Combined time- and space-resolved Raman spectrometer for the non-invasive depth profiling of chemical hazards

A time-resolved inverse spatially offset Raman spectrometer was constructed for depth profiling of Raman-active substances under both the lab and the field environments. The system operating principles and performance are discussed along with its advantages relative to traditional continuous wave spatially offset Raman spectrometer. The developed spectrometer uses a combination of space- and time-resolved detection in order to obtain high-quality Raman spectra from substances hidden behind coloured opaque surface layers, such as plastic and garments, with a single measurement. The time-gated spatially offset Raman spectrometer was successfully used to detect concealed explosives and drug precursors under incandescent and fluorescent background light as well as under daylight. The average screening time was 50 s per measurement. The excitation energy requirements were relatively low (20 mW) which makes the probe safe for screening hazardous substances. The unit has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than previous picosecond-based systems, to provide a functional platform for in-line or in-field sensing of chemical substances.

Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

Toward non-invasive detection of concealed energetic materials in-field under ambient-light conditions

Spatially offset Raman spectroscopy (SORS) is demonstrated for the non-contact detection of energetic materials concealed within non-transparent, diffusely scattering containers. A modified design of an inverse SORS probe has been developed and tested. The SORS probe has been successfully used for the detection of various energetic substances inside different types of plastic containers. The tests have been successfully conducted under incandescent and fluorescent background lights as well as under daylight conditions, using a non-contact working distance of 6 cm. The interrogation times for the detection of the substances were less than 1 minute in each case, highlighting the suitability of the device for near real-time detection of concealed hazards in the field. The device has potential applications in forensic analysis and homeland security investigations.

Bicycling injuries and perceived risk

Introduction Government promotion of active transport has renewed interest in cycling safety. Research has shown that bicyclists are up to 20 times more likely to be involved in serious injury crashes than drivers. On-road cycling injuries are under-reported in police data, and many non-serious injuries are not recorded in any official database. This study aims to explore the relationships between rider characteristics and environmental factors that influence per kilometre risk of bicycle-related crash and non-crash injuries. Method A survey of 2,532 Queensland adults who had ridden at least once in the past year was conducted from October 2009 to March 2010, with most responses received online (99.3%). Riders were asked where they rode (footpath, bike path, road etc.), average travel speed, purpose of riding, type of bike ridden, how far and how often they rode in. Measures of rider experience, skill, safety perceptions, safety behaviours, crash involvement and demographic characteristics were also collected. RESULTS Increasing exposure and having more expensive bicycles were shown to reduce the risk per km of crash and non-crash injury rates, and to reduce perceived risk. Never wearing bright coloured clothing related to increased crash risk, use of fluorescent and reflective clothing had no effect on crash risk. Riding in low-speed environments, never using a front light, and riding in low-speed environments were associated with reduced non-crash injury risk. Perceived risk was influenced by exposure, use of conspicuity aids and helmets, riding for utilitarian reasons, and group-riding behaviours. DISCUSSION Perceived risk does not appear to influence injury rates and injury rates do not appear to influence the perceived risk of cycling. Riders who perceive cycling to be risky tend not to be commuters, do not engage in group riding and always wear helmets. Not all measures of conspicuity were associated with risk, with rear lights found to have no relationship to injury. The risks of experiencing a crash or non-crash injury were similar, therefore injury prevention strategies should expand their scope to include other factors such as the importance of bicycle set-up.

Real-time detection of concealed chemical hazards under ambient light conditions using Raman spectroscopy

Current concerns regarding terrorism and international crime highlight the need for new techniques for detecting unknown and hazardous substances. A novel Raman spectroscopy-based technique, spatially offset Raman spectroscopy (SORS), was recently devised for non-invasively probing the contents of diffusely scattering and opaque containers. Here, we demonstrate a modified portable SORS sensor for detecting concealed substances in-field under different background lighting conditions. Samples including explosive precursors, drugs and an organophosphate insecticide (chemical warfare agent surrogate) were concealed inside diffusely scattering packaging including plastic, paper and cloth. Measurements were carried out under incandescent and fluorescent light as well as under daylight to assess the suitability of the probe for different real-life conditions. In each case, it was possible to identify the substances against their reference Raman spectra in less than one minute. The developed sensor has potential for rapid detection of concealed hazardous substances in airports, mail distribution centers and customs checkpoints.

Photoclickable surfaces for profluorescent covalent polymer coatings

The nitrile imine-mediated tetrazole-ene cycloaddition reaction (NITEC) is introduced as a powerful and versatile conjugation tool to covalently ligate macromolecules onto variable (bio)surfaces. The NITEC approach is initiated by UV irradiation and proceeds rapidly at ambient temperature yielding a highly fluorescent linkage. Initially, the formation of block copolymers by the NITEC methodology is studied to evidence its efficacy as a macromolecular conjugation tool. The grafting of polymers onto inorganic (silicon) and bioorganic (cellulose) surfaces is subsequently carried out employing the optimized reaction conditions obtained from the macromolecular ligation experiments and evidenced by surface characterization techniques, including X-ray photoelectron spectroscopy and FT-IR microscopy. In addition, the patterned immobilization of variable polymer chains onto profluorescent cellulose is achieved through a simple masking process during the irradiation. Photoinduced nitrile imine-alkene 1,3-dipolar cycloaddition (NITEC) is employed to covalently bind well-defined polymers onto silicon oxide or cellulose. A diaryl tetrazole-functionalized molecule is grafted via silanization or amidification, respectively. Under UV light, a reactive nitrile imine rapidly forms and reacts with maleimide-functionalized polymers yielding a fluorescent linkage. Via a masking method, polymeric fluorescent patterns are achieved.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. © 2009 Blackwell Publishing Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Bicyclists overestimate their own night-time conspicuity and underestimate the benefits of retroreflective markers on the moveable joints

