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Background: Plitidepsin has shown potent preclinical activity against severe acute respiratory syndrome coronavirus 2 and was generally well tolerated in a phase I trial of hospitalized patients with coronavirus disease 2019 (COVID-19). NEPTUNO, a phase III, multicenter, randomized, controlled trial, was designed to evaluate the efficacy and safety of plitidepsin in the management of moderate COVID-19 in hospitalized adult patients., Methods: Included patients had documented severe acute respiratory syndrome coronavirus 2 infection, required oxygen therapy, and had adequate organ function. The planned sample size was 609 patients. Patients were randomized 1:1:1 to at least 3 days of dexamethasone plus either plitidepsin (1.5 mg/day or 2.5 mg/day, for 3 days) or standard of care (control). The primary endpoint was the time to sustained withdrawal of supplemental oxygen. Secondary endpoints included time to sustained hospital discharge, clinical status, duration of oxygen support, percentage of patients requiring admission to the intensive care unit, and safety., Results: After randomizing 205 patients, NEPTUNO was discontinued due to a notable drop in COVID-19-related hospitalizations. Available data suggest a 2-day improvement in the median time to sustained oxygen therapy discontinuation (5 vs 7 days) favoring both plitidepsin arms (hazard ratio, 1.37; 95% confidence interval, .96-1.96; P = .08 for plitidepsin 1.5 mg vs control; hazard ratio, 1.06; 95% confidence interval, .73-1.53; P = .78 for plitidepsin 2.5 mg vs control). Plitidepsin was generally well tolerated., Conclusions: Despite the trial limitations, these results suggest that plitidepsin may have a positive benefit-risk ratio in the management of patients requiring oxygen therapy. Further studies with plitidepsin, including those in immunosuppressed patients, are warranted.Results from this phase III trial suggest that plitidepsin, a first-in-class antiviral, may have a positive benefit-risk ratio in the management of hospitalized patients requiring oxygen therapy for moderate COVID-19., Competing Interests: Potential conflicts of interest. V. E. has received economic support for educational talks and advisory boards from Gilead Sciences and PharmaMar SA. M. M-M. has received grants and fees for scientific advice not related with this work; received in the past 36 months from Boehringer Ing, Roche, Ferrer, Chiesi. P. V. has received provision of study materials, travel support travel from PharmaMar SA, and payments from PharmaMar SA and GlaxoSmithKline (GSK). P. G-V. received speaker fees from FLS Science, PharmaMar SA (Madrid, Spain), Pfizer (Spain), and GSK (Spain); consulting fees from Angelini Pharma and PharmaMar SA; served as an advisory board member for Berlin Cures GmbH and PharmaMar SA; and received meeting grants from GSK and PharmaMar SA. A. L. has received occasional training fees from Gilead, is a member of a Data Monitoring Committee for a randomized controlled trial for Biophytis Biopharma. R. P. has received grants or contracts from MSD, ViiV Healthcare; consulting fees Pfizer, Gilead, AstraZeneca, PharmaMar SA, GSK, Atea. G. P. has received fees, as member advisory panels and consultancy, from Pfizer, MSD, Menarini, and Gilead. M. M. has received fees from Boehringer as scientific advisor and payments for lectures and educational events from GSK. J. F. has received grants and payments from Gilead, Pfizer, Shionogy, Astellas, and MSD, as well as travel support. P. R. has received fees and travel support from Gilead, Merck, VIV, and Gesida. D. L-M., E-M. S-L, F. M., J. G., and N. T. are a full-time employees of PharmaMar. J. J. holds stocks of Pangaea Oncology, has a nonremunerated role in the Scientific Advisory Board, and holds stocks of Promontory Therapeutics; is a full-time employee and shareholder of PharmaMar, S.A. (Madrid, Spain), and a co-inventor of 2 patents for Plitidepsin (WO99-42125). J. A. L.-M. is a full-time employee and shareholder of PharmaMar, S.A (Madrid, Spain), and a co-inventor of a patent for plitidepsin (WO2008135793A1). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: T-cell receptor (TCR-T) therapies are based on the expression of an introduced TCR targeting a tumor associated antigen (TAA) which has been studied in several trials in cutaneous melanoma. We conducted a systematic review and meta-analysis aiming to assess the primary efficacy of TCR-based adoptive cell therapy in cutaneous melanoma., Methods: We searched through PubMed electronic database from its inception until May 21, 2022. Primary endpoints were pooled objective response rate (ORR) and disease control rate (DCR). We conducted logistic regression analyses to identify potential predictive factors for tumor response., Results: From 187 patients, 50 showed an objective response (pooled ORR 28%; 95% CI, 20%-37%) and a pooled DCR of 38% (95% CI, 27%-50%). Median PFS was 2, 9 months (95% CI, 1.4-3.1). A trend toward higher PFS was demonstrated for patients treated with cancer/testis antigens targeting TCR-T cells (HR 0.91 95% CI, 0.64-1.3, P = .61) among whom, patients treated with NYESO-1 targeting TCR-T showed a significantly higher PFS (HR 0.63 95% CI, 0.64-0.98, P = .03). In addition, the number of infused cells was associated with a significantly higher likelihood of tumor response (OR 6.61; 95% CI, 1.68-21.6; P = .007)., Conclusion: TCR-T therapy shows promising results in terms of antitumor activity and survival similar to those reported for TILs with a significantly higher benefit for cancer/testis antigens targeting cells. Since TCR-based therapy shows advantages of great potential over classic ACT strategies, further research in solid cancers is warranted (PROSPERO ID CRD42022328011)., (© The Author(s) 2023. Published by Oxford University Press.)

