Real-time tracking of ionic nano-domains under shear flow

The behaviour of ions at solid–liquid interfaces underpins countless phenomena, from the conduction of nervous impulses to charge transfer in solar cells. In most cases, ions do not operate as isolated entities, but in conjunction with neighbouring ions and the surrounding solution. In aqueous solutions, recent studies suggest the existence of group dynamics through water-mediated clusters but results allowing direct tracking of ionic domains with atomic precision are scarce. Here, we use high-speed atomic force microscopy to track the evolution of Rb+, K+, Na+ and Ca2+ nano-domains containing 20 to 120 ions adsorbed at the surface of mica in aqueous solution. The interface is exposed to a shear flow able to influence the lateral motion of single ions and clusters. The results show that, when in groups, metal ions tend to move with a relatively slow dynamics, as can be expected from a correlated group motion, with an average residence timescale of ~ 1–2 s for individual ions at a given atomic site. The average group velocity of the clusters depends on the ions’ charge density and can be explained by the ion’s hydration state. The lateral shear flow of the fluid is insufficient to desorb ions, but indirectly influences the diffusion dynamics by acting on ions in close vicinity to the surface. The results provide insights into the dynamics of ion clusters when adsorbed onto an immersed solid under shear flow.

Efficient Fluorescence-Based Localization Technique for Tracking Endophytes Route in Host-Plants Colonization

Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence-based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host-plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC-10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid-mediated transformation efficiencies of antibiotic-resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA-tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence-based technique's high resolution and reproducibility make it a platform-independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host-plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately three months.

A method for low-cost, low-impact insect tracking using retroreflective tags

Current methods for direct tracking of individual bee movement behaviour have several limitations. In particular, the weight and size of some types of electronic tag may limit their use to larger species. Radars and other electronic systems are also often large and very expensive to deploy. A tool is needed that complements these electronic-tag methods. In particular one that is simple to use, low-cost, can have a high spatial resolution and can be used with smaller insects. This paper presents a candidate method that uses retroreflective tags. These are detected using a camera with a global electronic shutter, with which we take photos with and without a flash. The tags can be detected by comparing these two photos. The small retroreflective tags are simple and light-weight, allowing many bees to be tagged at almost no cost and with little effect on their behaviour. We demonstrate this retroreflector-based tracking system (RTS) with a series of simple experiments: Training and validation with a manually positioned tag; case studies of individual bees; tracking multiple bees as they forage in a garden; use of real-time monitoring to allow easy re-observation to enable a simple floral preference experiment; and a very brief experiment with 3D path reconstruction (integrating two devices). We found we could detect bees to a range of about 35 m with the current configuration.%; We finally compare tagged and untagged bee foraging to assess the effect of the tags on bee behaviour. We envisage the system will be used in future to increase detection rates in mark-re-observation studies; provide 3D flight path analysis; and for automated long-term monitoring. In summary, this novel tracking method has advantages that complement those of electronic-tag tracking which we believe will lead to new applications and areas of research.

Tracking interfacial single-molecule pH and binding dynamics via vibrational spectroscopy

Understanding single-molecule chemical dynamics of surface ligands is of critical importance to reveal their individual pathways and, hence, roles in catalysis, which ensemble measurements cannot see. Here, we use a cascaded nano-optics approach that provides sufficient enhancement to enable direct tracking of chemical trajectories of single surface-bound molecules via vibrational spectroscopy. Atomic protrusions are laser-induced within plasmonic nanojunctions to concentrate light to atomic length scales, optically isolating individual molecules. By stabilizing these atomic sites, we unveil single-molecule deprotonation and binding dynamics under ambient conditions. High-speed field-enhanced spectroscopy allows us to monitor chemical switching of a single carboxylic group between three discrete states. Combining this with theoretical calculation identifies reversible proton transfer dynamics (yielding effective single-molecule pH) and switching between molecule-metal coordination states, where the exact chemical pathway depends on the intitial protonation state. These findings open new domains to explore interfacial single-molecule mechanisms and optical manipulation of their reaction pathways.

Occurrence of the p019 gene in the blaKPC-harbouring plasmids: adverse clinical impact for direct tracking of KPC-producing Klebsiella pneumoniae by MALDI-TOF MS

MALDI-TOF MS has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated K. pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or non carbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by WGS and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid, but it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K) and IncX3. Besides, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

Direct tracking of single proviruses reveals HIV-1/LEDGF complexes excluded from virus-induced membraneless organelles

The ultimate goal of HIV-1 is integration into the host chromatin to optimize the release of high levels of viral progeny and discretely coexist with the host. To uncover the HIV-1 DNA fate in the nuclear landscape we directly tracked the viral DNA (vDNA) and the viral RNA (vRNA) by coupling HIV-1 ANCHOR technology with RNA FISH or MCP-MS2 RNA-tagging bacterial system. Our computational imaging analysis revealed that proviral forms are early located in proximity of the nuclear periphery of mitotic and non-mitotic cells. We also observed that HIV-1 infection prompts clustering formation of the host factor CPSF6 restructuring membraneless organelles enriched in both viral proteins and speckle factors. Interestingly, we observed that integrase proteins are retained in CPSF6 clusters, while the late retrotranscribed DNA was excluded from HIV-induced membranelless organelles (HIV-1 MLOs), indicating that those structures are not proviral sites, but orchestrate viral events prior to the integration step. HIV-1 MLOs are in the vicinity of pre-existing LEDGF clusters. Importantly, we identified actively transcribing proviruses localize, outside HIV-1 MLOs, in LEDGF-abundant regions, known to be active chromatin sites. This study highlights single functional host-proviral complexes in their nuclear landscape, which is markedly restructured by HIV-1 to favor viral replication.

Direct Tracking of the Pareto Front of a Multi-Objective Optimization Problem

In this paper, some methodologies aimed at the identification of the Pareto front of a multi-objective optimization problem are presented and applied. Three different approaches are presented: local sampling, Pareto front resampling and Normal Boundary Intersection (NBI). A first approximation of the Pareto front is obtained by a regular sampling of the design space, and then the Pareto front is improved and enriched using the other two above mentioned techniques. A detailed Pareto front is obtained for an optimization problem where algebraic objective functions are applied, also in comparison with standard techniques. Encouraging results are also obtained for two different ship design problems. The use of the algebraic functions allows for a comparison with the real Pareto front, correctly detected. The variety of the ship design problems allows for a generalization of the applicability of the methodology.