Quantifying Inflammatory Response and Drug-Aided Resolution in an Atopic Dermatitis Model with Deep Learning.

Noninvasive quantification of dermal diseases aids efficacy studies and paves the way for broader enrollment in clinical studies across varied demographics. Related to atopic dermatitis, accurate quantification of the onset and resolution of inflammatory flare ups in the skin remains challenging because the commonly used macroscale cues do not necessarily represent the underlying inflammation at the cellular level. Although atopic dermatitis affects over 10% of Americans, the genetic underpinnings and cellular-level phenomena causing the physical manifestation of the disease require more clarity. Current gold standards of quantification are often invasive, requiring biopsies followed by laboratory analysis. This represents a gap in our ability to diagnose and study skin inflammatory disease as well as develop improved topical therapeutic treatments. This need can be addressed through noninvasive imaging methods and the use of modern quantitative approaches to streamline the generation of relevant insights. This work reports the noninvasive image-based quantification of inflammation in an atopic dermatitis mouse model on the basis of cellular-level deep learning analysis of coherent anti-Stokes Raman scattering and stimulated Raman scattering imaging. This quantification method allows for timepoint-specific disease scores using morphological and physiological measurements. The outcomes we show set the stage for applying this workflow to future clinical studies.

Label-Free Quantification of Pharmacokinetics in Skin with Stimulated Raman Scattering Microscopy and Deep Learning.

The treatment of inflammatory skin conditions relies on a deep understanding of how drugs and tissue behave and interact. Although numerous methods have been developed that aim to follow and quantify topical drug pharmacokinetics, these tools can come with limitations, assumptions, and trade-offs that do not allow for real-time tracking of drug flow and flux on the cellular level in situ. We have developed a quantitative imaging toolkit that makes use of stimulated Raman scattering microscopy and deep learning-based computational image analysis to quantify the uptake of specific drug molecules in skin without the need for labels. Analysis powered by trained convolutional neural networks precisely identified features such as cells, cell junctions, and cell types within skin to enable multifactorial analysis of skin pharmacokinetics. We imaged and quantified the flow and flux of small molecule drugs through the layers and structures of ex vivo nude mouse ear skin and extracted pharmacokinetic parameters through convolutional neural network-based image processing, including relative area under the curve accumulation, time of maximum drug concentration, and in situ partition ratios. This approach, which facilitates the direct observation and quantification of pharmacokinetics, can be used to glean mechanistic insight into underlying phenomena in skin pharmacokinetics.

Imaging and quantifying drug delivery in skin - Part 2: Fluorescence andvibrational spectroscopic imaging methods.

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo: i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies.

Imaging and quantifying drug delivery in skin - Part 1: Autoradiography and mass spectrometry imaging.

In this two-part review we present an up-to-date description of different imaging methods available to map the localization of drugs on skin as a complement of established ex-vivo absorption studies. This first part deals with invasive methods which are grouped in two classes according to their underlying principles: i) methods using radioactivity such as autoradiography and ii) mass spectrometry methods such as MALDI and SIMS. For each method, a description of the principle is given along with example applications of imaging and quantifying drug delivery in human skin. Thanks to these techniques a better assessment of the fate of drugs is obtained: its localization on a particular skin structure, its potential accumulation, etc. A critical comparison in terms of capabilities, sensitivity and practical applicability is included that will help the reader to select the most appropriate technique depending on the particular problem to be solved.

TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC proteins.

The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Here, we show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef relieves restriction. HIV-1 Nef does not down-regulate the overall level of TIM-1 expression, but promotes its internalization from the plasma membrane and sequesters its expression in intracellular compartments. Notably, Nef mutants defective in modulating membrane protein endocytic trafficking are incapable of antagonizing TIM-mediated inhibition of HIV-1 release. Intriguingly, depletion of SERINC3 or SERINC5 proteins in human peripheral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, coexpression of SERINC3 or SERINC5 increases the expression of TIM-1 on the plasma membrane and potentiates TIM-mediated inhibition of HIV-1 production. Pulse-chase metabolic labeling reveals that the half-life of TIM-1 is extended by SERINC5 from <2 to ∼6 hours, suggesting that SERINC5 stabilizes the expression of TIM-1. Consistent with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antagonize SERINC-mediated diminishment of HIV-1 infectivity, also effectively counteract TIM-mediated inhibition of HIV-1 release. Collectively, our work reveals a role of Nef in antagonizing TIM-1 and highlights the complex interplay between Nef and HIV-1 restriction by TIMs and SERINCs.