To better understand the potential of drug repurposing in COVID-19, we analyzed control strategies over essential host factors for SARS-CoV-2 infection. We constructed comprehensive directed protein-protein interaction (PPI) networks integrating the top-ranked host factors, the drug target proteins and directed PPI data. We analyzed the networks to identify drug targets and combinations thereof that offer efficient control over the host factors. We validated our findings against clinical studies data and bioinformatics studies. Our method offers a new insight into the molecular details of the disease and into potentially new therapy targets for it. Our approach for drug repurposing is significant beyond COVID-19 and may be applied also to other diseases., (© The Author(s) 2021. Published by Oxford University Press.)

Objective: Frailty is a prevalent risk factor for adverse outcomes among patients with chronic lung disease. However, identifying frail patients who may benefit from interventions is challenging using standard data sources. We therefore sought to identify phrases in clinical notes in the electronic health record (EHR) that describe actionable frailty syndromes., Materials and Methods: We used an active learning strategy to select notes from the EHR and annotated each sentence for 4 actionable aspects of frailty: respiratory impairment, musculoskeletal problems, fall risk, and nutritional deficiencies. We compared the performance of regression, tree-based, and neural network models to predict the labels for each sentence. We evaluated performance with the scaled Brier score (SBS), where 1 is perfect and 0 is uninformative, and the positive predictive value (PPV)., Results: We manually annotated 155 952 sentences from 326 patients. Elastic net regression had the best performance across all 4 frailty aspects (SBS 0.52, 95% confidence interval [CI] 0.49-0.54) followed by random forests (SBS 0.49, 95% CI 0.47-0.51), and multi-task neural networks (SBS 0.39, 95% CI 0.37-0.42). For the elastic net model, the PPV for identifying the presence of respiratory impairment was 54.8% (95% CI 53.3%-56.6%) at a sensitivity of 80%., Discussion: Classification models using EHR notes can effectively identify actionable aspects of frailty among patients living with chronic lung disease. Regression performed better than random forest and neural network models., Conclusions: NLP-based models offer promising support to population health management programs that seek to identify and refer community-dwelling patients with frailty for evidence-based interventions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Novel tools for in silico design of RNA constructs such as riboregulators are required in order to reduce time and cost to production for the development of diagnostic and therapeutic advances. Here, we present MoiRNAiFold, a versatile and user-friendly tool for de novo synthetic RNA design. MoiRNAiFold is based on Constraint Programming and it includes novel variable types, heuristics and restart strategies for Large Neighborhood Search. Moreover, this software can handle dozens of design constraints and quality measures and improves features for RNA regulation control of gene expression, such as Translation Efficiency calculation. We demonstrate that MoiRNAiFold outperforms any previous software in benchmarking structural RNA puzzles from EteRNA. Importantly, with regard to biologically relevant RNA designs, we focus on RNA riboregulators, demonstrating that the designed RNA sequences are functional both in vitro and in vivo. Overall, we have generated a powerful tool for de novo complex RNA design that we make freely available as a web server (https://moiraibiodesign.com/design/)., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The environmental fate of many functional molecules that are produced on a large scale as precursors or as additives to specialty goods (plastics, fibers, construction materials, etc.), let alone those synthesized by the pharmaceutical industry, is generally unknown. Assessing their environmental fate is crucial when taking decisions on the manufacturing, handling, usage, and release of these substances, as is the evaluation of their toxicity in humans and other higher organisms. While this data are often hard to come by, the experimental data already available on the biodegradability and toxicity of many unusual compounds (including genuinely xenobiotic molecules) make it possible to develop machine learning systems to predict these features. As such, we have created a predictor of the "risk" associated with the use and release of any chemical. This new system merges computational methods to predict biodegradability with others that assess biological toxicity. The combined platform, named BiodegPred (https://sysbiol.cnb.csic.es/BiodegPred/), provides an informed prognosis of the chance a given molecule can eventually be catabolized in the biosphere, as well as of its eventual toxicity, all available through a simple web interface. While the platform described does not give much information about specific degradation kinetics or particular biodegradation pathways, BiodegPred has been instrumental in anticipating the probable behavior of a large number of new molecules (e.g. antiviral compounds) for which no biodegradation data previously existed., (© The Author(s) 2020. Published by Oxford University Press.)

