The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

Nutrient scarcity as a selective pressure for mast seeding.

Mast seeding is one of the most intriguing reproductive traits in nature. Despite its potential drawbacks in terms of fitness, the widespread existence of this phenomenon suggests that it should have evolutionary advantages under certain circumstances. Using a global dataset of seed production time series for 219 plant species from all of the continents, we tested whether masting behaviour appears predominantly in species with low foliar nitrogen and phosphorus concentrations when controlling for local climate and productivity. Here, we show that masting intensity is higher in species with low foliar N and P concentrations, and especially in those with imbalanced N/P ratios, and that the evolutionary history of masting behaviour has been linked to that of nutrient economy. Our results support the hypothesis that masting is stronger in species growing under limiting conditions and suggest that this reproductive behaviour might have evolved as an adaptation to nutrient limitations and imbalances.

16p11.2 Locus modulates response to satiety before the onset of obesity.

BACKGROUND: The 600 kb BP4-BP5 copy number variants (CNVs) at the 16p11.2 locus have been associated with a range of neurodevelopmental conditions including autism spectrum disorders and schizophrenia. The number of genomic copies in this region is inversely correlated with body mass index (BMI): the deletion is associated with a highly penetrant form of obesity (present in 50% of carriers by the age of 7 years and in 70% of adults), and the duplication with being underweight. Mechanisms underlying this energy imbalance remain unknown. OBJECTIVE: This study aims to investigate eating behavior, cognitive traits and their relationships with BMI in carriers of 16p11.2 CNVs. METHODS: We assessed individuals carrying a 16p11.2 deletion or duplication and their intrafamilial controls using food-related behavior questionnaires and cognitive measures. We also compared these carriers with cohorts of individuals presenting with obesity, binge eating disorder or bulimia. RESULTS: Response to satiety is gene dosage-dependent in pediatric CNV carriers. Altered satiety response is present in young deletion carriers before the onset of obesity. It remains altered in adolescent carriers and correlates with obesity. Adult deletion carriers exhibit eating behavior similar to that seen in a cohort of obesity without eating disorders such as bulimia or binge eating. None of the cognitive measures are associated with eating behavior or BMI. CONCLUSIONS: These findings suggest that abnormal satiety response is a strong contributor to the energy imbalance in 16p11.2 CNV carriers, and, akin to other genetic forms of obesity, altered satiety responsiveness in children precedes the increase in BMI observed later in adolescence.

Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Impact of effluent organic matter on low-pressure membrane fouling in tertiary treatment.

This study aims at comparing low-pressure membrane fouling obtained with two different secondary effluents at bench and pilot-scale based on the determination of two fouling indices: the total fouling index (TFI) and the hydraulically irreversible fouling index (HIFI). The main objective was to investigate if simpler and less costly bench-scale experimentation can substitute for pilot-scale trials when assessing the fouling potential of secondary effluent in large scale membrane filtration plants producing recycled water. Absolute values for specific flux and total fouling index for the bench-scale system were higher than those determined from pilot-scale, nevertheless a statistically significant correlation (r(2) = 0.63, α = 0.1) was obtained for the total fouling index at both scales. On the contrary no such correlation was found for the hydraulically irreversible fouling index. Advanced water characterization tools such as excitation-emission matrix fluorescence spectroscopy (EEM) and liquid chromatography with organic carbon detection (LC-OCD) were used for the characterization of foulants. On the basis of statistical analysis, biopolymers and humic substances were found to be the major contribution to total fouling (r(2) = 0.95 and r(2) = 0.88, respectively). Adsorption of the low molecular weight neutral compounds to the membrane was attributed to hydraulically irreversible fouling (r(2) = 0.67).

Ten persistent myths and the realities of membrane bioreactor technology for municipal applications.

Twelve years after the first full scale municipal application in Europe of membrane bioreactor (MBR) technology, the process is now accepted as a technology of choice for wastewater treatment, and the market is showing sustained growth. However early misconceptions about the technology are persistent and false statements are commonly encountered in articles and conferences, generating unnecessary research efforts or even fuelling either fascination or scepticism with regards to the technology, which is ultimately detrimental to the perception of the process by water professionals. We try to provide some factual and rational clarifications on ten issues which are often wrongly reported about MBR technology.

Performances and fouling control of a flat sheet membrane in a MBR pilot-plant.

