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The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo−maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo−maternal interface was prominent, highlighting a potential role of miRNAs in embryo−maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6–7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6–7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.

ABSTRACT Burkholderia cenocepacia J2315 is a member of the B. cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B. cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B. cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B. cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B. cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to “cepacia syndrome,” a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B. cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B. cenocepacia that are relevant for infection.

Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells.

Recently emerged fungal diseases, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are an increasing threat to amphibians worldwide. In Europe, the threat of Bsal to salamander populations is demonstrated by the rapid decline of fire salamander populations in Germany, the Netherlands and Belgium. Although most European urodelans are susceptible to infection in infection trials, recent evidence suggests marked interspecific differences in the course of infection, with potentially far reaching implications for salamander conservation. As a salamander's skin is the first line of defense against such pathogens, interspecific differences in innate immune function of the skin may explain differential susceptibility. Here we investigate if compounds present on a salamander's skin can kill Bsal spores and if there is variation among species. We used a non-invasive assay to compare killing ability of salamander mucosomes of four different species (captive and wild Salamandra salamandra and captive Ichtyosaura alpestris, Cynops pyrrhogaster and Lissotriton helveticus) by exposing Bsal zoospores to salamander mucosomes and determining spore survival. In all samples, zoospores were killed when exposed to mucosomes. Moreover, we saw a significant variation in this Bsal killing ability of mucosomes between different salamander host species. Our results indicate that mucosomes of salamanders might provide crucial skin protection against Bsal, and could explain why some species are more susceptible than others. This study represents a step towards better understanding host species variation in innate immune function and disease susceptibility in amphibians.

Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part (Pflieger et al., 2008. Mol. Cell. Proteomics, 7: 326–46 [1]; Wu et al., 2011. Nat. Methods, 8: 677–83 [2]). Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup and data mining, the proteome coverage was extended through gel fractionation, while an internal replicate measurement creates more stringent data analysis opportunities. The latter allows other researchers to set their own threshold for selecting potential phosphorylation events in the dataset presented here, depending on the biological question or corroboration under investigation. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data accompanying the manuscript on this approach (Glibert et al., 2015, J. Proteome Res. 14: 2015, 839–49 [5]) have been deposited to the ProteomeXchange with identifier PXD001574.

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Posttranslational histone modifications are essential features in epigenetic regulatory networks. One of these modifications has remained largely understudied: regulated histone proteolysis. In analogy to the histone H3 clipping during early mouse embryonic stem cell differentiation, we report for the first time that also in human embryonic stem cells this phenomenon takes place in the two different analyzed cell lines. Employing complementary techniques, different cleavage sites could be identified, namely A21, R26 and residue 31. The enzyme responsible for this cleavage is found to be a serine protease. The formation of cleaved H3 follows a considerably variable pattern, depending on the timeframe, culture conditions and culture media applied. Contrary to earlier findings on H3 clipping, our results disconnect the link between declining Oct4 expression and H3 cleavage.

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation.

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.

Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host's transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic/metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host's RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following pathogenic infection with CPV, while they also highlight the relative importance of RNAi as an antiviral mechanism.

Surface sediments are important systems for the removal of anthropogenically derived inorganic nitrogen in estuaries. They are often characterized by the presence of a microphytobenthos (MPB) biofilm, which can impact bacterial communities in underlying sediments for example by secretion of extracellular polymeric substances (EPS) and competition for nutrients (including nitrogen). Pyrosequencing and qPCR was performed on two intertidal surface sediments of the Westerschelde estuary characterized by a two-fold difference in MPB biomass but no difference in MPB composition. Doubling of MPB biomass was accompanied by a disproportionately (ten-fold) increase in total bacterial abundances while, unexpectedly, no difference in general community structure was observed, despite significantly lower bacterial richness and distinct community membership, mostly for non-abundant taxa. Denitrifier abundances corresponded likewise while community structure, both for nirS and nirK denitrifiers, remained unchanged, suggesting that competition with diatoms for nitrate is negligible at concentrations in the investigated sediments (appr. 1 mg/l NO3-). This study indicates that MPB biomass increase has a general, significantly positive effect on total bacterial and denitrifier abundances, with stimulation or inhibition of specific bacterial groups that however do not result in a re-structured community.

