Terebratalia transversa.Larval features such as the apical organ, apical ciliary tuft, and ciliated bands often complicate the evaluation of hypotheses regarding the origin of the adult bilaterian nervous system. Understanding how neurogenic domains form within the bilaterian head and larval apical organ requires expression data from animals that exhibit aspects of both centralized and diffuse nervous systems at different life history stages. Here, we describe the expression of eight neural-related genes during the larval development of the brachiopod, Tt-Six3/6 and Tt-hbn in the animal cap ectoderm. Tt-NK2.1 and Tt-otp are restricted to a central subset of these cells, and Tt-fez and Tt-FoxQ2 expression domains are already asymmetric at this stage. As gastrulation proceeds, the spatial expression of these genes is split between two anterior ectodermal domains, a more dorsal region comprised of Tt-Six3/6, Tt-fez, Tt-FoxQ2, and Tt-otp expression domains, and an anterior ventral domain demarcated by Tt-hbn and Tt-NK2.1 expression. More posteriorly, the latter domains are bordered by Tt-FoxG expression in the region of the transverse ciliated band. Tt-synaptotagmin 1 is expressed throughout the anterior neural ectoderm. All genes are expressed late into larval development. The basiepithelial larval nervous system includes three neurogenic domains comprised of the more dorsal apical organ and a ventral cell cluster in the apical lobe as well as a mid-ventral band of neurons in the mantle lobe. Tt-otp is the only gene expressed in numerous flask-shaped cells of the apical organ and in a subset of neurons in the mantle lobe.Radially symmetric gastrulae broadly express Tt-Six3/6, Tt-FoxQ2, and Tt-otp confirm some aspects of bilaterian-wide conservation of spatial partitioning within anterior neurogenic domains and also suggest a common origin for central otp-positive cell types within the larval apical organs of spiralians. However, the field of sensory neurons within the larval apical organ of Terebratalia is broader and composed of more cells relative to those of other spiralian larvae. These cellular differences are mirrored in the broader spatial and temporal expression patterns of Tt-FoxQ2 and Tt-otp. Corresponding differences in the expression of Tt-hbn, Tt-NK2.1, and Tt-FoxG are also observed relative to their respective domains within the cerebral ganglia of spiralians. Based on these data we argue that the anterior region of the bilaterian stem species included Six3/6, NK2.1, otp, hbn, fez, and FoxQ2 expression domains that were subsequently modified within larval and adult neural tissues of protostome and deuterostome animals.Our expression data for  Several hypotheses exist concerning the putative homology of various parts of larval and adult nervous systems found among bilaterian animals. Based on similar expression patterns of evolutionarily conserved transcription factors, some authors have concluded that the structure of the adult nervous system of the last common ancestor of the Bilateria included an anterior brain with three divisions and a distinct longitudinal ventral nerve cord -3, with homeobrain, rx, and orthopedia) are only expressed within oral ectoderm of cnidarians [Confounding issues regarding the origin of the adult bilaterian nervous system pertain to its spatial proximity and integration with components of a larval nervous system, particularly, the development of ciliated apical tuft cells, which numerous larval forms exhibit, and their relationship to the larval apical (sensory) organ where many neuronal cell bodies of larval forms are concentrated. Despite some shared developmental and structural features there is no uniform consensus regarding the homology of the larval aboral organs of cnidarian planulae with the larval apical organs of various bilaterians -15. One idarians . How theidarians -19). Theidarians ,21, testThe larval apical organs of phoronids and brachiopods are relevant to the evolutionary reconstruction of bilaterian brains. Although the evolutionary relationships within phoronids and brachiopods ,23 as weSix3/6, homeobrain, and NK2.1 [Considering all of these structural and biochemical differences among bilaterian larval apical organs, evaluating the homology of cell types and the complex neuronal centers they make up becomes problematic as there are no universally agreed upon criteria for discriminating homologous neuronal cell types (but see ). Some snd NK2.1 -45. EvenNK2.1; [Terebratalia transversa, by analyzing the expression patterns of eight genes known to have roles in specification and differentiation of anterior neural tissues in other bilaterian animals.Evaluating alternative viewpoints regarding either the wide-scale homology or independent origin of larval apical organs requires more developmental data focused on the molecular specification of various neurogenic tissue domains from additional bilaterian animals with structurally diverse larval nervous systems. Recent reports on the development and structure of the sensory cells and larval nervous system in brachiopods ,46 suggeNK2.1; ) differsNK2.1; ). HoweveTerebratalia Erebratalia transversa orthologs of the transcription factors forebrain zinc-finger (fez), Forkhead G (FoxG), Forkhead Q2 (FoxQ2), homeobrain (hbn), NK2.1, orthopedia (otp), and Six3/6, and the synaptic vesicle-localized transmembrane protein synaptotagmin 1, were isolated by rapid amplification of cDNA ends (RACE). Orthology of each gene to representatives from other metazoan taxa was confirmed by Bayesian analysis of phylogenetics  of centrally located flask-shaped cells near the apical tuft region  expand in an equatorial ring around the middle of the apical lobe that correlates with the position of the developing transverse ciliated band and some rows of cells anterior to it , as well as another neurogenic domain within the mid-ventral portion of the mantle lobe. Neuronal cells within the anterior dorsal domain include the broad sensory and supportive histaminergic epithelium of the apical organ [Tt-synaptotagmin 1 expression is more pronounced in early and late trilobed larval stages consistent with this gene's role in late neuronal differentiation [Tt-synaptotagmin 1 expression is mainly found in anterior neural tissues and the basiepithelial nerve rings of the transverse ciliated band.Despite the broad expression domains exhibited by genes such as al organ , the cilal organ , and theal organ . Tt-synantiation . Tt-synaTt-hbn and Tt-NK2.1, both of which overlap with and are bordered laterally by Tt-fez in the anterior dorsal portion of the apical lobe. On the ventral surface of the apical lobe, both Tt-hbn and Tt-NK2.1 are bordered posteriorly by the expression of Tt-FoxG within the region of the transverse ciliated band  was also observed in similar larval cells of Patella vulgata [otp-positive cells are present throughout much of the dorsal neurogenic domain and are likely beyond the limits of the Tt-FoxQ2 domain, overlapping with Tt-fez  neurogenic domain and the ventral nerve cord of annelids are conserved [Terebratalia larvae is not centralized, then aspects of both anterior-posterior patterning and centralization of neurogenic domains may be different between Platynereis and Terebratalia. Collectively, however, these gene expression and immunohistochemical domains in T. transversa larvae are still reminiscent of anterior and ventral neurogenic domains in spiralian larval forms , albeit complicated by the basiepithelial nature of the larval nervous system in T. transversa. To evaluate the putative homology of these neurogenic domains we review the known expression patterns for genes we have isolated in T. transversa in several larval forms among protostome and deuterostome animals in the following sections.Of the eight genes considered here, only e Figure . Althougonserved . HoweverTerebratalia to orthologous genes in similar developmental stages of various bilaterian animals in Figure Species-specific tissue types, differences in developmental stages, and derived anatomical features of particular larval types complicate comparisons of gene expression patterns among disparate animals. In terms of the developing nervous system, discerning discrete larval versus juvenile expression domains can be difficult in more gradually developing animals such as annelids and mollusks, as opposed to echinoderms in which dramatic distinctions between larval and adult structures can be made -59. Howeotp and NK2.1. In the trochophore larva of the limpet, Patella vulgata, Pv-otp is expressed in a 'U'-shaped field of ectodermal cells that surrounds the apical tuft region  in proximity to the expression of Pd-NK2.1 and especially within the structures that Tomer et al. [Homeobrain-like expression is known for another polychaete annelid, Capitella teleta, where Ct-hbn is expressed in a subset of cells in the developing cerebral ganglia  and the histaminergic cells within the ventral cluster in the apical lobe of the larva are homologous to the cerebral ganglion of spiralians.Expression data for these genes from molluscan trochophores are not well characterized, except for n Figure . These ctectable . The troal organ . Anterioe domain , but Pd-n Figure  mainly weceptors . It is nr et al.  called mSix3/6 dependent neurogenic domain [NK2.1, homeobrain, and FoxQ2 [otp is a marker for specific neuronal cell types in deuterostomes, trochozoans, and ecdysozoans [FoxG is expressed throughout the oral ectoderm in the blastula, but is restricted to the developing ciliated bands during gastrulation [Hemicentrotus pulcherrimus, suggests that Hp-fez is positively regulated by Hp-FoxQ2, and that the fez protein serves to maintain the size of the neurogenic animal ectoderm by modulating the inhibitory effects of BMP2/4 [Tt-fez are consistent with these observations as Tt-fez expression generally borders the Tt-FoxQ2 domain. Six3/6 also demarcates the anterior ectoderm in the development of the direct-developing hemichordate worm, Saccoglossus kowalevskii  is found in the developing apical organ, the anterior lip of the mouth, and within select neuronal cells of the neotroch  are progressively separated into dorsal and ventral domains during the gastrulation of true spiralians and other related taxa with trochozoan larval features such as T. transversa. Since Cl-otp is also expressed in a subset of ventral neural cells similar to Tt-otp, and these expression patterns are spatially similar to what is observed in the mesosome of the hemichordate worm, Saccoglossus kowalevskii [NK2.1 expression in the neural tube of the tunicate, C. intestinalis [NK-class genes in the ventral nerve cord of spiralians [Insight into the complexity and degree of centralization of the adult nervous system of the ancestor of protostomes and deuterostomes may be gained from studying the