How Escherichia coli can bias the results of molecular cloning: Preferential selection of defective genomes of hepatitis C virus during the cloning procedure

Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression. These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected. The aim of the present study was to express HCV structural proteins. However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins. The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription–PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector. The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products. Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis. The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72–92%). Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.

Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a γ-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a β-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in γ-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

ϕ-Divergence Based Procedure for Parametric Change-Point Problems

This paper studies the change-point problem for a general parametric, univariate or multivariate family of distributions. An information theoretic procedure is developed which is based on general divergence measures for testing the hypothesis of the existence of a change. For comparing the exact sizes of the new test-statistic using the criterion proposed in Dale (J R Stat Soc B 48–59, 1986), a simulation study is performed for the special case of exponentially distributed random variables. A complete study of powers of the test-statistics and their corresponding relative local efficiencies, is also considered.

Herbert Bayer’s Chromatics : “a pure artistic theory”

This paper examines the roots and properties of Herbert Bayer’s chromatic paintings completed between 1966 and 1976. This series of paintings is grounded in advanced theories of color and geometry, first introduced to Bayer as a young architecture apprentice and later developed at the Bauhaus. An investigation of Bayer’s mature reassessment of those early Bauhaus teachings in color theory and the development of his own color system is the underlying focus of this paper. The purpose of this study is to shed light on the significance of the chromatics that have received little attention to date.

Academie to atelier : the artistic education of American women in nineteenth-century Paris

This paper puts forth an alternate reading of the artistic climate in late nineteenth-century Paris than that which has traditionally been suggested. I propose that the expansion of creative opportunity during this time reveals a climate of communal support, consent, and progressive reform for women artists, rather than a struggle to undermine central (masculine) control, as many scholars have claimed. Specifically, I explore the work of American expatriates living in Paris, including but not limited to Cecilia Beaux, Anna Klumpke, Alice Kellogg, and Ellen Day Hale. The birth of the private academy in Paris offered women the chance to develop their artistic ability and assert their independence. The Académie Julian in particular provided a comparatively accepting and progressive environment where American women studying abroad could study from the nude model, receive proper training, and explore their full creative potential. Through an examination of a) these women’s self-portraits, and b) depictions of them painted by their contemporaries – both male and female – I further investigate the artistic education of American women in the highly-gendered cultural milieu of late nineteenth-century France.

Applying Translation Quality Standards to a Certification Procedure for U.S. Language Service Providers

Many translation quality standards have been implemented to regulate the provision and procurement of language services. However, in the absence of a standardized procedure to certify U.S. language service providers (LSPs), the industry lacks consensus with regard to requirements, procedures, and expectations. This project establishes the need for such a procedure and proposes an LSP Certification Procedure based on existing quality standards. Through a review and analysis of existing translation quality standards, an interview with a key stakeholder, and the presentation of an LSP Certification Procedure, this project concludes that the U.S. language services industry requires a procedure to certify LSPs and that such a procedure may be designed and implemented based on existing standards.

Assessment of a simplified experimental procedure to evaluate impact sound reduction of floor coverings

The impact noise reduction provided by floor coverings is usually obtained in laboratory, using the methodology described in the standard EN ISO 140-8, which requires the use of standard acoustic chambers. The construction of such chambers, following the requirements described in the EN ISO 140-1, implies a significant investment, and therefore only a limited number exists in each country. Alternatives to these standard methodologies, that allow a sufficiently accurate evaluation and require lower resources, have been interesting many researchers and manufacturers. In this paper, one such strategy is discussed, where a reduced sized slab is used to determine the noise reduction provided by floor coverings, following the procedure described in the ISO/CD 16251-1 technical document. Several resilient coverings, floating floors and floating slabs are tested and the results are compared with those obtained using the procedures described in the standards EN ISO 140-8 and EN ISO 717-2.

An improved procedure for obtaining and maintaining well characterized partial water saturation states on concrete samples to be used for mass transport tests

A conditioning procedure is proposed allowing to install into the concrete specimens any selected value of water saturation degree with homogeneous moisture distribution. This is achieved within the least time and the minimum alteration of the concrete specimens. The protocol has the following steps: obtaining basic drying data at 50 °C (water absorption capacity and drying curves); unidirectional drying of the specimens at 50 °C until reaching the target saturation degree values; redistribution phase in closed containers at 50 °C (with measurement of the quasi-equilibrium relative humidities); storage into controlled environment chambers until and during mass transport tests, if necessary. A water transport model is used to derive transport parameters of the tested materials from the drying data, i.e., relative permeabilities and apparent water diffusion coefficients. The model also allows calculating moisture profiles during isothermal drying and redistribution phases, thus allowing optimization of the redistribution times for obtaining homogeneous moisture distributions.

Procedure for calculating the chloride diffusion coefficient and surface concentration from a profile having a maximum beyond the concrete surface

Chlorides induce local corrosion in the steel reinforcements when reaching the bar surface. The measurement of the rate of ingress of these ions, is made by mathematically fitting the so called “error function equation” into the chloride concentration profile, obtaining so the diffusion coefficient and the chloride concentration at the concrete surface. However, the chloride profiles do not always follow Fick’s law by having the maximum concentration at the concrete surface, but often the profile shows a maximum concentration more in the interior, which indicates a different composition and performance of the most external concrete layer with respect to the internal zones. The paper presents a procedure prepared during the time of the RILEM TC 178-TMC: “Testing and modeling chloride penetration in concrete”, which suggests neglecting the external layer where the chloride concentration increases and using the maximum as an “apparent” surface concentration, called C max and to fit the error function equation into the decreasing concentration profile towards the interior. The prediction of evolution should be made also from the maximum.

Application and Reliability of the Retrospective Interview Procedure to Trace Physical Activity

This paper reports the results of two studies. The purpose of the first study was to determine if lifestyle variables and past involvement in physical activity was related to current activity levels in master athletes and sedentary older adults. Retrospective interviews were conducted with 12 master athletes and 12 sedentary older adults. Results demonstrated that education level, spouse participation, smoking, and recent physical activity levels were indicators of current involvement. The second study investigated the reliability of the data collected in the retrospective interviews. Similar to results with younger samples, we confirm that lifestyle variables and physical activity involvement could be accurately recalled for a period of 25 years, making this tool a useful addition for the study of physical activity in older adults.

Tracing the Development of Athletes Using Retrospective Interview Methods: A Proposed Interview and Validation Procedure for Reported Information

A new interview procedure is proposed for collecting valid information on the acquisition of high-level performance in sport. The procedure elicits verifiable information on the development of athletes' achievements in their primary sport, as well as factors that might influence performance, including involvement in other sporting activities, injuries, physical growth and quality of training resources. Interviewed athletes also describe their engagement in specific training and other relevant activities during each year of their development as well as how they experienced each type of activity. The collected information is then examined to identify those aspects of the athletes' recall of their development that meet criteria of reliability and validity. Recommendations to coaches and scientists are discussed for how retrospective interviews can uncover aspects of development that distinguish elite from less accomplished athletes.