Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis.

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. the advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

Using Microemulsions: Formulation Based on Knowledge of Their Mesostructure.

Microemulsions, as thermodynamically stable mixtures of oil, water, and surfactant, are known and have been studied for more than 70 years. However, even today there are still quite a number of unclear aspects, and more recent research work has modified and extended our picture. This review gives a short overview of how the understanding of microemulsions has developed, the current view on their properties and structural features, and in particular, how they are related to applications. We also discuss more recent developments regarding nonclassical microemulsions such as surfactant-free (ultraflexible) microemulsions or ones containing uncommon solvents or amphiphiles (like antagonistic salts). These new findings challenge to some extent our previous understanding of microemulsions, which therefore has to be extended to look at the different types of microemulsions in a unified way. In particular, the flexibility of the amphiphilic film is the key property to classify different microemulsion types and their properties in this review. Such a classification of microemulsions requires a thorough determination of their structural properties, and therefore, the experimental methods to determine microemulsion structure and dynamics are reviewed briefly, with a particular emphasis on recent developments in the field of direct imaging by means of electron microscopy. Based on this classification of microemulsions, we then discuss their applications, where the application demands have to be met by the properties of the microemulsion, which in turn are controlled by the flexibility of their amphiphilic interface. Another frequently important aspect for applications is the control of the rheological properties. Normally, microemulsions are low viscous and therefore enhancing viscosity has to be achieved by either having high concentrations (often not wished for) or additives, which do not significantly interfere with the microemulsion. Accordingly, this review gives a comprehensive account of the properties of microemulsions, including most recent developments and bringing them together from a united viewpoint, with an emphasis on how this affects the way of formulating microemulsions for a given application with desired properties.

IGF-1 regulates astrocytic phagocytosis and inflammation through the p110α isoform of PI3K in a sex-specific manner.

Insulin-like growth factor-I (IGF-I) signaling plays a key role in neuroinflammation. Here we show that IGF-1 also regulates phagocytosis of reactive astrocytes through p110α isoform of phosphatidylinositol 3-kinase (PI3K), differentially in both sexes. Systemic bacterial lipopolysaccharide (LPS)-treatment increased the expression of GFAP, a reactive astrocyte marker, in the cortex of mice in both sexes and was blocked by IGF-1 only in males. In primary astrocytes, LPS enhanced the mRNA expression of Toll-like receptors (TLR2,4) and proinflammatory factors: inducible nitric oxide synthase (iNOS), chemokine interferon-γ-inducible protein-10 (IP-10) and cytokines (IL-1ß, IL-6, and IL-10) in male and female. Treatment with IGF-1 counteracted TLR4 but not TLR2, iNOS, and IP10 expression in both sexes and cytokines expression in males. Furthermore, reactive astrocyte phagocytosis was modulated by IGF-1 only in male astrocytes. IGF-1 was also able to increase AKT-phosphorylation only in male astrocytes. PI3K inhibitors, AG66, TGX-221, and CAL-101, with selectivity toward catalytic p110α, p110ß, and p110δ isoforms respectively, reduced AKT-phosphorylation in males. All isoforms interact physically with IGF-1-receptor in both sexes. However, the expression of p110α is higher in males while the expression of IGF-1-receptor is similar in male and female. AG66 suppressed the IGF-1 effect on cytokine expression and counteracted the IGF-1-produced phagocytosis decrease in male reactive astrocytes. Results suggest that sex-differences in the effect of IGF-1 on the AKT-phosphorylation could be due to a lower expression of the p110α in female and that IGF-1-effects on the inflammatory response and phagocytosis of male reactive astrocytes are mediated by p110α/PI3K subunit.

Chronic IL-10 overproduction disrupts microglia-neuron dialogue similar to aging, resulting in impaired hippocampal neurogenesis and spatial memory.

The subgranular zone of the dentate gyrus is an adult neurogenic niche where new neurons are continuously generated. A dramatic hippocampal neurogenesis decline occurs with increasing age, contributing to cognitive deficits. The process of neurogenesis is intimately regulated by the microenvironment, with inflammation being considered a strong negative factor for this process. Thus, we hypothesize that the reduction of new neurons in the aged brain could be attributed to the age-related microenvironmental changes towards a pro-inflammatory status. In this work, we evaluated whether an anti-inflammatory microenvironment could counteract the negative effect of age on promoting new hippocampal neurons. Surprisingly, our results show that transgenic animals chronically overexpressing IL-10 by astrocytes present a decreased hippocampal neurogenesis in adulthood. This results from an impairment in the survival of neural newborn cells without differences in cell proliferation. In parallel, hippocampal-dependent spatial learning and memory processes were affected by IL-10 overproduction as assessed by the Morris water maze test. Microglial cells, which are key players in the neurogenesis process, presented a different phenotype in transgenic animals characterized by high activation together with alterations in receptors involved in neuronal communication, such as CD200R and CX3CR1. Interestingly, the changes described in adult transgenic animals were similar to those observed by the effect of normal aging. Thus, our data suggest that chronic IL-10 overproduction mimics the physiological age-related disruption of the microglia-neuron dialogue, resulting in hippocampal neurogenesis decrease and spatial memory impairment.

