Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2). METHODS: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O2.- and H2O2), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H2O2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function. CONCLUSIONS: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O2.- and H2O2). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

The prevalence of programmed death ligand-1 (PD-L1) expression in non-small cell lung cancer in an unselected, consecutive population.

Little is known about prevalence of PD-L1 expression in tumor cells of unselected patients with all stages of non-small cell lung cancer. The objective of this study is to assess the prevalence of PD-L1 positivity in patients with non-small cell lung cancer, to analyze the association between PD-L1 positivity and patients' clinicopathological characteristics, and to assess the use of immune-oncologic treatment in eligible patients. All non-small cell lung cancer patients diagnosed in a 10-month period in an unselected population of 1.7 million Caucasian inhabitants were evaluated with the PD-L1 IHC 22C3 pharmDx kit. A total of 819 patients were diagnosed with non-small cell lung cancer. Samples analyzable for PD-L1 expression were obtained from 97% of patients. In a multivariate analysis with cut-off at tumor proportion score ≥50%, lower stage was associated with lower prevalence of PD-L1 positivity with an odds ratio of 0.31 for stage I vs. stage IV. A significant difference in PD-L1 expression between squamous-cell carcinoma and adenocarcinoma was observed with odds ratio for adenocarcinoma 1.8. With cut-off tumor proportion score ≥1%, attenuated effects of the same direction were seen. For neither cut-off did type and location of material used for PD-L1 analysis, age, sex, smoking history, or performance status have statistically significant impact on the PD-L1 expression. Fifty four percent of the patients who were eligible for immune-oncologic treatment were actually treated in first-line with pembrolizumab monotherapy. In conclusion, 97% of the patients had material analyzable for PD-L1. If a patient in need of immuno-oncologic treatment has shifted stage, a negative or low positive PD-L1 test performed on a biopsy taken in a lower stage might not mirror the PD-L1 expression in the new metastatic lesion. Therefore, a re-biopsy should be considered.

Intramuscular injection of "site enhancement oil": forensic considerations.

The use of intramuscular injection of foreign substances for aesthetic purposes is well known. Complications are usually local to the site of injection but can be potentially lethal. Here, we present a case of "site enhancement oil" use in a 42-year-old man who died from asphyxia due to hanging. Macroscopic and microscopic changes as well as computed tomographic changes in injected musculature are described and the potentially lethal adverse effects after site enhancement oil use are warranted.

Profiling native pulmonary basement membrane stiffness using atomic force microscopy.

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d.

Technical Note: Measuring the thickness of histological sections by detecting fluorescence intensity of embedding foam.

Fluorescence intensity of embedding foam in paraffin blocks can be used to measure the thickness of histological microsections. We embedded samples of embedding foam and produced several microsections of varying thicknesses using routine processing and staining. Fluorescence intensity in the blue area of the embedding foam detected with a slide scanner was compared to absolute thickness as measured using confocal microscopy. Correlation analysis displayed a clear linear correlation with convincingly low prediction interval. The concept of measuring thickness of histological microsections by detecting fluorescence intensity of embedding foam is suggested as an approach to high-throughput measuring of histological sections applicable for a fully digitized pathology department. No acquisition of dedicated equipment is required and the method can be applied as a fully automated technique requiring no time consumption.