Devrieseasis caused by Devriesea agamarum is a highly prevalent disease in captive desert lizards, resulting in severe dermatitis and in some cases mass mortality. In this study, we assessed the contribution of autovaccination to devrieseasis control by evaluating the capacity of 5 different formalin-inactivated D. agamarum vaccines to induce a humoral immune response in bearded dragons (Pogona vitticeps). Each vaccine contained one of the following adjuvants: CpG, incomplete Freund's, Ribi, aluminium hydroxide, or curdlan. Lizards were administrated one of the vaccines through subcutaneous injection and booster vaccination was given 3 weeks after primo-vaccination. An indirect ELISA was developed and used to monitor lizard serological responses. Localized adverse effects following subcutaneous immunization were observed in all but the Ribi adjuvanted vaccine group. Following homologous experimental challenge, the incomplete Freund's as well as the Ribi vaccine were observed to confer protection in bearded dragons against the development of D. agamarum associated septicemia but not against dermatitis. Subsequently, two-dimensional gelelectrophoresis followed by immunoblotting and mass spectrometry was conducted with serum obtained from 3 lizards that showed seroconversion after immunisation with the Ribi vaccine. Fructose-bisphosphate aldolase and aldo-keto reductase of D. agamarum reacted with serum from the latter lizards. Based on the demonstrated seroconversion and partial protection against D. agamarum associated disease following the use of formalin-inactivated vaccines as well as the identification of target antigens in Ribi vaccinated bearded dragons, this study provides promising information towards the development of a vaccination strategy to control devrieseasis in captive lizard collections.

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100-144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained by all SNPs for two phenotypically-related neurobehavioral disorders, obsessive-compulsive disorder (OCD) and Tourette Syndrome (TS), using GCTA. Our analysis yielded a heritability point estimate of 0.58 (se = 0.09, p = 5.64e-12) for TS, and 0.37 (se = 0.07, p = 1.5e-07) for OCD. In addition, we conducted multiple genomic partitioning analyses to identify genomic elements that concentrate this heritability. We examined genomic architectures of TS and OCD by chromosome, MAF bin, and functional annotations. In addition, we assessed heritability for early onset and adult onset OCD. Among other notable results, we found that SNPs with a minor allele frequency of less than 5% accounted for 21% of the TS heritability and 0% of the OCD heritability. Additionally, we identified a significant contribution to TS and OCD heritability by variants significantly associated with gene expression in two regions of the brain (parietal cortex and cerebellum) for which we had available expression quantitative trait loci (eQTLs). Finally we analyzed the genetic correlation between TS and OCD, revealing a genetic correlation of 0.41 (se = 0.15, p = 0.002). These results are very close to previous heritability estimates for TS and OCD based on twin and family studies, suggesting that very little, if any, heritability is truly missing (i.e., unassayed) from TS and OCD GWAS studies of common variation. The results also indicate that there is some genetic overlap between these two phenotypically-related neuropsychiatric disorders, but suggest that the two disorders have distinct genetic architectures.

The objective of this study was to screen a dog population from Belgium, the Netherlands and Germany for the presence of mutant alleles associated with hip dysplasia (HD), degenerative myelopathy (DM), exercise-induced collapse (EIC), neuronal ceroid lipofuscinosis 4A (NCL), centronuclear myopathy (HMLR), mucopolysaccharidosis VII (MPS VII), myotonia congenita (MG), gangliosidosis (GM1) and muscular dystrophy (Duchenne type) (GRMD). Blood samples (K3EDTA) were collected for genotyping with Kompetitive Allele Specific PCR (n = 476). Allele and genotype frequencies were calculated in those breeds with at least 12 samples (n = 8). Hardy-Weinberg equilibrium was tested. Genetic variation was identified for 4 out of 9 disorders: mutant alleles were found in 49, 15, 3 and 2 breeds for HD, DM, EIC and NCL respectively. Additionally, mutant alleles were identified in crossbreeds for both HD and EIC. For HD, DM, EIC and NCL mutant alleles were newly discovered in 43, 13, 2 and 1 breed(s), respectively. In 9, 2 and 1 breed(s) for DM, EIC and NCL respectively, the mutant allele was detected, but the respective disorder has not been reported in those breeds. For 5 disorders (HMLR, MPS VII, MG, GM1, GRMD), the mutant allele could not be identified in our population. For the other 4 disorders (HD, DM, EIC, NCL), prevalence of associated mutant alleles seems strongly breed dependent. Surprisingly, mutant alleles were found in many breeds where the disorder has not been reported to date.

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.

BACKGROUND: Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES) molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4) by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. CONCLUSIONS/SIGNIFICANCE: The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.

ContextHashimoto's thyroiditis (HT) and Graves' disease (GD), two autoimmune thyroid diseases (AITD), occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases.ObjectiveAim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD.ParticipantsEleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained.MethodsFluorescence in situ hybridization (FISH) and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized.ResultsIn patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p=0.0061). In patients with HT, mainly fetal CD8(+) T cells were found, while in patients with GD, fetal B and CD4(+) T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (pConclusionThis study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.

Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias.

BackgroundCTX-M-producing Escherichia coli strains are regarded as major global pathogens.Methodology/principal findingsThe nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1) and bla(CTX-M-15). It shares more than 90% homology with a previously published bla(CTX-M)-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1) and bla(CTX-M-15), were found. Six resistance genes, bla(TEM-1), bla(CTX-M-15), bla(OXA-1), aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15)-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM)-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1), aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids.ConclusionsCollectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust’s use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.