Epilepsy is one of the most common chronic disorders affecting individuals of all ages. A greater understanding of pathogenesis in epilepsy will likely provide the basis fundamental for development of new antiepileptic therapies that aim to prevent the epileptogenesis process or modify the progression of epilepsy in addition to treatment of epilepsy symptomatically. Therefore, several investigations have embarked on advancing knowledge of the mechanism underlying epileptogenesis, understanding in mechanism of pharmacoresistance and discovering antiepileptogenic or disease-modifying therapy. Animal models play a crucial and significant role in providing additional insight into mechanism of epileptogenesis. With the help of these models, epileptogenesis process has been demonstrated to be involved in various molecular and biological pathways or processes. Hence, this article will discuss the known and postulated mechanisms of epileptogenesis and challenges in using the animal models. Epilepsy is a neurological disorder characterized by recurrent and unpredictable interruptions of normal brain function, called epileptic seizure . It is oAccording to the causal etiology, epilepsy can be divided into 3 categories: idiopathic, acquired (symp-tomatic) and cryptogenic (presumed symptomatic) -7. IdiopOn the other hand, the International League against Epilepsy has classified seizures into two major types namely generalized seizures which involve both hemispheres of brain and partial  seizures which begin locally in one hemisphere of the brain . MajoritPathogenesis of epilepsyDefinition of epileptogenesisCurrently, there is no universally accepted definition for epileptogenesis. The term epilepto-genesis is defined as a process that leads to the occurrence of the first spontaneous seizure and recurring epileptiform events after the brain insult  15). La. La15). Mechanism of epileptogenesisEpileptogenesis can involve various biological pathways or processes, structural and functional changes. In general, it is unclear which mechanisms are required or necessary for the genesis of epilepsy. However, several experimental studies have provided some insights into the actual and postulated mechanisms of epileptogenesis. Neurotransmission signaling pathwayGlutamate and γ-aminobutyric acid (GABA) are the two neurotransmitters that have been studied extensively in relation to epilepsy. Both glutam-atergic and GABAergic system play crucial roles in epileptic phenomena. It has been hypothesized that the neuronal hyperexcitability in epilepsy is due to imbalance between glutamate-mediated excitation and GABA-mediated inhibition . GlutamaA and GABAB receptors. GABAA receptors (ligand-gated ion channels) mediate rapid inhibitory presynaptic potentials by increasing influx of chloride, and GABAB receptors (G-protein-coupled receptors) mediate slow inhibitory presynaptic potentials by increasing the potassium conductance and decreasing the calcium entry  channels that may contribute to TLE  and abse neurons , 72. HCNtability , 72. Exptability , 73. HenNeurogenesis and rewiring pathway: Structural, neurochemical and cellular changesAberrant hippocampal neurogenesis, a process of new neurongeneration, and new circuit creation has been proposed as another important pathogenesis in epilepsy , 75. VarMultiple structural alterations in the hippocampus could occur after acute seizures, including degeneration of dentate hilar neurons and CA1 – CA3 pyramidal neurons, aberrant sprouting and synaptogenesis of mossy fibers and loss of inhibitory GABAergic interneurons . Mossy fApart from structural changes, acute seizures can also up regulate several neurotropic factors and other proteins in the hippocampus. These include nerve growth factor , 83, braCellular changes are also involved in the hippo-campus following acute seizures. These changes include increase of neurogenesis, abnormal migra-tion of newly born granule cells into dentate hilus and dentate molecular layer, and occurrence of hilar basal dendrites in newly added granule cells -76. All Immunological and inflammatory pathwayCytokines are polypeptide mediators that are associated with activation of immune system and inflammatory reactions . Recent However, there is concern on how the activated inflammatory pathway contributes to pathogenesis of epilepsy. It has been revealed that these inflammatory cytokines have deleterious effects on neurons via alteration of neuronal excitability, production of toxic mediators and increase impermeability of blood-brain barrier (BBB) ,94. IL-1In addition, Febene and colleagues  showed tApoptotic pathway Apoptosis is a programmed cell death process during normal growth and development in multicellular organisms for maintaining cell homeostasis . ExperimIn experimental models of epilepsy and epilepto-genesis, researchers have revealed that executioner caspase-3 and -6 are activated and actively expressed in the hippocampus , 110. BeGene and protein regulation jun, junB, junD) and the Fos family  are believed to be involved in pathogenesis of seizures  early repressor (ICER) also plays a role in epileptogenesis. ICER is a group of proteins produced from the cAMP-responsive element modulator (CREM) gene and ICER messenger RNAs that are transcribed by an internal promoter of the CREM . ICER seChanges of neuropeptides expression in hippo-campus following seizure have been observed in experimental studies. These neuropeptides include neuropeptide Y (NPY), somatostatin, cholecysto-kinin, neurokinin B, galanin, thyrotropin-releasing hormone and cortistatin -127. In Another neuropeptide, galanin also has been demonstrated to be involved in modulating seizure activity . GalaninAnimal models for epileptogenesisA variety of animal models have been developed to study epilepsy and epileptic seizures. Each animal model demonstrates different types of epilepsy  14, 44), 4414, 4Kindling is a model of chronic seizures which involves progressive intensification of brain excitability by repeated excitatory stimuli  that ultimately induce seizure disorder , 142. ElPost-SE model with spontaneous recurrent seizure also comprises electrically induced models  and chemically induced models (e.g. pilocarpine or kainate) , 147. ThChallenges in animal modelsUndoubtedly, animal models have provided useful information in addressing critical research issues, including mechanism of epileptogenesis, which are impossible to study in humans due to ethical concerns. However, what is the degree that these models reflect the actual condition in humans with epilepsy? The actual mechanisms underlying epilepsy may be more complicated. For example, in TLE studies, post-SE models involved an acute triggering SE process that was frequently followed by a latency period with subsequent development of spontaneous motor seizure , 149 andThe animal models have a different etiologic process compared to status epilepticus in human, which is usually associated with traumatic brain injury and ischemic stroke , 9. HencApplication of the animal models in understanding other epileptic related conditions such as cognitive and behavioral changes could be improved. Many patients with TLE suffer from cognitive and behavioral alterations, such as depression, anxiety, psychosis and memory loss, which may be associated with morphologic and functional alterations in the temporal lobe . These bThere is a substantial degree of variability among individual animals in response to epileptogenic or convulsive stimuli in a specific model. In rodents for example, strain, gender and age factors affected the response to epileptogenic stimuli , 156. ImCurrently, these animal models still remain valuable research tools in view of presence of an intact central nervous system, which provides an opportunity for studying the mechanism underlying epileptogenesis. There is no general agreement about which animal model may be most appropriate and relevant to human condition and none of the available models have been clinically validated . For a mA greater understanding of pathogenesis of epilepsy will likely provide the basis fundamental for development of new antiepileptic therapies that aim to prevent the epileptogenesis process, or modify the progression of epilepsy in addition to treatment of epilepsy symptomatically. Animal models play a crucial and significant role in providing additional insight into mechanism of epileptogenesis, predo-minantly kindling and post-SE with spontaneous recurrent seizure models ,140. Wit


Neurons throughout the brain show remarkable diversity in their firing patterns, ranging from the generation of single action potentials to repetitive firing characterized by distinct latencies, firing frequencies, and spike shapes in response to current injection -5. We pr


The physical and faunal characteristics of coastal wetlands are driven by dynamics of hydrological connectivity to adjacent habitats. Wetlands on estuary floodplains are particularly dynamic, driven by a complex interplay of tidal marine connections and seasonal freshwater flooding, often with unknown consequences for fish using these habitats. To understand the patterns and subsequent processes driving fish assemblage structure in such wetlands, we examined the nature and diversity of temporal utilisation patterns at a species or genus level over three annual cycles in a tropical Australian estuarine wetland system. Four general patterns of utilisation were apparent based on CPUE and size-structure dynamics: (i) classic nursery utlisation (use by recently settled recruits for their first year) (ii) interrupted peristence (iii) delayed recruitment (iv) facultative wetland residence. Despite the small self-recruiting ‘facultative wetland resident’ group, wetland occupancy seems largely driven by connectivity to the subtidal estuary channel. Variable connection regimes (i.e. frequency and timing of connections) within and between different wetland units  will therefore interact with the diversity of species recruitment schedules to generate variable wetland assemblages in time and space. In addition, the assemblage structure is heavily modified by freshwater flow, through simultaneously curtailing persistence of the ’interrupted persistence’ group, establishing connectivity for freshwater spawned members of both the ‘facultative wetland resident’ and ‘delayed recruitment group’, and apparently mediating use of intermediate nursery habitats for marine-spawned members of the ‘delayed recruitment’ group. The diversity of utilisation pattern and the complexity of associated drivers means assemblage