Accuracy of point-of-care ultrasound by pediatric emergency physicians for testicular torsion.

INTRODUCTION: Acute scrotum is a common presentation to the pediatric emergency department, and ultrasound is frequently used to narrow the differential diagnosis. Point-of-care ultrasound (POCUS) is increasingly used by urologists and emergency physicians and could potentially be used to detect pediatric testicular torsion. OBJECTIVES: This study aimed to determine the accuracy of POCUS by pediatric emergency physicians in diagnosing testicular torsion and the agreement between point-of-care ultrasound and final diagnosis for other causes of acute scrotum. STUDY DESIGN: A chart review of patients presenting to the study emergency department who received POCUS by a pediatric emergency physician, as well as radiology department ultrasound and/or surgery, was performed. Charts were reviewed for POCUS diagnoses, final diagnoses, and imaging time metrics. RESULTS: A total of 120 patients met study criteria, with 12 cases of testicular torsion. The diagnostic accuracy of POCUS for testicular torsion is described in the summary table. For all causes of acute scrotum, point-of-care ultrasound agreed with final diagnosis in 70% (95% confidence interval [CI] 62-78%) of cases, and more experienced point-of-care ultrasound users displayed higher agreement with final diagnosis. Point-of-care ultrasound results were generated a median of 73 min (Q1 = 51, Q3 = 112) before radiology department ultrasound results. DISCUSSION: Scrotal POCUS performed by pediatric emergency physicians appears to be an accurate tool to detect testicular torsion in children with acute scrotum and saves time compared with radiology ultrasound. The study results may not be generalizable to hospitals without a multidisciplinary POCUS system for quality assurance and image sharing. Future work on POCUS for acute scrotum should investigate its impact on patient outcomes, cost-effectiveness, and family satisfaction. CONCLUSION: Point-of-care ultrasound by pediatric emergency physicians is accurate for detecting testicular torsion in children with acute scrotum and could expedite diagnosis of this time-sensitive condition.

Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin.

Coelomocytes, the heterogeneous population of sea urchin putative immune cells, were found to express a complex set of transcripts featuring scavenger receptor cysteine-rich (SRCR) repeats. SRCR domains define a metazoan superfamily of proteins, many of which are implicated in development and regulation of the immune system of vertebrates. Coelomocytes transcribe multiple SRCR genes from among a multigene family encoding an estimated number of 1,200 SRCR domains in specific patterns particular to each individual. Transcription levels for given SRCR genes may range from pronounced to undetectable, yet all tested animals harbor the genomic loci encoding these genes. Analysis of several SRCR genes revealed multiple loci corresponding to each type. In the case of one SRCR type, a cluster of at least three genes was detected within a 133-kb bacterial artificial chromosome insert, and conserved as well as unique regions were identified in sequences of three genomic clones derived from a single animal. Array hybridizations with repeated samples of coelomocyte messages revealed substantial alterations in levels of expression of many SRCR genes, with fluctuations of up to 10-fold in 1 week and up to 30-fold over a period of 3 months. This report is the first demonstration of genomic and transcriptional complexity in molecules expressed by invertebrate coelomocytes. The mechanisms controlling SRCR gene expression and the functional significance of this dynamic system await elucidation.

Origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes.

Echinoderms share common ancestry with the chordates within the deuterostome clade. Molecular features that are shared between their immune systems and that of mammals thus illuminate the basal genetic framework on which these immune systems have been constructed during evolution. The immune effector cells of sea urchins are the coelomocytes, whose primary function is protection against invasive marine pathogens; here we identify six genes expressed in coelomocytes, homologues of which are also expressed in cells of the mammalian immune system. Three coelomocyte genes reported here encode transcription factors. These are an NFKB homologue (SpNFKB); a GATA-2/3 homologue (SpGATAc); and a runt domain factor (SpRunt-1). All three of these coelomocyte genes respond sharply to bacterial challenge: SpNFKB and SpRunt-1 genes are rapidly up-regulated, while transcripts of SpGATAc factor disappear within hours of injection of bacteria. Sham injection also activates SpNFKB and SpRunt, though with slower kinetics, but does not affect SpGATAc levels. Another gene, SpHS, encodes a protein related to the signal transduction intermediate HS1 of lymphoid cells. Two other newly discovered genes, SpSRCR1 and SpSRCR5, encode proteins featuring SRCR repeats. These genes are members of a complex family of SRCR genes all expressed specifically in coelomocytes. The SRCR repeats most closely resemble those of mammalian macrophage scavenger receptors. Remarkably, each individual sea urchin expresses a specific pattern of SRCR genes. Our results imply some shared immune functions and more generally, a shared regulatory architecture which underlies immune system gene expression in all deuterostomes. We conclude that the vertebrate immune system has evolved by inserting new genes into old gene regulatory networks dedicated to immunity.