TRPV2: A Key Player in Myelination Disorders of the Central Nervous System.

Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders.

Selection of insectary plants for the conservation of biological control agents of aphids and thrips in fruit orchards.

This study evaluated the potential of flowering plant species naturally occurring to promote the conservation and early establishment of key natural enemies of aphids and thrips in apple and peach orchards. Flowering plants present in the North East of Spain, a main fruit production area in Europe, were sampled to determine their flowering period and to identify potential natural enemies present on each plant species. Thirty-six plant species were found blooming from early March to late May and provided an array of flowers that might ensure food resources for natural enemies. Among them, six species - Eruca vesicaria (L.) Cav., Cardaria draba (L.) Desv., Euphorbia serrata (L.) S.G. Gmel., Malva sylvestris L., Anacyclus clavatus (Desf.) Pers. and Diplotaxis erucoides (L.) DC. - hosted a high diversity of potential natural enemies of aphids and thrips. Their blooming started early in the season and lasted for several sampling weeks and they were widely distributed. Moreover, they had available nectar even in those species with protected nectaries. Therefore, these plant species can be considered as promising candidates for inclusion in the ecological infrastructure designed for fruit orchards in the study area to promote the conservation of the biological control agents of aphids and thrips.

Direct Observation of Dynamic Tube Dilation in Entangled Polymer Blends: A Combination of Neutron Scattering and Dielectric Techniques.

We report a microscopic observation of the time-dependent dynamic tube dilation process on isofrictional bidisperse melts. By applying neutron spin echo (NSE) and dielectric techniques on blends of long polyisoprene (PI) chains with short PI additives with different topology, we access the dynamics of the tube dilation process on a molecular scale. The time-dependent tube dilation is directly revealed by NSE as an additional time dependence of the dynamic structure factor in the local reptation regime. We identify the characteristic time of tube dilation as the terminal time of the additive.

Facile Access to Completely Deuterated Single-Chain Nanoparticles Enabled by Intramolecular Azide Photodecomposition.

Access to completely deuterated single-chain nanoparticles (dSCNPs) has remained an unresolved issue. Herein, the first facile and efficient procedure to produce dSCNPs is reported, which comprises: i) the use of commercially available perdeuterated cyclic ether monomers as starting reagents, ii) a ring-opening copolymerization process performed in bulk to produce a neat dSCNP precursor, iii) a standard azidation reaction to decorate this precursor with azide moieties, and iv) a facile intramolecular azide photodecomposition step carried out under UV irradiation at high dilution providing with highly valuable, completely deuterated soft nano-objects from the precursor. dSCNPs are used to investigate by means of neutron-scattering measurements the form factor (radius of gyration, scaling exponent) of polyethylene oxide (PEO) chains in nanocomposites with different amounts of dSCNPs. Moreover, to illustrate the possibilities offered by the synthetic route disclosed in this communication for potential applications, the significant reduction in viscosity observed in a pure melt of polyether-based single-chain nanoparticles when compared to a melt of the corresponding linear polymer chains is shown.

Controlling Complex Nanoemulsion Morphology Using Asymmetric Cosurfactants for the Preparation of Polymer Nanocapsules.

