Material Flaw Populations and Component Strength Distributions in the Context of the Weibull Function.

A clear relationship between the population of brittle-fracture controlling flaws generated in a manufactured material and the distribution of strengths in a group of selected components is established. Assumptions regarding the strength-flaw size relationship, the volume of the components, and the number in the group, are clarified and the contracting effects of component volume and truncating effects of group number on component strength empirical distribution functions highlighted. A simple analytical example is used to demonstrate the forward prediction of population → distribution and the more important reverse procedure of empirical strength distribution → underlying flaw population. Three experimental examples are given of the application of the relationships to state-of-the-art micro- and nano-scale strength distributions to experimentally determine flaw populations: two on etched microelectromechanical systems (MEMS) structures and one on native and oxidized silicon nanowires. In all examples, the minimum threshold strength and conjugate maximum flaw size are very well estimated and the complete flaw population, including the minimum flaw size, are very poorly estimated, although etching, bimodal, and oxidation effects were clearly discernible. The results suggest that the best use of strength distribution information for MEMS manufacturers and designers might be in estimation of the strength threshold.

Quantitative Scanning Probe Microscopy for Nanomechanical Forensics.

Atomic force microscopy (AFM) was used to assess the indentation modulus Ms and pull-off force Fpo in four case studies of distinct evidence types, namely hair, questioned documents, fingerprints, and explosive particle-surface interactions. In the hair study, Ms decreased and Fpo increased after adding conditioner and bleach to the hair. For the questioned documents, Ms and Fpo of two inks were markedly different; ballpoint pen ink exhibited smaller variations relative to the mean value than printer ink. The fingerprint case study revealed that both maximum height and Fpo decreased over a three-day period. Finally, the study on explosive particle-surface interactions illustrated that two fabrics exhibited similar Ms, but different Fpo. Overall, it was found that AFM addresses needs in forensic science as defined by several federal agencies, in particular an improved ability to expand the information extracted from evidence and to quantify its evidentiary value.

Ultimate bending strength of Si nanowires.

Test platforms for the ideal strength of materials are provided by almost defect-free nanostructures (nanowires, nanotubes, nanoparticles, for example). In this work, the ultimate bending strengths of Si nanowires with radii in the 20-60 nm range were investigated by using a new bending protocol. Nanowires simply held by adhesion on flat substrates were bent through sequential atomic force microscopy manipulations. The bending states prior to failure were analyzed in great detail to measure the bending dynamics and the ultimate fracture strength of the investigated nanowires. An increase in the fracture strengths from 12 to 18 GPa was observed as the radius of nanowires was decreased from 60 to 20 nm. The large values of the fracture strength of these nanowires, although comparable with the ideal strength of Si, are explained in terms of the surface morphology of the nanowires.

Compressive stress effect on the radial elastic modulus of oxidized Si nanowires.

Detailed understanding and optimal control of the properties of Si nanowires are essential steps in developing Si nanoscale circuitry. In this work, we have investigated mechanical properties of as-grown and oxidized Si nanowires as a function of their diameter. From contact-resonance atomic force microscopy measurements, the effect of the compressive stress at the Si-SiO(2) interface was revealed in the diameter dependence of the elastic modulus of Si nanowires oxidized at 900 and 1000 degrees C. A modified core-shell model that includes the interface stress developed during oxidation captures the diameter dependence observed in the measured elastic moduli of these oxidized Si nanowires. The values of strain and stress as well as the width of the stressed transition region at the Si-SiO(2) interface agree with those reported in simulations and experiments.

The active site of ICP47, a herpes simplex virus-encoded inhibitor of the major histocompatibility complex (MHC)-encoded peptide transporter associated with antigen processing (TAP), maps to the NH2-terminal 35 residues.

The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2-35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.

Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease.

Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.

Elastic moduli of faceted aluminum nitride nanotubes measured by contact resonance atomic force microscopy.

A new methodology for determining the radial elastic modulus of a one-dimensional nanostructure laid on a substrate has been developed. The methodology consists of the combination of contact resonance atomic force microscopy (AFM) with finite element analysis, and we illustrate it for the case of faceted AlN nanotubes with triangular cross-sections. By making precision measurements of the resonance frequencies of the AFM cantilever-probe first in air and then in contact with the AlN nanotubes, we determine the contact stiffness at different locations on the nanotubes, i.e. on edges, inner surfaces, and outer facets. From the contact stiffness we have extracted the indentation modulus and found that this modulus depends strongly on the apex angle of the nanotube, varying from 250 to 400 GPa for indentation on the edges of the nanotubes investigated.

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions.

BACKGROUND: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. OBJECTIVES: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 5' untranslated region (5' UTR)/exon 1 of the tat gene of EIAV. STUDY DESIGN: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RT-PCR (RT-qPCR) along with the AGID test. METHODS: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 5' UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. RESULTS: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. MAIN LIMITATIONS: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. CONCLUSIONS: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or "serologically silent" equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions.

Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene.

In 2006, an outbreak of equine infectious anaemia (EIA) occurred in Ireland. The initial source of the outbreak is believed to have been contaminated plasma imported from Italy. This paper presents the nucleotide sequence of the gag gene of the virus identified in Ireland (EIAV(Ire)), the first for a European strain of EIAV. Comparison of the gag gene with North American and Asian strains of the virus showed that the gag gene is less well conserved than previously believed, and that EIAV strains can have similar phenotypes despite considerable variations in genotype. On the basis of the deduced sequence of the EIAV(Ire) gag gene, highly sensitive, specific and quantitative RT-PCR and PCR assays were developed, and used to quantify the EIAV nucleic acid in postmortem tissues, plasma and secretions of infected horses. This is the first report of the detection and quantification of EIAV in nasal, buccal and genital swabs by RT-PCR.

Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

In situ spectroscopic study of the plastic deformation of amorphous silicon under non-hydrostatic conditions induced by indentation.

Indentation-induced plastic deformation of amorphous silicon (a-Si) thin films was studied by in situ Raman imaging of the deformed contact region of an indented sample, employing a Raman spectroscopy-enhanced instrumented indentation technique. Quantitative analyses of the generated in situ Raman maps provide unique, new insight into the phase behavior of as-implanted a-Si. In particular, the occurrence and evolving spatial distribution of changes in the a-Si structure caused by processes, such as polyamorphization and crystallization, induced by indentation loading were measured. The experimental results are linked with previously published work on the plastic deformation of a-Si under hydrostatic compression and shear deformation to establish a sequence for the development of deformation of a-Si under indentation loading. The sequence involves three distinct deformation mechanisms of a-Si: (1) reversible deformation, (2) increase in coordination defects (onset of plastic deformation), and (3) phase transformation. Estimated conditions for the occurrence of these mechanisms are given with respect to relevant intrinsic and extrinsic parameters, such as indentation stress, volumetric strain, and bond angle distribution (a measure for the structural order of the amorphous network). The induced volumetric strains are accommodated solely by reversible deformation of the tetrahedral network when exposed to small indentation stresses. At greater indentation stresses, the increased volumetric strains in the tetrahedral network lead to the formation of predominately five-fold coordination defects, which seems to mark the onset of irreversible or plastic deformation of the a-Si thin film. Further increase in the indentation stress appears to initiate the formation of six-fold coordinated atomic arrangements. These six-fold coordinated arrangements may maintain their amorphous tetrahedral structure with a high density of coordination defects or nucleate as a new crystalline ß-tin phase within the a-Si network.

Designing a standard for strain mapping: HR-EBSD analysis of SiGe thin film structures on Si.

Patterned SiGe thin film structures, heteroepitaxially deposited on Si substrates, are investigated as potential reference standards to establish the accuracy of high resolution electron backscattered diffraction (HR-EBSD) strain measurement methods. The proposed standards incorporate thin films of tetragonally distorted epitaxial Si1-xGex adjacent to strain-free Si. Six films of three different nominal compositions (x=0.2, 0.3, and 0.4) and various thicknesses were studied. Film composition and out-of-plane lattice spacing measurements, by x-ray photoelectron spectroscopy and x-ray diffraction, respectively, provided independent determinations of film epitaxy and predictions of tetragonal strain for direct comparison with HR-EBSD strain measurements. Films assessed to be coherent with the substrate exhibited tetragonal strain values measured by HR-EBSD identical to those predicted from the composition and x-ray diffraction measurements, within experimental relative uncertainties of order 2%. Such films thus provide suitable prototypes for designing a strain reference standard.

Patterns of illicit drug use in the Army.

The authors collected detailed histories of illicit drug use in the Army in individual interviews with a stratified random sample of 262 enlisted men at six military posts across the United States. Approximately half of the sample (N equals 128) were classified as drug users; 90 of these individuals were identified as career multiple-drug users. Most of these subjects used a variety of drugs in frequently changing patterns. The authors emphasize the individualistic nature of drug use and question the appropriateness of an addiction model for most users of illicit drugs.

A nonradioactive micro-assay for released reverse transcriptase activity of a lentivirus.

A nonradioactive micro-assay procedure for detection of released reverse transcriptase activity from cells infected with equine infectious anemia virus is described. This procedure utilizes biotinylated-dUTP in conjunction with a streptavidin-alkaline phosphatase conjugate. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of Lumi Phos 530. This method, as with reverse transcriptase micro-assays employing 32P-labeled nucleotides, is suited to the processing of numerous samples, while having the advantages of safety and stability normally associated with nonradioactive methods of detection. Sensitivity is comparable to a reverse transcriptase micro-assay using 32P-dTTP.

Application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus.

Nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (EIAV) was investigated using a modification of the dideoxy fingerprinting (ddF) technique. PCR-amplified regions of the gag gene from EIAV isolates were ligated into plasmid vectors and used to transform bacteria. The single dideoxynucleotide sequencing step was performed using plasmid DNA prepared from individual bacterial colonies using an 35S end-labeled primer and Taq DNA polymerase. Analysis of the products of this reaction was conducted using non-denaturing polyacrylamide gel electrophoresis. Polymorphism within this gene was suggested by the presence of several distinct electrophoretic profiles. Significantly, each profile could be correlated with variations in nucleotide sequence, which demonstrates that cycle ddF (CddF) offers a rapid and sensitive approach to identify polymorphism in PCR-amplified products.

Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses.

An antigen capture indirect enzyme linked immunosorbent assay (ELISA) was developed to detect influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine/H3N8 viruses. Results from this assay were compared with conventional virus isolation in embryonated hens eggs.

Differences in sensitivity in haemagglutinin inhibition assays between A/equine/H3N8 viruses isolated in eggs and MDCK cells are linked to cleavage of the haemagglutinin molecule.

Two primary isolates of A/equine/H3N8 viruses were obtained in embryonated hens' eggs and in Madin-Darby canine kidney (MDCK) cells. Viruses isolated in MDCK cells were significantly more sensitive as antigens in haemagglutination inhibition (HI) tests. This sensitivity appeared to be primarily linked to the extent of cleavage of the haemagglutinin molecule.

The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus.

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).