Infarcts of the corpus callosum are rare and have not been well documented previously. As for a variety of signs and symptoms presented, alien hand syndrome (AHS) can be easily overlooked.In this report, we present a patient with a mixed types of AHS coexistence secondary to the corpus callosum infarction, including a motor type of AHS by intermanual conflict  and a sensory type of AHS by alien hand and left hemianesthesia (posterior AHS).Our case may contribute to the early recognition of AHS and to explore the abnormal neural mechanism of AHS. To our knowledge, rare reports have ever documented such mixed AHS coexisting secondary to the callosal lesion, based on advanced neuroimaging methods as in our case. Alien hand syndrome (AHS) is a rare clinical syndrome, characterized by involuntary, uncontrollable and purposeless movements of one upper limb, and the patient denies ownership of the limb when touching it without visual guidance . Such moA 71-year-old right-handed woman was admitted to our hospital because of urgent onset of paraphasia and slight weakness of her left hand for 13 days. The patient had a 20-year history of hypertension, coronary heart disease and insulin-dependent diabetes mellitus, with no history of drinking or smoking, and negative personal or family history of neurological diseases. Educational history of the patient was five years.She complained of involuntary and purposeless movements of the left hand and abnormal feelings in the left upper limb. She claimed that the left hand did not belong to herself but to her grandson . She also presented that her left hand was controlled by someone else instead of herself, and the two hands always "fought" with each other during bimanual coordination, as the left hand performed movements opposite to the right hand . For instance, when she used her right hand to unbutton her clothes, the left hand interfered by buttoning the clothes up. Also, she had visual hallucination at admission, such as her grandson standing beside her bed. Although frustrated with her alien hand, she was not afraid of her abnormal hand.On her bedside mental examination, she had no symptoms of amnesia, disorientation, agraphia, tactile naming in left hand, alexia in left visual field, hemispatial neglect and hemiballismus. The scale of the Mini-mental state examination was 24 when she was admitted to our hospital. She comprehended language well, but could not find appropriate words to answer questions, frequently using wrong words. Left hemiasomatognosia and apraxia were also noted. For instance, she did not perceive the existence of her left hand, with the abnormal perception that it was her grandson's hand (hemiasomatognosia). She had apraxia when miming how to use tools with her left hand, such as dressing, brushing teeth, combing her hair (ideomotor apraxia). She also had some difficulties in purposefully picking up objects (motor apraxia). A neurological examination showed mild left hemiparesis with slight clumsiness, and left hemianesthesia. Babinski sign was positive on the left. Activities of daily living (ADLs) were severely impaired, such as bathing, dressing and transferring, but the self feeding, bladder and bowel control were not significantly affected. The impairment for ADLs may be attributed to the left hemiparesis, apraxia, and intermanual conflict, with their disabling impact on everyday life.Carotid ultrasound demonstrated atherosclerosis plaques bilaterally in carotid artery. A brain 3-Tesla MRI was performed 3 days after admission, including conventional MR sequences: sagittal T1, axial T2-weighted, axial fast fluid attenuated inversion recovery (FLAIR), along with diffusion weighted imaging (DWI). Conventional sequences of MRI indicated regions of low signal intensity in the genu, body and splenium of the corpus callosum on the T1 weighted image Figure  and highTwo weeks later after treatment (platelet aggregation inhibitor and rehabilitation), the patient's aphasia, apraxia and hemiasomatognosia disappeared and ADLs were significantly improved. At discharge, this patient could perform routine activities independently and the behaviors of AHS were completely resolved.We described a recent case of an acute infarct of the corpus callosum presented with mixed types of AHS. AHS was originally described by Brion and Jedynac in 1972, characterized by the denial of ownership of one hand when it was held by the other hand behind the back, due to the lesions of the corpus callosum . Two typBased on previous classifications, Kato et al reported three types of AHS . The firAlthough different subtypes of AHS have been distinguished, these classifications obviously can not involve the wide clinical variety of abnormal behaviors of the upper limb. Up to now, the classifications of AHS are still inconsistent and have been remain disputable. It is regrettable because different pathophysiological mechanisms probably underlie the diverse behaviors in alien hand. As for lack of uniformity in assessment methods , Scepkowski indicated it may be difficult to establish clear subtypes of alien-hand phenomena .Our patient in this report presented with mixed types of AHS, including a motor type of AHS by the presence of intermanual conflict  and sensory type of AHS by sensory impairment such as alien hand and left hemianesthesia (posterior AHS). The neuroanatomic substrate of AHS and its explanatory models have remained controversial due to the noncircumscribed nature of cerebral injuries present in most cases . SeveralNeuroimaging studies in our case identified the responsible lesions for the interhemispheric disconnection syndrome in the corpus callosum. This report is explicit in manifestations and the neuroimaging results. The cause of this infarct in our case was possibly due to the atherosclerotic fibrous plaques of the carotid artery, with long-standing hypertension, coronary heart disease and diabetes mellitus of 20 years. As a result, the collective evidences of DWI, MRA should have the advantages of differentiating infarction from other pathologies. Meanwhile, the combination of modern advanced imaging techniques makes the accurate diagnosis of corpus callosal infarction possible, biopsy not necessary any more . In addiIn conclusion, infarcts of the corpus callosum are not common, which is most likely due to its rich blood supply from three main arterial systems, specifically the anterior cerebral, anterior communicating, and posterior cerebral arteries . The isoWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JLY examined, evaluated the patient and drafted the manuscript. SKW and XJG performed and interpreted the MRI studies. WLH participated in the design of the case-report and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/142/prepub


Food processing unit operations can have effects on food allergens and their capacity to provoke reactions. The vast majority of food allergens are proteins, although most individual proteins in foods do not possess allergenic activity. Thus, the physical removal of proteins e.g. oil refining can eliminate the allergenic activity as is well documented for highly refined peanut oil. Furthermore, the chemical and/or enzymatic hydrolytic destruction of proteins can dramatically decrease allergen risk as evidenced by the use of extensively hydrolyzed casein, a major milk allergen, in hypoallergenic infant formula intended for milk-allergic infants. However, since exposure to trace amounts of allergenic proteins can provoke reactions in some allergic individuals, the efficacy of these unit operations in eliminating allergen risk is of considerable concern. The assessment of the residual allergenicity of processed foods can be difficult. The methods employed in such assessments must yield results that can predict allergenicity upon ingestion of the processed food. Clearly, blinded oral challenges on allergic individuals are the ultimate approach to document the safety of a particular processed food product. However, this approach is arduous, expensive and potentially risky. Because the allergenic activity of a protein depends upon its ability to bind to specific IgE antibodies from the blood sera of allergic individuals, the effect of processing on the IgE-binding capacity of an extract of the processed food may offer some indication of its allergenic potential. However, IgE binding is often assessed by immunoassay, an approach that depends upon the solubility of the processing-modified protein. Processing can diminish the solubility of proteins and thereby decrease apparent IgE binding in immunoassays. But the possibility exists that insoluble allergens may remain allergenic after ingestion. A full understanding of the effects of processing on allergenicity, the specific allergens, and detection methods does not exist for any allergenic food.


It was further shown that pretreatment with thiol compounds, like L-Cysteine or D-Penicillamine, reduced EtOH and ACD-induced motivational effects, in fact preventing self-administration of both EtOH and ACD, thus suggesting a possible role for ACD as a biomarker useful in evaluating potential innovative treatments of alcohol abuse. These findings suggest a key role of ACD in the EtOH reinforcing effects. In the present paper we review the role of EtOH-derived ACD in the reinforcing effects of EtOH and the possibility that ACD may serve as a therapeutically targetable biomarker in the search for novel treatments in alcohol abuse and alcoholism.Acetaldehyde (ACD), the first metabolite of ethanol (EtOH), has been implicated in several actions of alcohol, including its reinforcing effects. Previously considered an aversive compound, ACD was useful in alcoholic’s pharmacological treatment aimed at discouraging alcohol drinking. However, it has recently been shown that EtOH-derived ACD is necessary for EtOH-induced place preference and self-administration, thereby suggesting a possible involvement of ACD in EtOH motivational properties. In addition, EtOH-stimulating properties on DA neurons are prevented by pharmacological blockade of local catalase H A recurring emergent theory in the alcohol field is that the reinforcing properties of alcohol are not produced by the ethanol (EtOH) molecule itself, but may depend upon the action of EtOH metabolites/products within the central nervous system (CNS)  within the CNS could mediate most, if not all, of the CNS effects of EtOH , ADH inhibitor, following ALDH inhibition by both disulfiram and cyanamide  and catalase H2O2 system. In the periphery, ACD is formed from EtOH through the action of ADH primarily in the liver. In the brain, ADH is inactive , cytochrome  Sippel, .A prerequisite for the involvement of ACD in EtOH behavioral effects is the occurrence of pharmacologically significant levels of ACD in the brain after alcohol consumption.in situ thereby producing effects that are difficult to ascribe to peripheral mechanisms.The levels of ACD in the CNS have profound effects in mediating the reinforcing actions of EtOH. ACD derived from the peripheral metabolism of EtOH penetrates from blood to brain with difficulty because of the metabolic barrier presented by ALDH across the Blood-Brain Barrier (BBB) , rats reduce their intake and preference for EtOH  also showed a dose-dependent reduction of ACD , which reduced catalase H2O2 system levels by 70 to 80%  paradigm, Rodd-Henricks et al.  establis2O2 system inhibitor, 3-AT, failed to produce its characteristic excitation of the VTA. Further, in exploring the mechanism of ACD excitation of VTA neurons, Melis et al. (A (Melis et al., h of VTA neurons (Brodie and Appel, 2O2 system in the VTA, and the authors of these studies suggest that, in general, EtOH actions on the VTA are mediated by ACD (Deehan et al., ACD has excitatory actions on neurons of the VTA as clearly demonstrated by the effects on DA release and on the firing frequency of individual VTA neurons. In experiments using in vivo recording methods, ACD was injected intravenously, and a dose-dependent increase in firing of dopaminergic VTA neurons was reported (Foddai et al., s et al.  examineds et al. . The autOverall, it seems most likely that ACD is a crucial component of the overall effects of EtOH on dopaminergic neurons of the VTA; the essential action of ACD could be parallel to EtOH, or it could enhance EtOH-induced changes. Blockade of the formation of ACD can reduce the response of dopaminergic VTA neurons to EtOH, and could serve as a platform for the development of agents that reduce the rewarding and reinforcing actions of EtOH.The results reviewed above suggest that enzymatic manipulations of EtOH metabolism would diminish its rewarding properties, possibly discouraging drinking Figure . There c2O2 system by enzymatic inhibition (Melis et al., 2O2 scavenging may reduce ACD formation in the CNS and the motivational properties of EtOH (Ledesma et al., 2O2, as a co-substrate to form compound I, the central production of ACD derived from the metabolism of EtOH in the brain (Cohen et al., 2O2, reduces the acquisition and reconditioning of ethanol-induced CPP in mice (Ledesma and Aragon, In addition, modulation of catalase HThese considerations suggest further experiments to probe the use of these molecules as potential experimental therapies and could help in devising new effective pharmacologic treatments in alcoholism.It is hypothesized that many neuropharmacological, neurochemical, neurotoxic, and behavioral effects of EtOH are mediated by the first metabolite of EtOH, ACD Hunt, . In addiThe important distinction between the central and the peripheral effects of ACD lays the groundwork for considering that many of the central effects of EtOH could in fact be dependent on the actions of its first metabolite, ACD. Although peripherally accumulated ACD is a potential toxic and deterrent substance, high levels of this substance can reach the brain and generate positive effects that can promote later consumption.At last, targeting ACD, instead of EtOH, may offer new potential biomarkers in the search for novel compounds to reduce excessive alcohol intake, abuse and ultimately alcoholism. In general, targeting drug metabolism may reveal new ways to treat addictive disorders not limited to alcohol abuse but possibly useful in other addictions such as tobacco (Pianezza et al., 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.


