orthologs. Further, clusters of homologous sequences were determined using a re-implementation of the TribeMCL algorithm  with most applied reconstruction methods, so that a formal proposal of higher order taxonomic groups within this phylogenetic branch requires further investigation.Phylogenetic analyses were based on a comprehensive dataset of 200 nearly complete 16S rRNA gene sequences resulting in an alignment with a minimum sequence length of 1115 nucleotides and 1172 distinct alignment patterns in the maximum likelihood analysis. In reconstructed trees containing representatives of most recognized families within the class Cellvibrionales is considerably higher than in the 16S rRNA gene based tree. To exclude the possibility that the improved stability of the RpoB tree topology is related to the reduced number of selected sequences compared to the 16S rRNA gene based tree shown in Supplementary Figure Cellvibrionales lineage was not retained using the reduced 16S rRNA gene dataset (data not shown). Hence, the improved reliability of the RpoB based phylogeny is independent of the number of used sequences and caused by the higher information content of this large conserved protein compared to the 16S rRNA. However, despite the improved stability of the phylogenetic reconstruction several subgroups of the Cellvibrionales, like the clade represented by Cellvibrio species and closely related taxa was still not resolved with high confidence values in the RpoB based tree. We used, therefore, genome-scale data to further improve the reliability of the reconstructed phylogenetic relationships.In this study a dataset of 60 complete RpoB proteins derived from the available genome sequences of members of the OMG group and related gammaproteobacteria was used Table . The resCellvibrionales lineage including the five identified subgroups. Interestingly, the close relationship of the Cellvibrionales to members of the Pseudomonadaceae that became apparent in the 16S rRNA gene based tree was not evident in the RpoB or supermatrix trees. This finding supports our proposal to separate members of the main lineage of the phylogenetically defined OMG group and related taxa from the Pseudomonadales or any other established order within the Gammaproteobacteria.The basic supermatrix comprised 10,059 genes and 2,801,974 characters (i); a further matrix containing 928 genes and 223,650 characters was obtained after cleaning with MARE (ii). The core-genes matrix contained 341 genes and 111,505 characters (iii). For all three matrices the selected model for maximum likelihood reconstructions was PROTCATLGF; that is the LG model of amino acid evolution , which provides a strong argument to separate this group from the pseudomonads in sensu stricto which are characterized by ubiquinone 9 (Q9) as major respiratory lipoquinone .In contrast to the lipid patterns, the guanine plus cytosine content of genomic DNA  showed only little variation between the five different phylogenetic groups, with the highest average value found in the OM60/NOR5 clade (52–66 mol%) and the lowest median represented by the Cellvibrionales lineage that becomes apparent in the gene-content analyses shown in Figure sox genes for the utilization of reduced inorganic sulfur compounds and photosynthesis genes for the harvesting of light in the euphotic zone of the oceans. However, it seems that only light, but not thiosulfate, is used by members of this phylogenetic group as additional energy source and stimulates growth  are characterized by short periods of fast growth that alternate with long periods of non-growth usually resulting in a low abundance in the environment interrupted by occasional blooms. These bacteria prefer nutrient-rich niches  and have a metabolism of low efficiency but high plasticity. In contrast, typical K-strategists (most oligotrophs) are highly abundant and have long phases of continuous growth characterized by a constant low growth rate. Niches occupied by these bacteria are usually poor in nutrients leading to substrate limitation and utilization of alternative energy sources. Their metabolism is highly efficient and specialized, but has a low flexibility.The dichotomy in the e Figure  are typie Figure . Especiad Harris  the advaK-strategists. In addition, we tried to identify genomic features, which allow a reliable differentiation of both trophic guilds within the Cellvibrionales. In Figure K-strategists on lipids as carbon source due to their higher energy yield compared to carbohydrates  indicating a high translational power and growth rate, whereas bins assigned to genomes of the highly abundant predicted K-strategists Luminiphilus syltensis NOR5-1B, HTCC2080, and HTCC2207 had a significantly lower proportion of expressed ribosomal proteins  indicating a low growth rate. In addition, bins related to the K-strategists Luminiphilus syltensis NOR5-1B, HTCC2080, and HTCC2207 showed a relatively high expression of genes involved in mixotrophic growth reflecting their effort to continue proliferation under conditions of substrate limitation. The metatranscriptomic data indicate also a metabolism specialized on fatty acid degradation for the bins of NOR5-1B and HTCC2080. A notable discrepancy to our predictions may represent the reported high expression of genes related to motility and carbohydrate utilization allocated to the HTCC2207 bin, however it is unclear if this expression pattern reflects only specific conditions encountered in the studied site or was even selected by the used sampling method which excluded particles and surface sediments. In this context it should be also noted that in the real world a continuum of ecological strategies exists. Hence, it is unrealistic to expect a clear assignment of all environmental bacteria to one or the other extreme .Gammaproteobacteria. Cells are gram-stain-negative, straight to bent rods, cocci or pleomorphic, non-endospore-forming, motile by means of flagella or non-motile. Aerobic, mesophilic, neutrophilic, chemoorganotrophic, respiratory type of metabolism. Most strains are positive in the oxidase test. Ubiquinone 8 (Q8) is the major respiratory lipoquinone in most species. The G + C content of the genomic DNA of most strains is in the medium range between 44 and 66 mol%. The definition of the order relies mainly on the reconstruction of phylogenetic relationships based on comparative sequence analyses of complete rpoB genes or whole proteomes. At present, the order contains the families Cellvibrionaceae, Halieaceae, Microbulbiferaceae, Porticoccaceae, and Spongiibacteraceae. The type genus of the order is Cellvibrio .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.