The putative sponge aggregation receptor. Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats.

Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting. Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest. All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains. The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa. The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide. To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E. coli. In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa). The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells. In a functional assay Fab' fragments of the antibodies suppressed AF-mediated cell-cell reaggregation. Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation. We conclude that the 220 kDa SRCR-containing protein of the sponge G. cydonium is very likely the AR.

Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium.

The receptor tyrosine kinase of the marine sponge Geodia cydonium features two extracellular Ig-like domains in which we recently documented RT-PCR polymorphism among individuals. Genomic-PCR analysis presented here revealed 14 unique sequences from four sponges, differing predominantly in the sequence of an intron which splits the Ig-like domains. Nevertheless, analysis of putative coding regions in 19 distinct clones (156-159 aa) from seven sponges revealed 69 positions of nucleotide substitutions, 67.6% of them non-synonymous, translating into 43 positions of divergent residues. Excluding aa deletions, these 19 sequences share pairwise aa identities of 89-99%. In three sponges, four or five unique Ig-like coding regions were scored. PCR amplification across this intron revealed multiple bands, polymorphic among five of six sponges. Further substantiated by Southern and Northern blots, it is evident that the genome of G. cydonium harbors multicopies of moderately divergent Ig-like domains. Presently, this only appears paralleled by the human KIR multigene family of NK cells MHC class I-specific receptors, which consist of two or three moderately divergent extracellular Ig-like domains.

A novel member of an ancient superfamily: sponge (Geodia cydonium, Porifera) putative protein that features scavenger receptor cysteine-rich repeats.

Proteins featuring scavenger receptor cysteine-rich (SRCR) domains are prominent receptors known from vertebrates and from one phylum of invertebrates, the echinoderms. In the present study we report the first putative SRCR protein from the marine sponge Geodia cydonium (Porifera), a member of the lowest phylum of contemporary Metazoans. Two forms of SRCR molecules were characterized, which apparently represent alternative splicing of the same transcript. The long putative SRCR protein, of 1536 aa, features twelve SRCR repeats, a C-terminal transmembrane domain and a cytoplasmic tail. The sequence of the short form is identical with the long form except that it lacks a coding region near the C terminus, thus the 1195 aa deduced protein consists of only the first ten SRCR domains and the last 26 C-terminal aa residues, without the transmembrane domain. Homology searches revealed that the sponge putative SRCR protein shares with bovine T-cell antigen WC1 29.2% identity in 1054 aa overlap, 33.9% identity in 475 aa overlap with sea urchin speract and 56% identity in 110 aa overlap with macrophage scavenger receptor type I. Based upon the number and location of the conserved Cys residues, the sponge SRCR domain repeats were classified as belonging to group A of the SRCR superfamily. With twelve SRCR repeats, one more than those in any of the previously described SRCR proteins, and several membrane-bound and soluble forms, it seems that the most primitive known member of this family may be the structurally most complex one among SRCR containing proteins.

A novel tunicate (Botryllus schlosseri) putative C-type lectin features an immunoglobulin domain.

We have cloned a putative C-type lectin of Botryllus schlosseri [Ascidiacea], whose deduced protein of 333 amino acids features three building blocks: (i) a Greek-key motif signature at the amino-terminus, (ii) a C-type lectin domain signature, and (iii) an immunoglobulin (Ig) domain at the carboxyl terminus. This C-type lectin was termed BSCLT. Similarity searches revealed that the Ig domain in BSCLT, which is evidently not polymorphic, is best classified as an Intermediate-type Ig domain. Rabbit antibodies, raised against recombinant BSCLT, cross-reacted in a Western blot with a 38-kD polypeptide in tunicate crude extract. Presumably, this bimodal tunicate protein is the first description of a soluble lectin that features besides the carbohydrate recognition domain also a complete Ig domain.

On the origin of Metazoan adhesion receptors: cloning of integrin alpha subunit from the sponge Geodia cydonium.

