Infrared nanoimaging of neuronal ultrastructure and nanoparticle interaction with cells.

Here we introduce scattering-type scanning near-field optical microscopy (s-SNOM) as a novel tool for nanoscale chemical-imaging of sub-cellular organelles, nanomaterials and of the interactions between them. Our setup uses a tuneable mid-infrared laser and a sharp scanning probe to image at a resolution substantially surpassing the diffraction limit. The laser can be tuned to excite vibrational modes of functional groups in biomolecules, (e.g. amide moieties), in a way that enables direct chemical mapping without the need for labelling. We, for the first time, chemically image neuronal ultrastructure, identify neuronal organelles and sub-organelle structures as small as 10 nm and validate our findings using transmission electron microscopy (TEM). We produce chemical and morphological maps of neurons treated with gold nanospheres and characterize nanoparticle size and intracellular location, and their interaction with the plasma membrane. Our results show that the label-free nature of s-SNOM means it has a 'true' chemical resolution of up to 20 nm which can be further improved. We argue that it offers significant potential in nanomedicine for nanoscale chemical imaging of cell ultrastructure and the subcellular distribution of nanomaterials within tissues.

The Potential of Subsampling and Inpainting for Fast Low-Dose Cryo FIB-SEM Imaging.

Traditional image acquisition for cryo focused ion-beam scanning electron microscopy (FIB-SEM) tomography often sees thousands of images being captured over a period of many hours, with immense data sets being produced. When imaging beam sensitive materials, these images are often compromised by additional constraints related to beam damage and the devitrification of the material during imaging, which renders data acquisition both costly and unreliable. Subsampling and inpainting are proposed as solutions for both of these aspects, allowing fast and low-dose imaging to take place in the Focused ion-beam scanning electron microscopy FIB-SEM without an appreciable loss in image quality. In this work, experimental data are presented which validate subsampling and inpainting as a useful tool for convenient and reliable data acquisition in a FIB-SEM, with new methods of handling three-dimensional data being employed in the context of dictionary learning and inpainting algorithms using a newly developed microscope control software and data recovery algorithm.

Interaction of amisulpride with GLUT1 at the blood-brain barrier. Relevance to Alzheimer's disease.

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates, GLUT1 inhibitors and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT mice, 5xFAD mice and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol) and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose hCMEC/D3 accumulation. [3H]amisulpride uptake into the brain (except supernatant) of 5xFAD mice compared to WT remained unchanged. TEM revealed brain capillary degeneration in human AD. There was no difference in GLUT1 or P-glycoprotein BBB expression between WT and 5xFAD mice. In contrast, caudate P-glycoprotein, but not GLUT1, expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1 and that BBB transporter expression is altered in AD. This suggests that antipsychotics could potentially exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts.

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

Does hyperthermic povidone-iodine lavage increase the apoptotic rate of residual cancer cell in patients with malignant pleural mesothelioma?-a prospective pilot study.

Background: Malignant pleural mesothelioma (MPM) is an incurable, late presenting primary cancer, conferring a survival of 8-14 months. Different intrapleural treatments have been tested as part of a multimodality approach to treat a select group of patients with limited disease, increasing survival. Recently, povidone-iodine has been shown to induce apoptosis in microscopic tumour cells in vitro, with no reported complications. This is the first in vivo study assessing the apoptotic rate caused by intraoperative hyperthermic betadine lavage using routine immunohistochemistry combined with transmission electron microscopy (TEM). Methods: We included surgically fit patients aged >18, undergoing minimally invasive video-assisted thoracoscopic surgery (VATS) pleural biopsy between December 2016 and February 2018, for confirmed or presumed pleural malignancy. Parietal pleural biopsies were obtained at 7.5, 15 and 30 minutes after hyperthermic betadine lavage, and compared to pre-lavage biopsy samples, for apoptotic changes. Viable tumour samples underwent histological, immunohistochemical and ultrastructural analysis as well as TEM for features of apoptosis. Results: N=6. Median age was 76 years. Median overall survival was 26.7 months. There was no statistical impact on survival of side of disease (left vs. right). There was no significant difference in expressions of markers of apoptotic index pre and post betadine treatment upon immunohistochemical analysis. There was no discernible effect on morphological features of apoptosis seen with betadine treatment, on TEM analysis. No side effects were identified post betadine lavage. Conclusions: Although hyperthermic betadine lavage is a safe antiseptic solution with no toxicity when performed intraoperatively, it confers no effect on apoptotic rate or necrosis. It is therefore unlikely that hyperthermic betadine lavage will have an impact on reducing the microscopic residual disease after pleurectomy decortication and enhancing survival.

