A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.

The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.

Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and ß-catenin signaling to promote adipocyte dedifferentiation.

BACKGROUND: Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. METHODS: To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of ß-catenin and applying BML-284, an agonist of ß-catenin. RESULTS: Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-ß-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-ß-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. CONCLUSIONS: Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/ß-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/ß-catenin signaling using BML-284.

Predicting Stroke and Mortality in Mitral Regurgitation: A Machine Learning Approach.

We hypothesized that an interpretable gradient boosting machine (GBM) model considering comorbidities, P-wave and echocardiographic measurements, can better predict mortality and cerebrovascular events in mitral regurgitation (MR). Patients from a tertiary center were analyzed. The GBM model was used as an interpretable statistical approach to identify the leading indicators of high-risk patients with either outcome of CVAs and all-cause mortality. A total of 706 patients were included. GBM analysis showed that age, systolic blood pressure, diastolic blood pressure, plasma albumin levels, mean P-wave duration (PWD), MR regurgitant volume, left ventricular ejection fraction (LVEF), left atrial dimension at end-systole (LADs), velocity-time integral (VTI) and effective regurgitant orifice were significant predictors of TIA/stroke. Age, sodium, urea and albumin levels, platelet count, mean PWD, LVEF, LADs, left ventricular dimension at end systole (LVDs) and VTI were significant predictors of all-cause mortality. The GBM demonstrates the best predictive performance in terms of precision, sensitivity c-statistic and F1-score compared to logistic regression, decision tree, random forest, support vector machine, and artificial neural networks. Gradient boosting model incorporating clinical data from different investigative modalities significantly improves risk prediction performance and identify key indicators for outcome prediction in MR.

Attendance-related Healthcare Resource Utilisation and Costs in Patients With Brugada Syndrome in Hong Kong: A Retrospective Cohort Study.

Understanding health care resource utilisation and its associated costs are important for identifying areas of improvement regarding resource allocations. However, there is limited research exploring this issue in the setting of Brugada syndrome (BrS).This was a retrospective territory-wide study of BrS patients from Hong Kong. Healthcare resource utilisation for accident and emergency (A&E), inpatient and specialist outpatient attendances were analyzed over a 19-year period, with their associated costs presented in US dollars. A total of 507 BrS patients with a mean presentation age of 49.9 ± 16.3 years old were included. Of these, 384 patients displayed spontaneous type 1 electrocardiographic (ECG) Brugada pattern and 77 patients had presented with ventricular tachycardia/ventricular fibrillation (VT/VF). At the individual patient level, the median annualized costs were $110 (52-224) at the (A&E) setting, $6812 (1982-32414) at the inpatient setting and $557 (326-1001) for specialist outpatient attendances. Patients with initial VT/VF presentation had overall greater costs in inpatient ($20161 [9147-189215] vs $5290 [1613-24937],P < 0.0001) and specialist outpatient setting ($776 [438-1076] vs $542 [293-972],P = 0.015) compared to those who did not present VT. In addition, patients without Type 1 ECG pattern had greater median costs in the specialist outpatient setting ($7036 [3136-14378] vs $4895 [2409-10554],p=0.019). There is a greater health care demand in the inpatient and specialist outpatient settings for BrS patients. The most expensive attendance type was inpatient setting stay at $6812 per year. The total median annualized cost of BrS patients without VT/VF presentation was 78% lower compared to patients with VT/VF presentation.

Sulfonylurea Is Associated With Higher Risks of Ventricular Arrhythmia or Sudden Cardiac Death Compared With Metformin: A Population-Based Cohort Study.

