Towards continuous monitoring of TNF-α at picomolar concentrations using biosensing by particle motion

The ability to continuously monitor cytokines is desirable for fundamental research studies and healthcare applications. Cytokine release is characterized by picomolar circulating concentrations, short half-lives, and rapid peak times. Here, we describe the characteristics and feasibility of a particle-based biosensing technique for continuous monitoring of TNF-α at picomolar concentrations. The technique is based on the optical tracking of particle motion and uses an antibody sandwich configuration. Experimental results show how the analyte concentration influences the particle diffusivity and characteristic response time of the sensor, and how the sensitivity range depends on the antibody functionalization density. Furthermore, the data clarifies how antibodies supplemented in solution can shorten the characteristic response time. Finally, we demonstrate association rate-based sensing as a first step towards continuous monitoring of picomolar TNF-α concentrations, over a period of 2 h with delay times under 15 min. The insights from this research will enable the development of continuous monitoring sensors using high-affinity binders, providing the sensitivity and speed needed in applications like cytokine monitoring.</p

Sandwich Immunosensor Based on Particle Motion:How Do Reactant Concentrations and Reaction Pathways Determine the Time-Dependent Response of the Sensor?

To control and optimize the speed of a molecular biosensor, it is crucial to quantify and understand the mechanisms that underlie the time-dependent response of the sensor. Here, we study how the kinetic properties of a particle-based sandwich immunosensor depend on underlying parameters, such as reactant concentrations and the size of the reaction chamber. The data of the measured sensor responses could be fitted with single-exponential curves, with characteristic response times that depend on the analyte concentration and the binder concentrations on the particle and substrate. By comparing characteristic response times at different incubation configurations, the data clarifies how two distinct reaction pathways play a role in the sandwich immunosensor, namely, analyte binding first to particles and thereafter to the substrate, and analyte binding first to the substrate and thereafter to a particle. For a concrete biosensor design, we found that the biosensor is dominated by the reaction pathway where analyte molecules bind first to the substrate and thereafter to a particle. Within this pathway, the binding of a particle to the substrate-bound analyte dominates the sensor response time. Thus, the probability of a particle interacting with the substrate was identified as the main direction to improve the speed of the biosensor while maintaining good sensitivity. We expect that the developed immunosensor and research methodology can be generally applied to understand the reaction mechanisms and optimize the kinetic properties of sandwich immunosensors with particle labels.</p

Guidelines for Identifying Homologous Recombination Events in Influenza A Virus

The rapid evolution of influenza viruses occurs both clonally and non-clonally through a variety of genetic mechanisms and selection pressures. The non-clonal evolution of influenza viruses comprises relatively frequent reassortment among gene segments and a more rarely reported process of non-homologous RNA recombination. Homologous RNA recombination within segments has been proposed as a third such mechanism, but to date the evidence for the existence of this process among influenza viruses has been both weak and controversial. As homologous recombination has not yet been demonstrated in the laboratory, supporting evidence, if it exists, may come primarily from patterns of phylogenetic incongruence observed in gene sequence data. Here, we review the necessary criteria related to laboratory procedures and sample handling, bioinformatic analysis, and the known ecology and evolution of influenza viruses that need to be met in order to confirm that a homologous recombination event occurred in the history of a set of sequences. To determine if these criteria have an effect on recombination analysis, we gathered 8307 publicly available full-length sequences of influenza A segments and divided them into those that were sequenced via the National Institutes of Health Influenza Genome Sequencing Project (IGSP) and those that were not. As sample handling and sequencing are executed to a very high standard in the IGSP, these sequences should be less likely to be exposed to contamination by other samples or by laboratory strains, and thus should not exhibit laboratory-generated signals of homologous recombination. Our analysis shows that the IGSP data set contains only two phylogenetically-supported single recombinant sequences and no recombinant clades. In marked contrast, the non-IGSP data show a very large amount of potential recombination. We conclude that the presence of false positive signals in the non-IGSP data is more likely than false negatives in the IGSP data, and that given the evidence to date, homologous recombination seems to play little or no role in the evolution of influenza A viruses

Improved detection of artifactual viral minority variants in high-throughput sequencing data

textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs)

Widespread transfer of resistance genes between bacterial species in an intensive care unit: implications for hospital epidemiology

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Effects of Media Ratings on Children and Adolescents: A Litmus Test of the Forbidden Fruit Effect

Media ratings serve to inform parents about and protect minors from violent or otherwise harmful media content. Most of these systems use age pictograms and content warning pictograms for entertainment products. An experiment was conducted to investigate whether these pictograms, contrary to their purpose, have an appealing effect on children and adolescents. Compared to prior studies into this ‘‘forbidden fruit effect’’ concerning DVDs and games, more realistic materials were used. In the experiment, 322 elementary school students (9–11 years) and 335 high school students (13–15 years) were exposed to 10 DVD or game covers and asked to judge the products’ attractiveness. The results show that the pictograms did not increase the attractiveness of games and DVDs for children and adolescents

Triple Combination of Amantadine, Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza

A randomized trial predicting response to cognitive rehabilitation in multiple sclerosis:Is there a window of opportunity?

BACKGROUND: Cognitive training elicits mild-to-moderate improvements in cognitive functioning in people with multiple sclerosis (PwMS), although response heterogeneity limits overall effectiveness. OBJECTIVE: To identify patient characteristics associated with response and non-response to cognitive training. METHODS: Eighty-two PwMS were randomized into a 7-week attention training (n = 58, age = 48.4 ± 10.2 years) or a waiting-list control group (n = 24, age = 48.5 ± 9.4 years). Structural and functional magnetic resonance imaging (MRI) was obtained at baseline and post-intervention. Twenty-one healthy controls (HCs, age = 50.27 ± 10.15 years) were included at baseline. Responders were defined with a reliable change index of 1.64 on at least 2/6 cognitive domains. General linear models and logistic regression were applied. RESULTS: Responders (n = 36) and non-responders (n = 22) did not differ on demographics, clinical variables and baseline cognition and structural MRI. However, non-responders exhibited a higher baseline functional connectivity (FC) between the default-mode network (DMN) and the ventral attention network (VAN), compared with responders (p = 0.018) and HCs (p = 0.001). Conversely, responders exhibited no significant baseline differences in FC compared with HCs. Response to cognitive training was predicted by lower DMN-VAN FC (p = 0.004) and DMN-frontoparietal FC (p = 0.029) (Nagelkerke R(2) = 0.25). CONCLUSION: An intact pre-intervention FC is associated with cognitive training responsivity in pwMS, suggesting a window of opportunity for successful cognitive interventions

Destination shapes antibiotic resistance gene acquisitions, abundance increases, and diversity changes in Dutch travelers

BACKGROUND: Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel\u27s effects on the gut resistome remain largely unknown. METHODS: Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations. RESULTS: Travel markedly increased the abundance and α-diversity of AMR genes in the travelers\u27 gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including qnr fluoroquinolone resistance genes, bla CONCLUSIONS: Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes