Scyliorhinus canicula, focusing on ScFoxg1a, ScShh, ScNkx2.1, ScDlx2/5, ScOtp, and ScTbr1 expression profiles and on the identification α-acetylated-tubulin-immunoreactive (ir), TH-ir, 5-HT-ir, and GFAP-ir structures by means of immunohistochemistry. Analysis of the results within the updated prosomeric model framework support the existence of alar and basal histogenetic compartments in the hypothalamus similar to those described in the mouse, suggesting the ancestrality of these subdivisions in jawed vertebrates. These data provide new insights into hypothalamic organization in cartilaginous fishes and highlight the generality of key features of the prosomeric model in jawed vertebrates.The hypothalamus has been a central topic in neuroanatomy because of its important physiological functions, but its mature organization remains elusive. Deciphering its embryonic and adult organization is crucial in an evolutionary approach of the organization of the vertebrate forebrain. Here we studied the molecular organization of the hypothalamus and neighboring telencephalic domains in a cartilaginous fish, the catshark,  Testing their conservation across vertebrates is a powerful approach for the correct establishment of homologies between embryonic territories beyond amniotes . The undScyliorhinus canicula, and analyzed them under the updated prosomeric framework. We have integrated data from neuroepithelial specification codes , and from the distribution of α-acetylated-tubulin-immunoreactive (-ir) and TH-ir cell groups, neuron-fiber tracts (5-HT-ir) and glial-processes (GFAP-ir). In the search of conserved traits among jawed vertebrates, we compared our data in S. canicula with that obtained in murine models. Our analysis reveals a strikingly high degree in the conservation of hypothalamic histogenetic compartments between chondrichthyan and murine models. Furthermore, we identified some of the boundaries and confirmed some of the assumptions predicted by the prosomeric model. However, some differences and discrepancies also exist mainly concerning the neuroepithelial specification genetic codes of the basal hypothalamus (BHy). Similar studies are required in other basal species to figure out if these differences should prompt the model update or they are the consequence of shark specialization.Latest updates of the model provide novel views on the organization of the rostral-most (secondary) prosencephalon, and its telencephalic and hypothalamic moieties . DetaileS. canicula sequences were retrieved by tblastn searches in transcriptomic databases obtained by Sanger and Illumina sequencing of embryonic and adult cDNA libraries (stages 8–25 and mixed adult tissues). Maximum-likelihood trees were inferred using the PhyML program version 3, the LG-F+Γ12+I substitution model and the SPR algorithm. Posterior probabilities (PPs) supporting groupings were calculated using the aLRT algorithm implemented in PhyML and are displayed as percentages at the corresponding nodes. Only PP >80% are indicated. The trees were viewed and edited using Mega6.Sequence alignments of the sequences listed in Table S1 were constructed using the alignment editor Seaview 3.0 and the MUSCLE algorithm. S. canicula) were supplied by the Marine Biological Model Supply Service of the CNRS UPMC Roscoff Biological Station (France) and the Estación de Bioloxía Mariña da Graña . Additional embryos were kindly provided by the Aquaria of Gijón , O Grove  and the Aquarium Finisterrae . Embryos were staged by their external features according to ∘C), pH (7.5–8.5) and salinity (35 g/L). Adequate measures were taken to minimize animal pain or discomfort. All procedures conformed to the guidelines established by the European Communities Council Directive of 22 September 2010 (2010/63/UE) and by the Spanish Royal Decree 53/2013 for animal experimentation and were approved by the Ethics Committee of the University of Santiago de Compostela.Some embryos of the catshark  in seawater and separated from the yolk before fixation in 4% paraformaldehyde (PFA) in elasmobranch’s phosphate buffer  for 48–72 h depending on the stage of development. Subsequently, they were rinsed in phosphate buffer saline (PBS), cryoprotected with 30% sucrose in PB, embedded in OCT compound , and frozen with liquid nitrogen-cooled isopentane. Parallel series of sections (12–20 μm thick) were obtained in transverse and sagittal planes on a cryostat and mounted on Superfrost Plus  slides.∘C and allowed to cool for 20–30 min at room temperature (RT). Sections were then rinsed twice in 0.05 M Tris-buffered saline  for 5 min each and incubated overnight with the primary antibody . Appropriate secondary antibodies  were incubated for 2 h at RT. For double immunohistochemistry (IHC) experiments, cocktails of primary antibodies were mixed at optimal dilutions and subsequently detected by using mixtures of appropriate secondary antibodies. Sections were rinsed in distilled water (twice for 30 min), allowed to dry for 2 h at 37∘C and mounted in MOWIOL 4-88 Reagent . All dilutions were made with TBS containing 15% donkey normal serum , 0.2% Triton X-100 (Sigma) and 2% bovine serum albumin . Double IHC with primary antibodies raised in the same species was performed as described in For heat-induced epitope retrieval, sections were pre-treated with 0.01 M citrate buffer (pH 6.0) for 30 min at 95∘C for 2 days, embryos were washed in 0.9% NaCl in distilled water, dehydrated in graded series of methanol solutions  and stored at -20∘C. Samples to be stained were placed on ice in 2 mL of dimethyl sulfoxide (DMSO)/methanol (1/1) until they sank. Then, 0.5 mL of 10% Triton X-100/distilled water was added, and the embryos were incubated for 30 min at RT. After washing in 0.05 M TBS with 0.1% Triton X-100  the samples were sequentially blocked using spin-clarified aqueous 1% periodic acid and 5% non-fat dried milk in TST (TSTM). Primary antibody  was diluted in TSTM containing 0.1% sodium azide for 2–4 days at RT with gently agitation on a shaking platform. The secondary antibody HRP-conjugated goat anti-rabbit, BIORAD, dilution 1:200 in TSTM) was incubated overnight. After a final washing in TST, the embryos were pre-incubated with 0.25 mg/mL diaminobenzidine tetrahydrochloride  in TST with 2.5 mg/mL nickel ammonium sulfate for 1 h, and then allowed to react with DAB in TST containing 2.5 mg/mL nickel ammonium sulfate and 0.00075% H2O2 for 20–40 min at RT. The reaction was stopped using Tris-HCL buffered saline and specimens were post-fixed with 4% PFA overnight at 4∘C. Epidermis and mesodermic derivatives were carefully removed and specimens were rinsed in graded series of glycerol  in order to directly observe the neural tube under the stereomicroscope.For whole mounts embryos were prepared as previously described in in situ hybridization (ISH) results were similar to those obtained by IHC, and therefore validate the specificity of the anti-Shh antibody used here.No immunostaining was detected when primary or secondary antibodies were omitted during incubations. Controls and specificity of anti-TH and anti-5-HT were performed as described in ScFoxg1a, ScShh (ScNkx2.1 (ScDlx5 (ScOtp (ScTbr1 (ScDlx2 (S. canicula embryonic cDNA library (mixed stages 9–22), constructed in pSPORT1, and submitted to high throughput EST sequencing. cDNA fragments were cloned in pSPORT vectors. Sense and antisense digoxigenin-UTP-labeled and fluorescein-UTP-labeled probes were synthesized directly by in vitro transcription using as templates linearized recombinant plasmid DNA or cDNA fragments prepared by PCR amplification of the recombinant plasmids. ISH in whole mount and on cryostat sections was carried out following standard protocols (∘C (in sections) or 70∘C (whole mount) and incubated with the alkaline phosphatase-coupled anti-digoxigenin and anti-fluorescein antibody  overnight at 4∘C. The color reaction was performed in the presence of BM-Purple (Roche). Control sense probes did not produce any detectable signal.We applied ISH for a, ScShh , ScNkx2.ScNkx2.1 , ScDlx5  .Light field images were obtained with an Olympus BX51 microscope equipped with an Olympus DP71 color digital camera. Dlx gene repertoire have been previously published . In each case, phylogenies were constructed from alignments containing deduced amino acid sequences of all paralogous sequences retrieved from catshark transcriptomic databases and from a representative sampling of actinopterygians and sarcopterygians. The trees were rooted using a Branchiostoma floridae sequence, except in the case of Otp which could not be found in the amphioxus Ensembl database. In the case of Foxg1, three strongly supported classes (posterior probability or PP > 90%), each containing a catshark and several osteichthyan sequences, were retrieved, highlighting for the first time the presence of three gnathostome Foxg1 classes . These classes were termed Foxg1a, Foxg1b, and Foxg1c, respectively. One coelacanth and several actinopterygian sequences, but no amphibian or amniote sequence, were found in the Foxg1b and Foxg1c classes, suggesting a loss of their representatives in tetrapods. We focused the expression analysis on ScFoxg1a, the catshark orthologue of the only Foxg1 gene retained in all major gnathostome lineages including tetrapods. The tree topology obtained for the Hedgehog family confirmed the presence of the three gnathostome classes, corresponding to the Indian Hedgehog, Desert Hedgehog and Sonic Hedgehog classes already reported in osteichthyans, and confirmed ScShh as the representative of the latter . Concerning the Nkx2.1/Nkx2.4 family, a single catshark gene could be identified and it was unambiguously assigned to the Nkx2.1 class based on the strongly supported grouping of its deduced amino acid sequence with teleost, chick, and human Nkx2.1 sequences . This gene is therefore referred to as ScNkx2.1 hereafter. A single catshark Otp related sequence, termed ScOtp, could be found and as expected, it clustered with the elephant shark sequence annotated as Otp in the reconstruction shown in Figure 1D. Finally, the Tbr1, Tbx21, and Eomes classes were retrieved with high statistical support  within the Tbr1/Tbx21/Eomes family. Each class contained a single catshark sequence at the expected position, allowing an unambiguous identification of the ScTbr1 gene analyzed in this study .Exhaustive phylogenetic characterizations of the catshark ublished , confirmFigure 2 summarizes the general architecture of the hypothalamus in mouse according to the updated prosomeric model .The prosomeric model establishes that hypothalamus and telencephalon are part of the secondary prosencephalon, which is understood as a segmental unit at the rostral-most point of the neural tube, the hypothalamus being located ventral to the telencephalon and rostral to the diencephalon , should be considered as caudal