Characterising the chemical and physical properties of phase-change nanodroplets.

Phase-change nanodroplets have attracted increasing interest in recent years as ultrasound theranostic nanoparticles. They are smaller compared to microbubbles and they may distribute better in tissues (e.g. in tumours). They are composed of a stabilising shell and a perfluorocarbon core. Nanodroplets can vaporise into echogenic microbubbles forming cavitation nuclei when exposed to ultrasound. Their perfluorocarbon core phase-change is responsible for the acoustic droplet vaporisation. However, methods to quantify the perfluorocarbon core in nanodroplets are lacking. This is an important feature that can help explain nanodroplet phase change characteristics. In this study, we fabricated nanodroplets using lipids shell and perfluorocarbons. To assess the amount of perfluorocarbon in the core we used two methods, 19F NMR and FTIR. To assess the cavitation after vaporisation we used an ultrasound transducer (1.1 MHz) and a high-speed camera. The 19F NMR based method showed that the fluorine signal correlated accurately with the perfluorocarbon concentration. Using this correlation, we were able to quantify the perfluorocarbon core of nanodroplets. This method was used to assess the content of the perfluorocarbon of the nanodroplets in solutions over time. It was found that perfluoropentane nanodroplets lost their content faster and at higher ratio compared to perfluorohexane nanodroplets. The high-speed imaging indicates that the nanodroplets generate cavitation comparable to that from commercial contrast agent microbubbles. Nanodroplet characterisation should include perfluorocarbon concentration assessment as critical information for their development.

Cytosine Deaminase Base Editing to Restore COL7A1 in Dystrophic Epidermolysis Bullosa Human: Murine Skin Model.

Recessive dystrophic epidermolysis bullosa is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1, which encodes type VII collagen, the main component of anchoring fibrils at the dermal-epidermal junction. Although conventional gene therapy approaches through viral vectors have been tested in preclinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. The delivery of suitable repair templates for the repair of DNA cleaved by Cas9 is still a major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We show highly targeted and efficient cytidine deamination and molecular correction of a defined recessive dystrophic epidermolysis bullosa mutation (c.425A>G), leading to restoration of full-length type VII collagen protein expression in primary human fibroblasts and induced pluripotent stem cells. Type VII collagen basement membrane expression and skin architecture were restored with de novo anchoring fibrils identified by electron microscopy in base-edited human recessive dystrophic epidermolysis bullosa grafts recovered from immunodeficient mice. The results show the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations.

Multi-synaptic boutons are a feature of CA1 hippocampal connections in the stratum oriens.

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block-face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from postnatal day 22 [P22] to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.

Structural investigations into colour-tuneable fluorescent InZnP-based quantum dots from zinc carboxylate and aminophosphine precursors.

Fluorescent InP-based quantum dots have emerged as valuable nanomaterials for display technologies, biological imaging, and optoelectronic applications. The inclusion of zinc can enhance both their emissive and structural properties and reduce interfacial defects with ZnS or CdS shells. However, the sub-particle distribution of zinc and the role this element plays often remains unclear, and it has previously proved challenging to synthesise Zn-alloyed InP-based nanoparticles using aminophosphine precursors. In this report, we describe the synthesis of alloyed InZnP using zinc carboxylates, achieving colour-tuneable fluorescence from the unshelled core materials, followed by a one-pot ZnS or CdS deposition using diethyldithiocarbamate precursors. Structural analysis revealed that the "core/shell" particles synthesised here were more accurately described as homogeneous extended alloys with the constituent shell elements diffusing through the entire core, including full-depth inclusion of zinc.

Cortical wiring by synapse type-specific control of local protein synthesis.

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.

Nanostars Carrying Multifunctional Neurotrophic Dendrimers Protect Neurons in Preclinical In Vitro Models of Neurodegenerative Disorders.

A challenge in neurology is the lack of efficient brain-penetrable neuroprotectants targeting multiple disease mechanisms. Plasmonic gold nanostars are promising candidates to deliver standard-of-care drugs inside the brain but have not been trialed as carriers for neuroprotectants. Here, we conjugated custom-made peptide dendrimers (termed H3/H6), encompassing motifs of the neurotrophic S100A4-protein, onto star-shaped and spherical gold nanostructures (H3/H6-AuNS/AuNP) and evaluated their potential as neuroprotectants and interaction with neurons. The H3/H6 nanostructures crossed a model blood-brain barrier, bound to plasma membranes, and induced neuritogenesis with the AuNS, showing higher potency/efficacy than the AuNP. The H3-AuNS/NP protected neurons against oxidative stress, the H3-AuNS being more potent, and against Parkinson's or Alzheimer's disease (PD/AD)-related cytotoxicity. Unconjugated S100A4 motifs also decreased amyloid beta-induced neurodegeneration, introducing S100A4 as a player in AD. Using custom-made dendrimers coupled to star-shaped nanoparticles is a promising route to activate multiple neuroprotective pathways and increase drug potency to treat neurodegenerative disorders.

The PTPIP51 coiled-coil domain is important in VAPB binding, formation of ER-mitochondria contacts and IP3 receptor delivery of Ca2+ to mitochondria.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of fundamental physiological processes. This signaling involves close physical contacts between the two organelles that are mediated by the VAPB-PTPIP51 ″tethering" proteins. The VAPB-PTPIP51 tethers facilitate inositol 1,4,5-trisphosphate (IP3) receptor delivery of Ca2+ from ER to mitochondria. Damage to the tethers is seen in Alzheimer's disease, Parkinson's disease and frontotemporal dementia with related amyotrophic lateral sclerosis (FTD/ALS). Understanding the mechanisms that regulate the VAPB-PTPIP51 interaction thus represents an important area of research. Recent studies suggest that an FFAT motif in PTPIP51 is key to its binding to VAPB but this work relies on in vitro studies with short peptides. Cellular studies to support this notion with full-length proteins are lacking. Here we address this issue. Immunoprecipitation assays from transfected cells revealed that deletion of the PTPIP51 FFAT motif has little effect on VAPB binding. However, mutation and deletion of a nearby coiled-coil domain markedly affect this binding. Using electron microscopy, we then show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on ER-mitochondria contacts. Finally, we show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on the IP3 receptor-mediated delivery of Ca2+ to mitochondria. Thus, the coiled-coil domain is essential for PTPIP51 ER-mitochondria signaling functions.

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.

Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.

Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling.

Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

Mutations in EPHB4 cause human venous valve aplasia.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Preparing Lamellae from Vitreous Biological Samples using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Impacts of Metabolism and Organic Acids on Cell Wall Composition and Pseudomonas aeruginosa Susceptibility to Membrane Active Antimicrobials.

Reliable antimicrobial susceptibility testing is essential in informing both clinical antibiotic therapy decisions and the development of new antibiotics. Mammalian cell culture media have been proposed as an alternative to bacteriological media, potentially representing some critical aspects of the infection environment more accurately. Here, we use a combination of NMR metabolomics and electron microscopy to investigate the response of Escherichia coli and Pseudomonas aeruginosa to growth in differing rich media to determine whether and how this determines metabolic strategies, the composition of the cell wall, and consequently susceptibility to membrane active antimicrobials including colistin and tobramycin. The NMR metabolomic approach is first validated by characterizing the expected E. coli acid stress response to fermentation and the accompanying changes in the cell wall composition, when cultured in glucose rich mammalian cell culture media. Glucose is not a major carbon source for P. aeruginosa but is associated with a response to osmotic stress and a modest increase in colistin tolerance. Growth of P. aeruginosa in a range of bacteriological media is supported by consumption of formate, an important electron donor in anaerobic respiration. In mammalian cell culture media, however, the overall metabolic strategy of P. aeruginosa is instead dependent on consumption of glutamine and lactate. Formate doping of mammalian cell culture media does not alter the overall metabolic strategy but is associated with polyamine catabolism, remodelling of both inner and outer membranes, and a modest sensitization of P. aeruginosa PAO1 to colistin. Further, in a panel of P. aeruginosa isolates an increase between 2- and 3-fold in sensitivity to tobramycin is achieved through doping with other organic acids, notably propionate which also similarly enhances the activity of colistin. Organic acids are therefore capable of nonspecifically influencing the potency of membrane active antimicrobials.

More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.

Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.