compositions, and therefore ecosystem functioning, is likely to vary among years depending on variations in hydrological connectivity. Consequently, there is a need to incorporate this diversity into understandings of habitat function, conservation and management. Increasing knowledge of temporal utilisation patterns of functional groups, and of the underlying processes regulating their occurrence has led to great advances in our understanding of the functioning of estuarine fish assemblages Occurring adjacent to estuary channels worldwide are a variety of fringing wetlands with varying potential for fish utilisation. Vegetated intertidal wetlands  are prominent and iconic components of estuarine systems, and provide tidally available habitat for fauna inhabiting the estuary channel The dynamic regimes of hydrological connectivity characteristic of estuarine floodplain wetlands, featuring the interplay of tidal marine and freshwater connections, results in variable physical conditions, and simultaneously provides corridors for fish recruitment from both estuarine and freshwater systems Lates calcarifer. L. calcarifer spawn in coastal waters and mouths of estuaries during wet season months, coinciding with periods when connectivity and habitat availability of fragmented coastal wetlands is greatest L. calcarifer, wetland fish fauna are taxonomically diverse Estuarine pools have received some attention in Australia’s tropics To develop an understanding of the patterns and underlying processes driving the fish assembage of estuarine pools in the tropics, we examined the nature and diversity of temporal utilisation patterns  at a species level over three annual cycles that incorporated strong physical change. The study focused on a natural wetland system comprised of 20 discrete pools situated on a salt-marsh of the Ross River in North Queensland, Australia. Available data available from the main channels of estuaries in the region were used to provide a utilisation pattern ‘null model’, to investigate whether observed patterns were typical of estuary channel use, or if different sets of processes influenced floodplain wetland utilisation.This study was undertaken with the approval of James Cook University Animal Ethics Committee (permit A1466), in accordance with institutional, national, and international animal ethics guidelines, and under Queensland fisheries permit requirements (permit number: 114026). This study did not involve any endangered or protected species. No specific permits were required to access or conduct work on the field site, since the land is not protected nor privately owned.2Sporobolous virginicus salt-marsh system situated on the Ross River floodplain, 8 km’s upstream of the river mouth. Interspersed across the wetland are 20 discrete permanent pools, ranging in area from 80 m2–2500 m2, and in low-tide depth from 30 cm–130 cm. The pools are mostly unvegetated and generally lack permanent complex structure. They encompass a range of substrates, varying from fine mud to coarse rubble.The study was conducted in Annandale Wetland  , a 0.4 kWeather patterns in the study region can be divided into 4 periods Sampling of all 20 pools commenced after wet season floodwaters receded in March 2010, and was repeated on a monthly basis until the commencement of the following wet season in December. Monthly sampling in the first three months following draw-down was undertaken during 2011, to incorporate likely complexity associated with this transition period, followed by bi-monthly sampling until the end of the year. A third annual sample was collected for the first month after floods in April 2012.Oreochromis mossambicus, which was euthanized on site in accordance with fisheries requirements. Minimising mortalities through the rapid return of catch was integral to the study, as extraction may substantially influence catch in subsequent months. Salinity, water temperature, and visibility (Secchi depth) were recorded in each pool on each sampling occasion as potential explanatory variables of fish dynamics. Freshwater flow data over Aplin’s Weir were also provided by North Queensland Water.All samples were collected over the bottom quarter of the tidal cycle during the new moon phase to ensure consistent tidal regimes throughout the study. Sampling was conducted using a seine net , with an effective sampling width of 8 m. Some pools could be comprehensively sampled in a single seine haul. However, larger pools required up to 3 separate hauls to include the range of habitats present. Complete coverage of habitats was needed to fully represent each taxa in each pool, controlling for any within-pool microhabitat associations. Fish numbers and their sizes were recorded (measured in 10 mm fork length (FL) size classes, and reported as size-class minimums (e.g. 27 mm FL = 20 mm)). Fish <10 mm FL were excluded from analyses as a large proportion were below mesh selection size, and unlikely to be well represented. The catch was returned to the water alive, with the exception of the noxious pest species, Data collected across the complete lengths of 9 small North Queensland estuary channels as part of a previous study Lates calcarifer, Chanos chanos, Megalops cyprinoides, Elops hawaiensis), which commonly utilise off-channel habitats during early life-history stages The most commonly captured species’ were selected for analysis, along with some larger less-abundant species of commercial and recreational importance , for which the specified degrees of freedom were adjusted based on the size-class range. Where GAM curves were bimodal, two modal size classes were extracted for a single month. Size-class distributions were based on the sum of each 10 mm size-class increment across the 20 pools in each month (with the net of greatest abundance taken to represent each increment in the larger pools). For species sufficiently abundant in both the wetland and regional estuaries, CPUE vs. size-structure plots were qualitatively compared. Any large-scale disparities between the plots were considered as different utilisation patterns.Salinity in the wetland responded negatively to freshwater flow, ranging from 0–4 ppt directly after the wet season to >30 ppt pre-wet season . VisibilPseudomugil signifer and Hypseleotris compressa) which were unsuitable for analysis since they were below gear selection size for substantial proportions of their life-cycles. The remaining 8 taxa , together with the four commercial/recreational species, can be categorised into four groups based on CPUE vs. modal size-class plots  displayed a pattern of classic nursery utilisation (CNU), typified by cycles of recruitment at small size classes, followed by growth and then emigration. Taxa in the CNU group recruited as larvae and postlarvae  were caught exclusively as advanced juveniles  (N. erebi CPUE was relatively high from first sampling in March (C. chanos and N. erebi persisted for shorter periods than the other two species in this group.Three species (0 mm FL) , despite0 mm FL) ). These 0 mm FL) . In contin March , and desin March , observeH. castelnaui and Stolephorus spp.) recruited as larvae or post-larvae in the pre-wet season (the interrupted persistence (IP) group), illustrated by large peaks in CPUE dominated by size classes of 20–30 mm FL  displayed fluctuating CPUE’s through the year that matched with consistent size structures (facultative wetland resident (FWR) group). These trends reflected year-round occurrence of early post-settlement stages , in addsambicus . The sim90 mm FL ; Table 1channels , while OThere were diverse patterns of utilisation among the 12 taxa analysed, defined by taxa-to-taxa differences in the details of CPUE and size-structure dynamics. Despite differences in detail these taxa could be broadly categorised into four groups based on similar patterns of wetland usage. Most taxa demonstrated a surprising tolerance to the severe and abrupt shifts in salinity, although for many taxa, utilisation patterns were strongly modified by other effects of freshwater flow. In general, utilisation patterns reflected the relationship of life-history schedules, physical tolerances, and habitat requirements with variations in hydrological connectivity, physical conditions, and habitat availability mediated by the interplay of tidal and freshwater flow.L. equulus, Acanthopagrus spp., G. filamentosus, and E. hawaiensis) display CNU patterns, following cycles of post-larval recruitment, growth, and assumed emigration to other habitats upon reaching critical juvenile sizes E. hawaiensis, which has not been captured in abundance in previous studies sampling estuary channels across numerous systems in the region Four taxa  display a delayed recruitment to the wetland, arriving at advanced-size juvenile stages during wet or post-wet season months. Consequently, it is implicit that these species initially settle as post-larvae elsewhere. For N. erebi, settlement occurs in permanent freshwater reaches (e.g. above Aplin’s Weir), due to exclusive freshwater spawning N. erebi recruited as early post-settlement juveniles during the wet season sampling hiatus, recruitment to tidal wetlands is essentially decoupled from life-history schedule, and the exact size at recruitment is dependent on the relationship between timing of spawning and the timing of freshwater flows, which allow movement to the wetland. The other three species  spawn in coastal marine waters M. cyprinoides and C. chanos, L. calcarifer has a complex early-life history linking multiple coastal habitats. L. calcarifer and M. cyprinoides post-larvae recruit to shallow habitats associated with elevated wet season water levels, including supra-littoral depressions on saltpans and ephemeral freshwater and brackish swamps L. calcarifer into subtidal estuarine habitats synchronises with draw-down of these ephemeral habitats at the end of the wet season M. cyprinoides migrate upstream during post-wet season months Four species  were present in the wetland year-round both as young juveniles and adults. Continual presence of young juveniles suggests spawning may occur within the wetland or perhaps adjacent habitats. For A. vachelli these trends occur at the scale of the entire estuary  is indicative of re-colonisation from adjacent estuarine habitats, and subsequent spawning in the wetland. The shallow, sheltered nature and soft sediment common in the wetland appears to provide ideal habitat for the formation of breeding arenas  O. mossambicus in similar tropical estuaries suggest they are capable of spawning in seawater salinities, but