in this framework. Of note, the anterior commissure, located in the rostral-most roof plate, is a clear landmark of both the dorso-ventral and rostro-caudal axis .The model also postulates that the rostral-most point of the brain, referred as the acroterminal region (At), lies at the rostral border of the secondary prosencephalon. This region is restricted to the frontal border of the neural tube where left and right alar and basal plates meet. This border expands dorso-ventrally from the rostral-most roof plate  to the rostral-most floor plate . Thus, every structure classically considered being dorsal or ventral to these points : hp2  and hp1 . Each segment harbors telencephalic and hypothalamic derivatives . However, the telencephalon harbors only roof and alar plates while the hypothalamus harbors alar, basal, and floor plate derivatives. The existence of these segments is supported by several genes differentially expressed in the rostro-caudal axis, the location of commissures in the roof and floor plates , and the course of important tracts  running by a common path at the rostral border of hp1, through alar and basal plates. These data, in turn, support the existence of an intersegmental boundary that separates terminal and peduncular subdivisions of both telencephalon and hypothalamus, which is referred as the intrahypothalamic boundary . Caudally, the secondary prosencephalon limits with the diencephalon at the hypothalamic diencephalic border (HDB), another intersegmental limit among hp1 and p3, though it should be noticed that part of the caudal limit of the secondary prosencephalon does correspond to the telencephalon .The secondary prosencephalon presents two true segments rostro-caudally arranged . Of note, the POA also harbors the anterior commissure .The model considers the adult hypothalamic organization arranged in different histogenetic territories defined by neuroepithelial specification codes and radial units . These cFoxg1 is one of the earliest transcription factors expressed specifically in the part of the neural plate that gives rise to the telencephalon and it remains expressed throughout the telencephalon during embryonic development  up to a caudal point in the roof plate, which has been tentatively identified as the dorsal border between the telencephalon and the diencephalon . At stage 22, ScFoxg1a was observed in the telencephalon and in the nasal part of the optic cup . The expression in the telencephalon was maintained until late stages of development , which allowed identifying the border between the telencephalon and the hypothalamus.In mice, ment see . In an aScShh expression was detected during gastrulation (stage 12) in the caudal midline of the embryo (data not shown). At stage 14, during early neurulation, it has been detected in the axial mesoderm of the notochord and the prechordal plate and in the ectoderm of the caudal midline (data not shown). After the closure of the neural tube (stage 17), the signal was detected as a ventral longitudinal continuous band that extends from the caudal end of the spinal cord to the At of the forebrain, roughly at the level of the optic stalk . As in other vertebrates . At stage 19, ScShh expression became downregulated in the forebrain to progressively give rise to a caudal and a rostral domain . The narrow transverse and dorsally directed stripe of ScShh-expressing cells within the caudal domain was identified as the developing zona limitans intrathalamica . The rostral border of the ScShh caudal domain, in turn, was somewhat extended rostral to the HDB  and one (intermediate) negative for ScShh . At stage 24 , a new domain emerged within the telencephalon. This short domain  extended from a region located dorsally to the optic stalk without reaching the prospective territory of the anterior commissure . A clear gap of expression was observed between this telencephalic domain and the rostral hypothalamic one . The telencephalic domain was located medially while the hypothalamic one also expanded laterally (not shown). From stage 27 onward the zli expanded dorsally toward the roof plate . At stage 29 the medio-lateral histologic organization of the developing walls of the forebrain become more evident. As in previous developmental stages, Shh immunoreactivity was clearly identified in the basal plate of the diencephalon entering the caudo-ventral part of the BHy  and in the rostro-dorsal part of the BHy , so that a clear negative gap of Shh-immunoreactivity occupied most of the caudal BHy  and part of the rostral BHy (RBHy). In the telencephalon, Shh-immunoreactivity expanded caudally beyond the prospective territory of the anterior commissure . Of note, from late stage 30 onward, Shh-immunoreactivity is downregulated in the CBHy and basal diencephalon, except in the zli (data not shown).tebrates , the exp the HDB , which aScNkx2.1 was first detected at stage 18 in the rostro-ventral portion of the forebrain, in a longitudinal band which extended ventral to the optic stalk . At stage 23 ScNkx2.1 was expressed in most of the BHy . Differently from ScShh, ScNkx2.1 delimited the ABB even in the CBHy , though a small gap of expression was observed within the caudo-ventral part of the BHy . A second domain emerged in the telencephalon at this stage . This domain was restricted to the rostral-most portion of the telencephalon and extended from a region located dorsally to the optic stalk to the prospective territory of the anterior commissure . A clear gap of expression was observed between the telencephalic and the hypothalamic domains. At stage 25 , as in previous developmental stages, ScNkx2.1 expression was lacking in a small domain located within the caudo-ventral BHy . This region seems to fit with the rostral border of a basal α-acetylated-tubulin-ir tract [see mammillo-tegmental tract (MTT) in Figure 3I]. In the telencephalon, ScNkx2.1 expression became caudally expanded beyond the prospective territory of the anterior commissure . At stage 29, as in previous developmental stages, ScNkx2.1 was observed throughout most of the BHy, except in a small wedge-shaped domain within the caudo-ventral BHy . Groups of ScNkx2.1-expressing cells in the caudo-ventral portion of the hypothalamus were observed along the marginal zone . In the telencephalon, ScNkx2.1 expression expanded beyond the territory it occupied at previous developmental stages . Of note, Shh immunoreactivity was overlapping with ScNkx2.1 expression beyond the anterior commissure .The expression of ScNkx2.1 expression in the forebrain is dependent on Shh, we used in ovo injections of the Shh inhibitor cyclopamine. All control embryos (n = 4) exhibited the expected ScNkx2.1 signal in the rostral-most and ventral-most portion of the forebrain . This signal was lost in all embryos dissected following cyclopamine treatment (n = 3), supporting the conclusion that Shh signaling is required for the initiation of ScNkx2.1 expression in S. canicula as in osteichthyans.In order to test whether, as in osteichthyans, the initiation of ScDlx5 from stage 18 onward and the expression of ScDlx2 from stage 29 onward. Fairly identical results were observed with both markers in the brain of S. canicula from stage 29 onward, so we use ScDlx2/5 at these stages to refer indistinctly to both.We analyzed the expression of ScDlx5 expression and detailed profiles in the developing branchial arches have been previously described from stage 15 to stage 27 in ScDlx5 expression was found in the most anterior part of the neural tube , ScDlx5 expression spread caudally and ventrally  and reached the rostral-most portion of the optic stalk  and entered p3 . Therefore, this domain delineates the ABB along the hypothalamus. Of note, the longitudinal domain appeared to codistribute with α-acetylated-tubulin tracts . Although both ScDlx5 domains were continuous, a wedge-shaped area of reduced signal intensity was observed between the dorsal  and the ventral  domains . Two bands of cells were additionally observed, which were ventrally located with respect to the longitudinal domain . One was located at its caudal end and spread ventral-ward at the rostral end of p3. The other spread perpendicularly to the ABB from the caudo-dorsal part of the BHy up to the rostral hypothalamus . Of note, this ScDlx5-expressing domain appeared to delineate ScShh expression in the rostral hypothalamus . This pattern became more patent at stage 29 . At this stage, ScDlx2/5-expression was observed in the telencephalon . The telencephalic domain is almost continuous at medial levels  while a clear gap of expression was observed at the level of the anterior commissure in parasagittal sections . ScDlx2/5 expression was also observed in a longitudinal domain outlining the ABB, which crossed through the alar hypothalamus (AHy) and entered p3 . A wedge-shaped negative domain separated the telencephalic and hypothalamic-diencephalic domains . A transverse band of non-ventricular ScDlx2/5-expressing cells was observed extending ventral-ward from the alar plate , along the rostral-most p3Tg. This domain appeared to overlap with the ScShh-expressing domain from which the zli emerged . As observed previously, an additional domain  cut across the BHy perpendicularly to the ABB. This pattern was maintained until late stages of development .General features of ScOtp signal was detected in the rhombencephalon and in the rostral-most and ventral-most portion of the optic stalk  though a faint ScOtp expression was also observed in part of the BHy and in the AHy. At stage 25, three domains of ScOtp expression were observed in the forebrain. The rostral one was restricted to the rostral-most region of the forebrain, expanding ventrally from the optic stalk along the RBHy without reaching the prospective neurohypophysis. The second domain abutted the HDB at the intersection with the ABB and spread from this region up to the rostro-ventral hypothalamus. The third domain overlaid the ABB from the optic stalk up to the alar p3 and was poorly stained compared to the other two domains . From stage 28 onward these three domains were respectively identified in the RBHy , in an arched domain that spread from the CBHy  and in the AHy . In the BHy, a small domain containing ScNkx2.1 alone was identified caudal to the ScOtp-expressing domain . A novel domain of non-ventricular scattered ScOtp- expressing cells was also detected entering p3Tg from the most caudo-ventral part of the BHy . Scattered ScOtp-expressing cells were observed between the alar and basal domains of the caudal hypothalamus . In the AHy, ScOtp-expressing cells were only observed at parasagittal levels . Of note, non-ventricular ScOtp-expressing cells appeared to codistribute with the longitudinal band of ScDlx2/5 expression over the ABB . ScOtp-expressing cells were also observed in the telencephalon (data