Development of a liquid chromatography/tandem mass spectrometry method for monitoring of long-term exposure to parabens.

RATIONALE: Parabens, the alkyl esters of 4-hydroxybenzoic acid, are a family of compounds widely used as preservatives in cosmetic products, including for children, and some are permitted in foods. Parabens are known to be weak endocrine disruptors because they interfere with the function of endogenous hormones through binding to estrogen receptors. Therefore, the levels of parabens in biological samples indicate endocrine-disruptive exposure. In particular, hair samples can provide information on accumulated exposure to parabens. METHODS: For monitoring of long-term exposure to parabens, an improved analytical method for rapid and direct determination in hair sample was developed involving ultra-performance liquid chromatography-tandem mass spectrometry using on-line extraction. Five parabens (methyl-, ethyl-, propyl-, butyl- and benzylparaben) were separated within 10 min after incubation with 1 N HCl. Parabens were separated using a Waters BEH C18 column (2.1 mm × 100 mm, 1.7 µm) and a mobile phase consisting of 10 mM ammonium acetate in water and acetonitrile with a gradient program at a flow rate of 300 µL/min. The analysis of the separated parabens was monitored with electrospray negative ionization tandem mass spectrometry. RESULTS: The linearity of the method was demonstrated by r2  ≥ 0.994. The limits of detection as defined by a signal-to-noise ratio of 3 were 1-5 ng/g. The mean concentration of the five parabens in hair of human subjects was measured to be 55.6 ± 24.3 to 136.9 ± 48.5 ng/g. CONCLUSIONS: The levels of parabens in hair samples may play an important role in understanding probable endocrine-disruptive exposure, and the described method could be used to evaluate and monitor long-term exposure to parabens as endocrine disruptors.

Quantitative Correlation between Infectivity and Gp120 Density on HIV-1 Virions Revealed by Optical Trapping Virometry.

The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.

Simultaneous determination of volatile organic compounds with a wide range of polarities in urine by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry.

RATIONALE: Volatile organic compounds (VOCs) are ubiquitous environmental pollutants that have a high vapor pressure at room temperature. Some VOCs have been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC), because they can bind to DNA and cause cell mutations. Therefore, monitoring of VOCs in human urine is very important to evaluate the correlation between exposure to VOCs and human disease. METHODS: We have developed an improved analytical method for the simultaneous determination of VOCs with a wide range of polarities in human urine samples by headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography/mass spectrometry (GC/MS). In the improved method, a bi-polar carboxen-polydimethylsiloxane (CAR/PDMS) fiber was used for the optimized extraction of 15 VOCs with a wide range of polarities, including benzene, toluene, ethylbenzene, xylenes (BTEX), alkylbenzenes, cresols, and naphthalene, in human urine samples. Extracted VOCs from the human urine were effectively separated by GC using a mid-polarity column (DB-35, 35% phenylmethylpolysiloxane) and monitored by MS using extracted ion monitoring (EIM) mode. RESULTS: Under the optimized method, the linearity of the calibration curves was greater than 0.993. The limits of detection (LODs) at a signal-to-noise (S/N) ratio of 3 were 0.3-0.6 ng/mL. The coefficients of variation were in the range of 0.1-9.7% for within-day variation and 0.2-14.2% for day-to-day variation. CONCLUSIONS: The method was shown to be rapid and simple for the simultaneous determination of VOCs with a wide range of polarities in human urine and it could be applied to monitoring and to biomedical investigations to check exposure to VOCs. Copyright © 2017 John Wiley & Sons, Ltd.

Recovery style and stigma in psychosis: the healing power of integrating.

INTRODUCTION: Persecutory delusions are a very common symptom in psychotic disorders and represent a considerable cost for both patients and for society. The way in which a person faces their psychotic disorder (i.e., recovery style) has impact on their recovery. The impact of coping style as a moderator in the course of their illness has not been studied sufficiently in persecutory delusions. In addition, internalised stigma is a common process in psychosis that not only might affect emotional distress, but might also shape recovery style. The goal of this study was to examine the moderator role of recovery style between internalised stigma and emotional distress in people with persecutory delusions. METHODS: All 50 people with persecutory beliefs were assessed by the Recovery Style Questionnaire, the Beck Anxiety Inventory, Beck Depression Inventory, Second Edition, and Internalised Stigma of Mental Illness. RESULTS: Moderation analysis showed that participants with a sealing-over recovery style had high levels of depression when they experienced internalised stigma and low levels of depression only when internalised stigma was low. However, participants with an integration recovery style presented similar levels of depression regardless of the level of their internalised stigma. CONCLUSIONS: Findings suggest the moderator role of recovery style between internalised stigma and depression in people with persecutory delusions.

