LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.

Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.

Histidine-rich protein II nanoparticle delivery of heme iron load drives endothelial inflammation in cerebral malaria.

Histidine-rich protein II (HRPII) is secreted by Plasmodium falciparum during the blood stage of malaria infection. High plasma levels of HRPII are associated with cerebral malaria, a severe and highly fatal complication of malaria. HRPII has been shown to induce vascular leakage, the hallmark of cerebral malaria, in blood-brain barrier (BBB) and animal models. We have discovered an important mechanism for BBB disruption that is driven by unique features of HRPII. By characterizing serum from infected patients and HRPII produced by P. falciparum parasites in culture, we found that HRPII exists in large multimeric particles of 14 polypeptides that are richly laden with up to 700 hemes per particle. Heme loading of HRPII is required for efficient binding and internalization via caveolin-mediated endocytosis in hCMEC/D3 cerebral microvascular endothelial cells. Upon acidification of endolysosomes, two-thirds of the hemes are released from acid-labile binding sites and metabolized by heme oxygenase 1, generating ferric iron and reactive oxygen species. Subsequent activation of the NLRP3 inflammasome and IL-1ß secretion resulted in endothelial leakage. Inhibition of these pathways with heme sequestration, iron chelation, or anti-inflammatory drugs protected the integrity of the BBB culture model from HRPII:heme. Increased cerebral vascular permeability was seen after injection of young mice with heme-loaded HRPII (HRPII:heme) but not with heme-depleted HRPII. We propose that during severe malaria infection, HRPII:heme nanoparticles in the bloodstream deliver an overwhelming iron load to endothelial cells to cause vascular inflammation and edema. Disrupting this process is an opportunity for targeted adjunctive therapies to reduce the morbidity and mortality of cerebral malaria.

Design of an ultrafast pulsed ponderomotive phase plate for cryo-electron tomography.

Ponderomotive phase plates have shown that temporally consistent phase contrast is possible within electron microscopes via high-fluence static laser modes resonating in Fabry-Perot cavities. Here, we explore using pulsed laser beams as an alternative method of generating high fluences. We find through forward-stepping finite element models that picosecond or shorter interactions are required for meaningful fluences and phase shifts, with higher pulse energies and smaller beam waists leading to predicted higher fluences. An additional model based on quasi-classical assumptions is used to discover the shape of the phase plate by incorporating the oscillatory nature of the electric field. From these results, we find the transient nature of the laser pulses removes the influence of Kapitza-Dirac diffraction patterns that appear in the static resonator cases. We conclude by predicting that a total laser pulse energy of 8.7 µJ is enough to induce the required π/2 phase shift for Zernike-like phase microscopy.

Four-dimensional microED of conformational dynamics in protein microcrystals on the femto-to-microsecond timescales.

As structural determination of protein complexes approaches atomic resolution, there is an increasing focus on conformational dynamics. Here we conceptualize the combination of two techniques which have become established in recent years: microcrystal electron diffraction and ultrafast electron microscopy. We show that the extremely low dose of pulsed photoemission still enables microED due to the strength of the electron bunching from diffraction of the protein crystals. Indeed, ultrafast electron diffraction experiments on protein crystals have already been demonstrated to be effective in measuring intermolecular forces in protein microcrystals. We discuss difficulties that may arise in the acquisition and processing of data and the overall feasibility of the experiment, paying specific attention to dose and signal-to-noise ratio. In doing so, we outline a detailed workflow that may be effective in minimizing the dose on the specimen. A series of model systems that would be good candidates for initial experiments is provided.

UEMtomaton: A Source-Available Platform to Aid in Start-up of Ultrafast Electron Microscopy Labs.

The steady rise in the number of ultrafast electron microscopy (UEM) labs, in addition to the opacity and lack of detailed descriptions of current approaches that would enable point-by-point construction, has created an opportunity for sharing common methods and instrumentation for (for example) automating data acquisition to assist in efficient lab start-up and to learn about common and robust protocols. In the spirit of open sharing of methods, we provide here a description of an entry-level method and user interface (UI) for automating UEM experiments, and we provide access to the source code and scripts (source-available) for ease of implementation or as a starting reference point for those entering or seeking to enter the field (https://github.com/CEMSFlannigan/UEMtomaton/releases/tag/v1.0). Core instrumentation and physical connections in the UEM lab at Minnesota are described. Interface communication schemes consisting of duo server-client pairs between critical components - the optical delay stage and the UEM digital camera - are presented, with emphasis placed on describing the logic and communications sequence designed to conduct automated series acquisitions. An application designed and programmed with C++/CLI as Windows Forms in Microsoft Visual Studio - dubbed UEMtomaton - is also presented. Key to the UI layout is centralization of the automation tasks and establishment of communication within the software rather than by interfacing with each individual workstation. It is our hope that this note provides useful insight for current and future UEM researchers, particularly with respect to generalizability and portability of the approach to emerging labs. We note that while this basic, entry-level approach is certainly not the most sophisticated or comprehensive of those currently in use, we feel there is nevertheless value in clearly communicating a proven straightforward method to hopefully lower the barrier to entry into the field.

Socioeconomic and Racial Disparities in Cancer Stage at Diagnosis, Tumor Size, and Clinical Outcomes in a Large Cohort of Women with Breast Cancer, 2007-2016.

BACKGROUND: Socioeconomic and treatment factors contribute to diagnosis of early-stage (local-stage) breast cancer, as well as excess deaths among African American women. OBJECTIVES: We evaluated socioeconomic and treatment predictive factors for early-stage breast cancer among African American women compared to Caucasian women. A secondary aim evaluated predictors and overall risks associated with all-cause and breast cancer-specific mortality. METHODS: We used retrospective cohort population-based study data from the Surveillance, Epidemiology, and End Results (SEER) Program on 547,703 women aged ≥ 20 years diagnosed with breast cancer primary tumors from 2007 to 2016. Statistical analysis used logistic regression to assess predictors of early-stage breast cancer and Cox proportional hazards regression for mortality risks. RESULTS: African American women were more likely to be diagnosed at advanced-stage, had larger tumor size at diagnosis, and received less cancer-directed surgery, but more chemotherapy than Caucasian women. Insured women (> 50%) were more likely to be diagnosed at early-stage and to have smaller tumors (p < 0.05). Education level, poverty level, and household income had no impact on racial disparities or socioeconomic disparities in women diagnosed at early stage. We found increased risks for all-cause mortality (hazard ratio = 1.18; 95% confidence interval, 1.16-1.21) and breast cancer-specific mortality (HR = 1.22; 95% CI, 1.19-1.25) among African American women compared to Caucasian women after adjusting for demographic, socioeconomic, and treatment factors. CONCLUSIONS: In this population-based study using the most recent SEER data, African American women with breast cancer continued to exhibit higher all-cause mortality and breast cancer-specific mortality compared to Caucasian women.

Imaging phonon dynamics with ultrafast electron microscopy: Kinematical and dynamical simulations.

Ultrafast x-ray and electron scattering techniques have proven to be useful for probing the transient elastic lattice deformations associated with photoexcited coherent acoustic phonons. Indeed, femtosecond electron imaging using an ultrafast electron microscope (UEM) has been used to directly image the influence of nanoscale structural and morphological discontinuities on the emergence, propagation, dispersion, and decay behaviors in a variety of materials. Here, we describe our progress toward the development of methods ultimately aimed at quantifying acoustic-phonon properties from real-space UEM images via conventional image simulation methods extended to the associated strain-wave lattice deformation symmetries and extents. Using a model system consisting of pristine single-crystal Ge and a single, symmetric Lamb-type guided-wave mode, we calculate the transient strain profiles excited in a wedge specimen and then apply both kinematical- and dynamical-scattering methods to simulate the resulting UEM bright-field images. While measurable contrast strengths arising from the phonon wavetrains are found for optimally oriented specimens using both approaches, incorporation of dynamical scattering effects via a multi-slice method returns better qualitative agreement with experimental observations. Contrast strengths arising solely from phonon-induced local lattice deformations are increased by nearly an order of magnitude when incorporating multiple electron scattering effects. We also explicitly demonstrate the effects of changes in global specimen orientation on the observed contrast strength, and we discuss the implications for increasing the sophistication of the model with respect to quantification of phonon properties from UEM images.

Assessing the cutting efficiency of different burs on zirconia substrate.

