An ex vivo porcine spleen perfusion as a model of bacterial sepsis

An ex vivo, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. Constant perfusion of heparinized blood was performed for 6 hours. After injection of Streptococcus pneumoniae to the circuit, serial samples of both blood and spleen biopsies were collected and analyzed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damage in the organ architecture and no changes in the immune cell distribution other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed – in line with the 3Rs principles – has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology, and splenic architecture in particular, closely resemble those of humans

Deletion of the zinc transporter lipoprotein AdcAII causes hyperencapsulation of Streptococcus pneumoniae associated with distinct alleles of the Type I restriction modification system

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restrictionmodification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype

Draft Whole-Genome Sequences of Periodontal Pathobionts Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia Contain Phase-Variable Restriction-Modification Systems.

Periodontal disease comprises mild to severe inflammatory host responses to oral bacteria that can cause destruction of the tooth-supporting tissue. We report genome sequences for 18 clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, Gram-negative obligate anaerobes that play a role in the periodontal disease process.This work was in part funded by a grant from the BBSRC (BB/N002903/1) to M.R.O

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead,we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation,and suggest a prefusion rather than postfusion role for vesicular cation channels

Reduced occupancy of hedgehogs (Erinaceus europaeus) in rural England and Wales: the influence of habitat and an asymmetric intra-guild predator

Agricultural landscapes have become increasingly intensively managed resulting in population declines across a broad range of taxa, including insectivores such as the hedgehog (Erinaceus europaeus). Hedgehog declines have also been attributed to an increase in the abundance of badgers (Meles meles), an intra-guild predator. The status of hedgehogs across the rural landscape at large spatial scales is, however, unknown. In this study, we used footprint tracking tunnels to conduct the first national survey of rural hedgehog populations in England and Wales. Single and two-species occupancy modelling was used to quantify hedgehog occupancy in relation to habitat and predator covariates. Hedgehog occupancy was low (22% nationally), and significantly negatively related to badger sett density and positively related to the built environment. Hedgehogs were also absent from 71% of sites that had no badger setts, indicating that large areas of the rural landscape are not occupied by hedgehogs. Our results provide the first field based national survey of hedgehogs, providing a robust baseline for future monitoring. Furthermore, the combined effects of increasing badger abundance and intensive agriculture may have provided a perfect storm for hedgehogs in rural Britain, leading to worryingly low levels of occupancy over large spatial scales

Sodium lauryl ether sulfate (SLES) degradation by nitrate-reducing bacteria

The online version of this article (doi:10.1007/s00253-017-8212-x) contains supplementary material, which is available to authorized users.The surfactant sodium lauryl ether sulfate (SLES) is widely used in the composition of detergents and frequently ends up in wastewater treatment plants (WWTPs). While aerobic SLES degradation is well studied, little is known about the fate of this compound in anoxic environments, such as denitrification tanks of WWTPs, nor about the bacteria involved in the anoxic biodegradation. Here, we used SLES as sole carbon and energy source, at concentrations ranging from 50 to 1000 mg L1, to enrich and isolate nitrate-reducing bacteria from activated sludge of a WWTP with the anaerobic-anoxic-oxic (A2/O) concept. In the 50 mg L1 enrichment, Comamonas (50%), Pseudomonas (24%), and Alicycliphilus (12%) were present at higher relative abundance, while Pseudomonas (53%) became dominant in the 1000 mg L1 enrichment. Aeromonas hydrophila strain S7, Pseudomonas stutzeri strain S8, and Pseudomonas nitroreducens strain S11 were isolated from the enriched cultures. Under denitrifying conditions, strains S8 and S11 degraded 500 mg L1 SLES in less than 1 day, while strain S7 required more than 6 days. Strains S8 and S11 also showed a remarkable resistance to SLES, being able to grow and reduce nitrate with SLES concentrations up to 40 g L1. Strain S11 turned out to be the best anoxic SLES degrader, degrading up to 41% of 500 mg L1. The comparison between SLES anoxic and oxic degradation by strain S11 revealed differences in SLES cleavage, degradation, and sulfate accumulation; both ester and ether cleavage were probably employed in SLES anoxic degradation by strain S11.This research was supported by the Spanish Ministry of Education and Science (contract project CTQ2007-64324 and 447 CONSOLIDER-CSD 2007-00055). The Regional Government of Castilla y Leon (Ref. GR76) is also gratefully acknowledged. MRD is supported by the WIMEK graduate school (project BAdaptive capacity and functionality of multi-trophic aquatic ecosystems^). AJMS is supported by the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO). AJMS and AJC are supported by an European ResearchCouncil (ERC) Grant (Project 323009).Thisstudywassupported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was alsosupportedbythePortugueseFoundationforScienceandTechnology (FCT) under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). Joana Alves from University of Minho (Portugal) is acknowledged for support with the molecular techniques.info:eu-repo/semantics/publishedVersio

The Population Genetics of dN/dS

Evolutionary pressures on proteins are often quantified by the ratio of substitution rates at non-synonymous and synonymous sites. The dN/dS ratio was originally developed for application to distantly diverged sequences, the differences among which represent substitutions that have fixed along independent lineages. Nevertheless, the dN/dS measure is often applied to sequences sampled from a single population, the differences among which represent segregating polymorphisms. Here, we study the expected dN/dS ratio for samples drawn from a single population under selection, and we find that in this context, dN/dS is relatively insensitive to the selection coefficient. Moreover, the hallmark signature of positive selection over divergent lineages, dN/dS>1, is violated within a population. For population samples, the relationship between selection and dN/dS does not follow a monotonic function, and so it may be impossible to infer selection pressures from dN/dS. These results have significant implications for the interpretation of dN/dS measurements among population-genetic samples

Reproductive biology of the pampas deer (Ozotoceros bezoarticus): a review

The pampas deer (Ozotoceros bezoarticus) is a South American grazing deer which is in extreme danger of extinction. Very little is known about the biology of the pampas deer. Moreover, most information has not been published in peer-reviewed scientific journals, and is only available in local publications, theses, etc. Therefore, our aim was to update and summarize the available information regarding the reproductive biology of the pampas deer. Moreover, in most sections, we have also included new, unpublished information. Detailed descriptions are provided of the anatomy of both the female and the male reproductive tract, puberty onset, the oestrous cycle and gestational length. Birthing and the early postpartum period are described, as are maternal behaviour and early fawn development, seasonal distribution of births, seasonal changes in male reproduction and antler cycle, reproductive behaviour, semen collection, and cryopreservation. Finally, an overview is given and future directions of research are proposed

Prediction and estimation of effective population size

Effective population size (Ne) is a key parameter in population genetics. It has important applications in evolutionary biology, conservation genetics, and plant and animal breeding, because it measures the rates of genetic drift and inbreeding and affects the efficacy of systematic evolutionary forces such as mutation, selection and migration. We review the developments in predictive equations and estimation methodologies of effective size. In the prediction part, we focus on the equations for populations with different modes of reproduction, for populations under selection for unlinked or linked loci, and for the specific applications to conservation genetics. In the estimation part, we focus on methods developed for estimating the current or recent effective size from molecular marker or sequence data. We discuss some underdeveloped areas in predicting and estimating Ne for future research