The superfamily Pterioidea is a morphologically and ecologically diverse lineage of epifaunal marine bivalves distributed throughout the tropical and subtropical continental shelf regions. This group includes commercially important pearl culture species and model organisms used for medical studies of biomineralization. Recent morphological treatment of selected pterioideans and molecular phylogenetic analyses of higher-level relationships in Bivalvia have challenged the traditional view that pterioidean families are monophyletic. This issue is examined here in light of molecular data sets composed of DNA sequences for nuclear and mitochondrial loci, and a published character data set of anatomical and shell morphological characters.The present study is the first comprehensive species-level analysis of the Pterioidea to produce a well-resolved, robust phylogenetic hypothesis for nearly all extant taxa. The data were analyzed for potential biases due to taxon and character sampling, and idiosyncracies of different molecular evolutionary processes. The congruence and contribution of different partitions were quantified, and the sensitivity of clade stability to alignment parameters was explored.Pinctada provided further insights into the on-going controversy regarding the taxonomic identity of major pearl culture species.Four primary conclusions were reached: (1) the results strongly supported the monophyly of the Pterioidea; (2) none of the previously defined families (except for the monotypic Pulvinitidae) were monophyletic; (3) the arrangement of the genera was novel and unanticipated, however strongly supported and robust to changes in alignment parameters; and (4) optimizing key morphological characters onto topologies derived from the analysis of molecular data revealed many instances of homoplasy and uncovered synapomorphies for major nodes. Additionally, a complete species-level sampling of the genus  Since the lower Middle Ordovician some 470 million years ago, pterioidean bivalves have inhabited a remarkable diversity of marine epifaunal and semi-infaunal environments around the globe, typically confined to cryptic habitats and forming byssal attachments to various substrata. For a much shorter part of their history-from the dawn of human culture-they have become the primary source of pearls and nacre -3. PearlPteria and Electroma, evolved specialized associations with hydroid, scleractinian, and alcyonarian substrata. Most species of Pinctada (pearl oysters) inhabit sandy and hard bottoms, some living in association with commensal fishes and crustaceans. Several species of Pteria and Pinctada are cultured for commercial pearl production. Most species of a malleid genus Malleus are irregularly shaped to conform to narrow crevices in hard coral and rocky substrata, and develop elongated extensions of the hinge for stabilization in soft sediment. Species of the second malleid genus, Vulsella, are adapted for living within sponges. Isognomonids are characterized by the distinct morphology of the ligament that contains multiple grooves (resilifers) for ligamental attachments arranged sequentially along the hinge line (multivincular ligament). Species of the genus Isognomon often co-occur with species of Malleus in crevices and on soft muddy bottoms, but also are gregarious on mangrove roots, whereas species of Crenatula, the second isognomonid genus, live inside sponges . Similarly to isognomonids, pulvinitids possess multivincular ligament, but are distinguished from isognomonids by the presence of the foramen, an opening in the right valve through which the byssus is protruded. Living pulvinitids are represented by the genus Pulvinites, the sole extant species of which, Pulvinites exempla, lives byssally attached to vertical hard substrata at 200-400 m depths.The present-day diversity of the superfamily Pterioidea Gray, 1847  encompasPteria¬-like Paleozoic ancestor . Th. ThPterie Figure  18]. In. InPteria Figure  [18]. Inc Figure  19]. Wh. WhPteris Figure  [19].Pteria and Pinctada, whereas Isognomon was invariably monophyletic. The monophyly of the family Isognomonidae and the genera Electroma and Malleus were not tested, and representatives of the family Pulvinitidae and the genus Crenatula were not included in the analyses. The only study that focused exclusively on the Pterioidea was based on morphological characters and included representatives of all four families and nine genera  were recovered monophyletic. Species-level sampling in that study was insufficient to test the assumption of monophyly of the remaining genera and did not include type species for most nominal supraspecific taxa. Despite these recent challenges on both molecular and morphological grounds, no consensus on pterioidean relationships was reached and no revision of the existing classification was proposed.In summary, molecular studies, varying greatly in sampling and analytical techniques, produced inconsistent hypotheses of pterioidean relationships with low resolution and support. These findings, however, agreed on the monophyly of the superfamily and suggested that the families Pteriidae and Malleidae were not monophyletic. The results were conflicting with regard to the monophyly of the genera a Figure  15]. It. ItPteriThe identity of the pterioidean sister group remains uncertain, favoring the oysters (Ostreoidea), pen shells (Pinnoidea), or a clade composed of these two groups, currently classified together with the Pterioidea in the order Pterioida. Most analyses based on morphological data for extinct, extant, or both kinds of taxa, have suggested a sister relationship of the Pterioidea and Pinnoidea -24, excePinctada.The present analysis is the first study to produce a nearly comprehensive species-level phylogeny for the Pterioidea based on DNA sequence data. The principal specific goals of this study include (1) testing the monophyly of the Pterioidea, and all its families and genera; (2) reviewing the consequences of these results for the homology of important morphological characters; and (3) resolving species-level phylogenetic relationships within the commercially important pearl oyster genus http://www.ncbi.nlm.nih.gov/Genbank/index.html. Collection localities and museum catalog numbers for the specimens used in this study are listed in Table Most specimens were obtained from museum collections worldwide, with additional material gathered during fieldwork in the western Atlantic , the eastern Pacific , the Mediterranean , and the Indo-Pacific  regions. Living animals were collected in mangroves and shallow intertidal waters, fixed in 85-95% ethanol, and subsequently transferred to 75-95% ethanol. Collected specimens were deposited at the American Museum of Natural History  and the Field Museum of Natural History . DNA sequence data were obtained for 77 of the 100 terminal taxa. The remaining sequences were acquired from GenBank Hyotissa, and the ostreid Ostrea and Crassostrea exemplars were guided by their placement in a recently proposed phylogenetic hypothesis , the sole living member of the subgenus. Two exemplars of Vulsella, corresponding to the type species V. vulsella, one of possibly only two living species, were included in the analysis. The genus Pulvinites was represented by one exemplar of the only extant pulvinitid species, P. exempla.The sample of the genus diolaris ). The geGenome-wide sampling of multiple independently evolving genes is instrumental in overcoming the incongruence among trees derived from individual gene analyses, and producing a resolved and strongly supported phylogeny. Because this study involves the analysis of taxa of disparate levels of divergence, a data set was assembled that consisted of nuclear loci  and a mitochondrial ribosomal RNA locus, that in combination span a broad spectrum of variation. The ribosomal DNA sequences display a substantial difference in evolutionary rates among and within the genes , and are easily amplifiable due to the presence of multiple copies per genome and the pattern of concerted evolution . The fol®(Qiagen)-preserved tissues. Total genomic DNA was isolated from adductor muscle, foot, or the entire body using DNeasy® Tissue Kit (Qiagen). DNA amplifications were carried out using GeneAmp® PCR System 9700 (Applied Biosystems) using standard procedures. The PCR primers are listed in Table ® PCR Product Purification Kit  on a Biomek® 2000 Laboratory Automation Workstation (Beckman Coulter), and sequenced in both directions using the ABI Prism® BigDye™ Terminator Cycle Sequencing Reaction Kit (Applied Biosystems) on ABI Prism® 3700/3730xl DNA Analyzers (Applied Biosystems) at AMNH.Original DNA sequence data have been obtained for most sampled terminals; remaining sequences were obtained from the GenBank database Table . DNA washttp://www.ncbi.nlm.nih.gov. The new sequences have been deposited in GenBank under accession numbers HQ329247-HQ329509 . The sequence coverage varied from 2× to 6× depending on the quality of the material. The resultant sequences were checked for potential contamination by BLAST searches ,35 as im09 Table .i.e. multiple alignments).The ribosomal DNA sequences, the principal source of character data in the present study, vary considerably in length, creating uncertainty in the inference of nucleotide-level homology. To circumvent alignment ambiguity, all potential nucleotide homologies were evaluated in the framework of dynamic homology ,37, as iDNA sequences were partitioned into fragments to reduce computation time and to constrain the assignment of insertions and deletions (indels) to unambiguously homologous regions. None of the regions were excluded from the analyses except for the flanking primer sequences. Initially, multiple alignments were constructed using ClustalX 2.0.5  under de2 test as implemented in PAUP* 4.0d105 )Phylogenetic analyses of DNA sequence data were performed using direct optimization , a heuriParsimony analyses under direct optimization, as implemented in POY 4.1 , were caThe ML analyses were performed using RAxML 7.0.4  for 250 The ensemble consistency index  and theThe resulting trees were visualized using POY 4.1  and FigTa priori  for the reduced molecular data set.The significance of the length difference between alternative topologies was tested by the Kishino-Hasegawa test using resampling of estimated log-likelihoods (RELL) bootstrap  withsensu ).Substitution saturation in DNA sequences can potentially underestimate the actual amount of evolutionary change and reduce the phylogenetic signal due to excessive multiple hits in the same nucleotide positions resulting in extensive homoplasy . DespitHeterogeneity in substitution rates among sites within a nucleotide sequence can have considerable impact on sequence divergence ,119-121.Pinctada and Pteria), are rhythmical protandrous hermaphrodites, with occasional instances of protogyny and simultaneous hermaphroditism, and mtDNA appears to be maternally inherited , th, th15], Due to high sensitivity to alignment parameters and taxon sampling, uncertainty remains regarding the identity of the sister group of the Pterioidea. The paired-sites tests, applied to the alternative topologies differing only in the placement of the outgroup taxa, showed that the trees were not significantly different. Consequently, the sister-group of the Pterioidea could not be unequivocally inferred in light of the present results. Based on the results of the combined-data analyses under uniform weighting and predominant support , the Ostreoidea is tentatively proposed here as the most likely pterioidean sister group.It has long been acknowledged that extensive homoplasy is a recurrent theme in bivalve phenotypic evolution -142 thatA duplicated outer fold of the mantle edge (a "four-fold" condition) has been considered a derived trait that