Identification of age-associated proteins and functional alterations in human primary retinal pigment epithelium cells

Background: Retinal pigmented epithelium (RPE) has essential functions to nourish and support the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE in aging remain poorly defined. Methods: We isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10 - 67 years). A quantitative proteomic analysis was performed to analyze their intracellular and secreted protein changes, and potential age-associtated mechanisms were validated by ARPE-19 and hRPE cells. Results: Age-stage related subtypes and age-associtated proteins and functional alterations were revealed. Proteomic data and verifications showed that RNF123 and RNF149 related ubiquitin-mediated proteolysis might be an important clearance mechanism in elimination of oxidative damaged proteins in aged hRPE. In older hRPE cells, apoptotic signaling related pathways were up-regulated and endoplasmic reticulum organization was down-regulated both in intracellular and secreted proteome. Conclusions: This work paints a detailed molecular picture of human RPE in aging process and provides new insights for molecular characteristics of RPE in aging and related clinical retinal conditions.

Emerging Roles of Astrocyte Kir4.1 Channels in the Pathogenesis and Treatment of Brain Diseases

Inwardly rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K+) buffering, a clearance mechanism for excessive extracellular K+, in tripartite synapses. In addition to K+ homeostasis, astrocytic Kir4.1 channels also play an essential role in regulating extracellular glutamate levels via coupling with glutamate transporters. Moreover, Kir4.1 channels act as novel modulators of the expression of brain-derived neurotrophic factor (BDNF) in astrocytes. Specifically, inhibition of astrocytic Kir4.1 channels elevates extracellular K+ and glutamate levels at synapses and facilitates BDNF expression in astrocytes. These changes elevate neural excitability, which may facilitate synaptic plasticity and connectivity. In this article, we summarize the functions and pharmacological features of Kir4.1 channels in astrocytes and highlight the importance of these channels in the treatment of brain diseases. Although further validation in animal models and human patients is required, astrocytic Kir4.1 channel could potentially serve as a novel therapeutic target for the treatment of depressive disorders and epilepsy.

Extracellular Vesicles and Thrombosis: Update on the Clinical and Experimental Evidence

Extracellular vesicles (EVs) compose a heterogenous group of membrane-derived particles, including exosomes, microvesicles and apoptotic bodies, which are released into the extracellular environment in response to proinflammatory or proapoptotic stimuli. From earlier studies suggesting that EV shedding constitutes a cellular clearance mechanism, it has become evident that EV formation, secretion and uptake represent important mechanisms of intercellular communication and exchange of a wide variety of molecules, with relevance in both physiological and pathological situations. The putative role of EVs in hemostasis and thrombosis is supported by clinical and experimental studies unraveling how these cell-derived structures affect clot formation (and resolution). From those studies, it has become clear that the prothrombotic effects of EVs are not restricted to the exposure of tissue factor (TF) and phosphatidylserines (PS), but also involve multiplication of procoagulant surfaces, cross-linking of different cellular players at the site of injury and transfer of activation signals to other cell types. Here, we summarize the existing and novel clinical and experimental evidence on the role and function of EVs during arterial and venous thrombus formation and how they may be used as biomarkers as well as therapeutic vectors.

Correlation between Reflux Symptom Index and Reflux Finding Score in Laryngopharyngeal Reflux

Introduction: Laryngopharyngeal reflux is an extra esophageal variant of gastro esophageal reflux disease and is characterized by change in voice, recurrent throat clearing, chronic cough, discomfort in throat, globus. The larynx and pharynx are devoid of the normal acid clearance mechanism even three episodes of reflux per week seems to be associated with a significant disease. Aims: The aim of the study was to evaluate the correlation between the reflux symptom index and reflux finding score in patients with Laryngopharyngeal reflux. Methods: This prospective analytical study was conducted from November 2019 to October 2020 in total of 65 patients presented in department of Otorhinolaryngology, Nepalgunj Medcial College and Teaching Hospital, Nepalgunj. Reflux symptom index questionnaire with nine Questions were answered by patients on a 5 point scale. Reflux symptom index of more than 13 out of total score of 45 was considered to indicate Laryngopharyngeal reflux were as, reflux finding score was based on laryngoscopic findings after evaluating 8 items. Score more than 7 out of 26 was taken as an indicator for presence of Laryngopharyngeal reflux. Results: The reflux symptom index was more than 13 on 22 patients withmean11.85±3.48 and reflux finding score was more than 7 on 11 patients with mean 5.02±3.23 with statistically moderate correlation between reflux symptom index and reflux finding score (p=0.000,r=0.595). Conclusion: There is moderate correlation between the reflux symptom index and reflux finding score. The combined use of these questionnaires and laryngoscopic findings can be more precise, practical and cost effective in the diagnosis of laryngopharyngeal reflux.

A Quantitative Interspecies Comparison of The Respiratory Mucociliary Clearance Mechanism

Background: Collectively coordinated ciliary activity constantly propels the airway surface liquid, which lines the luminal surface of the vertebrate respiratory system, in cranial direction – constituting mucociliary clearance, the primary defence mechanism of our airways. Our contemporary understanding on how the quantitative characteristics of the metachronal wave field determines the resulting mucociliary transport is still limited, which is partly due to the sparse availability of quantitative observational data. Methods: We employed high-speed video reflection contrast microscopy to simultaneously image and quantitatively characterize the metachronal wave field as well as the mucociliary transport in excised bovine, porcine, ovine, lapine, turkey and ostrich samples of the luminal tracheal wall. Advanced image processing techniques were used to determine the ciliary beating frequency (CBF), the velocity and the wavelength of the metachronal wave as well as the mucociliary transport velocity. Results: The mucociliary transport direction was found to strongly correlate with the mean wave propagation direction in all six species. The CBF yielded similar values (10−15 Hz) for all six species. Birds were found to exhibit considerably higher transport speeds (130−260 μm/s) than mammals (20−80 μm/s). While the average transport direction significantly deviates from the tracheal long axis (TLA) in mammals, no significant deviation from the TLA was found in birds. In comparison to mammals, longer metachronal wavelengths were found in birds. Finally, the metachronal waves were found to propagate at about 4−8 times the speed of mucociliary transport in mammals, whereas the metachronal waves propagate at about the speed of mucociliary transport in birds. Conclusions: The tracheal mucociliary clearance mechanism is based on a symplectic metachronsim in all examined species. The mucociliary transport in birds is fast and roughly follows the TLA, whereas the transport is slower and proceeds along a left-handed spiral in mammals. The longer wavelengths and the lower ratio between the metachronal wave speed and the mucociliary transport speed provide further evidence that the mucociliary clearance mechanism operates differently in birds than in mammals.

New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury

We characterized the tissue repair response after penetrating traumatic brain injury (pTBI) in this study. Seventy specific pathogen-free Kunming mice were randomly divided into the following groups: normal control, 1, 3, 7, 15, 21, and 30 days after pTBI. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were performed to examine and monitor brain tissue morphology, and the distribution and expression of lymphatic-specific markers lymphatic vessel endothelial receptor-1 (LYVE-1), hematopoietic precursor cluster of differentiation 34 (CD34) antigen, and Prospero-related homeobox-1 (PROX1) protein. H&E staining revealed that damaged and necrotic tissues observed on day 1 at and around the injury site disappeared on day 7, and there was gradual shrinkage and disappearance of the lesion on day 30, suggesting a clearance mechanism. We explored the possibility of lymphangiogenesis causing this clearance as part of the post-injury response. Notably, expression of lymphangiogenesis markers LYVE-1, CD34, and PROX1 was detected in damaged mouse brain tissue but not in normal tissue. Moreover, new lymphatic cells and colocalization of LYVE-1/CD34 and LYVE-1/PROX1 were also observed. Our findings of the formation of new lymphatic cells following pTBI provide preliminary insights into a post-injury clearance mechanism in the brain. Although we showed that lymphatic cells are implicated in brain tissue repair, further research is required to clarify the origin of these cells.

The role of aldehyde oxidase in the metabolic clearance of substituted benzothiazoles

Introduction: A group of substituted benzothiazoles from a research project was found to have low microsomal clearance. However, these compounds had very high clearance in vivo. Experimental: In the present study, the clearance mechanism of two of the structural analogs, was investigated in vitro and in vivo. Results and Discussion: In vitro studies showed the formation of corresponding non-P450 dependent oxidative metabolites in S9, cytosol, and hepatocytes. The in vitro formation of these metabolites was observed in mouse, rat, non-human primates, and human. Dog did not form the corresponding metabolites in any of the matrices. Inhibition studies with S9 fraction and incubation with human recombinant aldehyde oxidase (AO) showed that the formation of the corresponding metabolites was AO dependent. To investigate the role of this pathway in vivo, mice were dosed with compound A and bile and plasma were analyzed. Most of the metabolites in bile contained the AO dependent oxidized benzothiazole moiety, indicating that metabolism involving AO was probably the main pathway for clearance. The same metabolites were also observed circulating in plasma. Mass spectrometric analysis of the metabolite showed that the oxidation was on the benzothiazole moiety, but the exact position could not be identified. Isolation of the metabolite of compound A and analysis by NMR confirmed the structure of the metabolite as C2 carbon oxidation of the thiazole ring resulting in carboxamide moiety. Further comparison of both metabolites with corresponding authentic standards confirmed the structures. Conclusion: To our knowledge, such an observation of in vitro and in vivo oxidation of substituted benzothiazole by AO has not been reported before. The results helped the medicinal chemists design compounds that avoid AO mediated metabolism and with better ADME property.

Dispersion as a waste-clearance mechanism in flow through penetrating perivascular spaces in the brain

Accumulation of metabolic wastes in the brain is correlated with several neurodegenerative disorders, including Alzheimer’s disease. Waste transport and clearance occur via dispersion, the combined effect of diffusion and advection by flow of fluid. We examine the relative contributions of diffusion and advection in the perivascular spaces (PVSs) that surround penetrating cortical blood vessels and are filled with cerebrospinal fluid (CSF). To do so, we adapt prior analytic predictions of dispersion to the context of PVSs. We also perform advection-diffusion simulations in PVS-like geometries with parameters relevant to transport of amyloid-$$\beta$$ β (associated with Alzheimer’s) in a variety of flows, motivated by in vivo measurements. Specifically, we examine solute transport in steady and unsteady Poiseuille flows in an open (not porous) concentric circular annulus. We find that a purely oscillatory flow enhances dispersion only weakly and does not produce significant transport, whereas a steady flow component, even if slow, clears waste more effectively.

DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo.Abstract Graphic