Fine mapping of loci on BTA8 associated to antibody response to Mycobacterium avium paratuberculosis in cattle

Paratuberculosis (ParaTB) or Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis commonly known as MAP in cattle, is a chronic gastroenteritis characterized by diarrhoea, decreased milk production and ultimately death. MAP is responsible for huge economic losses, particularly in dairy cattle herds. Susceptibility to MAP infection has been found to be heritable with heritability estimates ranging from 0.06 to 0.102. The definition of an infected animal can be based either on the presence of anti-MAP antibodies in the serum, or by direct demonstration of MAP in tissue or faeces by culture or PCR. Several studies have addressed the identification of genetic loci associated with MAP susceptibility. The objective of this study was to refine a locus associated with antibody response to Mycobacterium avium paratuberculosi (MAP). Using a genome- wide association analysis, a single nucleotide polymorphism on Bos taurus autosome BTA8 namely the SNP rs43161947 at posi- tion 35398490 with a p-value of 7.02 e-05, has previously been identified by the authors as associated with MAP infection. Fine mapping of the region was conducted with 100 single nucleotide polymorphisms spanning a region between BTA8: 34422912 and BTA8: 364553881 covering 2 Mega bases (Mb) designed in to cover 1 Mb ahead and after the SNP identified on BTA8. The 2 Mb region on BTA8 was evaluated within a group of 966 Holstein cows collected from routine ParaTB screening in the province of Lodi in Italy, in an area with a high prevalence of ParaTB. Animals were defined as ParaTB positive based on the detection of serum antibodies produced in response to MAP infection using the ID-screen\uae ELISA test (ID VET Montpellier, France). Of the 966 samples, 483 were MAP antibody positive (cases) and 483 MAP antibody negative (MAP negative controls). All animals were female, and cases and MAP negative controls were from the same farm tested on the same day.Using a single marker associ- ation analysis, conducted within the R statistical environment, we identified 3 different QTLs within the 2 Mega base region, under the main QTL on BTA8 associated with antibody response to MAP, in position 34.700.000, 35.800.000 and 36.400.000 bp. This reveals the complexity of the genetic architecture of thetrait and confirms the need to further explore the genome with fine mapping approaches, or by the use of whole genome sequencing to investigate complex traits, such as disease resistance

Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica

Background: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. Methodology/Principal Findings: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. Conclusions/Significance: These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases. © 2013 Corvo et al

Loco-regional adjuvant radiation therapy in breast cancer patients with positive axillary lymph-nodes at diagnosis (CN2) undergoing preoperative chemotherapy and with complete pathological lymph-nodes response. Development of GRADE (Grades of recommendation, assessment, Development and Evaluation) recommendation by the Italian Association of radiation therapy and Clinical Oncology (AIRO)

Objective: To perform a meta-analysis to determine the effect of loco-regional radiation therapy (RT) compared to no loco-regional RT for operated patients in clinical stage cN2 breast cancer at diagnosis and ypN0 after preoperative chemotherapy (PST). Material and Methods: Eligible studies were identified through a systematic search of the medical literature performed independently by two researchers using a validated search strategy. An electronic search of Medline via PubMed and Embase (Breast cancer AND preoperative chemotherapy AND radiation therapy) was conducted with no language or publication status restrictions. The effect of loco-regional RT on overall (OS), disease free (DFS), loco-regional recurrence-free (LRRFS) survival and local recurrence was evaluated. An electronic search of Medline via PubMed and Embase (Toxicity AND radiation therapy breast cancer AND preoperative therapy; toxicity AND breast surgery AND preoperative chemotherapy) was conducted for outcomes of harm: major acute and late skin toxicity, lymphedema and cardiac events. Results: Of 333 studies identified, 4 retrospective studies reporting on a total of 1107 patients were included in the meta-analysis. Six and 3 reported data of acute and late skin toxicity, while 2 studies provided information on cardiac events. Pooled results showed no difference in terms of hazard ratio for loco-regional RT versus no loco-regional RT [hazard ratio (HR) = 0.82, 95% confidence interval (CI) 0.63\u20131.68]. Loco-regional RT was associated with an OS benefit in the subgroup analysis: IIIB-C (loco-regional RT 79.3% vs no loco-regional RT 71.2%, p = 0.027) and T3-T4 (loco-regional RT 82.6% vs no loco-regional RT 76.6%, p = 0.025). No difference was shown in terms of 5-year DFS (loco-regional RT 91.2% vs no loco-regional RT 83%, p = 0.441) and LRRFS (loco-regional RT 98.1% vs no loco-regional RT 92.3%, p = 0.148). There was no significant difference between the groups in terms of acute and late skin toxicities, lymphedema and cardiac events. Conclusions: Because of the limitations due to the small number of studies and heterogeneity in the analysis, the present study does not allow to draw any definitive conclusion, highlighting the need for well-controlled trials to determine the effect of loco-regional RT in patients with cN2 having a pathological complete response in the axillary nodes after preoperative chemotherapy

