A protocol for dual calcium-voltage optical mapping in murine sinoatrial preparation with optogenetic pacing

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice

m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Abstract Background N6-methyladenosine (m6A) has been confirmed to function critically in acute myeloid leukemia (AML) progression. Hitherto, the subtyping and prognostic predictive significance of m6A-correlated genes in AML is unclear. Method From The Cancer Genome Atlas (TCGA-LAML), Therapeutically Applicable Research to Generate Effective Treatments (TARGET-AML) and Gene Expression Omnibus (GEO, GSE71014) databases, we collected the sequencing data of AML patients. The batch effect was removed via limma package for TCGA-LAML and TARGET-AML, and the aggregated samples were AML cohorts. Samples in the AML cohort identified m6A models in AML by consensus clustering based on 23-m6A-related modulators. M6A-related differentially expressed genes (m6ARDEGs) influencing the overall survival (OS) of AML were determined by performing differential expression analysis and univariate COX analysis, and consensus-based clustering was utilized to access AML molecular subtypes. LASSO and multivariate COX analyses were performed to obtain the optimized m6ARDEGs to construct the m6A Prognostic Risk Score (m6APR_Score). Whether the model was robust was evaluated according to Kaplan–Meier (K-M) and receiver operator characteristic (ROC) curves. Further, the abundance of immune cell infiltration was explored in different m6A modification patterns and molecular subtypes and m6APR_Score groupings. Finally, nomogram was constructed to predict OS in AML. Quantitative real-time polymerase chain reaction (RT-qPCR) and cell counting kit-8 (CCK-8) assay were used to validate the genes in m6APR_Score in AML cells. Results The m6A models (m6AM1, m6AM2, m6AM3) and molecular subtypes (C1, C2, C3) were identified in the AML cohort, exhibiting different prognosis and immunoreactivity. We recognized novel prognostic biomarkers of AML such as CD83, NRIP1, ACSL1, METTL7B, OGT, and C4orf48. AML patients were grouped into high-m6APR_Score and low-m6APR_Score groups, with the later group showing a better prognosis than former one. Both the AML cohort and the validation cohort GSE71014 demonstrated excellent prediction. Finally, the nomogram accurately predicted the survival of patients suffering from AML. Further, the decision curves showed that both nomogram and m6APR_Score showed excellent prediction. It was confirmed in vitro experiments that mRNA expressions of NRIP1, ACSL1, METTL7B and OGT were elevated, while CD83 and C4orf48 mRNA expressions downregulated in AML cells. A significant increase in the viability of U937 and THP-1 cell lines after inhibition of CD83, while siMETTL7B had contrast results. Conclusion Our study demonstrated that m6APR_Score and CD83, NRIP1, ACSL1, METTL7B, OGT, and C4orf48 potentially provided novel and promising prognostic support for AML patients

Synergistic effects on oxidative stress, apoptosis and necrosis resulting from combined toxicity of three commonly used pesticides on HepG2 cells

The widespread use of pesticides performs a vital role in safeguarding crop yields and quality, providing the opportunity for multiple pesticides to co-exist, which poses a significant potential risk to human health. To assess the toxic effects caused by exposures to individual pesticides (chlorpyrifos, carbofuran and acetamiprid), binary combinations and ternary combinations, individual and combined exposure models were developed using HepG2 cells and the types of combined effects of pesticide mixtures were assessed using concentration addition (CA), independent action (IA) and combination index (CI) models, respectively, and the expression of biomarkers related to oxidative stress, apoptosis and cell necrosis was further examined. Our results showed that both individual pesticides and mixtures exerted toxic effects on HepG2 cells. The CI model indicated that the toxic effects of pesticide mixtures exhibited synergistic effects. The results of the lactate dehydrogenase (LDH) release and apoptosis assay revealed that the pesticide mixture increased the release of LDH and apoptosis levels. Moreover, our results also showed that individual pesticides and mixtures disrupted redox homeostasis and that pesticide mixtures produced more intense oxidative stress effects. In conclusion, we have illustrated the enhanced combined toxicity of pesticide mixtures by in-vitro experiments, which provides a theoretical basis and scientific basis for further toxicological studies

Additional file 4 of m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Additional file 4

Additional file 2 of m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Additional file 2: Supplementary Figure 2. Identification of the m6A molecular subtypes. (A) CDF curves in consensus clustering (B) Clustering consistency at k = 2-10 (C) Heatmap of sample consistency for optimal clustering groupings

Additional file 3 of m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Additional file 3: Supplementary Figure 3. m6APR_Score independence analysis and evaluation of Nomogram predictive performance. (A) 1-year, 3-year, 5-year calibration curves for Nomogram (B) Decision curves for Nomogram, m6APR_Score

Additional file 5 of m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Additional file 5

Additional file 1 of m6A genotypes and prognostic signature for assessing the prognosis of patients with acute myeloid leukemia

Additional file 1: Supplementary Figure 1. Identification of m6A models. (A) CDF curves in consensus clustering (B) Clustering consistency at k = 2-10 (C) Heatmap of sample consistency for optimal clustering groupings

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models