not shown). This pattern is maintained until late stages of development. At stage 30 , a gap of ScOtp expression was observed at parasagittal levels that divide the AHy in rostral and caudal domains.At stage 19, ScTbr1 signal was detected at stage 25 in dispersed cells that spread the telencephalic vesicle , except for a rostral domain that extended from the optic stalk to the prospective territory of the anterior commissure . Of note, α-acetylated-tubulin-ir tracts reaching the telencephalon seem to define the boundary between telencephalic ScTbr1 positive and negative domains . ScTbr1 expression was additionally observed in the dorsal most part of the alar p3, abutting the ScDlx5 domain that entered p3 . At stage 29, the extension of the ScTbr1-expressing domain became decreased in the telencephalic vesicle (not shown).Figure 4I). Besides, some fibers were detected ascending throughout the AHy  toward the telencephalon  from the caudal part of the hypothalamus. This pathway seemed to concur with negative domains for ScDlx2/5 and ScOtp genes at the same developmental stage . A group of decussating fibers was detected close to the most caudo-ventral region of the hypothalamus  that coincides with the caudal-most and ventral-most domain of ScNkx2.1 in the BHy  and also with the rostral end of the ventral-most α-acetylated-tubulin-ir tract at stage 25, that could represent a pioneering tract, the mammillo-tegmental tract . Of note, from stage 30 onward, Shh immunoreactivity was only observed in the rostral portion of the zli but not in the caudo-ventral part of the BHy nor the diencephalic basal plate .Anti-5-HT immunoreactivity was coanalyzed with anti-Shh immunoreactivity at stage 30 to better understand the segmental organization of different fiber bundles, which in turn contributes to the understanding of the organization of the hypothalamus. Immunoreactivity for both markers was examined at stage 30 when the first 5-HT-ir fibers reach the telencephalon . PositivFigures 4O,P). Ascending fibers to the telencephalon were detected in a similar pathway to that described above for 5-HT . Some GFAP-ir processes were also observed in the same point where 5-HT-ir fibers decussate in the caudo-ventral part of the BHy . GFAP-ir processes were also observed in the subpallium .Glial fibrillary acidic protein immunoreactivity was also analyzed in the forebrain at stage 31. Radial and longitudinal GFAP-ir processes were detected through the whole forebrain  the AHy, together with the telencephalon, are the rostral-most regions of the alar plate. The AHy is located ventral to the Foxg1-expressing telencephalon, dorsal to the BHy , and rostral to the alar p3 (characterized by the complementary expression of Dlx and Tbr1 genes). Within the territory delimitated by the above-mentioned genes, two longitudinal  histogenetic domains are defined based on the complementary expression of Dlx and Otp genes. The dorsal-most domain is termed paraventricular domain (Pa), and expresses Otp but not Dlx genes. The ventral-most is the subparaventricular domain (SPa), which expresses Dlx genes but not Otp. Dlx is expressed beyond the HDB in the alar p3. The alar HDB is defined by the clear-cut expression among genes restricted to the AHy  or to the alar p3 . The alar p3, besides, includes the prethalamic eminence (PThE) and express genes in a complementary manner . Moreover, according with the prosomeric model both, Pa and SPa domains, present terminal (hp2) and peduncular (hp1) domains . However these subdomains cannot be genetically identified without additional markers. Thus, the AHy presents at least four different histogenetic domains although some work on the development of hypothalamic peptidergic cells point to much more subdivision . Thus, ScFoxg1a was expressed from early stages of development in the presumptive telencephalon , leading to the identification of the telencephalon-AHy border . ScNkx2.1 was expressed in most of the BHy  and delineates the ABB . The caudal border of the AHy could be delimited at stage 25 by the caudal-most expression of ScOtp at parasagittal levels . Both ScOtp and ScDlx5 were expressed from stage 25 onward within the AHy, though the intensity of the expression increases in late stages of development. At stage 29, both ScOtp and ScDlx2/5 were observed in the AHy. Of note, two mutually exclusive histogenetic domains dorso-ventrally arranged could be readily observed at ventricular levels , though non-ventricular ScOtp-expressing cells were also observed within the ScDlx2/5 domain. These observations allowed us to identify the TPa/PPa-like and the TSPa/PSPa-like domains . The former, ScOtp-expressing one, abutted a ScTbr1 domain that occupied the dorsal-most part of p3 , which is also negative for ScDlx2/5 . The latter, ScDlx2/5-expressing one, as in mouse, was continuous with the transverse ScDlx2/5-expressing domain in p3 .In the shark, we have studied the expression of Tbr1 in the mantle of PPa .In the mouse, and using markers homologous to those studied here, termino-peduncular compartments can be differentiated only by the late expression of e of PPa . While SFigure 6A). The BHy is characterized by the expression of Nkx2.1 in its whole extension with the exception of its caudal-most region. It harbors three longitudinal domains dorso-ventrally arranged: tuberal/retrotuberal region (Tu/RTu); perimammillary/periretromammillary region (PM/PRM) and mammillary/retromammillary region (MM/RM). These domains harbor terminal and peduncular parts separated by the IHB. The peduncular domains are distinctly referred in this basal region with the prefix “retro” while the terminal lack prefix. In the mouse , the Tu/RTu compartment is characterized by the expression of Dlx5 although it also presents subdomains expressing Shh (in part of the Tu and the whole RTu); the PM/PRM is characterized by the expression of Nkx2.1 and Otp although Shh is also expressed in the PRM; the MM only expresses Nkx2.1  while the RM express Shh and other genes in a complementary manner to Nkx2.1  or to the p3Tg . Within this territory, ScOtp was expressed in a restricted stripe at the most rostro-dorsal part of the basal plate spreading ventral-ward from the ABB without reaching the neurohypophysis . ScShh overlapped with this domain, extended caudally and abutted a ScDlx2/5-expressing domain that extended from the ABB up to the neurohypophysis. Therefore, in sharks, the Tu/RTu-like domain would be composed by four subdomains: a rostro-dorsal subdomain coexpressing ScShh, ScOtp and ScNkx2.1; a second subdomain expressing ScShh and ScNkx2.1; a third subdomain expressing ScDlx2/5 and ScNkx2.1; and a ventral subdomain expressing ScNkx2.1 alone . As in mouse, and with the markers studied here, it was not possible to establish a clear distinction between the Tu-like and the RTu-like compartment. It is noteworthy that the Tu/RTu-like compartment has a different histogenetic identity to that described in the mouse. On one hand, ScShh was down-regulated in a large portion of the BHy. On the other, ScDlx2/5 expression appeared much more restricted than in mouse, and different subdomains could be delineated, including a domain expressing ScNkx2.1 alone in midsagittal sections . In mouse, the single domain in the BHy that contains Nkx2.1 expression alone was identified as the MM region. The ScNkx2.1 expressing region ventral to ScDlx2/5 is unlikely to correspond to the MM for two reasons. First, it is not the only compartment where we detect the expression of ScNkx2.1 alone. Indeed, in mid-sagittal sections, a small domain can be observed ventral to ScOtp, which expressed ScNkx2.1 alone . Second, the presence of a MM-like domain at this location would imply the interruption of longitudinal compartments  and the redefinition of terminal domains within the BHy. Since it has been previously reported that, in mouse, some tuberal areas lack Dlx gene signal . At its caudal-most portion, this domain seems to express ScOtp only in the ventricular zone , while in its rostral-most portion ScOtp is mainly expressed on mantle cells. This ScOtp-expressing domain (including either ventricular or mantle cells) was therefore interpreted as a PM/PRM-like domain, where the PRM-like domain is likely to correspond to caudal ScOtp expression in cells at the ventricular zone, and the PM-like would mainly correspond to rostral ScOtp expression in mantle cells.According to the prosomeric model, in mouse, adjacent to the Tu/RTu area, a distinct PM/PRM histogenetic domain exists in which ScNkx2.1 alone was identified ventral to the ScOtp-expressing domain . This domain abutted a ScShh-expressing domain which expanded caudally through the diencephalic basal plate . Based on these expression similarities, the ScNkx2.1 and ScShh-expressing territories were respectively identified as the MM-like and the RM-like domains. The caudal border of the RM-like domain abutted a transverse band of non-ventricular ScDlx2/5-expressing cells at the p3Tg .These ScDlx2/5-expressing cells are just ventral to the ScDlx2/5-expressing domain in the alar p3, supporting their assignment to p3Tg. Furthermore, these cells are in the same position that Pax6-ir cells in the caudal posterior tuberculum of the shark, described by Dlx-expressing cells have not been described in the mouse but Dlx2- and Pax6-expressing cells have been described in the basal plate of zebrafish as belonging to the preglomerular complex (p3Tg), suggesting that their presence may be an ancestral characteristic of jawed vertebrates lost in mammals . A posterior tuberculum harboring TH-ir cells has been classically related to the hypothalamus (ScShh-expressing) domain while the caudal-most located portion of these TH-ir cells and fibers belongs to p3Tg . Thus, the rostral-most portion of TH-ir cells should be understood as hypothalamic. This configuration seems to fit parsimoniously with the prosomeric model under the light of the following facts. TH-ir ascending fibers from the rostral posterior tuberculum to the telencephalon seem to arise from these TH-ir hypothalamic groups  just before TH-ir cells emerge in the rostral posterior tuberculum , which support a PM/PRM-like origin for ScOtp-expressing cells in p3Tg.The shark hypothalamus and the posterior tuberculum have been analyzed before, mainly under neurochemical and topographical approaches. The posterior tuberculum in chondrichthyans extends caudally from the posterior recess (or mammillary recess). This recess lies just at the caudal and ventral border of thalamus  althoughthalamus . Our preberculum . Interesr groups . Finallynd mouse , scatterOtp, and that TH-ir cells of the RM-like domain send ascending projections to the telencephalon caudally to the IHB.Thus, it appears that, among different vertebrates, at least some populations of the RM-like and p3Tg emerge from hypothalamic