Conspicuity limitations make bicycling at night dangerous. This experiment quantified bicyclists’ estimates of the distance at which approaching drivers would first recognize them. Twenty five participants (including 13 bicyclists who rode at least once per week, and 12 who rode once per month or less) cycled in place on a closed-road circuit at night-time and indicated when they were confident that an approaching driver would first recognize that a bicyclist was present. Participants wore black clothing alone or together with a fluorescent bicycling vest, a fluorescent bicycling vest with additional retroreflective tape, or the fluorescent retroreflective vest plus ankle and knee reflectors in a modified ‘biomotion’ configuration. The bicycle had a light mounted on the handlebars which was either static, flashing or off. Participants judged that black clothing made them least visible, retroreflective strips on the legs in addition to a retroreflective vest made them most visible and that adding retroreflective materials to a fluorescent vest provides no conspicuity benefits. Flashing bicycle lights were associated with higher conspicuity than static lights. Additionally, occasional bicyclists judged themselves to be more visible than did frequent bicyclists. Overall, bicyclists overestimated their conspicuity compared to previously collected recognition distances and underestimated the conspicuity benefits of retroreflective markings on their ankles and knees. Participants mistakenly judged that a fluorescent vest that did not include retroreflective material would enhance their night-time conspicuity. These findings suggest that bicyclists have dangerous misconceptions concerning the magnitude of the night-time conspicuity problem and the potential value of conspicuity treatments.

The Use of a Nitroxide Probe in DMSO to Capture Radicals in Particulate Pollution

A profluorescent nitroxide was used to evaluate the oxidative potential of pollution derived from a compression ignition engine fuelled with biodiesel. The reaction products responsible for the observed fluorescence increase when a DMSO solution of nitroxide was exposed to biodiesel exhaust were determined by using HPLC/MS. The main fluorescent species was identified as a methanesulfonamide adduct arising from the reaction of the nitroxide with DMSO-derived sulfoxyl radicals.

Mid-gestation intra-amniotic infection with Ureaplasma parvum is resolved within spiny mice (Acomys cahirinus) by term delivery: but caused chronic infection of fetal lungs and placentae

Background: Ureaplasma species in amniotic fluid at the time of second-trimester amniocentesis increases the risk of preterm birth, but most affected pregnancies continue to term (Gerber et al. J Infect Dis 2003). We aimed to model intra-amniotic (IA) ureaplasma infection in spiny mice, a species with a relatively long gestation (39 days) that allows investigation of the disposition and possible clearance of ureaplasmas in the feto-placental compartment. Method: Pregnant spiny mice received IA injections of U. parvum serovar 6 (10µL, 1x104 colony-forming-units in PBS) or 10B media (10µL; control) at 20 days (d) of gestation (term=39d). At 37d fetuses (n=3 ureaplasma, n=4 control) were surgically delivered and tissues were collected for; bacterial culture, ureaplasma mba and urease gene expression by PCR, tissue WBC counts and indirect fluorescent antibody (IFA) staining using anti-ureaplasma serovar 6 (rabbit) antiserum. Maternal and fetal plasma IgG was measured by Western blot. Results: Ureaplasmas were not detected by culture or PCR in fetal or maternal tissues but were visualized by IFA within placental and fetal lung tissues, in association with inflammatory changes and elevated WBC counts (p<0.0001). Anti-ureaplasma IgG was detected in maternal (2/2 tested) and fetal (1/2 tested) plasma but not in controls (0/3). Conclusions: IA injection of ureaplasmas in mid-gestation spiny mice caused persistent fetal lung and placental infection even though ureaplasmas were undetectable using standard culture or PCR techniques. This is consistent with resolution of IA infection, which may occur in human pregnancies that continue to term despite detection of ureaplasmas in mid-gestation.

Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

Platelet-derived endothelial cell growth factor expression and angiogenesis in cervical intraepithelial neoplasia and squamous cell carcinoma of the cervix

Growth and metastatic spread of invasive carcinoma depends on angiogenesis, the formation of new blood vessels. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic growth factor for a number of solid tumors, including lung, bladder, colorectal, and renal cell cancer. Cervical intraepithelial neoplasia (CIN) is the precursor to squamous cell cervical carcinoma (SCC). Mean vessel density (MVD) increases from normal cervical tissue, through low- and high-grade CIN to SCC. We evaluated PD-ECGF immunoreactivity and correlated its expression with MVD in normal, premalignant, and malignant cervical tissue. PD-ECGF expression was assessed visually within the epithelial tissues and scored on the extent and intensity of staining. MVD was calculated by counting the number of vessels positive for von Willebrand factor per unit area subtending normal or CIN epithelium or within tumor hotspots for SCC. Cytoplasmic and/or nuclear PD-ECGF immunoreactivity was seen in normal epithelium. PD-ECGF expression significantly increased with histologic grade from normal, through low- and high-grade CIN, to SCC (P < .02). A progressive significant increase in the microvessel density was also seen, ranging from a mean of 28 vessels for normal tissue to 57 for SCC (P < .0005). No correlation was found between PD-ECGF expression and MVD (P = .45). We conclude that PD-ECGF expression and MVD increase as the cervix transforms from a normal to a malignant phenotype. PD-ECGF is thymidine phosphorylase, a key enzyme in the activation of fluoropyrimidines, including 5-fluorouracil. Evaluation of PD-ECGF thymidine phosphorylase expression may be important in designing future chemotherapeutic trials in cervical cancer. Copyright (C) 2000 by W.B. Saunders Company.