Cutting dental zirconia for endodontic access preparation is difficult. Therefore, this study aimed to determine cutting efficiency of various burs when cutting this material. An air turbine handpiece was used in a customised test rig to cut sintered zirconia specimens, using a conventional blue band diamond, two different zirconia-cutting diamond and a zirconia-cutting tungsten carbide bur. Position and speed of the bur were continuously determined using wireless data acquisition over two successive five-minute runs. Differences in cutting efficiency were statistically analysed. Burs were examined using light and scanning electron microscopy (SEM). All diamond burs cut zirconia more efficiently than the tungsten carbide bur. Overall, all burs showed decreasing cutting efficiency over time. SEM images showed discernible wear and damage to the cutting portion of each bur head. It is concluded that zirconia-cutting burs are advantageous regarding durability, and carbide burs are rather ineffective against carbide substrate.

A Single-Dose, Crossover-Design Bioequivalence Study Comparing Two Nicotine Gum Formulations in Healthy Subjects.

INTRODUCTION: Nicotine replacement therapy (NRT) benefits smokers who wish to quit; nicotine gum represents one NRT. New formulations of nicotine gum have been developed to consider consumer preferences and needs. A new mint-flavored nicotine gum with a different texture was developed that may provide a more appealing taste and chewing experience. This study evaluated this new nicotine gum (2 and 4 mg strengths) for bioequivalence versus the original flavor sugar-free nicotine gum at corresponding dosages. METHODS: All subjects randomized in this crossover study received a single dose of all treatments, i.e., 2 and 4 mg doses of test and reference gums, separated by 2-7 days of washout between treatments. Subjects' maximal plasma nicotine concentration (Cmax) and extent of nicotine absorption (AUC0-t) following the administration of each treatment were calculated from plasma nicotine concentrations. Ratios of test/reference for Cmax and AUC0-t were calculated to evaluate bioequivalence between the two products. RESULTS: Both 2 and 4 mg doses of the new mint-flavored nicotine gum were bioequivalent to the dose-matched reference product as determined by the ratio of the geometric means and their 90% confidence intervals for Cmax and AUC0-t as well as secondary pharmacokinetic parameters. The safety profiles of the test and reference gums were similar; all treatments were well tolerated. CONCLUSIONS: A new mint-flavored nicotine gum with modified taste and texture is bioequivalent to the original flavor sugar-free nicotine gum at both the 2 and 4 mg dosage strengths and has a similar safety profile. FUNDING: GlaxoSmithKline. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01847443.

Mechanisms of Local Stress Sensing in Multifunctional Polymer Films Using Fluorescent Tetrapod Nanocrystals.

Nanoscale stress-sensing can be used across fields ranging from detection of incipient cracks in structural mechanics to monitoring forces in biological tissues. We demonstrate how tetrapod quantum dots (tQDs) embedded in block copolymers act as sensors of tensile/compressive stress. Remarkably, tQDs can detect their own composite dispersion and mechanical properties with a switch in optomechanical response when tQDs are in direct contact. Using experimental characterizations, atomistic simulations and finite-element analyses, we show that under tensile stress, densely packed tQDs exhibit a photoluminescence peak shifted to higher energies ("blue-shift") due to volumetric compressive stress in their core; loosely packed tQDs exhibit a peak shifted to lower energies ("red-shift") from tensile stress in the core. The stress shifts result from the tQD's unique branched morphology in which the CdS arms act as antennas that amplify the stress in the CdSe core. Our nanocomposites exhibit excellent cyclability and scalability with no degraded properties of the host polymer. Colloidal tQDs allow sensing in many materials to potentially enable autoresponsive, smart structural nanocomposites that self-predict impending fracture.

Comparison of nicotine oral soluble film and nicotine lozenge on efficacy in relief of smoking cue-provoked acute craving after a single dose of treatment in low dependence smokers.