Complex nanoemulsions, comprising multiphase nanoscale droplets, hold considerable potential advantages as vehicles for encapsulation and delivery as well as templates for nanoparticle synthesis. Although methods exist to controllably produce complex emulsions on the microscale, very few methods exist to produce them on the nanoscale. Here, we examine a recently developed method involving a combination of high-energy emulsification with conventional cosurfactants to produce oil-water-oil (O/W/O) complex nanoemulsions. Specifically, we study in detail how the composition of conventional ethoxylated cosurfactants Span80 and Tween20 influences the morphology and structure of the resulting complex nanoemulsions in the water-cyclohexane system. Using a combination of small-angle neutron scattering and cryo-electron microscopy, we find that the cosurfactant composition controls the generation of complex droplet morphologies including core-shell and multicore-shell O/W/O nanodroplets, resulting in an effective state diagram for the selection of nanoemulsion morphology. Additionally, the cosurfactant composition can be used to control the thickness of the water shell contained within the complex nanodroplets. We hypothesize that this degree of control, despite the highly nonequilibrium nature of the nanoemulsions, is ultimately determined by a competition between the opposing spontaneous curvature of the two cosurfactants, which strongly influences the interfacial curvature of the nanodroplets as a result of their ultralow interfacial tension. This is supported by a correlation between cosurfactant compositions that produces complex nanoemulsions and those that produce homogeneous mixed micelles in equilibrium surfactant-cyclohexane solutions. Ultimately, we show that the formation of complex O/W/O nanoemulsions is weakly perturbed upon the addition of hydrophilic polymer precursors, facilitating their use as templates for the formation of polymer nanocapsules.

Accommodative stimulus-response curves to low-pass filtered natural images.

PURPOSE: To assess how the monocular steady-state accommodative stimulus-response curve is modified when viewing low-pass filtered natural images. METHODS: Eighteen adult subjects participated in the study. The accommodative stimulus-response curve was objectively assessed by means of a Hartmann-Shack wavefront sensor. Measurements were taken at different accommodative demands. Target images were low-pass filtered versions of a natural image that were obtained applying different digital spatial filters that limited the spatial frequency content of the natural image. Cutoff spatial frequencies were set at 30, 21, and 15 cycles per degree (cy/deg). RESULTS: Mean data obtained for each target were fitted to linear models. For the low-pass filtered natural image with a cutoff spatial frequency at 30 cy/deg, the slope for the averaged stimulus-response curve was 0.614, while the slopes obtained for the other two low-pass filtered images corresponding to cutoff frequencies at 21 and 15 cy/deg were 0.613 and 0.619, respectively (p < 0.01). The determination coefficient was R2 ≥ 0.988 for all targets. An ANCOVA analysis revealed that these slopes were not significantly different from each other (p = 0.82). Additionally, accommodative error indexes were obtained to measure both the discrepancy between the regression lines and the ideal lines and the degree of correlation between the stimulus and the response. CONCLUSIONS: Our study reveals that the low and middle spatial frequencies (< 15 cy/deg) are the most important to guide to the needed levels the accommodative response to natural images.

Synthesis of Oil-Laden Poly(ethylene glycol) Diacrylate Hydrogel Nanocapsules from Double Nanoemulsions.

Multiple emulsions have received great interest due to their ability to be used as templates for the production of multicompartment particles for a variety of applications. However, scaling these complex droplets to nanoscale dimensions has been a challenge due to limitations on their fabrication methods. Here, we report the development of oil-in-water-in-oil (O1/W/O2) double nanoemulsions via a two-step high-energy method and their use as templates for complex nanogels comprised of inner oil droplets encapsulated within a hydrogel matrix. Using a combination of characterization methods, we determine how the properties of the nanogels are controlled by the size, stability, internal morphology, and chemical composition of the nanoemulsion templates from which they are formed. This allows for identification of compositional and emulsification parameters that can be used to optimize the size and oil encapsulation efficiency of the nanogels. Our templating method produces oil-laden nanogels with high oil encapsulation efficiencies and average diameters of 200-300 nm. In addition, we demonstrate the versatility of the system by varying the types of inner oil, the hydrogel chemistry, the amount of inner oil, and the hydrogel network cross-link density. These nontoxic oil-laden nanogels have potential applications in food, pharmaceutical, and cosmetic formulations.

Investigation of the dynamics of aqueous proline solutions using neutron scattering and molecular dynamics simulations.

We applied quasielastic neutron scattering (QENS) techniques to samples with two different contrasts (deuterated solute/hydrogenated solvent and the opposite label) to selectively study the component dynamics of proline/water solutions. Results on diluted and concentrated solutions (31 and 6 water molecules/proline molecule, respectively) were analyzed in terms of the susceptibility and considering a recently proposed model for water dynamics [Arbe et al., Phys. Rev. Lett., 2016, 117, 185501] which includes vibrations and the convolution of localized motions and diffusion. We found that proline molecules not only reduce the average diffusion coefficient of water but also extend the time/frequency range of the crossover region ('cage') between the vibrations and purely diffusive behavior. For the high proline concentration we also found experimental evidence of water heterogeneous dynamics and a distribution of diffusion coefficients. Complementary molecular dynamics simulations show that water molecules start to perform rotational diffusion when they escape the cage regime but before the purely diffusive behavior is established. The rotational diffusion regime is also retarded by the presence of proline molecules. On the other hand, a strong coupling between proline and water diffusive dynamics which persists with decreasing temperature is directly observed using QENS. Not only are the temperature dependences of the diffusion coefficients of both components the same, but their absolute values also approach each other with increasing proline concentration. We compared our results with those reported using other techniques, in particular using dielectric spectroscopy (DS). A simple approach based on molecular hydrodynamics and a molecular treatment of DS allows rationalizing the a priori puzzling inconsistency between QENS and dielectric results regarding the dynamic coupling of the two components. The interpretation proposed is based on general grounds and therefore should be applicable to other biomolecular solutions.