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Individuals may reduce competition by temporally partitioning their use of a shared resource. Behavioral differences between sexes in ungulates may encourage segregation as individuals attempt to avoid antagonistic interactions. However, dominant sex and age groups may reduce subordinates' access to food resources, regardless of the subordinate's sex. We hypothesized that white-tailed deer Odocoileus virginianus temporally segregated at supplemental feeding sites based on social rank (subordinate: yearling males and adult females; dominant: adult males) and that segregation was affected by phase of the breeding season and diel cycle. If deer temporally segregate according to social rank, we predicted that the resulting activity patterns would manifest in one social class being relatively more susceptible to hunter-induced mortality. We used a multi-state modeling approach to quantify temporal segregation and calculated the probability that a feeding site was in a particular state during diurnal and nocturnal hours for each of the 3 phases of the breeding season. We determined that transition probabilities differed by season and diel cycle and dominant and subordinate social classes clearly avoided each other, with <1% co-occurrence at feeding sites. During the pre-breeding season, the probability of a subordinate being present during diurnal hours was 3.0 × more likely than a dominant being present, but did not differ during nocturnal hours. There was no difference for dominants and subordinates during diurnal or nocturnal hours during the breeding season. In the post-breeding season, subordinates were 1.7 × more likely to occur at the feeding site than a dominant during diurnal hours but they did not differ during nocturnal hours. Our results indicate that dominance status influences temporal segregation at feeding sites and is affected by the phase of the breeding season. Therefore, the resulting activity patterns may increase subordinates' risk to human predation during the pre-breeding and post-breeding seasons.

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major pest of pome fruit worldwide. Incorporation of semiochemicals, including the main sex pheromone (codlemone), into codling moth IPM programs has drastically reduced the amount of chemical insecticides needed to control this orchard pest. Odorant receptors located in sensory neuron membranes in the antennae are key sensors in the detection of semiochemicals and trigger downstream signaling events leading to a behavioral response. CpomOR1 is an odorant receptor belonging to the pheromone receptor subfamily in codling moth, and is a prime candidate for being a codlemone receptor based on its high expression levels in male antennae. In this study, the CpomOR1 gene was targeted using CRISPR/Cas9 genome editing to knockdown functional OR1 protein production to determine physiological function(s). By injecting early stage eggs, mutations were successfully introduced, including both deletions and insertions. When attempting to create stable populations of codling moth through mating of males with females containing mutations of the CpomOR1 gene, it was found that fecundity and fertility were affected, with edited females producing nonviable eggs. The role of CpomOR1 in fecundity and fertility in codling moth is unknown and will be the focus of future studies., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.)

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake ( Oryza sativa ssp japonica ), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations., (© 2017 American Society of Plant Biologists. All rights reserved.)