evolved twice independently in the Pterioidea based on the analysis of morphological data  and in aIn the analysis of morphological data, homorhabdic ctenidia (simple gills characterized by a single type of filaments) evolved once from heterorhabdic ctenidia . Molecular results, on the other hand, implied that the gill grades had evolved iteratively and the ancestral condition could not be unequivocally resolved. The presence of food grooves in the outer demibranchs showed the same pattern.Vulsella and Crenatula [Crenatula with Electroma, a relationship supported by a single unambiguously optimized nonhomoplastic apomorphy: a T-shaped (in cross-section) typhlosole, a longitudinal fold of the intestine. Molecular results were ambiguous with regard to the immediate relatedness of the Vulsella and the Crenatula/Electroma clades due to alignment uncertainty, and suggested a possibility that the evolution of adaptation for living inside sponges could have been two separate events. This supposition is corroborated by prior ecological observations that the host specificity, and the nature of mutualistic association of Vulsella and Crenatula species with their demosponge hosts are different: the former is a facultative mutualist with keratosid sponges, whereas the latter is an obligatory mutualist with monaxonid sponges [Vulsella possess a relatively thick and elongated shell with an extremely abbreviated hinge line and a narrow prismatic margin that provide a rigid "endo-skeleton" for the sponge, hence facilitating the host's growth. Species of Crenatula, on the other hand, have a very thin shell with a very wide prismatic margin, which appears to be influenced by the sponge growth, and produce highly irregular shell shapes without consistent orientation. The particular form of the multivincular ligament of Crenatula allows for curvature of the extensive hinge line, which facilitates attachment along the hinge line to irregular surfaces. The loss of postlarval byssus in distantly related Malleus albus  also supports the likelihood of convergence.The loss of postlarval byssus and posterior pedo-byssal retractor muscles has been previously considered as evidence for sister-group relationship of renatula . Both fe sponges ,148. SpeThe symmetry and relative placement of abdominal sense organs (ASOs), paired mechanoreceptors typically found on the surface of the adductor muscle, have been previously shown to be phylogenetically informative within Autolamellibranchiata ,150 and,Pulvinites, Isognomon, and Crenatula. Morphological data alone also identified an independent origin of multivincular ligament in Crenatula, but suggested a sister-group relationship of Pulvinites and Isognomon on the basis of shared ligament morphology and two other homoplastic characters. It is noteworthy that despite apparent similarity between multivincular systems in Pulvinites and Isognomon, the pattern of ligostracum deposition and the ontogenetic sequence of origination of ligamental layers along the hinge line differ between the genera. Even though not entirely congruent, the results of the molecular and morphological studies reject the long-standing assumption of close relatedness based on a single origin of multivincular ligament in the Pterioidea.A set of characters that has historically been used for higher-level systematics of pterioidean and other autolamellibranchiate taxa is associated with the types of ligament and their support structures. According to the molecular-based topology, the multivincular ligament characterizes three distantly related genera: Pulvinites is consistent with its basal placement in the Pterioidea as the same condition is displayed by the outgroup taxa. In contrast, in the analysis based on morphological data, the lack of the funnel was inferred as a loss. The molecular and morphological analyses agree that the anal funnel is subtriangular in outline in the more basal pterioidean clades, whereas it is characteristically lanceolate and rounded in two most derived clades, Pinctada and Vulsella/Crenatula/Electroma, respectively.The present molecular evidence confirmed the taxonomic significance of the shape of the anal funnel, an ear-shaped membranous structure situated at the posterior extremity of the rectum that presumably facilitates expulsion of fecal pellets. The absence of the anal funnel in Given the uncertainty in the identity of pterioidean sister group, it is instructive to explore the effect of alternative combinations of outgroup taxa on the distribution of diagnostic morphological characters using the best available hypothesis of the ingroup relationships. To this end, morphological characters were optimized on three trees constrained to the identical arrangement of the ingroup taxa  but differing in the placement of the outgroups.a priori criteria to differentially weigh the characters supporting each of the possibilities, the arrangement supported by the largest number of apomorphies and yielding the shortest tree length, that is (Pinnoidea(Ostreoidea/Pterioidea)), is the preferred hypothesis of relationship.The Pinnoidea(Ostreoidea/Pterioidea) relationship  was defined by five unambiguously optimized, non-homoplastic apomorphic characters that remained unchanged above this node: (1) the descending intestine produced towards the posteroventral side of the posterior adductor muscle; (2) the loss of the anterior adductor muscle (monomyary); (3) the absence of the pseudonymphae; (4) the presence of a resilifer throughout ontogeny; and (5) the placement of the posterior adductor muscle entirely within the boundary of the inner shell layer. Previously, Steiner and Hammer  had mappPinctada, a commercially important and the most exhaustively sampled group.Despite the fact that the focus of the present study was to establish higher-level relationships within the Pterioidea, it provided significant insights into species-level taxonomy of the genus Pinctada imbricata, P. fucata, and P. radiata are distinct genealogical units. The taxonomic status of the P. imbricata/fucata/radiata species complex previously remained unsettled due to extensive variation in shell characters within and among populations, extremely wide geographical distribution, and transport and hybridization by humans [P. imbricata in the western Atlantic region, P. radiata in the eastern Indian Ocean and the Red Sea regions, and P. fucata in the Indo-Pacific region. The Japanese populations have frequently been regarded as a distinct species, P. martensii, or a subspecies, P. fucata martensii. Because of its major role in perliculture, the species complex had become a major research focus in recent years. A large number of comparative genetic analyses using allozyme and DNA sequence data indicated significant levels of gene flow over large geographic distances among populations of Indian Ocean and Indo-Pacific regions, resulting in low levels of genetic differentiation [The present study suggests that y humans . Traditintiation ,32. The ntiation ,152. Shentiation ,154, matntiation , and comntiation -158 failP. imbricata/fucata/radiata species complex, comparable to the levels of divergence of conspecific exemplars of other Pinctada species. However, the three groups were reciprocally monophyletic showing an apparent clinal geographical structure. Therefore, it might be meaningful to treat these individual populations as evolutionary significant units (ESUs). Taking into account the comparatively low divergence values and the lack of diagnostic morphological characters distinguishing these groups, from the taxonomic standpoint it is advocated here to provisionally recognize them at a subspecies level under the senior synonym, P. imbricata . The final word on the taxonomic status of these taxa will depend on the future population-level analysis with dense sampling throughout the entire distribution range and using genetic markers more sensitive for resolving recent divergencies.The present results showed shallow levels of divergence among the members of the Pinctada albina and P. nigra, generally distinguished by shell color (as their names imply), represent two colormorphs of the same species. This possibility has been previously suggested based on extremely low levels of genetic divergence between two exemplars of P. albina and one of P. nigra [This study also suggested that P. nigra . This fiP. nigra -161. TheP. mazatlanica invariably nested among representatives of P. margaritifera or members of both species formed an unresolved clade, the present study provides preliminary support for conspecificity of these two species. These findings need to be investigated further and cannot be definitively tested given the present sample size.In addition, based on the fact that The most significant conclusion from the phylogenetic analyses presented here is that the state of pterioidean systematics is no longer in chaos. The present results establish a solid-however unconventional- framework for the higher-level taxonomy of the group, focusing future revisionary systematic effort and providing a better grasp of standing alpha-diversity. The phylogeny is crucial for understanding processes and patterns of diversification and morphological evolution of the Pterioidea through time. To this end, the major future challenge lies in integrating the knowledge on the Recent pterioideans with information on the extensive, well-documented fossil record of the group. As throughout their history, pterioideans remain a globally distributed, relatively common group of bivalves found in most warm, shallow marine environments around the globe, and are likely to display patterns of diversity typical of other marine benthic macrofauna. Therefore, the insights gained from the phylogenetically-informed studies of pterioidean bivalves might be broadly applicable across marine invertebrates.DNA sequence divergence. Word DOC file containing DNA sequence divergence.Click here for filePartitioned analyses. Word DOC file displaying Partitioned analyses.Click here for file


Intersectionality theory, a way of understanding social inequalities by race, gender, class, and sexuality that emphasizes their mutually constitutive natures, possesses potential to uncover and explicate previously unknown health inequalities. In this paper, the intersectionality principles of "directionality," "simultaneity," "multiplicativity," and "multiple jeopardy" are applied to inequalities in self-rated health by race, gender, class, and sexual orientation in a Canadian sample.The Canadian Community Health Survey 2.1  provided nationally representative data that enabled binary logistic regression modeling on fair/poor self-rated health in two analytical stages. The additive stage involved regressing self-rated health on race, gender, class, and sexual orientation singly and then as a set. The intersectional stage involved consideration of two-way and three-way interaction terms between the inequality variables added to the full additive model created in the previous stage.From an additive perspective, poor self-rated health outcomes were reported by respondents claiming Aboriginal, Asian, or South Asian affiliations, lower class respondents, and bisexual respondents. However, each axis of inequality interacted significantly with at least one other: multiple jeopardy pertained to poor homosexuals and to South Asian women who were at unexpectedly high risks of fair/poor self-rated health and mitigating effects were experienced by poor women and by poor Asian Canadians who were less likely than expected to report fair/poor health.Although a variety of intersections between race, gender, class, and sexual orientation were associated with especially high risks of fair/poor self-rated health, they were not all consistent with the predictions of intersectionality theory. I conclude