Human eyes do not need monochromatic aberrations for dynamic accommodation.

PURPOSE: To determine if human accommodation uses the eye's own monochromatic aberrations to track dynamic accommodative stimuli. METHODS: Wavefront aberrations were measured while subjects monocularly viewed a monochromatic Maltese cross moving sinusoidally around 2D of accommodative demand with 1D amplitude at 0.2 Hz. The amplitude and phase (delay) of the accommodation response were compared to the actual vergence of the stimulus to obtain gain and temporal phase, calculated from wavefront aberrations recorded over time during experimental trials. The tested conditions were as follows: Correction of all the subject's aberrations except defocus (C); Correction of all the subject's aberrations except defocus and habitual second-order astigmatism (AS); Correction of all the subject's aberrations except defocus and odd higher-order aberrations (HOAs); Correction of all the subject's aberrations except defocus and even HOAs (E); Natural aberrations of the subject's eye, i.e., the adaptive-optics system only corrected the optical system's aberrations (N); Correction of all the subject's aberrations except defocus and fourth-order spherical aberration (SA). The correction was performed at 20 Hz and each condition was repeated six times in randomised order. RESULTS: Average gain (±2 standard errors of the mean) varied little across conditions; between 0.55 ± 0.06 (SA), and 0.62 ± 0.06 (AS). Average phase (±2 standard errors of the mean) also varied little; between 0.41 ± 0.02 s (E), and 0.47 ± 0.02 s (O). After Bonferroni correction, no statistically significant differences in gain or phase were found in the presence of specific monochromatic aberrations or in their absence. CONCLUSIONS: These results show that the eye's monochromatic aberrations are not necessary for accommodation to track dynamic accommodative stimuli.

Oil-in-Water-in-Oil Multinanoemulsions for Templating Complex Nanoparticles.

Complex nanoemulsions involving nanodroplets with a defined inner structure have great potential for encapsulation and templating applications. We report a method to form novel complex oil-in-water-in-oil nanoemulsions using a combination of high-energy processing with mixed nonionic surfactants that simultaneously achieve ultralow interfacial tension and frustrated curvature of the water-oil interface. The method produces multinanoemulsions possessing morphologies resembling water-swollen reverse vesicles with core-shell and multicore-shell morphologies of water in cyclohexane. A combination of macroscopic and microscopic characterization conclusively verifies and quantifies the complex morphologies, which vary systematically and reproducibly with water content for water volume fractions between 0.01 and 0.10. The complex morphologies are stable tens of hours, providing a route for their use as liquid templates for internally structured nanoparticles. As a demonstration, we test the complex nanoemulsions' ability to template complex polymer nanogels.

Optical Characterization Method for Tilted or Decentered Intraocular Lenses.

PURPOSE: To test and validate a method for measuring the optical quality and optical power of monofocal intraocular lenses (IOLs) in the presence of a tilt or a decentration as well as its optical power. METHODS: The experimental system consists of an artificial eye (wet-cell) and a commercial aberrometer with a Shack-Hartmann sensor. Optical image quality parameters such as point spread function (PSF), modulation transfer function (MTF), and the simulations of the retinal image of an extended object are computed from the in vitro wavefront data of an IOL. Repeatability and reproducibility of the system are tested. Mathematical and ray-tracing simulations are used to ascertain the precision and accuracy of the method. Comparison with a standard single-pass method of measurement is also made. Optical properties of four commercial monofocal IOLs are measured under tilts of 0°, 2°, and 4°, and vertical decentrations of 0, 0.2, and 0.4 mm; the results of such measurements are compared with a ray-tracing simulation. RESULTS: Precision and accuracy of the system are in good agreement with theoretical calculations. Reproducibility and repeatability are within standard ISO norms. MTFs obtained with this method and with the standard method are very similar. The precision of the estimation of the IOL's optical power is higher than 98.6%. Commercially tested IOLs show a decrease of optical quality in the presence of decentrations and tilts, and their susceptibility to tilts and decentrations depends on the asphericity. These results are in concordance with the results obtained by ray-tracing simulation. CONCLUSIONS: A simple wavefront-based method for in vitro measurements of the wavefront aberrations and power of an IOL is proposed and tested showing accurate and precise results.

