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PubMed1982

Cosmid DNA packaging in vivo.

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.

Bacteriophage lambda↗
PubMed1985

EcoK restriction during in vitro packaging of coliphage lambda DNA.

The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.

Bacteriophage lambda↗
PubMedDate not supplied

Optimizing detection of microbial sepsis: a comparison of culture systems using packaged sets with directions for blood collection.

Detection of microbial sepsis is an important aspect of medical practice. To facilitate the recovery of bacteria and fungi we evaluated 10,933 complete blood culture sets integrating the automated ESP system (Difco) with a manual system using the Isolator (Wampole) and Thiol broth bottle (Difco). To improve compliance with the recommended procedure for submitting specimens, we packaged the three components as a unit that included brief instructional material. A three-component (one ESP bottle, one Thiol bottle, and one Isolator tube) package containing specific instructions was used. The aerobic ESP performance was compared with the Isolator, and the anaerobic ESP with the Thiol bottle. A hypothetical assessment involving all three components was also made. The cultures were processed under routine laboratory conditions. Charts were reviewed from specimens with discrepant results to ascertain true positives versus contaminant cultures in the discordant culture sets. There were 896 organisms recovered from 393 patients. Of these, 421 were in the aerobic compared with 475 in the anaerobic portion of the study. ESP enhanced the recovery of Streptococcus pneumoniae (P < .05). The Isolator was superior for the recovery of yeasts (.05 < P < .1). Using the packaged system, the compliance rate for complete three-component culture sets was 95.2%, with a 34.4% resultant increase in positive blood cultures from only a 5.7% increase in sets. Packaged culture media including instructions enhance appropriate collection of complete sets, increase the number of recovered organisms, and improve medical practice.

Aerobiosis↗
PubMed2004

Psychological morbidity after myocardial infarction in an area of deprivation in the UK: evaluation of a self-help package.

BACKGROUND: Psychological morbidity after an acute myocardial infarction (AMI) is known to be common, but can be addressed by appropriate rehabilitation. The area in which this research was conducted experiences high rates of deprivation and of coronary heart disease and limited access to hospital-based rehabilitation. Responding to concern about psychological needs of AMI patients, a self-help package was introduced and evaluated alongside standard hospital-based cardiac rehabilitation. AIMS: To evaluate the impact of a home-based self-help package (the Heart Manual), alongside existing cardiac rehabilitation provision, on psychological morbidity and health status after AMI. A secondary aim was to assess the suitability of the Heart Manual for older patients aged over 80 years. METHODS: A controlled observational study, comparing two cohorts of patients discharged from hospital after AMI. The intervention group was given the self-help package in addition to standard care. The control group received standard care alone. Outcome measures used were the Hospital Anxiety and Depression Scale and the EuroQol. RESULTS: The intervention group showed significant improvement in anxiety and depression scores after 3 months and nonsignificant improvement in general health status. Patients who attended hospital-based rehabilitation classes, and those aged over 80 years, also benefited from the intervention. CONCLUSION: A home-based self-help rehabilitation package is an effective tool alongside hospital-based rehabilitation classes and can be given to all age groups.

Aftercare↗
PubMed2004

The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging.

Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium. We have determined the complete sequence of its 39,044 bp genome. The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome. It has chromosomal regions that encode proteins >80% identical with at least 15 different previously characterized lambdoid phages and prophages, but 43% of the genome, including the virion assembly genes, is homologous to the genome of one phage, HK620. An analysis of the nucleotide differences between Sf6 and HK620 indicates that even these similar regions are highly mosaic. This mosaicism suggests ways in which the virion structural proteins might interact with each other. The Sf6 early operons are arranged like a typical lambdoid phage, with "boundary sequences" often found between functional modules in the "metabolic" genome domain. By virtue of high degree of similarity in the encoding genes and their DNA target sites, we predict that the integrase, early transcription anti-terminator, CI and Cro repressors, and CII protein of Sf6 have DNA binding specificities very similar to the homologous proteins encoded by phages HK620, lambda, 434 and P22, respectively. The late operon contains two tRNA genes. The Sf6 terminase genes are unusual. Analysis of in vivo initiation of the DNA packaging series showed that the Sf6 apparatus that recognizes DNA for packaging appears to cleave DNA for initiation of packaging series at many sites within a large region of about 1800 bp that includes a possible pac site. This is unlike previously characterized phage packaging mechanisms.

