New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

Facial fluctuating asymmetry is not associated with childhood ill-health in a large British cohort study

The idea that symmetry in facial traits is associated with attractiveness because it reliably indicates good physiological health, particularly to potential sexual partners, has generated an extensive literature on the evolution of human mate choice. However, large-scale tests of this hypothesis using direct or longitudinal assessments of physiological health are lacking. Here, we investigate relationships between facial fluctuating asymmetry (FA) and detailed individual health histories in a sample (n = 4732) derived from a large longitudinal study (Avon Longitudinal Study of Parents and Children) in South West England. Facial FA was assessed using geometric morphometric analysis of facial landmark configurations derived from three-dimensional facial scans taken at 15 years of age. Facial FA was not associated with longitudinal measures of childhood health. However, there was a very small negative association between facial FA and IQ that remained significant after correcting for a positive allometric relationship between FA and face size. Overall, this study does not support the idea that facial symmetry acts as a reliable cue to physiological health. Consequently, if preferences for facial symmetry do represent an evolved adaptation, then they probably function not to provide marginal fitness benefits by choosing between relatively healthy individuals on the basis of small differences in FA, but rather evolved to motivate avoidance of markers of substantial developmental disturbance and significant pathology.     

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro: Simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swift recognition and removal of apoptotic cells is important for normal tissue homeostasis and failure in the underlying clearance mechanisms has pathological consequences associated with inflammatory and auto-immune diseases. Cell cultures in vitro usually lack the capacity for removal of nonviable cells because of the absence of phagocytes and, as such, fail to emulate the healthy in vivo micro-environment from which dead cells are absent. While a key objective in cell culture is to maintain viability at maximal levels, cell death is unavoidable and non-viable cells frequently contaminate cultures in significant numbers. Here we show that the presence of apoptotic cells in monoclonal antibody-producing hybridoma cultures has markedly detrimental effects on antibody productivity. Removal of apoptotic hybridoma cells by macrophages at the time of seeding resulted in 100% improved antibody productivity that was, surprisingly to us, most pronounced late on in the cultures. Furthermore, we were able to recapitulate this effect using novel super-paramagnetic Dead-Cert™ Nanoparticles to remove non-viable cells simply and effectively at culture seeding. These results (1) provide direct evidence that apoptotic cells have a profound influence on their non-phagocytic neighbors in culture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy for improving antibody manufacture in vitro.Key words: apoptosis, hybridoma, phagocytosis, viability, cell-culture, cell-death, antibody, nanoparticles     

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

Summary Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with ¹³C and ¹⁵N isotopes. We report the first such uniform labeling of a glycoprotein using a universal ¹³C-and ¹⁵N-labeling medium to express ¹³C, ¹⁵N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The ¹³C, ¹⁵N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.To whom correspondence should be addressed.     

The accumulation of deleterious mutations within the frozen niche variation hypothesis

The frozen niche variation hypothesis proposes that asexual clones exploit a fraction of a total resource niche available to the sexual population from which they arise. Differences in niche breadth may allow a period of coexistence between a sexual population and the faster reproducing asexual clones. Here, we model the longer term threat to the persistence of the sexual population from an accumulation of clonal diversity, balanced by the cost to the asexual population resulting from a faster rate of accumulation of deleterious mutations. We use Monte-Carlo simulations to quantify the interaction of niche breadth with accumulating deleterious mutations. These two mechanisms may act synergistically to prevent the extinction of the sexual population, given: (1) sufficient genetic variation, and consequently niche breadth, in the sexual population; (2) a relatively slow rate of accumulation of genetic diversity in the clonal population; (3) synergistic epistasis in the accumulation of deleterious mutations.     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

Control of Boophilus annulatus (Acari: Ixodidae) on cattle using injectable microspheres containing ivermectin

The efficacy of an injectable microsphere formulation of ivermectin for control of the cattle tick, Boophilus annulatus (Say), was tested on 2 groups of 6 Hereford heifers held on separate 7-ha, tick-infested, buffel grass pastures. Cattle in one pasture were injected subcutaneously in the neck with a controlled-release microsphere formulation of ivermectin at the rate of 2.4 mg AI/kg body weight; the other group was injected with carrier only. Beginning 4 wk after injection and continuing throughout the remainder of the test (16 wk), no engorged ticks (> or = 5.5 mm) were found on any of the treated cattle, whereas large numbers of engorged ticks were found on the untreated controls. During this period, a few ticks were recovered from untreated sentinel animals placed in the treatment pasture during 7-8 wk after treatment, but none were recovered from animals exposed from 11-12 wk or 14-15 wk. Large numbers of B. annulatus ticks were found on untreated sentinel cattle placed in the control pasture during these same periods. Although the cattle, pastures, and tick habitat were approximately equal, the treated cattle gained an average of 77 kg compared with an average of 42 kg for the control group. This technology offers a possible alternative to the current official program of dipping and vacating pastures for eradication of Boophilus sp. infestations from the quarantine zone in southern Texas. Larger scale testing is needed to determine the potential of the injectable microsphere formulation and to optimize its use in eradication or control strategies.     

A Randomized Controlled Trial of Innovative Postpartum Care Model for Mother-Baby Dyads

Objective

To evaluate the efficacy, safety, and maternal satisfaction of a newly established integrative postpartum community-based clinic providing comprehensive support for mothers during the first month after discharge from the hospital. Our primary interests were breastfeeding rates, readmission and patient satisfaction.

Methods

A randomized controlled trial was conducted in Ottawa, Canada, where 472 mothers were randomized via a 1:2 ratio to either receive standard of care (n = 157) or to attend the postpartum breastfeeding clinic (n = 315). Outcome data were captured through questionnaires completed by the participants at 2, 4, 12 and 24 weeks postpartum. Unadjusted and adjusted logistic regression models were conducted to determine the effect of the intervention on exclusive breastfeeding at 12 weeks (primary outcome). Secondary outcomes included breastfeeding rate at 2, 4 and 24 weeks, breastfeeding self-efficacy scale, readmission rate, and satisfaction score.

Results

More mothers in the intervention group (n = 195, 66.1%) were exclusively breastfeeding at 12 weeks compared to mothers in the control group (n = 81, 60.5%), however no statistically significant difference was observed (OR = 1.28; 95% CI:0.84–1.95)). The rate of emergency room visits at 2 weeks for the intervention group was 11.4% compared to the standard of care group (15.2%) (OR = 0.69; 95% CI: 0.39–1.23). The intervention group was significantly more satisfied with the overall care they received for breastfeeding compared to the control group (OR = 1.96; 95% CI: 3.50–6.88)).

Conclusion

This new model of care did not significantly increase exclusive breastfeeding at 12 weeks. However, there were clinically meaningful improvements in the rate of postnatal problems and satisfaction that support this new service delivery model for postpartum care. A community-based multidisciplinary postpartum clinic is feasible to implement and can provide appropriate and highly satisfactory care to mother-baby dyads. This model of care may be more beneficial in a population that is not already predisposed to breastfeed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02043119","term_id":"NCT02043119"}}NCT02043119