that an intersectionality theory well suited for explicating health inequalities in Canada should be capable of accommodating axis intersections of multiple kinds and qualities. For example, people of the same race will experience race differently depending upon their location in the class structure as working class, professional managerial class or unemployed; in the gender structure as female or male; and in structures of sexuality as heterosexual, homosexual or bisexual" [10:326-7].Thus "multiplicativity" should supplant additivity . Racism "Race, class, gender and sexuality are not reducible to individual attributes to be measured and assessed for their separate contribution in explaining social outcomes, an approach that Elizabeth Spelman calls "pop-bead metaphysics," where a woman's identity consists of the sum of parts neatly divisible from one another. The matrix of domination seeks to account for the multiple ways that women experience themselves as gendered, raced, classed and sexualized" [10:327].Experiences of gender are racialized, sexualized, and classed; experiences of class are gendered, racialized, and sexualized, etc.From the abovementioned principles of directionality, simultaneity, and multiplicativity arise new versions of double jeopardy and triple jeopardy, renamed "multiple jeopardy" by Deborah King , whereinDespite the immense popularity of intersectionality theory in humanities and social sciences circles and the large and growing body of intersectionality research that includes applications of both qualitative and quantitative methodologies, very little quantitative research has explicitly applied intersectionality theory to health outcomes. However, many health determinants researchers have unintentionally addressed simultaneity and multiplicativity by identifying two-way statistical interactions between axes of inequality in regression modeling. In Canada, Zheng Wu and colleagues  identifiModeling the main effects of inequality identities (additivity) and then statistical interactions between them (multiplicativity) in multivariate regression models on health can establish whether two-way or three-way statistical interactions (intersections) between axes of inequality contribute to explaining variability in health above and beyond the additive approach to health inequalities that currently dominates health determinants research. This paper uses a two-stage analytical strategy, the first additive and the second multiplicative, applied to a large representative survey dataset from Canada in order to investigate health outcomes associated with intersections between race, gender, class, and sexual orientation.First, the strength and direction of the main effects in additive regression models such as Race + Gender + Class + Sexual Orientation = Health addresses the principles of simultaneity and directionality. Simultaneity suggests that all four identities will make significant contributions to these models before and/or after controlling for one another while directionality implies that non-Whites, women, lower-class people, and non-heterosexuals will manifest the poorer health outcomes.2 values between regression models with and without the cross-product terms can be used to assess the magnitude of their contributions to explaining variability in health above and beyond the contributions of the main effects.Second, simultaneity and multiplicativity imply that the inequality identities should interact meaningfully with one another as predictors of health, that is, statistical interactions between the inequality variables of race, gender, class, and sexual orientation should manifest significant effects above and beyond their main effects in the abovementioned additive models. The existence of interactions speaks to multiplicativity. The qualities of the interactions themselves speak to multiple jeopardy and directionality. At least three multiplicative scenarios are possible for a given statistical interaction: 1. two or more axes of inequality manifest directions of some kind or other in additive models and then display an aggravating effect in the interaction between them, 2. two or more axes manifest given directions in additive models and then display a mitigating effect in their interaction, and 3. an interaction manifests itself between two or more axes but not all of the axes display independent effects in additive models. Aggravating effects support the assumption of multiple jeopardy and reinforce the directionality identified in the additive models whereas non-aggravating effects run contrary to the assumption of multiple jeopardy and complicate directionality. Finally, contributions to predicted variability in the models address multiplicativity by providing an indication of the "value added" of the statistical interactions; comparisons of RCanadian Community Health Survey 2.1 dataset was collected by Statistics Canada in 2003. The target population for this cross-sectional survey was all persons 12 years of age and older residing in Canada, excluding individuals living on Indian Reserves and on Crown Lands, institutional residents, fulltime members of the Canadian Armed Forces, and residents of some remote regions. Sampling considered province or territory and health region of residence and applied three sampling frames  to select the sample of households. One person was chosen randomly from each household to complete the survey. A total of 134,072 usable responses were obtained, representing a national response rate of 80.7%. Final person estimation weights were provided by Statistics Canada.The This investigation focuses on survey respondents who were aged 25 and older at the time of the survey. Table Survey respondents were asked the following question: "People living in Canada come from many different cultural and racial backgrounds. Are you: White? Chinese? South Asian ? Black? Filipino? Latin American? Southeast Asian ? Arab? West Asian ? Japanese? Korean? Aboriginal ? Other - specify." The interviewer was instructed to read all of the possible responses and record all of them that applied. Due to small sample sizes for some responses this variable was recoded as follows: Aboriginal, Asian , Black, South Asian, and White, as well as a residual category created by combining the remaining categories, including the original "other" category, into a single un-interpretable category labeled "other."Highest educational attainment and household income were used to assess class standing. Statistics Canada asked a series of survey questions pertaining to educational attainment to generate the education variable described in Table Sexual orientation was assessed as follows: "Do you consider yourself to be: Heterosexual? ; Homosexual, that is lesbian or gay? ; Bisexual? " Approximately 0.6% of women and 0.5% of men self-reported as bisexual and 0.7% of women and 1.2% of men self-reported as homosexual, values that are slightly lower than numbers reported by similar studies in the United States , AustralGlobal self-rated health, a variable known to encompass both physical and mental well-being and to reliably predict other, more objective, measures of health  as well 2, a rough measure of the proportion of variability explained by a logistic regression model, was presented for each additive model.Self-rated health was dichotomized so that fair and poor responses were contrasted with good, very good, and excellent responses and binary logistic regression modeling was then used to predict fair/poor health. Each nominal independent variable in a regression model was treated as a set of dummy variables with one (missing) dummy variable serving as the reference. Because the N for a reference category should be large in order to provide a stable reference point, "White" was assigned the reference category for race and "heterosexual" was assigned the reference category for sexual orientation. In addition, "male" was assigned the reference category for gender and "postgraduate degree" was assigned the reference category for education. This strategy facilitated ready interpretation of how the other identities fare relative to what are generally considered the more privileged identities in Canadian society. Nagelkerke pseudo RIntroducing cross-product terms to hierarchically well-ordered models is a common approach to investigating statistical interactions in the context of logistic regression . Alpha wThe logistic regression models were implemented in SPSS 15.0. Because the sampling design for the CCHS 2.1 was complex, the 500 bootstrapping weights and BOOTVAR program created for the CCHS 2.1 by Statistics Canada were used to generate more reliable variance estimates and thus more reliable tests of significance and confidence intervals for individual variables within regression models. Due to the limitations of BOOTVAR, results from omnibus tests of significance for categorical variables and interaction terms comprised of sets of dummy variables and Model Chi-square tests of significance for logistic regression models in their entirety could not be generated.Table 2 values indicate that education and income followed by race were the strongest predictors of self-rated health. Education and income were also implicated in some of the "hidden" explained variability in the regression models (results not shown). Regarding the overall contributions of the main effects to predicted variability in health, as a set the five inequality variables produced an increase in Nagelkerke R2 of 0.061 over the regression model on self-rated health containing age alone.Comparisons of odds ratios and Nagelkerke RIn summary, the additive models of Table Two-way and three-way interactions between the five inequality variables were individually added to the final additive model of Table Neither of the three-way interactions had a statistically significant effect on self-rated health. However, each of gender, race, and sexual orientation manifested significant two-way interactions with class and gender interacted significantly with race Table . ConsideThe marked change for the worse in risk of fair/poor health from the additive model to the multiplicative model for poor homosexuals depicted in Figure 2. Two-way interactions between the four axes of inequality therefore contributed less than one percent predicted variability in self-rated health.Adding all of the two-way cross-product terms to the final model of Table In summary, each of the four axes of inequality interacted significantly with at least one other, suggesting that all four axes belong to the pantheon of intersectionality axes of inequality that contribute to health inequalities in Canada. The only instances of multiplicative disadvantage pertained to poor homosexuals and to South Asian women who were at an especially high risk of fair/poor self-rated health. Mitigating effects pertained to lower class women and to poorer Asians who were less likely to report fair/poor health than expected. Lastly, the multiplicative models contributed relatively little to overall predicted variability in self-rated health over and above the contribution of the full additive model.From the perspective of intersectionality theory, by focusing on a subset of the inequality identities or by treating multiple axes of inequality as distinct rather than intersected processes, a social researcher is in danger of misunderstanding the nature of social experiences and identities manifested in specific contexts and thus in danger of producing results and interpretations that are as misleading as they are incomplete. If this is true then much of the literature on health effects of inequalities pertaining to race, gender, class, and/or sexual orientation is incomplete, and some of it may even be misleading.The Canadian Community Health Survey dataset