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At least 1,459 records · Page 81Linked to original sources
PubMed2001

Development of a packaging cell line for propagation of replication-deficient adenovirus vector.

A human embryonic kidney cell line 293 is widely used for adenovirus production and propagation. With this cell line, however, replication-competent virus (RCV) is frequently generated, especially during large-scale production and successive propagation because 293 cells contain not only E1 gene but also non-E1 adenovirus gene. Homologous recombination between non-E1 region of 293 genomic DNA and its homologous region in the recombinant adenoviral vector generate RCV. To overcome this problem, we developed a new packaging cell line, Hela-E1, which contains minimum E1 region and from which non-E1 adenoviral region that is homologous with recombinant adenovirus vector was excluded. No RCV was detected during adenovirus propagation in Hela-E1 compared to in 293. In addition, adenovirus-p53 produced in HeLa-E1 was able to overexpress p53 protein when introduced into an ovarian cancer cell line, SKOV3. These results may have a significant impact on the development of packaging cell lines for replication-deficient adenovirus production.

Adenoviridae↗
PubMed2001

Teaching undergraduates about familial breast cancer: comparison of a computer assisted learning (CAL) package with a traditional tutorial approach.

We have developed a computer assisted learning package for teaching clinical medical students about familial breast cancer. It explains the principles of genetic predisposition to breast cancer, the association with other cancers, the principles of family history taking and confirmation, risk assessment and possible interventions. Clinical medical students were randomised to either conventional teaching or CAL, 48 students attended the evaluation session. Students randomised to conventional teaching received a 20 min mini-lecture, those randomised to CAL completed the package with technical, but not academic support available. At the end of the intervention both groups of students completed a short written assessment of acceptability and knowledge and understanding of breast cancer genetics. There was no significant difference between the CAL and mini-lecture groups in terms of marks or acceptability. Thus CAL appears to be an acceptable and effective method of teaching clinical medical students about familial breast cancer. Although time consuming to develop, CAL can be used in a variety of settings to increase curriculum flexibility. Methods of motivating students to complete the CAL, and of providing educational support are being explored.

Breast Neoplasms↗
PubMed2006

Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in cell factories.

The clinical implementation of gene therapy requires large-scale production of viral vector stocks (VS) derived from packaging cell lines. Upon scaling-up, maintenance of high viral titers and filtration of the VS become significantly challenging. Thus, production schemes amenable to straightforward validation must be developed. To this end, we have established a semi-closed process to manufacture batches of 7 l or more of clinical-grade oncoretroviral VS using 10-tray Cell Factories. Using a peristaltic pump, the VS are collected on 3 consecutive days, filtered, pooled and stored frozen. To ensure the absence of viable vector-producing cells (VPCs) from each VS unit-dose, we undertook an orthogonal log-removal validation study to demonstrate the ability of both the filtration system to remove viable cells and the VS freezing process to inactivate them. We demonstrate a total VPC-reduction of 11.6 log, thus insuring the absence of contaminating VPCs in transduced clinical samples. We also show that this production process generates stable VS that can be stored at -80 degrees C for more than 3 years. Importantly, this relatively simple and affordable process can be customized to generating large volume of VS for small animal or non-human primate studies. This methodology is not limited to the generation of cell-free clinical oncoretroviral VS, and can be applied to other types of vectors produced in packaging cell lines, such as lentiviral vectors.

Animals↗
PubMed2006

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Animals↗
PubMed2006

Advances in methods and algorithms in a modern quantum chemistry program package.

Advances in theory and algorithms for electronic structure calculations must be incorporated into program packages to enable them to become routinely used by the broader chemical community. This work reviews advances made over the past five years or so that constitute the major improvements contained in a new release of the Q-Chem quantum chemistry package, together with illustrative timings and applications. Specific developments discussed include fast methods for density functional theory calculations, linear scaling evaluation of energies, NMR chemical shifts and electric properties, fast auxiliary basis function methods for correlated energies and gradients, equation-of-motion coupled cluster methods for ground and excited states, geminal wavefunctions, embedding methods and techniques for exploring potential energy surfaces.

Algorithms↗
PubMed2006

Chitosan-mediated in situ biomolecule assembly in completely packaged microfluidic devices.

We report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.

Biosensing Techniques↗
PubMed2003

Assessing the learning needs of college tutors in anaesthesia: proposals for an introductory learning package.

At present there is no nationwide formal training course at which college tutors in anaesthesia can develop the specific skills required to perform their important role effectively. The purpose of this study was to ascertain whether there is a need for an introductory learning package for college tutors and if so, what learning needs ought to be addressed in such a package. A needs assessment was performed involving the use of individual interviews and the administration of two sequential questionnaires. The questionnaire was completed by 208 college tutors, a return rate of 83%. Most college tutors (93%) said they had had no formal training to perform their role and 94% felt there was a need for an introductory course for college tutors in anaesthesia. Of those, 77% said they would be interested in taking such a course. The study identified a number of learning needs.

Anesthesiology↗
PubMed2003

Food and waterborne infections associated with package holidays.