Background Commonly prescribed diabetic medications such as metformin and sulfonylurea may be associated with different arrhythmogenic risks. This study compared the risk of ventricular arrhythmia or sudden cardiac death between metformin and sulfonylurea users in patients with type 2 diabetes. Methods and Results Patients aged ≥40 years who were diagnosed with type 2 diabetes or prescribed antidiabetic agents in Hong Kong between January 1, 2009, and December 31, 2009, were included and followed up until December 31, 2019. Patients prescribed with both metformin and sulfonylurea or had prior myocardial infarction were excluded. The study outcome was a composite of ventricular arrhythmia or sudden cardiac death. Metformin users and sulfonylurea users were matched at a 1:1 ratio by propensity score matching. The matched cohort consisted of 16 596 metformin users (47.70% men; age, 68±11 years; mean follow-up, 4.92±2.55 years) and 16 596 sulfonylurea users (49.80% men; age, 70±11 years; mean follow-up, 4.93±2.55 years). Sulfonylurea was associated with higher risk of ventricular arrhythmia or sudden cardiac death than metformin hazard ratio (HR, 1.90 [95% CI, 1.73-2.08]). Such difference was consistently observed in subgroup analyses stratifying for insulin usage or known coronary heart disease. Conclusions Sulfonylurea use is associated with higher risk of ventricular arrhythmia or sudden cardiac death than metformin in patients with type 2 diabetes.

Comparisons of the risk of myopericarditis between COVID-19 patients and individuals receiving COVID-19 vaccines: a population-based study.

BACKGROUND: Both COVID-19 infection and COVID-19 vaccines have been associated with the development of myopericarditis. The objective of this study is to (1) analyse the rates of myopericarditis after COVID-19 infection and COVID-19 vaccination in Hong Kong, (2) compared to the background rates, and (3) compare the rates of myopericarditis after COVID-19 vaccination to those reported in other countries. METHODS: This was a population-based cohort study from Hong Kong, China. Patients with positive RT-PCR test for COVID-19 between 1st January 2020 and 30th June 2021 or individuals who received COVID-19 vaccination until 31st August were included. The main exposures were COVID-19 positivity or COVID-19 vaccination. The primary outcome was myopericarditis. RESULTS: This study included 11,441 COVID-19 patients from Hong Kong, four of whom suffered from myopericarditis (rate per million: 326; 95% confidence interval [CI] 127-838). The rate was higher than the pre-COVID-19 background rate in 2019 (rate per million: 5.5, 95% CI 4.1-7.4) with a rate ratio of 55.0 (95% CI 21.4-141). Compared to the background rate, the rate of myopericarditis among vaccinated subjects in Hong Kong was similar (rate per million: 5.5; 95% CI 4.1-7.4) with a rate ratio of 0.93 (95% CI 0.69-1.26). The rates of myocarditis after vaccination in Hong Kong were comparable to those vaccinated in the United States, Israel, and the United Kingdom. CONCLUSIONS: COVID-19 infection was associated with significantly higher rate of myopericarditis compared to the vaccine-associated myopericarditis.

Knock-In of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System.

The non-viral delivery of the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) nuclease system provides promising solutions for gene therapy. However, traditional chemical and physical delivery approaches for gene knock-in are confronted by significant challenges to overcome the drawbacks of low efficiency and high toxicity. An alternative method for directly delivering CRISPR components into single cells is microinjection. Here, we present the high-throughput robotic microinjection of CRISPR machinery plasmids to produce gene insertions. We demonstrate that the microinjection of CRISPR/Cas9 with an enhanced green fluorescent protein (eGFP) donor template into single HepG2 cells can achieve reporter gene knock-in targeting the adeno-associated virus site 1 locus. Homology-directed repair-mediated knock-in can be observed with an efficiency of 41%. Assessment via T7E1 assay indicates that the eGFP knock-in cells exhibit no detectable changes at potential off-target sites. A case study of injecting the eGFP knock-in cells into zebrafish (Danio rerio) embryos to form an in vivo tumor model is conducted. Results demonstrate the efficiency of combining microinjection with the CRISPR/Cas9 system in achieving gene knock-in.

Metformin versus sulphonylureas for new onset atrial fibrillation and stroke in type 2 diabetes mellitus: a population-based study.