Motivation: RNA thermometers (RNATs) are cis-regulatory elements that change secondary structure upon temperature shift. Often involved in the regulation of heat shock, cold shock and virulence genes, RNATs constitute an interesting potential resource in synthetic biology, where engineered RNATs could prove to be useful tools in biosensors and conditional gene regulation., Results: Solving the 2-temperature inverse folding problem is critical for RNAT engineering. Here we introduce RNAiFold2T, the first Constraint Programming (CP) and Large Neighborhood Search (LNS) algorithms to solve this problem. Benchmarking tests of RNAiFold2T against existent programs (adaptive walk and genetic algorithm) inverse folding show that our software generates two orders of magnitude more solutions, thus allowing ample exploration of the space of solutions. Subsequently, solutions can be prioritized by computing various measures, including probability of target structure in the ensemble, melting temperature, etc. Using this strategy, we rationally designed two thermosensor internal ribosome entry site (thermo-IRES) elements, whose normalized cap-independent translation efficiency is approximately 50% greater at 42 °C than 30 °C, when tested in reticulocyte lysates. Translation efficiency is lower than that of the wild-type IRES element, which on the other hand is fully resistant to temperature shift-up. This appears to be the first purely computational design of functional RNA thermoswitches, and certainly the first purely computational design of functional thermo-IRES elements., Availability: RNAiFold2T is publicly available as part of the new release RNAiFold3.0 at https://github.com/clotelab/RNAiFold and http://bioinformatics.bc.edu/clotelab/RNAiFold, which latter has a web server as well. The software is written in C ++ and uses OR-Tools CP search engine., Contact: clote@bc.edu, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press.)

Glucagon-like peptide-1 (GLP-1) receptor agonists, used for the treatment of type 2 diabetes, have caused hyperplasia/neoplasia of thyroid C cells in rodent carcinogenicity studies. Studies in monkeys have not identified an effect of GLP-1 receptor agonists on thyroid C cells; however, group sizes were small. Dulaglutide is a once-weekly, long-acting human GLP-1 receptor agonist recently approved in the United States and the European Union. The objective of this study was to determine whether dulaglutide altered C-cell mass in monkeys. Male cynomolgus monkeys (20 per group) were sc injected with dulaglutide 8.15 mg/kg (∼500-fold maximum human plasma exposure) or a vehicle control twice weekly for 52 weeks. Basal and calcium gluconate-stimulated serum calcitonin concentrations were obtained at 3, 6, 9, and 12 months. Thyroid glands were weighed, fixed, and sectioned at 500-μm intervals. C-cell volumes were measured using an automated image analysis. C-cell proliferation was estimated using Ki67/calcitonin colabeling and cell counting. Administration of dulaglutide 8.15 mg/kg twice weekly for 52 weeks did not increase serum calcitonin in monkeys or affect thyroid weight, histology, C-cell proliferation, or absolute/relative C-cell volume. This study represents a comprehensive evaluation of the monkey thyroid C cells after dosing with a GLP-1 receptor agonist, with a large group size, and measurement of multiple relevant parameters. The lack of effect of dulaglutide on C cells is consistent with other studies in monkeys using GLP-1 receptor agonists and suggests that nonhuman primates are less sensitive than rodents to the induction of proliferative changes in thyroid C cells by GLP-1 receptor agonists.

The tumorigenic potential of dulaglutide was evaluated in rats and transgenic mice. Rats were injected sc twice weekly for 93 weeks with dulaglutide 0, 0.05, 0.5, 1.5, or 5 mg/kg corresponding to 0, 0.5, 7, 20, and 58 times, respectively, the maximum recommended human dose based on plasma area under the curve. Transgenic mice were dosed sc twice weekly with dulaglutide 0, 0.3, 1, or 3 mg/kg for 26 weeks. Dulaglutide effects were limited to the thyroid C-cells. In rats, diffuse C-cell hyperplasia and adenomas were statistically increased at 0.5 mg/kg or greater (P ≤ .01 at 5 mg/kg), and C-cell carcinomas were numerically increased at 5 mg/kg. Focal C-cell hyperplasia was higher compared with controls in females given 0.5, 1.5, and 5 mg/kg. In transgenic mice, no dulaglutide-related C-cell hyperplasia or neoplasia was observed at any dose; however, minimal cytoplasmic hypertrophy of C cells was observed in all dulaglutide groups. Systemic exposures decreased over time in mice, possibly due to an antidrug antibody response. In a 52-week study designed to quantitate C-cell mass and plasma calcitonin responses, rats received twice-weekly sc injections of dulaglutide 0 or 5 mg/kg. Dulaglutide increased focal C-cell hyperplasia; however, quantitative increases in C-cell mass did not occur. Consistent with the lack of morphometric changes in C-cell mass, dulaglutide did not affect the incidence of diffuse C-cell hyperplasia or basal or calcium-stimulated plasma calcitonin, suggesting that diffuse increases in C-cell mass did not occur during the initial 52 weeks of the rat carcinogenicity study.

Unlabelled: Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules., Availability: the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic by products from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore,genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment—two stresses relevant to industrial bioethanol production—pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains.

Synthetic biology and nanotechnology are poised to make revolutionary contributions to the 21st century. In this article, we describe a new web server to support in silico RNA molecular design. Given an input target RNA secondary structure, together with optional constraints, such as requiring GC-content to lie within a certain range, requiring the number of strong (GC), weak (AU) and wobble (GU) base pairs to lie in a certain range, the RNAiFold web server determines one or more RNA sequences, whose minimum free-energy secondary structure is the target structure. RNAiFold provides access to two servers: RNA-CPdesign, which applies constraint programming, and RNA-LNSdesign, which applies the large neighborhood search heuristic; hence, it is suitable for larger input structures. Both servers can also solve the RNA inverse hybridization problem, i.e. given a representation of the desired hybridization structure, RNAiFold returns two sequences, whose minimum free-energy hybridization is the input target structure. The web server is publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold, which provides access to two specialized servers: RNA-CPdesign and RNA-LNSdesign. Source code for the underlying algorithms, implemented in COMET and supported on linux, can be downloaded at the server website.

A direct detection method for anti-doping control of recombinant human erythropoietin (rHuEPO) abuse in racehorses is proposed. This method involves screening of plasma (or serum) by an enzyme-linked immunosorbent assay specific for human EPO and confirmation in urine samples by characterization of the urinary EPO isoelectric profile. This method was tested on horses that were administered epoetin alpha (rHuEPO) and the hyper-glycosylated form of this drug (darbepoetin alpha).

A patient with a long history of seropositive rheumatoid arthritis presented with a progressive papular eruption on the trunk. A skin biopsy showed a neutrophilic and palisaded granulomatous dermatitis compatible with rheumatoid papules. The eruption failed to respond to topical and systemic corticosteroids and the patient was treated with dapsone with complete resolution of the rash. Rheumatoid papules are a rare disorder seen in a variety of collagen vascular diseases. The literature concerning the treatment of rheumatoid papules is scanty. In this patient, dapsone was an effective treatment.

Given the recent surge in obesity, effective dietary strategies for weight management are required. Because fruits and vegetables are high in water and fiber, incorporating them in the diet can reduce energy density, promote satiety, and decrease energy intake. Although few interventions have specifically addressed fruit and vegetable consumption, evidence suggests that coupling advice to increase intake of these foods with advice to decrease energy intake is a particularly effective strategy for weight management. This approach may facilitate weight loss because it emphasizes positive messages rather than negative, restrictive messages.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

In an effort to produce phytases cost-effectively, and to determine the efficiency of a novel expression system, the genes for Aspergillus awamori ( phyA) phytase and Aspergillus fumigatus ( phyA) phytase (a putative thermostable enzyme) were cloned and overexpressed in A. awamori. Regulation of phytase expression was achieved by separately placing the genes under the transcriptional control of the glucoamylase A ( glaA) promoter of A. awamori. A gene fusion strategy was employed that involved the insertion of a hexapeptide Kex-2 protease cleavage site between the native glucoamylase and heterologous proteins and allowed for the efficient secretion and processing of the resultant chimeric proteins produced in this system by an endogenous Kex-2 protease. The genes for both of the above-mentioned phytases have already been cloned; however, this is the first report of either of the two phytases being fused with the glucoamylase gene, placed under the transcriptional control of the glaA promoter and overexpressed in A. awamori. Following transformation of A. awamori with separate expression vectors (one for each phytase), induction of phytase expression in submerged culture was effected by utilisation of a starch-containing medium. Optimisation of heterologous protein production in small shake-flask cultures involved changes in medium constituents. Maximum phytase expression levels of 200 phytase units (PU) ml(-1) and 62 PU ml(-1) for recombinantly expressed phytases from A. awamori and A. fumigatus, respectively, were obtained in crude fermentation extracts. Subsequent process scale-up to 4 l batch fermentation yielded phytase production levels comparable to those obtained on small scale. The enzyme yields herein reported demonstrate that the expression system developed and the host strain utilised were capable of expressing phytase at levels comparable to, or exceeding, previously reported data.

