Identification of Keratoconus-Related Phenotypes in Three Ppip5k2 Mouse Models.

Purpose: It is necessary to establish a mouse model of keratoconus (KC) for research and therapy. We aimed to determine corneal phenotypes in 3 Ppip5k2 mouse models. Methods: Central corneal thickness (CCT) was determined using spectral domain optical coherence tomography (SD-OCT) in Ppip5k2+/K^ (n = 41 eyes), Ppip5k2K^/K^ (n = 17 eyes) and 2 knock-in mice, Ppip5k2S419A/+ (n = 54 eyes) and Ppip5k2S419A/S419A (n = 18 eyes), and Ppip5k2D843S/+ (n = 42 eyes) and Ppip5k2D843S/D843S (n = 44 eyes) at 3 and 6 months. Pachymetry maps were generated using the Mouse Corneal Analysis Program (MCAP) to process OCT images. Slit lamp biomicroscopy was used to determine any corneal abnormalities, and, last, hematoxylin and eosin (H&E) staining using corneal sections from these animals was used to examine morphological changes. Results: CCT significantly decreased from 3 to 6 months in the Ppip5k2+/K^ and Ppip5k2K^/K^ mice compared to their littermate controls. OCT-based pachymetry maps revealed abnormally localized thinning in all three models compared to their wild-type (WT) controls. Slit lamp examinations revealed corneal abnormalities in the form of bullous keratopathy, stromal edema, stromal scarring, deep corneal neovascularization, and opacities in the heterozygous/homozygous mice of the three models in comparison with their controls. Corneal histological abnormalities, such as epithelial thickening and stromal layer damage, were observed in the heterozygous/homozygous mice of the three models in comparison with the WT controls. Conclusions: We have identified phenotypic and histological changes in the corneas of three mouse lines that could be relevant in the development of animal models of KC.

Evaluation of Novel Nasal Mucoadhesive Nanoformulations Containing Lipid-Soluble EGCG for Long COVID Treatment.

Following recovery from the acute infection stage of the SARS-CoV-2 virus (COVID-19), survivors can experience a wide range of persistent Post-Acute Sequelae of COVID-19 (PASC), also referred to as long COVID. According to the US National Research Action Plan on Long COVID 2022, up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to these symptoms, leading to a USD 50 billion annual loss of salary. Neurological symptoms associated with long COVID result from persistent infection with SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation in the central nervous system (CNS). As of today, there is no evidence that vaccines or medications can clear the persistent viral infection in olfactory mucosa. Recently published clinical data demonstrate that only 5% of long COVID anosmia patients have fully recovered during the past 2 years, and 10.4% of COVID patients are still symptomatic 18 months post-infection. Our group demonstrated that epigallocatechin-3-gallate-monopalmitate (EC16m) nanoformulations possess strong antiviral activity against human coronavirus, suggesting that this green-tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug targeting the persistent SARS-CoV-2 infection, as well as inflammation and oxidative stress in the CNS, leading to restoration of neurologic functions. The objective of the current study was to evaluate the mucociliary safety of the EC16m nasal nanoformulations and their efficacy against human coronavirus. METHODS: Nanoparticle size and Zeta potential were measured using the ZetaView Nanoparticle Tracking Analysis system; mucociliary safety was determined using the MucilAir human nasal model; contact antiviral activity and post-infection inhibition against the OC43 viral strain were assessed by the TCID50 assay for cytopathic effect on MRC-5 cells. RESULTS: The saline-based EC16 mucoadhesive nanoformulations containing 0.005 to 0.02% w/v EC16m have no significant difference compared to saline (0.9% NaCl) with respect to tissue integrity, cytotoxicity, and cilia beat frequency. A 5 min contact resulted in 99.9% inactivation of ß-coronavirus OC43. OC43 viral replication was inhibited by >90% after infected MRC-5 cells were treated with the formulations. CONCLUSION: The saline-based novel EC16m mucoadhesive nasal nanoformulations rapidly inactivated human coronavirus with mucociliary safety properties comparable to saline, a solution widely used for nasal applications.

The impact of ischemic stroke on bone marrow microenvironment and extracellular vesicles: A study on inflammatory and molecular changes.