development of acoelomorph 'flatworms' , dependi 'brain' ,6. Withithin it , none of which originate from the larval ciliary bands [The cellular morphology of the bilaterian anterior larval nervous systems may include an apical organ consisting of a single group or bilateral masses of numerous (thirty of more) bipolar serotonergic neurons, some of which are sensory, as found in the larvae of particular classes of echinoderms and hemichordates -77. Spirry bands -81. AlthTt-otp within a subset of cells of the developing apical organ of late gastrula stage Terebratalia show striking similarities to the morphology of the central (serotonergic) flask cells of larval entoprocts, annelids, and some mollusks [Terebratalia coincides with late differentiation of neuronal and other ciliated cell types . This interpretation is supported by numerous flask-shaped cells that express Tt-otp in the apical organ of the trilobed larval stage and also correlates with the widening of the Tt-FoxQ2 domain at this stage. Expression of Tt-Six3/6, Tt-fez, Tt-FoxQ2, and Tt-otp in the dorsal neural ectoderm that gives rise to the apical organ continues into the late larval stage. These expression patterns are also reflected in the numerous sensory neurons and other supportive cells labeled by anti-acetylated α-tubulin and anti-histamine only at the late larval stage.Our results of the expression of mollusks -81, suppmollusks . Howevermollusks , as indimollusks . Larval FoxQ2, COE, and FGFa1 are expressed within the region of the aboral ciliary tuft in planula larvae of the hydrozoan, Clytia hemisphaerica, and the anthozoan, Nematostella vectensis [Nematostella planulae, the oral-aboral axis of cnidarians is believed to be homologous to the anterior-posterior axis in bilaterians, and the bilaterian dorsal-ventral axis is homologous to the cnidarian directive axis [NK2.1, homeobrain, noggin1, and otp expression domains and several other bilaterian anterior-related genes [Six 3/6 that is present in the aboral region of the larva  homeobrain, otp, and NK2.1 expression. How these separate expression domains became coupled at the animal pole of bilaterian embryos, the adult head, and the bilaterian larval apical organ remains unclear  where i were dredged from the waters adjacent to San Juan Island, Washington, USA during the early spring of 2005 and the winter of 2008. Adults were maintained in a free-flowing seawater table (8 to 10°C) at Friday Harbor Laboratories until needed. Gametes were gathered and larval cultures were created using the methods of Reed [Adults of  of Reed . BrieflyTt-fez, Tt-FoxG, FoxQ2, Tt-homeobrain, Tt-NK2.1 and Tt-otp, were identified from Expressed Sequence Tags (ESTs) clones sequenced for a previous phylogenomic analysis [http://www.ncbi.nlm.nih.gov/projects/dbEST/). Fragments of Tt-synaptotagmin 1 and Tt-Six3/6 were amplified by degenerate PCR using cDNA generated from mRNA isolated from mixed embryological stages. Nested primer sets for Tt-Six3/6 were Six3/6 F1 FLSWSLP 5' TTYYTNTGGWSNYTNCC 3', Six3/6 R1 QRDRAA 5'GCNGCNCKRTCNCKYTG 3', Six3/6 R2 NWFKNRRQ 5'TGNCKNCKRTTYTTRAACCARTT 3', Six3/6 F2 GPVDKYRV 5'GGNCCNGTNGAYAARTAYMGNGT 3'. Nested primer sets for Tt-synaptotagmin were Syn F1: 5' TYAAYCCNGTNTTYAAYGA 3', Syn F2: 5' TAYGAYTTYGAYMG/ideoxyI/TT 3', Syn R1: 5' TCRTTRTARTANGGRTT 3', Syn R2: 5' SWRAARCADATRTC/ideoxyI/CC 3'. Full-length cDNAs were obtained by rapid amplification of cDNA ends using the SMART RACE kit  using sequence specific primers.analysis  publiclyTerebratalia forebrain embryonic zinc-finger, Forkhead G, orthopedia, otx, NK2.1, homeobrain, synaptotagmin 1, and Six3/6 along with those for representative related proteins from other taxa, retrieved from NCBI  and Joint Genome Institute , were aligned with MUSCLE [The deduced amino acid sequences for h MUSCLE  or MacVeh MUSCLE ,97, withIn situ hybridizations were conducted using an established protocol [protocol . Probes = 7.4) or filtered seawater. Larvae were removed from this solution and larval tissues were permeablized with 0.1 M Sørenson's phosphate buffer and 0.1% to 0.3% Triton-X detergent (PTA solution) for 24 hours at 4°C before proceeding with the staining protocol. All further steps were carried out on a rotary shaker table. Nonspecific sites were blocked with 4% normal goat serum  in PTA solution (GS-PTA) for 24 hours at 4°C. The primary antibody  or anti-histamine  was diluted 1:500 with 4% GS-PTA and incubated with the larvae for 48 hours at 4°C. Specimens were washed (three times) in PTA for a total of 24 hours. Larvae were exposed to the secondary antibody, goat anti-mouse or rabbit AlexaFluor 488 or 568 immunoglobin , at a dilution of 1:50 or 1:100 for 24 hours. Deleting the primary antibody from the protocol produced negative controls. Some specimens were then stained for either fibrous actin or nucleic acids. Nucleic acids were stained with a 1:1,000 dilution of sytox green  for 10 minutes or a 1:500 dilution of propidium iodide  for 10 to 15 minutes. Specimens were adhered to clean glass slides coated with a poly-L-lysine solution  and put through an alcohol dehydration series using 2-propanol within four minutes. Finally, specimens were cleared in a solution of benzyl benzoate and benzyl alcohol (2:1) for two minutes and mounted in the same solution. Slides were kept in the dark at 4°C until viewed with a BioRad Radiance 2100 laser confocal system and a Nikon E800 microscope  or a Zeiss 710 Confocal Laser Scanning Microscope . Confocal z-series were gathered as 1 μm sections. Z-projections and volume renderings of embryos and larvae were created using OsiriX  or FIJI.Fixation and immunohistochemical procedures of embryos and larvae followed Santagata . BrieflyBranchiostoma Fez (ADK13096.1); Caenorhabditis Y38H8A.5 (NP_502594.2); Capitella fez (18104); Capitella Gfi (45287); Drosophila earmuff (NP_608631.1); Daphnia fez (EFX89329.1); Homo fez1 (NP_001019784.2); Homo fez2 (NP_060478.3); Homo Gfi-1b (NP_004179.3); Homo ZF430 (AAP30885.1); Homo ZF85 ; Lottia Gfi1 (129344); Lottia Gfi2 (83709); Mus fez1 (NP_082738.1); Mus fez2 (NP_536681.2); Nematostella 12000017 (228271); Nematostella 265000001 (230810); Nematostella 30000108 (201757); Nematostella e_gw.3.372.1 (80425); Nematostella Gfi (182742); Saccoglossus fez (NP_001158457.1); Schmidtea fez (XP_002575460.1); Schmidtea Gfi (XP_002580588.1); Trichoplax fez (7089); Trichoplax Gfi (63664); Terebratalia fez (JQ88195)Clytia FoxQa (ABG21224.1); Clytia FoxQb (ABG21225.1); Capitella FoxA (169665); Capitella FoxAB (131123); Capitella FoxB (225366); Capitella FoxC (199610); Capitella FoxD (126386); Capitella FoxF (50240); Capitella FoxG (139421); Capitella FoxGa (182306); Capitella FoxI (154409); Capitella FoxJ1 (222987); Capitella FoxJ2/3 (137131); Capitella FoxK (23732); Capitella FoxL1 (49410); Capitella FoxL2 (88179); Capitella FoxM (115253); Capitella FoxN1/4 (129521); Capitella FoxN2/3 (102038); Capitella FoxO (91312); Capitella FoxP (173180); Capitella FoxQ1 (175391); Capitella FoxQ2a (111555); Capitella FoxQ2b (148596); Nematostella (110212); Nematostella (118122); Nematostella (120142); Nematostella (121754); Nematostella (123903); Nematostella (125256); Nematostella (132285); Nematostella (138488); Nematostella (150900); Nematostella (161006); Nematostella (165261); Nematostella (165603); Nematostella (18324); Nematostella (187332); Nematostella (192525); Nematostella (19405); Nematostella (200222); Nematostella (200356); Nematostella (201028); Nematostella (213966); Nematostella (218419); Nematostella (228732); Nematostella (38679); Nematostella (39596); Nematostella (39632); Nematostella (5001); Nematostella (58039); Nematostella (59063); Nematostella (65438); Nematostella (67043); Nematostella (67209); Nematostella (88569); Nematostella (93177); Nematostella (96685); Lottia FoxA (183845); Lottia FoxAB (99760); Lottia FoxB (186344); Lottia FoxC (117369); Lottia FoxD (137594); Lottia FoxF (117350); Lottia FoxG (59807); Lottia FoxH (134143); Lottia FoxJ1 (69660); Lottia FoxJ1 (59864); Lottia FoxJ2/3 (98413); Lottia FoxK (183124); Lottia FoxL1 (178394); Lottia FoxL2 (89841); Lottia FoxN2/3 (138633); Lottia FoxP (54435); Lottia FoxQ2 (79770); Mus FoxA1 (NP_032285.2); Mus FoxA2 (NP_034576.2); Mus FoxB1 (NP_071773.2); Mus FoxB2 (NP_032049.1); Mus FoxC1 (NP_032618.2); Mus FoxC2 (NP_038547.2); Mus FoxD2 (NP_032619.1); Mus FoxD3 (NP_034555.3); Mus FoxD4 (NP_032048.1); Mus FoxE1 (NP_899121.1); Mus FoxE3 (NP_056573.1); Mus FoxF1 (NP_034556.1); Mus FoxF2 (NP_034355.2); Mus FoxG1 (NP_032267.1); Mus FoxH1 (NP_032015.1); Mus FoxI1 (NP_076396.3); Mus FoxI2 (NP_899016.1); Mus FoxJ1 (NP_032266.3); Mus FoxJ2 (NP_068699.1); Mus FoxJ3 (NP_766287.1); Mus FoxK1 (NP_951031.2); Mus FoxK2 (NP_001074401.2); Mus FoxM1 (NP_032047.4); Mus FoxN1 (NP_032264.1); Mus FoxN2 (NP_851305.2); Mus FoxN4 (NP_683737.2); Mus FoxO1 (NP_062713.2); Mus FoxO3 (NP_062714.1); Mus FoxO6 (NP_918949.1); Mus FoxP1 (NP_444432.1); Mus FoxP2 (NP_997600.1); Mus FoxP3 (NP_473380.1); Mus FoxP4 (NP_083043.2); Mus FoxQ1 (NP_032265.3); Mus FoxS1 (NP_034356.1); Platynereis FoxG (ADG26725.1); Ptychodera FoxQ2 (ADZ61650.1); Rattus FoxR1 (XP_243815.4); Rattus FoxR2 (XP_228808.3); Strongylocentrotus FoxI (ABB89485.1); Strongylocentrotus Fox_L1 (ABB89488.1); Strongylocentrotus FoxA (ABE68834.1); Strongylocentrotus FoxAB-like (ABB89474.1); Strongylocentrotus FoxB (NP_999797.1); Strongylocentrotus FoxC (ABB89478.1); Strongylocentrotus FoxD (ABB89476.1); Strongylocentrotus FoxF (ABB89479.1); Strongylocentrotus FoxG (ABB89477.1); Strongylocentrotus FoxJ1 (ABB89480.1); Strongylocentrotus FoxK (ABB89486.1); Strongylocentrotus FoxL2 (ABB89483.1); Strongylocentrotus FoxM (ABB89490.1); Strongylocentrotus FoxN1/4 (ABB89491.1); Strongylocentrotus FoxN2/3 (ABB89482.1); Strongylocentrotus FoxO (ABB89484.1); Strongylocentrotus FoxP (ABB89487.1); Strongylocentrotus FoxQ1 (ABB89489.1); Strongylocentrotus FoxQ2 (ABB89473.1); Terebratalia FoxG (JQ88193); Terebratalia FoxQ2 (JQ88200)Capitella Lbx (ACI26672.1); Capitella NK-like-1a (ACH70609.1); Capitella NK-like-1b (ACI26669.1); Capitella NK-like-2.1a (ACH89430.1); Capitella NK-like-2.1b (ACH89431.1); Capitella NK-like-2.2a (ACH89432.1); Capitella NK-like-2.2b (ACH89433.1); Capitella NK-like-3 (ACI26670.1); Capitella NK-like-4a (ACH89434.1); Capitella NK-like-4b (ACH89435.1); Capitella NK-like-5 (ACH89437.1); Capitella NK-like-5b (ACH88440.1); Capitella NK-like-6 (ACI26668.1); Capitella NK-like-7_ACI26671.1); Capitella Tlx (ACH89436.1); Drosophila bap (NP_732637.1); Drosophila C15 (NP_476873.2); Drosophila dll (NP_726486.1); Drosophila drop (NP_477324.1); Drosophila H6 (NP_732244.3); Drosophila lbe (NP_524435.2); Drosophila lbl (NP_524434.2); Drosophila scro (NP_001015473.1); Drosophila slou (NP_476657.1); Drosophila tinman (NP_524433.1); Drosophila vnd (NP_001036253.1); Mus Dlx1 (NP_034183.1); Mus Dlx2 (NP_034184.1); Mus Dlx3 (NP_034185.1); Mus Dlx4 (NP_031893.3); Mus Dlx5 (NP_034186.2); Mus Dlx6 (NP_034187.1); Mus HMX1 (NP_034575.1); Mus HMX2 (NP_666110.1); Mus HMX3 (NP_032283.3); Mus Lbx1 (NP_034821.2); Mus Lbx2 (NP_034822.1); Mus Msx1 (NP_034965.2); Mus Msx2 (NP_038629.2); Mus Msx3 (NP_034966.1); Mus Nk2.5 (NP_032726.1); Mus Nk3.2 (NP_031550.2); Mus Nkx-3.1 (NP_035051.1); Mus Nkx-6.1 (NP_659204.1); Mus Nkx-6.2 (NP_899071.2); Mus Nkx-6.3 (NP_083278.1); Mus Nkx1.1 (XP_001473685.1); Mus Nkx1.2 (NP_033149.1); Mus Nkx2.1 (NP_033411.3); Mus Nkx2.2 (NP_035049.1); Mus Nkx2.3 (NP_032725.1); Mus Nkx2.4 (NP_075993.1); Mus Nkx2.6 (NP_035050.2); Mus Nkx2.8 (NP_032727.2); Mus Tlx1 (NP_068701.1); Mus Tlx2 (NP_033418.1); Mus Tlx3 (NP_064300.2); Platynereis Dlx (CAJ38799.1); Platynereis Lbx (ABQ10642.1); Platynereis Msx (CAJ38810.1); Platynereis Nk1 (CAJ38797.1); Platynereis NK2.1 (CAJ38809.1); Platynereis NK2.2 (ABO93209.1); Platynereis NK3 (ABQ10641.1); Platynereis NK4 (ABQ10640.1); Platynereis NK5 (ABQ10644.1); Platynereis Tlx (ABQ10643.1); Terebratalia NK2.1 (JQ88197)Crepidula Pitx (ADI48168); Ciona Prop (XP (002119699); Ciona otp (NP (001072023); Ciona otx (NP (001027662); Capitella EBX (ABC58683); Capitella Gsx (AAZ23124); Capitella Pax3-7 (ABC68267); Capitella Xlox (AAZ95509); Capitella cdx (AAZ95508); Drosophila Pitx (NP (733410); Drosophila Vsx1 (NP (572232); Drosophila Vsx2 (NP (001033832); Drosophila aristaless (NP (722629); Drosophila dll (NP (523857); Drosophila gsc (NP (476949); Drosophila homeobrain (NP (788420); Drosophila otd (P22810); Drosophila otp (P56672); Drosophila repo (NP (477026); Drosophila rx (NP (726006); Hydroides otx (ABK76302); Platynereis Arx (ADG26723); Platynereis Cdx (ACH87546); Platynereis Gsx (ACH87540); Platynereis Pax6 (CAJ40659); Platynereis Xlox (ACH87551); Platynereis dlx (CAJ38799); Platynereis dlx (CAJ387991); Platynereis gsc (CAC19336); Platynereis otp (ABR68849); Platynereis otx (CAC19028); Platynereis rx (AAU20320); Patella gsc (CAD45551); Patella otp (AAM33145); Patella otx (AAM33144); Saccoglossus Prop (NP (001161635); Saccoglossus hbn (XP (002731203); Saccoglossus otd (NP (001158360); Saccoglossus otp (NP (001158374); Strongylocentrotus hbn (XP (781057); Strongylocentrotus otp (XP (784599); Strongylocentrotus otx (NP (999753); Terebratalia Pax6 (ADZ24784); Terebratalia homeobrain (JQ88198); Terebratalia otp(JQ88194); Terebratalia otx (ADZ24785)Capitella 180297 (180297); Capitella 180301 (180301); Capitella 180303 (180303); Capitella 226834 (226834); Capitella 227938 (227938); Drosophila optix (NP_524695.2); Drosophila sine-oculis (NP_476733.1); Drosophila Six4_NP_649256.10; Lottia 115798 (115798); Lottia 129577 (129577); Lottia 179424 (179424); Mus Six1 (NP_033215.2); Mus Six2 (NP_035510.1); Mus Six3 (NP_035511.2); Mus Six4 (NP_035512.1); Mus Six5 (NP_035513.1); Mus Six6 (NP_035514.1); Nematostella 126214 (126214); Nematostella 130873 (130873); Nematostella 138693 (138693); Platynereis Six2 (CAC86663.1); Platynereis Six3 (CAR66435.1); Saccoglossus Six1 (XP_002735213.1); Saccoglossus Six3 (NP_001158378.1); Saccoglossus Six4 (XP_002735606.1); Strongylocentrotus Six1 (XP_001181583.1); Strongylocentrotus Six3 (XP_781696.1); Strongylocentrotus Six4 (XP_001181543.1); Terebratalia Six3/6 (JQ88196)Capitella_Dblc2 (195440); Capitella_Esyt2 (160561); Capitella_Rph (238583); Capitella_Syt12 (238576); Capitella_Syt15 (238580); Capitella_Syt16 (238581); Capitella_Syt17 (238578); Capitella_Syt1var1 (183139); Capitella_Syt1var2 (183138); Capitella_Syt4 (238579); Capitella_Syt44 (238582); Capitella_Syt7 (175638); Capitella_Syt9 (181126); Capitella_Sytalpha (238577); Lottia_Dblc2 (236949); Lottia_Esyt2var1 (249373); Lottia_Esyt2var2 (249374); Lottia_Rph (249375); Lottia_Syt12 (249365); Lottia_Syt15a (249370); Lottia_Syt15b (249367); Lottia_Syt16 (249369); Lottia_Syt17 (249364); Lottia_Syt18 (249372); Lottia_Syt1var1 (249360); Lottia_Syt1var2 (249359); Lottia_Syt21 (249371); Lottia_Syt4 (249363); Lottia_Syt47 (249366); Lottia_Syt7 (249362); Lottia_Syt9 (249361); Lottia_Sytalpha (249358); Mus_Dblc2 (NP_034199.1); Mus_Rph (NP_035416.1); Mus_Syt1 (NP_033332.1); Mus_Syt1 (NP_061274.2); Mus_Syt10 (NP_061273.1); Mus_Syt12 (NP_598925.1); Mus_Syt13 (NP_109650.1); Mus_Syt14 (NP_853524.1); Mus_Syt15 (NP_852682.1); Mus_Syt16 (NP_766392.2); Mus_Syt17 (NP_619590.1); Mus_Syt2 (NP_033333.2); Mus_Syt3 (NP_057872.2); Mus_Syt4 (NP_033334.2); Mus_Syt5 (NP_058604.1); Mus_Syt6 (NP_061270.2); Mus_Syt7 (NP_061271.1); Mus_Syt8 (NP_061272.2); Mus_Syt9 (NP_068689.2); Terebratalia_Syt1 (JQ88199)2; NCBI: National Center for Biotechnology Information; NBT: nitroblue tetrazolium chloride; PCR: polymerase chain reaction.BCIP: 5-bromo-4-chloro-3-indolyl phosphate; ESTs: Expressed Sequence Tags; FMRFamide: Phenylalanine-Methionine-Arginine-Phenylalanine-NHThe authors declare that they have no competing interests.SS designed the study, conducted the immunohistochemistry and microscopic imaging, contributed to the gene expression protocols, and drafted the paper. CR participated in molecular cloning, gene expression protocols, microscopic imaging, and assisted in drafting the manuscript. AH participated in molecular cloning, gene expression protocols, microscopic imaging, and assisted in drafting the manuscript. MQM assisted in drafting the manuscript. YJP conducted the molecular cloning and gene expression protocols, participated in microscopic imaging, and assisted in the drafting of the paper. All authors read and approved the final manuscript.Tt-hbn and Tt-otpPhylogenetic analysis of . Phylogram of Tt-hbn, Tt-otp, and related paired-class homeodomain proteins, supporting the orthology assignments of Tt-hbn and Tt-otp. Posterior probability for the homeobrain clade, including Tt-hbn, is 95 percent. Posterior probability for the otp clade, including Tt-otp, is 99 percent. The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 10,000,000 generations each.Click here for fileTt-Six3/6Phylogenetic analysis of . Phylogram of Tt-Six3/6 and related Six-class homeodomain proteins, supporting the orthology assignment of Tt-Six3/6. Posterior probability for the Six3/6 clade, including Tt-Six3/6, is 100 percent. The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 10,000,000 generations each.Click here for fileTt-fezPhylogenetic analysis of . Phylogram of Tt-fez and related zinc-finger proteins, supporting the orthology assignment of Tt-fez. Posterior probability for the Fez clade, including Tt-fez, is 100 percent. The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 10,000,000 generations each.Click here for fileTt-NK2.1Phylogenetic analysis of . Phylogram of Tt-NK2.1 and related NK-class homeodomain proteins, supporting the orthology assignment of Tt-NK2.1. Posterior probability for the NK2.1 clade, including Tt-NK2.1, is 70 percent. The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 10,000,000 generations each.Click here for fileTt-FoxG and Tt-FoxQ2Phylogenetic analysis of . Phylogram of Tt-FoxG, Tt-FoxQ2, and related Forkead box proteins, supporting the orthology assignments of Tt-FoxG and Tt-FoxQ2. Posterior probability for the FoxG clade, including Tt-FoxG, is 100 percent. Posterior probability for the FoxQ2 clade, including Tt-FoxQ2, is 94 percent. The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 20,000,000 generations each.Click here for fileTt-synaptotagmin 1Phylogenetic analysis of . Phylogram of Tt-synaptotagmin 1 and related synaptotagmin proteins, supporting the orthology assignment of Tt-synaptotagmin 1. Posterior probability for the synaptotagmin 1 clade, including Tt-synaptotagmin 1, is 100 percent . The phylogram is a consensus of the last 2,000,000 generations from a Bayesian likelihood analysis with four independent runs of 10,000,000 generations each.Click here for file


Spermatocytic Seminoma is an unusual germ cell tumour known to arise from testis only. It is associated with good prognosis. Testicular tumours as such are uncommon in Asia as compared to western countries. In the literature only five cases of bilateral synchronous Spermatocytic Seminoma have been reported. Fifty years male patient presented to us with bilateral scrotal swelling and evaluation revealed neoplastic aetiology of bilateral testicular enlargement. Left side radical orchidectomy was performed initially which histopathologically revealed spermatocytic seminoma. Subsequently right side radical orchidectomy was performed after intra-op frozen section confirmation of neoplastic nature. Histopathology revealed same pathology as on left side. Immunohistochemistry of specimen from both testes was again conclusive of spermatocytic seminoma. We hereby report this rare case of Bilateral Synchronous Spermatocytic Seminoma. This is the first case report from entire Asian continent except for Japan. Spermatocytic seminoma is an uncommon germ cell neoplasm. It accounts for less than 1% of all testicular tumours and usually arises in older men with a mean age of 53.6 years [3, non tender, non fluctuant, transillumination negative and uniformly firm in consistency. Testicular sensation was typically absent. Right testis measured 5 x 4 x 3 cm3, with similar palpatory findings as left testis. Bilateral groin examination, per abdomen and systemic examination was normal. Ultrasound (USG) scrotum showed bilateral testicular neoplasm with increase vascularity and altered echogenicity and bilateral minimal hydrocele. Ultrasound of abdomen was normal. Tumor markers viz. alpha fetoprotein (AFP), beta HCG, lactate dehydrogenase (LDH) were within normal limits. CT scan abdomen and pelvis showed uniformly enlarged both testis (Fifty years male presented with bilateral scrotal swelling. Left side was larger and noticed since five years, and Right side since one and half year. On examination, left testis measured 13 x 8 x 6 cmh testis  and no eh testis . Histopah testis  was doneh testis . The hisTesticular tumours are uncommon in Asia compared to Western countries. In Asia, the incidence of testicular tumours is as low as 0.4 per 100,000 population . SpermatIt is well known that spermatocytic seminoma is exclusively a testicular tumor, which has never been observed in ovarian or ectopic testis. Spermatocytic seminoma is not associated with any known risk factors for germ cell tumors like cryptorchidism, infertility or gonadal dysgenesis . The metTesticular malignancy as such are unusual. Spermatocytic seminoma is a rare malignancy, and its synchronous bilateral presentation is the rarest. It should be considered, especially while evaluating germ cell tumor in older men. It is distinct in its histologic appearance with three different cell types, lack of cytoplasmic glycogen and sparse or absent lymphocytic infiltrate. Since it rarely metastasizes, correct histologic diagnosis and treatment i.e. orchidectomy can have great impact on prognosis.