Nanoparticle-cell interactions induced apoptosis: a case study with nanoconjugated epidermal growth factor.

In addition to the intrinsic toxicity associated with the chemical composition of nanoparticles (NP) and their ligands, biofunctionalized NP can perturb specific cellular processes through NP-cell interactions and induce programmed cell death (apoptosis). In the case of the epidermal growth factor (EGF), nanoconjugation has been shown to enhance the apoptotic efficacy of the ligand, but the critical aspects of the underlying mechanism and its dependence on the NP morphology remain unclear. In this manuscript we characterize the apoptotic efficacy of nanoconjugated EGF as a function of NP size (with sphere diameters in the range 20-80 nm), aspect ratio (A.R., in the range of 4.5 to 8.6), and EGF surface loading in EGFR overexpressing MDA-MB-468 cells. We demonstrate a significant size and morphology dependence in this relatively narrow parameter space with spherical NP with a diameter of approx. 80 nm being much more efficient in inducing apoptosis than smaller spherical NP or rod-shaped NP with comparable EGF loading. The nanoconjugated EGF is found to trigger an EGFR-dependent increase in cytoplasmic reactive oxygen species (ROS) levels but no indications of increased mitochondrial ROS levels or mitochondrial membrane damage are detected at early time points of the apoptosis induction. The increase in cytoplasmic ROS is accompanied by a perturbation of the intracellular glutathione homeostasis, which represents an important check-point for NP-EGF mediated apoptosis. Abrogation of the oxidative stress through the inhibition of EGFR signaling by the EGFR inhibitor AG1478 or addition of antioxidants N-acetyl cysteine (NAC) or tempol, but not trolox, successfully suppressed the apoptotic effect of nanoconjugated EGF. A model to account for the observed morphology dependence of EGF nanoconjugation enhanced apoptosis and the underlying NP-cell interactions is discussed.

Membrane Fluidity Sensing on the Single Virus Particle Level with Plasmonic Nanoparticle Transducers.

Viral membranes are nanomaterials whose fluidity depends on their composition, in particular, the cholesterol (chol) content. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of sensing the membrane fluidity on the single-virus level are required. In this manuscript, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature, for the first time on the single-VLP level. Chol extraction studies with different methyl-ß-cyclodextrin (MßCD) concentrations yielded a gradual decrease in polarization fluctuations as a function of time. The rate of chol extraction for individual VLPs showed a broad spread, presumably due to differences in the membrane composition for the individual VLPs, and this heterogeneity increased with decreasing MßCD concentration.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. IMPORTANCE: Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.

Access of HIV-2 to CD169-dependent dendritic cell-mediated trans infection pathway is attenuated.

The mechanisms behind the low viral loads and lower mortality rates of HIV-2(+) individuals remain unknown. We hypothesized that reduced interaction of HIV-2 with CD169, the primary HIV-1 attachment factor on monocyte-derived dendritic cells (DCs) that targets captured virus particles to the trans infection pathway, contributes to its diminished pathogenic phenotype in vivo. We observed a significant decrease in capture of HIV-2 Gag-eGFP virus-like particles (VLPs) and infectious GFP-containing HIV-2 particles compared to corresponding HIV-1 particles by CD169(+) mature DCs. Interestingly, there was decreased co-localization of HIV-2 with HIV-1 Gag at plasma membrane microdomains in virus producer cells which correlated with reduced incorporation of GM3, the CD169 ligand, in HIV-2 virions, and reduction in mature DC-mediated HIV-2 trans infection compared to HIV-1. We conclude that limited interaction of HIV-2 with CD169 diminishes virus access to the mature DC-mediated trans infection pathway and might result in attenuated HIV-2 dissemination in vivo.

Plasmonic nanoshell functionalized etched fiber Bragg gratings for highly sensitive refractive index measurements.

A novel fiber optical refractive index sensor based on gold nanoshells immobilized on the surface of an etched single-mode fiber including a Bragg grating is demonstrated. The nanoparticle coating induces refractive index dependent waveguide losses, because of the variation of the evanescently guided part of the light. Hence the amplitude of the Bragg reflection is highly sensitive to refractive index changes of the surrounding medium. The nanoshell functionalized fiber optical refractive index sensor works in reflectance mode, is suitable for chemical and biochemical sensing, and shows an intensity dependency of 4400% per refractive index unit in the refractive index range between 1.333 and 1.346. Furthermore, the physical length of the sensor is smaller than 3 mm with a diameter of 6 µm, and therefore offers the possibility of a localized refractive index measurement.