RATIONALE: Pilot study results suggested that a new form of nicotine oral soluble film relieved smoking cue-provoked acute craving faster than nicotine lozenge or gum. The new nicotine film may provide smokers another choice to relieve acute craving. OBJECTIVES: This study compared the efficacy of the 2.5 mg nicotine oral soluble film to 2 mg nicotine lozenge for acute relief of smoking cue-provoked craving. METHODS: A randomized, open label, active comparator controlled, parallel group study was conducted with 322 smokers enrolled. After 4 h of abstinence from smoking, eligible subjects were exposed to smoking cues as provocation. Immediately after the post-provocation baseline craving assessment using a 0-100 mm visual analogue scale (VAS), subjects took a randomized single dose of either the 2.5 mg nicotine film or the 2 mg nicotine lozenge. Craving assessments were completed at 50 s, 3 min, 5 min, 7 min, 15 min, 20 min, 25 min and 30 min after drug administration. RESULTS: Both treatments reduced cue-induced craving and had similar maximum effects on craving relief. However, the 2.5 mg nicotine film relieved cue-induced craving to a greater degree than the 2 mg nicotine lozenge at 50 s (mean difference: -4.9, p = 0.014), 3 min (mean difference: -6.7, p = 0.011), and 5 min (mean difference: -5.6, p = 0.049) post-treatment. CONCLUSIONS: The study confirmed the results from the pilot study. The 2.5 mg nicotine film relieved cue-provoked craving much quicker than the 2 mg nicotine lozenge while both having similar maximum effects. Nicotine film could be useful to provide quick craving relief for low dependence smokers.

A clinical pharmacokinetic study comparing two azelastine hydrochloride nasal formulations in a single-dose design.

Azelastine hydrochloride is a potent second-generation antihistamine, available in Europe and the USA as a nasal spray formulation for the treatment of allergic rhinitis symptoms. GlaxoSmithKline (GSK) Consumer Healthcare has developed a new nasal formulation of azelastine hydrochloride. The present study was aimed at comparing the clinical pharmacokinetic profiles and assessing the bioequivalence of the new formulation of azelastine hydrochloride with a marketed reference nasal spray product. This was a randomized, two-way crossover, two-stage, single-dose pharmacokinetic study with 2 weeks washout between the two treatment periods. A dosage of 0.28 mg of the test and reference products was administered as a single dose to healthy volunteers according to the crossover design. Twenty-three subjects (15 subjects from stage 1 and 8 subjects from stage 2) were enrolled in the study. Adjusted mean values for AUC0-t were 1,526.8 h pg/mL for the test drug and 1,441.5 h pg/mL for the reference drug; for C max the values were 61.59 pg/mL for the test drug and 58.21 pg/mL for the reference drug. The 94.12 % CI of geometric mean ratios (test/reference) were 0.99-1.13 and 0.95-1.18 for AUC0-t and C max. This met the predefined criteria for bioequivalence between test and reference drugs. Secondary pharmacokinetic parameters for azelastine and for the metabolite desmethyl azelastine, AUC(0-∞) and t max, were numerically similar between the two study treatments. Both test and reference azelastine hydrochloride formulations were well tolerated at single dose. This study demonstrated the bioequivalence between the new azelastine hydrochloride nasal spray formulation and the marketed reference Allergodil(®) after single-dose administration.

Pantolactams as androgen receptor antagonists for the topical suppression of sebum production.

A series of pantolactam based compounds were identified as potent antagonists for the androgen receptor (AR). Those that possessed properties suitable for topical delivery were evaluated in the validated Hamster Ear Model. Several compounds were found to be efficacious in reducing wax esters, a major component of sebum, initiating further preclinical work on these compounds.

Targeted delivery of a poorly water-soluble compound to hair follicles using polymeric nanoparticle suspensions.

This study explored the utility of topically applied polymeric nanoparticle suspensions to target delivery of poorly water-soluble drugs to hair follicles. Several formulations of amorphous drug/polymer nanoparticles were prepared from ethyl cellulose and UK-157,147 (systematic name (3S,4R)-[6-(3-hydroxyphenyl)sulfonyl]-2,2,3-trimethyl-4-(2-methyl-3-oxo-2,3-dihydropyridazin-6-yloxy)-3-chromanol), a potassium channel opener, using sodium glycocholate (NaGC) as a surface stabilizer. Nanoparticle suspensions were evaluated to determine if targeted drug delivery to sebaceous glands and hair follicles could be achieved. In in vitro testing with rabbit ear tissue, delivery of UK-157,147 to the follicles was demonstrated with limited distribution to the surrounding dermis. Delivery to hair follicles was also demonstrated in vivo, based on stimulation of hair growth in tests of 100-nm nanoparticles with a C3H mouse model. The nanoparticles were well-tolerated, with no visible skin irritation. In vivo tests of smaller nanoparticles with a hamster ear model also indicated targeted delivery to sebaceous glands. The nanoparticles released drug rapidly in in vitro nonsink dissolution tests and were stable in suspension for 3 months. The present results show selective drug delivery to the follicle by follicular transport of nanoparticles and rapid release of a poorly water-soluble drug. Thus, nanoparticles represent a promising approach for targeted topical delivery of low-solubility compounds to hair follicles.

