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Neurotrophic Tyrosine Receptor Kinase (NTRK) genes undergo chromosomal translocations to create novel open reading frames coding for oncogenic fusion proteins; the N-terminal portion, donated by various partner genes, becomes fused to the tyrosine kinase domain of either NTRK1, NTRK2, or NTRK3. NTRK fusion proteins have been identified as driver oncogenes in a wide variety of tumors over the past three decades, including Pediatric Gliomas, Papillary Thyroid Carcinoma, Spitzoid Neoplasms, Glioblastoma, and additional tumors. Importantly, NTRK fusions function as drivers of pediatric sarcomas, accounting for approximately 15% of childhood cancers including Infantile Fibrosarcoma (IFS), a subset of pediatric soft tissue sarcoma (STS). While tyrosine kinase inhibitors (TKIs), such as larotrectinib and entrectinib, have demonstrated profound results against NTRK fusion-positive cancers, acquired resistance to these TKIs has resulted in the formation of gatekeeper, solvent-front, and compound mutations. We present a comprehensive compilation of oncogenic fusions involving NTRKs focusing specifically on pediatric STS, examining their biological signaling pathways and mechanisms of activation. The importance of an obligatory dimerization or multimerization domain, invariably donated by the N-terminal fusion partner, is discussed using characteristic fusions that occur in pediatric sarcomas. In addition, examples are presented of oncogenic fusion proteins in which the N-terminal partners may contribute additional biological activities beyond an oligomerization domain. Lastly, therapeutic approaches to the treatment of pediatric sarcoma will be presented, using first generation and second-generation agents such as selitrectinib and repotrectinib.

Cholangiocarcinoma, originating from the biliary duct, represents a subset of liver cancer. With about 8000 new cases of cholangiocarcinoma diagnosed annually in the U.S., these fall into three categories: intrahepatic, peri-hilar, and extrahepatic cholangiocarcinoma. Arising from the epithelium of the bile duct, intrahepatic cholangiocarcinoma (ICC) is a universally fatal malignancy with very few treatment options. The poor prognosis and lack of molecular targeted therapies highlights ICC as a critical unmet medical need. With advances in sequencing technology, numerous chromosomal translocations have been discovered as drivers in cancer initiation and progression. Particularly in ICC, chromosomal translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) have been frequently identified, resulting in the creation of oncogenic fusion proteins. At the N-terminus, these fusion proteins share a nearly-identical FGFR2 moiety retaining an intact kinase domain and, at the C-terminus, a dimerization/oligomerization domain provided by different partner genes, including: Periphilin 1 (PPHLN1), Bicaudal family RNA binding protein 1 (BICC1), Adenosylhomocysteinase Like 1 (AHCYL1), and Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3). A number of pre-clinical and clinical trials have shown the effectiveness of FGFR inhibitors in treating FGFR2 fusion-positive ICC patients. However, the efficacy of these inhibitors may be short-lived due to acquired resistance. In this review, we provide an overview of FGFR2 fusions, comparing their structures and mechanism of dimerization, examining the importance of FGFR2 as a partner gene, as well as highlighting the significance of alternative splicing of FGFR2 in these fusion proteins. In addition, we discuss various therapeutic options and their associated potencies in targeting these translocation-induced ICCs.

Traditional evolutionary scenarios posit that land plants emerged from land plant-like relatives, the charophytes. New phylogenies suggest a closer affinity to simpler pond scum relatives, and evidence the gradual assembly of the land plant genome, revealing a phenotypic simplification from the complex ancestors envisaged by traditional scenarios.

SUMMARY High-quality protein feeds are needed in ecological and organic poultry production. Feeds that are rich in methionine (MET) are particularly important because synthetic amino acids are generally prohibited in organic livestock production. Bigheaded carp present an opportunity as feed because they are an invasive fish species that are high in protein and MET. A carp-based meal (CBM) was prepared using innovative dry extrusion, and proximate analysis and nutritional analyses were conducted. The digestible amino acids and true metabolizable energy (TMEn) of the CBM were determined using a colony of cecetomized adult roosters. Starter and grower diets were formulated with CBM and tested in a broiler feeding trial. Diets were high overall in protein, but formulated without synthetic MET. Day-old chicks were randomly assigned to pens of 30 chicks belonging to 1 of 2 treatments (CBM or control containing synthetic MET), with 8 replications. Body weights, feed intake, and feed conversion ratio (FCR) for starter (0–21 d), grower (22–42 d), and overall periods (0–42 d) were analyzed using 1-way ANOVA. The weight gains, feed intake, and FCR did not differ between treatments (P > 0.05) for the overall period (0–42 d). The results indicate that invasive bigheaded carp can be harvested and dry extruded into a novel meal, providing not only a natural and sustainable source of MET, but also an environmental service for damaged aquatic ecosystems in the U.S.

Micro-CT provides a means of nondestructively investigating the internal structure of organisms with high spatial resolution and it has been applied to address a number of palaeontological problems that would be undesirable by destructive means. This approach has been applied successfully to characterize the cranial anatomy of Shuyu, a 428 million-year-old galeaspid (jawless stem-gnathostome) from the Silurian of Changxing, Zhejiang Province, China. Here, we use the synchrotron X-ray tomographic microscopy (SRXTM) to further describe the circulatory system of the head of Shuyu. Our results indicate that the circulatory system of galeaspids exhibits a mosaic of primitive vertebrate and derived gnathostome characters, including a number of derived gnathostome characters that are absent from osteostracans — the group conventionally interpreted as the sister lineage of jawed vertebrates. Our study provides a rich source of information that can be used to infer and reconstruct the early evolutionary history of the vertebrate cardiovascular system.

Two new studies that consider the timing of origin of angiosperms are poles apart in their estimates. However, their partisan molecular and palaeontological perspectives may hold the key to establishing a unified evolutionary timescale for flowering plants.

