Epigenetic Reprogramming Drives Epithelial Disruption in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) remains a major public health challenge that contributes greatly to mortality and morbidity worldwide. Although it has long been recognized that the epithelium is altered in COPD, there has been little focus on targeting it to modify the disease course. Therefore, mechanisms that disrupt epithelial cell function in patients with COPD are poorly understood. In this study, we sought to determine whether epigenetic reprogramming of the cell-cell adhesion molecule E-cadherin, encoded by the CDH1 gene, disrupts epithelial integrity. By reducing these epigenetic marks, we can restore epithelial integrity and rescue alveolar airspace destruction. We used differentiated normal and COPD-derived primary human airway epithelial cells, genetically manipulated mouse tracheal epithelial cells, and mouse and human precision-cut lung slices to assess the effects of epigenetic reprogramming. We show that the loss of CDH1 in COPD is due to increased DNA methylation site at the CDH1 enhancer D through the downregulation of the ten-eleven translocase methylcytosine dioxygenase (TET) enzyme TET1. Increased DNA methylation at the enhancer D region decreases the enrichment of RNA polymerase II binding. Remarkably, treatment of human precision-cut slices derived from patients with COPD with the DNA demethylation agent 5-aza-2'-deoxycytidine decreased cell damage and reduced air space enlargement in the diseased tissue. Here, we present a novel mechanism that targets epigenetic modifications to reverse the tissue remodeling in human COPD lungs and serves as a proof of concept for developing a disease-modifying target.

Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease.

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SCV2), which has resulted in higher morbidity and mortality rate than other respiratory viral infections, such as Influenza A virus (IAV) infection. Investigating the molecular mechanisms of SCV2-host infection vs IAV is vital in exploring antiviral drug targets against SCV2. We assessed differential gene expression in human nasal cells upon SCV2 or IAV infection using RNA sequencing. Compared to IAV, we observed alterations in both metabolic and cytoskeletal pathways suggestive of epithelial remodeling in the SCV2-infected cells, reminiscent of pathways activated as a response to chronic injury. We found that spike protein interaction with the epithelium was sufficient to instigate these epithelial responses using a SCV2 spike pseudovirus. Specifically, we found downregulation of the mitochondrial markers SIRT3 and TOMM22. Moreover, SCV2 spike infection increased extracellular acidification and decreased oxygen consumption rate in the epithelium. In addition, we observed cytoskeletal rearrangements with a reduction in the actin-severing protein cofilin-1 and an increase in polymerized actin, indicating epithelial cytoskeletal rearrangements. This study revealed distinct epithelial responses to SCV2 infection, with early mitochondrial dysfunction in the host cells and evidence of cytoskeletal remodeling that could contribute to the worsened outcome in COVID-19 patients compared to IAV patients. These changes in cell structure and energetics could contribute to cellular resilience early during infection, allowing for prolonged cell survival and potentially paving the way for more chronic symptoms. IMPORTANCE COVID-19 has caused a global pandemic affecting millions of people worldwide, resulting in a higher mortality rate and concerns of more persistent symptoms compared to influenza A. To study this, we compare lung epithelial responses to both viruses. Interestingly, we found that in response to SARS-CoV-2 infection, the cellular energetics changed and there were cell structural rearrangements. These changes in cell structure could lead to prolonged epithelial cell survival, even in the face of not working well, potentially contributing to the development of chronic symptoms. In summary, these findings represent strategies utilized by the cell to survive the infection but result in a fundamental shift in the epithelial phenotype, with potential long-term consequences, which could set the stage for the development of chronic lung disease or long COVID-19.

Upregulation of SYNGAP1 expression in mice and human neurons by redirecting alternative splicing.

The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency.

Participation and adherence to mammography screening in the Capital Region of Denmark: The importance of age over time.

Mammography screening's effectiveness depends on high participation levels. Understanding adherence patterns over time is important for more accurately predicting future effectiveness. This study analyzed longitudinal adherence to the biennial invitations in the Capital Region of Denmark from 2008-2017. We analyzed participation rates for five-year age groups along with their percent changes in each invitation round using linear regressions. Participation in the mammography screening program increased from 73.1% to 83.1% from 2008-2017. The participation rate among all age groups increased from the first to the fifth round, with the oldest age group having the largest increase (average percent change = 3.66; p-value = 0.03).

Maintained physical activity and physiotherapy in the management of distal arm pain: a randomised controlled trial.