domains expressing Figure 6A). This boundary is supported by (i) the existence of the anterior and retromammillary commissures in the roof and floor plates, respectively, (ii) the restricted expression of several genes to one or other segment, and (iii) the course of main tracts  separating both segments. Thus, there is a correlation among histogenetic data and fiber tract data. In fact, it was argued that the course of tracts is a powerful test for brain models since they are also guided by mechanisms related to those involved in histogenetic patterning , the IHB is also supported by (i) the existence of commissures in the alar and floor plates, (ii) the restricted expression of ScShh and ScNkx2.1 in particular subdomains within either the rostral or the caudal segment, and (iii) the presence of different neurochemical populations of fibers, topologically homologue to those described in the model, which divide the hypothalamus in a caudal  and a rostral  domain. Thus, we have tentatively defined an IHB-like based on genetic or histogenetic data and partial neurochemical or fiber tract data. We also discuss the congruence between the two kinds of data, when possible.In the shark  and GFAP-immunoreactivity at late development . In the floor plate, the IHB coincides with the border between MM and RM-like subdomains. In mouse, the IHB at this point can be associated with the retromammillary commissure  where fx tracts cross at its rostral-most portion. This commissure seems to be continuous and indistinguishable from the prethalamic (p3Tg) commissure, a bit more caudally arranged, where fibers (partly p3Tg and partly from the raphe nuclei) cross  connects hypothalamic cell masses, fibers of the tract of the saccus vasculossus decussate in the caudal part .In the roof plate, according to the prosomeric model, the IHB ends caudal to the anterior commissure. This commissure has been identified in ross see . Based oSix3, Neurog3, Six6, Nkx2.6 or de rostral border of Tbr1, Uncx4.1, Sim1, Olig2, Foxb1, Nr5a1, which is the same point whereby the mfb, lfb, and fx course .Either the expression of different genes (that would help identifying different histogenetic domains) or, accordingly, the course of the IHB through the telencephalon, has not been analyzed here. In the AHy, the prosomeric model proposes that the IHB can be delineated just caudal to the optic stalk, at the caudal border of expression of genes like e fx run . TherefoS. canicula, as discussed above, no evidence for subdivisions along the rostro-caudal axis could be found on the basis of ScOtp or ScDlx2/5 expression. In the BHy, two distinct territories could be inferred from ScNkx2.1 and ScShh expressions, the MM-like  and the RM-like  domains , which is consistent with the predictions of the prosomeric model. According to the prosomeric model, the IHB can be delineated between both domains. The other compartments (Tu/RTu and PM/PRM) are presumably divided by the IHB, but any other gene among those used here serves as a caudal (hp1) or rostral (hp2) marker, except for ScShh in the Tu/RTu-like domain that, in S. canicula (but not in mouse) seems to be restricted to the rostral (hp2) domain.In the AHy of fasciculus basalis telencephali in chondrichthyans literature [the sot in zebrafish literature . Of note, 5-HT-ir and GAD-ir cells have been also identified in the posterior tuberculum of the shark and other vertebrates. Whether ascending projections from this source coursed to the telencephalon has not been determined to date . The most conspicuous of these tracts were 5-HT-ir tracts recognizable at stage 30 . This path is also followed by GFAP-ir processes which, besides, cross at the anterior commissure . Since, as reported in mouse, they coursed just caudal to the optic stalk, we propose that these tracts in S. canicula course caudally to the IHB, separating the TPa and TSPa (hp2) from the PPa and PSPa . While, with the markers used here, we cannot ascertain if this boundary in the AHy is also supported by histogenetic domains, these tracts appear to course through gaps of ScDlx2/5 and ScOtp expression in the telencephalon .In the alar plate, 5-HT-ir, GAD-ir, TH-ir and GFPA-ir fibers of the mfb, arising from different points of the brain, have been observed ascending to the telencephalon by a common path just caudal to the optic stalk, which could correspond to the alar IHB  to the postinfundibular commissure  through the mfb. Note that, in contrast to mouse, the IHB in S. canicula can be traced by partial genetic and fiber tract evidences, since tracts coursing through the telencephalon to the floor plate have not been demonstrated. In the alar plate the mfb courses topologically caudal to the optic stalk and is known to be composed by different neurochemical systems arising from different points of the brain. In the basal plate the mfb is known to be composed, at least, of TH-ir fibers which arise upward from the rostral posterior tuberculum.To summarize, our data support the conclusion that, in S. canicula, within a prosomeric and histogenetic framework. These data reveal striking similarities with the organization described in the mouse by means of the updated prosomeric model , the Xunta de Galicia , European Community-Research Infrastructure Action under the FP7 “Capacities” Specific Programme (ASSEMBLE 227799), the Région Centre, Région Bretagne , National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d’Orléans and Université Pierre et Marie Curie. GNSD would like to thank Spanish SEPE for its funding support.http://www.frontiersin.org/journal/10.3389/fnana.2015.00037/abstractThe Supplementary Material for this article can be found online at: Click here for additional data file.


RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5′-maturation, is ubiquitous in all domains of life, but the evolution of its protein components  is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs , we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs. RNase P, a ubiquitous endonuclease responsible for tRNA 5′ maturation in all three domains of life ,4,5,6,7,multiple RPPs , indicating the broad functional importance of the zinc-ribbon motif. Pfam’s seed alignment of 291 archaeal and eukaryotic RPP21 homologs (pfam04032) confirms near-universal conservation of both cysteine motifs but not of the arginines; the latter residues are substituted with lysine at high frequency. A recent study that examined an alignment of eukaryotic RPP21 homologs also observed conservation of these cysteine motifs, though the motif sequences were slightly different .,34.20,34ormation . This rePho RNase P [Pfu and Pho. Nevertheless, such differences only heighten the importance of the few conserved positions between archaea and eukaryotes  for strong protein–protein interactions.Residues E16 and R17 in RPP21 form prominent intermolecular salt bridges with residues H46 and E47 in RPP29, respectively  also identifies R17, Y18, R71, S103, G104, and T105 as near-universally conserved.Pho RPP30 meet the 80% sequence identity threshold, but this number increases to 11 when we use a 75% threshold. Upon mutation in Pho RNase P, R90A and R176A exhibit two- and three-fold decreases in activity, respectively, indicating their importance for function [Of the RPPs examined, RPP30 shows the least conservation—only five out of 212 residues in function . HoweverMEME analysis indicates that while only one out of eight possible motifs is conserved between archaeal and eukaryotic RPP30, this motif includes R176 and G189. An alignment of 40 archaeal and eukaryotic RPP30 sequences in Pfam (pfam01876) confirms the conservation of R176.Pho POP5•RPP30  helix with Watson-Crick base pairs, a three-nucleotide bulge, and a 3′ non-canonical (NC) helix with two consecutive airs see C, inset.airs see C. MutatiOur sequence analyses identified residues conserved in only archaea  as well An unexpectedly large number of glycines are highly conserved in the RPPs. The ability of glycine to adopt a wide range of φ and ψ angles makes it well suited for turns where other residues with side chains are less favored. For example, G47 in POP5 is universally conserved, likely due to its key role in facilitating the sharp turn between α-helices H1 and H2 A. In somi.e., convergent evolution). This claim is supported by the finding that, while dissimilar in amino acid sequence, POP5 and the bacterial RPP share a similar tertiary fold [Given the structural and functional similarities of the RPR in all three domains of life ,18,65, iary fold ,27.Thermoplasma acidophilum, NanoarchaeotaNst1). It is difficult to conclude whether these instances reflect a smaller suite of RPPs or if the “missing” RPPs have diverged extensively, thereby escaping detection by sequence similarity searches. However, at least in a few cases, the change in RPP number may reflect a corresponding change in the RPR. For example, the RPR, which typically comprises a specificity and a catalytic domain, is somewhat smaller in Thermoproteaceae (phylum Crenarchaeota) due to an abbreviated specificity domain [Nst1 RPR has undergone remodeling. Once these computationally predicted thematic variations in the make-up of archaeal RNase P are experimentally validated, it would be beneficial to investigate whether such rewiring of the RNase P holoenzyme (through changes to the RPR and/or RPPs) reflects either coevolutionary adaptations to a specific suite of substrates (pre-tRNAs or otherwise) and or the need to network with other macromolecular assemblies.Consistent with our postulate above, we predict that new variants of archaeal RNase P will be uncovered as additional phyla in unique niches are studied. Indeed, in our analyses, we found instances where the genes for some but not all RPPs are present (e.g., y domain . Thermopy domain , a findiy domain ,25,26,28y domain . Since Py domain ,25,26,28The presence of L7Ae in archaeal RNase P, snoRNPs, and the ribosome, which are all macromolecular machines involved in some aspect of translation, suggests coordinate regulation of these complexes. The expectation for such crosstalk is heightened by the presence of RPP29 in an operon encoding ribosomal proteins. In fact, this appears to be a recurring theme in all three domains of life: bacterial RPP is co-transcribed with the ribosomal protein L34, and yeast RNase P has been implicated in the processing of antisense RNAs from genes that encode ribosomal proteins ,69. The Based on our analysis of an extensive collection of archaeal genomes, we propose that the last common ancestor of archaeal and eukaryotic RNase P consisted of one RPR and five RPPs–RPP21, RPP29, POP5, RPP30, and L7Ae. Moreover, by examining sequence conservation in the context of the high-resolution structures, we recommend residues for mutational analysis to help uncover structure-function relationships in these RPPs.