is especially well suited to investigating the applicability of intersectionality theory to health disparities in Canada. It is the first and only Statistics Canada survey dataset to assess sexual orientation, distinguishing between bisexuals, homosexuals, and heterosexuals, and unlike most Canadian survey datasets it is large enough to produce a multi-category measure of race. The analysis described herein is therefore unique by virtue of its consideration of intersections between all four key inequality axes of intersectionality theory, its consideration of bisexual identities as well as homosexual and heterosexual identities, and its consideration of racialized identities such as Aboriginal, Asian, and South Asian as well as Black and White. In addition, the application of central principles of intersectionality theory to Canada, close neighbor to the United States, can contribute to future speculation about the portability of intersectionality assumptions across borders. Cross-contextual comparisons are essential in light of the fact that institutionalized race relations, gender relations, etc. are historically and contextually specific . HoweverThe intersectionality principle of simultaneity maintains that all four axes of inequality should be considered in an analysis while the principle of multiplicativity maintains that intersections between axes should overshadow or supplant the individual axes themselves in their effects. Although we carry our identities into every social situation, not all of them are necessarily salient in or relevant to a particular encounter . Even soor less than  cumulative double or triple jeopardy. This multiplicity of multiplicative possibilities demands a kind of conceptual fluidity that is not accommodated by the principle of multiple jeopardy as it is depicted it in the introduction to this paper.The intersectionality assumption of multiple jeopardy maintains that meaningful intersections manifest multiplicative - inordinate amounts of - disadvantage or advantage. While two intersections were to indeed to the further detriment of certain complex social locations, i.e., of poor homosexuals and South Asian women, two demonstrated a mitigating quality for certain complex locations, i.e., for lower class women and poor Asian Canadians. Many other possible interactions were not large or statistically significant. It therefore appears that, with regards to self-rated health in Canada at least, multiple jeopardy can be more Bart Landry  argues tadvantages to social invisibility in that marginal members of marginal groups may be able to elude the more active forms of oppression which are directed at "prototypical" members of marginal groups. The multiplicity of multiplicative possibilities described in my analyses begs for further investigation from an intersectional invisibility perspective. For example, characteristics of workplaces and occupations, health behaviors, residential segregation, experiences with systemic, institutional, and interpersonal discrimination, adherence to different norms of masculinity and femininity, and encounters with the health care system may identify advantages and disadvantages adhering to various complex social locations and explicate varying risks of poor health in Canada by intersecting axes of inequality. However, acknowledging with Weber and Parra-Medina [The theory of "invisible intersectionality" has this potential. Valerie Purdie-Vaughns and Richard Eibach  argue tha-Medina  that intFrom an additive, non-intersectional perspective, poor self-rated health outcomes were reported by respondents claiming Aboriginal, Asian, or South Asian affiliations, lower class respondents, and bisexual respondents. However, from an intersectional perspective, each axis of inequality interacted significantly with at least one other: multiple jeopardy pertained to poor homosexuals and (possibly) South Asian women who were at an unexpectedly high risk of fair/poor self-rated health and mitigating effects were experienced by poor women and by poor Asians who were less likely than expected to report fair/poor health. I conclude from these varied results that the intersectionality theory best suited for explicating health inequalities in Canada should be theoretically capable of accommodating axis intersections of multiple kinds and qualities.The author declares that he has no competing interests.


We evaluated the direct and indirect influence of climate, land use, phylogenetic structure, species richness and endemism on the distribution of New World threatened amphibians.We used the WWF’s New World ecoregions, the WWFs amphibian distributional data and the IUCN Red List Categories to obtain the number of threatened species per ecoregion. We analyzed three different scenarios urgent, moderate, and the most inclusive scenario. Using path analysis we evaluated the direct and indirect effects of climate, type of land use, phylogenetic structure, richness and endemism on the number of threatened amphibians in New World ecoregions. In all scenarios we found strong support for direct influences of endemism, the cover of villages and species richness on the number of threatened species in each ecoregion. The proportion of wild area had indirect effects in the moderate and the most inclusive scenario. Phylogenetic composition was important in determining the species richness and endemism in each ecoregion. Climate variables had complex and indirect effects on the number of threatened species.Land use has a more direct influence than climate in determining the distribution of New World threatened amphibians. Independently of the scenario analyzed, the main variables influencing the distribution of threatened amphibians were consistent, with endemism having the largest magnitude path coefficient. The importance of phylogenetic composition could indicate that some clades may be more threatened than others, and their presence increases the number of threatened species. Our results highlight the importance of man-made land transformation, which is a local variable, as a critical factor underlying the distribution of threatened amphibians at a biogeographic scale. The worldwide decline of amphibian populations has become one of the main priorities on the conservation agenda. Amphibians are protagonists in the current biodiversity crisis, with one third of species threatened with extinction risk Recently, Ellis & Ramankutty The current rate of biodiversity loss has challenged ecologists to develop predictive models which summarize important ecological and evolutionary processes and, most importantly, to provide recommendations for on the ground conservation action that can be readily assimilated by decision and policy makers We evaluated the direct and indirect influences of climate, land use, phylogenetic structure, richness and endemism on the distribution of threatened amphibians across the New World using path analysis i.e. the anthropogenic biomes), phylogenetic structure, species richness and endemism on the distribution of threatened amphibians throughout the New World ecoregions. From the 289 New World ecoregions described by Olson et al. We analyzed the direct and indirect influence of climate, land use (We classified amphibian species following the extinction risk categories proposed by the IUCN Red List Categories and Criteria 2), villages (327 persons/km2), croplands (33 persons/km2), rangelands (7 persons/km2), seminatural (1person/km2) and wild (0 person/km2). The cover proportion of each anthrome category per ecoregion was treated as an independent variable in the path analysis (see below). The spatial distribution of the cover of different land uses along the ecoregions can be visualized in We used Ellis & Ramankutty’s To describe the climate in each ecoregion, we used nine environmental variables: altitude, annual mean temperature, temperature seasonality , maximum temperature of the warmest month, minimum temperature of the coldest month, annual mean rainfall, rainfall seasonality , precipitation of the wettest month and precipitation of the driest month. We decomposed each climatic variable into mean value and the range, totaling 18 climatic variables. All variables were compiled from the WorldClim 1.4 database F) based on node counting for the genera contained in matrix W was computed using the software Mesquite 2.73 To generate a phylogenetic tree of amphibians inhabiting the New World ecoregions we adopted the phylogenetic tree built by Pyron & Wiens F were used in terms of their complement as similarities (SF). Then, phylogenetic similarities in SF were used to weigh the number of species per genera in matrix W. This procedure generated a matrix P containing phylogeny-weighted genera composition for each ecoregion. Accordingly, those j taxa most phylogenetically related to i (e.g. from the same genus) received a proportionally higher fraction of the presence of i in that ecoregion than more phylogenetically distant taxa (e.g. from a different genus), which will receive a proportionally lower fraction, and so on. Note that the sum of the number of species per genera (i.e. species richness) in an ecoregion belonging to W will remain exactly the same in P after phylogenetic fuzzy-weighting. Matrix P expresses the phylogenetic composition in the set of ecoregions.We scaled-up the phylogenetic relationships between species to the site level, generating a matrix describing the phylogeny-weighted genera composition of each ecoregion, which was defined using the phylogenetic fuzzy-weighting method developed by Pillar & Duarte By performing a PCoA To remove the effect of area and geographical position of each ecoregion, we did a set of multiple linear regressions between all the variables included in path analysis with latitude, longitude and area. Then, the residuals obtained from these regressions were used to build a causal model linking the different types of variables. Considering that, the final results of the analysis will represent the effect of climate, land use, phylogenetic structure, species richness, endemism on threatened amphibian distribution, with no influence of the area and geographical position of the ecoregions.We evaluated the influence of the two climatic axes, six anthromes and the three phylogenetic filters in the distribution of threatened amphibians using model selection based on Akaike’s information criterion AIC, , separatFurther, we evaluated causal connections between the selected climatic axes, anthromes, phylogenetic structure, species richness, endemism and threatened amphibian distribution using path analysis We built the path model in several steps using the model selection based on AIC. First, using the pre-selected climatic and phylogenetic variables plus species richness, endemism and threatened amphibian distribution, we built a hypothetical model establishing all possible and plausible causal relationships between variables . For thiOur second analytical step consisted in running a model selection to find which variables directly determined the variation in threatened amphibian distribution, based on AIC modeling. After that, we proceeded to iteratively find the explanatory variables determining each endogenous predictor of threatened amphibian distribution. That is, each variable found to determine threatened amphibian distribution was taken as a response variable, and their respective predictors were determined using model selection P values of each path coefficient were calculated by using randomization test We obtained path coefficients for the so-built models by linear multiple/simple regressions, being the standardized regression coefficient (β) equivalent to the path coefficient P generated 239 PCPS. The first three PCPS contained, respectively, 41%, 12% and 6% of the total variation of