Applications of a transportable Raman spectrometer for the in situ detection of controlled substances at border controls

A transportable Raman spectrometer was tested for the detection of illicit drugs seized during border controls. In a first step, the analysis methodology was optimized using reference substances such as diacetylmorphine (heroin), cocaine and amphetamine (as powder or liquid forms). Adequate focalisation distance and times of analysis, influence of daylight and artificial light sources, repeatability and limits of detection were studied. In a second step the applications and limitations of the technique to detect the illicit substances in different mixtures and containers was evaluated. Transportable Raman spectroscopy was found to be adequate for a rapid screen of liquids and powders for the detection and identification of controlled substances. Additionally, it had the advantage over other portable techniques, such as ion mobility spectrometry, of being non-destructive and capable of rapid analysis of large quantities of substances through containers such as plastic bags and glass bottles.

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

Comparison between a high-resolution single-stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs.

The capabilities of a high-resolution (HR), accurate mass spectrometer (Exactive-MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50,000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive-MS was compared with that of a triple quadrupole mass spectrometer (TQ-MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole-N-oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR-MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR-MS offers several benefits over TQ-MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR-MS delivers comparable results to TQ-MS in routine quantitative drug analyses. Considering the advantages of HR-MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed.

A cross-sectional survey of attitudes to HIV risk and rapid HIV testing among clients of sex workers in Switzerland.

OBJECTIVES: To assess attitudes to HIV risk and acceptability of rapid HIV testing among clients of street-based female sex workers (FSW) in Lausanne, Switzerland, where HIV prevalence in the general population is 0.4%. METHODS: The authors conducted a cross-sectional study in the red light district of Lausanne for five nights in September of 2008, 2009 and 2010. Clients of FSW were invited to complete a questionnaire in the street assessing demographic characteristics, attitudes to HIV risk and HIV testing history. All clients interviewed were then offered anonymous finger stick rapid HIV testing in a van parked on-site. RESULTS: The authors interviewed 112, 127 and 79 clients in 2008, 2009 and 2010, respectively. All were men, average age 32-37 years old; 40-60% were in a stable relationship. History of unprotected sex was higher with non-commercial partners (33-50%) than with FSW (6-11%); 29-46% of clients had never undergone an HIV test. Anonymous rapid HIV testing was accepted by 45-50% of clients. Out of 109 HIV tests conducted during the three study periods, none was reactive. CONCLUSIONS: On-site HIV counselling and testing is acceptable among clients of FSW in this urban setting. These individuals represent an unquantified population, a proportion of which has an incomplete understanding of HIV risk in the face of high-risk behaviour, with implications for potential onward transmission to non-commercial sexual partners.

Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes.

Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.

Rapid evolution of cancer/testis genes on the X chromosome.

BACKGROUND: Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole. RESULTS: To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes. CONCLUSION: Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Microcalorimetry Assay for Rapid Detection of Voriconazole Resistance in Aspergillus fumigatus.

We describe a calorimetric assay for detection of voriconazole-resistant Aspergillus fumigatus within 8 h. Among 27 genetically distinct strains, all 21 resistant and all 6 susceptible strains were correctly identified by measurement of fungal heat production in the presence of voriconazole. This proof-of-concept study demonstrates the potential of microcalorimetry for rapid detection of azole resistance in A. fumigatus.

Mass spectrometry as a rapid and powerful alternative to antibodies for detecting LPXTG wall-associated proteins of Staphylococcus aureus

Functional characterization of transformed or natively present bacterial virulence proteins can be achieved employing various model systems. A prerequisite is to verify the correct expression of the transformed protein or the presence of the native protein in the microbe. Traditionally, antibodies are raised against the protein or a peptide thereof, followed by Western blot analysis or by fluorescence-activated cell sorting. Alternatively, the protein-coding gene can be fused with a downstream reporter gene, the expression of which reports the simultaneous expression of the upstream recombinant protein. Although being powerful, these methods are time consuming, especially when multiple proteins must be assessed. Here we describe a novel way to validate the expression of Gram-positive surface proteins covalently attached to the peptidoglycan. Eighteen out of the 21 known LPXTG-motif carrying cell wall-associated proteins of Staphylococcus aureus were cloned in Lactoccocus lactis either alone, in combinations or as truncated forms, and their correct expression was assessed by liquid chromatography coupled to mass spectrometry (LC-MS). The method is rapid, sensitive and precise. It can identify multiple proteins in transformed constructs without the time and cost needed for raising and testing multiple sets of antibodies.