distributions are limited to torpid waters in the upper estuary or enclosed water bodies In contrast to the nursery-orientated utilisation of the rest of the assemblage, two facultative wetland residents (FWR)  and physical integrity  of connections to the estuary channel are likely to be the sole regulators of wetland utilisation pattern. In Annandale Wetland estuary channel-to-pool connections were established through most tidal cycles, and utilisation of several taxa mirrored patterns in the estuary channel. However, in reality regimes of estuary connection across estuarine floodplains are highly variable from wetland to wetland, occurring on scales of days, weeks, months, and sometimes years N. erebi and O. mossambicus from permanent freshwater sources. Meanwhile, the extent of freshwater flooding will regulate use of ephemeral wetlands that certain members of the DR group initially recruit to. Effective use of these intermediate habitats is likely to modify the extent, timing, and size of recruitment of these larger and mostly predatory species  to estuarine pools.Beyond the simple effect of enhancing connection depths and durations, other effects of wet season freshwater flows appear to modify wetland utilisation patterns and assemblage structures. Flows move certain planktivorous species (IP group) out of the wetland system, and simultaneously donate many Despite the presence of Aplin’s Weir directly upstream of the study site, the wet season flow dynamics observed in study are similar to dynamics in unregulated river systems The pivotal role of freshwater flow in mediating key physical and biological processes of estuarine floodplain wetlands adds a profound layer of variability to wetland functioning since wet season rainfall in dry tropical and sub-tropical regions is inter-annually inconsistent, following a loose cycle of wet and dry climactic periods spanning multiple years, largely associated with ENSO cycle Due to their diversity of form and connectivity, several additional processes operating at finer spatio-temporal and conceptual scales are likely to further complicate assemblage structure and dynamics of estuarine floodplain wetlands. This includes taxonomic and ontogenetic differences in locomotory capabilities This study demonstrates the diversity of utilisation pattern and complexity of associated drivers inherent in a coastal nursery habitat characterised by dynamic physical conditions and a high taxonomic diversity. It is evident that the processes regulating the occurrences of fish are not mutual across the assemblage, but vary among taxa, with different species responding differently to the same hydrological connectivity event. Therefore any future change in hydrological regime in this system, driven by natural fluctuation, climate change or water regulation, will have differing impacts on different members of the assemblage. Consequently, the assemblage composition and ecological function of estuarine floodplain wetlands is prone to variation among years, and there is a need to incorporate the diversity of assemblage drivers into understandings of habitat function, conservation and management.Appendix S1Pseudomugil signifier and Hypseleotris compressa whose small body sizes limited interpretation of size-structure dynamics under the applied techniques.Summary of catch - raw abundance across all 20 pools summed over the complete sampling period featuring taxa collectively constituting 99.2% of the total catch. Taxa which alone constitute >1% of the catch are highlighted in bold, and were selected for analysis, *with the exception of (DOC)Click here for additional data file.


Quantitatively one of the principal products of the phenylpropanoid pathway, lignin confers high mechanical strength and hydrophobicity to plant walls, thus enabling erect growth and high-pressure water transport in the vessels. Lignin is characterized by a high natural heterogeneity in its composition and abundance in plant secondary cell walls, even in the different tissues of the same plant. A typical example is the stem of fibre crops, which shows a lignified core enveloped by a cellulosic, lignin-poor cortex. Despite the great value of fibre crops for humanity, however, still little is known on the mechanisms controlling their cell wall biogenesis, and particularly, what regulates their spatially-defined lignification pattern. Given the chemical complexity and the heterogeneous composition of fibre crops’ secondary walls, only the use of multidisciplinary approaches can convey an integrated picture and provide exhaustive information covering different levels of biological complexity. The present review highlights the importance of combining high throughput  Plant cell walls are indeed valuable natural products which find a wide array of industrial applications, spanning from conversion into energy to material development. The food-oriented agricultural industry is progressively being redirected towards one ensuring also raw material for pulp and paper, textiles, construction and biofuels [Medicago sativa), that has been selected for centuries for a decreasing lignin-content, to increase the digestibility of this queen of forages.In the list of highly sought-after goals for a sustainable exploitation of natural resources, the use of plant-based material to replace fossil carbon-derived products ranks undoubtedly among the top. The increase in the world population, together with rapid industrial development, push towards the depletion of petrochemicals and the subsequent need of finding sustainable sources of raw materials. Plant-sourced raw material, biofuels –3. This biofuels ,5, much biofuels ), which biofuels ,3 are emLinum usitatissimum L.), hemp (Cannabis sativa L.), ramie (Boehmeria nivea L.), jute (Corchorus capsularis L. and Corchorus olitorius L.), kenaf (Hibiscus cannabinus L.), sisal . Other plants that are of much less importance on a global scale, but are nonetheless cultivated for their fibres, include nettle (Urtica spp. [Abelmoschus esculentus [Musa textilis). The most important fibre crop, when looking at global annual production, is without any doubt cotton; however cotton fibres are enlarged trichomes and thus have an origin completely different from the fibres discussed in this review.Fibre crops comprise flax (ica spp. ), okra (culentus ), bananaThe fibrous mass of these plants is characterized by a low lignin content and by the occurrence of fibres highly enriched in cellulose. These features make them extremely appealing not only as models to study secondary cell wall formation, but also as sources of valuable raw materials used in different industrial sectors. Their fibres  show physical properties (namely tensile strength) which make them optimal for the woven textile industry, but at the same time their low lignin content is a desirable feature for biomass saccharification. Their woody core fibres (a.k.a hurd or shives) are excellent for paper production, as insulating material, as fibrous component in concrete and wall coatings and once crushed to powder they can be used to make biodegradable plastics. It is hence clear that progressing in an integrated study of the mechanisms which regulate the tissue-specific secondary cell wall composition of fibre crops can help devise strategies to maximize the exploitation of their lignocellulosic biomass.i.e., plants’ mechanical tissue [Arabidopsis thaliana which has delivered milestone data concerning, for instance, the cascade regulating the transcriptional wiring of secondary cell wall formation [A. thaliana offers a wide collection of cell wall mutants’ [Arabidopsis can be overcome with poplar, which has contributed to gain a deep understanding on the formation of woody fibres in trees [M. truncatula [Plant lignocellulosic fibres hold great value both for the environment and the economy, as they favor the shift towards a bio-based economy. Moreover, they shape important plant features, as the mechanical properties of wood and the quality of textiles and paper. True fibres are cells of the sclerenchyma, l tissue , charactl tissue ,11; howeormation –26. Moremutants’ –37 and bmutants’ ), it is mutants’ . The limin trees –45. The in trees , togethein trees , makes puncatula –51, whicFibre crops, principally hemp and flax for which the genomes are available ,53, are -omics data is still missing. In the last decade the approach to biological studies has changed and these are now conducted at the systems level [i.e., integrative systems biology). The integration of all these data flows into a general model which, applied to fibre crops, can provide a depth of analysis never so far attained.The use of an integrated biology approach can help to shed light on the molecular processes which intervene in the two tissues and determine the contrasting nature of secondary cell walls’ composition. Although some molecular studies (mainly concerning flax and hemp tissues) are available in literature , redox homeostasis (chlorogenic acid), reproduction (flavonoids as flower pigments) and many other aspects of plant biology [Plant secondary metabolism comprises an array of complex pathways which shape the biochemical plasticity of plants. Considered dispensable for plant survival, it fuels cells with important organic compounds which mediate relevant events, as for instance defense and structural support. Within plant secondary metabolism, the phenylpropanoid pathway undoubtedly occupies a central position, as it generates the common precursor,  biology ,57. The  biology –63. The  biology –71, or t biology –74.Lignin, a polyaromatic, hydrophobic, heterogeneous biopolymer, is the main product of plant secondary metabolism, and is in mass globally produced second only after cellulose. Its structural complexity confers an intrinsic “chaotic” nature and often theoretical analyses spanning from fractal geometry to deterministic chaos concepts are invoked to describe the multi-level intricacy of its organization . This suLignin constitutes an inspiring model for the creation of biomimetic materials with desirable physical properties and this is an issue of particular relevance for the construction sector, which is shifting towards an eco-friendly building concept, through the use of plant-sourced materials instead of petrochemicals. Lignin contributes indeed to the elastic modulus of timber while conferring strength, and is water-proof.The physiological role of lignin is that of biological “glue” for wall polysaccharides, and its presence impairs saccharification of plant biomass, as it impregnates cellulose, thus