A fossil femur excavated by Eugène Dubois between 1891–1900 in the Lower/Middle Pleistocene bonebed of the Trinil site  was recognised by us as that of a Hylobatidae. The specimen, Trinil 5703 of the Dubois Collection , has the same distinctive form of fossilization that is seen in many of the bonebed fossils from Trinil in the collection. Anatomical comparison of Trinil 5703 to a sample of carnivore and primate femora, supported by morphometric analyses, lead to the attribution of the fossil to gibbon. Trinil 5703 therefore provides the oldest insular record of this clade, one of the oldest known Hylobatidae fossils from Southeast Asia. Because living Hylobatidae only inhabit evergreen rain forests, the paleoenvironment within the river drainage in the greater Trinil area evidently included forests of this kind during the Lower/Middle Pleistocene as revealed here. Pithecanthropus erectus Dubois (Homo erectus since Mayr Semnopithecus - (Kleine soort) – l. femur” [Translated: Semnopithecus -  – left femur]. Following examination of the specimen it became clear that it was attributable to a Hylobatidae, rather than to Semnopithecus.The site of Trinil  is famous for being the locality that yielded the type specimen of Homo erectus occupation. After describing the fossil femur anatomically, we compare it with SEA mammals of similar size in order to establish the taxonomic attribution of Trinil 5703.Small apes are very scarce in the fossil record Hauptknochenschicht. Following de Vos and Sondaar Homo erectus, originating from this bonebed.Since its first description, two major amendments have been made to the Trinil fauna. First, Badoux De Vos et al. 40Ar/39Ar date of 0.8 myr that is obtained from the fossil-bearing beds at Ngebung 2, recently excavated by modern archaeological methods and referred to as Final Trinil H.K. fauna Stegodon trigonocephalus and absence of Elephas hysudrindicusAxis lydekkeri ngebungensis that differs on subspecies level with Axis lydekkeri and has therefore been used to define this association as a Final Trinil H.K. fauna These new biostratigraphic zones (biozones) have been dated radiometrically Trachypithecus auratus robustus and the Cercopithecinae Macaca fascicularis  discovered in the Jetis biozone at Sangiran Dome. Hooijer Hylobates in the Jetis and Trinil faunas  and very small ones (Tarsius and Nycticebus)  . The TriBunopithecus, Hylobates, Nomascus, Symphalangus), langurs  odd-nosed monkeys  and macaques (Macaca fascicularis and Macaca nemestrina).We further compared our measurements on Trinil 5703 with those taken from the femora of extant Southeast Asian primates housed at the Muséum national d'Histoire naturelle  and the Naturalis Biodiversity Center , again excluding the very small and very large genera. This sample comprises 70 adults, 10 sub-adults and 6 juveniles when available in the collections for a total of 86 individuals from nine genera and 18 species . For somEight linear measurements, taken with a digital calliper , are used for metrical comparisons. The length of the neck was measured from the line of fusion of the head to the lesser trochanter (referred to as the LNW distance) and from the line of fusion of the head to the medial aspect of the greater trochanter (UNW). The other six measurements were the antero-posterior width of the neck (ANW), the minimum proximo-distal height of the neck (NH), the antero-posterior and medio-lateral shaft widths just below the lesser trochanter , and the maximum antero-posterior and corresponding medio-lateral shaft widths . We also measured the total length of the femur in our comparative sample in order to estimate the total length (TL) of Trinil 5703 by means of linear regression equations.Our measurements of the specimens and the fossils  are presBecause the Javanese Pleistocene is characterized by numerous changes in mammal body size Hylobatidae are characterized by their very specialized locomotor behaviour, known as suspensory brachiation. Their anatomy reflects this specialization, especially in particularly slender and elongated limbs. Therefore, two characteristics may be regarded as important in the identification of femurs of small apes, which are the length of the femoral neck and the rectilinear shaft of the femur. Geometric morphometric methods have been successfully applied to long-bone curvature in primates by All the analyses were performed with R software Pardoxurus and Prionailurus; (b) the shaft and neck are narrower than those of Arctictis and Cuon; (c) the neck is much longer than in all the carnivores in Paradoxurus, Prionailurus and Arctictis.Morphological comparison of Trinil 5703 to femora of Southeast Asia carnivores and primates of similar size indicates clearly that the fossil