Neuromodulation by oxytocin and vasopressin in the central nervous system as a basis for their rapid behavioral effects

The last several years have seen an increasing number of studies that describe effects of oxytocin and vasopressin on the behavior of animals or humans. Studies in humans have reported behavioral changes and, through fMRI, effects on brain function. These studies are paralleled by a large number of reports, mostly in rodents, that have also demonstrated neuromodulatory effects by oxytocin and vasopressin at the circuit level in specific brain regions. It is the scope of this review to give a summary of the most recent neuromodulatory findings in rodents with the aim of providing a potential neurophysiological basis for their behavioral effects. At the same time, these findings may point to promising areas for further translational research towards human applications.

Withholding antimalarials in febrile children who have a negative result for a rapid diagnostic test.

BACKGROUND: The availability of a rapid diagnostic test for malaria (RDTm) allows accurate diagnosis at all levels of health facilities. The objective of the present study was to evaluate the safety of withholding antimalarials in febrile children who have a negative test result. METHODS: We conducted a prospective 2-arm longitudinal study in areas of Tanzania that are moderately and highly endemic for malaria. Children with a history of fever were managed routinely by resident clinicians of 2 health facilities, except that no antimalarials were prescribed if the RDTm result was negative. Children were followed up at home on day 7. The main outcome was the occurrence of complications in children with negative RDTm results; children with positive RDTm results were followed up for the same outcomes for indirect comparison. RESULTS: One thousand children (median age, 24 months) were recruited. Six hundred three children (60%) had a negative RDTm result. Five hundred seventy-three (97%) of these children were cured on day 7. Forty-nine (8%) of the children with negative RDTm results spontaneously visited the dispensary before day 7, compared with 10 (3%) of the children with positive RDTm results. All children who had negative initial results had negative results again when they were tested either at spontaneous attendance or on day 7 because they were not cured clinically, except for 3 who gave positive results on days 2, 4, and 7 respectively but who did not experience any complication. Four children who had negative initial results were admitted to the hospital subsequently, all with negative results for malaria tests upon admission. Two of them died, of causes other than malaria. CONCLUSIONS: Not giving antimalarial drugs in febrile children who had a negative RDTm result was safe, even in an area highly endemic for malaria. Our study provides evidence for treatment recommendations based on parasitological diagnosis in children <5 years old.

Genotype 3 is associated with rapid histological progression in chronic HCV infection

While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of the present study was to assess independent predictors for fibrosis progression. Methods: We identified 1540 patients from the Swiss Hepatitis C Cohort database with at least one liver biopsy prior to antiviral treatment. Factors associated with fibrosis stage, steatosis and histological activity were assessed in univariate and multivariate regression models. Fibrosis progression rate per year was calculated in a subgroup of 1263 patients, in whom risk factors were assessed by cumulative incidence curves, logistic and linear regression models. Results: Independent risk factors for rapid fibrosis progression included male sex (OR = 1.66, 95% CI 1.25-2.21, P <0.001), age at infection (OR = 1.08, 95% CI 1.06-1.10, P <0.001), histological activity (OR = 2.14, 95% CI 1.61-2.85, P <0.001) and genotype 3 (OR = 1.97, 95% CI 1.43-2.72, P <0.001). Genotype 2 was associated with slow progression (OR = 0.51, 95% CI 0.30-0.89, P = 0.02), but this observation may be due to the decreased prevalence of genotype 2 over the last decades, leading to an overrepresentation of subjects with genotype 2 with a slow progression rate. Conclusion: This study shows a significant association of genotype 3 with accelerated fibrosis. While assessing risk factors for fibrosis progression, the changing epidemiology of HCV genotypes over time needs to be taken into account.