Optical coherence tomography in the assessment of acute changes in cutaneous vascular diameter induced by heat stress.

There are limited imaging technologies available that can accurately assess or provide surrogate markers of the in vivo cutaneous microvessel network in humans. In this study, we establish the use of optical coherence tomography (OCT) as a novel imaging technique to assess acute changes in cutaneous microvessel area density and diameter in humans. OCT speckle decorrelation images of the skin on the ventral side of the forearm up to a depth of 500 μm were obtained prior to and following 20-25 mins of lower limb heating in eight healthy males (30.3±7.6 yrs). Skin red blood cell flux was also collected using laser Doppler flowmetry probes immediately adjacent to the OCT skin sites, along with skin temperature. OCT speckle decorrelation images were obtained at both baseline and heating time points. Forearm skin flux increased significantly (0.20±0.15 to 1.75±0.38 CVC, P<0.01), along with forearm skin temperature (32.0±1.2 to 34.3±1.0°C, P<0.01). Quantitative differences in the automated calculation of vascular area densities (26±9 to 49±19%, P<0.01) and individual microvessel diameters (68±17 to 105±25 μm, P<0.01) were evident following the heating session. This is the first in vivo within-subject assessment of acute changes in the cutaneous microvasculature in response to heating in humans and highlights the use of OCT as an exciting new imaging approach for skin physiology and clinical research

Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al

Functional human α7 nicotinic acetylcholine receptor (nAChR) generated from escherichia coli

Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design

Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism

Parametric imaging of attenuation by optical coherence tomography: review of models, methods, and clinical translation

SIGNIFICANCE: Optical coherence tomography (OCT) provides cross-sectional and volumetric images of backscattering from biological tissue that reveal the tissue morphology. The strength of the scattering, characterized by an attenuation coefficient, represents an alternative and complementary tissue optical property, which can be characterized by parametric imaging of the OCT attenuation coefficient. Over the last 15 years, a multitude of studies have been reported seeking to advance methods to determine the OCT attenuation coefficient and developing them toward clinical applications. AIM: Our review provides an overview of the main models and methods, their assumptions and applicability, together with a survey of preclinical and clinical demonstrations and their translation potential. RESULTS: The use of the attenuation coefficient, particularly when presented in the form of parametric en face images, is shown to be applicable in various medical fields. Most studies show the promise of the OCT attenuation coefficient in differentiating between tissues of clinical interest but vary widely in approach. CONCLUSIONS: As a future step, a consensus on the model and method used for the determination of the attenuation coefficient is an important precursor to large-scale studies. With our review, we hope to provide a basis for discussion toward establishing this consensus

Highly tunable ultra-narrow-resonances with optical nano-antenna phased arrays in the infrared

We report our recent development in pursuing high Quality-Factor (high-Q factor) plasmonic resonances, with vertically aligned two dimensional (2-D) periodic nanorod arrays. The 2-D vertically aligned nano-antenna array can have high-Q resonances varying arbitrarily from near infrared to terahertz regime, as the antenna resonances of the nanorod are highly tunable through material properties, the length of the nanorod, and the orthogonal polarization direction with respect to the lattice surface,. The high-Q in combination with the small optical mode volume gives a very high Purcell factor, which could potentially be applied to various enhanced nonlinear photonics or optoelectronic devices. The 'hot spots' around the nanorods can be easily harvested as no index-matching is necessary. The resonances maintain their high-Q factor with the change of the environmental refractive index, which is of great interest for molecular sensing.Comment: 8 pages, appears in Proc. SPIE 9163, Plasmonics: Metallic Nanostructures and Their Optical Properties XII, 91630R (September 10, 2014

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Funder: Medical Research CouncilSince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules

Engineering the Photonic Density of States with metamaterials

The photonic density of states (PDOS), like its' electronic coun- terpart, is one of the key physical quantities governing a variety of phenom- ena and hence PDOS manipulation is the route to new photonic devices. The PDOS is conventionally altered by exploiting the resonance within a device such as a microcavity or a bandgap structure like a photonic crystal. Here we show that nanostructured metamaterials with hyperbolic dispersion can dramatically enhance the photonic density of states paving the way for metamaterial based PDOS engineering

DPEP1 Inhibits Tumor Cell Invasiveness, Enhances Chemosensitivity and Predicts Clinical Outcome in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. To identify biologically relevant genes with prognostic and therapeutic significance in PDAC, we first performed the microarray gene-expression profiling in 45 matching pairs of tumor and adjacent non-tumor tissues from resected PDAC cases. We identified 36 genes that were associated with patient outcome and also differentially expressed in tumors as compared with adjacent non-tumor tissues in microarray analysis. Further evaluation in an independent validation cohort (N = 27) confirmed that DPEP1 (dipeptidase 1) expression was decreased (T: N ratio ∼0.1, P<0.01) in tumors as compared with non-tumor tissues. DPEP1 gene expression was negatively correlated with histological grade (Spearman correlation coefficient = −0.35, P = 0.004). Lower expression of DPEP1 in tumors was associated with poor survival (Kaplan Meier log rank) in both test cohort (P = 0.035) and validation cohort (P = 0.016). DPEP1 expression was independently associated with cancer-specific mortality when adjusted for tumor stage and resection margin status in both univariate (hazard ratio = 0.43, 95%CI = 0.24–0.76, P = 0.004) and multivariate analyses (hazard ratio = 0.51, 95%CI = 0.27–0.94, P = 0.032). We further demonstrated that overexpression of DPEP1 suppressed tumor cells invasiveness and increased sensitivity to chemotherapeutic agent Gemcitabine. Our data also showed that growth factor EGF treatment decreased DPEP1 expression and MEK1/2 inhibitor AZD6244 increased DPEP1 expression in vitro, indicating a potential mechanism for DPEP1 gene regulation. Therefore, we provide evidence that DPEP1 plays a role in pancreatic cancer aggressiveness and predicts outcome in patients with resected PDAC. In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments