Primer on Pulsed Electrical Field Ablation: Understanding the Benefits and Limitations

Pulsed electrical field (PEF) energy is a promising technique for catheter ablation of cardiac arrhythmias. In this article, the key aspects that need to be considered for safe and effective PEF delivery are reviewed, and their impact on clinical feasibility is discussed. The most important benefit of PEF appears to be the ability to kill cells through mechanisms that do not alter stromal proteins, sparing sensitive structures to improve safety, without sacrificing cardiomyocyte ablation efficacy. Many parameters affect PEF treatment outcomes, including pulse intensity, waveform shape, and number of pulses, as well as electrode configuration and geometry. These physical and electrical characteristics must be titrated carefully to balance target tissue effects with collateral implications (muscle contraction, temperature rise, risk of electrical arcing events). It is important to note that any combination of parameters affecting PEF needs to be tested for clinical efficacy and safety. Applying PEF clinically requires knowledge of the fundamentals of this technology to exploit its opportunities and generate viable, durable health improvements for patients.

Stromal Protein-Mediated Immune Regulation in Digestive Cancers

The stromal tumor microenvironment (TME) consists of immune cells, vascular and neural structures, cancer-associated fibroblasts (CAFs), as well as extracellular matrix (ECM), and favors immune escape mechanisms promoting the initiation and progression of digestive cancers. Numerous ECM proteins released by stromal and tumor cells are crucial in providing physical rigidity to the TME, though they are also key regulators of the immune response against cancer cells by interacting directly with immune cells or engaging with immune regulatory molecules. Here, we discuss current knowledge of stromal proteins in digestive cancers including pancreatic cancer, colorectal cancer, and gastric cancer, focusing on their functions in inhibiting tumor immunity and enabling drug resistance. Moreover, we will discuss the implication of stromal proteins as therapeutic targets to unleash efficient immunotherapy-based treatments.

Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

Background Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. Results With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. Conclusions Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

Extensive Intratumor Proteogenomic Heterogeneity Revealed by Multiregion Sampling in High-Grade Serous Ovarian Tumor Specimens

Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten primary high-grade serous ovarian tumors and analyzed using proteomics (mass spectrometry and reverse phase protein microarray) and RNA-sequencing analyses. Comparative analyses of transcript and protein abundances revealed independent clustering of enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering between purified collections, driven by overall tumor purity. Comparison of historic prognostic molecular subtypes for HGSOC revealed protein and transcript expression from tumor epithelium correlated most strongly with the differentiated molecular subtype, whereas stromal proteins and transcripts most strongly correlated with mesenchymal subtype. Protein and transcript abundance in tumor epithelium and stromal collections from neighboring sections exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial protein and transcript expression heterogeneity within the tumor microenvironment that directly bears on prognostic signatures and underscore the need to enrich cellular subpopulations for expression profiling.