hindering its accessibility to hydrolytic enzymes. However, its function is to ensure vascular integrity, fortify the cell wall against pathogen attack and to confer increased hydrophobicity and mechanical rigidity.Plant secondary cell walls show a sort of “promiscuity” towards lignin structures: They can tolerate variation in the polymer macrostructure, therefore providing extreme flexibility in situations of environmental stress or in case of transgene-induced modifications. The enzymes which intervene in the lignin biosynthetic pathway, the efficiency in energy and carbon retention of which were recently calculated , constitArabidopsis cytochrome P450 catalyzing the hydroxylation of the phenolic ring of monolignols [Metabolic channeling in the phenylpropanoid pathway has been demonstrated for phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase (C4H) ,103 and olignols . The enzAlthough the availability of different plant mutants defective in genes involved in the lignin deposition route –107 has A great heterogeneity in wall lignification exists in nature: Among different species, in the different tissues within the same plant  and in rC. sativa), hemicelluloses (5%–16%) and pectin (1%–4%) [Bast fibres are cortical extraxylary sclerenchymatous structures which provide mechanical support to the conductive elements of the phloem and are typically present in bundles held together by pectins and lignin. Their cell walls are composed primarily of cellulose (which can make up 75%–80% of the dry mass in  (1%–4%) , while l (1%–4%) ,115. Fori.e., tension wood) in response to a mechanical stimulus and can generate high tensional stress. However bast fibres do not play the same contractile role as tension wood [G-layers are usually found in reaction wood . High throughput analyses can deliver a wealth of data, but generally only convey partial information [i.e., a certain phenotype) by combining high throughput -omics data [-omics data . The avnections . This mepe-level ,132. DoEpe-level .i.e., orthologs of AtMYB4, EgMYB1, ZmMYB31/42 [Only a handful of studies are available in literature on fibre crops’ transcriptomics (summarized in MYB31/42 ). The esMYB31/42 , has offMYB31/42 . ArabinoMYB31/42 .de novo transcriptome assembly to identify crucial cell wall-related genes, as cellulose synthases (CesAs) [Arabidopsis) responsive to mechanical stress exist, and that they are associated with the biosynthesis of cell wall metabolites [The availability of next generation sequencing (NGS) techniques is now only starting to be applied to fibre crops to perform for instance genome-wide SNPs discovery (which can facilitate (Quantitative Trait Loci) QTL identification ) and to  (CesAs) . Being a (CesAs) . NGS tec (CesAs) ,139,140,abolites . The senet al. reported the identification of 152 predicted cell wall proteins in flax stems using a sequential salt extraction [As for transcript and metabolite profiling, most work on the proteome abundance in fibre crops has been focused on flax. Recently Day traction . The majtraction . Of the traction ,143, thetraction . One of traction .versus the surrounding cortical tissue. From the 240 spots that were more intensely stained in the fibres, corresponding to enriched proteins, 51 were identified [While the previous studies isolated the different tissues or compared the proteome of the stems at different developmental stages, the most targeted study on fibre proteome was reported by Hotte and Deyholos . In thisentified . This stAlthough proteomics has proven its power to contribute to the elucidation of pathways activated during cell wall development, the enzymes responsible for the biosynthesis of secondary metabolites, mainly the phenylpropanoid pathway with monolignols as major product, are of low abundance and therefore generally not detected. Furthermore key steps in phenylpropanoid metabolism are catalyzed by cytochrome P450-like membrane-anchored proteins ,145, mak-omics analyses with accurate experimental design, bioinformatic tools for meta-analysis and co-visualization of the data and mathematically-supported models inference. The integrated knowledge thus obtained can be applied to test the models through functional genomics: Modifications of cell wall metabolic networks can be engineered and used to improve the extractability of fibres , the agronomic traits of existing cultivars, or to create application-specific fibres.The stem of fibre crops represents one of the best examples in nature of contrasting lignification pattern existing within the same plant. This heterogeneous secondary cell wall composition allows the use of fibre crops’ lignocellulosic biomass for different industrial sectors. Although recent studies have contributed to shed light on some aspects controlling the different lignification profile in these plants, an integrative study obtained by simultaneously interrogating the organisms at different levels of biological complexity is still missing. To fully understand the molecular events responsible for secondary cell wall biogenesis in fibre crops, it will be necessary to integrate high throughput


The importance of cardiac fibroblasts in the regulation of myocardial remodelling following myocardial infarction (MI) is becoming increasingly recognised. Studies over the last few decades have reinforced the concept that cardiac fibroblasts are much more than simple homeostatic regulators of extracellular matrix turnover, but are integrally involved in all aspects of the repair and remodelling of the heart that occurs following MI. The plasticity of fibroblasts is due in part to their ability to undergo differentiation into myofibroblasts. Myofibroblasts are specialised cells that possess a more contractile and synthetic phenotype than fibroblasts, enabling them to effectively repair and remodel the cardiac interstitium to manage the local devastation caused by MI. However, in addition to their key role in cardiac restoration and healing, persistence of myofibroblast activation can drive pathological fibrosis, resulting in arrhythmias, myocardial stiffness and progression to heart failure. The aim of this review is to give an appreciation of both the beneficial and detrimental roles of the myofibroblast in the remodelling heart, to describe some of the major regulatory mechanisms controlling myofibroblast differentiation including recent advances in the microRNA field, and to consider how this cell type could be exploited therapeutically. At the cellular level, heart tissue constitutes cardiomyocytes, cardiac fibroblasts, vascular and neuronal cells, as well as inflammatory cells under certain pathological conditions. In the healthy heart, cardiac fibroblasts are the most prevalent cell type, accounting for up to 70% of cells, depending on the species in question ,2. AlthoFibroblasts are a heterogeneous population of cells, reflecting both their multiple developmental origins and their exposure to differential physical and chemical microenvironments. Fibroblasts derived from different anatomical sites have been proposed to effectively represent distinct differentiated cell types as they exhibit unique transcriptional signatures that probably reflect phenotypic differences . Such diAlthough fibroblasts have the capacity to proliferate, migrate and regulate ECM turnover to maintain cardiac homeostasis, they are also able to undergo differentiation into a more contractile and synthetic myofibroblast phenotype to aid with cardiac repair following myocardial infarction (MI) -9. MyofiAppreciating the dual roles of cardiac myofibroblasts in the myocardial remodelling process is important, as they can be perceived to be both beneficial and detrimental depending on their prevalence and their temporal and spatial location. The infarct scar is not a simple acellular structure comprising structural ECM molecules; on the contrary, it contains myofibroblasts that maintain a viable, dynamic scar important for maintaining myocardial integrity against a background of continuous mechanical forces associated with the pumping of the heart . MyofibrThe differential origin of myofibroblasts in the remodelling heart has become a hot topic in recent years ,23. Althin vitro on rigid plastic surfaces; hence studies on cultured cardiac fibroblasts are generally indicative of myofibroblast behaviour [Phenotypic conversion of resident cardiac fibroblasts to myofibroblasts requires integration of both mechanical and biochemical stimuli. Fibroblasts are mechanosensitive and are therefore able to detect the loss of integrity of the ECM that occurs following MI. In response to increased mechanical stress and platelet-derived growth factor, fibroblasts adopt a partially differentiated phenotype known as the proto-myofibroblast . Conversehaviour ,27. TGF-ehaviour . Howeverehaviour , and thiin vitro. The latter study also employed an experimental MI model with TRPC6 knockout mice to show that myofibroblast differentiation was attenuated in vivo and this manifested in reduced infarct size, increased ventricular dilatation, reduced cardiac function and increased mortality due to ventricular wall rupture [A number of additional stimuli that promote differentiation to the myofibroblast phenotype have been reported, including specific cytokines, growth factors and ECM molecules; several of which elicit their effects through up regulation of TGF-β activity and/or signalling . There i rupture .TGF-β-induced myofibroblast differentiation can be opposed by proinflammatory cytokines  that may contribute to the temporal and spatial regulation of myofibroblast function in the transition from inflammatory to granulation and maturation phases of infarct healing . Basic fAlthough myofibroblasts play key roles in scar formation, in most tissues  they usually undergo apoptotic cell death once the scar has matured and the healing process is resolved . In the in vitro evidence obtained using porcine aortic valve myofibroblasts suggests that fully differentiated myofibroblasts may also have the capacity to revert back to quiescent fibroblasts when substrate rigidity is reduced [Recent  reduced . Further reduced . These sImportantly, while reducing apoptosis of myofibroblasts in the scar may deliver short-term beneficial effects, persistence of myofibroblasts in remote regions of the heart away from the scar area is detrimental. This is particularly relevant to nonischaemic cardiac remodelling such as left ventricular hypertrophy associated with pressure overload, in which myofibroblast persistence drives a profibrotic state leading to ventricular wall stiffening, neurohormonal