Claudin 13, a Member of the Claudin Family Regulated in Mouse Stress Induced Erythropoiesis

Mammals are able to rapidly produce red blood cells in response to stress. The molecular pathways used in this process are important in understanding responses to anaemia in multiple biological settings. Here we characterise the novel gene Claudin 13 (Cldn13), a member of the Claudin family of tight junction proteins using RNA expression, microarray and phylogenetic analysis. We present evidence that Cldn13 appears to be co-ordinately regulated as part of a stress induced erythropoiesis pathway and is a mouse-specific gene mainly expressed in tissues associated with haematopoietic function. CLDN13 phylogenetically groups with its genomic neighbour CLDN4, a conserved tight junction protein with a putative role in epithelial to mesenchymal transition, suggesting a recent duplication event. Mechanisms of mammalian stress erythropoiesis are of importance in anaemic responses and expression microarray analyses demonstrate that Cldn13 is the most abundant Claudin in spleen from mice infected with Trypanosoma congolense. In mice prone to anaemia (C57BL/6), its expression is reduced compared to strains which display a less severe anaemic response (A/J and BALB/c) and is differentially regulated in spleen during disease progression. Genes clustering with Cldn13 on microarrays are key regulators of erythropoiesis (Tal1, Trim10, E2f2), erythrocyte membrane proteins (Rhd and Gypa), associated with red cell volume (Tmcc2) and indirectly associated with erythropoietic pathways (Cdca8, Cdkn2d, Cenpk). Relationships between genes appearing co-ordinately regulated with Cldn13 post-infection suggest new insights into the molecular regulation and pathways involved in stress induced erythropoiesis and suggest a novel, previously unreported role for claudins in correct cell polarisation and protein partitioning prior to erythroblast enucleation.     

A molecular phylogeny of the nightjars (Aves: Caprimulgidae) suggests extensive conservation of primitive morphological traits across multiple lineages

We report a molecular re-assessment of the classification of the nightjars which draws conclusions that are strongly at odds with the traditional, morphology-based classifications. We used maximum likelihood and Bayesian methods to compare the cytochrome b gene for 14 species from seven of the 15 genera of the Caprimulgidae and partial cytochrome b sequence data was available for a further seven species including three further genera. We found that within the Caprimulgidae there were four geographically isolated clades with bootstrap support greater than 70%. One of these clades contained just Chordeiles species, the remaining three clades each contained a mixture of genera including Caprimulgus sp. A clade of exclusively South American nightjars included the genera Caprimulgus, Uropsalis, Eleopthreptus and Hydropsalis. A clade of African and Eurasian birds included Caprimulgus and Macrodipteryx. Phalaenoptilus nuttallii and Caprimulgus vociferous formed a clade of North American birds. Two ecological factors appear to make morphological classification potentially misleading: first, the apparent retention of primitive anti-predator and foraging-related traits across genetically divergent groups; second, rapid divergence in other traits, especially those related to mating, which generate high levels of morphological divergence between species that are genetically very similar. The cytochrome b data suggests that the genus Caprimulgus is not monophyletic and is restricted to Africa and Eurasia and that Caprimulgus species from outside this area have been misclassified as a consequence of retention of primitive adaptations for crepuscular/nocturnal living. Some other genera also appear to have little support from the cytochrome b data.     

Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. We show in mouse that Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. We provide new morphologic data on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, we show that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems.     

Intraspecific nuclear ribosomal DNA divergence and reticulation in sexual diploid Erigeron strigosus (Asteraceae)

Apomictic complexes in flowering plants often harbor multiple sexual taxa, which must be studied to interpret accurately the origin and diversity of apomictic derivatives. The daisy fleabane group in North America (Erigeron sect. Phalacroloma) includes the widespread polyploid apomictic taxa E. annuus, E. strigosus, and E. tenuis, as well as recently discovered sexual diploid populations in the southeastern United States (E. strigosus var. calcicola, E. strigosus var. dolomiticola, sexual E. strigosus var. strigosus). Phylogenetic analysis for 35 sexual populations was conducted using sequences from the internal and external transcribed spacers (ITS and ETS) of ribosomal DNA. Results indicate that the three groups of sexual plants form separate monophyletic clades and that edaphic specialization is ancestral in the group. Sequence analysis for sexual E. strigosus var. strigosus was confounded by the fact that 13 of 31 samples (42%) possessed nucleotide polymorphisms at 1.0-1.8% of sites. However, analysis of the genomic sequence for plants with low polymorphism levels in conjunction with analysis of cloned PCR products for plants with high levels of intra-individual polymorphism indicated that three combinations of haplotypes (N I + N II, two plants; N I + N III, five plants; N II + N III, six plants) accounted for the majority of sequence polymorphisms encountered. These data are consistent with historical diversification and subsequent reticulate evolution at the diploid level in sexual E. strigosus var. strigosus.     

Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health

Reports that elasmobranchs (sharks, skates, and rays) may havea low incidence of disease have stimulated interest in understandingthe role of their immune system in this apparent resistance.Although research in this area may potentially translate intoapplications for human health, a basic understanding of theelasmobranch immune system components and how they functionis essential. As in higher vertebrates, elasmobranch fishespossess thymus and spleen, but in the absence of bone marrowand lymph nodes, these fish have evolved unique lymphomyeloidtissues, namely epigonal and Leydig organs. As conditions forshort-term culture of elasmobranch immune cells have becomebetter understood, the opportunity to examine functional activityof cytokine-like factors derived from conditioned culture mediumhas resulted in the identification of growth inhibitory activityagainst a variety of tumor cell lines. Specifically, the mediumenriched by short term culture of bonnethead shark (Sphyrnatiburo) epigonal cells (epigonal conditioned medium, ECM) hasbeen shown to inhibit the growth of mammalian tumor cell lines,including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-celllymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer(PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinomacell lines (MCF7, HCC38, Hs578T). Of the cell lines tested,WEHI-164, A375.S2, Daudi, and Jurkat cells were among the mostsensitive to growth inhibitory activity of ECM whereas PANC-1and NIH:OVCAR-3 cells were among the least sensitive. In addition,ECM demonstrated preferential growth inhibition of malignantcells in assays against two different malignant/non-malignantcell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separationof protein components of ECM using SDS-PAGE resulted in a veryreproducible pattern of three major bands corresponding to molecularsizes of approximately 40–42 kD, 24 kD, and 17 kD. Activityis lost after heating at 75°C for 30 min, and can be diminishedby treatment with proteinase K and protease. Activity is notaffected by treating with trypsin, DNase I or RNase A.     

Novel SNP Discovery in African Buffalo,Syncerus caffer,Using High-Throughput Sequencing

The African buffalo, Syncerus caffer, is one of the most abundant and ecologically important species of megafauna in the savannah ecosystem. It is an important prey species, as well as a host for a vast array of nematodes, pathogens and infectious diseases, such as bovine tuberculosis and corridor disease. Large-scale SNP discovery in this species would greatly facilitate further research into the area of host genetics and disease susceptibility, as well as provide a wealth of sequence information for other conservation and genomics studies. We sequenced pools of Cape buffalo DNA from a total of 9 animals, on an ABI SOLiD4 sequencer. The resulting short reads were mapped to the UMD3.1 Bos taurus genome assembly using both BWA and Bowtie software packages. A mean depth of 2.7× coverage over the mapped regions was obtained. Btau4 gene annotation was added to all SNPs identified within gene regions. Bowtie and BWA identified a maximum of 2,222,665 and 276,847 SNPs within the buffalo respectively, depending on analysis method. A panel of 173 SNPs was validated by fluorescent genotyping in 87 individuals. 27 SNPs failed to amplify, and of the remaining 146 SNPs, 43–54% of the Bowtie SNPs and 57–58% of the BWA SNPs were confirmed as polymorphic. dN/dS ratios found no evidence of positive selection, and although there were genes that appeared to be under negative selection, these were more likely to be slowly evolving house-keeping genes.     