AIMS: To gain insights on the cardiovascular effects of metformin and sulphonylurea, the present study compares the rates of incident atrial fibrillation, stroke, cardiovascular mortality and all-cause mortality between metformin and sulphonylurea users in type 2 diabetes mellitus. METHODS: This was a retrospective population-based cohort study of type 2 diabetes mellitus patients receiving either sulphonylurea or metformin monotherapy between January 1, 2000, and December 31, 2019. The primary outcome was new-onset AF or stroke. Secondary outcomes were cardiovascular, non-cardiovascular and all-cause mortality. Propensity score matching (1:2 ratio) between sulphonylurea and metformin users was performed, based on demographics, CHA-DS-VASc score, past comorbidities and medication use. Cox regression was used to identify significant risk factors. Competing risk analysis was conducted using cause-specific and subdistribution hazard models. Sensitivity analyses using propensity score stratification, high-dimensional propensity score and inverse probability of treatment weighting were conducted. Subgroup analyses were conducted for age and gender in the matched cohort. RESULTS: A total of 36,228 sulphonylurea users and 72,456 metformin users were included in the propensity score-matched cohort. Multivariable Cox regression showed that sulphonylurea users had higher risks of incident AF (hazard ratio [HR]: 2.89, 95% confidence interval [CI]: 2.75-3.77; P < 0.0001), stroke (HR: 3.23, 95% CI: 3.01-3.45; P < 0.0001), cardiovascular mortality (HR: 3.60, 95% CI: 2.62-4.81; P < 0.0001) and all-cause mortality (HR: 4.35, 95% CI: 3.16-4.75; P < 0.0001) compared to metformin users. Similarly, significant results were observed using cause-specific and subdistribution hazard models. Sensitivity analysis using techniques based on the propensity score also yielded similar results. CONCLUSIONS: Sulphonylurea use was associated with higher risks of incident AF, stroke, cardiovascular mortality and all-cause mortality compared to metformin. Males and patients older than 65 years with sulphonylurea use were exposed to the highest risks.

Measures of repolarization variability predict ventricular arrhythmogenesis in heptanol-treated Langendorff-perfused mouse hearts.

BACKGROUND: Time-domain and non-linear methods can be used to quantify beat-to-beat repolarization variability but whether measures of repolarization variability can predict ventricular arrhythmogenesis in mice have never been explored. METHODS: Left ventricular monophasic action potentials (MAPs) were recorded during constant right ventricular 8 â€‹Hz pacing in Langendorff-perfused mouse hearts, in the presence or absence of the gap junction and sodium channel inhibitor heptanol (0.1, 0.5, 1 or 2 â€‹mM). RESULTS: Under control conditions, mean action potential duration (APD) was 39.4 â€‹± â€‹8.1 â€‹ms. Standard deviation (SD) of APDs was 0.3 â€‹± â€‹0.2 â€‹ms, coefficient of variation was 0.9 â€‹± â€‹0.8% and the root mean square (RMS) of successive differences in APDs was 0.15 â€‹± â€‹0.14 â€‹ms. Poincaré plots of APDn+1 against APDn revealed ellipsoid morphologies with a SD along the line-of-identity (SD2) to SD perpendicular to the line-of-identity (SD1) ratio of 4.6 â€‹± â€‹2.1. Approximate and sample entropy were 0.61 â€‹± â€‹0.12 and 0.76 â€‹± â€‹0.26, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.49 â€‹± â€‹0.27 and 0.81 â€‹± â€‹0.36, respectively. Heptanol at 2 â€‹mM induced ventricular tachycardia in five out of six hearts. None of the above parameters were altered by heptanol during which reproducible electrical activity was observed (KW-ANOVA, P â€‹> â€‹0.05). Contrastingly, SD2/SD1 decreased to 2.03 â€‹± â€‹0.41, approximate and sample entropy increased to 0.82 â€‹± â€‹0.12 and 1.45 â€‹± â€‹0.34, and short-term fluctuation slope decreased to 0.82 â€‹± â€‹0.19 during the 20-s period preceding spontaneous ventricular tachy-arrhythmias (n â€‹= â€‹6, KW-ANOVA, P â€‹< â€‹0.05). CONCLUSION: Measures of repolarization variability, such as SD2/SD1, entropy, and fluctuation slope are altered preceding the occurrence of ventricular arrhythmogenesis in mouse hearts. Changes in these variables may allow detection of impending arrhythmias for early intervention.

Metformin accelerates zebrafish heart regeneration by inducing autophagy.

Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Toxicity effects of hydrophilic algal lysates from Coolia tropicalis on marine medaka larvae (Oryzias melastigma).