activation, systolic and diastolic dysfunction and, eventually, heart failure ,47.Recent advances in a number of laboratories have revealed a role for epigenetics in influencing the differentiation process of myofibroblasts and resultant fibrogenesis (reviewed in ). These Differentiated myofibroblasts play central roles in fibrogenesis through their ability to synthesise increased quantities of ECM proteins. However, recent new data suggest that differential expression of miRs, and specifically elevated levels of miR-125b, can regulate the process of cardiac EndMT to a fibroblast-like phenotype in murine cells and promote profibrotic signalling . AnotherDifferentiation of fibroblasts in the stressed myocardium confers ability to upregulate ECM turnover towards augmented fibrosis. A number of miRs have emerged as important regulators in this mechanism, and miR-21 and miR-29 have proven to be of particular interest. Whilst miR-21 expression is prominent in cardiac fibroblasts and substantially weaker in myocytes, increased miR-21 expression observed in failing murine hearts has been shown to be of fibroblast origin . In thatIn the heart, miR-29 is mainly expressed in fibroblasts and has been shown to be downregulated in viable myocardium after experimental MI . In the An observed correlation between miR-24 expression and fibrosis in hypertrophic hearts was pursued in a recent study in which miR-24 was shown to be downregulated after MI and related to ECM remodelling . Myocardin vivo by miR-30 requires verification in functional studies.In the healthy heart, it appears that expression of miR-133a and miR-30 are able to control ECM turnover by maintaining low secreted levels of the profibrotic cytokines TGF-β and connective tissue growth factor (CTGF/CCN2); a number of reports have demonstrated that miR-133a and miR-30 are downregulated in rodent and human heart failure ,60. miR-The challenge of post-MI therapeutic regulation at the level of the cardiac myofibroblast is to encourage optimum myofibroblast recruitment and retention in the scar (reparative fibrosis), whilst reducing myofibroblast activity in remote non-infarcted areas of the myocardium (reactive fibrosis). Several commonly prescribed drugs for MI patients that deliver beneficial effects on adverse cardiac remodelling appear to do so in part by directly influencing cardiac fibroblast and myofibroblast behaviour. Such drug classes include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β-blockers, statins and thiazolidinediones . What about strategies designed specifically to target fibrogenesis? In light of our current knowledge of the mechanisms involved in post-MI repair and remodelling, some important considerations include: precisely controlling temporal administration of antifibrotic therapies to optimise scar formation, but ameliorate subsequent reactive fibrosis; targeting individual molecules that play differential roles in reparative and reactive fibrosis; and selective targeting of myofibroblast populations derived from different sources that play diverse roles in post-MI remodelling. Some such strategies are discussed briefly hereafter.TGF-β is one of the most important inducers of the myofibroblast phenotype, as well as being an important profibrotic signalling molecule in its own right . InhibitThe ability of TGF-β to induce fibroblast differentiation and fibrogenesis can also be regulated by matricellular proteins; a group of ECM-associated regulatory proteins that are expressed only under pathological conditions in the heart . Ongoingin vitro[The Wnt/Frizzled signalling pathway has been shown to be an important modulator of the migration and differentiation of cardiac fibroblasts in vitro. Moreovein vitro. The potin vitro.FN-ED-A is an important contributor to the innate inflammatory response, as well as being a major driver of myofibroblast differentiation ,78. FN-EMyocardin-related transcription factor A (MRTF-A) appears to be a key inducer of gene programmes that mediate both cardiomyocyte hypertrophy  and fibrAs discussed earlier, myofibroblasts in the remodelling heart are derived not only from resident cardiac fibroblasts, but also from endothelial cells (via EndMT), epithelial cells, mesenchymal stem cells, bone marrow-derived fibrocytes, smooth muscle cells and pericytes ,23. Ther+/CD45+  myofibroblasts with resultant loss of interstitial fibrosis [+/CD45+ fibrocytes into myofibroblasts in an ischaemic cardiomyopathy model [For example, monocyte chemotactic protein 1 (MCP-1/CCL2) is important for fibrocyte recruitment . Cardiacfibrosis . Rho kinhy model . Hearts hy model . One shohy model , and thuhy model .Nevertheless, as our knowledge on the origins of myofibroblasts in the heart increases, this will hopefully reveal novel therapeutic targets in addition to those described above. For example, it would be interesting to determine the effects of modulating miR-125b, as this has been shown to be important for regulating EndMT in the heart . StrategThe development and/or progression of many human pathologies is now widely accepted to be attributed to dysregulation of miRs, and understanding their functional relevance will advance exploitation of these molecules as therapeutic targets. Moreover, the tightly regulated cell type specificity of miR expression makes these molecules amenable to modulating function of individual cell types. Whilst current pharmacological therapies used in the treatment of adverse cardiac remodelling and failure are known to retard its progression, mortality rates remain high and there is a clear need for new therapies . Whilst Molecular tools for manipulating miR levels (through inhibition or mimicry) have been an area of rapid development and ongoing refinement . As discProgressive expansion of our knowledge concerning dysregulation of miRs in cardiac (myo)fibroblast phenotype and function will undoubtedly lead to strategies that optimise targeted delivery of miR therapeutics. The ability to deliver therapies directly to selected cell types is indeed a realistic option for future medicine.Cardiac myofibroblasts represent a unique, yet developmentally diverse, population of cells that play key roles in post-MI infarct healing, but also in adverse cardiac remodelling, fibrosis and progression to heart failure. Improved understanding of not only the origins of myofibroblasts in the post-MI heart, but also the capacity to assign specific roles and regulatory mechanisms to them, creates optimism for the future that this multifunctional cell type can be manipulated therapeutically to optimise infarct scar formation, whilst ameliorating reactive fibrosis.CTGF: Connective tissue growth factor; ECM: Extracellular matrix; EndMT: Endothelial–mesenchymal transition; FN-ED-A: Fibronectin extra domain A splice variant; IL: Interleukin; MCP-1: Monocyte chemotactic protein 1; MI: Myocardial infarction; miR: microRNA; MRTF-A: Myocardin-related transcription factor-A; TNF: Tumour necrosis factor; TGF-β: Transforming growth factor betaThe authors declare that they have no competing interests.Both NAT and KEP contributed to the writing of the manuscript and approved its final submission.


PIWI proteins, a subclade of the Argonaute family proteins, are expressed predominantly in the germline and bind to PIWI-interacting RNAs (piRNAs), which are 25–31 nucleotides in length. The PIWI/piRNA pathway plays critical roles in germline development by regulating transposons and other targets to maintain genome integrity. While the functions of PIWI in the germline have been extensively investigated, recent studies have accumulated evidence that the human PIWI proteins, HIWI and HILI, are aberrantly expressed in a variety of cancers. This review summarizes our knowledge of PIWI expression in cancer and discusses its possible role in tumorigenesis. Arabidopsis thaliana, and PIWI, named after the Drosophila protein PIWI , and AGO3  have been receiving increased attention. CTAs are a category of tumor antigens whose normal expression is restricted to male germ cells in the testis  has been shown to have multiple variant forms. PIWIL2-like (PL2L) proteins have been identified in a wide variety of cancers  and directs HP1a localization to heterochromatin formation  fraction and also associates with mRNA cap-binding complexes (Grivna et al., Drosophila deficient for PIWI proteins accumulate γ-H2Av foci, a sign of double-stranded DNA breaks, suggesting a positive contribution of PIWI proteins to the repair of DNA damage (Kuramochi-Miyagawa et al., Thirdly, PIWI proteins may be involved in genomic instability, one of the most common occurrences in cancer. Genomic instability is attributable to an extra copy of genomic DNA or a chromosome, chromosomal translocation, chromosomal deletion, or single-stranded or double-stranded breaks in genomic DNA (Cassidy and Venkitaraman, aub Drosophila mutant. Vasa is a conserved germline DEAD-box RNA helicase that plays diverse roles in the regulation of mRNA translation, germline differentiation, germ granule assembly, and piRNA-mediated transposon silencing (Liang et al., Fourthly, PIWI proteins may promote cell proliferation in cancer and cause aneuploidy during mitosis. Pek and Kai  have recA growing number of reports have revealed the aberrant expression of PIWI proteins in various cancers, and it appears highly plausible that PIWI proteins are involved in tumorigenesis. However, data for elucidating the detailed molecular role of PIWI proteins in tumorigenesis is very limited, in part because a considerable portion of the experimental results is derived from studies of patient samples, with limited opportunities for experimental manipulation. Further basic studies with more manipulable materials, such as cell lines and experimental animals, are urgently needed to address the possibility of PIWI as a therapeutic target. An immediate focus is to identify PIWI protein-associating RNAs to determine whether piRNAs and/or other RNA species specifically interact with PIWI proteins in cancer cells. It is also imperative to investigate how PIWI proteins are involved in the biological functioning of cancer cells, such as transposon silencing, transcriptional or post-transcriptional regulation, DNA repair, and chromosome condensation and segregation, and to determine the roles of PIWI proteins in tumorigenesis. Such investigations will significantly advance our understanding of tumorigenesis and may lead to novel therapeutic applications targeting PIWI proteins and their molecular functions.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.