This paper deals with the performance and the optimisation of the hydraulic operating conditions of the A3 Water Solutions flat sheet membrane technology in a MBR pilot-plant to achieve a satisfying fouling control and also a reduction in the required aeration. Two vertically stacked modules were tested at pilot-scale at Anjou Recherche under typical biological operating conditions (mixed liquor suspended solids concentration (MLSS) =10 g/l; sludge retention time (SRT) =28 days; food to microorganism ratio (F/M)=0.12 kg COD/kg MLSS/d). The use of a double-deck and of specific backwashes for this membrane technology enabled to achieve satisfying membrane performances for a net flux of 25 L h(-1) m(-2), 20 degrees C at a low specific aeration demand per membrane surface (SADm = 0.2 Nm(3) h(-1) m(-2)) which corresponds to a specific aeration demand per permeate volume unit (SADp) of 8 Nm(3) air/m(3) permeate, which is lower than reported for many commercial membrane systems. The mixed liquor characteristics (foaming, MLSS concentration) appeared to influence the fouling behaviour of the membranes but no correlation was found with the fouling rate. However, with the new operating conditions, the system is robust and can cope with fouling resulting from biological stress and daily peak flows for MLSS concentrations in the membrane tank up to 18 g/l.

Proteomes and transcriptomes of the Apicomplexa--where's the message?

The Apicomplexa have some of the most comprehensive and integrated proteome datasets of all pathogenic micro-organisms. Coverage is currently at a level where these data can be used to help predict the potential biological function of proteins in these parasites, without having to defer to measurement of mRNA levels. Transcriptomic data for the Apicomplexa (microarrays, expressed sequence tag (EST) collections, serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) tags) are also copious, enabling us to investigate the extent to which global mRNA levels correlate with proteomic data. Here, we present a proteomic and transcriptomic perspective of gene expression in key apicomplexan parasites, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum, Neospora caninum and Theileria spp., and discuss the alternative views of gene expression that they provide. Although proteomic evidence does not exist for every gene, many examples of readily detected proteins whose corresponding genes display little or no detectable transcription, are seen across the Apicomplexa. These examples are not easily explained by the "guilt by association", or "stock and go" hypotheses of gene transcription. With the advent of ultra-high-throughput sequencing technologies there will be a quantum shift in transcriptional analysis which, combined with improving quantitative proteome datasets, will provide a core component of a systems-wide approach to studying the Apicomplexa.

In the search of alternative cleaning solutions for MBR plants.

The objective of the study was to identify alternative cleaning reagents to chlorine for membrane permeability regeneration in MBR applications. Indeed, chlorine is prohibited in some countries because of the formation of by-products such as THM. The study was focused on the comparison of ten cleaning reagents performances and in particular on their ability to remove irreversible fouling. The tests were carried on with the A3 Water Solutions' Maxflow membrane (flat sheet membrane). A specific experimental protocol was defined at lab scale to develop an irreversible fouling by filtering sludge supernatant. The more promising reagents at lab scale were then tested on the A3 membrane continuously immersed in a MBR pilot plant functioning under typical biological conditions (MLSS=11 g/l; SRT=28 days). A full scale test was finally performed with hydrogen peroxide, one of the best reagents. Chlorine was taken as reference for all performed tests. The cleaning performances of the selected reagents were different at the different scales, probably due to the difficulty to obtain an irreversible membrane fouling at larger scales. This testing procedure will be reproduced with other membrane materials to have a better understanding of interactions between irreversible fouling, material nature and chemical reagents.

The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Evaluation of fluorenhymustine as a rationally designed novel anticancer agent.

AIM: To develop a rationally designed new nitrogen mustard namely Fluorenhymustine (compound 2), where N,N'-bis(2chloro-ethyl)amino group, the established anticancer functionality, is attached to the 2-ethyl fluorenone hydantoin moiety. MATERIALS AND METHODS: Starting from fluorenone hydantoin, a 3-step synthetic procedure was followed to obtain the title compound. 4-(4-Nitrobenzyl)pyridine was used to assess its chemical alkylating activity. Murine tumors (Ehrlich ascites carcinoma (EAC) and Sarcoma-180 (S-180)) were used to assess its in vivo activity. Its cytotoxicity was determined in vitro in MCF-7 human breast tumor cell line, toxicity - in vivo in normal and EAC bearing mice. 3H-Thymidine and 3H-Uridine were employed to study its inhibitory effect on DNA and RNA synthesis respectively in S-180 tumor cells in vitro. RESULTS: Alkylating activity of fluorenmustine exceeded that of N-di(2-chloroethyl)amine used as a standard alkylating compound. It has displayed an excellent and reproducible antitumor activity in vivo against EAC and S-180 comparable to that of 5-fluorouracil judging by the increase in median survival times of treated animals. It also significantly increased the life span of mice bearing advanced tumors for 6 days before the drug challenge. However in vitro screening in MCF-7 did not reveal any significant cytotoxicity. The compound did not adversely affect hematopoiesis at its optimum dose. Drug-induced hepatotoxicity and nephrotoxicity were also not detected. It inhibited the synthesis of DNA and RNA in S-180 tumor cells at 8 microM concentration. CONCLUSION: Results indicated promising chemotherapeutic potential of Fluorenhymustine.