matrix P. Only the first three PCPS were submitted to model selection procedure, since most variation in phylogeny-weighted species composition (≅ 60%) was concentrated in these three orthogonal axes. The correlation of phylogenetic clades distribution and PCPS1, PCPS2 and PCPS3 is shown in the From the 2472 amphibian species present in the ecoregions, 1886 belong to some threat category, 4 species were classified as Extinct, 221 as Critically Endangered, 326 as Endangered, 246 as Vulnerable. It means that the urgent scenario contained 225 species, the moderate scenario 551 species and the most inclusive 797 species. The maps showing the spatial distribution of species richness, endemism and the number of threatened species in each scenario is presented in the Across the New World ecoregions, endemism was the best predictor in our urgent scenario, followed by the proportion of the village anthrome and species richness . PhylogeIn our moderate scenario, endemism, the village anthrome and species richness were also the main predictors of threatened amphibian distribution . The modThe most inclusive scenario showed climatic factors as determinant of the threatened amphibian distribution, apart from the importance of endemism, proportion of villages and species richness variables . The effDespite the reported influence of climate on amphibian distribution Our results point toward a better outcome of amphibian conservation efforts if they are to be founded on land-use policies not only at the landscape level, but also at broader spatial scales. However, most current amphibian conservation actions are generally either species or site-specific. Our analysis has a particular caveat when applied to local actions. Although our results indicate that land-use change could drive diversity patterns not only at the landscape level It is largely known that human activities impact amphibian diversity Amphibian species with small geographical ranges are more prone to extinction than those with broad distributions, since they are more likely to be exposed to threatening process throughout their entire range, generally present a low abundance and they are often habitat or environment specialists Furthermore, land use was important not only via direct effects, but also through indirect effects by determining the spatial distribution of amphibian phylogenetic lineages. That is to say, closely-related species tended to be assembled by similar land use types, suggesting phylogenetic habitat filtering In conclusion, our results showed that land use was directly more important than climate in determining the distribution of threatened amphibian species across the New World. Nonetheless, a considerable portion of the effect of land use on species threat was phylogenetically structured, meaning that human impact on amphibian distribution affects not only species individually, but may also define the fate of entire lineages of this imperiled group.Figure S1Scaling-up of phylogenetic data from species to the site level employed in this study. Matrices are: SF with phylogenetic pairwise similarities of species, Q′ is a transposed matrix with degrees of species belonging to every other species based on SF, standardized within columns, W with presence of species in sites, P with phylogeny-weighted species composition. Principal coordinates analysis of P using an appropriate dissimilarity measure generates a matrix of principal coordinates of phylogenetic structure (PCPS) composed of sites described by eigenvectors (EV). (TIF)Click here for additional data file.Table S1Raw data of richness of amphibian species, endemism and number of threatened amphibian species according to the three different scenarios  for each ecoregion, which were used in the analysis. The urgent scenario, containing only CR species and those EW and EX, the moderate scenario containing all EN species and those at threat categories higher than EN , and the most inclusive scenario, which included all VU species and those at higher threat categories. The percentage values were calculated in relation to the total ecoregion richness.(PDF)Click here for additional data file.Table S2Pearson correlation coefficient between the number of threatened amphibian species according to the three different scenarios , richness of amphibian species, endemism, two climatic axes, proportion of cover of villages and wildlands and the three axes of phylogenetic structure, all in residual form, which were used in the path analyses.(PDF)Click here for additional data file.


Bone is a common site for cancer metastasis. To create space for their growth, cancer cells stimulate bone resorbing osteoclasts. Cytokine RANKL is a key osteoclast activator, while osteoprotegerin (OPG) is a RANKL decoy receptor and an inhibitor of osteoclastogenesis. Consistently, systemic application of OPG decreases metastatic tumor burden in bone. However, OPG produced locally by cancer cells was shown to enhance osteolysis and tumor growth. We propose that OPG produced by cancer cells causes a local reduction in RANKL levels, inducing a steeper RANKL gradient away from the tumor and towards the bone tissue, resulting in faster resorption and tumor expansion. We tested this hypothesis using a mathematical model of nonlinear partial differential equations describing the spatial dynamics of OPG, RANKL, PTHrP, osteoclasts, tumor and bone mass. We demonstrate that at lower expression rates, tumor-derived OPG enhances the chemotactic RANKL gradient and osteolysis, whereas at higher expression rates OPG broadly inhibits RANKL and decreases osteolysis and tumor burden. Moreover, tumor expression of a soluble mediator inducing RANKL in the host tissue, such as PTHrP, is important for correct orientation of the RANKL gradient. A meta-analysis of OPG, RANKL and PTHrP expression in normal prostate, carcinoma and metastatic tissues demonstrated an increase in expression of OPG, but not RANKL, in metastatic prostate cancer, and positive correlation between OPG and PTHrP in metastatic prostate cancer. The proposed mechanism highlights the importance of the spatial distribution of receptors, decoys and ligands, and can be applied to other systems involving regulation of spatially anisotropic processes. Breast and prostate cancers commonly metastasize to bone. To create more space for their expansion, metastatic tumors activate osteoclasts, the only cells capable of bone destruction. The main osteoclast stimulator is the cytokine RANKL, while osteoprotegerin (OPG) acts as a RANKL inhibitor. Systemic application of OPG leads to a decrease in tumor-associated bone destruction, but surprisingly, OPG produced locally by metastasizing cancer cells can enhance bone destruction and tumor growth. Here, we provide a novel explanation for these apparently contradictory experimental results: the osteolysis-promoting effect of OPG is due to a local reduction in RANKL levels, resulting in a spatial RANKL gradient oriented from tumor towards bone tissue. At low rates of OPG expression by cancer cells, such gradients result in the correct orientation of osteoclast movement and intensified bone resorption. We positively test our hypothesis by means of a partial differential equations model, and further substantiate our results with a meta-analysis of gene expression. Even though developed for the specific problem of bone metastases, our model naturally applies to other systems operating within a geometrically anisotropic environment. Primary cancers develop metastatic tumors in distant sites and tissues of the body, and frequently, fatal outcome is due to those secondary rather than the primary tumors Once a secondary tumor starts growing in the bone environment, its expansion is geometrically constrained by the presence of inelastic bone tissue. Physiologically, bone is remodeled through the process where old or damaged tissue is resorbed by cells specialized in bone destruction, osteoclasts, and new bone is produced by specialized bone-forming osteoblasts Based on the model described above, the presence of osteoprotegerin in bone metastases should lead to reduced bone destruction and tumor growth. In agreement with this prediction, the systemic application of OPG leads to a decrease in tumor burden Altogether, these results indicate that osteoprotegerin plays a controversial role in bone metastases: while a large set of experimental data supports its osteoclast- and hence tumor-inhibiting role, in certain situations osteoprotegerin is documented to stimulate osteolysis and tumor growth.1) When OPG is applied globally , it uniformly reduces RANKL levels, thus acting as an inhibitor of osteoclast formation and tumor growth. 2) When OPG is produced locally by cancer cells, the outcome is determined by the rate of OPG expression. At low expression rates, OPG enhances the chemotactic RANKL gradient responsible for osteoclast movement, thus stimulating osteolysis and tumor growth. At high expression rates, the RANKL-inhibiting effect of OPG becomes predominant and results in an overall decrease in osteolysis and tumor burden. The distinction of the two regimes for tumor-derived osteoprotegerin provides a potential explanation of the differential experimental outcomes in We have recently demonstrated a potential role of OPG in enhancing RANKL gradients in silico experiments focusing on the following main questions: 1) How does the impact of systemic OPG compare to the impact of cancer–cell derived OPG production? 2) How is indirect stimulation of RANKL production via PTHrP different from direct production of RANKL by tumor cells?To test this hypothesis we developed a mathematical model of tumor-osteoclast interactions, including the cytokine fields of RANKL, OPG and PTHrP, and examined the model predictions by means of appropriate We adapted a previously developed nonlinear partial differential equations model of bone remodeling We model a single trabecula exposed to bone marrow and pre-existing cancer cells. Hemi-osteonal, trench-like remodeling of trabecular bone movement . By choocancer cells below), we assume that the dynamics at the tumor–bone interface are dominated by the processes of bone resorption and tumor growth. Therefore, we focus here on the interaction of osteoclasts with the tumor, and do not take into account the much slower osteoblast dynamics.Metastasizing cancer cells cannot resorb bone tissue themselves and hence the outcome of the metastasis depends on the tumor's ability to stimulate osteoclasts. Even in the case of metastases which promote osteoblastic activity, bone resorption precedes bone formation Text S1 as well as We assume that osteoclastic differentiation and activity are controlled by the RANK/RANKL/OPG pathway. We assume that RANKL is produced by host tissue cells, including osteocytes, bone-lining cells, and stromal cells The mechanisms underlying osteoclast initiation are complex and remain poorly understood to this date. In lieu of speculation, we assume that initiation has taken place prior to the beginning of the scenarios considered in this study, and that active osteoclasts are already present at time bone homeostasis). Therefore, the rate of tumor growth within the osseous tissue is expected to be limited by the availability of newly freed space, which in turn is limited by the rate of bone resorption. In keeping with this hierarchy of time scales, we assume that as soon as new space becomes available through bone resorption, proliferating cancer cells rapidly expand into the cavity. In other words, the tumor density Tumor expansion is a complex process, and the mathematical modeling of tumor growth dynamics constitutes an active field of research Model Assumptions, the tumor density The mathematical model consists of 6 state variables: osteoclast population density  is the subject of Scenario 