SUMMARY Organic poultry production is based on ecological processes, such as nutrient cycling, and reduces the use of synthetic inputs. High-quality protein feeds are needed to provide essential amino acids for poultry, particularly methionine. Although synthetic methionine is currently allowed in poultry feeding in the US organic program, sources that are natural are needed. Fishmeal is a high-quality protein and contains methionine; however, fishmeal sources are limited and may not be sustainable or local. A possible source of fishmeal in the USA is Asian carp fishmeal. Asian carp have invaded the Mississippi river basin with negative impacts on native fish and riparian ecosystems, and harvesting the carp may be a management strategy to help control it. Additional options for high-quality protein feeds include invertebrate meals from mass insect production, such as black soldier fly larvae, and worms. These can be raised sustainably for poultry feed by using crop by-products, residues, or recycled food to reduce food waste. Alternative feeding methods may be helpful to utilize these innovative feeds. Alternative protein feeds can contribute to ecological poultry production and have an important role under certified organic standards.

SUMMARY Currently, there is little scientific information regarding the effect of differing housing types and production methods on the final quality of broiler meat. The purpose of this study was to evaluate meat quality characteristics of commercial fast-growing broiler chickens when raised in portable vs. fixed housing, with or without access to pasture. Fast-growing broilers are being grown by many small farm poultry producers due to their favorable attributes, including greater breast meat yields, a more uniform carcass, and higher feed efficiency than heritage or slower growing breeds. The pasture land used in this study closely matched the lands common to this local area. It was a pasture composed of a mix of tall fescue, Bermuda grass, and a lesser amount of legumes and forbs. The experiment had 4 treatment groups: (1) small, portable hoop houses with access to pasture, (2) small, portable hoop houses without access to pasture, (3) a fixed house with access to the outdoors, and (4) a fixed house without access to the outdoors. The study was conducted during spring and fall within the same yr to evaluate if any seasonal effects on carcass or meat quality were present. Overall, the access to pasture did not alter meat quality parameters such as moisture, protein, fat percentage, pH, color, texture, cooking loss, or shear energy when compared to birds without pasture access. Since forages contribute to the nutritive value of the meat and may help in reducing costs, the type and quality of forage choices in pastures are important considerations for these production methods and need to be explored further for pasture production systems.

Summary We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes., Graphical Abstract, Highlights • Ganoin emerged in actinopterygians; true enamel arose in sarcopterygians • Dental enamel, acrodin, and enameloid in actinopterygians are related to ganoin • SCPP5 evolved in association with ganoin, whereas AMEL evolved with true enamel • Shifts in SCPP gene expression explain the evolution of hypermineralized tissues, Evolutionary Biology; Evolutionary Processes; Phylogenetics; Paleobiology

Aim of study Chromosomal translocations such as t(10;12)(q26,q12) are associated with intrahepatic cholangiocarcinoma, a universally fatal category of liver cancer. This translocation creates the oncogenic fusion protein of Fibroblast Growth Factor Receptor 2 joined to Periphilin 1. The aims of this study were to identify significant features required for biological activation, analyze the activation of downstream signaling pathways, and examine the efficacy of the TKIs BGJ398 and TAS-120, and of the MEK inhibitor Trametinib. Methods These studies examined FGFR2-PPHLN1 proteins containing a kinase-dead, kinase-activated, or WT kinase domain in comparison with analogous FGFR2 proteins. Biological activity was assayed using soft agar colony formation in epithelial RIE-1 cells and focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways were examined for activation. Membrane association was analyzed by indirect immunofluorescence comparing proteins altered by deletion of the signal peptide, or by addition of a myristylation signal. Results Biological activity of FGFR2-PPHLN1 required an active FGFR2-derived tyrosine kinase domain, and a dimerization domain contributed by PPHLN1. Strong activation of canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways was observed. The efficacy of the tyrosine kinase inhibitors BGJ398 and TAS-120 was examined individually and combinatorially with the MEK inhibitor Trametinib; heterogeneous responses were observed in a mutation-specific manner. A requirement for membrane localization of the fusion protein was also demonstrated. Concluding statement Our study collectively demonstrates the potent transforming potential of FGFR2-PPHLN1 in driving cellular proliferation. We discuss the importance of sequencing-based, mutation-specific personalized therapeutics in treating FGFR2 fusion-positive intrahepatic cholangiocarcinoma.

Diving beetles and their allies are a virtually ubiquitous group of freshwater predators. Knowledge of the phylogeny of the adephagan superfamily Dytiscoidea has significantly improved since the advent of molecular phylogenetics. However, despite recent comprehensive phylogenomic studies, some phylogenetic relationships among the constituent families remain elusive. In particular, the position of the family Hygrobiidae remains uncertain. We address these issues by re-analyzing recently published phylogenomic datasets for Dytiscoidea, using approaches to reduce compositional heterogeneity and adopting site-heterogeneous mixture models. We obtained a consistent, well-resolved, and strongly supported tree, robust to analyses of various sizes of datasets. Consistent with previous studies, the monophyly of the geographically disjunct Aspidytidae is strongly supported. Our analyses support that Aspidytidae are the sister group of Amphizoidae, and more importantly, Hygrobiidae are sister to the diverse Dytiscidae, as convincingly demonstrated by morphology-based phylogenies. Our new results are congruent with recent morphology-based phylogenies. The phylogeny of Dytiscoidea can be resolved by reducing the effect of among-site compositional heterogeneity and adopting a better-fitting model accommodating site-specific amino acid preferences. Our analyses provide a backbone phylogeny of Dytiscoidea, which lays the foundation for better understanding the evolution of morphological characters, life habits, and feeding behaviors of dytiscoid beetles.

Campylobacter is one of the leading causes of foodborne illness worldwide and is associated with consumption of poultry products. One potential strategy to reduce Campylobacter colonization in poultry is the use of probiotics. In an attempt to reduce Campylobacter in poultry, we isolated 117 GRAS (generally recognized as safe) bacterial isolates from the ceca of healthy birds and evaluated their efficacy against Campylobacter, using a soft agar overlay technique. Twenty-six isolates (Bacillus and Lactobacillus spp.) with greatest ability to inhibit Campylobacter in vitro, were tested in birds. In these studies, bacterial isolates were administered orally on day-of-hatch and, chicks were challenged with a four-strain mixture of wild type C. jejuni (∼106 CFU/mL) on day 7. Cecal samples were collected on day 14 for Campylobacter enumeration. Out of 26 isolates, 3 isolates demonstrated a reduction in Campylobacter counts (1 to 2 logs). In an attempt to improve the efficacy of these 3 isolates, follow-up studies were conducted with various doses of 2 prebiotics (fructooligosaccharide [FOS] or mannan oligosaccharide [MOS]) in the feed. Campylobacter counts were not reduced for any of the FOS treatments (0.125%, 0.25%, or 0.5%) or the MOS treatments (0.04%, 0.08%, or 0.16%), but were reduced for the combination of isolate 3 with 0.04% MOS in feed. The results of this study indicate that the selection and administration of bacterial isolates in combination with selected prebiotics may reduce enteric Campylobacter counts in preharvest poultry.