Mycobacterium avium subsp. paratuberculosis were assessed in 17 infected herds . Additionally, the level of knowledge of herd managers about the disease was assessed. In a case–control study with 4 matched negative control herds per infected herd, the association of potential risk factors with the infection status of the herd was investigated.Many studies have been conducted to define risk factors for the transmission of bovine paratuberculosis, mostly in countries with large herds. Little is known about the epidemiology in infected Swiss herds and risk factors important for transmission in smaller herds. Therefore, the presence of known factors which might favor the spread of paratuberculosis and could be related to the prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis of 6.1% was found in infected herds, whereby shedders younger than 2 years of age were found in 46.2% of the herds where the young stock was available for testing. Several factors related to contamination of the heifer area with cows’ feces and the management of the calving area were found to be significantly associated with the within-herd prevalence. Animal purchase was associated with a positive herd infection status .Exposure of the young stock to feces of older animals was frequently observed in infected and in control herds. The farmers’ knowledge about paratuberculosis was very limited, even in infected herds. An overall prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis from adult animals to the young stock were observed in infected Swiss dairy and beef herds, which may be amenable to improvement in order to control the disease. Important factors were contamination of the heifer and the calving area, which were associated with higher within-herd prevalence of fecal shedding. The awareness of farmers of paratuberculosis was very low, even in infected herds. Animal purchase in a herd was significantly associated with the probability of a herd to be infected and is thus the most important factor for the control of the spread of disease between farms.Numerous risk factors favoring the spread of  Mycobacterium avium ssp. paratuberculosis (MAP). Clinical signs include profuse diarrhea unresponsive to treatment and significant weight loss in spite of good appetite. Once these symptoms are established, the disease is characterized by chronic wasting with a fatal outcome. Paratuberculosis results in substantial economic losses through decreased production [Paratuberculosis (PTB) or Johne’s disease is a chronic enteritis of ruminants caused by an infection with oduction , increasoduction .Infection with MAP typically occurs during the first year of life because calves are more susceptible to infection than older animals . HoweverAs no effective treatment exists against PTB, efforts are focused on preventive strategies. Several studies have investigated the control of PTB in affected dairy farms -12, but Little is known about the MAP infection status of cattle herds in Switzerland. In particular, there are no recent studies which describe the prevalence of the disease in the Swiss cattle population or within infected herds. Within-herd seroprevalences were estimated between 4.2% and 21.5% in the early 1990s . AnotherThe aim of the present study was to acquire further knowledge on MAP infection in affected Swiss dairy and beef herds, to assess the level of disease awareness of herd managers, to identify risk factors associated with high within-herd prevalence, and to identify risk factors which are more common in infected herds than in herds without a PTB history.Twelve infected dairy and nine infected beef herds were identified based on the records of the Ruminant Clinics of the Vetsuisse Faculty in Berne and Zurich, of veterinary practitioners and of state veterinary officers. The farmers were then invited to join the study by phone call. The main inclusion criterion was the presence of at least one confirmed case  of clinical PTB during the five years preceding the start of the study. The confirmed cases had to have been born on the farm.The 21 herds included in the study were visited between February 2011 and July 2011. During these visits, the husbandry practices by age group, the feed storage, water supply, and manure storage were recorded. Furthermore, a questionnaire was completed through an interview with the farmers to assess potential risk factors for PTB on the farm, with different questionnaires for dairy and beef farms. Both questionnaires had previously been evaluated on test farms for applicability and clarity before they were used on the study farms. They included open and closed questions related to general characteristics of the farms, such as size, cattle breed(s) and production level, as well as targeted questions referring to potential risk factors for MAP transmission, chosen based on available information from the literature , and queA fecal sample of each animal of one year of age or older was collected for MAP testing. Cattle that had been bought from other farms only for fattening and were kept separately from the herds were excluded from the sampling. Obstetric gloves used for fecal sample collection were changed between animals. The samples were sent to the laboratory overnight and reached it within 24 hours. After arrival, they were frozen at -20°C if immediate processing was not possible. The duration between sampling and processing ranged from one day to 3.3 months.Fecal samples from three animals of the same herd  were pooled for MAP analysis. The pools were analyzed according to established methods, both by culture  and F57 Four control herds per infected herd were enrolled to evaluate whether classical risk factors for MAP transmission were more often present on infected farms than on PTB-free farms. These herds had to be without any history of PTB for the ten years preceding the start of the study. They were matched to the individual infected herds by type (dairy/beef), size, cattle breed and region. The questionnaires used for the visits of the control farms were similar to the questionnaires for the infected farms, except for the questions about the PTB history of the herd which were deleted, as well as some general questions about the farms not directly related to PTB transmission, as personnel, forage, disease prevention, herd health issues, and future of the farm.The study was designed as a case–control study for the identification of risk factors in the management of infected farms as compared with control herds, while risk factors associated with high prevalence levels were evaluated in relation to the MAP excretion levels within infected herds.. The significance level was set at p < 0.05. Because the control herds had not been confirmed as truly negative other than based on history, a second analysis was undertaken with the assumption that 10%  could still be positive in reality. Briefly, a Monte-Carlo simulation was carried out. Each of the control herds was randomly assigned a disease-positive status with a probability of 10%. The conditional logistic regression was repeated using the modified data set and a new set of modified parameters was estimated. This was repeated 10,000 times to build up a probability distribution of the modified parameters in the model and statistical inference was calculated on this probability distribution. Thus there were 10,000 replicates of the parameters and from these 10,000 replicates the median and 95 percentiles could be calculated.For comparison of infected herds with control herds, univariable analysis using logistic regression was applied as an initial screening test to identify potential risk factors associated with the herds’ infection status. This was followed by conditional logistic regression that included all variables with a p-value of < 0.2 in the univariable analysis. Three analyses were performed, first for the factors that were present in both dairy and beef herds, and subsequently for potential risk factors associated with infection in dairy or beef herds separately. Competing models were analyzed using the Akaike Information Criterion (AIC) and the best model was selected on the basis of the lowest AICFurther analyses were performed to investigate the relation between potential risk factors and the prevalence of MAP shedding within infected herds using logistic regression. However, in this case, only a univariable analysis was conducted as multivariable analysis was not possible due to the small number of infected herds participating in the study. Likewise, three analyses were performed: one for all herds, one for dairy and one for beef herds separately. For calculation of the within-herd prevalence, the infection status of animals aged two years or older only was considered, in order to have a comparable sample population across all farms, as the heifers younger than two years of several herds were kept on separate farms under rearing contract and therefore were not available for testing.This study is reported in conformity with the STROBE statement for reporting observational studies . All staThe study was conducted in accordance with the animal welfare legislation of Switzerland, all  aspects of the study had been previously approved by the Swiss Veterinary Office. All farmers were thoroughly informed about the project prior to the herd visit and gave their informed consent for the sampling of fecal specimens from their animals and for completion of a questionnaire regarding herd management practices.Twelve dairy herds and nine beef herds were enrolled in the study between January and July 2011. Two dairy and two beef herds were later excluded from further analyses because all fecal cultures were negative for MAP. The four excluded herds were small with 11 to 21 tested animals.A total of 1120 animals older than one year were tested for MAP shedding in the 17 remaining herds. The number of tested animals per herd ranged from 25 to 130 , with an average of 66 animals older than one year (73 for dairy and 56 for beef herds) or 49 cows (55 for dairy and 41 for beef herds), respectively. Several breeds were represented in the infected herds. The dairy herds included three Red Holstein herds, three Holstein Friesian herds, one Jersey herd, one mixed herd with Red Holstein and Swiss Fleckvieh, one mixed herd with Holstein Friesian and Brown Swiss, and one Brown Swiss herd with some Jerseys. In the latter herd, PTB cases had been recorded in the Jersey population only. The beef cattle population under study consisted of four Limousin herds, two Angus herds and one Charolais herd.The most important data about general management practices are listed in Table Calving of over 90% of the cows in individual calving pens was observed in only three farms 18%). In the other farms, it could happen that two cows shared the same calving pen, that they had group calving pens, or that calvings took place in the barn  received colostrum from their own dam only. In the remaining 20%, calves from first-calf heifers occasionally received colostrum from an older cow. After the colostrum, all were fed bulk tank milk, except on one farm where they were fed a milk replacer. Waste milk containing antibiotics or high somatic cell counts was also fed in 78% of the farms feeding milk.In the beef herds, the calves usually stayed in the herd with their dams for ten months. During that time, a straw-bedded area to which the adult did not have access was available for the calves in all farms.In 40% of the dairy farms, the young stock went to contract heifer-rearing facilities at an age between one and four months. In contrast, only one beef farmer sent roughly 10% of his heifers at the age of 14 months to another farm. Dairy and beef heifers generally stayed at the contract rearing farm until approximately one month before calving.During the summer, 20% of young dairy cattle aged less than a year and 71% of all (dairy and beef) heifers aged more than a year were kept on pastures that were either pastured by cows or where slurry from the cow herd had been spread during the same season. In all beef herds, with one exception, slurry was also spread during the pasturing season.In general, the farmers’ knowledge about PTB was limited on infected farms and very low on control farms Table . Little In total, 1120 animals were tested for MAP excretion. As the young stock was kept on rearing contract farms in some of the herds, only the 913 animals (602 from dairy herds and 311 from beef herds) aged two years