4. Finally, the most comprehensive situation is captured in Scenario 5, where cancer cells produce both PTHrP and OPG.We performed five different numerical experiments, referred to as We first assess how different levels of RANKL in the host tissue influence tumor growth. We solve Next, we investigate the impact of systemically administered OPG by introducing The simulations in Video S1). In comparison to the control case with no OPG expression  in absence of the PTHrP equation, set Text S1 for details), i.e. we replace the reaction term in the osteoclast equation of (6) bySince high levels of RANKL in the tissue are important for the osteolysis-enhancing effects of OPG, we assess now if cancer cells could promote osteolysis by directly producing RANKL. We model this situation by adding a tumor-derived RANKL source to the We consider now the case where cancer cells produce PTHrP (but no OPG), which in turn promotes RANKL production by osteoblastic and stromal cells. More precisely, we consider system (6) in absence of the oduction , , switc 90 days , . Moreoon front , , the emor mass , . Thus,We assess now the impact of simultaneous production of OPG and PTHrP by cancer cells. This leads to the most comprehensive scenario considered, and is captured by the complete system (6) with www.oncomine.org), and processed data as described in Text S2 for details. We found that expression of osteoprotegerin was significantly increased in samples from metastatic prostate cancer compared to normal prostate  that diffuses across the tissue before triggering the expression of RANKL on osteoblastic cells. The involvement of such a mediator assures that the RANKL gradient is correctly oriented to induce osteoclast movement away from the tumor into unresorbed bone tissue. In accordance, the meta-analysis of gene expression reveals that osteoprotegerin expression in metastatic prostate tissue is positively correlated with the expression of PTHrP, but not RANKL.Due to the large number of a priori unknown parameters in the model, our results are predominantly of qualitative nature. While the emergence of two distinct regimes of OPG action is observed across a large span of parameter values, and is in particular independent of the production rate of parathyroid hormone-related protein PTHrP by cancer cells, further experimental investigation will be indispensable for a full validation of our hypothesis. Thereby, an experimental assessment of the diffusion rates of different molecules in tumor and bone tissues would be useful. In addition, the potential role of OPG in stimulating osteoclast movement could be studied in vitro, and the metastatic capacity of prostate cancer cells expressing different levels of OPG could be assessed in vivo. In the meantime, our qualitative predictions are valuable in suggesting a new conceptual model which is consistent with all the experimental data available to date. In particular, our model suggests that future experimental designs should take into account the directional movement of the constituent cells, as well as the geometry of the tumor-bone interface.The proposed mechanism emphasizes the importance of the spatial configuration of molecular densities, and thus may be relevant to other systems where distinct spatial patterns are imperative. An interesting example is the regulation of immune cell migration by chemokines. It has recently been shown that in addition to signaling receptors, there exist several decoy receptors that bind to chemokines, but do not induce any cellular changes Text S1Model development, numerics, and parameter estimation.(PDF)Click here for additional data file.Text S2Meta-analysis of gene expression.(PDF)Click here for additional data file.Video S1Dynamics of Scenario 2 as described in the caption of (AVI)Click here for additional data file.


Erugosquilla massavensis. For documentation we used the autofluorescence capacities of the specimens, resulting in a significant contrast between sclerotized and membranous areas.We describe the tagmatization pattern of the anterior region of the extant stomatopod E. massavensis can be grouped into three tagmata. Tagma I, the sensorial unit, comprises the segments of the eyes, antennules and antennae. This unit is set-off anteriorly from the posterior head region. Ventrally this unit surrounds a large medial sclerite, interpreted as the anterior part of the hypostome. Dorsally the antennular and antennal segments each bear a well-developed tergite. The dorsal shield is part of tagma II, most of the ventral part of which is occupied in the midline by the large, partly sclerotized posterior part of a complex combining hypostome and labrum. Tagma II includes three more segments behind the labrum, the mandibular, maxillulary and maxillary segments. Tagma III includes the maxillipedal segments, bearing five pairs of sub-chelate appendages. The dorsal sclerite of the first of these tagma-III segments, the segment of the first maxillipeds, is not included in the shield, so this segment is not part of tagma II as generally thought. The second and third segments of tagma III form a unit dorsally and ventrally. The tergites of the segments of tagma III become progressively larger from the anterior to the posterior, possibly resulting from a paedomorphic effect during evolution, which caused this reversed enlargement.The anterior body region of The described pattern of tagmosis differs from current textbook knowledge. Therefore, our re-description of the anterior body area of stomatopods is of considerable impact for understanding the head evolution of Stomatopoda. Likewise, it has a bearing upon any comparisons with fossil stomatopods, as mainly sclerotized areas are fossilized, and, on a wider scale, upon larger-scale comparisons with other malacostracans and eucrustaceans in general. Malacostraca includes all the more commonly known and larger crustacean species with a more strongly calcified cuticle . Among them the mantis shrimps (Stomatopoda) are probably the most impressive representatives. This is due to their possession of astonishing morphological features such as their extremely complex visual system ,2 or theTheir unusual morphology is, however, partly bridged by a number of fossil representatives  and relaThe study of tagmatization is an important field of research within arthropod evolutionary biology. The division-of-labor principle has even been assumed to be one of the major factors for the evolutionary success of this animal lineage. Additionally, some of the major (eu)arthropod groups have rather ‘fixed’ patterns of tagmosis, i.e., there is only limited variation of these patterns within their in-groups. Examples are the insects with six head segments, three main appendage-bearing thoracomeres and a posterior body region of ≤ 11 segments, and Malacostraca with six head segments, 14 appendage-bearing segments uniformly divided into an eight-segmented and a six-segmented part (only phyllocarids have one more) -17. SignTagmatization in fossil arthropods is more difficult to understand, not least because incomplete preservation often prohibits access to important structures. Yet, studying fossils for these aspects is very important as they may exhibit patterns of tagmosis not developed in extant representatives ,16,18-20Erugosquilla massavensis , including the application of state-of-the-art documentary techniques [Pseudosquillopsis cerisii , for which dissection was permitted. Our contribution focuses on morphological structures that are to a certain degree probably known to some in-group experts, but we will discuss them in the broader context of the evolutionary history of Stomatopoda. This will facilitate better comparisons with various fossil species in the future, as will be exemplified in part here, but, furthermore, improve comparative studies on a broader scale of, e.g., Malacostraca and Eucrustacea in general.In this paper, we aim at contributing to the understanding of the evolution of tagmatization within Stomatopoda by re-evaluating the tagmosis of extant mantis shrimps. For this purpose, we investigated the external morphology of adults of hniques -33. SuppThe body of the investigated stomatopods Figure  can be g1) Dorsally or ventrally conjoined segments 2) Similar dorsal morphology (mostly tergites)3) Similar appendage morphology (implying similar function) or related function 4) Close spatial association of segments versus long distance to other segments.The five tagmata identified here are the sensorial unit (tagma I), the anterior food-processing unit (tagma II), the posterior food-processing unit (tagma III), the walking-appendage area (tagma IV), and the pleon plus tail fan (tagma V). The anterior three functional units are described here Figure A; tagmatThis most anterior tagma is recognized based on a partly conjoined ventral area (criterion 1), a related function of the appendages of its segments (criterion 3), and the close spatial association of its segments (criterion 4). Tagma I includes the three anterior segments, the eye segment , the antennular and the antennal segment. It therefore can be termed the sensorial unit. Tagma I is set off from the dorsal shield , a related function of the appendages of its segments (criterion 3), and a close spatial association of its segments (criterion 4). Tagma II is involved in food handling and intake. Dorsally the tagma forms the sub-rectangular, dorso-ventrally flattened shield Figure B, ventraThe anterior ventral region, extending to about two thirds of the entire length of the tagma, is completely occupied by a large, elongate triangular, slightly elevated and partly sclerotized structure Figure A. This sE. massavensis the paragnaths are lobe-shaped and medially equipped with about 20 setae.The posterior region of tagma II is formed by the segments of the mandibles, maxillulae and maxillae. The mandibles lie directly behind the hypostome-labrum complex and are tightly followed by a complex that is composed of the paragnaths, the flat and anteriorly concave maxillulae and the similarly shaped maxillae Figure B. The maThe maxillulae are smaller than the mandibles Figure D. They cThe maxillae appear to be composed of four elements Figure D, but itTagma III behind the maxillary segment is characterized by a similar morphology of the appendages of its segments (criterion 3) as well as a similarity of the dorsal morphology of the segments (criterion 2), yet the dorsal morphology obviously changes gradually from anterior to posterior. Furthermore, the segments of this tagma  are very closely associated spatially (criterion 4). Tagma III forms the posterior food-processing unit. The situation of the dorsal area of this tagma is difficult to interpret. Therefore, we discuss here first the ventral details and then the dorsal side (the appendage morphology will only be touched very briefly).The sternitic regions of all five segments are very short, and by this the entire region appears rather ‘squeezed’ in anterior-posterior direction Figure A. All fiThe first pair of appendages inserts relatively far laterally. Its sub-chelae are very small to indistinguishable Figure A, B. TheThe second appendage of tagma III, the raptorial appendage, also inserts relatively far laterally, but has a larger insertion area due to its size. It is the largest appendage of the set and bears a prominent ‘meral’ saddle on the third element  resemble each other in shape and size. They share the principal morphology of the raptorial appendage, but are much shorter and slimmer than it, and they lack a ‘meral’ saddle Figure . Their dE. massavensis has a well-developed tergite, which extends laterally into elongate-oval, bifid tergopleurae . Closer Figure E.The detailed investigation of sclerotized and membranous areas as conducted here highlights a data set