Bacteriophages↗
PubMed2005

APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

APOBEC-3G Deaminase↗
PubMed2004

XMIPP: a new generation of an open-source image processing package for electron microscopy.

X-windows based microscopy image processing package (Xmipp) is a specialized suit of image processing programs, primarily aimed at obtaining the 3D reconstruction of biological specimens from large sets of projection images acquired by transmission electron microscopy. This public-domain software package was introduced to the electron microscopy field eight years ago, and since then it has changed drastically. New methodologies for the analysis of single-particle projection images have been added to classification, contrast transfer function correction, angular assignment, 3D reconstruction, reconstruction of crystals, etc. In addition, the package has been extended with functionalities for 2D crystal and electron tomography data. Furthermore, its current implementation in C++, with a highly modular design of well-documented data structures and functions, offers a convenient environment for the development of novel algorithms. In this paper, we present a general overview of a new generation of Xmipp that has been re-engineered to maximize flexibility and modularity, potentially facilitating its integration in future standardization efforts in the field. Moreover, by focusing on those developments that distinguish Xmipp from other packages available, we illustrate its added value to the electron microscopy community.

Algorithms↗
PubMed2006

Health insurance benefit packages prioritized by low-income clients in India: three criteria to estimate effectiveness of choice.

We applied a decision tool for rationing choices, with a predetermined budget of about 11 US dollars per household per year, to identify priorities of poor people regarding health insurance benefits in India in late 2005. A total of 302 individuals, organized in 24 groups, participated from a number of villages and neighborhoods of towns in Karnataka and Maharashtra. Many individuals were illiterate, innumerate and without insurance experience. Involving clients in insurance package design is based on an implied assumption that people can make judicious rationing decisions. Judiciousness was assessed by examining the association between the frequency of choosing a package and its perceived effectiveness. Perceived effectiveness was evaluated by comparing respondents' choices to the costs registered in 2049 illness episodes among a comparable cohort, using three criteria: 'reimbursement' (reimbursement regardless of the absolute level of expenditure), 'fairness' (higher reimbursement rate for higher expenses) and 'catastrophic coverage' (insurance for catastrophic exposure). The most frequently chosen packages scored highly on all three criteria; thus, rationing choices were confirmed as judicious. Fully 88.4% of the respondents selected at least three of the following benefits: outpatient, inpatient, drugs and tests, with a clear preference to cover high aggregate costs regardless of their probability. The results show that involving prospective clients in benefit package design can be done without compromising the judiciousness of rationing choices, even with people who have low education, low-income and no previous experience in similar exercises.

Choice Behavior↗
PubMed2005

Encapsidation of minute virus of mice DNA: aspects of the translocation mechanism revealed by the structure of partially packaged genomes.

Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3' end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3' sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1-2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3') third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem-loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5' end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.

Cell Line↗
PubMed2005

Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.

Base Sequence↗
PubMed2005

The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1-3 synthesis.

Alphaviruses are regarded as attractive systems for expression of heterologous genes and development of recombinant vaccines. Venezuelan equine encephalitis virus (VEE)-based vectors are particularly promising because of their specificity to lymphoid tissues and strong resistance to interferon. To improve understanding of the VEE genome packaging and optimize application of this virus as a vector, we analyzed in more detail the mechanism of packaging of the VEE-specific RNAs. The presence of the RNAs in the VEE particles during serial passaging in tissue culture was found to depend not only on the presence of packaging signal(s), but also on the ability of these RNAs to express in cis nsP1, nsP2 and nsP3 in the form of a P123 precursor. Packaging of VEE genomes into infectious virions was also found to be more efficient compared to that of Sindbis virus, in spite of lower levels of RNA replication and structural protein production.

Animals↗
PubMed2005

The genomic RNA packaging scheme of Red clover necrotic mosaic virus.

Red clover necrotic mosaic virus (RCNMV) is a small icosahedral plant virus with a bipartite RNA genome. While the RCNMV genome consists of two RNAs, it has not been definitively established whether these RNAs are co-packaged into a single virion or packaged individually into separate virions. Biochemical evidence exists to support both hypotheses. To determine the genomic RNA complement within RCNMV, virions were subjected to heat treatments and UV crosslinking. A stable RNA-1:RNA-2 heterodimer was formed with both treatments establishing that RCNMV genomic RNAs are co-packaged into a single virion. Furthermore, RNA-2 homodimer and homotrimers were also observed indicating that some virions contain multiple copies of RNA-2 exclusively. These results indicate that RCNMV virions consist of two distinct populations: (i) virions containing both genomic RNAs; and (ii) virions with multiple copies of RNA-2. This type of hybrid packaging arrangement was unexpected and appears to be unique among the multipartite RNA viruses.

