Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide variety of essential cell-cell and cell-matrix processes ranging from immune recognition to cancer development. The first general protein glycosylation (pgl)¹ pathway was discovered in the epsilonproteobacterium Campylobacter jejuni (1). The organism transfers a conserved heptasaccharide en bloc to asparagine residues within the sequon D/E- X₁-N-X₂-S/T (X₁, X₂ ≠ P) of >60 glycoproteins (2–4). Furthermore, the pathway can be functionally transferred into Escherichia coli, and the oligosaccharyltransferase (OTase), PglB, is capable of adding foreign sugars to acceptor proteins (5–7). C. jejuni PglB also possesses hydrolase activity, influenced by the cellular growth phase and osmotic environment, releasing free oligosaccharides (fOS) into the periplasmic space in a 10:1 ratio relative to the amount of heptasaccharide N-linked to protein (8, 9).The C. jejuni N-linked heptasaccharide is conserved in structure in both C. jejuni and C. coli, the two most commonly isolated pathogenic Campylobacter species and major causes of human enteritis worldwide (10, 11). All campylobacters, but one, possess conserved pgl genes required for N-linked protein glycosylation ((12) and this study). This post-translational modification in C. jejuni influences DNA uptake, chicken and mouse colonization, epithelial cell adherence and invasion, recognition by human sera, and binding to the macrophage galactose lectin (MGL) receptor on dendritic cells (2, 13–17). Several Campylobacter species have now been recognized as emerging pathogens and causative agents of human gastroenteritis (e.g. C. upsaliensis and C. hyointestinalis), gingivitis, periodontitis, and human abortions (e.g. C. rectus, C. concisus, C. gracilis, C. showae, and C. upsaliensis) and inflammatory bowel disease in children (e.g. C. concisus) (18). Other species cause venereal disease and infertility in cattle (C. fetus subsp. venerealis; Cfv) or abortions in sheep (C. fetus subsp. fetus; Cff) (19).In this study, we used phylogenetic, immunological, structural and glycoproteomic studies to compare the N-glycosylation systems of 29 Campylobacter species and identified unexpected variations. Thus, although the pathway is a common feature within this genus, variability in the N-glycans and fOS at the species level suggests that each species possess a unique array of glycosyltransferases, which correlate with their phylogenetic relatedness.     

A comparison of national approaches to setting ecological status boundaries in phytobenthos assessment for the European Water Framework Directive: results of an intercalibration exercise

The European Union (EU)’s Water Framework Directive (WFD) requires that all Member States participate in intercalibration exercises in order to ensure that ecological status concepts and assessment levels are consistent across the EU. This paper describes one such exercise, performed by the countries in the Central/Baltic Geographical Intercalibration Group stretching from Ireland in the west to Estonia in the east and from the southern parts of Scandinavia to the northern regions of Spain and Italy (but excluding alpine regions, which were intercalibrated separately). In this exercise, methods used to measure ecological status of rivers using benthic diatoms were compared. Ecological status is estimated as the ratio between the observed value of a biological element and the value expected in the absence of significant human impact. Approaches to defining the ‘reference sites’, from which these ‘expected’ values were derived, varied from country to country. Minimum criteria were established as part of the exercise but there was still considerable variation between national reference values, reflecting typological differences that could not be resolved during the exercise. A simple multimetric index was developed to compare boundary values using two widely used diatom metrics. Boundary values for high/good status and good/moderate status set by each participant were converted to their equivalent values of this intercalibration metric using linear regression. Variation of ±0.05 EQR units around the median value was considered to be acceptable and the exercise provided a means for those Member States who fell significantly above or below this line to review their approaches and, if necessary, adjust their boundaries. Handling editor: J. Padisak     