Pachycephalosaurs were bipedal herbivorous dinosaurs with bony domes on their heads, suggestive of head-butting as seen in bighorn sheep and musk oxen. Previous biomechanical studies indicate potential for pachycephalosaur head-butting, but bone histology appears to contradict the behavior in young and old individuals. Comparing pachycephalosaurs with fighting artiodactyls tests for common correlates of head-butting in their cranial structure and mechanics.Stegoceras validum and Prenocephale prenes. Finite element analyses (FEA), incorporating bone and keratin tissue types, determined cranial stress and strain from simulated head impacts. Recursive partition analysis quantified strengths of correlation between functional morphology and actual or hypothesized behavior. Strong head-strike correlates include a dome-like cephalic morphology, neurovascular canals exiting onto the cranium surface, large neck muscle attachments, and dense cortical bone above a sparse cancellous layer in line with the force of impact. The head-butting duiker Cephalophus leucogaster is the closest morphological analog to Stegoceras, with a smaller yet similarly rounded dome. Crania of the duiker, pachycephalosaurs, and bighorn sheep Ovis canadensis share stratification of thick cortical and cancellous layers. Stegoceras, Cephalophus, and musk ox crania experience lower stress and higher safety factors for a given impact force than giraffe, pronghorn, or the non-combative llama.Computed tomographic (CT) scans and physical sectioning revealed internal cranial structure of ten artiodactyls and pachycephalosaurs Anatomy, biomechanics, and statistical correlation suggest that some pachycephalosaurs were as competent at head-to-head impacts as extant analogs displaying such combat. Large-scale comparisons and recursive partitioning can greatly refine inference of behavioral capability for fossil animals. Ovis canadensis) vigorously impact each other on the apices of their heads between the horn cores Many animals strike with their heads at conspecifics, in ritualized flank-butting, head-to-head shoving matches and head-butting combat. Correlates cited for head-butting in modern ungulates include cranial sinuses Dome function in pachycephalosaurs has been controversial, with trabeculae within the dome interpreted as developmental traces inconsistent with head-butting Cephalophus leucogaster, musk ox Ovibos moschatus and giraffe Giraffa camelopardalisStegoceras validum. As controls we examine specimens of other artiodactyls that engage in a spectrum of combative behaviors. Bighorn sheep [Ovis canadensis) butt heads, and regress in horn-horn contact as these ornaments take a greater display function in older rams Antilocapra americana collide with cranial ornaments but do not butt heads Cervus canadensis, peccary Tayassu tajacu and llama (Lama glama). Expanding on previous studies of pachycephalosaur crania, we perform 3D finite element analysis (FEA) of simulated head impacts with models based on CT scans.We use CT (computed tomographic) scanning and finite element analysis (FEA) to compare structural capabilities of crania in head-striking artiodactyls , and yield stress is that of permanent deformation (beginning the inelastic portion of the stress-strain curve). Ultimate stress is usually higher than yield stress, and results in full breakage or crushing of a structure or its constituents. A brittle material, with little porosity to dissipate energy of cracking, often has similar strength and yield stresses. Bone has both a brittle mineralized component and ductile, flexible collagen, whereas keratin is highly ductile. Safety factor refers to ultimate or yield stress (or strain) divided by the experienced value.When FEA reveals safety factors under given loads, we can predict and compare forces necessary to break the structures. A cranial element with a higher safety factor can experience higher magnitudes of impact force before it breaks, which would potentially damage critical soft tissues.  The strain threshold of fatal soft tissue injury dictates the maximum force of a strike, which can be higher if extensive stiff and compliant hard tissues diminish impact stress and absorb strain energy. The current comparison quantifies relative capability for head-strikes of hard tissues of the head, a major step for circumscribing behavioral hypotheses. In the absence of trace evidence, inference of actual behavior of fossil animals requires novel phylogenetic and statistical methods.A potentially useful method for inferring behavior is recursive partition analysis (RPA), an algorithm used to guide diagnoses of illness based on correlations between an affliction and its symptoms. Analogously to a patient with an unknown illness and presented symptoms, an extinct animal can be diagnosed for an unknown behavior based on morphological traits. Correlation strengths for animals with known behavior can test for likelihood of the behavior in an extinct taxon. Hieronymous and colleagues Stegoceras morphology would suit it for combat relative to extant head-butting taxa. For an extant animal with strong linkages between structure and behavior, the strengths of these correlations will decrease when an incorrect behavior is assigned in RPA. We can test for a hypothetical behavior in an extinct animal by varying its behavioral assignment. If the alternate hypothesis changes correlation strength by the same amount as for the mis-assigned extant animal, the extinct animal can be inferred as an equivalently probable candidate for the behavior.We also introduce an extension of RPA called correlate disruption, which examines how strongly adult Stegoceras, Cephalophus, and juvenile and adult Ovibos. Superficially the dome of Stegoceras is highly and uniformly dense  adjacent to the braincase, and cancellous and compact layers superficially  are especially similar between the specimens. We predict that more mature specimens of Ovis canadensis will possess a thicker compact zone 3, as seen in this Stegoceras and other pachycephalosaur specimens.Similarly to nadensis  shows denadensis . The deeOvibos crania display bone density patterns grossly similar to those in Cephalophus and Stegoceras , but this region is not particularly deep. The frontals of Antilocapra are thin between the flanking horn cores, but much denser than the equivalent region of Cephalophus. This inter-horn bone in Antilocapra is the densest seen in any of the artiodactyl crania, aside from bone of some auditory bullae,The other artiodactyls vary substantially in distribution of cranial sinuses, trabecular bone, and compact bone  and 11. rn sheep . Giraffern sheep , as in tegoceras  and Prene prenes . The mede prenes  has simipecimens  lack extStegoceras validum (UA 2).Stegoceras dome diminish rapidly deep to the area of loading .Ovibos than in Cephalophus, but both stress and strain diminish more rapidly deep to the force application. Stress and strain are higher in struts traversing the parietal sinus than in struts less in line with the impacts. Compact bone of the skull roof experiences low stress. Expected from its lower elastic modulus, cancellous bone over the brain and especially in the occipital condyles experiences higher strain than compact bone. The dorsal surface of the endocranial cavity experiences lower stresses and strains than in any of the other artiodactyls .Giraffa experience high stress relative to the impacting structures in Ovibos. Stress is also greater in struts within frontal sinuses of Giraffa than in similar struts of Ovibos. Stress and strain are low in compact bone over the brain cavity, but substantially higher in impacts to the median ossicone than when all three are loaded .Antilocapra differed greatly from those of the other specimens .Lama model experienced high bending stresses and strains, with primarily compressive stress on the skull roof and tensile stress on the dorsal surface of the endocranial cavity. The latter stresses are particularly high compared with those in the other examined taxa, despite artificial thickening of the model necessary for successful FE meshing. Peak von Mises stress reached 60 MPa for a distributed impact  by the same amount; the morphology of Ovibos is a slightly better fit. In contrast, Giraffa disrupts G2 values less than the other taxa, indicating that giraffe morphology is better classified outside that of the animals that strike their heads together directly.Correlate disruption suggests that Stegoceras. Both Ovibos and Giraffa have dome-like structures that dissipate force, yet in Ovibos cancellous bone lies over the brain as compliant protection, and in the giraffe the point of impact lies anterior to the brain cavity and above struts of the frontal sinus. The Antilocapra model experiences tension in the thin skull roof. The domes of Stegoceras and Prenocephale resemble Ovibos heads in general shape and occurrence of cancellous bone, and domes of Cephalophus leucogaster, Ovis canadensis and giraffe ossicones in stratification of compact and trabecular regions.Extant combative ungulates vary in adaptations for head-butting, and pachycephalosaurs possessed a combination of their respective traits. In extant ungulates, recursive partition analysis shows strong correlations between head-butting and cranial morphologies that also occur in Ovis canadensis and especially Cephalophus leucogaster  cancellous strain, and dramatic reduction of stress distal to the impact on the dome surface. The cortical portion of the dome experiences peak stresses of 8–46 MPa, below their ultimate levels  covering the crania of head-butting artiodactyls and hornbills. The struts in Stegoceras are interpretable simultaneously as vascular conduits feeding development of a keratin covering The scanned adult Tayassu, and present in head-butting suids that Barghusen Our findings, and those of Farke Cephalophus, CapraOvisStegoceras) would be possible to model using 2D analyses The forces applied thus far to FE models of pachycephalosaurs, Ovis canadensis) was sectioned mid-sagittally with a bone saw. Crania of Stegoceras validum, Ovibos moschatus, Giraffa camelopardalis, Cervus canadensis (elk), Lama glama, Antilocapra