Outcomes of a 2-year investigation on enhanced biological nutrients removal and trace organics elimination in membrane bioreactor (MBR).

Two configurations of membrane bioreactors were identified to achieve enhanced biological phosphorus and nitrogen removal, and assessed over more than two years with two parallel pilot plants of 2m3 each. Both configurations included an anaerobic zone ahead of the biological reactor, and differed by the position of the anoxic zone: standard pre-denitrification, or post-denitrification without dosing of carbon source. Both configurations achieved improved phosphorus removal. The goal of 50 microgP/L in the effluent could be consistently achieved with two types of municipal wastewater, the second site requiring a low dose of ferric salt ferric salt < 3 mgFe/L. The full potential of biological phosphorus removal could be demonstrated during phosphate spiking trials, where up to 1 mg of phosphorus was biologically eliminated for 10 mg BOD5 in the influent. The post-denitrification configuration enabled a very good elimination of nitrogen. Daily nitrate concentration as low as 1 mgN/L could be monitored in the effluent in some periods. The denitrification rates, greater than those expected for endogenous denitrification, could be accounted for by the use of the glycogene pool, internally stored by the denitrifying microorganisms in the anaerobic zone. Pharmaceuticals residues and steroids were regularly monitored on the two parallel MBR pilot plants during the length of the trials, and compared with the performance of the Berlin-Ruhleben WWTP. Although some compounds such as carbamazepine were persistent through all the systems, most of the compounds could be better removed by the MBR plants. The influence of temperature, sludge age and compound concentration could be shown, as well as the significance of biological mechanisms in the removal of trace organic compounds.

Evaluation of naphthalmustine, a nitrogen mustard derivative of naphthalimide as a rationally-designed anticancer agent.

Naphthalmustine, 2-[2-[bis-(2-chloroethyl)amino]ethyl]-1H-benz[de]isoquinoline-1,3-dione (Compound 1) has been synthesized as a rationally designed new anticancer agent from N-(2-bromoethyl)naphthalimide. Its chemical alkylating activity exceeded that of nor-HN2 used as standard compound for comparison. Its antitumour efficacy was assessed in vivo in two murine ascites tumours namely Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. The clinical drug cyclophosphamide and the experimental compound mitonafide were used as positive controls for comparison. Compound 1 has displayed substantial and reproducible antitumoural activity in these tumours since very high remission times of treated animals were observed. Significant increase in the life span of mice bearing highly advanced tumour for 10 days before the drug challenge was also noted after its treatment. Its LD50 value was 200 mg/Kg by single i.p. injection. Its toxicity was also assessed in vivo in normal and in S-180 bearing mice by measuring drug-induced changes in hematological parameters, femoral bone marrow and splenic cellularity sequentially on days 9, 15 and 21 following drug treatment at the optimum dose of 12 mg/kg from day 1 to 7. The results indicated that the compound did not adversely affect hematopoiesis. Drug-induced hepatotoxicity and nephrotoxicity were also evaluated on those days but no such toxicities were detected. Naphthalmustine inhibits the synthesis of DNA and RNA in S-180 tumour cells. It was further screened in vitro in 4 different human tumour cell lines but no significant activity was observed in those lines.

Synthesis and evaluation of substituted naphthalimide nitrogen mustards as rationally designed anticancer compounds.

Bromonapmustine 4a and chloronapmustine 4b, two new nitrogen mustards of substituted naphthalimides, have been synthesized as mixed-function anticancer compounds from 4-bromo- and 4-chloro-N-(2-hydroxyethyl)-naphthalimide respectively following a three-step process. Their chemical alkylating activity exceeded that of nor-HN2. Their antitumour efficacy were assessed in vivo in two murine ascites tumours, namely Ehrlich ascites carcinoma (EAC) and Sarcoma-180 (S-180) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. Two standard clinical drugs, namely endoxan (cyclophosphamide) and 5-fluorouracil (5-FU) were used as positive controls for comparison. Both of them have displayed substantial and reproducible antitumoural activity in these tumours comparable with 5-FU. These compounds inhibit the synthesis of DNA and RNA in S-180 tumour cells. These were further screened in vitro in 3 different human tumour cell lines but no significant activity was observed in those lines.

A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production.

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13.

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.