Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

Background  

The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq.     

Mucosal dendritic cell diversity in the gastrointestinal tract

The discovery of dendritic cells (DCs) in skin by Paul Langerhans in 1868 identified a cell type which has since been recognized as a key link between innate and adaptive immunity. DCs originate from bone marrow and disseminate through blood to all tissues in the body, and distinct DC subpopulations have been identified in many different tissues. DC diversity is apparent throughout all mucosal surfaces of the body, but the focus of this review article is DC diversity throughout the gastro-intestinal tract (GIT). DC subpopulations have been well characterized in the oral cavity and small intestine, but DC characterization in other regions, such as the esophagus and stomach, is limited. Substantial research has focused on DC function during disease, but understanding the regulation of inflammation and the induction of acquired immune responses requires combined phenotypic and functional characterization of individual DC subpopulations. Furthermore, little is known regarding mucosal DC subpopulations in the GIT of the neonate and how these DC populations change following colonization by commensal microflora. The current review will highlight mucosal DC diversity and discuss factors that may influence mucosal DC differentiation.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.

     

Cerebral oxygen availability by NIR spectroscopy during transient hypoxia in humans

The effects of mild hypoxia on brain oxyhemoglobin, cytochrome a,a3 redox status, and cerebral blood volume were studied using near-infrared spectroscopy in eight healthy volunteers. Incremental hypoxia reaching 70% arterial O2 saturation was produced in normocapnia [end-tidal PCO2 (PETCO2) 36.9 +/- 2.6 to 34.9 +/- 3.4 Torr] or hypocapnia (PETCO2 32.8 +/- 0.6 to 23.7 +/- 0.6 Torr) by an 8-min rebreathing technique and regulation of inspired CO2. Normocapnic hypoxia was characterized by progressive reductions in arterial PO2 (PaO2, 89.1 +/- 3.5 to 34.1 +/- 0.1 Torr) with stable PETCO2, arterial PCO2 (PaCO2), and arterial pH and resulted in increases in heart rate (35%) systolic blood pressure (14%), and minute ventilation (5-fold). Hypocapnic hypoxia resulted in progressively decreasing PaO2 (100.2 +/- 3.6 to 28.9 +/- 0.1 Torr), with progressive reduction in PaCO2 (39.0 +/- 1.6 to 27.3 +/- 1.9 Torr), and an increase in arterial pH (7.41 +/- 0.02 to 7.53 +/- 0.03), heart rate (61%), and ventilation (3-fold). In the brain, hypoxia resulted in a steady decline of cerebral oxyhemoglobin content and a decrease in oxidized cytochrome a,a3. Significantly greater loss of oxidized cytochrome a,a3 occurred for a given decrease in oxyhemoglobin during hypocapnic hypoxia relative to normocapnic hypoxia. Total blood volume response during hypoxia also was significantly attenuated by hypocapnia, because the increase in volume was only half that of normocapnic subjects. We conclude that cytochrome a,a3 oxidation level in vivo decreases at mild levels of hypoxia. PaCO is an important determinant of brain oxygenation, because it modulates ventilatory, cardiovascular, and cerebral O2 delivery responses to hypoxia.     

RepSeq – A database of amino acid repeats present in lower eukaryotic pathogens

Background  

Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses.     

Prestress as an optimal biomechanical parameter for needle penetration

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.