The attributional style of outpatients with schizophrenia with and without persecutory delusions was investigated. Thirty individuals with schizophrenia were divided into persecutory-deluded and non-persecutory-deluded groups based on a score of 5 or higher on the suspiciousness item from the Expanded Brief Psychiatric Rating Scale (BPRS-E). The two resulting groups, and a nonclinical control group, were administered a battery of attributional measures, and their attributional responses were coded by both the subjects themselves and a pair of independent raters. The results showed evidence of a self-serving bias for subjects with persecutory delusions; however, this bias was not unique to those with persecutory delusions, and it disappeared when independent raters evaluated subjects' causal statements on a reliable measure of attributional style. Subjects with persecutory delusions tended to show a stronger bias toward blaming others rather than situations for negative outcomes, and there was a linear association between persecutory ideation and a self-serving attributional style. Finally, there were significant discrepancies between the attributional ratings of the persecutory-deluded subjects and those of independent judges. Implications for future research are discussed.

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.

Background: The technical skill of surgical trainees is not well assessed. This study aimed (1) to compare the reliability of three scoring systems, (2) to compare live and bench formats and (3) to assess construct validity of a test of operative skill., Methods: Parallel examinations of operative skill, one using live animals and one using simulations, were developed. Performance was graded using operation-specific checklists, detailed global rating forms and pass/fail judgements. Twenty surgical residents each took both formats., Results: Disattenuated correlations between live and bench scores were high (0.69-0.72). Mean interrater reliability across stations ranged from 0.64 to 0.72. Internal consistency was moderate to high (alpha: 0.61-0.74) for the live format using the checklist and for live and bench formats using global ratings. Global ratings discriminated between resident levels for both formats (bench: F(2,17) = 4.45, P < 0.05; live: F(2,17) = 3.55, P < 0.05), checklists did not., Conclusion: This preliminary study suggests that the Objective Structured Assessment of Technical Skill can reliably and validly assess surgical skills. Global ratings are a better method of assessment than task-specific checklists. Bench model simulation gives equivalent results to use of live animals for this test format.

Neurofibrillary tangle (NFT)-rich brain samples from patients with progressive supranuclear palsy (PSP) or Alzheimer's disease (AD) were probed with a large panel of anti-tau antibodies to compare the species of tau present in PSP and AD NFTs by immunohistochemistry and Western blot methods. These antibodies have been shown to recognize phosphate-independent or -dependent epitopes that extend from the amino to the carboxy terminal domains of normal brain tau and the abnormal tau in the paired helical filaments (PHFs) of AD NFTs (PHFtau). The immunohistochemical studies showed that all of the tau epitopes detected in brainstem PSP NFTs also were found in hippocampal AD NFTs and vice versa. While Western blots demonstrated 2 PHFtau-like immunobands in PSP brainstem, a triplet of PHFtau proteins were seen in the AD and PSP hippocampus. Despite differences in the distribution, ultrastructure and immunoblot profile of NFTs in PSP and AD, the same constellation of tau epitopes is present in the abnormal tau proteins in PSP and AD NFTs. Thus, the generation of abnormal tau proteins in PSP (PSPtau) and AD (PHFtau) may have similar adverse biological consequences in both diseases.

The usual battery of skin tests employed for determining penicillin allergy may fail to detect allergic reactions to side chain-specific beta-lactam agents. We report the cases of six patients who experienced anaphylactic reactions after treatment and challenge with amoxicillin but who tolerated parenteral challenges with benzylpenicillin, aztreonam, and ceftazidime. Results of skin tests for amoxicillin (10 mg/mL) were positive for four of the six patients.

Thirty-one psoriatic patients 14 of whom were being treated with methotrexate (MTX), participated in a controlled study to evaluate the influence of MTX therapy on the intestinal absorption of folate. Low serum and red cell folate levels in the MTX treated group confirmed previously reported findings. However, analysis of the intestinal absorption profile yielded no significant difference between the two groups. It was concluded that MTX therapy for psoriasis does not adversely affect the intestinal absorption of folate and the observed low body folate status is probably mainly the result of enhanced renal excretion.