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.

Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus-A Transcriptomic Profiling Study.

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFß1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFß1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70-90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFß1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFß1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFß1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFß1, thirteen were responsive to CMS, and six were responsive to TGFß1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFß1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFß1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFß1 treatment under CMS, suggesting a potential role of TGFß1 and biomechanical stretch in KC development.

Effects of functional mandibular lateral shift on craniofacial growth and development in growing rats.

BACKGROUND: Unilateral posterior crossbite, one of the most frequent malocclusions, is often associated with functional lateral shift of the mandible. Although the effects of functional lateral shift on the mandible and temporomandibular joint have been examined in various animal experiments, cranial and maxillary changes have received less attention. OBJECTIVE: The aim of this study was to investigate the effects of functional lateral shift on the craniofacial complex in growing rats. METHODS: Eighty 5-week-old male Sprague-Dawley rats were randomly divided into an experimental group (n = 40), which received an oblique guide appliance that shifted the mandible to the left during closure, and a control group (n = 40). The rats were scanned by cone-beam computed tomography at 3 days and 1, 2, 4 and 8 weeks. The dimensions of the mandibular bone, condyle, maxilla and cranium were measured. RESULTS: The mandibles of rats in the experimental group were smaller than those of the rats in the control group and were asymmetrical. The condyles of the rats in the experimental group were thinner than those of the control rats. The condylar length on the ipsilateral side was shorter and wider than that on the contralateral side from 4 to 8 weeks. No significant differences in cranial length or height were observed between the experimental and control groups. The height of the upper first molar and alveolar bone on the contralateral side was significantly smaller than that on the ipsilateral side and in the controls from 4 to 8 weeks. CONCLUSION: Functional shift in the mandible produces morphological asymmetries in the mandible and maxillary region and may cause bilateral condylar degenerative changes.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.

Differential DNA methylation patterns in human Schlemm's canal endothelial cells with glaucoma.

Purpose: Schlemm's canal (SC) endothelial cells derived from donors with or without glaucoma showed different mechanical properties and gene expression. As an important contributor to the regulation of intraocular pressure (IOP) and pathogenesis of primary open-angle glaucoma (POAG), the heritable key epigenetic changes, methylation may play an important role in the physiologic function of SC cells. This study aims to identify differentially methylated CpG sites (DMSs) in primary cultures of human SC cells with or without glaucoma. Methods: We examined the methylation pattern of seven strains of primary human cells (two glaucoma and five normal SC cell samples), which were isolated and characterized using established protocols. DNA methylation was profiled using Illumina Human Methylation 450 BeadChip. Raw data were extracted and exported using Illumina GenomeStudio software. After quantile normalization, DNA methylation data were analyzed using R package RnBeads in Bioconductor. DMSs were filtered with p ≤ 1E-5, methylation change ≥ 0.1, and false discovery rate ≤ 0.05. The closest genes and the location of each CpG site were annotated using R package FDb.InfiniumMethylation.hg19. Gene Ontology and pathway analysis was performed using WebGestalt. Selected DMSs were validated using the Zymo qMethyl kit. Results: We used five non-glaucoma and two glaucomatous SC cell samples to profile genome-wide DNA methylation using Illumina Infinium Methylation BeadChips. Principle component analysis showed the separation between the glaucoma and control samples. After quality control and differential analysis, we identified 298 highly significant DMSs (p ≤ 1E-5). Among them, 221 DMSs were within 1 kb of a nearby gene. Gene Ontology analysis demonstrated significant enrichment in positive regulation of cell migration, negative regulation of endothelial cell proliferation, and stress fiber and actin filament bundles. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in cell adhesion and gap junctions. Several glaucoma-related genes were identified, including TGFBR3, THBS1, PITX2, DAXX, TBX3, TNXB, ANGPT1, and PLEKHA7. We also examined differentially methylated regions (DMRs) near these CpG sites and identified significant DMRs in TBX3, TNXB1, DAXX, and PITX2. Conclusions: This study represents the first genome-wide DNA methylation profiling in cultured human primary SC cells. The DMSs were enriched in the pathways related to outflow resistance. Several DMRs were validated in glaucoma-associated genes, further suggesting the role of DNA methylation in glaucoma development. This study could provide comprehensive understanding of DNA methylation in glaucoma and its effect on aqueous humor outflow.