Scaling-up the delivery of dog vaccination campaigns against rabies in Tanzania

An increasing number of countries are committing to meet the global target to eliminate human deaths from dog-mediated rabies by 2030. Mass dog vaccination is central to this strategy. To interrupt rabies transmission from dogs to humans, the World Health Organization recommends that vaccination campaigns should be carried out every year in all dog-owning communities vaccinating 70% of their susceptible dogs. Monitoring and evaluation of dog vaccination campaigns are needed to measure progress towards elimination. In this study, we measured the delivery performance of large-scale vaccination campaigns implemented in 25 districts in south-east Tanzania from 2010 until 2017. We used regression modelling to infer the factors associated with, and potentially influencing the successful delivery of vaccination campaigns. During 2010–2017, five rounds of vaccination campaigns were carried out, vaccinating in total 349,513 dogs in 2,066 administrative vaccination units (rural villages or urban wards). Progressively more dogs were vaccinated over the successive campaigns. The campaigns did not reach all vaccination units each year, with only 16–28% of districts achieving 100% campaign completeness (where all units were vaccinated). During 2013–2017 when vaccination coverage was monitored, approximately 20% of vaccination units achieved the recommended 70% coverage, with average coverage around 50%. Campaigns were also not completed at annual intervals, with the longest interval between campaigns being 27 months. Our analysis revealed that districts with higher budgets generally achieved higher completeness, with a twofold difference in district budget increasing the odds of a vaccination unit being reached by a campaign by slightly more than twofold (OR: 2.29; 95% CI: 1.69–3.09). However, higher budgets did not necessarily result in higher coverage within vaccination units that were reached. We recommend national programs regularly monitor and evaluate the performance of their vaccination campaigns, so as to identify factors hindering their effective delivery and to guide remedial action.     

TGFβ Activated Kinase 1 (TAK1) at the Crossroad of B Cell Receptor and Toll-Like Receptor 9 Signaling Pathways in Human B Cells

B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR), receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R) and the innate receptor, Toll-like receptor 9 (TLR9). However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF) and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs), ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1) is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.     

Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

Interstitial lung disease and pulmonary fibrosis in Hermansky-Pudlak syndrome type 2, an adaptor protein-3 complex disease

Pulmonary fibrosis develops in Hermansky-Pudlak syndrome (HPS) types 1 and 4. Limited information is available about lung disease in HPS type 2 (HPS-2), which is characterized by abnormal function of the adaptor protein-3 (AP-3) complex. To define lung disease in HPS-2, one child and two adults with HPS-2 were evaluated at the National Institutes of Health on at least two visits, and another child was evaluated at the University of Texas Health Science Center San Antonio. All four subjects with HPS-2 had findings of interstitial lung disease (ILD) on a high-resolution computed tomography scan of the chest. The predominant feature was ground glass opacification. Subject 1, a 14-year-old male, and subject 4, a 4-year-old male, had severe ILD, pulmonary fibrosis, secondary pulmonary hypertension and recurrent lung infections. Lung biopsy performed at 20 months of age in subject 1 revealed interstitial fibrosis and prominent type II pneumocyte hyperplasia without lamellar body enlargement. Subject 2, a 27-year-old male smoker, had mild ILD. Subject 3, a 22-year-old male nonsmoker and brother of subject 2, had minimal ILD. Severe impairment of gas exchange was found in subjects 1 and 4 and not in subjects 2 or 3. Plasma concentrations of transforming growth factor-β1 and interleukin-17A correlated with severity of HPS-2 ILD. These data show that children and young adults with HPS-2 and functional defects of the AP-3 complex are at risk for ILD and pulmonary fibrosis.     

Exploring Beneficial/Virulence Properties of Two Dairy-Related Strains of Streptococcus infantarius subsp. infantarius

The genus Streptococcus includes various species, remarkably different in their behavior, applications, virulence, and safety. Taxonomically Streptococcus infantarius subsp. infantarius belonging to the Streptococcus bovis group, which includes several pathogen species, however, has been found as predominant species in some African dairy products that are widely consumed and considered to be safe. Streptococcus infantarius subsp. infantarius’ safety may be questioned due to the association of this species with clinical cases. In this study, isolates from dairy origin were selected based on their bacteriocinogenic potential and differentiated by their RAPD-PCR profiles. Two strains were identified by 16S rRNA sequencing as St. infantarius subsp. infantarius and investigated regarding their potential beneficial properties and factors related to virulence and safety. A series of in vitro tests included properties related to survival in the gastrointestinal tract and beneficial intestinal activities. Production of bacteriocin/s, detection of related genes, and partial characterization of expressed antimicrobial protein were evaluated. Genes related to folate biosynthesis were detected in both studied strains. Evaluation of physiological tests related to strains virulence, adhesion, and resistance to antibiotics and detections of virulence and biogenic amines production-related genes were also investigated. Taking in consideration all the aspects of the specific nature of St. infantarius subsp. infantarius K1–4 and K5–1 (beneficial properties and virulence characteristics), both strains cannot be considered safe for human or other animals application, even though they have been isolated from dairy products. This study is highlighting the importance of evaluation for presence of potential virulence factors in newly characterized strains in order to be confident in their safety.