Dynasore inhibition on productive infection of HIV-1 in commonly used cell lines is independent of transferrin endocytosis.

The route of HIV-1 entry for productive infection in CD4+ host cells is a fundamental question for the molecular understanding of HIV-1 infection and transmission. Although direct fusion has long been thought to be the mode of entry, recent studies have suggested that productive entry of HIV-1 may actually occur through dynamin-dependent endocytosis. In several of these studies, dynasore, a noncompetitive inhibitor of the GTPase activity of dynamin, has been used to support this conclusion. Here we show that dynasore does produce inhibitory effects on the productive infection of HIV-1 in several commonly used cell lines. This effect is present regardless of the methods used to facilitate the infection of HIV-1. However, transferrin uptake remains fully functional in these cell lines upon dynasore treatment. Therefore, the inhibition on HIV-1 infection by dynasore in these cell lines is due to an effect that is independent of transferrin endocytosis. The use of dynasore in probing the role of endocytosis in HIV-1 infection should be corroborated by other methods.

Using optical trap to measure the refractive index of a single animal virus in culture fluid with high precision.

The refractive index (RI) is a fundamental parameter of materials that can be used to distinguish and sort materials of different nature. Although the RI of a virus is required for many optics-based biosensing applications, RIs of animal viruses have never been measured. Here we have developed a technique that can measure the RI of individual viruses in aqueous media with high precision. This technique is based on optical trapping of single virions and works by relating the size and RI of a single virus to the stiffness of an optical trap. We have derived an analytic expression to quantitatively describe the optical trapping of these particles. We have validated this equation using nanoparticles of known RI, and measured the RI of individual human immunodeficiency viruses type-1, which yielded a value of 1.42 at 830 nm with less than 2% coefficient of variation. This value is much lower than the RI typically assumed for viruses, but very close to that of 2.0 M sucrose solution in water. To the best of our knowledge, this is the first report on the experimental measurement of the RI for a single animal virus in aqueous media. This technique does not require prior knowledge on the diameter of the nanoparticles, and can be applied to other viruses or nanoparticles for accurate measurement of RI that is critical for the label-free detection of these particles in various settings.

Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution.

Optical tweezers use the momentum of photons to trap and manipulate microscopic objects, contact-free, in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application to study viruses has been very limited, largely due to their small size. Here, using optical tweezers that can simultaneously resolve two-photon fluorescence at the single-molecule level, we show that individual HIV-1 viruses can be optically trapped and manipulated, allowing multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at the single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

Optimized Infectivity of the Cell-Free Single-Cycle Human Immunodeficiency Viruses Type 1 (HIV-1) and Its Restriction by Host Cells.

The infectivity of retroviruses such as HIV-1 in plasma or cultured media is less than 0.1% in general, the mechanisms of which are not yet fully understood. One possible explanation among others is the potential presence of large numbers of defective virions in a virus pool, which limits the apparent infectivity of HIV virions. To test this hypothesis, we have varied the culture conditions used to generate single-cycle HIV-1 virions. Among these culture variables, virion harvest time, media change after transfection, and envelope plasmid input can all improve HIV-1 infectivity by reducing the number of defective virions. A harvest time of 18-24 hours post transfection as opposed to 48 hours, and a media change six hours post transfection both improve viral infectivity. An optimal quantity of envelope plasmid input during transfection was also found. Collectively, these conditions increased the infectivity of HIV-1 virions by sevenfold compared to normally reported values in TZM-bl indicator cell lines. These conditions also increased the infectivity of HIV-1 in CD4(+) T cells, suggesting that these conditions work by increasing the intrinsic infectivity of a virus pool. Nevertheless, these improvements on virion infectivity were marginal compared to the impact of host cells on HIV infection, which can decrease the apparent infectivity by 19-fold even for the most optimized viruses. These results suggest that the infectivity of HIV-1 virions can be optimized by reducing the number of defective virions; however, viral-cell interactions may pose a major barrier for HIV-1 infectivity.