A novel self-replicating chimeric lentivirus-like particle

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.     

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis.Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3–26.6% for tetracycline and 33.3–36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.     

Variation in basal heat shock protein 70 is correlated to core temperature in human subjects

Heat shock proteins are highly conserved proteins and play an important chaperone role in aiding the folding of nascent proteins within cells. The heat shock protein response to various stressors, both in vitro and in vivo, is well characterised. However, basal levels of heat shock protein 70 (Hsp70) have not previously been investigated. Monocyte-expressed Hsp70 was determined every 4 h, over a 24 h time period, in 17 healthy male subjects (177 ± 6.4 cm, 75.7 ± 10.9 kg, 19.8 ± 4.3 years) within a temperature and activity controlled environment. Core temperature was measured at 5-min intervals during the 24 h period. Hsp70 showed significant diurnal variation (F = 7.4; p < 0.001), demonstrating peaks at 0900 and 2100 hours, and a nadir at 05.00. Core temperature followed a similar temporal trend (range = 35.96–38.10°C) and was significantly correlated with Hsp70 expression (r _s = 0.44; p < 0.001). These findings suggest a high responsiveness of Hsp70 expression in monocytes to slight variations in core temperature.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Use of conductimetry to rapidly determine relative stress sensitivity in Salmonella isolates

Aims:  To compare conventional plate counting and indirect conductimetry as techniques for ranking the resistance of Salmonella spp. to processing stressors.
Methods and Results:  Forty Salmonella isolates were subjected to three separate stressors used in food processing; irradiation, heat and high hydrostatic pressure (HHP). Total viable counts (TVC) using conventional plate counts and time to detection (TTD) using indirect conductimetry were determined. A significant negative correlation between TVC and TTD was seen with irradiation ( P  < 0·01) and heat ( P  < 0·05) but not HHP.
Conclusions:  For a group of salmonellas, indirect conductimetry can rapidly determine a ranking of isolate sensitivity to irradiation and heat. However, for HHP, the results indicated that conventional plate counting alone cannot be used to determine sensitivity.
Significance and Impact of the Study:  The resistance of micro-organisms to processing systems must be ranked to allow the selection of appropriate isolates for process validation. TTD measurements allow rapid screening of salmonellas to rank isolates for resistance to irradiation and heat stress. However, following HHP, the TVC of survivors is independent of the time required for growth to a set cell density and therefore it cannot be used as the sole measure of relative stress resistance.     

Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif''s effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.Pseudomonas aeruginosa is a Gram-negative bacterium that acts as an opportunistic pathogen. In colonizing the urinary tract, eye, and lung, as well as the surfaces of implanted medical devices, it forms antibiotic-resistant biofilms (12). In nosocomial infections, such as ventilator-associated pneumonia, P. aeruginosa is the second most common bacterial agent, and it represents the leading cause of death due to hospital-acquired infection (1). Among patients with compromised pulmonary function, P. aeruginosa frequently establishes persistent lung infections, exacerbating outcomes in chronic obstructive pulmonary disease (42) and cystic fibrosis (30). Overall, nearly 80% of patients with cystic fibrosis have a chronic P. aeruginosa infection in the lung by age 18 (22). Preventing infection by limiting exposure to the pathogen is difficult due to its ubiquitous distribution in the environment (47). On the other hand, the treatment of chronic lung infections is similarly challenging due to the formation of antibiotic-resistant biofilms. As a result, there currently is no effective treatment to eradicate a chronic P. aeruginosa infection from the lung once established (15, 54).P. aeruginosa secretes a multitude of virulence factors that assist the bacterium during the initial process of airway colonization and biofilm formation (32), in some instances acting directly on host cells. In particular, it was shown recently in a coculture model that the presence of P. aeruginosa causes a decrease in the quantity of cystic fibrosis transmembrane conductance regulator (CFTR) at the apical membrane of human airway epithelial cells (49). CFTR is the ion channel responsible for chloride secretion into the airway surface liquid (ASL) in the lung. The removal of CFTR from the cell surface leads to reduced chloride efflux, ASL dehydration, and decreased mucociliary clearance, thus facilitating the establishment of a bridgehead for bacterial infection.The downregulation of plasma membrane CFTR is mediated by a single secreted protein, the CFTR inhibitory factor (Cif), which is encoded at the PA14_26090 or cif locus (37) and is delivered into the host cell by outer membrane vesicles (5). The CFTR inhibitory effect also can be replicated by the application of purified, recombinant Cif protein directly to the apical surface of human airway epithelial cells. Within an hour after treatment with Cif, the levels of CFTR in the apical membrane are significantly reduced (37). While the mechanism of Cif action is incompletely understood, Cif has been shown to inhibit the recycling of CFTR to the apical membrane following endocytic uptake (49). Additional work has shown that Cif treatment causes a similar effect on some ABC transporters while having no effect on others (56). The mechanism by which Cif is able to generate this selectivity currently is unknown.Based on sequence comparisons, Cif was predicted to belong to the α/β hydrolase family (37), which contains several different classes of enzymes with closely related sequences. Specifically, Cif showed the greatest degree of sequence similarity to the class of epoxide hydrolases (EHs), which catalyze the conversion of epoxide moieties to vicinal diols (Fig. ​(Fig.1).1). The EHs are conserved between bacteria and mammals and are used to detoxify products of oxidative metabolism as well as xenobiotic compounds. In mammals, they metabolize potent chemical signal mediators (9). As a family, they also are of potential biocatalytic interest (46). EHs have not previously been reported as bacterial virulence factors, but Pseudomonas is adept at exploiting a wide variety of biochemical strategies to subvert host cell functions.Open in a separate windowFIG. 1.Epoxide hydrolase activity. The EH class of enzymes is responsible for the catalytic addition of a water molecule to an epoxide ring, creating a vicinal diol.Preliminary mutagenesis experiments targeting Cif suggested a link between EH activity and host cell effects, but these studies relied on low-identity sequence alignments and activity assays performed with an artificial EH substrate (37) that also is susceptible to esterase activity (18). Sequence alignment of Cif with known EHs revealed substitutions in several conserved EH motifs thought to be required for the formation of the enzyme active site. Furthermore, sequence relationships suggest that several haloacetate dehalogenases (HADs) previously had been misclassified as EHs, all of which cluster in the EH subgroup that includes Cif (52). As a basis for a detailed structure-function analysis of Cif, we have determined its crystal structure and assayed its activity against a variety of candidate substrates for EHs and related α/β hydrolases both for wild-type protein and for a mutation that abrogates Cif''s host cell activity.     

The GAMY Project: young people’s attitudes to genetics in the South Wales valleys

This paper explores young people’s attitudes to genetics. It describes a qualitative study involving a group of teenagers in a deprived South Wales valley town over a period of 18 months. The GAMY (Genetics and Merthyr Youth) Project involved a series of interactions with participants, including 2 interviews, 4 group days and 4 genetics tasks through which these young people learned about, and then reflected upon, issues relating to genetics and health. We have gathered data about the informed attitudes of teenagers to genetics based on deliberative learning and reflection over a long period of time, and as such this paper provides useful insights into the underlying values that are guiding young people’s views and the factors that are shaping their responses to new genetic technologies. Attitudes to genetics are complex and not easily generalisable. There were low levels of familiarity with, and knowledge of, genetics from the outset. Most young people did not have pre-existing attitudes towards genetics and had given little or no thought to the topic before the project began. However, levels of awareness and general genetic literacy increased as the project progressed. This study suggests that over time young people can develop an awareness of genetics that makes sense to them; they demonstrate that they can think creatively about genetics, and they are able to engage in considering genetic and other risk factors when thinking about health and disease.     

Preparation of reconstituted cytochrome oxidase vesicles with defined trans-membrane protein orientations employing a cytochrome c affinity column

Reconstituted cytochrome oxidase systems in which the majority of the vesicles contain a single oxidase dimer can be prepared. It is shown that, when these are passed through a cytochrome c affinity column, only those vesicles oriented outwards (such that the active site is available to external cytochrome c) are bound to the support matrix. Protein-free vesicles and vesicles containing an inwardly oriented enzyme are eluted in the void volume. Subsequently, vesicles containing an outwardly oriented enzyme can be eluted from the column at high salt concentrations. This protocol has been used successfully to resolve vesicles of either oxidase orientation when the enzyme is reconstituted with a variety of lipid mixtures. The recovery of oxidase activity from the column ranged between 75 and 94%.     

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.