Coolia tropicalis is a species of benthic and epiphytic toxic algae, which can produce phycotoxins that intoxicate marine fauna. In this study, the potential toxic effects of C. tropicalis on fish were investigated using larval marine medaka (Oryzias melastigma) as a model to evaluate fish behavior, physiological performance, and stress-induced molecular responses to exposure to two sublethal concentrations (LC10 and LC20) of hydrophilic algal lysates. Exposure to C. tropicalis lysates inhibited swimming activity, activated spontaneous undirected locomotion, altered nerve length ration, and induced early development abnormalities, such as shorter eye diameter, body as well as axon length. Consistent with these abnormalities, changes in the expression of genes associated with apoptosis (CASPASE-3 and BCL-2), the inflammatory response (IL-1ß and COX-2), oxidative stress (SOD), and energy metabolism (ACHE and VHA), were also observed. This study advances our understanding of the mechanisms of C. tropicalis toxicity in marine fish in the early life stages and contributes to future ecological risk assessments of toxic benthic dinoflagellates.

Territory-Wide Chinese Cohort of Long QT Syndrome: Random Survival Forest and Cox Analyses.

Introduction: Congenital long QT syndrome (LQTS) is a cardiac ion channelopathy that predisposes affected individuals to spontaneous ventricular tachycardia/fibrillation (VT/VF) and sudden cardiac death (SCD). The main aims of the study were to: (1) provide a description of the local epidemiology of LQTS, (2) identify significant risk factors of ventricular arrhythmias in this cohort, and (3) compare the performance of traditional Cox regression with that of random survival forests. Methods: This was a territory-wide retrospective cohort study of patients diagnosed with congenital LQTS between 1997 and 2019. The primary outcome was spontaneous VT/VF. Results: This study included 121 patients [median age of initial presentation: 20 (interquartile range: 8-44) years, 62% female] with a median follow-up of 88 (51-143) months. Genetic analysis identified novel mutations in KCNQ1, KCNH2, SCN5A, ANK2, CACNA1C, CAV3, and AKAP9. During follow-up, 23 patients developed VT/VF. Univariate Cox regression analysis revealed that age [hazard ratio (HR): 1.02 (1.01-1.04), P = 0.007; optimum cut-off: 19 years], presentation with syncope [HR: 3.86 (1.43-10.42), P = 0.008] or VT/VF [HR: 3.68 (1.62-8.37), P = 0.002] and the presence of PVCs [HR: 2.89 (1.22-6.83), P = 0.015] were significant predictors of spontaneous VT/VF. Only initial presentation with syncope remained significant after multivariate adjustment [HR: 3.58 (1.32-9.71), P = 0.011]. Random survival forest (RSF) model provided significant improvement in prediction performance over Cox regression (precision: 0.80 vs. 0.69; recall: 0.79 vs. 0.68; AUC: 0.77 vs. 0.68; c-statistic: 0.79 vs. 0.67). Decision rules were generated by RSF model to predict VT/VF post-diagnosis. Conclusions: Effective risk stratification in congenital LQTS can be achieved by clinical history, electrocardiographic indices, and different investigation results, irrespective of underlying genetic defects. A machine learning approach using RSF can improve risk prediction over traditional Cox regression models.

Arrhythmogenic Mechanisms in Hypokalaemia: Insights From Pre-clinical Models.

Potassium is the predominant intracellular cation, with its extracellular concentrations maintained between 3. 5 and 5 mM. Among the different potassium disorders, hypokalaemia is a common clinical condition that increases the risk of life-threatening ventricular arrhythmias. This review aims to consolidate pre-clinical findings on the electrophysiological mechanisms underlying hypokalaemia-induced arrhythmogenicity. Both triggers and substrates are required for the induction and maintenance of ventricular arrhythmias. Triggered activity can arise from either early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs). Action potential duration (APD) prolongation can predispose to EADs, whereas intracellular Ca2+ overload can cause both EADs and DADs. Substrates on the other hand can either be static or dynamic. Static substrates include action potential triangulation, non-uniform APD prolongation, abnormal transmural repolarization gradients, reduced conduction velocity (CV), shortened effective refractory period (ERP), reduced excitation wavelength (CV × ERP) and increased critical intervals for re-excitation (APD-ERP). In contrast, dynamic substrates comprise increased amplitude of APD alternans, steeper APD restitution gradients, transient reversal of transmural repolarization gradients and impaired depolarization-repolarization coupling. The following review article will summarize the molecular mechanisms that generate these electrophysiological abnormalities and subsequent arrhythmogenesis.