The four receptor tyrosine kinases (RTKs) within the family of Fibroblast Growth Factor Receptors (FGFRs) are critical for normal development but also play an enormous role in oncogenesis. Mutations and/or abnormal expression often lead to constitutive dimerization and kinase activation of FGFRs, and represent the primary mechanism for aberrant signaling. Sequencing of human tumors has revealed a plethora of somatic mutations in FGFRs that are frequently identical to germline mutations in developmental syndromes, and has also identified novel FGFR fusion proteins arising from chromosomal rearrangements that contribute to malignancy. This review details approximately 200 specific point mutations in FGFRs and 40 different fusion proteins created by translocations involving FGFRs that have been identified in human cancer. This review discusses the effects of these genetic alterations on downstream signaling cascades, and the challenge of drug resistance in cancer treatment with antagonists of FGFRs.

Outdoor access is an important part of organic and free-range poultry production, yet limited information exists on the effect of various housing and production systems on growth performance and colonization of food-borne pathogens. Therefore, the primary purpose of the current study was to evaluate the influence of different housing systems, particularly fixed versus small, portable houses, with and without outdoor access to pasture, on seasonal growth performance, leg quality, and presence of bacterial food-borne pathogens. In the current study, we used fast-growing broilers, as many small producers use commercial broilers due to their carcass conformation and high breast yield compared with slow-growing hybrids or standard heritage breeds. Although interest in alternative genetics exists because they may be more adapted to outdoor production, they require longer growing periods, with accompanying labor and cost. A pasture containing a mixture of forages was used to simulate the conditions common for small farms in the local region. The experiment had 4 treatment groups: (1) small, portable hoop houses with access to pasture, (2) small, portable hoop houses without access to pasture, (3) a fixed house with access to the outdoors, and (4) a fixed house without access to the outdoors. The present study was repeated at different times of the year to determine if a seasonal effect on the consumption of pasture and carcass quality was present. Overall, raising birds in hoop houses resulted in a reduced growth rate compared with birds raised in the fixed house. None of the production systems altered bone strength or feed conversion. Food-borne pathogens commonly associated with poultry were not found in any of the environments tested. Seasonal production was an issue in the small hoop house birds, as extreme heat in the summer resulted in early termination of that trial. Expanding on forage choice in pastures and customizing hoop houses to deal with weather fluctuations, especially in regions where extreme heat may affect production, are important considerations for these systems.

microRNAs (miRNAs) are short noncoding regulatory genes that perform important roles in plant development and physiology. With the increasing power of next generation sequencing technologies and the development of bioinformatic tools, there has been a dramatic increase in the number of studies surveying the miRNAomes of plant species, which has led to an explosion in the number of described miRNAs. Unfortunately, very many of these new discoveries have been incompletely annotated and thus fail to discriminate genuine miRNAs from small interfering RNAs (siRNAs), fragments of longer RNAs, and random sequence. We review the published repertoire of plant miRNAs, discriminating those that have been correctly annotated. We use these data to explore prevailing hypotheses on the tempo and mode of miRNA evolution within the plant kingdom.

SUMMARY Interest in small- and medium-scale free-range poultry production for local and regional markets is growing, and alternative feeding methods should be considered. Free-choice feeding is a method that offers birds separate feedstuffs, such as grains, protein concentrates, and natural vitamin and mineral sources, from which they can self-select a diet suited to their changing needs. Free-choice feeding may prove useful in production systems with outdoor access because nutrient needs change widely due to temperature fluctuations and bird activity. Many small producers do not have access to the nutritional services that large producers do and may have specific goals in regard to nutrition, such as using farm-raised feed ingredients and pasture forage to provide nutrients. In addition, most organic programs do not permit the use of synthetic amino acids in feed. Free-choice methods have been used historically and can be useful for alternative producers, making use of farm-raised feeds to improve savings and increase nutrient cycling. In the current study, a fully formulated diet and free-choice diet were compared in a free-range system using slow-growing meat chickens. The formulated diet was a commercial product (20% CP), whereas the free-choice diet chosen by birds was much lower in CP (13%). Final live weights did not differ between treatments; however, ready-to-cook yield and breast yields were higher in the birds from the formulated treatment, most likely due to amino acid supplements in the formulated feed. The diet chosen by free-choice birds was less expensive than the formulated diet.

Duplications of the X chromosome are rare cytogenetic findings, and have been associated with an abnormal phenotype in the male offspring of apparently normal or near normal female carriers. We report on the prenatal diagnosis of a duplication on the long arm of chromosome X from chromosomal band Xq13.2 to q21.31 in a male fetus with increased nuchal translucency in the first trimester and polyhydramnios at 22 weeks of gestation. Amniocentesis was undertaken and cytogenetic analysis revealed additional chromosomal material in the long arm of chromosome X at position Xq13. Analysis with high resolution array CGH revealed the additional material is in fact a duplication of the region Xq13.2–q21.13. The duplication is 14.8 Mb in size and includes fourteen genes: SLC16A2, KIAA2022, ABCB7, ZDHHC15, ATRX, MAGT1, ATP7A, PGK1, TBX22, BRWD3, POU3F4, ZNF711, POF1B and CHM. Analysis of the parents revealed the mother to be a carrier of the same duplication. After elected termination of the pregnancy at 28 weeks a detailed autopsy of the fetus allowed for genotype–phenotype correlations.