Rational design of a topical androgen receptor antagonist for the suppression of sebum production with properties suitable for follicular delivery.

A novel nonsteroidal androgen receptor antagonist, (R)-4-(1-benzyl-4,4-dimethyl-2-oxopyrrolidin-3-yloxy)-2-(trifluoromethyl)benzonitrile (1), for the topical control of sebum production is reported. This compound, which is potent, selective, and efficacious in the clinically validated golden Syrian hamster ear animal model, was designed to be delivered to the pilosebaceous unit, the site of action, preferentially by the follicular route.

Diphenyl ethers as androgen receptor antagonists for the topical suppression of sebum production.

A series of diphenyl ethers was prepared and evaluated for androgen receptor antagonist activity in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro activities were evaluated for topical in vivo efficacy in the Golden Syrian Hamster ear model. Several compounds showed reduction in wax esters in this validated animal model.

4-(Alkylthio)- and 4-(arylthio)-benzonitrile derivatives as androgen receptor antagonists for the topical suppression of sebum production.

The first examples of thioether-substituted benzonitriles as potential soft-drug androgen receptor antagonists are reported. A number of 4-(alkylthio)- and of 4-(arylthio)-benzonitrile analogs were evaluated in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro binding and cellular activities were evaluated for topical in vivo efficacy in the Golden Syrian hamster ear model. Analogs from both the 4-(alkylthio)- and of 4-(arylthio)-benzonitrile series showed moderate reduction of wax esters in vivo.

Rational design and synthesis of 4-((1R,2R)-2-hydroxycyclohexyl)-2(trifluoromethyl)benzonitrile (PF-998425), a novel, nonsteroidal androgen receptor antagonist devoid of phototoxicity for dermatological indications.

4-((1 R,2 R)-2-Hydroxycyclohexyl)-2(trifluoromethyl)benzonitrile [PF-0998425, (-)- 6a] is a novel, nonsteroidal androgen receptor antagonist for sebum control and treatment of androgenetic alopecia. It is potent, selective, and active in vivo. The compound is rapidly metabolized systemically, thereby reducing the risk of unwanted systemic side effects due to its primary pharmacology. (-)- 6a was tested negative in the 3T3 NRU assay, validating our rationale that reduction of conjugation might reduce potential phototoxicity.

Synthesis and biological evaluation of amino-pyridines as androgen receptor antagonists for stimulating hair growth and reducing sebum production.

A series of amino-pyridines were synthesized and evaluated for androgen antagonist activities. Among these compounds, (R)-(+)-6-[methyl-(1-phenyl-ethyl)-amino]-4-trifluoromethyl-nicotinonitrile was the most active example of this class. This compound displayed potent androgen receptor antagonist activity as well as favorable pharmacokinetic characteristics for a potential topical agent. It also demonstrated remarkable potency for stimulating hair growth in a male C3H mouse model as well as reducing sebum production in the male Syrian hamster ear model.

Preparation of 4-aryl-2-trifluoromethylbenzonitrile derivatives as androgen receptor antagonists for topical suppression of sebum production.

A series of substituted 4-aryl-2-trifluoromethylbenzonitrile analogs were evaluated in the human androgen receptor binding and cellular functional assays. Analogs with sufficient in vitro binding and cellular potency (IC(50)<200 nM) were tested in the progesterone receptor binding assay for selectivity and in the Golden Syrian hamster ear model for in vivo efficacy. Within the series, compound 4 e was identified to be the most active analog in vivo (wax ester inhibition=86%).