The surveillance and prevention of food and waterborne infections in package holiday tourists relies more on common sense and experience rather than evidence based scientific facts. In spite of the major economic value to both sending and receiving countries it is a problem that is largely ignored by health departments at both local and national levels. Package holiday tourism is a growing industry with over 20 million holidays sold every year in the UK. Destinations are in every continent including countries with poorly developed, as well as those with an advanced, public health infrastructure. The incidence of gastrointestinal infection is not reflected in official surveillance programmes as they largely fail to capture information on travel associated infections. Outbreaks of food and waterborne infections in these resorts are largely not investigated. Major British tour operators have responded by developing a crude but effective continuous surveillance system for subjective travellers' diarrhoea. The importance of food and water hygiene is, however, not ignored and proactive preventative programmes are being developed and implemented in some resort as well as by the tourist industry and international agencies. There is a need for further cooperation and partnership between workers in different countries, different disciplines and between the public and private sectors.

Commerce↗
PubMed1998

Evaluation of a training package in the assessment and management of depression in primary care.

This study aimed to evaluate the impact on the behaviour and attitudes of experienced general practitioners of a 10-hour training package in the assessment and management of depression. Twenty general practitioners participated. Both subjective and objective assessments were carried out which suggested significant improvements in both assessment and management skills. However, subjectively reported changes were not always supported by the objective data obtained from rating role-played interviews. The role-played patients rated the doctors as better communicators after training. All participants felt attending the course was beneficial. They all felt more confident in their abilities to deal with depression and said the skills they had learnt on the course would be useful to them in their future work. An outcome study is now underway in order to assess whether the training package, which has been demonstrated to have an impact on the behaviour, skills and attitudes of doctors, has an impact on the health of patients.

Adult↗
PubMed2003

[Information regarding adverse drug effects and treatment indications ("package inserts") exemplified by cervistatin (Lipobay)].

A worldwide frequently applied HMG-CoA-reductase-inhibitor ("statin") recently has been withdrawn from the market. This obviously has been caused by serious adverse events. The question is whether the package inserts sufficiently inform about the risks. But, although they list up numerous side effects, they lack informations concerning the frequency of them, so the patients when reading the package inserts become confused. Thus they have to rely on their doctors choice regarding the danger outlined in the inserts. In that respect packing inserts could be improved.

Adverse Drug Reaction Reporting Systems↗
PubMed1986

Adjusting for confounding with statistical software packages.

Much computer use in support of clinical research involves the execution of standard statistical packages. Prenatal risks, such as maternal substance use, may be related to outcomes, such as lowered birthweight for gestational age, using multiple regression. In standard stepwise multiple regression, variance shared by two risks is attributed to the first variable entered in the equation, potentially underestimating the effect size of subsequently entered variables. A novel solution to this problem, adapted from the epidemiologic technique of calculating attributable risk, is applied in the multivariate setting and produces more appropriate estimates of the effect sizes of independent risks. An example of this method, using a standard statistical package, is presented.

Alcohol Drinking↗
PubMed2004

Dynamics of polymer packaging.

We use the stochastic rotation dynamics algorithm to investigate the packaging of flexible and semiflexible polymers into a capsid that is permeable to solvent molecules. The model takes into account hydrodynamic interactions arising due to local flow. The flexible chain maintains a random configuration as it is being fed into the capsid, in contrast to the semiflexible chain, whose configuration is initially spool-like, becoming more random at high packing. We measure the packing rate, which is found to decrease with the percentage of the chain packed and highlight the difference between the flexible and semiflexible chains. Reflecting experiments, we find pauses in the packing process for individual chains as the motor loses grip of the fluctuating beads. We also find that hydrodynamics is important, in that the packaging rate is faster when flow is included.

Journal Article↗
PubMed2005

Replication and packaging of Norwalk virus RNA in cultured mammalian cells.

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.

Animals↗
PubMed2002

Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection.

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) (32)P-labeled riboprobe generated by in vitro transcription of the U(S)8.5 ORF bound three proteins identified as the products of U(S)11, U(L)47, and U(L)49 (VP22) genes. (ii) Viral RNA was bound to U(L)47 or U(S)11 proteins immune precipitated from cells transduced with baculoviruses expressing U(L)47 or U(S)11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U(S)8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U(S)8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in approximately 1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U(S)8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U(L)47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

Animals↗
PubMed2001

DNA packaging and ejection forces in bacteriophage.

We calculate the forces required to package (or, equivalently, acting to eject) DNA into (from) a bacteriophage capsid, as a function of the loaded (ejected) length, under conditions for which the DNA is either self-repelling or self-attracting. Through computer simulation and analytical theory, we find the loading force to increase more than 10-fold (to tens of piconewtons) during the final third of the loading process; correspondingly, the internal pressure drops 10-fold to a few atmospheres (matching the osmotic pressure in the cell) upon ejection of just a small fraction of the phage genome. We also determine an evolution of the arrangement of packaged DNA from toroidal to spool-like structures.

Bacteriophages↗
PubMed1977

Packaging recombinant DNA molecules into bacteriophage particles in vitro.

Recombinant phage genomes made in reactions with purified enzymes may be recovered directly by packaging into phage heads in vitro. The process is efficient and nonselective and offers containment in initial stages of handling recombinant DNA. Ligase [poly(deoxyribonucleotide):poly-(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1] reaction products can recombine with endogenous phage DNA during packaging, but UV-irradiation eliminates the biological activity of the endogenous DNA.

Coliphages↗
PubMed1981

Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena.

A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes. This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules. Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length. The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion. Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion. The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

Cell Nucleus↗