Distributed Computing Grid Experiences in CMS

The CMS experiment is currently developing a computing system capable of serving, processing and archiving the large number of events that will be generated when the CMS detector starts taking data. During 2004 CMS undertook a large scale data challenge to demonstrate the ability of the CMS computing system to cope with a sustained data-taking rate equivalent to 25% of startup rate. Its goals were: to run CMS event reconstruction at CERN for a sustained period at 25 Hz input rate; to distribute the data to several regional centers; and enable data access at those centers for analysis. Grid middleware was utilized to help complete all aspects of the challenge. To continue to provide scalable access from anywhere in the world to the data, CMS is developing a layer of software that uses Grid tools to gain access to data and resources, and that aims to provide physicists with a user friendly interface for submitting their analysis jobs. This paper describes the data challenge experience with Grid infrastructure and the current development of the CMS analysis system

Preclinical Studies in Anti-Trypanosomatidae Drug Development

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 μM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity

Insights in the genome of mycobacterium avium subsp. paratuberculosis by Next Generation Sequencing approaches

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis - or Johne's disease \u2013 that affects farmed and wild animals worldwide, causing negative economic consequences particularly relevant in the livestock sector of dairy cattle and beef (1). Recent estimates say that more than 50 % of the herds in Europe and North America are infected (3). In Italy, a study conducted in the Lombardy and Veneto regions reveals that about 70 % of dairy herds are infected (2). The disease shows high variability in the progression and symptoms that may be due to the genetic variability of the host, the pathogen, or a combination of the two. Understanding the mechanism responsible of this variability could be of paramount importance for the control of the disease (1, 3). Aim of this work was to study the genomic variability of MAP isolated from dairy cattle from different farms distributed in several Italian regions through the use of Next Generation Sequencing (NGS) techniques. The preliminary results on 15 strains are presented

VarGoats project: a dataset of 1159 whole-genome sequences to dissect Capra hircus global diversity

Background: Since their domestication 10,500 years ago, goat populations with distinctive genetic backgrounds have adapted to a broad variety of environments and breeding conditions. The VarGoats project is an international 1000-genome resequencing program designed to understand the consequences of domestication and breeding on the genetic diversity of domestic goats and to elucidate how speciation and hybridization have modeled the genomes of a set of species representative of the genus Capra. Findings: A dataset comprising 652 sequenced goats and 507 public goat sequences, including 35 animals representing eight wild species, has been collected worldwide. We identified 74,274,427 single nucleotide polymorphisms (SNPs) and 13,607,850 insertion-deletions (InDels) by aligning these sequences to the latest version of the goat reference genome (ARS1). A Neighbor-joining tree based on Reynolds genetic distances showed that goats from Africa, Asia and Europe tend to group into independent clusters. Because goat breeds from Oceania and Caribbean (Creole) all derive from imported animals, they are distributed along the tree according to their ancestral geographic origin. Conclusions: We report on an unprecedented international effort to characterize the genome-wide diversity of domestic goats. This large range of sequenced individuals represents a unique opportunity to ascertain how the demographic and selection processes associated with post-domestication history have shaped the diversity of this species. Data generated for the project will also be extremely useful to identify deleterious mutations and polymorphisms with causal effects on complex traits, and thus will contribute to new knowledge that could be used in genomic prediction and genome-wide association studies

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP’s Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

Observation of two new Ξb−\Xi_b^- baryon resonances

Two structures are observed close to the kinematic threshold in the $\Xi_b^0 \pi^-$ mass spectrum in a sample of proton-proton collision data, corresponding to an integrated luminosity of 3.0 fb$^{-1}$ recorded by the LHCb experiment. In the quark model, two baryonic resonances with quark content $bds$ are expected in this mass region: the spin-parity $J^P = \frac{1}{2}^+$ and $J^P=\frac{3}{2}^+$ states, denoted $\Xi_b^{\prime -}$ and $\Xi_b^{*-}$. Interpreting the structures as these resonances, we measure the mass differences and the width of the heavier state to be $m(\Xi_b^{\prime -}) - m(\Xi_b^0) - m(\pi^{-}) = 3.653 \pm 0.018 \pm 0.006$ MeV$/c^2$, $m(\Xi_b^{*-}) - m(\Xi_b^0) - m(\pi^{-}) = 23.96 \pm 0.12 \pm 0.06$ MeV$/c^2$, $\Gamma(\Xi_b^{*-}) = 1.65 \pm 0.31 \pm 0.10$ MeV, where the first and second uncertainties are statistical and systematic, respectively. The width of the lighter state is consistent with zero, and we place an upper limit of $\Gamma(\Xi_b^{\prime -}) < 0.08$ MeV at 95% confidence level. Relative production rates of these states are also reported.Comment: 17 pages, 2 figure