Dimerization↗
PubMed2006

Evaluation of replicative capacity and genetic stability of West Nile virus replicons using highly efficient packaging cell lines.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

Animals↗
PubMed1999

Consumption of foods from the WIC food packages of Chinese prenatal patients on the US west coast.

OBJECTIVE: To assess consumption of foods from food packages provided by the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) among 80 Chinese prenatal WIC recipients living in Oakland or San Francisco, Calif. DESIGN: During a single interview, a food frequency questionnaire was used to assess WIC food consumption by the prenatal patients for the period before and after receipt of WIC vouchers. SETTING: The study was conducted at Asian Health Services, Oakland, and the Chinatown Public Health Center of the San Francisco Department of Public Health in California. SUBJECTS: Eighty low-income Chinese prenatal women with limited education and limited ability to speak English, aged 21 to 43 years, with gestational stages of 15 to 38 weeks. STATISTICAL ANALYSES PERFORMED: Descriptive statistics (frequency and percent distribution) were used to report the findings of the study. RESULTS: Subjects reported that before receiving WIC vouchers, milk, eggs, and juice were the only foods in their WIC food package that had been consumed frequently (> 5 times/week) in their daily diet. Other WIC foods, including cheese, peanut butter, dried beans, and hot and cold cereals, were consumed infrequently (0 to 1 time/month). Subjects reported that with the availability of WIC vouchers, milk, eggs, and juice remained frequently consumed with 81% to 100% of monthly supply as the most prevalent reported consumption rate. Dried beans and hot and cold cereals were also consumed frequently. Cheese remained poorly consumed (0% to 20% of the monthly supply). As many as 74 of 80 subjects stated that they had shared foods from their own WIC package, except milk, with their families. The use of any WIC foods provided to other children in the family was not assessed in this study. APPLICATIONS: Data from this study indicate that most WIC foods were well used by Chinese prenatal patients. The most notable exception was cheese, which was poorly consumed. The ready consumption of milk by pregnant Chinese WIC recipients in this study suggests that milk may be readily consumed by these women, even though it is atypical of the Asian diet. The WIC food package for Chinese prenatal patients may be improved by omitting cheese and substituting more milk and/or foods such as tofu and dark green leafy vegetables.

Adult↗
PubMed1997

The conformation of DNA packaged in bacteriophage G.

When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix. The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, undirectional winding of the DNA, or a combination of both folding and winding. To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals standards of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid. However, these strands are not visible toward the center of the zone of packaged DNA. 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at corners of the polyhedral bacteriophage G capsid. 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unravelling at multiple positions uniformly distributed around the compacted DNA region. The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings.

Bacteriophages↗
PubMed2003

Direct quantitation of volatile organic compounds in packaging materials by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

The quantification of volatile organic compounds (VOCs) in flexible multilayer packaging materials using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was studied. The analytes imclude 22 compounds such as aldehydes. ketones, carboxylic acids and hydrocarbons formed by thermooxidative degradation of polyethylene during the extrusion coating process in the manufacture of the packaging, and many of them are involved in the unpleasant and undesirable odour of these materials. External standard calibration using a solution of the analytes in an appropriate solvent was the first approach studied. Aqueous solutions of the analytes provided low reproducibility and the reduction of aldehydes to alcohols under the HS-SPME conditions. Hexadecane was chosen as the solvent since its polarity is similar to that of polyethylene and its volatility is lower than that of the analytes. However, hexadecane should be added to the sample before the analysis as it modifies the absorption capacity of the fibre. A 75-microm Carboxen-poly(dimethylsiloxane) fibre was used to extract the VOCs from the headspace above the packaging in a 15-ml sealed vial at 100 degrees C after 5 min of preincubation. The influence of the extraction time on the amount extracted was studied for a standard solution of the analytes in hexadecane, together with the influence of the volume of the standard solution and the amount of the sample placed in the vial. Standard addition and multiple HS-SPME were also studied as calibration methods and the results obtained in the quantitative analysis of a packaging material were compared.

Calibration↗
PubMed2003

The structures of bovine herpesvirus 1 virion and concatemeric DNA: implications for cleavage and packaging of herpesvirus genomes.

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I(L)) segments have been reported for some class D herpesviruses. Inefficient formation of class D I(L) genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I(L) genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I(L) orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I(L) genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur.

Animals↗