Airway Delivery of Hydrogel-Encapsulated Niclosamide for the Treatment of Inflammatory Airway Disease

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl⁻ currents and intracellular Ca²⁺ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca²⁺ activated Cl⁻ channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca²⁺ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.     

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.     

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia—a locus for craniofacial sensory nerves—were used to examine how NGF affects the Ca²⁺-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca²⁺-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca²⁺-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181–197 and 198–206) to support CAP-evoked Ca²⁺-dependent exocytosis at different intensities of stimulation.     

TBP,PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.     

Precipitation of asphaltenes from solvent-diluted heavy oil and thermodynamic properties of solvent-diluted heavy oil solutions

Ratios of n-heptane (hep) to toluene (tol) affect the solubility of the asphaltenes in heavy oil extraction processes. Consequently phase changes and time after mixing n-heptane and heavy oil in toluene are important for understanding produced emulsions. The kinetics of phase change when n-heptane is added to toluene-diluted heavy oils, and the thermodynamic properties of partially deasphalted heavy oils were studied. The methods used were monitoring precipitation in time using light microscopy, quantitative asphaltenes analysis by near infrared spectroscopy, refractive index and densities measurements, and calculated solubility parameters of mixtures. At critical mass ratios of hep/tol from 1.37 to 2.0 in diluted heavy oil the precipitated asphaltene particles were observed under the microscope after lag times from 2 h to instantly. Lag times were longer at low initial oil concentration. The floc growth time decreased as heavy oil concentration in toluene increased. The growth patterns in time appeared as dots to beads (strings) to clusters (fractal-like flocs). Final wt% precipitated asphaltenes vs. mass fraction (hep+tol)/heavy oil followed sigmoidal relationships. Curves showing wt% soluble asphaltenes vs. mass fraction hep/tol after 24 h initially followed the same shape as time zero curves and diverged at the onset ratios of hep/tol. Slope for precipitated asphaltenes vs. solubility parameters curve showed a break at 16.4 MPa^1/2. Linear correlations were established for concentrations of soluble asphaltenes in residual oils and density, for refractive index and density and for refractive index and solubility parameter. The latter correlation was in accordance with Lorenz-Lorentz theory. These equations provided a means by which oil density, refractive index and solubility parameter can be predicted when these measurements are difficult to measure practically.     

Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.     

Diabetes and Ischemic Stroke: An Old and New Relationship an Overview of the Close Interaction between These Diseases

Diabetes mellitus is a comprehensive expression to identify a condition of chronic hyperglycemia whose causes derive from different metabolic disorders characterized by altered insulin secretion or faulty insulin effect on its targets or often both mechanisms. Diabetes and atherosclerosis are, from the point of view of cardio- and cerebrovascular risk, two complementary diseases. Beyond shared aspects such as inflammation and oxidative stress, there are multiple molecular mechanisms by which they feed off each other: chronic hyperglycemia and advanced glycosylation end-products (AGE) promote ‘accelerated atherosclerosis’ through the induction of endothelial damage and cellular dysfunction. These diseases impact the vascular system and, therefore, the risk of developing cardio- and cerebrovascular events is now evident, but the observation of this significant correlation has its roots in past decades. Cerebrovascular complications make diabetic patients 2–6 times more susceptible to a stroke event and this risk is magnified in younger individuals and in patients with hypertension and complications in other vascular beds. In addition, when patients with diabetes and hyperglycemia experience an acute ischemic stroke, they are more likely to die or be severely disabled and less likely to benefit from the one FDA-approved therapy, intravenous tissue plasminogen activator. Experimental stroke models have revealed that chronic hyperglycemia leads to deficits in cerebrovascular structure and function that may explain some of the clinical observations. Increased edema, neovascularization, and protease expression as well as altered vascular reactivity and tone may be involved and point to potential therapeutic targets. Further study is needed to fully understand this complex disease state and the breadth of its manifestation in the cerebrovasculature.