Integrins are prominent receptors known from vertebrates and the higher phyla of invertebrates. Until now, no evidence has been provided for the existence of integrins in the lowest Metazoa, the sponges (Porifera). We have isolated and characterized a cDNA clone encoding the alpha subunit of integrin from the marine sponge Geodia cydonium (GCINTEG). The open reading frame encodes a polypeptide of 1,086 residues (118 kDa). The intracellular domain features the sequence Tyr-Phe-x-Gly-Phe-Phe-x-Arg, which is different in one residue from the characteristic consensus pattern for integrin alpha subunits. We conclude that sponges, the oldest multicellular animal phylum, already utilize the structural elements which are required for a tuned and controlled interaction among cells, and between cells and the extracellular matrix.

Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium.

In humans, the 30,000 M(r) Rhesus (Rh) polypeptide D (RhD) is a dominant antigen (Ag) of the Rh blood group system. To date, an Rh-like protein has been found in chimpanzees, gorillas, gibbons, and rhesus monkeys. Related to the 30,000 M(r) Rh Ag protein are two polypeptides of 50,000 M(r), the human 50,000 M(r) Rh Ag and the RhD-like protein from Caenorhabditis elegans. The function of all these proteins is not sufficiently known. Here we characterize a cDNA clone (GCRH) encoding a putative 57,000 M(r) polypeptide from the marine sponge Geodia cydonium, which shares sequence similarity both to the RhD Ag and the Rh50 glycoprotein. The sponge Rh-like protein comprises 523 aa residues; hydropathy analysis hints at the presence of ten transmembrane domains. An N-terminal hydrophobic cleavage signal sequence is missing, suggesting that the first membrane-spanning domain of the sponge Rh-like protein acts as a signal-anchor sequence. The sponge Rh-like protein, like the human Rh50, lacks the CLP motif which is characteristic of the 30,000 M(r) RhD. In addition, the hydropathy profile of the sponge Rh-like protein is of a similar size and shape as that of human Rh50. This data indicates that the RhD and its structurally related Rh50 glycoprotein, which are highly immunogenic in humans, share a common ancestral molecule with the G. cydonium Rh-like protein.

Molecular cloning and sequence analysis of two cDNAs coding for putative anionic trypsinogens from the colonial Urochordate Botryllus schlosseri (Ascidiacea).

Botryllus schlosseri is a colonial marine invertebrate that belongs to the subphylum Urochordata. Previously we analyzed the activity of a serine protease in this species, and cloned a tunicate chymotrypsin-like molecule. In the present study we further analyzed the protease activity of this animal, and found biochemical evidence also for specific trypsin-like activity. Subsequently we utilized a degenerate polymerase chain reaction (PCR) primer to clone two B. schlosseri cDNAs coding for two different putative trypsinogens, each 243 amino acids long, that differ within the coding region in 42 amino acids and 99 nucleotides. Both clones feature the characteristics of animal anionic trypsinogens. Sequence analysis of the tunicate putative trypsinogens revealed the invertebrate characteristics of three disulfide bridges, and higher similarity to invertebrate than to vertebrate trypsinogens. We therefore propose that the typical characteristics of vertebrate trypsinogens evolved after the divergence of Urochordates and Cephalochordates.

A urochordate putative homolog of human EB1, the protein which binds APC1.

The human EBI protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EBI homolog from Botryllus schlosseri (Urochordata. Ascidiacea). The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EBI and 24-25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EBI homologs is indicative of an essential regulatory mechanism in eukaryotic cells.

A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system.

The cDNA for the full-length lectin from the marine sponge Geodia cydonium was cloned. Analysis of the deduced aa sequence revealed that this lectin belongs to the group of galectins. The full-length galectin, which was obtained also in a recombinant form, has an M(r) of 20,877; in the processed form it is a 15 kDa polypeptide. The enriched aggregation factor from G.cydonium also was determined to contain, besides minimal amounts of the galectin, a 140 kDa polypeptide which is involved in cell-cell adhesion. Monoclonal antibodies have been raised against this protein; Fab' fragments prepared from them abolished cell-cell reaggregation. Cell reaggregation experiments revealed that the aggregation factor is an essential component in the aggregation process but it requires likewise the presence of the galectin. Antibodies against the galectin blocked the aggregation factor-mediated cell adhesion. A plasma membrane component was identified (a 29 kDa polypeptide) which interacted with the aggregation factor in the presence of galectin; binding could be blocked both by antibodies against the galectin as well as against the aggregation factor. Immunohistochemical analysis revealed that spherulous cells contain the galectin but not the aggregation factor. By laser scanning microscopy, it is shown that both the aggregation factor and the galectin are located at the rim of the cells. From these data we conclude that the G.cydonium aggregation factor binds to the cells via a galectin bridge.

Cloning of sponge (Geodia cydonium) and tunicate (Botryllus schlosseri) proteasome subunit epsilon (PRCE): implications about the vertebrate MHC-encoded homologue LMP7 (PRCC).

Proteasomes are large protein complexes that play a major role in selective degradation of intracellular proteins. Eukaryotes feature seven different alpha and beta subunits. Two of the vertebrate housekeeping beta-subunits have MHC-encoded homologues that can substitute for the housekeeping counterparts upon interferon-gamma induction. In the present study we report the cloning of invertebrate beta-subunit proteasome epsilon (PRCE), from the marine sponge Geodia cydonium and from the colonial tunicate Botryllus schlosseri. Sequence comparisons revealed that the sponge and tunicate proteins are strikingly similar to vertebrate and yeast PRCEs and their MHC-linked counterparts the PRCCs (also termed LMP7), and to a lesser degree also to archaebacterial proteasome subunit beta. Based on this comparison we suggest that all eukaryotic PRCEs and PRCCs feature a cleavable N-terminal propeptide, including the two mammalian PRCEs which appear to have been wrongly predicted from incomplete cDNAs. Our comparative analysis outlines 25 amino acid positions which appear to be unique for PRCCs, distinct from the corresponding residues in metazoan PRCEs.

Polymorphism in the immunoglobulin-like domains of the receptor tyrosine kinase from the sponge Geodia cydonium.

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cydonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatible. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5'-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.

Molecular evolution of the metazoan protein kinase C multigene family.

Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues; they respond to second messengers, e.g., Ca2+ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced from Geodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One sponge G. cydonium PKC, GCPKC1, belongs to the "novel" (Ca2+-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hallmarks of the "conventional" (Ca2+-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted aa sequences for these cDNAs with respective segments from previously reported sequences, revealed highest homology to PKCs from animals but also distant relationships to Ser/Thr kinases from protozoa, plants, and bacteria. However, a comparison of the complete structures of the sponge PKCs, which are-already-identical to those of nPKCs and cPKCs from higher metazoa, with the structures of protozoan, plant, and bacterial Ser/Thr kinases indicates that the metazoan PKCs have to be distinguished from the nonmetazoan enzymes. These data indicate that metazoan PKCs have a universal common ancestor which they share with the nonmetazoan Ser/Thr kinases with respect to the kinase domain, but they differ from them in overall structural composition.

Coexistence and Possible Parasitism of Somatic and Germ Cell Lines in Chimeras of the Colonial Urochordate Botryllus schlosseri.

Fusion between conspecifics (chimerism) is a well-documented phenomenon in a variety of taxa. Chimerism and the subsequent mixing of genetically different stem cell lines may lead to competition between cell lineages for positions in the germ line and to somatic and germ cell parasitism. It is suggested that somatic compatibility systems evolved to alleviate the costs and the threat of such cell lineage competition. Allogeneic colonies of the ascidian Botryllus schlosseri form vascular chimeras based on matching in one or both alleles on one highly polymorphic fusibility haplotype. Thereafter, one of the partners is completely or partially resorbed. Here we used a polymorphic molecular marker (PCR typing at a microsatellite locus) to follow somatic and gametic consequences of chimera formation. Twenty-two chimeras and subclone samples were established from 12 different genotype combinations, in which blood cells, zooids, and gonads were typed 45-130 days thereafter. Somatic coexistence of both partners was recorded in 73% of the subcloned chimeras (83% of chimeric entities) up to 100 days after disconnection between genotypes and in all chimeras where colony-resorption was completed. Both genotypes were present in 23% of the sampled gonads (in 33% of the chimeras), and in 22% of the cases, germ cells of the second partner only were detected. Injection of allogeneic but compatible blood cells into three recipient colonies revealed proliferation of the donor cells in one case, 100 days after injection. To further evaluate somatic and germ cell parasitism in chimeric organisms, we propose four key features that characterize cell lineage competition processes. These include the somatic embryogenesis mode of development, the capability for independent existence of stem cells, the disproportionate share of gametic output within chimeras, and the existence of hierarchial responses.

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily.

Mammalian tumor necrosis factor (TNF)-alpha degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-alpha, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs.1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs.1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the aa similarity (up to 50.5%) of Bs.1 with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs.1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.