Over the past several years, there has been newly emerging and overlapping fields of research investigating CSF pressure and flow pulsatility (CSFp), and cerebral blood flow (CBF). It is not known whether CSFp is merely a passive epiphenomena or alternatively, plays a physiologically significant role in cerebral compliance and blood flow. Thus, the relationship between CSFp and CBF is not clearly understood. In order to study the effect of CSF pulsatility on blood flow, we have developed a novel method and device for controlling  pulsatility.In 15 canine subjects, a small (0.3-3.0cc), custom-made polyurethane bladder was surgically implanted in either the cranial or spinal epidural space such that it would alter the underlying CSF space. CSFp were controlled via an oscillating air pump gated to the cardiac cycle timed to deflate (ICP reduction) or inflate (ICP augmentation) during systole. Measures for ICP (fiberoptic probes) and CBF  were obtained intra-operatively under different acute and chronic conditions.CSPp reduction or augmentation was successfully achieved via cardiac-gated oscillation of a bladder device. Alteration of ICP-waveform was global and measured remotely on the contralateral side and with device activation in the spinal space. Under specific inflation cycles and physiological conditions, operation of the system increased CBF up to 15 mL/min*100gm, or by as much as 40%. Mean systemic pressure, cardiac output, and mean ICP did not significantly change with system activation.We have developed a method of altering CSF pressure pulsatility using cardiac-gated oscillating bladder which dynamically alters CSF space volume. The ability of the system to increase CBF without affecting cerebral perfusion pressure (MAP or ICP) suggests it may work through alteration of cerebrovascular compliance and impedance. Understanding the effect of CSFp on cranial compliance may ultimately allow a new therapy for increasing blood flow in acute and chronic states, including hydrocephalus, where CBF and CSFp are abnormal.SMD, MGL serve as Scientific Advisors for CSF Therapeutics, Inc.


Granulocyte colony-stimulating factors (G-CSFs), filgrastim and lenograstim, are recognised to be useful in accelerating engraftment after autologous stem cell transplantation. Several forms of biosimilar non-glycosylated G-CSF have been approved by the European Medicines Agency, with limited published data supporting the clinical equivalence in peripheral blood stem cell mobilisation and recovery after autologous stem cell transplantation.With the aim of comparing cost-effective strategies in the use of G-CSF after autologous stem cell transplantation, we retrospectively evaluated 32 patients consecutively treated with biosimilar filgrastim XM02 (Tevagrastim) and 26 with lenograstim. All patients received G-CSF (biosimilar or lenograstim) at a dosage of 5 mcg/kg/day subcutaneously from day 5 to absolute neutrophil count of 1500/mmc for three days.p< 0.001). The median days of febrile neutropenia were 1.5 and 1 for filgrastim XM02 and lenograstim, respectively. No adverse event related to the use of XM02 filgrastim was recorded.The median time to absolute neutrophil count engraftment was 11 days for the filgrastim XM02 group and 12 days for the lenograstim group. As for platelets recovery, the median time was 12 days in both groups. The median number of G-CSF vials used for patients was 9.5 for Tevagrastim and 10.5 for lenograstim, reflecting a mean estimated cost of about 556.1 euros for Tevagrastim versus 932.2 euros for lenograstim (In our experience, filgrastim XM02 and lenograstim showed comparable efficacy in shortening the period of neutropenia after cytoreduction and autologous stem cell transplantation, with a favourable cost effect for filgrastim XM02. Escherichia colidiffering from lenograstim in being non-glycosylated and in having an extra methionine group at the N-terminal end of the peptide chain [Granulocyte colony-stimulating factors (G-CSFs), lenograstim and filgrastim, are biological growth factors that promote the proliferation, differentiation, and activation of neutrophils in the bone marrow. Lenograstim is derived from Chinese hamster ovary cells consisting of 174 amino acids with 4% carbohydrate, indistinguishable from native G-CSF, whereas filgrastim is produced in de chain .Both are recognised to be useful in the treatment in chemotherapy-induced febrile neutropenia, in peripheral blood stem cells (PBSCs) mobilisation and in promoting recovery after autologous stem cell transplantation , 2, 4–8.The American Society of Clinical Oncology’s 2006 update on the use of white blood cell growth factors provides recommendations for primary and secondary G-CSF prophylaxis –12; theyThe clinical efficacy of lenograstim and filgrastim has been compared in a systematic review of 16 studies, showing no differences between the two growth factors in any of the approved indications .Biosimilars are non-identical versions of originator biopharmaceuticals. They differ from originator drugs in the size of the active substance, complexity, and nature of the manufacturing process. Several biosimilar G-CSFs are approved in Europe: Biograstim®/Filgrastim, ratiopharm/Ratiograstim®/Tevagrastim® (XM02), Zarzio® and Nivestim®. Recently, biosimilars have been routinely introduced in clinical practice, in particular in the treatment of cancer neutropenia . On the Here, we report our experience of the use of G-CSF biosimilar Tevagrastim (filgrastim XM02), compared with branded lenograstim (Myelostim) in the recovery after PBSC transplantation, taking into account their cost-efficacy profile.From November 2010 to December 2011, 26 consecutive patients with haematological disease  and four patients with solid tumours (testicular seminoma) underwent PBSC transplantation in our haematoncology division and received lenograstim after PBSC reinfusion to accelerate engraftment. The median age was 58 (range 18–73). Thirty-two patients with haematological disease , with similar median age  received G-CSF biosimilar  at the same dosage. The conditioning regimen was ICE  for testicular seminoma, R-FEAM or R-BEAM  for high-grade B cell lymphomas or for Hodgkin lymphoma, Melaphalan for multiple myeloma, and Rituximab–Novantrone–melphalan for follicular lymphomas. Clinical characteristics are reported in 3for three days.All patients received Tevagrastim or lenograstim after PBSC reinfusion at the dosage of 5 mcg/kg/day subcutaneously from day +5 until absolute neutrophil count (ANC) of 1500/mmEngraftment was defined as ANC > 500/mmc and platelet count > 20,000/mmc.3) with an axillary temperature ≥ 38.5 °C or two or more febrile episodes at > 38 °C within a 12-h period.Febrile neutropenia was defined as grade 4 neutropenia  and tabulated with 95% confidence intervals. The hazard ratios have been calculated taking lenograstim as reference. All other treatment comparisons were done using the two-sample Wilcoxon test or the unpaired t-test as appropriate. All tests were considered statistically significant at the 5% level and two tailed. Statistical analyses were performed using the SAS 9.2 .The patients’ characteristics have been summarised and tabulated using either counts and percentages for categorical data or count, mean, median standard error, min and max for continuous variables. The median days to reach the ANC cut-off values of 0.5 x 106/kg (2.0–9.5) and 3.84 x 106/kg (2.0–10.0) in the lenograstim group and filgrastim group, respectively (p= 0.437).The median number of CD34+ cells reinfused was 3.05 x 10p= 0.055) (The median number of days to ANC recovery was 12 days (10–12) in the lenograstim group and 11 (10–11) in the tevagrastim group without statistically significant difference (= 0.055) . Regardipns). With a median follow-up of 14 months (range 3–23), neither systemic or local side effects nor immunogenicity related to the filgrastim was observed.The median number of days of febrile neutropenia was 1 (range 0–10) for the lenograstim group and 1.5 (range 0–5) for the tevagrastim group without statistically significant difference; the median days of antibiotic therapy was 3.5 (range 0–12 ) for the filgrastim group and 4 (range 0–22) for the tevagrastim group (p0.027). Performing a cost analysis evaluation, we found a mean estimated cost about of 932.2 ± 54.4 euros for filgrastim and 556.1 ± 20.0 euros for tevagrastim (p< 0.001)  and 9.5 (9.6 ±0.3), with a statistically significant difference (< 0.001) .et al[Different studies have evaluated the impact of lenograstim in the treatment of chemotherapy-induced febrile neutropenia , in periet al performeBiosimilars are non-identical versions of originator biopharmaceuticals. From their introduction to clinical practice, a cost-efficient impact has been evaluated in different onco-haematological fields.et al[et al[et al[The first application of biosimilars was the chemotherapy-induced febrile neutropenia. Salesi et al reported al[et al in a com al[et al in theirRegarding the application of biosimilars in peripheral stem cells mobilisation, our group reported their experience on the use of Plerixafor in 28 patients treated with the G-CSF originator or with G-CSF biosimilar (Tevagrastim) in patients with lymphoma or myeloma candidate to autologous stem cell transplantation. In this report, no difference was observed with the use of G-CSF or biosimilar Tevagrastim, and no major side effect was reported either with G-CSF or biosimilar Tevagrastim .p0.26) and platelet engraftment , without any increased toxicity with the biosimilar ratiopharm.Finally, the impact of biosimilars was evaluated in the engraftment after PBSC reinfusion. Publicover  demonstret al[6CD34+ cells/kg . The benefit of biosimilar G-CSF in PBSC mobilisation with regard to safety and cost saving was unquestionable due to the identical median consumption of G-CSF doses and leukapheresis procedures noted to reach the target of a minimal count.Lefrère et al enrolledet al[Finally, Czerw et al showed tIn our analysis, patients with haematological and non-haematological disease have been included. Two different agents have been compared in the post-autologous transplantation: the originator lenograstim, (myelostim) and biosimilar filgrastim (Tevagrastim) used in our institution.In our experience, tevagrastim and myelostim were both used at 5 mcg/kg/die. In particular, we did not find a significant statistical difference in terms of ANC recovery between filgrastim biosimilar and lenograstim originator. Nevertheless, this result has been obtained with a lower median number of G-CSF biosimilar vials (9.5) than G-CSF originator (10.5) with a statistically significant difference.Both treatments were safe. With a median follow-up of 14 months we did not observe any deaths related to the autologous procedure.According to multivariate analysis, no statistically significant differences were observed in the two groups in terms of number of febrile neutropenia; engraftment and days of hospitalisation were similar. Nevertheless, the cost between biosimilar G-CSF and originator is very different, biosimilars being cheaper than G-CSF originators. This could allow a significant cost reduction at our centre.To the best of our knowledge, this is the first experience comparing lenograstim versus biosimilar filgrastim, showing no differences in activity and toxicity, with a 27% cost savings in favour of biosimilar.The authors have declared no conflicts of interest.The cost of publication was kindly donated by Gruppo Italiano Infermieristico Mobilizzazione e Aferesi (GIIMA).


