APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

Chemorheology of Aqueous-Based Alumina-Poly(vinyl alcohol) Gelcasting Suspensions

A new gelcasting system based on aqueous-based alumina-poly(vinyl alcohol) (PVA) suspensions cross-linked by an organotitanate coupling agent has been developed. The chemorheological properties of this system exhibited a strong compositional dependence. A sol-gel phase diagram was established, which yielded the critical titanium concentration ([Ti]_c) required for gelation at a given PVA volume fraction as well as the minimum PVA volume fraction (φ^min_PVA= 0.0245) and titanium concentration ([Ti]_min= 9.984 × 10^-4 g of Ti/mL) below which gelation was not observed irrespective of solution composition. The gelation time of suspensions of constant PVA volume fraction (φ^soln_PVA) decreased with increased cross-linking agent concentration, temperature, and solids volume fraction. The steady-state viscosity and elastic modulus of polymer solutions (φ^soln_PVA= 0.05) of varying [Ti] were well described by the percolation model, giving scaling exponents of 0.84 and 1.79, respectively. The steady-state elastic modulus of gelcasting suspensions, which provided a measure of their handling strength in the as-gelled state, increased with increased solids volume fraction.     

Simple and Straightforward Synthesis of Porous Ionosilica for Efficient Chromate Adsorption

We report a very simple synthesis strategy for the formation of moderately porous ionosilica materials containing ammonium groups. We synthesized an ionosilica xerogel and an ionosilica aerogel, either via conventional or supercritical work‐up. The synthesis scheme of both precursor and ionosilica materials could easily be up‐scaled to a 100 g scale. All syntheses and work‐up procedures are sustainable as no additional agent and very low quantities of solvents were used, and no additional purifications steps were necessary. Although both materials show excellent anion exchange properties for chromate adsorption, noticeable differences were found regarding the thermodynamics of the exchange process. We attribute these differences to different surface chemistries of the materials, induced by the different work‐up procedures. Due to the easy availability of high quantities of material, ionosilicas can be implemented as functional ion exchange materials in larger scale processes, thus opening new fields of applications.     

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.     

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject’s brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5^-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5^-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.     

Pial Vessel-Associated Microglia/Macrophages Increase in Female Dahl-SS/Jr Rats Independent of Pregnancy History

As the resident immune cells of the central nervous system, microglia have a wide range of functions such as surveillance, phagocytosis, and signaling through production of chemokines and cytokines. Recent studies have identified and characterized macrophages residing at the meninges, a series of layers surrounding the brain and spinal cord. While perivascular microglia within the brain parenchyma increase following chronic hypertension, there are no reports of changes at the meninges, and specifically, associated with the pial vasculature. Thus, we used female Sprague Dawley and Dahl salt-sensitive (SS/Jr) rat brains, stained for ionized calcium-binding adapter molecule (Iba1), and characterized microglia/macrophages associated with pial vessels in the posterior brain. Results indicate that Iba1⁺ pial vessel-associated microglia (PVAM) completely surrounded the vessels in brains from the Dahl-SS/Jr rats. PVAM density was significantly higher and distance between PVAMs lower in Dahl-SS/Jr compared to the Sprague Dawley rat brains. Pregnancy history did not affect these findings. While the functional role of these cells are not known, we contextualize our novel findings with that of other studies assessing or characterizing myeloid cells at the borders of the CNS (meninges and choroid plexus) and perivascular macrophages and propose their possible origin in the Dahl-SS/Jr model of chronic hypertension.     

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.     

As you like it: Understanding the relationship between packing design and accessibility

Accessible packaging is continually receiving greater attention as an ageing population becomes a primary driver behind inclusive design. As we age, our strength dexterity and some cognitive functions decline. For this reason, the ease of interaction with simple tasks in daily living becomes an ever increasing concern. ISO 17480 “Packaging Guidelines” consider capability issues relating to packaging and ageing to broadly come under three areas, namely, strength, dexterity, and cognition. Significant previous work has been undertaken looking at the issue of strength on packaging accessibility, with fewer studies looking at the relationship between the physical demands of dexterity and the understanding of how to open the pack. In this previous work, there has been little attempt to quantify the effect of the affordances, perceptual information and symbology, and the physical demands of the pack and how this relates to accessibility. Hence, this exploratory study seeks to use motion capture and a dexterity test, along with a cognitive demand test to study this relationship and any changes with age. This research indicates that the dexterous demands of a task are linked to the cognitive demands; and thus in reaching a level of comprehension of the pack and how to access the product a subject may interact with a pack effectively.     

Peaks Over Thresholds Modeling With Multivariate Generalized Pareto Distributions

When assessing the impact of extreme events, it is often not just a single component, but the combined behavior of several components which is important. Statistical modeling using multivariate generalized Pareto (GP) distributions constitutes the multivariate analogue of univariate peaks over thresholds modeling, which is widely used in finance and engineering. We develop general methods for construction of multivariate GP distributions and use them to create a variety of new statistical models. A censored likelihood procedure is proposed to make inference on these models, together with a threshold selection procedure, goodness-of-fit diagnostics, and a computationally tractable strategy for model selection. The models are fitted to returns of stock prices of four UK-based banks and to rainfall data in the context of landslide risk estimation. Supplementary materials and codes are available online.     

Improved scanning laser fundus imaging using polarimetry

We present a polarimetric technique to improve fundus images that notably simplifies and extends a previous procedure [Opt. Lett.27, 830 (2002)]. A generator of varying polarization states was incorporated into the illumination path of a confocal scanning laser ophthalmoscope. A series of four images, corresponding to independent incoming polarization states, were recorded. From these images, the spatially resolved elements of the top row of the Mueller matrix were computed. From these elements, images with the highest and lowest quality (according to different image quality metrics) were constructed, some of which provided improved visualization of fundus structures of clinical importance (vessels and optic nerve head). The metric values were better for these constructed images than for the initially recorded images and better than averaged images. Entropy is the metric that is most sensitive to differences in the image quality. Improved visualization of features could aid in the detection, localization, and tracking of ocular disease and may be applicable in other biomedical imaging.