A COL11A2 Mutation in Labrador Retrievers with Mild Disproportionate Dwarfism

We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2). It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA) and collagen alpha-2(XI) chain gene (COL11A2), respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.     

Effects of Population Based Screening for Chlamydia Infections in The Netherlands Limited by Declining Participation Rates

Background

A large trial to investigate the effectiveness of population based screening for chlamydia infections was conducted in the Netherlands in 2008–2012. The trial was register based and consisted of four rounds of screening of women and men in the age groups 16–29 years in three regions in the Netherlands. Data were collected on participation rates and positivity rates per round. A modeling study was conducted to project screening effects for various screening strategies into the future.

Methods and Findings

We used a stochastic network simulation model incorporating partnership formation and dissolution, aging and a sexual life course perspective. Trends in baseline rates of chlamydia testing and treatment were used to describe the epidemiological situation before the start of the screening program. Data on participation rates was used to describe screening uptake in rural and urban areas. Simulations were used to project the effectiveness of screening on chlamydia prevalence for a time period of 10 years. In addition, we tested alternative screening strategies, such as including only women, targeting different age groups, and biennial screening. Screening reduced prevalence by about 1% in the first two screening rounds and leveled off after that. Extrapolating observed participation rates into the future indicated very low participation in the long run. Alternative strategies only marginally changed the effectiveness of screening. Higher participation rates as originally foreseen in the program would have succeeded in reducing chlamydia prevalence to very low levels in the long run.

Conclusions

Decreasing participation rates over time profoundly impact the effectiveness of population based screening for chlamydia infections. Using data from several consecutive rounds of screening in a simulation model enabled us to assess the future effectiveness of screening on prevalence. If participation rates cannot be kept at a sufficient level, the effectiveness of screening on prevalence will remain limited.     

The crystal structure of the secreted dimeric form of the hemophore HasA reveals a domain swapping with an exchanged heme ligand

To satisfy their iron needs, several Gram-negative bacteria use a heme uptake system involving an extracellular heme-binding protein called hemophore. The function of the hemophore is to acquire free or hemoprotein-bound heme and to transfer it to HasR, its specific outer membrane receptor, by protein-protein interaction. The hemophore HasA secreted by Serratia marcescens, an opportunistic pathogen, was the first to be identified and is now very well characterized. HasA is a monomer that binds one b heme with strong affinity. The heme in HasA is highly exposed to solvent and coordinated by an unusual pair of ligands, a histidine and a tyrosine. Here, we report the identification, the characterization and the X-ray structure of a dimeric form of HasA from S. marcescens: DHasA. We show that both monomeric and dimeric forms are secreted in iron deficient conditions by S. marcescens. The crystal structure of DHasA reveals that it is a domain swapped dimer. The overall structure of each monomeric subunit of DHasA is very similar to that of HasA but formed by parts coming from the two different polypeptide chains, involving one of the heme ligands. Consequently DHasA binds two heme molecules by residues coming from both polypeptide chains. We show here that, while DHasA can bind two heme molecules, it is not able to deliver them to the receptor HasR. However, DHasA can efficiently transfer its heme to the monomeric form that, in turn, delivers it to HasR. We assume that DHasA can function as a heme reservoir in the hemophore system.     

Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families

The gene encoding the α-agarase from “Alteromonas agarilytica” (proposed name) has been cloned and sequenced. The gene product (154 kDa) is unrelated to β-agarases and instead belongs to a new family of glycoside hydrolases (GH96). The α-agarase also displays a complex modularity, with the presence of five thrombospondin type 3 repeats and three carbohydrate-binding modules.     

Bilberry xyloglucan--novel building blocks containing beta-xylose within a complex structure

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.     

The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.     

Comparative evaluation of in-house manual, and commercial semi-automated and automated DNA extraction platforms in the sample preparation of human stool specimens for a Salmonella enterica 5'-nuclease assay

In the present study, three methods (NucliSens miniMAG [bioMérieux], MagNA Pure DNA Isolation Kit III Bacteria/Fungi [Roche], and a silica-guanidiniumthiocyanate {Si-GuSCN-F} procedure for extracting DNA from stool specimens were compared with regard to analytical performance (relative DNA recovery and down stream real-time PCR amplification of Salmonella enterica DNA), stability of the extracted DNA, hands-on time (HOT), total processing time (TPT), and costs. The Si-GuSCN-F procedure showed the highest analytical performance (relative recovery of 99%, S. enterica real-time PCR sensitivity of 91%) at the lowest associated costs per extraction (euro 4.28). However, this method did required the longest HOT (144 min) and subsequent TPT (176 min) when processing 24 extractions. Both miniMAG and MagNA Pure extraction showed similar performances at first (relative recoveries of 57% and 52%, S. enterica real-time PCR sensitivity of 85%). However, when difference in the observed Ct values after real-time PCR were taken into account, MagNA Pure resulted in a significant increase in Ct value compared to both miniMAG and Si-GuSCN-F (with on average +1.26 and +1.43 cycles). With regard to inhibition all methods showed relatively low inhibition rates (< 4%), with miniMAG providing the lowest rate (0.7%). Extracted DNA was stable for at least 1 year for all methods. HOT was lowest for MagNA Pure (60 min) and TPT was shortest for miniMAG (121 min). Costs, finally, were euro 4.28 for Si-GuSCN, euro 6.69 for MagNA Pure and euro 9.57 for miniMAG.     

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.     

Comparison of Contact Patterns Relevant for Transmission of Respiratory Pathogens in Thailand and the Netherlands Using Respondent-Driven Sampling

Understanding infection dynamics of respiratory diseases requires the identification and quantification of behavioural, social and environmental factors that permit the transmission of these infections between humans. Little empirical information is available about contact patterns within real-world social networks, let alone on differences in these contact networks between populations that differ considerably on a socio-cultural level. Here we compared contact network data that were collected in the Netherlands and Thailand using a similar online respondent-driven method. By asking participants to recruit contact persons we studied network links relevant for the transmission of respiratory infections. We studied correlations between recruiter and recruited contacts to investigate mixing patterns in the observed social network components. In both countries, mixing patterns were assortative by demographic variables and random by total numbers of contacts. However, in Thailand participants reported overall more contacts which resulted in higher effective contact rates. Our findings provide new insights on numbers of contacts and mixing patterns in two different populations. These data could be used to improve parameterisation of mathematical models used to design control strategies. Although the spread of infections through populations depends on more factors, found similarities suggest that spread may be similar in the Netherlands and Thailand.