Epithelioid hemangioendothelioma (EHE) is a rare, well-differentiated endothelial tumor with intermediate malignancy which develops more frequently from the peripheral veins, generally in the lower limb. Bone EHE comprises less than 1% of the bone neoplasms. We present the case of a young man, 24-year-old, with EHE of the forearm with secondary involvement of the distal radius. The location and the extension of the tumor allowed a wide excision, without the reconstruction of radius, followed by adjuvant radiotherapy, with a subsequent favorable evolution. Based on the clinical, radiographic, and pathological features of the EHE review, we concluded that it is difficult to adopt a standardized therapeutic approach due to the extremely low incidence of the bone involvement in EHE and the variable tendencies towards malignancy of this tumor. To our knowledge this is the third case of EHE with the involvement of the radius. Epithelioid hemangioendothelioma (EHE) is a rare, well-differentiated endothelial tumor with a wide spectrum of behavior. EHE, also known as: low degree anaplastic angiosarcoma, cellular hemangioma, histiocytoid hemangioma or hemangioendothelioma, was considered a term meant to describe tumors that range between hemangiomas and angiosarcomas.It represents 1% of all vascular neoplasms and it is locally aggressive [Generally speaking, EHE as an extremely rare, intermediate-malignant vascular tumor, that develops from the peripheral veins of the lower limb, especially from the iliac and femoral veins . The epiA 24-year-old, right-handed male, machinist, with no pathological personal or family history, was admitted to evaluate a right forearm mass that has slowly increased in the past 4 years. The patient described vague forearm pain, weakness and an oval mass (of approximately 7/4 cm) of hard-elastic consistency that was found on palpation of the extensor part of the forearm. No numbness or functional impairment of the hand were present and the neurovascular examination revealed a normal distal extremity. No history of trauma or other significant systemic diseases was found. Routine laboratory tests were in between normal range. The anteroposterior (AP) radiograph of the distal forearm showed the presence of lamellar periostosis of the radius bone associated with diffuse changes in the bone structure and an aria of reduced bone density in the distal radius Figure . The ultThe patient was scheduled for elective surgical resection. Intrasurgically we found an oval mass that was delimitated by the extensor digitorum muscle, and the extensor brevis of the thumb muscle, and the tendinous portion of the extensor carpi radialis brevis muscle. Figure . The tumThe resected mass measured 6.8 cm × 3.5 cm and was surrounded by healthy tissue with grossly negative margins.The submitted material was fixed in 10% formalin, paraffin-embedded and cut at 5 μm. Slides were stained with hematoxylin and eosin (H&E stain), Masson trichrome, with monoclonal antibody against CD31 , as well as the standard avidin-biotin complex immunostaining method for Ki67.Microscopic examination revealed the proliferation of venous channels with dilated lumina, partially filled with thrombi. Papillary endothelial proliferation was focally present. On this background, a particular finding was the presence of a solid intraluminal nodule, formed by spindled cells that infiltrated the venous wall and extended into the surrounding fibrous and adipose tissue, focally reaching the bone surface Figure . The tumThe patient was referred to the Oncology Centre for local adjuvant radiotherapy having a total dose of 60 Gray/23 fractions/43 days. The tolerance of the treatment was good.No regional or distant metastasis was found. No local recurrence has been observed at the 6, 12 and 24 month follow-up.Besides EHE, the goup of hemangioendotheliomas includes the kaposiform Dabska tumor , spindle-cell, retiform (hobnail hemangioendothelioma), and composite -5EHE is a rare neoplasm of the vascular endothelium, which was described for the first time in 1982 by Weiss and Enzinger as a subgroup of the hemangioendotheliomas having an important endothelial or histiocytic component . Weiss aClinically, EHE is accompanied by pain and swelling. On radiological examination, EHE is an expansive, osteolytic and poorly demarcated lesion. EHE occurs in male\ adults during the second or third decade of life, 60-80% involving the femur, tibia, fibula, or metatarsals ,11. MultThe preoperative evaluation of the EHE must be as complete as possible. The clinical examination should be completed with extensive imagistic investigations. Ultrasonography emphasizes its vascularization and allows a differential diagnosis vis-à-vis an arteriovenous malformation or even an aneurysm. Taking into consideration the rarity of the malignant character of bone EHE, especially at the upper limb level, the soft tissue sarcoma is the closest diagnosis that would come into discussion. The radiology examination and the MRI are necessary in order to determine the degree of the neoformation, the relations with the surrounding tissues, the possible existence of a cleavage plan, and the morphological data about the lesion .The definite EHE diagnosis is established after a careful histopathological extensive differential diagnosis with Langerhans cell histiocytosis, angiosarcoma, infection, myeloma, metastasis, and lymphoma, intranodal palisaded myofibroblastoma, and other tumors of the hand and forearm region -14. SincBecause the cytology prior to the surgical treatment cannot always confirm the malignant character, the treatment of choice is the complete local excision of the tumor with or without regional lymphadenectomy . The ampThe particularity of our case consists in the surgical solution that did not require radius reconstruction or even upper limb amputation, similar to the other two cases. The subsequent favorable evolution in time of our case is an argument in favor of the correctness of the selected complex diagnostic and therapeutical attitude, the latter of which combined surgery and radiotherapy. To the best of our knowledge, we are describing the third case of EHE with distal radius involvement.A standardized therapeutic approach is difficult to adopt due to the extremely low incidence of bone involvement or primitive bone EHE  and also because of the variable tendencies thereof towards malignancy .The role of radiotherapy remains controversial, because of the same reason very few series of reported patients. In the series of Kleer et al.  4 out ofIn summary, bone EHE, the destructive local level tumor with a variable evolution, is remarkable because of the diagnostic difficulties, both from a clinical and a histopathological point of view, resulting in a high frequency of initial misdiagnosis. Its behavior cannot be predicted based only on the histological characteristics, the most important criteria in the appreciation of an unfavorable prognosis seems to be the visceral implication . Under tWritten informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CT: Computer Tomography; EHE: Epithelioid hemangioendothelioma; MRI: Magnetic resonance imaging.The authors declare that they have no competing interests.CG conceived and designed the case report and drafted the manuscript. DF supplied clinical data and contributed to the revisions and editing of the manuscript. All authors have read and approved the final manuscript.


