Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Report on a review of selected general and application controls over the Iowa Department of Human Services’ Targeted Case Management System (TCM) for the period March 31, 2014 through April 18, 2014

Report on a review of selected general and application controls over the Iowa Department of Human Services’ Targeted Case Management System (TCM) for the period March 31, 2014 through April 18, 2014

In pancreatic carcinoma, dual EGFR/HER2 targeting with cetuximab/trastuzumab is more effective than treatment with trastuzumab/erlotinib or lapatinib alone: implication of receptors' down-regulation and dimers' disruption.

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2(low) human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab')(2) fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

Health consultation : Allied Products Corporation Targeted Brownfields Assessment 13th Avenue and E Street, Charles City, Floyd County, Iowa (2005)

The Iowa Department of Natural Resources (IDNR) has requested the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program to evaluate environmental data collected at former farm equipment manufacturing facility located in Charles City, Iowa. The site, most recently operated by Allied Products Corporation, is a 70-acre site located at 13th Street and E Street in Charles City, Iowa (Figure 1). The site is undergoing a Targeted Brownfields Assessment conducted by the Contaminated Sites Section of the IDNR. This health consultation addresses potential health risks to people from future exposure to the soil within the property boundary, and any health impacts resulting from contaminated groundwater beneath the site property. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.

Health consultation : Chicago Milwaukee and St. Paul rail yard targeted brownields assessment, Spring Valley Township, Perry, Dallas County, Iowa (2007)

The Iowa Department of Natural Resources (IDNR) has requested the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program evaluate future health impacts of exposures at the formerly utilized Chicago Milwaukee St. Paul Rail Yard located on the west side of Perry, Iowa. This site has undergone a Targeted Brownfields Assessment conducted by the Contaminated Sites Section of the IDNR. This health consultation assesses potential health risks to people from future exposure to soil and surface water within the property boundary, and any health impacts resulting from contaminated groundwater beneath the site property from an evaluation of the data collected during the Targeted Brownfields Assessment. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.

Health consultation : IBP Inc./tire chop facility targeted brownfields assessment, 1525 "O" Avenue, Fort Dodge, Webster County, Iowa (2007)

The Iowa Department of Natural Resources (IDNR) has requested the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program to evaluate future health impacts of exposures at the formerly utilized IBP Inc./Tire Chop facility located at 1525 “O” Avenue, Fort Dodge, Iowa. This site has undergone a Targeted Brownfields Assessment conducted by the Contaminated Sites Section of the IDNR. This health consultation addresses potential health risks to people from future exposure to soil within the property boundary, and any health impacts resulting from contaminated groundwater beneath the site property from an evaluation of the data collected during the Targeted Brownfields Assessment. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Health consultation : Rolfe aboveground storage tank petroleum bulk plant targeted brownfields assessment, Railroad Street and 300th Avenue, Rolfe, Pocahontas County, Iowa (2005)

The Iowa Department of Natural Resources (IDNR) has requested the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program evaluate future health impacts of exposures at a former aboveground storage tank site located in Rolfe, Iowa. The former aboveground storage tank site is located to the southwest of the intersection of Railroad Street and 300th Avenue in Rolfe, Iowa. This site is undergoing a Targeted Brownfields Assessment conducted by the Contaminated Sites Section of the IDNR. This health consultation addresses potential health risks to people from future exposure to the soil within the property boundary, and any health impacts resulting from contaminated groundwater beneath the site property. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.

Stratégies destinées a optimiser l'utilisation des antibiotiques en réanimation [Strategies targeted at optimising antimicrobial therapy in critically ill patients]

Antibiotics are widely used in critical care and adequate empirical treatments has a significant impact on the outcome of many patients. Most nosocomial infections may be due to multidrug-resistant pathogens and requires empirical borad spectrum coverage before identification of the etiologic agents. This is associated with overuse of antibiotics which contributes to the further increase in multidrug-resistances. In this context, new strategies targeted at antibiotic control, such as guidelines and de-escalation are needed to control this evolution.

Multiplex targeted high-throughput sequencing for Mendelian cardiac disorders.

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.