or older were considered for the determination of prevalence in order to have a valid comparison across all herds. Of the animals aged two years or older, 46 (5.0%) were positive based on the results of individual cultures and 11 (1.2%) were positive in individual PCR. In addition, samples of 10 pools that had been positive in the culture gave negative results in individual culture. Two positive animals in such pools could be identified later, one by serology and one by PCR of a fresh fecal sample. For the 8 other pools, the presence of one positive animal in each positive pool was assumed for further calculations. With these additional animals, the total number of positive animals was determined to be 56, which corresponds to an overall prevalence of 6.1% (6.0% dairy and 6.4% beef cattle). Of the animals between one and two years of age not included for further analyses, eight animals (3.9%) from 6 farms (out of 13 where the young stock was available for testing) were positive in the culture, resulting in a herd prevalence of 46.2%, whereas none was positive in the PCR. The results of culture and PCR and the characteristics of the herds with their sizes and prevalence rates are listed in Table Because culture sensitivity was higher compared to PCR and because all PCR positive animals were also positive in the culture, only culture results were used for statistical analyses. Detailed results on the performance of culture and PCR will be published elsewhere.Table The results of the univariable analysis investigating the relation between the risk factors observed on the farms and the within-herd prevalence in infected herds are shown in Table The present study allowed for identifying the management parameters in Swiss cattle herds that are significantly associated with the infection status in regard to PTB  and with the prevalence of fecal MAP excretion within infected herds . Similar within-herd prevalence values were observed in beef and in dairy operations. A further important observation is that the knowledge level of the farmers about PTB was extremely low.Regarding the infection status of a herd, we discuss mostly the influence of animal purchase, which was the most important factor identified upon comparison of 17 infected herds (10 dairy and 7 beef) with 68 matched uninfected control herds (i.e. 4 control herds per infected herd), as well as shared pastures, herd size and sample size. The discussion of risk factors associated with the within-herd prevalence includes direct and indirect contact of heifers and calves with fecal material from adult animals, as well as management and hygiene in the calving area. Finally, the low level of knowledge of the farmers regarding PTB in infected and control herds and the consequences thereof are addressed at the end of the discussion.The analysis of management factors in infected herds in comparison to control herds showed that a positive infection status was associated with higher intensity of cattle movement, i.e. with regular purchase and introduction of animals into the herds. Farms that bought more than one animal per year had a higher risk of being infected than farms that bought an average of one animal per year or less. The fact that animal purchase was the only parameter remaining statistically significant in all models used for comparison of infected and control herds suggests an important effect on the PTB infection status of Swiss cattle herds, which is in accordance with reports from other countries -30. In bOne feature particular to Switzerland and the surrounding alpine countries is the common pasturing of cattle from several herds on mountain pastures. It is especially common for heifers to graze on high pastures in remote areas. Common pasturing of animals from several herds could potentially become a source of infection for negative herds, particularly for beef cow herds as the calves share their pasture with the adult cows; indeed, 2 of the beef herds (29%) shared mountain pastures with at least one other herd during the summer. A potential risk of infection may also exist for the 60% of dairy heifers which shared their summer pastures with heifers from other herds. A significant effect of shared pastures on the herd infection status was, however, not observed.The present results did not allow determining if the absence of other significant risk factors regarding the infection status of the herds under study was due to the limited sample size or to the fact that management practices are fairly uniform in Switzerland. Indeed, only very few differences were observed between the infected and the control herds.The average size of the infected herds participating in the study  was more than twice as big as the average herd size in Switzerland . Although this observation must be interpreted with caution because the herds included in the study were not necessarily representative of the entire population of herds affected with PTB in Switzerland, it is in accordance with studies conducted elsewhere ,29. WhetAlthough the pooling of three samples for culture and PCR might have led to a slight underestimation of the true prevalence in the infected herds, the observed prevalence values were generally low, with a mean within-herd prevalence of 6.0% in dairy and 6.4% in beef herds, respectively. While only 1 dairy herd out of 10 had a prevalence above 10%, a prevalence higher than 10% was observed in 2 out of 7 beef herds (28.6%). This is in contrast to other reports of lower prevalence values in beef than in dairy herds in North America ,33. ThisHigher within-herd prevalence was primarily associated with potential for contamination of the heifer area with feces from adult animals . This is in contrast to the results of a systematic review of literature regarding risk factors for MAP transmission  which shThe level of significance was not reached for the same contamination pathways for younger calves, although it is generally admitted that calves play a greater role in the transmission cycle of MAP . In the Shedding of MAP into the colostrum and milk of infected cows has been described as a risk factor for infection of young calves . In the In general, the knowledge level about PTB of the herd managers was extremely low, especially in the control farms. Almost two thirds of the managers of control herds stated that they had never heard of the disease. This suggests that PTB is not an important issue for Swiss farmers. The level of knowledge about the disease was also low in infected farms. While 100% of the herd managers knew the cardinal symptoms of diarrhea and emaciation, only 80% (dairy) and 43% (beef) farmers were aware of the reduction in milk yield associated with MAP infection. The epidemiology of the disease, especially the infection pathways that can be influenced by preventive strategies were understood by only 14%  to 90%  of the managers in infected herds, i.e. farmers who had already had at least one confirmed clinical case of the disease in their farm. This indicates that increased efforts aimed at raising the awareness of PTB of the Swiss veterinarians are necessary in order to improve the transfer of this knowledge to the farmers.Some results of the risk factor analysis within infected herds suggest that control measures were more likely to have been implemented in herds with high prevalence of MAP shedding. Indeed, cleaning the calving pen with a high-pressure washer > 1x/year was surprisingly associated with a higher MAP prevalence in the analysis of the dairy herds. This can likely be explained by the higher awareness of farmers having suffered the greatest financial losses due to PTB about the importance of hygiene, especially in the calving pens. This was confirmed by the farmers’ answers to the questionnaire (data not shown). The “protective” OR calculated for increased intensity of animal purchase in dairy herds can likely be explained by the same reason, beside the fact that this factor is rather associated with the herd status (infected or not) than with the within-herd prevalence. Likewise, using the calving pen as a sick cow pen appeared to reduce the risk of MAP transmission, which is also rather associated with the increased awareness of farmers suffering a higher prevalence of the disease. Indeed, the two herds with the highest prevalence of MAP excretion in their category  had separated pens for calvings and sick cows in their barns, which might explain this unexpected result due to the large influence of single herds given the small sample size in the present study.Despite the limited number of herds included, this study allows for a first description and evaluation of the situation in MAP infected herds under traditional farming conditions in smaller farms, i.e. in an agricultural system different from those mostly described in the literature from North America or Australia. While the presence of classical risk factors for PTB associated e.g. with calving management practices was confirmed in small infected cattle herds, unexpectedly numerous significant risk factors associated with the management of heifers older than one year were identified. This underlines the importance of avoiding contamination with feces from adult animals not only for young calves but also for older breeding stock. Furthermore, the fact that animal purchase has been shown to be the most significant factor determining the infection status of a herd, this parameter should be taken into consideration for the development of programs designed to control the spread of PTB among herds in the future. Based on the limited knowledge of farmers about PTB observed in the present study, increased information efforts toward Swiss herd managers and veterinarians are needed to raise their level of awareness about the disease and its control.Mycobacterium avium subsp. paratuberculosis; AIC: Akaike Information Criterion; OR: Odds ratio; CI: Confidence Interval.PTB: Paratuberculosis; MAP: The authors declare that they have no competing interests.RK coordinated the study, conducted the farm visits, analyzed the data and drafted the manuscript; PT planned and performed the statistical analyses; SK performed the MAP cultures and PCR; MW and RS participated in the planification of the study and supervised the laboratory work; GKB participated in the conception of the study and the redaction of the manuscript; BB participated to the farm visits and to the drafting of the manuscript; MM conceived and coordinated the study, helped to draft and finalized the manuscript; All authors have read and approved the manuscript.


Assembly of primary cilia relies on vesicular trafficking towards the cilium base and intraflagellar transport (IFT) between the base and distal tip of the cilium. Recent studies have identified several key regulators of these processes, including Rab GTPases such as Rab8 and Rab11, the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances, the exact molecular mechanisms by which these proteins interact and target to the basal body to promote ciliogenesis are not fully understood.We surveyed the human proteome for novel ASPM, SPD-2, Hydin (ASH) domain-containing proteins. We identified the TRAPP complex subunits TRAPPC8, -9, -10, -11, and -13 as novel ASH domain-containing proteins. In addition to a C-terminal ASH domain region, we predict that the N-terminus of TRAPPC8, -9, -10, and -11, as well as their yeast counterparts, consists of an α-solenoid bearing stretches of multiple tetratricopeptide (TPR) repeats. Immunofluorescence microscopy analysis of cultured mammalian cells revealed that exogenously expressed ASH domains, as well as endogenous TRAPPC8, localize to the centrosome/basal body. Further, depletion of TRAPPC8 impaired ciliogenesis and GFP-Rabin8 centrosome targeting.Our results suggest that ASH domains confer targeting to the centrosome and cilia, and that TRAPPC8 has cilia-related functions. Further, we propose that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery. The primary cilium is a microtubule-based sensory organelle that extends from the mother centriole, projecting from the cell surface into the extracellular environment . The assPrimary cilia have become the focus of mounting studies owing to their role in coordinating cellular signaling processes during development and tissue homeostasis, and consequently, their association with a constellation of genetic diseases and syndromes called ciliopathies ,7. TheseCurrent evidence indicates that the yeast TRAPPII