SUMMARY As parasites become resistant to available anthelmintics, new methods of control are needed. New drugs take a long time to develop in addition to being expensive; therefore, there is increasing interest in finding and using natural alternatives. Additionally, natural remedies are needed for the organic sector because synthetic drugs are not allowed and birds with outdoor access are likely to encounter parasites. Sericea lespedeza [SL; Lespedeza cuneata (Dum. Cours.) G. Don.] is a common perennial legume found in pastures across the southern United States that has been shown to be effective at controlling parasitic nematodes in small ruminants because of its condensed tannin content. Diets high in condensed tannins are often unpalatable to poultry; however, growers report that chickens maintained on pastures will consume SL. These reports and the level of consumption have not been verified. Therefore, before determining its potential in controlling parasites in poultry, we confirmed in a preliminary study that birds on pasture consumed SL (92% of birds examined had SL in crops), and in a subsequent study, dried SL leaves were added to a commercial broiler feed to determine the palatability of SL at various concentrations. Diets included 0 (control), 5, 10, or 20% SL (DM weight) and were fed from hatch until slaughter at 6 wk of age. Male broilers (n = 80) were randomly divided into 8 groups and fed 1 of the 4 diets in replicate. Body weight gains were similar between the control and SL at 5%. Including more than 5% SL in the diet reduced individual BW (P < 0.05). At the end of the feeding period, the digestive organs as a percentage of body weight of birds fed 20% SL were larger than those of control birds. Feed conversion was higher in the 20% SL treatment (2.31) than in the control treatment (1.63; P < 0.05). Palatability of SL did not appear to be a problem because all treatment groups consumed a similar amount of feed. Therefore, the effects of SL on parasite control will be evaluated in subsequent studies.

SUMMARY In this study, we investigated the therapeutic efficacy of the plant compounds trans-cinnamaldehyde (TC) and eugenol (EG) on reducing Salmonella enterica serovar Enteritidis (SE) in commercial, market-age broiler chickens. Straight run, day-old commercial broiler chicks (N = 84) were randomly grouped into 6 groups of 14 birds each (n = 14/group): a negative control (no SE, no TC or EG), EG control (no SE, 1% EG), TC control (no SE, 0.75% TC), a positive control (SE, no TC or EG), an EG challenge group (SE, 1% EG), and a TC challenge group (SE, 0.75% TC). Before the start of each experiment, the flock was screened for any inherent Salmonella (n = 12 birds/experiment). Birds were given ad libitum access to Salmonella-free feed and water. On d 30, birds were challenged with a 4-strain mixture of SE (8 log10 cfu/bird). Two birds from each group were killed after 24 h (d 31) to check for colonization of SE in the cecum. Birds were given feed supplemented with TC (0.75%) or EG (1%) for 5 d before slaughter on d 42 (n = 10 birds/group) for determination of SE populations in the cecum and cloaca. The experiment was repeated 2 times. The trans-cinnamaldehyde and EG consistently reduced SE in the samples in both experiments (P 0.05). On the basis of histological analysis, no abnormal changes in the liver were observed because of supplementation of plant compounds. Therefore, TC and EG supplemented through the feed could reduce SE colonization in market-age chickens.

Campylobacter is one of the most common causes of foodborne illness, and poultry are considered a primary source of Campylobacter infections. Caprylic acid, an 8-carbon fatty acid, has been shown in previous studies to reduce enteric cecal Campylobacter concentrations in poultry when administered in the feed. For greater ease of application for producers, a water-soluble form of caprylic acid, sodium octanoate, was evaluated for efficacy against enteric Campylobacter. The first trial consisted of 70 birds in 7 groups (n = 10 chicks/group): an untreated control and 6 other groups that were challenged with Campylobacter at d 3 and that received 0, 0.175, 0.35, 0.7, 1.4, or 2.8% water-soluble caprylic acid in water 3 d before necropsy at d 14. The second trial consisted of 80 birds in 8 groups (n = 10 chicks/group): an untreated negative control and 7 other groups, all of which were challenged with Campylobacter at d 3 and received 0, 0.044, 0.088, 0.175, 0.35, 0.7, or 1.4% water-soluble caprylic acid for 3 d before necropsy at d 14. In trial 1, only the 0.175% dose caused a reduction in cecal Campylobacter counts in comparison with the positive control (approximately a 3-log reduction). In trial 2, no treatment reduced Campylobacter counts compared with the positive control. Unlike the efficacy of caprylic acid in feed, treatment with caprylic acid in water had an inconsistent effect on intestinal Campylobacter counts.

Summary The emergence of multicellular animals was associated with an increase in phenotypic complexity and with the acquisition of spatial cell differentiation and embryonic development. Paradoxically, this phenotypic transition was not paralleled by major changes in the underlying developmental toolkit and regulatory networks. In fact, most of these systems are ancient, established already in the unicellular ancestors of animals [1, 2, 3, 4, 5]. In contrast, the Microprocessor protein machinery, which is essential for microRNA (miRNA) biogenesis in animals, as well as the miRNA genes themselves produced by this Microprocessor, have not been identified outside of the animal kingdom [6]. Hence, the Microprocessor, with the key proteins Pasha and Drosha, is regarded as an animal innovation [7, 8, 9]. Here, we challenge this evolutionary scenario by investigating unicellular sister lineages of animals through genomic and transcriptomic analyses. We identify in Ichthyosporea both Drosha and Pasha (DGCR8 in vertebrates), indicating that the Microprocessor complex evolved long before the last common ancestor of animals, consistent with a pre-metazoan origin of most of the animal developmental gene elements. Through small RNA sequencing, we also discovered expressed bona fide miRNA genes in several species of the ichthyosporeans harboring the Microprocessor. A deep, pre-metazoan origin of the Microprocessor and miRNAs comply with a view that the origin of multicellular animals was not directly linked to the innovation of these key regulatory components., Highlights • The animal-specific miRNA Microprocessor is discovered in unicellular Ichthyosporea • The origin of the animal miRNA machinery was independent of animal multicellularity • The Microprocessor is lost in ctenophores and is not an ancestral animal trait • Several ichthyosporeans harboring the Microprocessor express bona fide miRNAs, In animals, microRNAs and the miRNA biogenesis machinery are essential for correct organismal development. Bråte et al. demonstrate that the core of this machinery, the Microprocessor, is not an animal innovation but originated among their unicellular relatives. Several unicellular species harboring the Microprocessor also express bona fide miRNAs.