americana (pronghorn), and Tayassu tajacu (peccary) were scanned on a General Electric Lightspeed CT scanner , at settings for diagnoses of bone pathology. The Stegoceras validum specimen was also scanned on a high-resolution x-ray CT at the University of Texas at Austin. Ovibos specimens represent a juvenile and an adult, as determined by examining tooth eruption patterns. A cranium scan of the duiker Cephalophus leucogaster was provided by Andrew Farke, also scanned on a General Electric Lightspeed medical scanner http://www.digimorph.org/specimens/Giraffa_camelopardalis/skull/, accessed August 17, 2009). For anatomical comparison with Stegoceras, we examined scans of a large specimen of the Mongolian pachycephalosaur Prenocephale prenes , then on loan to Philip Currie (University of Alberta).Specimen numbers, lengths, and forces for included taxa are listed in Prenocephale specimen, which was rotated slightly out of anatomical neutral pose on the scanner bed, and for anteroventrally sloping sections of two artiodactyls. The latter angled sections are of an un-impacted control region through the braincase of Antilocapra, and lateral ossicones of the female Giraffa specimen.Except for the male giraffe (TMM M6815), all CT data were in DICOM format. We used OsiriX® for 2- and 3D visualization of structure and density, to evaluate internal density distribution and guide reconstruction of finite element geometry and material properties. Densities were assessed primarily with the full-color NIH lookup table, which visualizes density gradations more clearly than do grayscale palettes. OsiriX® can section CT volumes in transverse, coronal, and sagittal planes. In other planes, we used the scissor tool to remove unwanted rendered bone, and produce sections approximately two mm thick. This was necessary for the Stegoceras, Cephalophus, adult Ovibos, Giraffa, Lama, and Antilocapra. Mimics®  facilitated construction of most 3D models , for compatibility with Strand7® (Strand7 Pty Ltd) finite element analysis software. We used Avizo® (Visage Imaging) to construct the finite element model of Cephalophus. The surface mesh was refined initially with Avizo®'s RemeshSurface function, and remaining errors corrected with the software's surface editor. As with the Mimics®-based artiodactyl models, Avizo® yielded a high resolution tetrahedral model with larger struts and nasal conchae intact.We produced cranial finite element models of D models  from denStegoceras, Antilocapra, Giraffa, and Lama specimens, Strand7® and Mimics® produced error-free tetrahedral solid meshes, suitable for FEA, from a triangular surface mesh that included surfaces around internal cavities  to better approximate original surface contours. This grouping retains anatomical details such as struts of bone, but results in a blocky external appearance in parts of the model. Even with varying element shape, a strictly hexahedral mesh is less “smooth" and accurate than a combination of hexahedral and tetrahedral elements, as Jasinoski et al. From these surface models and masks, we constructed two types of FE models. For the cavities , 15, 16.Stegoceras cranium was created based on the Austin CT scan, which had twice the transverse resolution and 4.5 times the anteroposterior resolution of the original medical scan. A tetrahedral mesh of 2.2 million elements was created in Avizo®. This model became the primary one for analyses of the Stegoceras cranium.An additional FE model of the 3Stegoceras densities could not be fully automated . In the absence of data on forces, it is reasonable to apply a unit magnitude to all FE models in a comparison, and directly scale stress and strain magnitudes when realistic forces are determined. However, we scaled forces to puts them in a biological context, and to open our assumptions to criticism. The baseline force for  m Capra . The forInferring behavior in fossil animals is possible by phylogenetic comparisons 2) of correlation between a category (such as behavior) and a potentially influencing factor . We included comparative taxa whose morphological traits we could observe directly. These included specimens of the cape buffalo Synceras caffer which clash through the flat portion of their horns, the helmeted hornbill Buceras vigil which collide in flight with keratin-covered osseous domes Equus caballus which do not engage in head strikes.In RPA We chose not to include phylogeny as an influencing factor in recursive partitioning. Analogous morphology and behavior can arise in distantly related groups, yet behavior can differ between closely related clades even at the species level. Our application of RPA strictly assessed correlation between morphology and known or hypothesized behavior. Because behavior in an extinct taxon is usually unknown, phylogeny might unduly bias the strength of the taxon's behavioral assignment. An example would be bias towards head butting in a thin-skulled extinct bovid, if “Bovidae" is a trait that otherwise correlates well with ramming behavior. However, RPA results can be informative in later studies that optimize co-evolution of behavior and morphology onto known phylogenies.2) from the sum of “correct" G2 values (CD = ΣG2 correct-ΣG2 incorrect), which conversely indicates the fit of an animal to assigned behavior.To assess the strength of morphology-behavior correlation for individual taxa, we used correlate disruption as described in the


Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are currently the preferred methods of malaria vector control. In many cases, these methods are used together in the same households, especially to suppress transmission in holoendemic and hyperendemic scenarios. Though widespread, there has been limited evidence suggesting that such co-application confers greater protective benefits than either ITNs or IRS when used alone. Since both methods are insecticide-based and intradomicilliary, this article hypothesises that outcomes of their combination would depend on effects of the candidate active ingredients on mosquitoes that enter or those that attempt to enter houses. It is suggested here that enhanced household level protection can be achieved if the ITNs and IRS have divergent yet complementary properties, e.g. highly deterrent IRS compounds coupled with highly toxic ITNs. To ensure that the problem of insecticide resistance is avoided, the ITNs and IRS products should preferably be of different insecticide classes, e.g. pyrethroid-based nets combined with organophosphate or carbamate based IRS. The overall community benefits would however depend also on other factors such as proportion of people covered by the interventions and the behaviour of vector species. This article concludes by emphasizing the need for basic and operational research, including mathematical modelling to evaluate IRS/ITN combinations in comparison to IRS alone or ITNs alone. Few vector control methods can be considered as effective against malaria mosquitoes as insecticide-treated nets (ITNs) and house spraying with residual insecticides (IRS). In recent years, endemic countries using the two methods singly or in combination have reported significant declines in malaria related morbidity and mortality -4. A revToday, universal coverage with long lasting insecticide-treated nets (LLINs) or IRS is actively promoted as the main prevention strategy under the WHO endorsed malaria control and elimination plan ,10. WherIn this paper, recent trends of using ITNs and IRS are explored with special emphasis on: 1) significance of the two methods in current malaria control agenda, 2) potential benefits of combining the methods and 3) important research issues that should be considered to support decision making regarding combination of these two methods.Other than intermittent preventive treatment (IPT), artemisinin-based combination therapy (ACT) and improved case detection by rapid malaria diagnostic tests (RDTs), recent declines of malaria are mostly attributable to expanded use of ITNs and IRS -4,17,18.Global Malaria Action Plan, recently launched by the WHO-Roll Back Malaria Partnership [The current tnership , targetstnership ,11,19. ROn one hand, this new roadmap may be considered a realistic proposition given the proven effectiveness -6,20,21 The WHO has provided guidelines for individual countries to use when prioritizing IRS, ITNs or both ,39. For Combining ITNs and IRS for malaria control has increasingly become a common practice in Africa. At national level in sub-Saharan Africa, nearly all malaria endemic countries have adopted ITNs, IRS or both. Based on the latest world malaria report  more thaTypically, ITNs and IRS are not usually used in a mutually exclusive way. IRS is not always restricted to only households where ITNs are not already being used, and the application of IRS itself does not always preclude use of ITNs. Instead, the two methods are commonly used together in the same communities or households. For example, a common application of IRS is in the mitigation of malaria epidemics ,13, wherBased on local evidence on malaria endemicity and other factors, such as financial costs and availability of storage and distribution systems, endemic countries often prioritize which regions should preferentially receive the different interventions. For example in Zambia, use of ITNs is targeted primarily in rural areas, while IRS is targeted primarily in urban and peri-urban areas , where sDespite the widespread implementation of ITNs and IRS and the likelihood of interactions between their properties, little is known about their impacts when they are used together. WHO has suggested that the two methods should be co-implemented to reduce transmission especially in hyperendemic and holoendemic scenarios ,38. Howeet al evaluated the national malaria control programme between 2000 and 2004, there was no added advantage of using IRS and ITNs as opposed to using either method alone [Anopheles arabiensis was endophillic (indoor resting), and was, therefore, redundantly affected by ITNs and IRS since these interventions are both used indoors. Elsewhere, in a retrospective evaluation of control operations between 1993 and 1999 in the Solomon