complex consists of a core containing the seven TRAPPI subunits  as well as the four TRAPPII-specific subunits Trs65, Trs120, Trs130, and Tca17 . FunctioSimilar to yeast TRAPP complexes, mammalian TRAPP has been implicated in various vesicle trafficking pathways . For exaWe here revisit the ciliary family of ASH domain-containing proteins in humans and yeast and unveil the existence of ASH domains in several members of the TRAPP complex family of proteins. Our database searches identify conserved ASH domains in the C-terminus of mammalian TRAPPC8, -9, -10, -11, and -13, as well as in the yeast TRAPPII complex proteins Trs65, Trs120, and Trs130, none of which were previously assigned any protein structure. We find that the ASH domain in most of these proteins is preceeded by an N-terminal region containing long α-helical matrices exhibiting high levels of helicity and TPR repeat propensity. We further provide evidence that the ASH domains of TRAPPC10 and TRAPPC11 localize to the centrosome when expressed as fusion proteins in mammalian cells and that their expression leads to defects in microtubule organization. Finally, we find that endogenous TRAPPC8 localizes to the centrosome/basal body in cultured cells, and that TRAPPC8 depletion impairs ciliogenesis and targeting of GFP-Rabin8 to the centrosome. Our results corroborate the notion that the ASH domain is involved in microtubule- and cilia-related processes and provide insight into the mechanism by which mammalian TRAPPII homologs target to the centrosome/basal body. Our results further suggest that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery .http://pfam.sanger.ac.uk) were performed using HHpred [Profile-to-profile hidden Markov model (HMM)-HMM searches against the PFAMA database  and reverse  primers containing EcoR1 and Kpn1 restriction sites , respectively. The PCR products were cloned into pCMV-Myc  and transformed into Escherichia coli DH10α using standard procedures. Plasmids from recombinant bacteria were purified using endotoxin-free plasmid DNA purification kit (NucleoBond Xtra Midi EF) from Macherey-Nagel and the inserts sequenced at Eurofins MWG Operon.For generation of plasmids coding for Myc-tagged ASH domains from human TRAPPC10 (amino acid residues 1000-1259) and TRAPPC11 , the corresponding cDNA regions were PCR-amplified from retinal pigment epithelial cell cDNA  by standThe retinal pigment epithelial (RPE) cells used (lab stock) were derived from the immortalized hTERT RPE-1 cell-line and cultured as described previously .Glued . To study the localization of endogenous TRAPPC8 RPE cells were seeded on coverslips and incubated in serum-depleted medium for 24 h to induce cilia formation. Cells were fixed with methanol and subjected to immunofluorescence microscopy as described [Glued . Imaging was performed with a motorized Olympus BX63 upright microscope equipped with a DP72 color, 12.8 megapixel, 4140 × 3096 resolution camera and differential interference contrast (DIC). The software used was Olympus CellSens dimension. Images were processed for publication using Adobe Photoshop CS4 version 11.0.For immunofluorescence microscopy analysis of cells expressing ASH domain fusion proteins RPE cells were seeded on coverslips, transfected with plasmids encoding Myc-TRAPPC10-ASH or Myc-TRAPPC11-ASH (see above) and serum starved for 24 h. Cells were fixed with methanol or 4% PFA and subjected to immunofluorescence microscopy as described  using raescribed  using raGlued antibody as described above, and the number of GFP-positive centrosomes in GFP-Rabin8 expressing cells was scored.For TRAPPC8 knock-down experiments, RPE cells were seeded and subjected to transfection with 100 nM esiRNA specifically targeting TRAPP8C  or control siRNA  using DharmaFECT Duo Transfection Reagent (Thermo Scientific) essentially as described . Cells whttp://toolkit.tuebingen.mpg.de/hhpred)  [-5). This indicates that the family of TRAPPII proteins, encompassing metazoan TRAPPC9 homologs and yeast Trs120, bear homology by sequence to the ASH domain. Indeed, a reciprocal profile-to-profile search seeded with, for example, yeast Trs120 (amino acids 660-948) identified, besides TRAPPC9-Trs120, the PapD family as the highest scoring hit . Likewise, using a longer stretch of the Trs120 C-terminus (amino acids 486-1166) as a search query, employing three MSA generation iterations, we identified a portion of human Hydin (amino acids 361-892) containing three ASH domains , supporting the existence of the ASH domain in Trs120/TRAPPC9. Interestingly, this search also identified remote sequence similarities to additional or putative TRAPPII subunits, including human TRAPPC13/C5orf44 , C4orf44/Gryzun (TRAPPC11)  as well as TRAPPC8 . These findings raise the possibility that mammalian TRAPP possesses multiple paralogous ASH domain-bearing components.Sequence homologies between the ASH domain and yeast TRAPPII-specific components and their mammalian counterparts were readily attained by searching the PFAM database of protein families  using thfile -5) . Since whttp://toolkit.tuebingen.mpg.de/hhpred). Remarkably, we were able to show the presence of one or two ASH domains in three of the subunits of yeast TRAPPII  and their human counterparts TRAPPC9, -10, -11, and -13 as well as TRAPPC8 . Collectively, these searches show that almost all the known and putative TRAPPII-specific components exhibit genuine homology to the ASH/MSP domain  we could predict a tertiary structure model of each identified ASH domain that was compatible with the solved 3D structure of the human OCRL1 ASH domain [Encouraged by the above findings, and to uncover the full repertoire of Trs120/TRAPPC9 homologs bearing ASH domains in humans and yeast, we decided to validate the occurrence of this domain in each of the known or putative TRAPPII-specific subunits. In each instance, the minimal C-terminal portion displaying sequence homology to ASH was retrieved and used as a query in HHpred  that consist of short tracts of tetratricopeptide (TPR) repeats, indicating that Trs120 bears TPR repeats in its N-terminus. To further examine the TRAPPII complex subunits for the co-occurrence of TPR repeat conformations, the N-terminal portion of each polypeptide sequence was probed for the presence of high α-helical content coinciding with TPR repeat propensity. With the exception of TRAPPC13, all human and yeast TRAPPII subunits were predicted to harbor amino termini containing long α-helical matrices exhibiting high levels of helicity (>50%) and high TPR repeat propensity, as judged by secondary degrees structure prediction algorithms at the HNN server  and searGlued to mark the microtubule cytoskeleton, cilia, and centrosome, respectively. As shown in Figure Since the ASH domain seems to be confined to proteins that localize to the centrosome, Golgi, and the cilium , and to Glued, EB3, or acetylated tubulin . Among the subunits that adopt such a TPR-ASH bipartite arrangement, we find the yeast TRAPPII Trs120, Trs130, and Trs65 subunits as well as human TRAPPC8, 9, 10, and 11. In addition, we find that these TRAPP components, as with the MSP modules of VAPB , show reDrosophila ASPM homolog abnormal spindle, asp, binds microtubules within a 512 amino acid region that contains the ASH domain [Our immunofluorescence microscopy analysis showing that heterologously expressed TRAPPC10-ASH and TRAPPC11-ASH localize to the centrosome and affect microtubule organization Figure , as wellH domain . AlternaH domain , and TRAH domain , a GTP eH domain . FinallyTRAPPC9, leading to formation of a truncated TRAPPC9 polypeptide ending at amino acid 967, in two patients with a Prader-Willi-like phenotype [TRAPPC11 leading to a single amino acid change in the region between the two predicted ASH domains . From red meaning very significant to blue meaning not significant.Graphical output showing results of the initial HHpred search with human DLEC1 (residues 769-980) as search query. The amino acid sequence of human DLEC1 (residues 769-980) was used as a search query in HHpred. Profile-to-profile searches were obtained by three iterative PSI-BLAST searches against the PFAM database of HMM profiles. The figure shows a bar graph summarizing the positions and color-coded significances of the database matches with more than 40% probability. The bars are color-coded according to the significance of the hits (For details see Click here for file


Streptococcus pneumoniae and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a scavenger receptor cysteine-rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the macrophage-associated receptor with collagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however, the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO’s SRCR domain, which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together, these findings indicate a common origin of the SRCR domain within the cA-SRs; however, different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.The class A scavenger receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria, such as  Theshia coli , and Mycrculosis .Neisseria meningitidis and showed that MARCO and SR-A were able to bind different target proteins , one of eight classes of SRs, are membrane-associated phagocytic receptors, which reside on the surface of immune cells . The cA-proteins . MARCO h in SR-A . In addiacterium . In addiacterium . These sacterium .E. coli and Staphylococcus aureus , over 1200 SRCR domains were identified, often with several domains found in tandem repeats (The SRCR domain is an evolutionarily conserved 90–110 amino acid long domain that is characterized by 6–8 cysteine residues . Within  repeats . It has  repeats . SRCR do repeats . These c repeats . Three o repeats . However repeats .Petromyzon marinus (sea lamprey) and Callorhinchus milii (ghost shark), we can now study the evolution of the cA-SRs before the divergence of teleost fish. In this study, we test the hypothesis that MARCO, SR-A, and SCARA5 share a common ancestor containing the SRCR domain using various phylogenetic approaches. In addition, we reconstruct a hypothetical ancestral SRCR domain and analyze the evolution of two motifs within the SRCR domain. We also test the hypothesis that MARCO’s SRCR domain is under different selective pressure when compared to that of SCARA5 and SR-A due to its direct role in host defense. These data will provide new insight on the origin of the SRCR domain and also its role in ligand binding within the Class A Scavenger Receptors.Previous work has hypothesized that the cA-SR family members were created through multiple duplication events of an ancestral gene . Of the 22.1Amino acid sequences of the 5 cA-SRs were searched for using the National Center for Biotechnology Information (NCBI) and the ENSEMBL databases . Full-le2.2In order to study the evolutionary history of the receptors and their SRCR domains, Bayesian phylogenetic analyses were performed using the software MrBayes . SeveralAnalysis of the SRCR domain of SR-A, MARCO, and SCARA5 was carried out using the WAG model with invariable (I) sites and gamma (G) distributed rates for 10 million generations. A combined tree of all 5 cA-SRs was performed using a Jones Thorton and Taylor (JTT) model with an IG distribution for 15 million generations and displayed with midpoint rooting. Finally, an analysis of all 5 cA-SRs with an outgroup was performed in MrBayes using JTT with invariable gamma distribution for 20 million generations. All MrBayes output trees were visualized using TRACER  to ensurGeodia cydonium (sea sponge) SRCR-containing protein (GCSRCR) (NCBI ID: CAA75175.1) were used as outgroups. The GCSRCR protein’s ninth repeat has been shown to share sequence similarity to both MARCO and SR-A’s SRCR domains (S. purpuratus as the outgroup (NCBI ID: NP_001073014.1). Colmedin is a membrane spanning protein containing a transmembrane domain, multiple collagenous domains, and an olfactomedin  and tenth repeat (1153–1255) of the  domains . We also domains . Finally042–1142 ctomedin . Due to Predicted ancestral SRCR domain sequences were reconstructed using the FastML webserver . The MrB2.3Mus musculus) and a WGTICDD motif (amino acids 452–458 in M. musculus) of interest; SCARA5 contains an EGRVEVYH motif (position 399–406) in M. musculus and a WGTVCDD motif (position 410–416) in M. musculus. We included the RGRAEVYY motif in our analysis due to its known functional role in ligand binding within MARCO (P. marinus (sea lamprey), 