Ultrasound for pectinase modification: an investigation into potential mechanisms.

BACKGROUND: Today, ultrasound is increasingly utilized in enzyme modification. Strongly dependent on the specific operational conditions, the modification effect brought by ultrasound can be activation and inactivation of enzymes. This work aims to study the ultrasound mechanisms under different conditions, to investigate the respective roles of free radical effect and mechanical effect in pectinase activation and inactivation, and to reveal the influence of pectinase concentration on the ultrasound-modification effect. RESULTS: When ultrasound was introduced to a liquid system, generation of free radicals was positively correlated with ultrasound intensity and treatment duration, but negatively correlated with temperature. Thiourea with a concentration of 4 mmol L-1 was selected as a free radical scavenger to effectively shield ultrasound free radicals. The highest enzyme activity of pectinase solutions at 0.1, 1.0, and 10.0 mg mL-1 was obtained at the same ultrasound intensity of 4.50 W mL-1 and time of 15 min, where the enzyme activity was increased by 68.24%, 20.98% and 18.83%, respectively. Furthermore, the addition of thiourea enhanced the enzyme activity at each tested ultrasound intensity and time, especially those exceeding the best conditions; it also eliminated the redshift phenomenon that was previously presented in the fluorescence spectra of pectinase samples. CONCLUSION: Pectinase concentrations did not change the optimum ultrasound conditions for enzyme modification, but pectinase with a low concentration was more vulnerable to ultrasound treatment. During modification, ultrasound mechanical effects dominated in the pectinase activation, while free radical effects dominated in the inactivation process. © 2020 Society of Chemical Industry.

Differential in vivo biodistribution of 131I-labeled exosomes from diverse cellular origins and its implication for theranostic application.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 µm syringe filter and 100 k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers.

Retinal pigment epithelium (RPE) alterations in age-related macular degeneration occur in patches, potentially involving long-distance communication between damaged and healthy areas. Communication along the epithelium might be mediated by extracellular vesicles (EVs). To test this hypothesis, EVs were collected from supernatants of polarized ARPE-19 and primary porcine RPE monolayers for functional and biochemical assays. EVs from oxidatively stressed donor cells reduced barrier function in recipient RPE monolayers when compared to control EVs. The effect on barrier function was dependent on EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Mass spectrometry-based proteomic analysis of EVs identified HDAC6, which is known to reduce tight junction stability. Activity assays confirmed the presence of HDAC6 in EVs, and EV transfer assays using HDAC6 inhibitors confirmed its effect in monolayers. These findings demonstrate that EVs can communicate stress messages to healthy RPE cells, potentially contributing to RPE dysfunction.

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.

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.

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.

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.

Atractylodin alleviates nonalcoholic fatty liver disease by regulating Nrf2-mediated ferroptosis.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Oxidative stress is one of the main inducers of NAFLD. Atractylodin (ART), a major active ingredient of Atractylodes lancea, possesses potential antioxidant and anti-inflammatory activity in many types of disease. In the current study, the underlying mechanism by which ART alleviates the progression of NAFLD was explored. The function of ART in facilitating NAFLD was investigated in vitro and in vivo. Functionally, ART attenuated high-fat diet (HFD)-induced NAFLD in mice and palmitic acid (PA)-induced oxidative stress in HepG2 cells. Furthermore, our data verified that ART attenuated HFD-induced NAFLD by inhibiting ferroptosis of hepatocyte cells, as evidenced by decreased Fe2+ concentration, reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and increased glutathione (GSH) content. The protective effect of ART on the cell viability of hepatocytes was blocked by a specific ferroptosis inhibitor (ferrostatin-1). Mechanistically, ART treatment promoted the translocation of nuclear factor erythroid 2-related Factor 2 (NFE2L2/NRF2) and thus increased glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11) expression. Taken together, ART alleviates NAFLD by regulating Nrf2-mediated ferroptosis.

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells.

Clinical trials have shown that electric stimulation (ELSM) using either cardiac resynchronization therapy (CRT) or cardiac contractility modulation (CCM) approaches is an effective treatment for patients with moderate to severe heart failure, but the mechanisms are incompletely understood. Extracellular vesicles (EV) produced by cardiac mesenchymal stem cells (C-MSC) have been reported to be cardioprotective through cell-to-cell communication. In this study, we investigated the effects of ELSM stimulation on EV secretion from C-MSCs (C-MSCELSM). We observed enhanced EV-dependent cardioprotection conferred by conditioned medium (CM) from C-MSCELSM compared to that from non-stimulated control C-MSC (C-MSCCtrl). To investigate the mechanisms of ELSM-stimulated EV secretion, we examined the protein levels of neutral sphingomyelinase 2 (nSMase2), a key enzyme of the endosomal sorting complex required for EV biosynthesis. We detected a time-dependent increase in nSMase2 protein levels in C-MSCELSM compared to C-MSCCtrl. Knockdown of nSMase2 in C-MSC by siRNA significantly reduced EV secretion in C-MSCELSM and attenuated the cardioprotective effect of CM from C-MSCELSM in HL-1 cells. Taken together, our results suggest that ELSM-mediated increases in EV secretion from C-MSC enhance the cardioprotective effects of C-MSC through an EV-dependent mechanism involving nSMase2.

Oral submucous fibrosis induced by graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) is one of the most common and serious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). About 45%∼83% of patients develop GVHD in the oral cavity. There has no medical records of oral submucous fibrosis (OSF) induced by GVHD after allo-HSCT, which should be brought to the attention of dentists.

White Sponge Nevus Caused by Keratin 4 Gene Mutation: A Case Report.

White sponge nevus (WSN) is a rare autosomal dominant disease with a family history, often caused by mutations of the keratin 4 (K4) and keratin 13 (K13) genes in patients. It is characterized by frequently occurred white corrugated folds in the bilateral buccal mucosa with soft texture. On histopathological examination, hyperkeratosis of epithelial cells, edema, and vacuolar changes in the spinous cells are observed in the lesions, despite a normal layer of basal cells. WSN should be differentiated from other oral white spot diseases, mainly oral lichen planus, oral candidiasis, oral white edema, and Heck's disease, to reduce misdiagnosis and unnecessary treatment. At present, there is no specific treatment method. The purpose of this study was to report the clinical data of four WSN patients of the same family with the K4 gene mutation. The occurrence of WSN in a pair of monozygotic twins with very similar clinical presentations was identified for the first time. The gene sequencing results showed that there was a heterozygous deletion (C. 438_440delCAA) in exon 1 of the K4 gene, resulting in an aspartic acid loss in both the proband and his father. Finally, the etiology, pathogenesis, pathological manifestations, clinical manifestations, diagnosis, differential diagnosis, and related treatment methods are discussed to provide a reference for clinical treatment of the disease.

Explore the Mechanism of ß-Asarone on Improving Cognitive Dysfunction in Rats with Diabetic Encephalopathy.

Background: The number of people with diabetes is increasing, and many patients have significantly impaired cognitive function. For patients with diabetic encephalopathy (DE), simply lowering blood sugar does not improve learning and memory. Studies have shown that ß-asarone can significantly improve cognitive impairment in patients with DE, but the specific mechanism of action is unclear. Objective: This experiment hopes to use a variety of experimental methods to clarify the protective effect and mechanism of ß-asarone on brain neurons during the development of DE disease. Methods: A high-sugar and high-fat diet and streptozotocin injection-induced DE rat model was used. ß-asarone was administered for four weeks. The experiment used the Morris water maze test, biochemical index detection, and many methods to evaluate the neuroprotective effect of ß-asarone on DE rats from various aspects and understand its mechanism. Results: ß-asarone reduced neuronal cell damage and significantly improved the learning and memory ability of DE rats. In addition, ß-asarone can reduce the oxidative stress response and amyloid-ß accumulation in the brain of DE model rats and increase the content of brain-derived neurotrophic factor (BDNF) in the brain tissue, thereby reducing neuronal cell apoptosis and playing a protective role. Conclusion: ß-asarone can reduce the accumulation of oxidative stress and amyloid-ß in the brain, increase the content of BDNF, reduce the apoptosis of neuronal cells, and exert neuronal protection, thereby improving the learning and memory ability of DE model rats.

Experimental functional shift-induced osteoarthritis-like changes at the TMJ and altered integrin expression in a rat model.

Mandibular deviation affects the biomechanical environment of the temporomandibular joint (TMJ) and causes thinning of cartilage on the deviated side. We aimed to evaluate, using a rat model, the effect of mandibular functional deviation on the TMJ in relation to the functional roles of integrin ß family members. The effects of experimental functional deviation on the TMJ of 6-week-old Sprague-Dawley female rats, randomly assigned to control (n = 42) and experimental groups (n = 42), were evaluated at 3 days and 1, 2, 4, and 8 weeks by histological staining, immunofluorescence, real-time quantitative polymerase chain reaction, and micro-computed tomography. The results showed that the experimental functional shift changed the shape of condyles, thinned the cartilage, and increased the proportion of the hypertrophic layer on the deviated sides of condyles. In addition, the extracellular matrix of the condyle cartilage exhibited degradation at 1 week and subchondral trabecular bone was lost at 4 and 8 weeks. Osteoarthritis (OA)-like changes occurred in the left and right condyles of rats in the experimental group and were aggravated over time. Integrin ß family expression, especially integrin ß2 , was altered from week 1, possibly related to the OA-like changes. These data may provide insight into the onset of TMJ OA.

Utilising Network Pharmacology to Explore Underlying Mechanism of Astragalus membranaceus in Improving Sepsis-Induced Inflammatory Response by Regulating the Balance of IκBα and NF-κB in Rats.

Objective: The purpose of the present study was to explore the mechanism of Astragalus membranaceus in the treatment of sepsis. Methods: We searched the active components and targets of Astragalus membranaceus using the TCMSP and BATMAN databases. Then, the GeneCards, MalaCards, and OMIM databases were used to screen out relevant targets of sepsis. The common targets of the former two gene sets were uploaded to the STRING database to create an interaction network. DAVID was used to perform KEGG enrichment analysis of the core targets. Based on the results of KEGG and previous studies, key pathways for the development of sepsis were identified and experimentally validated. Result: We obtained 3,370 sepsis-related targets in databases and 59 active components in Astragalus membranaceus through data mining, corresponding to 1,130 targets. The intersection of the two types of targets led to a total of 318 common targets and 84 core targets were obtained after screening again. The KEGG and previous studies showed that these 84 core targets were involved in sepsis by regulating TNF, MAPK, and PI3K pathways. TNF, MAPK8, NF-κB, and IκBα are crucial in sepsis. Experimental validation demonstrated that some markers in sepsis model rats were improved after the intervention with Astragalus granules and their chemical components. Among them, IL-1ß, IL-6, and TNF-α in rat serum were reduced. The mRNA and protein expression of TNF-α, IL-6, MMP9, MAPK8, and NF-κB were reduced in rat blood. However, the mRNA and protein expression of IκBα and PI3K were increased in rat blood. Conclusion: The AST could affect the TNF, PI3K, and MAPK pathway cascade responses centred on IκBα and NF-κB, attenuate the expression of IL-6 and MMP9, and interfere with the inflammatory response during sepsis.