An ischemic stroke (IS) is caused due to the lack of blood flow to cerebral tissue. Most of the studies have focused on how stroke affects the localized tissue, but it has been observed that a stroke can cause secondary complications in distant organs, such as Bone Marrow (BM). Our study focused on the effect of ischemic strokes on the bone marrow microenvironment. Bone marrow (BM) is a vital organ that maintains inflammatory homeostasis and aids in the repair of damaged tissue after injury/IS. We used the middle cerebral artery occlusion (MCAO) model of ischemic stroke on adult mice (6 months) and investigated the changes in the BM environment. BM cells were used for western blot and RT-PCR, and the BM supernatant was used for cytokine analysis and extracellular vesicle (EVs) isolation. We observed a significant increase in the total cell number within the BM and an increase in TNF-alpha and MCP-1, which are known for inducing a pro-inflammatory environment. Western blots analysis on the whole BM cell lysate demonstrated elevated levels of inflammatory factors (IL-6, TNF-alpha, and TLR-4) and senescence markers (p21 p16). EVs isolated from the BM supernatant showed no change in size or concentration; however, we found that the EVs carried increased miRNA-141-3p and miRNA-34a. Proteomic analysis on BM-derived EVs showed an alteration in the protein cargo of IS. We observed an increase in FgB, C3, Fn1, and Tra2b levels. The signaling pathway analysis showed mitochondrial function is most affected within the bone marrow. Our study demonstrated that IS induces changes in the BM environment and EVs secreted in the BM.

Feasibility Study of Developing a Saline-Based Antiviral Nanoformulation Containing Lipid-Soluble EGCG: A Potential Nasal Drug to Treat Long COVID.

A recent estimate indicates that up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to associated symptoms, leading to a USD 50 billion annual loss of salary. Post-COVID (Long COVID) neurologic symptoms are due to the initial robust replication of SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation of the olfactory epithelium (OE) and the central nervous system (CNS), and the OE becoming a persistent infection site. Previously, our group showed that Epigallocatechin-3-gallate-palmitate (EC16) nanoformulations possess strong antiviral activity against human coronavirus, suggesting this green tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug to eliminate the persistent SARS-CoV-2 infection, leading to restored olfactory function and reduced inflammation in the CNS. The objective of the current study was to determine the compatibility of the nanoformulations with human nasal primary epithelial cells (HNpECs). METHODS: Nanoparticle size was measured using the ZetaView Nanoparticle Tracking Analysis (NTA) system; contact antiviral activity was determined by TCID50 assay for cytopathic effect on MRC-5 cells; post-infection inhibition activity was determined in HNpECs; and cytotoxicity for these cells was determined using an MTT assay. The rapid inactivation of OC43 (a ß-coronavirus) and 229E (α-coronavirus) viruses was further characterized by transmission electron microscopy. RESULTS: A saline-based nanoformulation containing 0.1% w/v EC16 was able to inactivate 99.9999% ß-coronavirus OC43 on direct contact within 1 min. After a 10-min incubation of infected HNpECs with a formulation containing drug-grade EC16 (EGCG-4' mono-palmitate or EC16m), OC43 viral replication was inhibited by 99%. In addition, all nanoformulations tested for their effect on cell viability were comparable to normal saline, a regularly used nasal irrigation solution. A 1-min incubation of an EC16 nanoformulation with either OC43 or 229E showed an altered viral structure. CONCLUSION: Nanoformulations containing EC16 showed properties compatible with nasal application to rapidly inactivate SARS-CoV-2 residing in the olfactory mucosa and to reduce inflammation in the CNS, pending additional formulation and safety studies.

Unimolecular Cascaded Multienzyme Conjugates Modulate the Microenvironment of Diabetic Wound to Promote Healing.

An abnormal microenvironment underlies poor healing in chronic diabetic chronic wounds. However, effectively modulating the microenvironment of the diabetic wound remains a great challenge due to sustained oxidative stress and chronic inflammation. Here, we present a unimolecular enzyme-polymer conjugate that demonstrates excellent multienzymatic cascade activities. The cascaded enzyme conjugates (CECs) were synthesized by grafting poly(N-acryloyl-lysine) (pLAAm) from the glycan moieties of glucose oxidase (GOx) via glycan-initiated polymerization. The resulting CECs exhibited multiple enzymatic properties of GOx, superoxide dismutase mimic, and catalase mimic activities simultaneously. The CECs facilitated the depletion of high blood glucose, ROS scavenging, bacteria-killing, anti-inflammatory effects, and sustained oxygen generation, which restored the microenvironment in diabetic wounds. In vivo results from a diabetic mouse model confirmed the capacity and efficiency of the cascade reaction for diabetic wound healing. Our findings demonstrate that the three-in-one enzyme-polymer conjugates alone can modulate the diabetic microenvironment for wound healing.