1 sequence from C. milii (ghost shark), 23 sequences from various mammals, 7 sequences from reptiles, amphibians and birds, and 8 sequences from fish. WebLogos for SCARA5 were made using 1 sequence from P. marinus , 26 sequences from various mammals, 6 sequences from reptiles, birds, and amphibians, and 7 fish sequences.Consensus sequences of SCARA5 and MARCO were generated for mammals, birds, fish, and other species using Jalview  and reprin MARCO . WebLogo2.4M. musculus as the outgroup. MARCO, SR-A, and SCARA5 sequences from M. musculus, Homo sapiens, Pan troglodytes, and Gorilla gorilla were used. Protein alignments were performed using MAFFT  package, we tested whether MARCO is under a different selective pressure from SCARA5 and SR-A . Using cng MAFFT  and wereng MAFFT . We used33.1The evolutionary history of the five cA-SRs has been studied previously . In thisP. marinus (sea lamprey). The domains of MARCO varied among species, with different numbers of collagen repeats found across different taxa. Within the SRCR domain of MARCO, the RGRAEVYY motif was highly conserved across mammals but less conserved in birds, reptiles, and fish. The SRCR domain of P. marinus, however, did not possess the conserved RGRAEVYY motif characteristic of MARCO. SR-A was found in mammals exclusively, except for a sequence found in Xenopus tropicalis (western clawed frog). We included it in our analysis and found that the domains of SR-A were highly conserved in both the collagenous and SRCR regions of mammals and the X. tropicalis sequence, 70 and 78% conservation, respectively. SCARA5 was found in birds, reptiles, fish, mammals, and as well in P. marinus. The collagenous and SRCR domains of SCARA5 were fairly conserved across the 40 species with 69 and 75% conservation, respectively.To test this hypothesis, our analysis includes additional protein sequences from divergent taxa and includes sequences from more diverse species. We are able to expand upon this previous work due to recent genome sequencing projects including the sea lamprey and ghost shark. This allowed for our analysis to include in total, 40 MARCO, 25 SR-A, 40 SCARA5, 40 SCARA4, and 40 SCARA3 protein sequences for this analysis. MARCO was found in birds, reptiles, fish, and mammals and a partial sequence was found in X. tropicalis SR-A sequence, we repeated our analysis excluding the sequence and found no difference in our findings (data not shown). These data suggest that MARCO shares a more recent common ancestor with SR-A and SCARA5 than with SCARA3 and SCARA4.Using Bayesian phylogenetics, we generated a new phylogenetic tree of the cA-SR family. Here, we show using midpoint rooting that the non-SRCR-containing proteins are more closely related, while the SRCR-containing proteins branch together . Within the SRCR-containing proteins, SCARA5 and SR-A cluster together while MARCO appears to have diverged from them before early teleost fish and possibly before the sea lamprey. We constructed a second phylogenetic tree with the addition of the colmedin sequence as an outgroup. Colmedin was used as it possesses a transmembrane domain and multiple collagenous domains Figure C. Using 3.2Our current knowledge of functional motifs within the SRCR domain is limited to the RGRAEVYY motif within MARCO, which contains a positive cluster essential for ligand binding . AlthougM. musculus) contain the RGRAEVYY motif. The motif is highly conserved within mammals, but is less conserved in fish, where it takes the form of QGRVEVFH , tyrosine (Y), and histidine (H) are somewhat variable Figure . We alsoM. musculus. This newly identified motif is highly conserved across the SRCR domains of MARCO, SR-A, and SCARA5, and we hypothesize that the motif may have a functional role due its proximity to a conserved cysteine residue (C1) within the SRCR domain. In contrast to the RGRAEVYY motif, the WGTICDD motif within MARCO is highly conserved across all the different taxa examined  in some fish species. Based on our multiple sequence alignment, SCARA5 possesses a homologous, highly conserved, WGTVCDD  to synonymous substitutions (dS) is free to vary along both the branches and the sites of the phylogeny. Using this model, we found evidence for positive selection along several sites within MARCO’s SRCR domain. We confirmed our results with a Likelihood Ratio Test (Δ LRT) at a 95% significance level. The sites identified as under positive selection were 442, 452, and 477 , or with the non-SRCR-containing receptors (SCARA3 and SCARA4) . In addiS. purpuratus, also shows the same relationship. This suggests that an ancestral cA-SR containing an SRCR domain was duplicated to produce an ancestral MARCO and a SCARA5/SR-A like precursor. Following this duplication event, the SCARA5/SR-A like precursor underwent a duplication event to produce modern SCARA5 and SR-A sequences. Based on the available sequence data, it still remains unknown if the ancestral gene to all five cA-SRs lacked or contained the SRCR domain. It is possible that the ancestral gene terminated at its collagenous domain and resembled SCARA3 and later acquired the SRCR domain in the MARCO/SCARA5/SR-A precursor. Equally likely, the ancestral gene may have contained the SRCR domain and it was lost in a SCARA3/SCARA4 precursor. Additional sequence data are required to fully uncover the origin of these proteins.Using Bayesian methods, we generated a new phylogenetic tree of all five cA-SRs. Our phylogenetic tree shows that MARCO shares a more recent common ancestor with SCARA5 and SR-A than with SCARA3 and SCARA4. The addition of an outgroup sequence, colmedin from To investigate the origin of the SRCR domains, we utilized Bayesian methods to construct a phylogenetic tree and subsequently reconstructed ancestral SRCR domains. Based on our analysis, MARCO shares a recent common ancestor with SCARA5 and SR-A. Using FastML, we reconstructed the SRCR domains at ancestral nodes between SCARA5, MARCO, and SR-A. We focused on two motifs within SCARA5 and MARCO; the RGRAEVYY motif within MARCO and a downstream WGTICDD motif. SCARA5 possesses two motifs similar to these, as it contains an EGRVEVYH motif and a WGTVCDD motif. Based on our multiple sequence alignment, the RGRAEVYY motif is specific to MARCO while the WGTVCDD motif is shared between the three SRCR domains. Furthermore, the ancestral motif to all three cA-SR SRCR domains resembled SCARA5’s EGRVEVYH and WGTVCDD motifs. This suggests that MARCO’s SRCR domain originally resembled SCARA5, and may have undergone purifying selection. Since SCARA5 and the ancestral domain lack the RGRAEVYY motif, we hypothesize the ancestral SRCR domain did not play a functional role in ligand binding due to the functional importance of the RGRAEVYY motif within MARCO. In invertebrate species, the SRCR domains are thought to play a role in cellular recognition as opposed to ligand binding . This thGiven MARCO’s role in bacterial binding and clearance within the immune system, we hypothesized that its SRCR domain may be under positive selection. Here, we have shown that MARCO has several sites under positive selection including positions 442, 452, and 477. Two of the sites are adjacent to the highly conserved WGTVCDD motif within the SRCR domain, and may have a biological function. However, we did not detect positive selection acting on the RGRAEVYY motif, despite the domain having a known role in ligand binding. Future experiments will look to identify the functional relevance of the positively selected sites and the WGTVCDD motif. We hypothesize that the WGTVCDD motif may have a structural role within the SRCR domain due to the inclusion of a conserved cysteine within the motif. Studying WGTICDD motif will further our understanding of the SRCR domain and its biological relevance in MARCO’s ability to facilitate bacterial binding, phagocytosis, and induction of T-cell tolerance.Manuscript planned and written by all. NY conducted and implemented all experiments.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.http://journal.frontiersin.org/article/10.3389/fimmu.2015.00342The Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.Click here for additional data file.


RVU and 153.63RVU for experimental samples compared with 392.35RVU obtained for the control. Most of the sensory properties for the experimental samples were significantly different from the control. This study showed that bread with better quality and acceptability can be produced from wheat–unripe banana blends.Bread was produced from wheat flour and fermented unripe banana using the straight dough method. Matured unripe banana was peeled, sliced, steam blanched, dried and milled, and sieved to obtain flour. The flour was mixed with water and made into slurry and allowed to stand for 24 h after which it was divided into several portions and blended with wheat flour in different ratios. Proximate and mineral compositions as well as functional, pasting, and sensory characteristics of the samples were determined. The results of proximate analysis showed that crude fiber ranged between 1.95% and 3.19%, carbohydrate was between 49.70% and 52.98% and protein was 6.92% and 10.25%, respectively, while iron was between 27.07 mg/100 g and 29.30 mg/100 g. Swelling capacity of the experimental samples showed a significant difference from that of control. Peak viscosity ranged between 97.00 Bread is the loaf that results from the baking of dough which is obtained from a mixture of flour, salt, sugar, yeast, and water. Other ingredients like fat, milk, milk solids, egg, anti‐oxidants, etc. may be added. Bread is an important food whose consumption is steady and increasing in Nigeria. It is, however, relatively expensive because it is made from wheat flour which has to be imported  constitutes a rich energy source with carbohydrate accounting for 22–32% of the fruit weight. It is rich in vitamins A, B6, and C as well as minerals particularly potassium, magnesium, phosphorus, and folate , using the RVA general pasting method.The sensory analysis was carried out using the scoring test as described by Akinjayeju . The senAll analysis was conducted in duplicate and the data were all subjected to ANOVA  as described by Akinjayeju  while thTable Table 3 with sample OAV having the highest value while sample TAD has the lowest amount. This means the higher the bulk density, the denser the flour (Eke‐Ejiofor and Kiin‐Kabari Table The pasting properties of the flour samples are shown in table The Table Addition of unripe banana was found to increase the crude fiber, ash, iron, and zinc content of the bread samples. Meanwhile, the products were acceptable by the sensory panelists although a significant difference was observed between the control and the experimental samples used in this study.Authors hereby declare that no conflict of interest exists.


Myeloid Derived Suppressor Cells (MDSCs) heavily infiltrate in a variety of solid tumors and suppress anti-tumor T-cell activity. Our recent studies have demonstrated the ability of monocytic, Ly6C+ MDSCs to promote glioma growth through the activation of cyclooxygenase (COX)-2 pathway, which is responsible for prostaglandin-synthesis. ONO-AE3-208 is an antagonist of the prostaglandin E (EP)-4 receptor, which is an important positive feedback regulator of the COX-2 pathway. We thus examined the ability of ONO-AE3-208 to suppress MDSC activity in gliomas. ONO-AE3-208 treatment in mice bearing established GL261-quad glioma in the brain resulted in complete and persistent rejection of the tumors. Flow cytometric analysis revealed that gliomas in the ONO-AE3-208-treated mice were infiltrated by fewer numbers of Ly6C+ MDSCs compared with non-treated animals. We subsequently isolated glioma-infiltrating Ly6C+ MDSCs by flow-sorting to address their functions. RT-PCR analysis revealed that the Ly6C+ MDSCs derived from ONO-AE3-208 treated mice expressed lower levels of the Arg1 and Cox2 expression compared to control animals. Consistently, brain infiltrating leukocytes in ONO-AE3-208 treated tumor-bearing mice demonstrated enhanced IFN-g expression compared with control mice, suggestive of enhanced T-cell activity. Importantly, ONO-AE3-208 inhibited glioma growth and promoted immune activity in 2 additional murine glioma models: the Sleeping Beauty de novo glioma model and the SB28 glioma cell line model. Our data demonstrate that ONO-AE3-208 may be useful in the treatment of glioma patients to suppress Ly6C+ MDSCs and promote anti-tumor immunity.