SUMMARY Salmonella enterica serovar Enteritidis and Campylobacter jejuni are 2 major foodborne pathogens transmitted through poultry products. These pathogens colonize the chicken cecum, leading to contamination of carcasses during slaughter and subsequent processing operations. We investigated the antimicrobial efficacy of 4 GRAS (generally recognized as safe) status plant-derived molecules, namely, trans-cinnamaldehyde, eugenol, carvacrol, and thymol, against Salmonella Enteritidis and C. jejuni in chicken cecal contents in vitro. The plant molecules were added at different concentrations (ranging from 10 to 75 mM for Salmonella Enteritidis and 10 to 30 mM for C. jejuni) to autoclaved chicken cecal contents inoculated with approximately 7.0 log10 cfu/mL of Salmonella Enteritidis or approximately 5.0 log10 cfu/mL of C. jejuni. The pathogen populations in the cecal contents after 15 s, 8 h, and 24 h of incubation at 40°C were determined. Duplicate samples of treatments and the control were included, and the study was replicated 3 times. Campylobacter jejuni was more sensitive to all the molecules than Salmonella Enteritidis (P ≤ 0.05). All molecules were highly bactericidal, with the lowest concentration of trans-cinnamaldehyde (10 mM) reducing (P ≤ 0.05) Salmonella Enteritidis populations by approximately 6.0 log10 cfu/mL after 8 h and >8.0 log10 cfu/mL after 24 h of incubation. Trans-cinnamaldehyde at 25 mM eliminated detectable (P ≤ 0.05) Salmonella Enteritidis counts by 8 h of incubation. In addition, trans-cinnamaldehyde at all tested concentrations (10, 20, and 30 mM) reduced C. jejuni counts to

A framework for exploring regional-scale trade-offs among ecosystem services and biodiversity protection has been established for some time, and it is clear that optimizing these trade-offs provides a strategy to address targets for a reduced rate of biodiversity loss. Recent trade-off studies have highlighted the need for better biodiversity measures, to complement measures of ecosystem services. Biodiversity typically has been linked in this context to existence and other non-use values. We argue that biodiversity will have a stronger role in such trade-off analyses if measures of biodiversity better reflect additional current and future services. These ‘evosystem services’ have been, and, if we are careful, can continue to be provided by the evolutionary process. Some services have been provided through evolution operating in the past, and a phylogenetic diversity measure can help us to quantify these current and potential future benefits derived from the tree of life. Furthermore, a variety of evosystem services are delivered through ongoing contemporary evolution, and value should therefore be placed on the maintenance of healthy evosystems. We argue that the concept of evosystem services could be useful as a complement to the traditional concept of ecosystem services. Together, these reflect a fuller range of the services supported by biodiversity, and thereby provide a sounder basis for conservation planning and decision-making.

Genetic elements determine phenotypes of organisms by interacting with environments. Despite genetic diversity within and between species being the fundamental basis of biological diversity, its contribution has been long neglected in biodiversity studies. This situation is rapidly changing as quantification of genetic diversity, from intraspecific up to the ecosystem level, has become more accessible owing to the development of next-generation sequencing technologies (NGSTs). Whole-genome sequencing techniques provide two specific approaches for accessing genetic diversity at large scales: metagenomics (environmental genomics) and EST (Expressed Sequence Tag) comparisons. The former has been applied successfully in the profiling of different microbial biomes, and it is particularly interesting in understanding their ecosystem structure and function. The latter is particularly useful in the studies of adaptation and the assessment of functional traits. Unquestionably, advances in the genomic sciences combined with a new generation of ecological and evolutionary science will boost new approaches to global and local assessments of biodiversity changes, and more importantly, will surely reframe the questions we are asking in biodiversity science.

Campylobacter is a leading cause of foodborne illness in the United States, and epidemiological evidence indicates poultry products to be a significant source of human Campylobacter infections. Caprylic acid, an eight-carbon medium-chain fatty acid, reduces Campylobacter colonization in chickens. How caprylic acid reduces Campylobacter carriage may be related to changes in intestinal microflora. To evaluate this possibility, cecal microbial populations were evaluated with denaturing gradient gel electrophoresis from market-age broiler chickens fed caprylic acid. In the first trial, chicks (n = 40 per trial) were assigned to four treatment groups (n = 10 birds per treatment group): positive controls (Campylobacter, no caprylic acid), with or without a 12-h feed withdrawal before slaughter; and 0.7% caprylic acid supplemented in feed for the last 3 days of the trial, with or without a 12-h feed withdrawal before slaughter. Treatments were similar for trial 2, except caprylic acid was supplemented for the last 7 days of the trial. At age 14 days, chicks were orally challenged with Campylobacter jejuni, and on day 42, ceca were collected for denaturing gradient gel electrophoresis and Campylobacter analysis. Caprylic acid supplemented for 3 or 7 days at 0.7% reduced Campylobacter compared with the positive controls, except for the 7-day treatment with a 12-h feed withdrawal period. Denaturing gradient gel electrophoresis profiles of the cecal content showed very limited differences in microbial populations. The results of this study indicate that caprylic acid's ability to reduce Campylobacter does not appear to be due to changes in cecal microflora.

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

Salmonella Enteritidis and Campylobacter jejuni are 2 major foodborne pathogens in the United States, estimated to cause more than 3 million cases of human illness annually. Chickens are the natural hosts of these bacteria, and their drinking water can be a source of S. Enteritidis and C. jejuni, contributing to the colonization of birds. In this study, trans-cinnamaldehyde (TC), a natural, generally recognized as safe ingredient in cinnamon oil was evaluated for its efficacy to inactivate S. Enteritidis and C. jejuni in the drinking water of chickens. Well water containing 0, 0.016, 0.03, and 0.06% TC was inoculated with a 5-strain mixture of S. Enteritidis or C. jejuni (˜6 log10 cells/mL). Water samples containing 1% chicken feces or feed were also included. The samples were incubated at 12.5 or 25°C for 7 d and analyzed for bacterial populations on d 0, 1, 3, 5, and 7. Duplicate samples of treatments and control were included, and the study was replicated 3 times. Trans-cinnamaldehyde at 0.06% inactivated Salmonella completely after 24 h in water with 1% feces at both temperatures. In water containing 1% feed, TC (0.06%) reduced S. Enteritidis to undetectable levels after 3 d at 12.5°C or 7 d at 25°C. Presence of feed or feces in water reduced the antibacterial effect (P 0.001) on the antibacterial property of TC on C. jejuni. Results indicate that TC is effective in killing S. Enteritidis and C. jejuni in chicken drinking water and may decrease the likelihood that water will contribute to colonization of chickens by these pathogens.