Posthumous organ procurement is hindered by the consenting process. Several consenting systems have been proposed. There is limited information on public relative attitudes towards various consenting systems, especially in Middle Eastern/Islamic countries.We surveyed 698 Saudi Adults attending outpatient clinics at a tertiary care hospital. Preference and perception of norm regarding consenting options for posthumous organ donation were explored. Participants ranked  the following, randomly-presented, options from 1 to 11: no-organ-donation, presumed consent, informed consent by donor-only, informed consent by donor-or-surrogate, and mandatory choice; the last three options ± medical or financial incentive.vs. 11, respectively, p = 0.002). Compared to females, males more perceived donor-or-surrogate informed consent as the norm , more preferred mandated choice with financial incentive option , and less preferred mandated choice with medical incentive option . There was no association between consenting options ranking scores and age, health status, education level, or knowing an organ donor or recipient.Mean(SD) age was 32(9) year, 27% were males, 50% were patients’ companions, 60% had ≥ college education, and 20% and 32%, respectively, knew an organ donor or recipient. Mandated choice was among the top three choices for preference of 54% of respondents, with an overall median ranking score of 3, and was preferred over donor-or-surrogate informed consent , donor-only informed consent , and presumed consent . The addition of a financial or medical incentive, respectively, reduced ranking of mandated choice to 7, p < 0.001, and 5, p < 0.001; for donor-or-surrogate informed consent to 7, p < 0.001, and 5, p = 0.004; and for donor-only informed consent to 8, p < 0.001, and 5, p = 0.56. Distribution of ranking score of perception of norm and preference were similar except for no-organ donation  most respondents were in favor of posthumous organ donation, 2) mandated choice system was the most preferred and presumed consent system was the least preferred, 3) there was no difference between preference and perception of norm in consenting systems ranking, and 4) financial  and medical  incentives reduced preference. Organ transplantation has enabled many patients to have longer and better quality of life, which resulted in an increased demand for a limited supply of organs . PosthumShort of mandated “donation” or conscription, several consenting systems have been proposed/used. Informed (or explicit or ‘opting in’) consent by the donor or his/her family is used in several countries worldwide including Saudi Arabia ,4. A preOrgan donation has been generally based on altruistic donation, however, incentives have also been considered as a means to increase donation rates . FinanciPublic opinion regarding the various consenting systems is crucial, is expected to be culture-specific , and reqThe aim of this study was to survey the Saudi public preference and perception of norm on several consenting systems for posthumous organ donation and determine if they are related to demographic data.This cross sectional survey was based on a convenience sample of a tertiary care hospital attendees and was conducted in accordance with the ethical principles contained in the Declaration of Helsinki and after approval of the Research Ethics Committee (REC) of the King Faisal Specialist Hospital and Research Center (KFSH&RC) in the period from November 2007 to November 2010. A request for waiver of written informed consent was approved by the REC and all respondents gave verbal informed consent.Saudi adult individuals in the waiting areas of the outpatients’ clinics were approached by research coordinators. The number of individuals invited from each waiting area was prorated based on individual clinic load. The questionnaire was self-administered in Arabic language with research coordinator’s support as requested by respondents . The following demographic data were collected, age, gender, reason of visit , perceived health status , education level , knowing an organ donor , and knowing an organ recipient .The questionnaire was developed by the authors in Arabic language based on literature review. After initial development, the questionnaire was presented for comments to 5 physicians and revised accordingly (minor changes in language usage to have consistency throughout the questionnaire). Face validity was assessed by interviewing 10 respondents after completing the questionnaire. The final version was pilot-tested on 10 other respondents for acceptability, comprehensibility in the setting, clarity, and stability (2–3 days); and found suitable. The pilot results were not included in this report. An English translation (accuracy confirmed by back translation) of the questionnaire and the instructions given to respondents are available in the Additional file The study was exploratory, and the sample size and sampling method were conveniently determined. The response rate was calculated as the number of usable questionnaires divided by the number of individuals approached. Data were verified by double entry and validity checks were undertaken. The mean (SD) and median [25% and 75%] score for each consenting option was determined. We used Kendall’s W test to compare median ranking scores among 3 consenting options  and Wilcoxon signed ranks test for pairwise comparisons. We used the Mann–Whitney test to examine if median ranking scores differed according to gender, health status, reason for visit, or knowledge of an organ donor or recipient; and the Kruskal-Wallis test to examine if they differed according to education level. Correlation between age and ranking scores of each option and between consenting options was studied using Spearman’s test. A 2-tailed p value of <0.01 was considered significant. Analyses were conducted by one of the author (MMH) with SPSS for Windows software . 2-tailed p values are reported.1003 individuals were approached; 46 refused to participate, 60 did not understand the study, and 199 did not return a usable questionnaire. The main reasons for not completing the questionnaire were: people were expecting to be called for their appointment, were occupied with children, did not feel well, or did not want to be bothered. Thus responses from 698 (70%) individuals were available for analysis. The demographics of 698 respondents are shown in Table 655 respondents to the preference questionnaire ranked the no-organ donation option. 64% assigned to this option the last 3 ranks 9–11), 18% the five middle ranks (4–8), and 17% the first 3 ranks (1–3), with a median 25%, 75%] ranking score of 11 , respectively, p = 0.002), indicating that even a higher percentage of respondents perceived organ donation as the norm.The distribution of perception of norm ranking scores followed the same pattern Table  and therAs shown in Table vs. 4 , respectively, p = 0.09), donor-or-surrogate informed consent , donor-only informed consent , presumed consent options , mandated choice with medical incentive , donor-or-surrogate informed consent with financial , or medical incentive , or donor-only with medical incentive . However, there was significant difference between personal preference and perception of norm in ranking score for mandated choice with financial incentive  and donor-only informed consent with financial incentive , indicating higher acceptance of the addition of financial incentive as the norm .The distribution of ranking scores of perception of norm followed the same preference pattern Table , and theThe addition of a financial incentive reduced the preference for mandated choice option from 3  to 7  (p <0.001), for donor-or-surrogate informed consent from 4  to 7  (p <0.001), and for donor-only informed consent from 5  to 8  (p <0.001).The addition of a medical incentive had a similar but smaller affect. It reduced the preference for mandated choice option to 5  (p <0.001), for donor-or-surrogate informed consent to 5  (p = 0.004), but not for donor-only informed consent . Figure There was no significant correlation between ranking scores for each of the 11 consenting options and age  for no-organ donation to 0.94 for donor-only informed consent with financial incentive; for perception of norm: p value ranged from 0.11 for no-organ donation to 0.98 for donor-or-surrogate informed consent with financial incentive).There was no difference between subgroups based on perceived health status , or reported reason for hospital visit , or education level .vs. 5  for those who did, p = 0.03) and no-organ donation option . Similarly, there was no significant difference between subgroups based on knowing an organ recipient .There was no significant difference between subgroups based on knowing an organ donor  except of border line significance for presumed consent option , less preferred mandated choice with medical incentive option , and more perceived donor-or-surrogate informed consent as the norm . Mandated choice option continued to be significantly the most preferred and best perceived as norm and financial and medical incentives continued to significantly have a negative effect when analysis was restricted to females. Similar results were obtained when analysis was restricted to males, except that the significant difference between mandated choice and donor-or-surrogate informed consent options was lost both for personal preference (p = 0.17) and perception of norm (p = 0.27).Gender had several significant associations Table . CompareThe aims of this study were to survey Saudi public preference and perception of norm on several consenting options for posthumous organ donation and determine if they are associated with certain demographic data. We studied a convenient sample of 698 adults in the outpatient setting at a tertiary healthcare center in Riyadh, Saudi Arabia. The study sample had a mean (SD) age of 32 (9) year, 27% were males, and 60% had college or higher education. The strengths of the study include a relatively large sample size, a high response rate, simultaneous examination of preference and perception of norm, directly comparing various consenting options, and uniquely addressing Islamic/Arab culture. We found that: 1) most respondents were in favor of posthumous organ donation, 2) mandated choice system was the most preferred and presumed consent system was the least preferred, 3) there was no difference between preference and perception of norm in consenting systems ranking, and 4) financial  and medical  incentives reduced preference for mandated choice and informed consent options.In trying to adopt a consenting system for posthumous organ donation, the following considerations are relevant.1) A common notion is that living people may have interests in the future when they have ceased to exist; although experiential interests are fulfilled only during life, critical interests, such as interest in a good reputation, confidentiality, and body organs after death, can be fulfilled after death ,16. Such2) Is the body a property? One of the fundamental rights is self ownership; the right of non-interference in one’s body without consent. In certain situations, this right is extended to accept payment for body usage , and it has been argued that body organs are akin to goods to which one can claim rights ,18. Howe3) Who “owns” the body of the deceased? The family has a limited property rights in the body of the deceased according to US court . Since t4) The magnitude of psychological harm from an erroneous donation under presumed consent may be more than that from an erroneous non-donation under informed consent; the disutility of losing may be more than the disutility of not getting, and expressing opposition may reflect deeper commitment than expressing agreement.5) People may be more likely to donate when they feel they retain control and people may not object to the act of donation but to the consenting system itself. An act that is freely chosen, regardless of whether it is objectively wrong, may have a greater moral value than an act that may be objectively good but has not been freely chosen ,20,21.The observed diversity in preferences suggests that a one-size-fits-all policy on posthumous organ donation may results in some degree of public dissatisfaction. The diversity in perceptions of norm may be due to an absence of a norm, that the norm is not well known to the public, or that there are several rather than a single norm. The later is more likely. There is of course no statement in Quran or Prophet Muhammad’s Sayings that directly address organ donation, consequently positions on organ donation are based on interpretation. The Saudi Senior Ulama Commission decree issued in 1982 permitted organ donation and transplantation from living and deceased donors . HoweverUnderstanding cultural expectations can provide insight into people preferences and perceptions. Just as secular Western societies continue to be influenced by Judo-Christian norms concerning social ethics , Arabic We found that only 13% and 8% of respondents selected the option of no-organ donation as the first choice for personal preference and perceived norm, respectively. This is consistent with previous studies in Saudi Arabia ,24,30,31vs. being a companion) in personal preference or perception of norm.It is not known if the degree of the expressed preference for donation would change during illness or impending death. Interestingly, we found no difference between subgroups based on perceived health status or reported reason for hospital visit (having a clinic appointment vs. 60% for presumed consent) [We found that the most favorable system  for consenting for posthumous organ donation was the mandated choice system. In this line, a survey of young adults in the USA indicated that 90% supported mandated choice (consent) . Furtherconsent) , which wconsent) .Mandated choice system falls between informed consent system on one hand and presumed consent and mandated donation systems on the other. Advocates of mandated donation system, a system based on the notion of normative consent  , and theThe implementation of a mandated choice system may not be easy as it requires a centralized data bank and may reduce organ availability; it was tested in Texas during the 1990’s, when forced to choose, almost 80% of the people chose not to donate . A mandaWe found that the presumed consent system is the most disfavored among the options studied (we did not explore family involvement or differentiate between stringent and lenient systems). A systemic review of 8 attitude surveys of the UK public to presumed consent reported 28-57% support before 2000, which increased to 64% in 2007 . SurveysThe presumed consent system has been the subject of major public and ethical debates because it put the utilitarian and rights and justice approaches to ethics in conflict. It is associated with higher donation rate ,45, and The expressed disfavoring of presumed consent that we found in our study could be due to distrust in the medical system  and to the feeling of losing control. Additionally, based on the virtue approach to ethics, one need to will the good act ,48. IslaWe found that adding a medical or financial incentive to a mandated choice, donor-only, or donor-or-surrogate system had a negative effect both from preference, and to a lower extent, norm perception points of view. Adding financial incentive had more negative effect than adding medical incentive, which was mainly due to increased less favorable ranking rather than intermediate ranking.Organ donation has long relied on altruism. However, financial incentives have been advocated . The majArguments in favor of adding incentives include an increase in organ supply based on basic economics , intrinsWe are not aware of published public surveys on medical incentives. Consistent with our results, a 2005 study in Saudi Arabia found that only 0.6% of the respondents agreed to donate their organs after death for financial reasons  and a stAlthough the public generally express favorable views toward organ donation ,60,61, fWe