Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Spontaneous Extrusion of a Conjunctivolith Containing Herpes Virus Confirmed by Electron Microscopy and Energy Dispersive Spectroscopy.

: This report describes the spontaneous extrusion from between the eyelids of a presumed conjunctivolith in a patient with resolving severe herpes zoster ophthalmicus. A 57-year-old man presented for ophthalmologic assessment and management due to severe left herpes zoster ophthalmicus. At one subsequent ophthalmologic assessment, a conjunctivolith spontaneously egressed the lateral commissure of the OS when the lateral fornix was inspected. The conjunctivolith was retrieved from the floor of the consulting room. Electron microscopic analysis and energy dispersive spectroscopy was undertaken to determine its composition. Scanning electron microscopy showed that the conjunctivolith was composed of carbon, calcium, and oxygen. Transmission electron microscopy diagnosed Herpes virus within the conjunctivolith. Conjunctivoliths, or possible lacrimal gland stones, are a very rare phenomenon, and their etiology is currently unclear. In this case, there was likely to have been an association between herpes zoster ophthalmicus and the conjunctivolith.

Controlling the Biological Behaviors of Polymer-Coated Upconverting Nanoparticles by Adjusting the Linker Length of Estrone Ligands.

The estrone ligand is used for modifying nanoparticle surfaces to improve their targeting effect on cancer cell lines. However, to date, there is no common agreement on the ideal linker length to be used for the optimum targeting performance. In this study, we aimed to investigate the impact of poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) linker length on the cellular uptake behavior of polymer-coated upconverting nanoparticles (UCNPs). Different triblock terpolymers, poly(poly (ethylene glycol) methyl ether methacrylate)-block-polymethacrylic acid-block-polyethylene glycol methacrylate phosphate (PPEGMEMAx-b-PMAAy-b-PEGMP3: x = 7, 15, 33, and 80; y = 16, 20, 18, and 18), were synthesized with different polymer linker chain lengths between the surface and the targeting ligand by reversible addition-fragmentation chain transfer polymerization. The estrone ligand was attached to the polymer via specific terminal conjugation. The cellular association of polymer-coated UCNPs with linker chain lengths was evaluated in MCF-7 cells by flow cytometry. Our results showed that the bioactivity of ligand modification is dependent on the length of the polymer linker. The shortest polymer PPEGMEMA7-b-PMAA16-b-PEGMP3 with estrone at the end of the polymer chain was found to have the best cellular association behavior in the estrogen receptor (ER)α-positive expression cell line MCF-7. Additionally, the anticancer drug doxorubicin•HCl was encapsulated in the nanocarrier to evaluate the 2D and 3D cytotoxicity. The results showed that estrone modification could efficiently improve the cellular uptake in ERα-positive expression cell lines and in 3D spheroid models.

2D Porphyrinic Metal-Organic Framework Nanosheets as Multidimensional Photocatalysts for Functional Materials.

Ultrathin porphyrinic 2D MOFs, ZnTCPP nanosheets (TCPP: 5,10,15,20-(tetra-4-carboxyphenyl) porphyrin) were employed as heterogeneous photocatalysts to activate PET-RAFT polymerization under various wavelengths ranging from violet to orange light. High polymerization rates, oxygen tolerance, and precise temporal control were achieved. The polymers showed narrow molecular weight distributions and good chain-end fidelity. The 2D ZnTCPP nanosheets were applied as photocatalysts in stereolithographic 3D printing in an open-air environment under blue light to yield well-defined 3D printed objects. Apart from providing an efficient catalytic system, 2D ZnTCPP nanosheets reinforced the mechanical properties of the 3D printed materials. The presence of ZnTCPP embedded in the materials conferred effective antimicrobial activity under visible light by production of singlet oxygen, affording 98 % and 93 % anti-bacterial efficiency against Gram-positive and Gram-negative bacteria, respectively.

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Antiviral potential and mode of action of Indigofera heterantha against HSV-2 by targeting the early stages of infection.

BACKGROUND: The development of antivirals against herpes simplex virus 2 (HSV-2) has a major public health importance because of the wide spectrum of associated clinical disease in both immunocompetent and immunocompromised populations. Even with the extensive use of acyclovir, issues such as emergence of drug-resistant strains, poor oral bioavailability and low effectiveness in recurrent infections have highlighted the requirement for alternate therapies. Plants, which are rich in metabolites and active against viruses, are being explored as one such source. We had earlier reported specific and potent anti-HSV-2 activity from the roots of the plant Indigofera heterantha. Herein, we describe the mechanism by which it exerts this antiviral potential against HSV-2. METHODS: MTT, plaque reduction and immunofluorescence techniques were used for in vitro antiviral studies. Animal studies were carried out in HSV-2-infected mice followed by plaque reduction assays. RESULTS: The extract was found to act at multiple steps of viral entry viz attachment, adsorption and penetration by blocking binding sites present on the viral envelope glycoproteins which eventually blocks its binding with the cell surface receptors present on the host cells. We also showed efficacy of PP9706642 topical application in prohibiting HSV-2 invasion to nearby organs from the site of infection, that is vagina in HSV-2 infected animals. CONCLUSIONS: The extract targets the early and late stages of HSV-2 viral life cycle and thus shows great promise as both a prophylactic as well as therapeutic phytopharmaceutical against HSV-2.

Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells.

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein-protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.