Quantifying lipid contents in enveloped virus particles with plasmonic nanoparticles.

Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1 ) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. Here, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different HIV-1 and Ebola virus-like particles (VLPs) using sample sizes of less than 3 × 10(6) particles is introduced. The assay evaluates both scattering intensity and resonance wavelength, and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. A correlation of the optical observables with absolute lipid contents is achieved by calibration of the plasmon coupling-based methodology with unilamellar liposomes of known PS or GM1 concentration. The performed studies reveal significant differences in the membrane of VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers.

Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells.

Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

Nanoconjugation: A Materials Approach to Enhance Epidermal Growth Factor Induced Apoptosis.

Apoptosis evasion is a hallmark of cancer that motivates the development of novel strategies for inducing cell death in a controlled fashion. The size-compatibility of nanoparticles (NPs) with cellular components provides new opportunities for regulating cellular processes, potentially including apoptosis. We investigated the impact of the covalent attachment of epidermal growth factor (EGF) to 40 nm diameter Au NPs on cellular apoptosis levels, quantified as caspase-3 activity, in two in vitro cancer cell lines: A431 and HeLa. Our studies show that nanoconjugation enhances EGF-induced apoptosis in EGF receptor (EGFR) overexpressing A431 and triggers a quantifiable increase in apoptosis in HeLa. The latter has physiological receptor expression levels and does not show apoptosis in response to free EGF. Endocytosis and trafficking are involved in key EGFR regulation processes, most prominently signal termination. Our experimental findings indicate that these processes can be manipulated through nanoconjugation to induce apoptosis.

Tailoring Plasmon Coupling in Self-Assembled One-Dimensional Au Nanoparticle Chains through Simultaneous Control of Size and Gap Separation.

We investigated the near- and far-field response of one-dimensional chains of Au nanoparticles (NPs) fabricated with high structural control through template guided self-assembly. We demonstrate that the density of polyethylene glycol (PEG) ligands grafted onto the NP surface, in combination with the buffer conditions, facilitate a systematic variation of the average gap width (g) at short separations of g<1.1nm. The overall size (n) of the cluster was controlled through the template. The ability to independently vary n and g allowed for a rational tuning of the spectral response in individual NP clusters over a broad spectral range. We used this structural control for a systematic investigation of the electromagnetic coupling underlying the superradiant cluster mode. Independent of the chain length, plasmon coupling is dominated by direct neighbor interactions. A decrease in coupling strength at separations ≲0.5nm indicates the presence of non-local and/or quantum mechanical coupling mechanisms.

Illuminating the lateral organization of cell-surface CD24 and CD44 through plasmon coupling between Au nanoparticle immunolabels.

CD44 and CD24 are important cell surface glycoproteins whose relative expression levels are used to identify so-called cancer stem cells (CSCs). While current diagnostic applications of CD44 and CD24 focus primarily on their expression levels, we demonstrate here that noble metal nanoparticle (NP) immunolabeling in combination with plasmon coupling microscopy (PCM) can reveal more subtle differences, such as the spatial organization of these surface species on subdiffraction limit length scales. We quantified both expression and spatial clustering of CD44 and CD24 on MCF7 and SKBR3 breast cancer cells through analysis of the labeling intensity and the electromagnetic coupling of the NP labels, respectively. The labeling intensity was well correlated with the receptor expression, but the inspection of the labeled cell surface in the optical microscope revealed that the NP immunolabels were not homogeneously distributed. Consistent with a heterogeneous spatial distribution of the targeted CD24 and CD44 in the plasma membrane, a significant fraction of the NPs were organized into clusters, which were easily detectable in the optical microscope as discrete spots with colors ranging from green to orange. To further quantify the spatial organization of the targeted proteins, we characterized individual NP clusters through spatially resolved elastic scattering spectroscopy. The statistical analysis of the single cluster spectra revealed a higher clustering affinity for CD24 than for CD44 in the investigated cancer models. This preferential clustering was removed upon lipid raft disruption through cholesterol sequestration. Overall, these observations confirm a preferential enrichment of CD24 in lipid rafts and a more random distribution of CD44 in the plasma membrane as cause for the observed differences in protein clustering.