Phylogenetic analyses of chloroplast DNA sequences, morphology, and combined data have provided consistent support for many of the major branches within the angiosperm clade Dipsacales. Here we use sequences from three mitochondrial loci to test the existing broad scale phylogeny and in an attempt to resolve several relationships that have remained uncertain. Parsimony, maximum likelihood, and Bayesian analyses of a combined mitochondrial data set recover trees broadly consistent with previous studies, although resolution and support are lower than in the largest chloroplast analyses. Combining chloroplast and mitochondrial data results in a generally well-resolved and very strongly supported topology but the previously recognized problem areas remain. To investigate why these relationships have been difficult to resolve we conducted a series of experiments using different data partitions and heterogeneous substitution models. Usually more complex modeling schemes are favored regardless of the partitions recognized but model choice had little effect on topology or support values. In contrast there are consistent but weakly supported differences in the topologies recovered from coding and non-coding matrices. These conflicts directly correspond to relationships that were poorly resolved in analyses of the full combined chloroplast-mitochondrial data set. We suggest incongruent signal has contributed to our inability to confidently resolve these problem areas.

Phylogenetic thinking has infiltrated many areas of biological research, but has had little impact on studies of global ecology or climate change. Here, we illustrate how phylogenetic information can be relevant to understanding vegetation-atmosphere dynamics at ecosystem or global scales by re-analyzing a data set of carbonic anhydrase (CA) activity in leaves that was used to estimate terrestrial gross primary productivity. The original calculations relied on what appeared to be low CA activity exclusively in C4 grasses, but our analyses indicate that such activity might instead characterize the PACCAD grass lineage, which includes many widespread C3 species. We outline how phylogenetics can guide better taxon sampling of key physiological traits, and discuss how the emerging field of phyloinformatics presents a promising new framework for scaling from organism physiology to global processes.

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

The immune system of neonatal chicks is functionally immature during the first week of life. Researchers have previously demonstrated that the avian humoral response can be increased with probiotics. Although the humoral response provides the chick with an effective mechanism to combat pathogens, sufficient antibody titers are not attained until 7 to 10 d postinfection. However, the innate immune system (i.e., heterophils) can respond much more quickly to pathogens. The objective of this study was to determine whether probiotic bacteria can also upregulate heterophil function. Heterophils were isolated from the peripheral blood of neonatal chickens by using a discontinuous density gradient. Oxidative burst and degranulation are bactericidal mechanisms used by heterophils to kill pathogens and were used in this study as indicators of heterophil function. We found that each of the 10 "generally recognized as safe" probiotic isolates (designated G1 to G11) tested in vitro were capable of increasing (P < 0.05) heterophil oxidative burst and degranulation when compared with unstimulated controls. Bacillus subtilis (G3), Lactococcus lactis lactis (G6), and Lactobacillus acidophilus (G8) isolates were determined to elicit the greatest heterophil response in vitro and were subsequently fed to chicks. Phosphate-buffered saline or 1 of these 3 probiotic isolates (approximately 2.5 x 10(8) cfu/chick; 50 chicks/treatment) resuspended in PBS was administered by oral gavage on the day of hatch. Heterophils were isolated from chicks from each of these 4 treatment groups 24 h posttreatment. Significant increases in heterophil degranulation and oxidative burst were observed with the G3-, G6-, and G8-treated chicks when compared with heterophils isolated from birds with no probiotic treatment. These data suggest that probiotic bacteria can significantly improve heterophil oxidative burst and degranulation in broilers. To our knowledge, this is the first study demonstrating a relationship between probiotics and avian heterophil function.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

In several areas of southern Sweden, limestone nodules, locally called Orsten occur within bituminous alum shales. These shales and nodules were deposited under dysoxic conditions at the bottom of what was most likely a shallow sea during the late Middle to Upper Cambrian (ca. 500 million years ago). Subsequently, the name 'Orsten' has been referred to particular, mainly arthropod, fossils from such nodules, and, in a wider sense, to the specific type of preservation of minute fossil through secondarily phosphatization. This preservation is exceptional in yielding uncompacted and diagenetically undeformed three-dimensional fossils. 'Orsten'-type preservation resulted from incrustation of a thin external layer and also by impregnation by calcium phosphate and, therefore, mineralization of the surface of the former animals during early diagenesis. Primarily, this type of preservation seems to have affected only cuticle-bearing metazoans such as cycloneuralian nemathelminths and arthropods. 'Orsten' preservation in this sense seems to be limited by size, in having yielded no partial or complete animals larger than 2 mm. On the other end of the scale, even larvae 100 μm long are preserved, often more complete than larger specimens, and details such as setules and pores smaller than 1 μm can be observed. Fossils preserved in such a manner are almost exclusively hollow carcasses, but can be filled secondarily; less common are completely phosphatized compact specimens. The high quality of preservation makes the Swedish 'Orsten' a typical Konservat-Lagerstätte. Yet, its special type of preservation is more widespread in time and geographical distribution than assumed initially, and the origin of the phosphate is not necessarily restricted just to one source. Subsequent to the first discoveries of limb fragments of Cambrian arthropods in 1975, animals in this special preservational type have been discovered in several continents and across a broad stratigraphic range including even Proterozoic strata. The latter have yielded early cleavage and metazoan embryonic stages, expanding knowledge on the preservational capacities of the 'Orsten'. Here, we report the recent status of our research on the 'Orsten' and give perspectives for future exploration on a worldwide scale, particularly in light of a recently formed international research group named Center of Orsten Research and Exploration (C.O.R.E.). © 2006.

Vertebrate evolution has been punctuated by three episodes of widespread gene or genome duplication, which have been linked with the origin of vertebrates, gnathostomes and teleosts, respectively. These three events coincide with bursts of character acquisition and increases in phenotypic complexity, and many researchers have suggested a causal relationship between the two. However, this pattern is derived from data for living taxa only; we argue here that, when fossils are taken into account, bursts of character acquisition disappear and gen(om)e duplication in vertebrate phylogeny can no longer be correlated with the origin of body plans. If patterns of character acquisition or morphological gaps between higher taxa are a reflection of phenotypic complexity, then more inclusive data sets incorporating fossil taxa provide no support for hypotheses linking gen(om)e duplications and the evolution of complexity in vertebrates.

Japanese quail provide an advantageous avian model for assessing long-term biological consequences of endocrine disrupting chemicals (EDCs). These studies examined route of exposure and vulnerability to biological impact of EDCs over the life cycle in a precocial avian model, the Japanese quail. Embryonic exposure occurs with maternal deposition and methoxychlor (MXC) accumulated with maternal exposure. Egg injections of MXC or estradiol at selected stages of development impacted hypothalamic neuroendocrine systems in hatchlings and affected sexual maturation, with evidence for long-term effects on neurotransmitters and male behavior. Two-generation dietary studies were conducted to examine transgenerational effects of EDCs. Adult quail (P1) were exposed to dietary MXC (0, 0.5 and 5 ppm), with continued exposure in their offspring (F1), and control diet for all F2 chicks. Toxicological end points, including fertility, hatching success, and 14-day viability were unaffected. F1 and F2 male offspring from MXC-treated pairs MXC had impaired mating behavior and altered plasma hormones. These studies confirm neuroendocrine and behavioral measures as reliable indices of exposure to an estrogenic EDC. Moreover, maternal deposition remains a primary route of EDC exposure, with potential deleterious consequences for field birds, especially precocial species that appear to be particularly sensitive to embryonic EDC exposure.

Ecology and historical (phylogeny-based) biogeography have much to offer one another, but exchanges between these fields have been limited. Historical biogeography has become narrowly focused on using phylogenies to discover the history of geological connections among regions. Conversely, ecologists often ignore historical biogeography, even when its input can be crucial. Both historical biogeographers and ecologists have more-or-less abandoned attempts to understand the processes that determine the large-scale distribution of clades. Here, we describe the chasm that has developed between ecology and historical biogeography, some of the important questions that have fallen into it and how it might be bridged. To illustrate the benefits of an integrated approach, we expand on a model that can help explain the latitudinal gradient of species richness.

DNA sequencing studies of the granule-bound starch synthase gene (GBSSI) indicate the presence of two loci in Viburnum. Gene trees from separate and combined phylogenetic analyses of the GBSSI paralogues are generally congruent with each other and with trees from previous analyses, especially those of Donoghue et al. [Syst. Bot. 29 (2004) 188] based on nuclear ribosomal ITS and chloroplast trnK intron DNA sequences. Specifically, our GBSSI trees confirm (i) the monophyly of some and non-monophyly of other traditionally recognized taxonomic sections, (ii) the presence of three major supra-sectional lineages within Viburnum, and (iii) the resolution of many species relationships within the section-level clades. Analyses of GBSSI also provide greater resolution of relationships within the largest supra-sectional lineage. Relationships at the base of the Viburnum phylogeny remain uncertain; in particular, the position of the root, relationships among the supra-sectional clades, and the exact placement of several smaller groups (e.g., Viburnum clemensiae, Viburnum urceolatum, and section Pseudotinus). In two lineages each GBSSI paralogue is represented by two distinct sequences. The presence of additional copies appears to be correlated with polyploidy in these clades. Placement of the homoeologues in our gene trees suggests the possibility of a hybrid origin for these polyploids.

Campylobacter and Salmonella are the most commonly reported pathogens causing foodborne illness in the United States. In turkeys, the potential that semen used for artificial insemination is contaminated with these foodborne pathogens has not been investigated. Because semen on turkey farms is pooled and then used to inseminate multiple hens, contaminated semen could easily spread these bacteria throughout entire flocks via artificial insemination. The objectives of this study were to 1) determine if semen from commercial turkey farms contained these foodborne pathogens and 2) if present, evaluate the efficacy of semen extenders to reduce or eliminate Campylobacter and Salmonella from semen. Semen was collected from randomized pools of ejaculates from 10 to 30 toms per farm from 6 flocks over a 7-wk period and, on occasion, was found to contain Campylobacter, Salmonella, or both. To evaluate the efficacy of semen extenders to reduce or eliminate pathogens, pooled ejaculates were challenged with Campylobacter or Salmonella and treated with commercial poultry extenders containing various concentrations of antibiotics or an antibiotic combination previously demonstrated to remove Campylobacter from mammalian semen. Results demonstrate that commercial turkey semen may contain Campylobacter or Salmonella, and the semen extenders tested either did not reduce the bacteria or reduced but did not eliminate these bacteria from semen. We concluded that semen may be a potential vehicle for Campylobacter transfer to hens, and, if this is true, development of a method for eliminating pathogens in semen before insemination could reduce the risk of colonization.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States. Recent evidence has demonstrated that Campylobacter is present in poultry semen and may contribute to the vertical transmission between the breeder hen and offspring. As Campylobacter is considered sensitive to oxygen and cold temperature, the objective of this study was to determine if aeration and storage temperature could reduce or eliminate Campylobacter in poultry semen. In 4 separate trials, pooled semen samples were collected from roosters or toms, diluted with a commercial poultry semen extender, and inoculated with an average of 10(7) cells/mL of a wild-type C. jejuni or C. coli semen isolate. Pooled ejaculates were then divided into 3 aeration treatments: Control (no aeration), air, or oxygen (gently bubbled for 20 min with atmospheric air or oxygen, respectively). Immediately after aeration, pooled semen samples were further divided to 3 test storage temperatures: 4, 23, or 42 degrees C. At 0, 2, 6, and 24 h of storage, samples were enumerated for Campylobacter. Aeration of the semen alone, or aeration with reduced temperatures (4 or 23 degrees C), did not reduce the amount of Campylobacter compared with the controls. Campylobacter concentrations were, however, reduced when stored at 42 degrees C for 24 h. This effect is associated with reduced sperm viability and is therefore not a practical treatment of reducing Campylobacter in semen. It appears alternative methods will be needed to eliminate Campylobacter from poultry semen.

Regulatory monitoring for most antibiotic residues in edible poultry tissues is often accomplished with accurate, although expensive and technically demanding, chemical analytical techniques. The purpose of this study is to determine if a simple, inexpensive bioassay could detect fluoroquinolone (FQ) residues in chicken muscle above the FDA established tolerance (300 ppb) comparable to a liquid chromatography-fluorescencemass spectrometryn method. To produce incurred enrofloxacin (ENRO) tissues (where ENRO is incorporated into complex tissue matrices) for the method comparison, 40-d-old broilers (mixed sex) were orally dosed through drinking water for 3 d at the FDA-approved dose of ENRO (50 ppm). At the end of each day of the 3-d dosing period and for 3 d postdosing, birds were sacrificed and breast and thigh muscle collected and analyzed. Both methods were able to detect ENRO at and below the tolerance level in the muscle, with limits of detection of 26 ppb (bioassay), 0.1 ppb for ENRO, and 0.5 ppb for the ENRO metabolite, ciprofloxacin (liquid chromatography-fluorescence-mass spectrometryn). All samples that had violative levels of antibiotic were detected by the bioassay. These results support the use of this bioassay as a screening method for examining large numbers of samples for regulatory monitoring. Positive samples should then be examined by a more extensive method, such as liquid chromatography-fluorescence-mass spectrometryn, to provide confirmation of the analyte.

