Oxidative Effects of Nanosecond Pulsed Electric Field Exposure in Cells and Cell-Free Media

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H2O2 and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage

Model organisms contribute to diagnosis and discovery in the Undiagnosed Diseases Network: Current state and a future vision

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support

Electroporation-Induced Electrosensitization

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency

Estimation of Pap-test coverage in an area with an organised screening program: challenges for survey methods

BACKGROUND: The cytological screening programme of Viterbo has completed the second round of invitations to the entire target population (age 25–64). From a public health perspective, it is important to know the Pap-test coverage rate and the use of opportunistic screening. The most commonly used study design is the survey, but the validity of self-reports and the assumptions made about non respondents are often questioned. METHODS: From the target population, 940 women were sampled, and responded to a telephone interview about Pap-test utilisation. The answers were compared with the screening program registry; comparing the dates of Pap-tests reported by both sources. Sensitivity analyses were performed for coverage over a 36-month period, according to various assumptions regarding non respondents. RESULTS: The response rate was 68%. The coverage over 36 months was 86.4% if we assume that non respondents had the same coverage as respondents, 66% if we assume they were not covered at all, and 74.6% if we adjust for screening compliance in the non respondents. The sensitivity and specificity of the question, "have you ever had a Pap test with the screening programme" were 84.5% and 82.2% respectively. The test dates reported in the interview tended to be more recent than those reported in the registry, but 68% were within 12 months of each other. CONCLUSION: Surveys are useful tools to understand the effectiveness of a screening programme and women's self-report was sufficiently reliable in our setting, but the coverage estimates were strongly influenced by the assumptions we made regarding non respondents

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome

AusTraits, a curated plant trait database for the Australian flora

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge