Unilateral zebrafish corneal injury induces bilateral cell plasticity supporting wound closure

The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration.

Comprehensive analysis of the long noncoding RNA-associated competitive endogenous RNA network in the osteogenic differentiation of periodontal ligament stem cells

Backgroud The mechanism implicated in the osteogenesis of human periodontal ligament stem cells (PDLSCs) has been investigated for years. Previous genomics data analyses showed that long noncoding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) have significant expression differences between induced and control human PDLSCs. Competing for endogenous RNAs (ceRNA), as a widely studied mechanism in regenerative medicine, while rarely reported in periodontal regeneration. The key lncRNAs and their ceRNA network might provide new insights into molecular therapies of periodontal regeneration based on PDLSCs. Results Two networks reflecting the relationships among differentially expressed RNAs were constructed. One ceRNA network was composed of 6 upregulated lncRNAs, 280 upregulated mRNAs, and 18 downregulated miRNAs. The other network contained 33 downregulated lncRNAs, 73 downregulated mRNAs, and 5 upregulated miRNAs. Functional analysis revealed that 38 GO terms and 8 pathways related with osteogenesis were enriched. Twenty-four osteogenesis-related gene-centred lncRNA-associated ceRNA networks were successfully constructed. Among these pathways, we highlighted MAPK and TGF-beta pathways that are closely related to osteogenesis. Subsequently, subnetworks potentially linking the GO:0001649 (osteoblast differentiation), MAPK and TGF-beta pathways were constructed. The qRT-PCR validation results were consistent with the microarray analysis. Conclusion We construct a comprehensively identified lncRNA-associated ceRNA network might be involved in the osteogenesis of PDLSCs, which could provide insights into the regulatory mechanisms and treatment targets of periodontal regeneration.

Integrin/TGF-Beta1 inhibitor GLPG-0187 blocks SARS-CoV-2 Delta and Omicron pseudovirus infection of airway epithelial cells which could attenuate disease severity

As COVID-19 continues to pose major risk for vulnerable populations including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced inhibition of pseudovirus infection by GLPG-0187. Because integrins activate TGF-beta; signaling, we compared plasma levels of active and total TGF-beta; in COVID-19+ patients. Plasma TGF-beta1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-beta1 levels correlated with activated TGF-beta1 levels. In our preclinical studies, Omicron infects lower airway lung cells less efficiently than other COVID-19 variants. Moreover, inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and may mitigate COVID-19 severity through decreased TGF-beta1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Reaction of the Liver upon Long-Term Treatment of Fluoxetine and Atorvastatin Compared with Alcohol in a Mouse Model

Background. Alcoholism is known to cause liver toxicity and is extensively researched. On the other hand, stress, depression, and obesity are interrelated conditions with alcoholism, and their medications would affect the liver itself. In this study, we investigated the effects of the drugs fluoxetine and atorvastatin on the liver and compared with those of alcohol in a mouse model. Methods. Comparisons of animals treated with the three drugs were carried out: serum aspartate transaminase (AST), alanine transaminase (ALT), and albumin were measured; liver tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta-1) levels were evaluated; proliferative cells were detected via immunohistochemistry (IHC) targeting on proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2); for apoptosis, IHC targeting on activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were employed; and histopathology was also documented in all groups. Results. For ALT, AST, albumin, and liver TNF alpha, only the ethanol group surged to significantly higher levels. For TGF beta-1, both ethanol and atorvastatin groups reached a significantly higher level. PCNA and MCM2 showed increased proliferation in the livers of all three groups, with the ethanol group having the highest number of positive cells followed by atorvastatin and then the fluoxetine group. As for cell death, both ethanol and fluoxetine groups showed significantly more apoptosis than control in TUNEL and activated caspase-3, while in the atorvastatin group, activated caspase-3 positive cells increased significantly, but the increase in TUNEL-positive cells did not reach statistical significance.

Disease-modifying immune-modulatory effects of the N-163 strain of Aureobasidium pullulans-produced 1,3-1,6 Beta glucans in young boys with Duchenne muscular dystrophy: Results of an open-label, prospective, randomized, comparative clinical study

Background: Duchenne muscular dystrophy (DMD) is an inherited neuromuscular disorder causing progressive muscle weakness and premature death. Steroids remain the mainstream approach for supportive care but have side effects; other targeted therapies and gene therapies are also being developed. As there is limited evidence on the use of disease modifying nutritional supplement adjuncts in DMD, this pilot trial is to evaluate the effects of supplementation of Aureobasidium pullulans-derived 1,3 1,6 beta glucan from the N163 strain in young patients with DMD. Methods: Twenty-seven patients with Duchenne muscular dystrophy (DMD), nine in the control arm (undergoing conventional therapies) participated. The patients were divided into groups: those not administered steroids (Steroid negative) (n = 5), those administered steroids (Steroid positive) (n = 4), and 18 in the treatment arm (N163 beta glucan supplement along with conventional therapies; N-163 Steroid negative and N-163 Steroid positive); they participated in the study for 45 days. Assessments of muscle function, disease status, and levels of IL6, IL13, TGF beta;, creatinine kinase (CK), titin, TNF Alpha;, haptoglobin, and dystrophin in the blood and myoglobin in the urine were performed at baseline and at the end of the study. Results: IL6 showed a significant decrease in the N163 Steroid negative group, from a baseline value of 7.2 pg/ml to 2.7 pg/ml. IL13 decreased in both treatment groups, from 157.76 pg/ml to 114.08 pg/ml (N-163 Steroid negative) and from 289.56 pg/ml to 255.56 pg/ml (N-163 Steroid positive). TGF beta levels showed a significant decrease in the N163 Steroid negative group, from a baseline value of 3302 ng/ml to 1325.66 ng/ml post intervention. Dystrophin levels increased by up to 32% in both Steroid positive and negative groups. Medical research council (MRC) grading showed muscle strength improvement in 12 out of 18 patients (67%) in the treatment group and four out of nine (44%) subjects in the control group. Conclusion: Supplementation with the N163 beta glucan food supplement produced disease-modifying beneficial effects: a significant decrease in inflammation and fibrosis markers, increase in dystrophin and improvement in muscle strength in DMD subjects over 45 days, thus making this a potential adjunct treatment for DMD after validation. A longer duration of follow up and further research on the mechanism of action and commonalities with other diseases provoked by hyperactive inflammation and/or fibrosis may pave the way for their extended applications in other dystrophinopathies and neuroinflammatory diseases.

Integrated Transcriptome and Multiple Activated Pathways in Endometrial Cancer

Because the incidence of endometrial cancer is notably increasing worldwide, it has become the leading gynecologic cancer in the United States. Standard treatment results in the loss of reproductive function in women of childbearing age. Furthermore, advanced cancer stages are associated with poor overall survival. The aim of this study was to explore the abnormal expression profile of genes during the development of endometrial cancer, which is essential to provide a better understanding of the mechanisms involved. Five pairs of endometrial cancer tissues and normal endometrial tissues were subjected to next-generation transcriptome sequencing technology. Quantitative real-time PCR (RT-qPCR) was performed to validate the expression profile of key differentially expressed genes (2.0-fold change, adj. p < 0.05) (DEGs) identified in the RNA-seq result. GO and KEGG pathways were used for bioinformatic analyses. The transcriptomic sequencing results showed 1153 DEGs, including 673 upregulated and 480 downregulated genes, in the EC specimens. Decreased expression of ID1, IGF1, GDF7, SMAD9, TGF-beta and WNT4, as well as GDF5, INHBA and ERBB4 overexpression, were confirmed in EC using RT-qPCR. Additionally, EC tissue exhibited marked enrichment in genes promoting cellular adhesion, proliferation, migration and plasma membrane. KEGG analysis revealed changes in various pathways, such as the TGF-beta, PI3K-Akt, Wnt, and estrogen pathways. Our data describe the molecular events involved in the pathogenesis of EC, which may be potential diagnostic markers and targets of therapeutic interventions.

The role of stromal immune microenvironment in the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer

Aim The first aim of the study was to compare the scores and types of stromal immune cells in 30 patients with primary DCIS and in the same patients after invasive breast recurrence in order to assess possible differences in both during tumor progression. The second aim was to evaluate possible differences in stromal cells of 30 patients with primary DCIS before progression and in the control group of 11 DCIS patients without recurrence during long-term follow-up. Material and methods Evaluation of tumor-infiltrating lymphocytes (TILs) and immunohistochemical stains for immune cell markers CD4, CD8, CD20, CD138, FOXP3, CD163 and TGF beta was performed on the stroma of primary DCIS before progression, invasive breast cancer of the same patients after progression and DCIS without progression. Results The comparison of stromal cells in 30 patients with initial DCIS and its invasive recurrence revealed an increased level of CD20 + immune cells (median score 5% vs. 17%, respectively, p < 0.001) and CD163 + cells (median score 1% vs. 5%, respectively, p < 0.001) in invasive breast cancer. The comparison of stromal cells in 30 patients with initial DCIS before recurrence and the control group of 11 patients with DCIS without recurrence showed statistically significant difference for CD138 + cells, which were more prevalent in patients with worse prognosis (median score 0 vs. 2%, respectively, p < 0.001). No similar relationship was found for the other tested cells as well as for TGF-beta. Conclusions CD138 + immune cells that were more prevalent in patients with a worse prognosis should be explored in further studies to confirm or exclude their role as a potential biological marker of DCIS invasive recurrence.