Learning curves, taking instructions, and patient safety: using a theoretical domains framework in an interview study to investigate prescribing errors among trainee doctors

ABSTRACT: BACKGROUND: Prescribing errors are a major source of morbidity and mortality and represent a significant patient safety concern. Evidence suggests that trainee doctors are responsible for most prescribing errors. Understanding the factors that influence prescribing behavior may lead to effective interventions to reduce errors. Existing investigations of prescribing errors have been based on Human Error Theory but not on other relevant behavioral theories. The aim of this study was to apply a broad theory-based approach using the Theoretical Domains Framework (TDF) to investigate prescribing in the hospital context among a sample of trainee doctors. METHOD: Semistructured interviews, based on 12 theoretical domains, were conducted with 22 trainee doctors to explore views, opinions, and experiences of prescribing and prescribing errors. Content analysis was conducted, followed by applying relevance criteria and a novel stage of critical appraisal, to identify which theoretical domains could be targeted in interventions to improve prescribing. RESULTS: Seven theoretical domains met the criteria of relevance: "social professional role and identity," "environmental context and resources," "social influences," "knowledge," "skills," "memory, attention, and decision making," and "behavioral regulation." From critical appraisal of the interview data, "beliefs about consequences" and "beliefs about capabilities" were also identified as potentially important domains. Interrelationships between domains were evident. Additionally, the data supported theoretical elaboration of the domain behavioral regulation. CONCLUSIONS: In this investigation of hospital-based prescribing, participants' attributions about causes of errors were used to identify domains that could be targeted in interventions to improve prescribing. In a departure from previous TDF practice, critical appraisal was used to identify additional domains that should also be targeted, despite participants' perceptions that they were not relevant to prescribing errors. These were beliefs about consequences and beliefs about capabilities. Specifically, in the light of the documented high error rate, beliefs that prescribing errors were not likely to have consequences for patients and that trainee doctors are capable of prescribing without error should also be targeted in an intervention. This study is the first to suggest critical appraisal for domain identification and to use interview data to propose theoretical elaborations and interrelationships between domains.     

Isolation, purification, and characterization of pregnancy-specific protein B from elk and moose placenta.

Pregnancy-specific protein B (PSPB) was isolated, purified, and partially characterized from elk and moose placenta. The procedure, which was monitored by bovine PSPB (bPSPB) RIA, included homogenization and extraction in aqueous solution, acidic and ammonium sulfate precipitation, and ion exchange, gel filtration, and affinity chromatographies. The estimated molecular sizes of moose PSPB (mPSPB) were 58 kDa and 31 kDa, and of elk PSPB (ePSPB) were 57 kDa, 45 kDa, and 31 kDa by SDS-PAGE. The isoelectric points of mPSPB were 4.8, 6.6, and 6.7, and of ePSPB were 4.8, 4.9, 6.1, and 6.2 as determined by isoelectric focusing and two-dimensional gel electrophoresis. The carbohydrate contents of mPSPB and ePSPB were approximately 3.15% and 4.98%, respectively. Although ePSPB and mPSPB were recognized by anti-bPSPB in an Ouchterlony double immunodiffusion test, they were found to share identical epitopes and partial identities compared to bPSPB. After treatment at different temperatures (20-60 degrees C) for 1 h, the immunoreactivities of ePSPB and mPSPB in serum were very stable. Only ePSPB in serum treated at 60 degrees C lost some immunoreactivity. After alteration of serum pH (pH 3-11) for 2 h, the immunoreactivities of ePSPB and mPSPB became lower at pH 3 and 4, and remained stable from pH 5 to 11. These data show that moose and elk PSPB have properties similar to those of bovine and ovine PSPB.     

STUDIES ON ENZYME ACTION : XXXV. LIPASE ACTIONS OF EXTRACTS OF TISSUES OF RABBITS AT DIFFERENT AGES.

The ester-hydrolyzing actions of extracts of a number of tissues of rabbits of different ages were studied under comparable conditions. The ages of the rabbits ranged from 8 days before birth to 2162 days. The esters used included phenyl acetate, glyceryl triacetate, methyl butyrate, benzyl acetate, methyl acetate, ethyl acetate, ethyl butyrate, methyl benzoate, ethyl benzoate, and isobutyl acetate. The following tissues were studied: kidney, liver, lung, skin, leg muscle, heart muscle, brain, spleen, stomach, and small intestine. The results, as in previous communications, are presented in the form of plots for the relative enzyme actions, and in tables for the absolute actions. The changes in the curves of the relative actions as the rabbits became older are considered in some detail. The relations between the embryonic state of certain tissues, as shown by their enzyme actions, and the adult state, are described, and compared with their physiological behavior. The probable reversion to a type approaching the embryonic for the oldest rabbits studied is indicated with some of the tissues. The changes in the absolute enzyme actions of the tissues as the rabbits became older are also discussed. The absolute actions do not form as regular a progression as do the relative actions but, at the same time, show marked regularities with increasing age of the rabbits.