The effect of longitudinal chromatic aberration on the lag of accommodation and depth of field.

PURPOSE: Longitudinal chromatic aberration is present in all states of accommodation and may play a role in the accommodation response and the emmetropisation process. We study the change of the depth of field (DOFi) with the state of accommodation, taking into account the longitudinal chromatic aberration. METHODS: Subjective DOFi was defined as the range of defocus beyond which the blur of the target (one line of optotypes of 0.1 logMAR shown on a black-and-white microdisplay, seen through different colour filters) was perceived as objectionable. The subject's eye was paralysed and different, previously-measured accommodative states (corresponding to the accommodative demands of 0D, 2D and 4D) were simulated with a deformable mirror. Different colour conditions (monochromatic red, green and blue and polychromatic (white) were tested. The DOFi was measured subjectively, using a motorised Badal system. RESULTS: Taking as reference the average accommodative response for the white stimulus, the blue response exhibits on average a lead of 0.45 ± 0.09D, the green a negligible lead of 0.07 ± 0.02D and red a lag of 0.49 ± 0.10D. The monochromatic DOFi, calculated by averaging DOFi over the red, green and blue colour conditions for each accommodative demand was 1.10 ± 0.10D for 0D, 1.20 ± 0.08D for 2D, and 1.26 ± 0.40D for 4D. The polychromatic white DOFi were greater than the average monochromatic DOFi by 19%, 9% and 14% for 0D, 2D, and 4D of accommodative demand, respectively. CONCLUSION: The longitudinal chromatic aberration causes a dioptric shift of the monochromatic accommodation response. The study did not reveal this shift to depend on the accommodative demand or to have an effect on the DOFi.

Opto-mechanical artificial eye with accommodative ability.

The purpose of this study was to describe the design and characterization of a new opto-mechanical artificial eye (OMAE) with accommodative ability. The OMAE design is based on a second-pass configuration where a small source of light is used at the artificial retina plane. A lens whose focal length can be changed electronically was used to add the accommodation capability. The changes in the OMAE's aberrations with the lens focal length, which effectively changes the accommodative state of the OMAE, were measured with a commercial aberrometer. Changes in power and aberrations with room temperature were also measured. The OMAE's higher-order aberrations (HOAs) were similar to the ones of the human eye, including the rate at which fourth-order spherical aberration decreased with accommodation. The OMAE design proposed here is simple, and it can be implemented in an optical system to mimic the optics of the human eye.

Phase Behavior of Nonionic Microemulsions with Multi-end-capped Polymers and Its Relation to the Mesoscopic Structure.

The polymer architecture of telechelic or associative polymers has a large impact on the bridging of self-assembled structures. This work presents the phase behavior, small angle neutron scattering (SANS), dynamic light scattering (DLS), and fluorescence correlation spectroscopy (FCS) of a nonionic oil-in-water (O/W) microemulsion with hydrophobically end-capped multiarm polymers with functionalities f = 2, 3, and 4. For high polymer concentrations and large average interdroplet distance relative to the end-to-end distance of the polymer, d/R(ee), the system phase separates into a dense, highly connected droplet network phase, in equilibrium with a dilute phase. The extent of the two-phase region is larger for polymers with similar length but higher f. The interaction potential between the droplets in the presence of polymer has both a repulsive and an attractive contribution as a result of the counterbalancing effects of the exclusion by polymer chains and bridging between droplets. This study experimentally demonstrates that higher polymer functionalities induce a stronger attractive force between droplets, which is responsible for a more extended phase separation region, and correlate with lower collective droplet diffusivities and higher amplitude of the second relaxation time in DLS. The viscosity and the droplet self-diffusion obtained from FCS, however, are dominated by the end-capped chain concentration.

Focus correction in an apodized system with spherical aberration.

We performed a theoretical and computational analysis of the through-focus axial irradiance in a system with a Gaussian amplitude pupil function and fourth- and sixth-order spherical aberration (SA). Two cases are analyzed: low aberrated systems, and the human eye containing significant levels of SA and a natural apodization produced by the Stiles-Crawford effect. Results show that apodization only produces a refraction change of the plane that maximized the Strehl ratio for eyes containing significant levels of negative SA.