found no significant correlation between ranking scores for each of the consenting options and age. However, older age was positively associated with ranking score for the no-organ donation option. This is consistent with previous studies showing that older people are less likely to donate and that younger age correlates positively with willingness to donate . However. We found no difference between subgroups based on perceived health status, perceived reason for hospital visit, or reporting knowing an organ recipient. However, interestingly, respondents who reported knowing an organ donor more favored the presumed consent option and more disfavored no-organ donation option (borderline significance). It is of note that although 55%of the current study population had clinic appointment, 73%were self-perceived as healthy, which has been noted in a previous study and may reflect adaption to the state of illness [There is some evidence that individuals who find themselves increasingly likely to need an organ more intensively perceive the benefits of organ donation . We foun illness .Finally, we found that the addition of financial incentive has a more negative effect on females’ preference and perception of norm than on males. The addition of medical incentive has the opposite affect. This may reflect a difference in gender view of the two types of incentives based on current social role, financial incentives may be seen more amoral and medical incentives less amoral by females because females carry less financial responsibility and are less affluent than males. Females were also less likely to perceive donor-or-surrogate informed consent (but not donor-only informed consent) as the norm, maybe unconsciously stressing self-based (rather than family-based) decision making. Some  but not The study was based on convenience sampling and was performed in a single tertiary health care institution in a major metropolitan city and thus the results may not be generalizable to the general public. Further, the study sample overrepresented females and people with higher education. However, it is of note that the institution is a governmental referral center for the entire country, restricting analysis to males or females did not change the main conclusions of the study, and subgroup analysis based on education level did not reveal significant differences . Since public opinion regarding the various systems for posthumous organ donation would be expected to continue to evolve, the results may not be extrapolateable in time. The study also addressed preferences and perceptions rather than actual choices and did not include the option of family veto.In the setting of outpatient clinics at a tertiary care hospital in Saudi Arabia, we found that: 1) Most respondents favored posthumous organ donation. 2) There is a considerable diversity regarding the most favored consenting system, which may indicate that a one-fits-all policy may result in public’ dissatisfaction and that there is a need for more public education/debates. 3) Distribution of ranking scores of preference and perception of norm were similar, suggesting a rather norm-desiring culture. 4) A mandatory choice system was most favored and a presumed consent system was least favored, suggesting a combination of a response to the duty of easy rescue and the importance to will/intend the act of donation. 5) The addition of financial and medical incentives had negative effect, suggesting prevalence of altruistic motive and belief in sacredness of the body. 6) There was no association between favoring a consenting system and age, perceived health status, education level, or knowing an organ donor or recipient.The authors declare that they have no competing interests.MMH designed the study, performed statistical analysis, performed in literature review, and wrote the manuscript. HMA, AE, HA, and AA participated in data collection. SH participated in statistical analysis and literature review and co-wrote the manuscript. KAC participated in study design and literature review. EAG participated in data collection and critically reviewed the initial draft. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6939/13/32/prepubConsenting Options for Organ Donation: A Survey of the Opinions and Preferences of Saudis.Click here for file


Ventricular hypertrabeculation/noncompaction is a morphologic and functional anomaly of myocardium characterized by prominent trabeculae accompanied by deep recessus. Dilated cardiomyopathy with left ventricular failure is observed in these patients, while the cause or pathophysiologic nature of this complication is not known. Anti-troponin antibodies are formed against circulating cardiac troponins after an acute coronary event or conditions associated with chronic myocyte necrosis, such as dilated cardiomyopathy. In present study, we aimed to investigate cardiac troponins and anti troponin autoantibodies in ventricular noncompaction/hypertrabeculation patients with/without reduced ejection fraction. A total of 50 patients with ventricular noncompaction and 23 healthy volunteers were included in this study. Noncompaction/hypertrabeculation was diagnosed with two-dimensional echocardiography using appropriate criteria. Depending on ejection fraction, patients were grouped into noncompaction with preserved EF  and noncompaction with reduced EF  groups. Troponin I, troponin T, anti-troponin I IgM and anti-troponin T IgM were measured with sandwich immunoassay method using a commercially available kit. Patients with noncompaction had significantly higher troponin I (28.98±9.21 ng/ml in NCNE group and 28.11±10.42 ng/ml in NCLE group), troponin T (22.17±6.97 pg/ml in NCNE group and 22.78±7.76 pg/ml in NCLE group) and antitroponin I IgM (1.92±0.43 µg/ml in NCNE group and 1.79±0.36 µg/ml in NCLE group) levels compared to control group, while antitroponin T IgM and IgG were only elevated in patients with noncompaction and reduced EF (15.81±6.52 µg/ml for IgM and 16.46±6.25 µg/ml for IgG). Elevated cardiac troponins and anti-troponin I autoantibodies were observed in patients with noncompaction preceding the decline in systolic function and could indicate ongoing myocardial damage in these patients. Venricular hypertrabeculation/noncompaction (NC/HT) is a primarily genetic cardiomyopathy characterized by prominent trabeculae with deep recessus separating trabeculations Both cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are components of myocardial troponin-tropomyosin complex and elevated serum levels are observed in dilated cardiomyopathy (DCM) patients due to myocardial necrosis, apoptosis or myocardial leakage. Elevated troponin levels are almost invariably related with poor prognosis in DCM patients While a few studies and report had shown elevated cTnT levels in NC/HT patients with accompanying neuromuscular disorders A total of 50 patients with a previous diagnosis of NC/HT and followed up by institutional heart failure and cardiac transplantation clinic between years 2006 and 2012 were included in this study. All enrolled patients were re-evaluated to confirm previous diagnosis of NC/HT as detailed below. Patients who had severe aortic stenosis or obstructive lesions at left ventricular outflow tract, complex congenital heart lesions, had existing diseases that might cause left ventricular dysfunction or other forms of cardiomyopathy, and patients with previous myocardial infarction were excluded from study. Patients with left ventricular noncompaction were separated in two groups based on their ejection fraction: those with a left ventricular EF of less than 35% (NCLE group) and those with a left ventricular EF of equal to or more than 50% (NCNE group). Additionally, twenty-three volunteers without known coronary artery disease or previous myocardial infarction and had an ejection fraction equal to or more than 50% served as healthy controls.For all subjects, demographic data were collected after inclusion to study. This study was approved by Dr. Siyami Ersek Hospital ethics committee and all patients gave their written informed consent. For patients below 18 years, a written consent was obtained from legal guardians.Echocardiographic examination was performed with an echocardiography platform  equipped with a 1.5–3.6 MHz phased-array transducer. Diagnosis of noncompaction was established when all of these conditions were satisfied from parasternal short-axis view: observation of three or more prominent trabeculations, deep recessus in contact with blood, and a noncompacted-to-compacted myocardium of more than 2 during systole Patients and healthy controls were recalled one day after echocardiographic examination with overnight fasting before collection of blood samples. Whole blood samples were obtained from subjects in the supine position from the antecubital vein with a 20-gauge needle by applying minimal tourniquet force. For all subjects, 8 cc blood was collected in a dry tube and measurements for cTnI, cTnT, antitroponin I IgM, antitroponin T IgM and antitroponin T IgG were made as detailed below. The laboratory was blinded for samples.A serum separator tube was used and samples were allowed to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000×g. Samples were then stored at a temperature between −20° and −80°C. Design of the assay was based on a sandwich enzyme linked immunosorbent assay (ELISA) using Dimension analyzer . The microtiter plate provided in the kit had been precoated with a monoclonal antibody specific to human troponin I or troponin T. Samples were pippetted into these wells. Unbound troponin and other components of the sample were removed by washing and biotin conjugated monoclonal antibody  specific to troponin I or troponin T was added. To quantitatively determine the amount of troponin I or troponin T present in the sample, avidin conjugated to horseradish peroxidase was then added to each microplate well. Next, a TMB-substrate solution was added to each well. Finally, a sulfiric acid solution was added and a resulting yellow colored product was formed. The optical density of wells was determined using a microplate reader set to 450 nm. The absorbance is directly proportional to the amount of captured troponin I or troponin T.The samples were kept at room temperature for 20 minutes to allow clotting, were centrifuged, and were then stored at −20 degrees. The values listed of marker represent the peak concentrations during each collection period. A sandwich enzyme immunologic assay (ELISA) method was used with a Dimension analyzer . Based on a technique using the streptavidin, the single step sandwich assay allowed serial determination of blood samples to be made within 2 hours. The test was carried out in microprocessor-controlled photometers and required streptavidin-coated tubes and two monoclonal antihuman cardiac anti troponin T and anti troponin I antibodies . During a 60 minute incubation period, the antigen was bound by one biotinylated and one peroxidase-labeled antibody. This complex adhered to the test tube wall because of the high affinity streptavidin-biotin interaction. After two washing steps, the substrate chromogen was added.2 and Fisher’s exact tests were used. Correlation analyses were performed using Spearman’s rho. A p value of <0.05 was accepted as significant.Statistical analysis was performed using SPSS 17.0 . Quantitative data was given as mean ± SD and categorical data was given as percentages. Distribution of scalar data was determined with Kolmogorov-Smirnov test. For scalar variables, comparisons between groups were performed with one-way ANOVA and post-hoc Scheffe test or Kruskal-Wallis and Mann-Whitney U tests, as appropriate. For categorical variables, χDemographic and clinical data, including previous medications and interventions regarding to study groups are given in Both troponin T and troponin I levels were significantly higher in both NC/HT groups  compared to controls . Anti-troponin I IgM antibodies were significantly higher in NC/HT patients (1.92±0.43 µg/ml for NCNE group and 1.79±0.36 µg/ml for NCLE group) compared to healthy volunteers . In contrast, anti-troponin T IgM levels are only elevated in patients with noncompaction and systolic dysfunction (15.81±6.52 µg/ml) compared to patients in NCNE group  and healthy controls . Anti-troponin T IgG levels were similar to that of IgM levels  .Troponin I and antitroponin I IgM levels showed a significant correlation with each other ; while both parameters did not show a significant correlation with LVEF, LVEDv or LVESv in noncompaction patients. Troponin T did not correlate with antitroponin T IgM or IgG levels , and both parameters lacked a significant correlation with LVEF, LVEDv or LVESv in noncompaction patients . No signCardiac troponins C, T and I are a part of troponin-tropomyosin complex, which control interaction of actin with myosin. Appearance of cardiac-specific troponins in circulation reflects myocardial damage. Chronic elevation of cardiac specific troponins is observed in conditions such as ischemic or dilated cardiomyopathies, presumably related with ongoing myocellular destruction Our results show that both cardiac troponin I and cardiac troponin T were elevated in patients with NC/HT, regardless of initial systolic function. In patients with DCM, elevated troponin I and T are associated with myocyte injury or death and a progression of heart failure Anti-troponin antibodies are formed after exposure of troponins to circulation. They could be observed in very low quantities in normal individuals We have shown that anti-cTnI IgM antibodies are elevated in patients with NC/HT, regardless of initial ventricular function. This finding is compatible with high cTnI levels in patients with NC/HT with or without reduction of ejection fraction. Since anti-cTnI antibodies are capable of inducing myocardial damage, it is possible that observed anti-cTnI antibody levels could initiate or progress systolic dysfunction in NC/HT patients. However, this hypothesis remains as a deduction and remains to be proven. In contrast, we observed that anti-cTnT IgM and IgG antibodies were elevated only in a subgroup of patients with NC/HT and reduced EF. Despite the fact that anti-cTnT antibodies could be produced after immunization with cTnT in mice, no damage to myocardial cells could be demonstrated Despite intense concern, left ventricular noncompaction remains an enigmatic disease. NC/HT can be observed in isolation or accompanying congenital anomalies or muscular dystrophies This study is designed solely to investigate troponin and antitroponin antibody levels in patients with NC/HT. Our results should be interpreted with caution, as the presence of antibodies themselves does not imply a causal relationship between left ventricular dysfunction and immunity-mediated damage to myocytes. The causative role of antitroponin antibodies in the development of DCM is also debated.A rise in antitroponin autoantibodies is not specific to NC/HT-related ventricular failure and was previously shown to be related with other causes of dilated cardiomyopathy Regrettably, we were unable to study anti-cTnI IgG antibodies, as we could not obtain necessary reagent. While anti-cTnT IgM and IgG levels were similar between groups, we could not verify whether anti-cTnI IgG levels followed a similar pattern with anti-cTnI IgM. While we expect a similar pattern between groups for anti-cTnI IgM and anti-cTnI IgG, as antigenic stimulation is continuous rather than intermittent, another study will be needed to confirm that.In conclusion, this is the first study that examines troponin levels and antitroponin autoantibodies in NC/HT. Our results show that both troponin I and T levels were elevated in patients with NC/HT, regardless of systolic function. Anti-troponin I levels were also elevated in both NC/HT groups, while antitroponin T levels were only elevated in patients with NC/HT and reduced EF. While these results point to ongoing myocardial damage and autoimmune response in NC/HT patients, further studies are required to ascertain the role of autoimmunity in NC/HT.


However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS). The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes  Because almost all proteins in eukaryotic cells are synthesized by cytosolic ribosomes, protein translocation across membranes is critical for organelle biogenesis. The invention of organelle-specific targeting systems in the cytosol was instrumental to facilitate correct translocation events and to avoid mistargeting. These pathways are usually complemented by machineries in the organelle lumen which provide driving force and ensure directionality. For example, in the endoplasmic reticulum (ER) and the mitochondrial matrix members of the Hsp70 family of chaperones bind incoming substrates and thereby prevent their backsliding (ratchet-like mechanism) . A simil3C proteins or twin-CX9C proteins  . So far more than 30 twin-CX9C family members have been identified in human cells, and some of them have been confirmed to be disulfide relay substrates [Most proteins that are imported into mitochondria contain either a mitochondrial targeting signal (MTS) or internal targeting sequences , 7, 8. Tno acid) . The memectively –16. Twinomplexes , 17–22. bstrates , 24.3C and twin-CX9C proteins several more complex substrates exist that rely on the mitochondrial disulfide relay for oxidation  [Translocation of IMS proteins takes place across the translocase of the outer membrane (TOM). Upon exposure of a recognition motif termed MISS or ITS  disulfide relay substrates are recognized by the protein Mia40  , 38, whitofilin)   –50. 3C and twin-CX9C proteins but their nature has to be clarified for the growing class of nonclassical substrates like Atp23. It contains hydrophobic residues, and in most cases, a single cysteine residue, that are positioned on the same side of an alpha helix [MISS/ITS motifs have been well defined for classical twin-CXha helix , 38  [in vivo no semioxidized intermediates of substrate proteins can be observed [The intermolecular disulfide bond between substrate and Mia40 is resolved by another nucleophilic attack of a thiolate anion in the substrate leaving an oxidized substrate molecule and a reduced Mia40 molecule . Thus, fhsErv1) , 53. Theobserved .c [in vitro cytochrome c appears to be the preferred electron acceptor [The sulfhydryl oxidase Erv1/ALR acts to reoxidize the CPC motif in Mia40 . It is ac , 62, 63.acceptor , 65. At acceptor . The idein vitro, too high amounts hamper substrate oxidation and Mia40 reoxidation by binding to reduced cysteines [In vitro substrate oxidation appears to yield side products with nonnative disulfides or substrates that are trapped in their mixed disulfide complex with Mia40 [in vivo as the CPC motif of Mia40 is affected by glutathione in intact cells [In addition to Mia40 and Erv1/ALR further factors modulate oxidative folding in the IMS—the protein helper of Tim protein  and the local glutathione pool  pathways. Mia40 was shown to be critical for mitochondrial dynamics and the hypoxia response  Figure . For Ervα (HIF1α) [α is continuously degraded by the proteasome after hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and subsequent ubiquitinylation by the E3 ligase VHL (von Hippel-Lindau) [α. Moreover, reactive oxygen species take part in the stabilization process by further inhibiting PHD [α by low oxygen concentrations can be mimicked by incubating cells with iron chelators as PHD activity depends on an iron cofactor [Mia40 is not only necessary for proper assembly of the respiratory chain but is also involved in the stabilization of hypoxia inducing factor 1 (HIF1α) , 74  –85. Undeting PHD , 87. Thecofactor –90.α stabilization at low oxygen concentration but not by treatment with iron chelators [α failed to accumulate under low oxygen conditions, while Mia40 overexpression enhanced HIF-1α stabilization under hypoxic conditions. Since the hypoxia response is critical for tumor growth Mia40 depletion effectively inhibited tumor growth and angiogenesis in vivo [α arises from a direct interaction or is indirectly mediated by an impaired respiratory chain remains unclear and is an exciting question for future research.The modulation of Mia40 levels affects HIF1helators . Upon de in vivo . In line in vivo . WhetherHuman patients with a homozygous mutation in Erv1/ALR exhibit respiratory-chain deficiency, myopathy, congenital cataract, sensorineural hearing loss, and delayed development , 91 Fig. In zebrMost of those physiological effects of Erv1/ALR likely derive directly or indirectly from its role in the mitochondrial disulfide relay Figures  and 4. Hα (TGF-α), or insulin [Erv1/ALR has been described to enhance the regenerative capacities of liver tissue –97. For  insulin . Binding insulin . in vitro using recombinant proteins or by overexpressing Erv1/ALR. As already stated above overexpression of IMS proteins leads to cytosolic or nuclear mislocalization [In a second model cytosolic Erv1/ALR acts independently of the MAPK pathway. Cytosolic Erv1/ALR thereby interacts with Jun-activating domain-binding protein 1 (JAB1) which promotes phosphorylation of c-Jun and therefore formation of the c-Jun/activator protein-1 (AP-1) transcription factor complex . C-Jun ilization . This is2+/calmodulin-dependent protein kinase 4). HSCs isolated from Camk4−/− mice possessed reduced levels of ALR and p27(kip) and were deficient in proliferation, which could be restored by ectopic expression of ALR [The interaction of Erv1/ALR with JAB1 is not limited to liver cells. Knockdown of Erv1/ALR in hematopoietic stem cells leads to an increased inhibition of the cyclin-dependent kinase inhibitor p27(kip) by JAB1 while overexpression of ALR leads to a decreased inhibition of p27(kip), probably because JAB1 is sequestered by ALR . Furthern of ALR .γ (interferon-gamma) levels [A complementing explanation for the enhancement of liver regeneration is that Erv1/ALR treatment decreases cytotoxicity of natural killer cells and decreases IFN-) levels , 104. Al) levels . However) levels . Recentl) levels . This in9C proteins by in silico approaches, a concise identification of disulfide relay substrates is still lacking. This becomes especially important because in recent years several proteins with complex structures have been identified as Mia40 substrates. Since these substrates do not adhere to classical cysteine patterns, they cannot be predicted by in silico approaches. This might indicate that the substrate range of the disulfide relay is much wider than previously anticipated. It might also include targets for thiol-dependent redox regulation that cycle between oxidized and reduced states and consequently adapt their activities.Mia40 and Erv1/ALR are well characterized regarding their functions as oxidoreductase and sulfhydryl oxidase of the mitochondrial disulfide relay, respectively. Still several open questions remain : first, Secondly, while we understand oxidative protein folding in the IMS in detail, little is known about the cytosolic processes that take place before translocation across the outer membrane. In previous studies, cytosolic factors were identified which facilitate the import of MTS-containing proteins. However, most disulfide relay substrates lack such targeting information and thus appear like cytosolic proteins. It will therefore be exciting to identify factors that interact with IMS proteins after their translation and guide them to mitochondria or mediate their degradation. Thirdly, the role of Mia40 and Erv1/ALR in processes that appear not directly linked to mitochondria such as hypoxia and liver regeneration is still mechanistically ill-defined. It especially remains unclear whether the functions of both proteins in each case are connected to their function in the disulfide relay or if they operate by completely different mechanisms. We think that it will be exciting to establish in detail the molecular mechanisms that underlie these potentially extra-mitochondrial functions and thus link the biochemistry of thiol oxidation with its physiological impact.


A 8 month old boy suffering from inconsolable cry and tenderness presented to our hospital. Ten hours had passed from the onset of his symptoms. Physical examination showed a tender mass on the left groin. Patient was evaluated with Doppler ultrasound and Technetium-99m pertechnetate testicular scintigraphy Differential diagnosis of torsion and inflammatory disease could not be made by adjunctive tests. The patient underwent surgery based on clinical findings and necrotic inguinal left gonad was shown. In this study, we discussed the scintigraphic pattern in a patient with torsion in undescended inguinal testicleConflict of interest:None declared. Testicular torsion is a well known urologic emergency that needs to be diagnosed and treated rapidly for the salvage of testis . SometimAn 8-month-old infant who has discomfort and inconsolable cry for ten hours was admitted to our hospital. Physical examination showed a tender mass on the left inguinal region. Previously, the patient had a diagnosis of left inguinal hernia and undescended testis.The left testis was found in the inguinal canal, 16x12 mm in diameter by Doppler ultrasound. This testis had significantly heterogeneous parenchyma compared to contralateral testis. Peritesticular tissues in this region were oedemateous and increased vascularity was shown at the left epididymal localization. There was no abnormality in the right testis. Differential diagnosis could not be made.Testicular scintigraphy was performed after the intravenous injection of 3 mCi Tecnetium-99m (Tc-99m) pertechnetate. Flow images and blood pool images of 500.000 counts were obtained in anterior view in a supine position with a large field of view gamma camera using low-energy high resolution parallel hole collimator. The left inguinal mass showed diffusely increased activity -b. ThereSince torsion was more likely suspected in the clinical evaluation, surgical exploration of the inguinal region has been decided. At surgery, the testis was seen as necrotic and twisted 720 degrees intravaginally. After detorsion and heating, there was no recovery for the testis and orchiectomy was performed. The patient's post operative course was uneventful.It is important to differentiate the diagnosis of testicular torsion from the inflammatory diseases such as epididymitis or orchitis in acute scrotum. Delayed diagnosis of testicular torsion may result in necrosis or atrophy. On the other hand, non-surgical clinical events could lead to unnecessary surgery. Although the normal anatomic localization of testes give the chance of easy examination, sometimes it might be difficult to differentiate the diagnosis of acute scrotum, especially in early diagnosis . It coulRadionuclide imaging and Doppler ultrasonography can be performed for differential diagnosis of testicular torsion. The sensitivity of the radionuclide imaging which has been used for this cause for over 30 years is reported within 90 to 100% ,12. DelaThere are a few published cases with torsion of undescended inguinal testis diagnosed with testicular scintigraphy based on photopenic area ,14. ScinAlthough rare, torsion of undescended testis should be considered in a child presenting with abdominal groin pain and empty ipsilateral scrotum. Nuclear physician should be very careful before eliminating the diagnosis of torsion.