is a primate . Trinil M. fascicularis.Trinil 5703 is generally similar to the primates shown in Trachypithecus or Presbytis .Equations have been calculated from linear regressions of the log-transformed TL over the log-transformed NH for each of the following clades: small apes, langurs and macaques . The equresbytis . The femis femur . TherefoOur morphometric analysis of Trinil 5703, summarized in Although the femoral head of Trinil 5703 is mostly lacking, the small portion preserved on the posterior aspect likely suggests a globular shape comparable to what is observe in small apes. The head typically extends on the posterior aspect of the neck in Cercopithecidae such as macaques, langurs and odd-nosed monkeys . This characteristic is confirmed in Trinil 5703 with the small portion of the head preserved which clearly overhangs the neck . The gloTrinil 5703 shows an elongated, thin and antero-posteriorly flattened femoral neck, similar to that found in all small apes . The proportions of the neck in the fossil are similar to young and sub-adult small apes. Although the neck-shaft angle cannot be measured, the angle formed is obtuse. The anteversion of the neck cannot be estimated here without the distal portion of the femur. There is no tuberculum on the posterior aspect of the neck . Such a tuberculum is always present in catharrines and occurs in most hominoid taxa, but is usually absent in small apes ligamentum teres during brachiating locomotion.The trochanteric fossa of Trinil 5703 femur is small and very rounded . Bacon Hylobates is oriented medially but of sub-adult Nomascus, posteriorly. Nevertheless, this age-related position of the lesser trochanter has to be confirmed by further studies with larger samples of young individuals, which is beyond the aim of this study. To summarize, the position of the lesser trochanter in Trinil 5703 is within the range of small apes, and may indicate a sub-adult developmental stage.The lesser trochanter is on the posterior face of the shaft of Trinil 5703. The position of the lesser trochanter appears to vary within primate populations Presbytis and Hylobates with a mean distance in baseline unit respectively of 0.010 and 0.014. The distance to the other primates is much greater, followed by Trachypithecus (0.037) and Nomascus (0.064), and finally the macaques M. fascicularis (0.073) and M. nemestrina (0.126). Moreover, there are features of Trinil 5703 that are seen in gibbons but not in other primates such as the shaft cross section morphology. While the cross section in female gibbons is circular, males have a spiral line, which resulting in an asymmetrical cross-section of the shaft. Trinil 5703 has a spiral line, although weakly developed.The shape of Trinil 5703 is typical for small apes. The femora in all small apes are straight in anterior view . Moreover, the small-ape femoral shafts are straight in lateral view, as Dubois linea aspera is salient at the contact with the greater trochanter. This salience is not seen in Trinil 5703, neither at the contact of the greater trochanter nor more distally along the shaft. The salience of the linea aspera in Trinil 5703 is intermediate between those of small apes, macaques and langurs, falling within the 95% confidence intervals of the three groups. The weak development of the linea aspera in the femoral diaphysis of small apes is confirmed by shaft thickness . In the LDA  with ferns and rain forest-refuges . Palynological results from Trinil, although scarce, also testify to an open environment with the presence of a seasonal forest The presence of a gibbon in Trinil H.K indicates that the closed forests were present near Trinil at the time of deposition. Small apes are mainly characterized by suspensory locomotion Evidence has been scarce in support of rain forest vegetation during Trinil H.K. The fossils that Eugène Dubois excavated from the Trinil site have drawn close scrutiny and often evoked controversy, because of their great importance to paleoanthropology. Some scholars have expressed concern about poor stratigraphic control during excavation, specifically that fossils from two geological formations might have been mixed Homo erectus and other mammals which have been known from the Trinil H.K. fauna for over a century.With our recognition of Trinil 5703 as the first Hylobatidae fossil from Trinil, we provide strong evidence that during the Lower/Middle Pleistocene the river basin upstream of the site contained a population of small-sized gibbons, who inhabited rain-forest, in addition to the Table S1Linear measurements used in this study.(XLS)Click here for additional data file.Table S2Shaft curvature 3D coordinates used for the baseline registration.(XLSX)Click here for additional data file.