Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeriamonocytogenes in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food sample

Nucleic Acid Lateral Flow Immunoassay for the Detection of Pathogenic Bacteria from Food

Nucleic acid lateral flow immunoassay (NALFIA) is a method combining molecular biological principle of detection with immunochemical principle of visualisation. Following isolation of DNA from the sample, a duplex PCR with two primer sets, of which one was labelled with biotin and the other with digoxigenin or fluorescein, respectively, was performed. The PCR solution and carbon particles conjugated with avidin are directly added to the nitrocellulose membrane with two test lines of immobilised antibodies specific for digoxigenin and fluorescein. The appearance of a black line indicates the presence of specific amplicon. We would like to present the NALFIA for the simultaneous detection of L. monocytogenes in particular and the genus Listeria in general, in food. Bacteria from the genus Listeria frequently contaminate a large variety of foods. Occurrence of Listeria strains in food may indicate errors in good hygienic and manufacturing practice, only L. monocytogenes is a significant human and animal pathogen responsible for the serious illness listeriosis. Conventional microbiological methods for L. monocytogenes detection are laborious and take several days to achieve a confirmed identification

Generation of vortices and observation of Quantum Turbulence in an oscillating Bose-Einstein Condensate

We report on the experimental observation of vortex formation and production of tangled vortex distribution in an atomic BEC of Rb-87 atoms submitted to an external oscillatory perturbation. The oscillatory perturbations start by exciting quadrupolar and scissors modes of the condensate. Then regular vortices are observed finally evolving to a vortex tangle configuration. The vortex tangle is a signature of the presence of a turbulent regime in the cloud. We also show that this turbulent cloud has suppression of the aspect ratio inversion typically observed in quantum degenerate bosonic gases during free expansion.Comment: to appear in JLTP - QFS 200

Arrays on disc for screening and quantification of pollutants

A rapid compact disc based methodology for screening and quantification of organic pollutants in mandarin juices is presented. The assay is established on the coating conjugate indirect competitive principle and developed under disc-array configuration. The detection is based on the acquisition of attenuated reflective signals that were proportional to optical density of the immunoreaction product. The competitive assay is applied to quantify simultaneously, in a selective manner, non-systemic insecticides in mandarin juices. The detection limits were 0.2 and 0.1 ¿g L¿1 and the sensitivity 2.1 and 1.5 ¿g L¿1, for chlorpyrifos and fenthion, respectively. Pollutants were directly quantified after sample dilution in a total time of 40 min. Also, the implementation of positive and negative controls into the array configuration served as an automatic quality control test. The effect of thermal treatment on pesticide dissipation was studied and found that it was insignificant under the studied conditions. Recovery intervals ranged from 96¿105% to 94¿103%, for chlorpyrifos and fenthion, respectively and were similar to those obtained with gas chromatography coupled to mass spectrometry. In the current configuration, 64 samples can be simultaneously analyzed on a disc at a very competitive value, demonstrating its potential for high-throughput multiplexed screening applications for controlled monitoring programs in low-level labs or outside the lab settingThis work was funded by the projects FEDER CTQ2010-15943 (CICYT, Spain), and PROMETEO 2010/008 and ACOMP/2012/158 (Generalitat Valenciana).Navarro, P.; Morais Ezquerro, SB.; Gavaldón, JA.; Pérez, AJ.; Puchades Pla, R.; Maquieira Catala, Á. (2013). Arrays on disc for screening and quantification of pollutants. Analytica Chimica Acta. 784:59-64. https://doi.org/10.1016/j.aca.2013.04.041S596478

Cell line-dependent variability in HIV activation employing DNMT inhibitors

Long-lived reservoirs of Human Immunodeficiency Virus (HIV) latently infected cells present the main barrier to a cure for HIV infection. Much interest has focused on identifying strategies to activate HIV, which would be used together with antiretrovirals to attack reservoirs. Several HIV activating agents, including Tumor Necrosis Factor alpha (TNFα) and other agents that activate via NF-kB are not fully effective in all latent infection models due to epigenetic restrictions, such as DNA methylation and the state of histone acetylation. DNA methyltransferases (DNMT) inhibitors like 5-aza-2'deoxycytidine (Aza-CdR) and histone deacetylase (HDAC) inhibitors like Trichostatin A (TSA) have been proposed as agents to enhance reactivation and have shown activity in model systems. However, it is not clear how the activities of DNMT and HDAC inhibitors range across different latently infected cell lines, potential models for the many different latently infected cells within an HIV patient. We determined HIV activation following treatment with TNFα, TSA and Aza-CdR across a range of well known latently infected cell lines. We assessed the activity of these compounds in four different Jurkat T cell-derived J-Lat cell lines (6.3, 8.4, 9.2 and 10.6), which have a latent HIV provirus in which GFP replaces Nef coding sequence, and ACH-2 and J1.1 (T cell-derived), and U1 (promonocyte-derived) cell lines with full-length provirus. We found that Aza-CdR plus TNFα activated HIV at least twice as well as TNFα alone for almost all J-Lat cells, as previously described, but not for J-Lat 10.6, in which TNFα plus Aza-CdR moderately decreased activation compared to TNFα alone. Surprisingly, a much greater reduction of TNFα-stimulated activation with Aza-CdR was detected for ACH-2, J1.1 and U1 cells. Reaching the highest reduction in U1 cells with a 75% reduction. Interestingly, Aza-CdR not only decreased TNFα induction of HIV expression in certain cell lines, but also decreased activation by TSA. Since DNMT inhibitors reduce the activity of provirus activators in some HIV latently infected cell lines the use of epigenetic modifying agents may need to be carefully optimized if they are to find clinical utility in therapies aimed at attacking latent HIV reservoirs

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4⁺ T-Cells to Recognition by Cytotoxic T-Lymphocytes

Resting CD4⁺ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8⁺ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8⁺ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8⁺ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8⁺ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8⁺ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam₃CSK₄. In contrast, we did not observe CD8⁺ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8⁺ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8⁺ T-cells in HIV eradication strategies.United States. National Institutes of Health (AI111860

HIV infection and HERV expression: a review

The human genome contains multiple copies of retrovirus genomes known as endogenous retroviruses (ERVs) that have entered the germ-line at some point in evolution. Several of these proviruses have retained (partial) coding capacity, so that a number of viral proteins or even virus particles are expressed under various conditions. Human ERVs (HERVs) belong to the beta-, gamma-, or spuma- retrovirus groups. Endogenous delta- and lenti- viruses are notably absent in humans, although endogenous lentivirus genomes have been found in lower primates. Exogenous retroviruses that currently form a health threat to humans intriguingly belong to those absent groups. The best studied of the two infectious human retroviruses is the lentivirus human immunodeficiency virus (HIV) which has an overwhelming influence on its host by infecting cells of the immune system. One HIV-induced change is the induction of HERV transcription, often leading to induced HERV protein expression. This review will discuss the potential HIV-HERV interactions

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch

Background: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4+ T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-aα, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4+ T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency