The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.     

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.     

Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin.

The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label's own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC : BSA 1 : 17-15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.     

Operative management and outcome of complex wounds following total knee arthroplasty.

This study describes the treatment protocol for and the outcome of the management of complex wounds around total knee replacements. An analysis of 28 patients (29 knees) with complex defects who had surgery between January 1, 1986, and July 30, 1996, was performed. A specific management protocol was applied to each knee on the basis of the size and depth of the wound, the presence of infection, and the quality of soft tissue. Primary treatment included local wound care, debridement, and skin grafting or coverage with a fasciocutaneous flap, pedicled muscle flap, or free muscle transfer. Postoperatively, knees were evaluated using the Knee Society objective score. Successful salvage of the lower extremity was obtained in 28 knees (97 percent) and of the knee prosthesis in 24 of 29 knees (83 percent). Secondary plastic surgery procedures were necessary in five knees (17 percent), and secondary orthopedic procedures were necessary in four knees (14 percent). Successful salvage of total knee arthroplasty in the presence of a complex wound requires early identification of infection, aggressive irrigation and debridement, and early appropriate soft-tissue coverage. The use of our proposed algorithm will facilitate management of these complex wounds.     

Influence of secondary structure on the decay kinetics of fluorescent donor-acceptor labelled peptides

Time-resolved fluorescence decays from a series of methoxynaphthalene labelled peptides in ethyl acetate were monitored over the temperature range −40 to 60°C. The quenching effect of a piperidone acceptor group placed at various positions along the peptide chain relative to the fluorescent methoxynaphthalene donor was studied. In this moderately polar solvent the mechanism of quenching is most likely electron transfer, although a Dexter exchange mechanism cannot be ruled out. Both donor and acceptor moieties were covalently attached to the side-chains of glutamic acid residues. These were either placed adjacently, in the case of a dipeptide, or separated by three and six amino acids within a 12 and 15 amino-acid oligopeptide, respectively. The presence of the piperidone group resulted in a reduction in the fluorescence lifetime and a change from a simple monoexponential decay to more complex behaviour. This was found to vary reversibly with temperature and not to be caused by impurities. Modelling of the fluorescence decays was carried out using either the sum of two exponentials or a distribution of decays. For the dipeptide the best fit was a distribution while in the case of the 12-mer two clearly distinguishable populations could be observed. The results for the 15-mer were equivocal. Importantly, regardless of the fitting method used the quenching rate was found to be fastest for the 12-mer. The slower quenching rates observed for the dipeptide compared to the oligopeptides provide strong evidence that secondary structure promotes better electronic coupling between the donor and acceptor. The biexponential fluorescence behaviour for the 12 amino-acid oligopeptide is ascribed to two slowly (>10ns) interconverting conformational states. Comparison with circular dichroism and infrared obtained in acetonitrile indicates these two conformers are likely to be an -helix and a 3(10)-helix with electronic coupling strongest in the latter case.     

A new system for understanding the biodiversity in different nature reserves:capacity,connectivity and quality of biodiversity

In this paper,we propose a new system for understanding the biodiversity in different conservation areas.It includes three aspects:the capacity,the connectivity and the quality.The capacity refers to the numbers of biodiversity,including absolute and relative richness of the vegetation types Nv and Dv = (Nv-1)/lnA,species numbers S and richness of species dGI = (S- 1)/lnA,and germ plasm resources within a nature reserve,and also the potential biological living space offered by the natural resource.It comprises the total biological resources in a nature reserve.The connectivity refers to the flux of biodiversity,including similarity and connected status of the vegetation types SILi = 2z/(x + y) and species numbers SIc = 2z/(x + y) among different nature reserves.The quality refers to the stability of biodiversity,including relative species richness index RSLi = d/dmax,relative vegetation richness index RVLi =Dv/Dmaxv,fastness to invasion species fLi = 1-Si/St,weighted values,representativeness and vulnerability of special vegetations,special species,CITES species and rare species as the protected targets.     

Disseminated simian varicella virus infection in an irradiated rhesus macaque (Macaca mulatta)

We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.