hitherto more or less neglected for taxonomic issues or in-group systematics of the stomatopod crown group (Verunipeltata). Part of these structures may already be known to experts of the taxon, but do not appear to have been specifically addressed in the literature. One main point of this paper, sclerotized versus membranous areas, cannot be simply extracted from existing publications, as there is no distinction visible in the line drawings usually provided ,40. Yet,In the following, we discuss the morphology of those structures that to our knowledge differ from available data. We also review which of the morphological details we describe here have already been found among fossil stomatopods, which ones we still have to identify, and how our results will facilitate this process.The segments of the eyes and antennulae of stomatopods are clearly separate from the shield, which has been known for a long time -43. AccoA hypostome is found in trilobites, agnostines , crustacDorsally several separate sclerites are visible Figure B, appareIn the ground pattern of Eucrustacea five appendage-bearing anterior body segments are incorporated into the head shield . Only inThe large sclerotic structure occupying most of the ventral area of tagma II is, because of the location of the mouth opening at its rear, interpreted as the posterior, extremely elongated part of the hypostome. The small lip-like structure at the posterior end of the hypostome is interpreted as the labrum, overhanging the mouth opening Figure A.In Eumalacostraca in general the distance between the antennae and the mandibles is very large, while the mouth opening is still close to the mandibles. Therefore, the morphology of the hypostome-labrum complex observed here is significantly different from that of earlier ground patterns. The entire complex is extremely elongated and appears to be bipartite: an anterior sclerotic part between the insertions of antennulae and antennae, connected by a small triangular sclerite with the strongly extended posterior part, at which end the mouth opening is overhung by the labrum. The partition of the hypostome-labrum complex into separate sclerites probably evolved because of the anterior kinesis of the head in stomatopods. With an unjointed hypostome between antennulae and the mouth opening, it would not be possible to move tagma I independently of tagma II. The evolution of this structural complex needs to be investigated further within Eumalacostraca.The investigation of the morphology of tagma III yielded some unexpected results. One peculiar finding was that the second and third segment of this tagma  have a common sclerotization surrounding the insertions of their appendages Figure C. This sTextbooks and monographs offer varying interpretations of the number of maxillipedal segments that should be incorporated into the “carapace” (shield); numbers range from one  to at lePseudosquillopsis cerisii in comparison to the adult condition in E. massavensis  and is also functionally coupled to them . AddFossil stomatopods have preserved several morphological features of the first tagma in quite some detail, while other features remain obscure. For example, eyes are frequently preserved Figure B, D 1212,14, buThe large hypostome, although well-sclerotized in the extant species, and the associated labrum have not yet been identified in any fossil stomatopod. In laterally and dorsally preserved specimens this region is usually covered by the well-sclerotized shield Figure E. The hyThe mouthparts are also almost unknown from the fossil representatives. The mandible has been found in a single Mesozoic specimen, which had the shield broken off in that area Figure A, C 1313. In soSternites of tagma II may have been preserved in some of the laterally preserved specimens, however, a clear identification is very difficult. The sternites are usually folded medially and then superimposed on each other. While the larger sternites of the pleon still retain some identifiable structures when preserved like this, the small and highly condensed sternites of the mandibular, maxillular and maxillary segments simply end up as an unsorted accumulation of small sclerites between remains of the appendages and maybe even covered by parts of the shield.Remarkably, the first sub-chelate appendage of tagma III, the grooming appendage in modern forms, has not been found in any of the fossil stomatopod specimens. The succeeding four pairs of maxillipeds are well-documented for many different species from Mesozoic and Palaeozoic strata ,13. The Erugosquilla massavensis .The first post-maxillary segment (that of the grooming appendage) is not incorporated into the head shield (feature of tagma II), but retains its own unsclerotized dorsal area within tagma III .The segments of the maxillipeds two and three (second and third segment of tagma III) lack tergites, but share a single, very short unsclerotized dorsal area separate from the head shield. The ventral sclerotization surrounding the insertion area of the second maxilliped appears to be continuous with that surrounding the insertion area of the third maxilliped.The tergite of the segment of the fourth maxilliped (fourth segment of tagma III) is very short and appears bipartite due to an unsclerotized median area. The interpretation of this bipartite condition as the result of paedomorphic heterochrony is based on two observations: 1) The corresponding tergite is fully developed in adult stomatopods from the Carboniferous. 2) In late larval stages of extant stomatopods also the tergite of the segment of the fifth maxilliped (fifth segment of tagma III) is bipartite, but becomes a continuous tergite in later stages.Most of these details have not yet been identified in fossil stomatopods. Therefore, this study may serve as a guideline on which structures might be detectable in fossil stomatopods. In this way it may be possible to identify the specific character states that are less derived from the ground pattern of Stomatopoda than those in extant species, which again facilitates the reconstruction of a more detailed evolutionary scenario. Studies similar to this one on other representatives of Malacostraca would be very helpful in amending our current approach.Erugosquilla massavensis was collected from Port Said harbour, the northern entrance of the Suez Canal on the Egyptian Mediterranean coast. Fresh specimens were obtained from the catch of trawling vessels and immediately preserved in 70% ethanol. The material of Pseudosquillopsis cerisii was provided by Björn van Reumont, Bonn, and collected by Nils Brenke, Wilhelmshaven, at the Great Meteor Seamount, Northeast Atlantic, during the Meteor Expedition M42/3. Comparative fossil material was loaned from the collections of the Staatliches Museum für Naturkunde Stuttgart (SMNS) and the Museum für Naturkunde Berlin (MB.A).The studied material of Extant specimens were immersed in 70% ethanol and carefully dissected with the aid of fine pincers, needles and knives. All parts of the specimens were stored in 70% ethanol and also documented directly within this fluid  and image stitching , or - as a combination of these methods - composite imaging ,31,60. HThe authors declare that they have no competing interests.Erugosquilla massavensis. JTH dissected and documented the extant material. CH documented the fossil material. CH and JTH processed the images. All authors participated in the morphological and evolutionary interpretations, drafted the manuscript, read and approved it.WSS collected the studied material of


Neurons residing in the gut-brain axis remain understudied despite their important role in coordinating metabolic functions. This lack of knowledge is observed, in part, because labeling gut-brain axis neurons and their connections using conventional neuroanatomical methods is inherently challenging. This article summarizes genetic approaches that enable the labeling of distinct populations of gut-brain axis neurons in living laboratory rodents. In particular, we review the respective strengths and limitations of currently available genetic and viral approaches that permit the marking of gut-brain axis neurons without the need for antibodies or conventional neurotropic tracers. Finally, we discuss how these methodological advances are progressively transforming the study of the healthy and diseased gut-brain axis in the context of its role in chronic metabolic diseases, including diabetes and obesity. We have this benefit by this sense, that as soon as anything troubles and vellicates the bowels, we being admonished thereof may look for help in time”–Ambroise Paré, circa 1579.The gut-brain axis comprises a network of autonomic neurons that connect the central nervous system (CNS)—specifically, the caudal brainstem and spinal cord—to the esophagus, gastrointestinal tract, liver, and pancreas  gene encodes the enzyme necessary for the synthesis of acetylcholine, the principal neurotransmitter of preganglionic parasympathetic and sympathetic neurons and a large proportion of enteric neurons  neurons have been stained in this mouse, but other gut-brain axis neurons have not been examined. More recently, ChATBAC-eGFP and ChATBAC-tau-GFP animals were generated using a BAC strategy , they appear to be useful for the study of cholinergic gut-brain axis neurons.The choline acetyltransferase (Melanocortin-4 receptor (MC4R) is an important molecular player in the regulation of feeding, metabolic, and autonomic functions Cone, . Not surA mouse that expresses eGFP under the ghrelin receptor (GHSR) promoter was recently characterized   play a critical role in vago-vagal reflexes  is a transcription factor that is critically involved in the early differentiation of autonomic and viscerosensory pathways  is a precursor of several pituitary and hypothalamic peptides that is important in neuroendocrine and metabolic functions  gene is responsible for the synthesis of noradrenaline in the autonomic nervous system ; however, the anatomical integrity of the entire vagus nerve can easily be gauged .Postganglionic sympathetic neurons express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis  are available  into the rat DMV using a lentivirus and an adenovirus  vs. tension receptors (intraganglionic laminar endings) from mucosal endings would be useful in clarifying the physiological role of each of the aforementioned vagal endings. It is particularly important considering that subpopulations of vagal afferents may have completely different roles in appetite regulation. Moreover, the ability to differentiate the non-neuronal cells implicated in the normal functioning of the gut-brain axis, such as glial cells and interstitial cells of Cajal, would be useful  function. Pharmacological techniques, including the administration of capsaicin to destroy small-diameter sensory neurons, have been used. However, this method also kills some neurons in the CNS without killing all vagal sensory neurons (Ritter and Dinh, The above considerations are meant only to illustrate how transgenic tools can be useful to further our understanding and to predict the beneficial and deleterious consequences of gut-brain axis activity modulation by surgical, pharmacological, or device-assisted means. As the number of transgenic reagents available to gut-brain axis scientists continues to grow and become more sophisticated, we predictable that these new tools will play a critical role in the advancement of our knowledge of the development, morphological plasticity, molecular phenotyping, and connectivity of the healthy and diseased gut-brain axis. In addition to performing neuroanatomy, one can easily imagine many applications for these transgenic tools, including, but not limited to, patch clamp (Bailey et al., 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.