Pédiatrie 5. Approche ciblée de l'ostéoporose chez l'enfant et l'adolescent [Targeted approach to osteoporosis for children and teenagers].

The osteoporosis of the child and the teenager is a pathological reality; its multifactorial pathogenesis often requires a collaborative approach and multidisciplinary. The osteoporosis characterized by a reduction in the bone mineral density is not a uniform pathology; it must be dealt with on all the levels by analyzing the factors of risks, by giving itself the diagnostic means and while insisting on the importance of a preventive approach as well as therapeutic.

Targeted sequence capture and Ultra High Throughput sequencing for gene discovery in inherited diseases

L'introduction des technologies de séquençage de nouvelle génération est en vue de révolutionner la médecine moderne. L'impact de ces nouveaux outils a déjà contribué à la découverte de nouveaux gènes et de voies cellulaires impliqués dans la pathologie de maladies génétiques rares ou communes. En revanche, l'énorme quantité de données générées par ces systèmes ainsi que la complexité des analyses bioinformatiques nécessaires, engendre un goulet d'étranglement pour résoudre les cas les plus difficiles. L'objectif de cette thèse a été d'identifier les causes génétiques de deux maladies héréditaires utilisant ces nouvelles techniques de séquençage, couplées à des technologies d'enrichissement de gènes. Dans ce cadre, nous avons développé notre propre méthode de travail (pipeline) pour l'alignement des fragments de séquence (reads). Suite à l'identification de gènes, nous avons réalisé une analyse fonctionnelle pour élucider leur rôle dans la maladie. Dans un premier temps, nous avons étudié et identifié des mutations impliquées dans une forme récessive de la rétinite pigmentaire qui est à ce jour la dégénérescence rétinienne héréditaire la plus fréquente. En particulier, nous avons constaté que des mutations faux-sens dans le gène FAM161A étaient la cause de la rétinite pigmentaire préalablement associé avec le locus RP28. De plus, nous avons démontré que ce gène avait des fonctions au niveau du cil du photorécepteur, complétant le large spectre des cilliopathies rétiniennes héréditaires. Dans un second temps, nous avons exploré la possibilité qu'un syndrome, relativement fréquent en pédiatrie de fièvre récurrente, appelé PFAPA (acronyme de fièvre périodique avec adénite stomatite, pharyngite et cervical aphteuse) puisse avoir une origine génétique. L'étiologie de cette maladie n'étant pas claire, nous avons tenté d'identifier le spectre génétique de patients PFAPA. Comme nous n'avons pas pu mettre à jour un nouveau gène unique muté et responsable de la maladie chez tous les individus dépistés, il semblerait qu'un modèle génétique plus complexe suggérant l'implication de plusieurs gènes dans la pathologie ait été identifié chez les patients touchés. Ces gènes seraient notamment impliqués dans des processus liés à l'inflammation ce qui élargirait l'impact de ces études à d'autres maladies auto-inflammatoires.

Targeted therapy against multi-resistant bacteria in leukemic and hematopoietic stem cell transplant recipients: guidelines of the 4th European Conference on Infections in Leukemia (ECIL-4, 2011).

The detection of multi-resistant bacterial pathogens, particularly those to carbapenemases, in leukemic and stem cell transplant patients forces the use of old or non-conventional agents as the only remaining treatment options. These include colistin/polymyxin B, tigecycline, fosfomycin and various anti-gram-positive agents. Data on the use of these agents in leukemic patients are scanty, with only linezolid subjected to formal trials. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for their use in these patient populations. Targeted therapy should be based on (i) in vitro susceptibility data, (ii) knowledge of the best treatment option against the particular species or phenotype of bacteria, (iii) pharmacokinetic/pharmacodynamic data, and (iv) careful assessment of the risk-benefit balance. For infections due to resistant Gram-negative bacteria, these agents should be preferably used in combination with other agents that remain active in vitro, because of suboptimal efficacy (e.g., tigecycline) and the risk of emergent resistance (e.g., fosfomycin). The paucity of new antibacterial drugs in the near future should lead us to limit the use of these drugs to situations where no alternative exists.