Islands [Anopheles punctulatus and the exophilic (outdoor resting), early evening feeding Anopheles farauti [In Eritrea, where Nyarango od alone . The aut Islands , where p farauti , it was  farauti . Though et al who evaluated the generally successful malaria control programme in the highlands of Burundi, where PermaNet 2.0™ nets, (deltamethrin treated LLINs), were deployed alongside very high coverage (90%) of deltamethrin and alpha-cypermethrin based IRS [There are also reports showing that even though combination of insecticidal nets with IRS lowered overall vector densities inside houses, there was no overall reduction in malaria transmission relative to situations where only one of the methods was used. Examples include reports by Protopopoff ased IRS ,47. In tased IRS -48.et al completed a review of studies involving both IRS and ITNs [More recently, Kleinschmidt and ITNs . Of the and ITNs , and fouand ITNs .et al [o = 11.1 down from an control baseline of 39.5), than 80% coverage with just the ITNs alone without the DDT (Ro = 0.1). Their explanations were that: 1) IRS compounds such as DDT, which have significant repellent properties reduce the likelihood that mosquitoes contact ITNs within the sprayed houses, and 2) ITNs prevent mosquitoes from blood feeding and, therefore, reduce the rate at which blood fed mosquitoes rest on the walls [Mathematical modelling is also increasingly being adopted as a way of estimating potential benefits of combined ITN-IRS interventions, thereby partly filling the evidence gap while awaiting controlled field trials, but also enabling informed decision making by policy makers in areas where such co-applications are already being implemented ,41,49. Iet al  recentlyhe walls . This thhe walls ,7.et al [Anopheles gambiae. It should be noted that whereas DDT is proven to have significant repellency against mosquitoes [et al found that humans using only ITNs are generally better protected than those with only IRS, and that even though the ITNs or IRS with DDT provided similarly high personal protection, neither of them alone could interrupt transmission on its own [Chitnis et al  also usesquitoes -52, bendsquitoes . Chitnis its own . Besides its own .Other than actual efficacy of individual insecticides, there are several other factors associated with the overall performance of these intradomicilliary interventions and their combinations. For example, a comprehensive model-based evaluation of interventions showed that in low endemicity areas, where people experience approximately three infectious mosquito bites per year  or less, LLINs alone can drive malaria transmission to levels below the 1% parasite prevalence threshold necessary to start pursuing elimination . Howeveret al in Burundi, where LLINs were provided to continue protecting people even after the residual activity of the IRS insecticides had ceased to be effective [et al reported that huts sprayed with both Malathion and DDT remained toxic to mosquitoes much longer and that these huts were less irritant against both Anopheles funestus and An. gambiae than huts sprayed with just DDT [Where ITN and IRS insecticides have overlapping modes of action, insecticide combinations may remain protective over longer times than in situations where only a single insecticide is used. Such an observation is exemplified in the work reported by Protopopoff ffective ,47. Thisjust DDT . There ajust DDT . Though just DDT ,55. Evenjust DDT , sustainjust DDT , which cBased on reports analysed above, it seems that at least in some cases, there are advantages of combining ITNs with IRS relative to using either method alone, but that this outcome may be different in certain situations, since there are numerous confounding factors that can affect the results. It is therefore certain that evidence to support or refute this strategy of combinations remains inconclusive and any generalizations for optimal strategies cannot be made.In practice, the decision to use IRS, ITNs or both methods should be based on existing epidemiological conditions, operational requirements and expected protective efficacy of the interventions. The protective efficacy is itself a function of several other factors including behaviour of the local mosquito populations and presence or absence of insecticide resistance among these vectors. Both IRS and ITNs are insecticide-based and they both target mosquitoes that enter or those that attempt to enter human dwellings Figure . The WHOAn. gambiae and An. funestus, in special experimental huts designed to mimic local human houses [Each insecticide elicits a distinct spectrum of behavioural and physiological outcomes on mosquitoes, implying that ITNs and IRS, if based on different insecticides could differentially affect vectors even if they are simultaneously used in the same house. In this section, data from previous studies on house spraying and insecticide treated nets are considered to enable a generalised description of these interventions on the basis of how each one of them can affect mosquitoes that enter or those that attempt to enter human occupied houses  whether the two methods complement or diminish beneficial effects of each other, 2) which insecticides are the most appropriate to use in co-applications, 3) what are the epidemiological and operational determinants necessary for optimal outcomes of such co-implementation, 4) whether co-application can be used to manage challenges like insecticide resistance and finally 5) how cost-effective would the strategy be.Clearly these questions will require different kinds of studies. Therefore, research on combined ITN-IRS use should include: 1) experimental hut investigations where efficacies of the combinations are directly assessed against wild free-flying malaria vectors in malaria endemic areas, 2) mathematical simulations incorporating characteristics of candidate insecticidal applications to estimate likely benefits of the combinations in different scenarios, 3) long-term community-wide studies to determine effectiveness of the combinations and 4) cost benefit analyses of the combinations compared to individual methods on their own and also to other existing interventions. The proposed linkages between these studies are illustrated in Figure As malaria control enters the phase of intensive and sustained vector control, health authorities must ensure that important gains so far achieved from existing interventions are not lost. Similarly, traditional control operations must shift dynamics to reflect the current goals of malaria elimination and eradication , and decThe LLIN-IRS combination strategy is mostly recommended for accelerating control in high transmission areas ,38,41,44a) that any insecticide can possess an array of properties which together determine its overall protective efficacy at household level, b) that these properties function at different stages along the path of a mosquito approaching the human inside the house  that maximizing the protective benefits at each of these stages of action is an essential process in any attempt to optimize benefits obtainable from combined ITN-IRS interventions  ts Figure . It shoumutation ,36. As sFinally, to achieve community level effects, this paper recognizes the importance of coverage, i.e. proportion of all residents who consistently use these interventions, as a crucial factor. While toxic insecticidal interventions can kill large numbers of disease vectors -97 thus It remains largely unclear whether using both ITNs and IRS would confer significant additional benefits relative to using either method alone. Even though there have been no specific studies that expressly tested this hypothesis, previous IRS and ITN trials and a number of mathematical models have resulted in mixed results showing improved benefits in some situations and redundancy in others. Nevertheless, there are still a number of reasons that theoretically justify combination of IRS and ITNs in households. For household level protection, it is strongly recommended that where residents use pyrethroid treated LLIN, the IRS product to be sprayed in houses to supplement the nets must be of completely different mode of action. The overall epidemiological outcome of such co-applications at community level would however depend on factors such as level of intervention coverage achieved, baseline epidemiological conditions, behaviour of malaria vectors, nature of insecticides used for IRS and the type of nets being used. Therefore, to maximize any possible additional benefits from IRS/ITN co-applications, rigorous field evidence, supported by mathematical modelling where necessary, should be pursued to support the entire process of decision making, including the selection of which insecticides to be used for IRS and what type of LLINs to use.The authors declare that they have no competing interests.FO conducted the review and drafted the manuscript. Both FO and SM wrote the final version of the manuscript. Both the authors have read and approved the final manuscript.Effects of insecticides commonly used for IRS in Africa. A table showing effects of insecticides commonly used for indoor residual spraying (IRS) in Africa, on mosquitoes that enter or those that attempt to enter human occupied huts. The effects are classified as deterrence, feeding inhibition, toxicity, and excess exit.Click here for fileProperties of insecticides commonly used in ordinary home-treated ITNs. A table showing properties of conventionally treated nets (ordinary home-treated ITNs) commonly used in Africa, on mosquitoes that enter or those that attempt to enter human huts. The effects are classified as deterrence, feeding inhibition, toxicity, and excess exit. The nets are grouped as per the active ingredients (insecticides) used to treat them.Click here for fileProperties of different long lasting insecticidal nets (LLINs) commonly used in Africa. A table showing properties of different long lasting insecticidal nets (LLINs) commonly used in Africa, on mosquitoes that enter or those that attempt to enter human occupied huts. The effects are classified as deterrence, feeding inhibition, toxicity, and excess exit.Click here for file