Recent genetic exchanges and admixture shape the genome and population structure of the zoonotic pathogen Cryptosporidium parvum.

Cryptosporidium parvum is a globally distributed zoonotic pathogen and a major cause of diarrhoeal disease in humans and ruminants. The parasite's life cycle comprises an obligatory sexual phase, during which genetic exchanges can occur between previously isolated lineages. Here, we compare 32 whole genome sequences from human- and ruminant-derived parasite isolates collected across Europe, Egypt and China. We identify three strongly supported clusters that comprise a mix of isolates from different host species, geographic origins, and subtypes. We show that: (1) recombination occurs between ruminant isolates into human isolates; (2) these recombinant regions can be passed on to other human subtypes through gene flow and population admixture; (3) there have been multiple genetic exchanges, and most are probably recent; (4) putative virulence genes are significantly enriched within these genetic exchanges, and (5) this results in an increase in their nucleotide diversity. We carefully dissect the phylogenetic sequence of two genetic exchanges, illustrating the long-term evolutionary consequences of these events. Our results suggest that increased globalization and close human-animal contacts increase the opportunity for genetic exchanges between previously isolated parasite lineages, resulting in spillover and spillback events. We discuss how this can provide a novel substrate for natural selection at genes involved in host-parasite interactions, thereby potentially altering the dynamic coevolutionary equilibrium in the Red Queens arms race.

Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates.

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/-2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

Mining Public Metagenomes for Environmental Surveillance of Parasites: A Proof of Principle.

Parasites often have complex developmental cycles that account for their presence in a variety of difficult-to-analyze matrices, including feces, water, soil, and food. Detection of parasites in these matrices still involves laborious methods. Untargeted sequencing of nucleic acids extracted from those matrices in metagenomic projects may represent an attractive alternative method for unbiased detection of these pathogens. Here, we show how publicly available metagenomic datasets can be mined to detect parasite specific sequences, and generate data useful for environmental surveillance. We use the protozoan parasite Cryptosporidium parvum as a test organism, and show that detection is influenced by the reference sequence chosen. Indeed, the use of the whole genome yields high sensitivity but low specificity, whereas specificity is improved through the use of signature sequences. In conclusion, querying metagenomic datasets for parasites is feasible and relevant, but requires optimization and validation. Nevertheless, this approach provides access to the large, and rapidly increasing, number of datasets from metagenomic and meta-transcriptomic studies, allowing unlocking hitherto idle signals of parasites in our environments.

Antisense Transcription in Loci Associated to Hereditary Neurodegenerative Diseases.

Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples.

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeqTM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeqTM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics.

Effects of Pin1 Loss in Hdh(Q111) Knock-in Mice.

Huntington's disease (HD) is a fatal, dominantly inherited, neurodegenerative disorder due to a pathological expansion of the CAG repeat in the coding region of the HTT gene. In the quest for understanding the molecular basis of neurodegeneration, we have previously demonstrated that the prolyl isomerase Pin1 plays a crucial role in mediating p53-dependent apoptosis triggered by mutant huntingtin (mHtt) in vitro. To assess the effects of the lack of Pin1 in vivo, we have bred Pin1 knock-out mice with Hdh(Q111) knock-in mice, a genetically precise model of HD. We show that Pin1 genetic ablation modifies a portion of Hdh(Q111) phenotypes in a time-dependent fashion. As an early event, Pin1 activity reduces the DNA damage response (DDR). In midlife mice, by taking advantage of next-generation sequencing technology, we show that Pin1 activity modulates a portion of the alterations triggered by mHtt, extending the role of Pin1 to two additional Hdh(Q111) phenotypes: the unbalance in the "synthesis/concentration of hormones", as well as the alteration of "Wnt/ß-catenin signaling". In aging animals, Pin1 significantly increases the number of mHtt-positive nuclear inclusions while it reduces gliosis. In summary, this work provides further support for a role of Pin1 in HD pathogenesis.

Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

RNA-Seq mapping and detection of gene fusions with a suffix array algorithm.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.

Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss.

BACKGROUND: The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. RESULTS: To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. CONCLUSIONS: Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides an example of a group of bacteria that appears to evolve through a loss of virulence rather than acquisition of virulence characteristics. While Listeria includes a number of species-like clades, many of these putative species include clades or strains with atypical virulence associated characteristics. This information will allow for the development of genetic and genomic criteria for pathogenic strains, including development of assays that specifically detect pathogenic Listeria strains.

The lymphoid cell surface receptor NTB-A: a novel monoclonal antibody target for leukaemia and lymphoma therapeutics.

NTB-A is a CD2-related cell surface protein expressed primarily on lymphoid cells including B-lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB-A and assessed their therapeutic potential for CLL. Selective mAbs to NTB-A were further tested in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB-A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB-A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB-A mAbs demonstrated anti-tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB-A as a potential new target for immunotherapy of leukaemia and lymphomas.

IGFL: A secreted family with conserved cysteine residues and similarities to the IGF superfamily.

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus. The IGF-like (IGFL) genes encode proteins of approximately 100 amino acids that contain 11 conserved cysteine residues at fixed positions, including two CC motifs. In H. sapiens, the family is composed of four genes and two pseudogenes that are referred as IGFL1 to IGFL4 and IGFL1P1 and IGFL1P2, respectively. Human IGFL genes are clustered together on chromosome 19 within a 35-kb interval. M. musculus has a single IGFL family member that is located on chromosome 7. Further, evolutionary analysis shows a lack of direct orthology between any of the four human members and the mouse gene. This relationship between the mouse and the human family members suggests that the multiple members in the human complement have arisen from recent duplication events that appear limited to the primate lineage. Structural considerations and sequence comparisons would suggest that IGFL proteins are distantly related to the IGF superfamily of growth factors. IGFL mRNAs display specific expression patterns; they are expressed in fetal tissues, breast, and prostate, and in many cancers as well, and this pattern is consistent with that of the IGF family members.

Synthesis of cell-permeable peptide nucleic acids and characterization of their hybridization and uptake properties.

Guanidine-based peptide nucleic acid (GPNA) monomers and oligomers containing all four natural (adenine (A), cytosine (C), guanine (G), and thymine (T)) and two unnatural (2-thiouracil (sU) and 2,6-diaminopurine (D)) nucleobases have been synthesized. Thermal denaturation study showed that GPNA oligomers containing alternate D-backbone configuration bind sequence-specifically to DNA and, when incubated with mammalian cells, localized specifically to the endoplasmic reticulum (ER).