The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.

An efficient multiresidue method for determination of fluoroquinolone antibiotics in eggs has been developed, in which quantitation and confirmation are achieved simultaneously using fluorescence and MS n . With this method, eight fluoroquinolones were analyzed in fortified egg samples at levels of 10–100 ng g −1 . Recoveries for desethylene ciprofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin and difloxacin were generally in the range 60–100%, with excellent relative standard deviations. The limits of quantitation ranged from 0.1 to 2 ng g −1 in mixed egg. Confirmation was achieved by monitoring the ratios of two prominent product ions in MS 2 (desethylene ciprofloxacin) or MS 3 (all others). The method was found to be applicable to the analysis of egg whites and also egg yolks, with limits of quantitation ranging from 0.1 to 7 ng g −1 (egg white) and 0.1–1.5 ng g −1 (egg yolk). ENRO-incurred mixed eggs, egg whites and egg yolks were also successfully analyzed using this method.

The Morinaceae (Dipsacales) contains 13 species placed in Acanthocalyx, Cryptothladia or Morina, and is distributed from the mountains of southeastern Europe through the Himalayas to the Qinghai-Tibetan Plateau, mainly in alpine habitats. Sequence data from two chloroplast regions (the trnK intron and the trnL-F region) and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships of Morinaceae and related Dipsacales. Both the nuclear and chloroplast datasets, as well as the combined data, provide strong support for relationships within the Valerina clade, placing Morinaceae as the sister group of a clade containing Valerianaceae and Dipsacaceae plus Triplostegia. The Morinaceae, Acanthocalyx, Cryptothladia, and a clade containing Morina and Cryptothladia, are all supported as monophyletic. However, Morina was found to be paraphyletic in several of our analyses, with Morina longifolia more closely related to Cryptothladia than to other Morina species. There is some evidence that Morina longifolia produces cleistogamous flowers, as do Cryptothladia species. Dispersal-vicariance analyses support the view that Valerina radiated initially within Asia, with subsequent movement into Europe in Morinaceae, Dipsacaceae, and Valerianaceae, and into the New World in Valerianaceae. For Morinaceae, as for a number of plant groups, the Brahmaputra river drainage marks a significant biogeographic divide, although this has been spanned within Acanthocalyx and the Morina-Cryptothladia lineage.

Two complementary methods for identifying and measuring sulfonamide residues in eggs were developed for use in surveying eggs for potential drug residues. The first method uses liquid chromatography–tandem mass spectrometry (LC–MS–MS) to confirm the presence of sulfonamide residues in eggs. During its validation the limit of confirmation was estimated to be 5–10 ng/g (ppb) depending on the drug. Also, a method for measuring residue level by liquid chromatography with ultraviolet detection (LC–UV) was validated using the same extraction procedure as the confirmatory method. The determinative method was validated over the 50–200 ppb range. Samples were prepared by homogenizing whole egg, extracting with acetonitrile, and cleaning up with a C18 solid-phase extraction cartridge. For confirmation, analytes were separated by gradient LC on a C18 column, ionized by electrospray ionization (ESI), and detected by MS–MS with an ion trap mass spectrometer. For determination, analytes were separated by a different gradient LC procedure and detected by UV at 287 nm. Fifteen drugs were dosed individually in laying hens, and residues of parent drug and/or metabolites were found in eggs for all the drugs. Validation was based on repetitive analyses of control samples, control samples fortified at 100 ppb sulfonamides, and samples of blended incurred eggs.

Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen was employed using the cytoplasmic kinase domain of FGFR3 as bait. We identified the adapter protein SH2-B as an FGFR3-interacting protein. Coimmunoprecipitation experiments demonstrate binding of the SH2-Bβ isoform to FGFR3 in 293T cells. Tyrosine phosphorylation of SH2-Bβ was observed when coexpressed with activated FGFR3 mutants such as the weakly activated mutant N540K or the strongly activated mutant K650E, both associated with human developmental syndromes. The extent of tyrosine phosphorylation of SH2-Bβ correlates with receptor activation, suggesting that FGFR3 activation mediates tyrosine phosphorylation of SH2-Bβ. Furthermore, two tyrosine phosphorylation sites of FGFR3, Tyr-724 and Tyr-760, are required for optimal binding of the Src homology-2 (SH2) domain of SH2-Bβ. We also demonstrate the phosphorylation and nuclear translocation of Stat5 by activated FGFR3, which increases in response to overexpression of SH2-Bβ. Taken together, our results identify SH2-Bβ as a novel FGFR3 binding partner that mediates signal transduction.

The visual cortex in primates is parcellated into cytoarchitectonically, physiologically, and connectionally distinct areas: the striate cortex (V1) and the extrastriate cortex, consisting of V2 and numerous higher association areas [1]. The innervation of distinct visual cortical areas by the thalamus is especially segregated in primates, such that the lateral geniculate (LG) nucleus specifically innervates striate cortex, whereas pulvinar projections are confined to extrastriate cortex [2–8]. The molecular bases for the parcellation of the visual cortex and thalamus, as well as the establishment of reciprocal connections between distinct compartments within these two structures, are largely unknown. Here, we show that prospective visual cortical areas and corresponding thalamic nuclei in the embryonic rhesus monkey ( Macaca mulatta ) can be defined by combinatorial expression of genes encoding Eph receptor tyrosine kinases and their ligands, the ephrins, prior to obvious cytoarchitectonic differentiation within the cortical plate and before the establishment of reciprocal connections between the cortical plate and thalamus. These results indicate that molecular patterns of presumptive visual compartments in both the cortex and thalamus can form independently of one another and suggest a role for EphA family members in both compartment formation and axon guidance within the visual thalamocortical system.