Objectives: The epidemiology of distal arm pain and back pain are similar. However, management differs considerably: for back pain, rest is discouraged, whereas patients with distal arm pain are commonly advised to rest and referred to physiotherapy. We hypothesised that remaining active would reduce long-term disability and that fast-track physiotherapy would be superior to physiotherapy after time on a waiting list. Methods: Adults referred to community-based physiotherapy with distal arm pain were randomised to: advice to remain active while awaiting physiotherapy (typically delivered after 6-8 weeks); advice to rest while awaiting physiotherapy, or immediate treatment. Intention-to-treat analysis determined whether the probability of recovery at 26 weeks was greater among the active advice group, compared with those advised to rest and/or among those receiving immediate versus usually timed physiotherapy. Results: 538 of 1663 patients invited between February 2012 and February 2014 were randomised (active=178; rest=182; immediate physiotherapy=178). 81% provided primary outcome data, and complete recovery was reported by 60 (44%), 46 (32%) and 53 (35%). Those advised to rest experienced a lower probability of recovery (OR: 0.54; 95% CI 0.32 to 0.90) versus advice to remain active. However, there was no benefit of immediate physiotherapy (0.64; 95% CI 0.39 to 1.07). Conclusions: Among patients awaiting physiotherapy for distal arm pain, advice to remain active results in better 26-week functional outcome, compared with advice to rest. Also, immediate physiotherapy confers no additional benefit in terms of disability, compared with physiotherapy delivered after 6-8 weeks waiting time. These findings question current guidance for the management of distal arm pain.

Altered activity patterns of transcription factors induced by endoplasmic reticulum stress.

BACKGROUND: The endoplasmic-reticulum (ER) responds to the burden of unfolded proteins in its lumen by activating intracellular signal transduction pathways, also known as the unfolded protein response (UPR). Many signal transduction events and transcription factors have been demonstrated to be associated with ER stress. The process in which ER stress affects or interacts with other pathways is still a progressing topic that is not completely understood. Identifying new transcription factors associated with ER stress pathways provides a platform to comprehensively characterize mechanism and functionality of ER. METHODS: We utilized a transcription factor (TF) activation plate array to profile the TF activities which were affected by ER stress induced by pharmacological agents, thapsigargin (TG) and tunicamycin (TM) at 1 h, 4 h, 8 h and 16 h respectively, in MiaPACA2 cells. The altered activity patterns were analyzed and validated using gel shift assays and cell-based luciferase reporter assay. RESULTS: The study has not only confirmed previous findings, which the TFs including ATF4, ATF6, XBP, NFkB, CHOP and AP1, were activated by ER stress, but also found four newly discovered TFs, NFAT, TCF/LEF were activated, and PXR was repressed in response of ER stress. Different patterns of TF activities in MiaPaCa2 were demonstrated upon TM or TG treatment in the time course experiments. The altered activities of TFs were confirmed using gel shift assays and luciferase reporter vectors. CONCLUSION: This study utilized a TF activation array technology to identify four new TFs, HIF, NFAT, TCF/LEF and PXR that were changed in their activity as a result of ER stress induced by TG and TM. The TF activity patterns were demonstrated to be diverse in response to the duration of TG or TM treatment. These new findings will facilitate further unveiling the complex mechanisms of the ER stress process and associated diseases.

Co-Eradication of Breast Cancer Cells and Cancer Stem Cells by Cross-Linked Multilamellar Liposomes Enhances Tumor Treatment.

The therapeutic limitations of conventional chemotherapeutic drugs have emerged as a challenge for breast cancer therapy; these shortcomings are likely due, at least in part, to the presence of the cancer stem cells (CSCs). Salinomycin, a polyether antibiotic isolated from Streptomyces albus, has been shown to selectively inhibit cancer stem cells; however, its clinical application has been hindered by the drug's hydrophobility, which limits the available administration routes. In this paper, a novel drug delivery system, cross-linked multilamellar liposomal vesicles (cMLVs), was optimized to allow for the codelivery of salinomycin (Sal) and doxorubicin (Dox), targeting both CSCs and breast cancer cells. The results show that the cMLV particles encapsulating different drugs have similar sizes with high encapsulation efficiencies (>80%) for both Dox and Sal. Dox and Sal were released from the particles in a sustained manner, indicating the stability of the cMLVs. Moreover, the inhibition of cMLV(Dox+Sal) against breast cancer cells was stronger than either single-drug treatment. The efficient targeting of cMLV(Dox+Sal) to CSCs was validated through in vitro experiments using breast cancer stem cell markers. In accordance with the in vitro combination treatment, in vivo breast tumor suppression by cMLV(Dox+Sal) was 2-fold more effective than single-drug cMLV treatment or treatment with the combination of cMLV(Dox) and cMLV(Sal). Thus, this study demonstrates that cMLVs represent a novel drug delivery system that can serve as a potential platform for combination therapy, allowing codelivery of an anticancer agent and a CSC inhibitor for the elimination of both breast cancer cells and cancer stem cells.