Spectroscopic and microscopic examination of teeth exposed to green tea at different temperatures.

Tea is a popular beverage consumed at different temperatures. The effect of tea on teeth at different temperatures has not been studied previously. The present study used an in vitro green tea immersed tooth model at different tea temperatures (hot and cold) compared to an in vivo tea administration model allowing rats to drink tea over the course of a week. The elements present in tea leaves were identified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and compared to the elements in teeth (enamel surface) using Laser-Induced Breakdown Spectroscopy (LIBS). Here, LIBS demonstrated in vivo and in vitro green tea treatments resulted in a significant increase in the mineral elements found in enamel. For the in vitro assessment, elements in enamel varied based on cold-tea and hot-tea treatment; however, hot water reduced the elements in enamel. Atomic force microscopy found the in vivo tea group had a higher roughness average (RA) compared with the in vivo water group. Cold tea and hot tea in vitro groups demonstrated lower RA than in vitro water controls. Scanning electron microscopy found hot water induced cracks more than 1.3µm in enamel while cold tea and hot tea promoted the adhering of extrinsic matter to teeth. Overall, teeth treated to high temperature lost the mineral phase leading to demineralization. Our results indicate that green tea protects enamel, but its protective action in dental structures is enhanced at cold temperature.

Association of NPAC score with survival after acute myocardial infarction.

BACKGROUND AND AIMS: Risk stratification in acute myocardial infarction (AMI) is important for guiding clinical management. Current risk scores are mostly derived from clinical trials with stringent patient selection. We aimed to establish and evaluate a composite scoring system to improve short-term mortality classification after index episodes of AMI, independent of electrocardiography (ECG) pattern, in a large real-world cohort. METHODS: Using electronic health records, patients admitted to our regional teaching hospital (derivation cohort, n = 1747) and an independent tertiary care center (validation cohort, n = 1276), with index acute myocardial infarction between January 2013 and December 2017, as confirmed by principal diagnosis and laboratory findings, were identified retrospectively. RESULTS: Univariate logistic regression was used as the primary model to identify potential contributors to mortality. Stepwise forward likelihood ratio logistic regression revealed that neutrophil-to-lymphocyte ratio, peripheral vascular disease, age, and serum creatinine (NPAC) were significant for 90-day mortality (Hosmer- Lemeshow test, p = 0.21). Each component of the NPAC score was weighted by beta-coefficients in multivariate analysis. The C-statistic of the NPAC score was 0.75, which was higher than the conventional Charlson's score (C-statistic = 0.63). Judicious application of a deep learning model to our dataset improved the accuracy of classification with a C-statistic of 0.81. CONCLUSIONS: The NPAC score comprises four items from routine laboratory parameters to basic clinical information and can facilitate early identification of cases at risk of short-term mortality following index myocardial infarction. Deep learning model can serve as a gatekeeper to facilitate clinical decision-making.

Glymphatic clearance of simulated silicon dispersion in mouse brain analyzed by laser induced breakdown spectroscopy.

Silicon-based devices, such as neural probes, are increasingly used as electrodes for receiving electrical signals from neural tissue. Neural probes used chronically have been known to induce inflammation and elicit an immune response. The current study detects and evaluates silicon dispersion from a concentrated source in the mouse brain using laser induced breakdown spectroscopy. Element lines for Si (I) were found at the injection site at approximately 288 nm at 3hr post-implantation, even with tissue perfusion, indicating possible infusion into neural tissue. At 24hr and 1-week post-implantation, no silicon lines were found, indicating clearance. An isolated immune response was found by CD68 macrophage response at 24hr post injection. Future studies should measure chronic silicon exposure to determine if the inflammatory response is proportional to silicon administration. The present type of protocol, coupling laser induced breakdown spectroscopy, neuroimaging, histology, immunohistochemistry, and determination of clearance could be used to investigate the glymphatic system and different tissue states such as in disease (e.g. Alzheimer's).

Role of PARP1 regulation in radiation-induced rescue effect.

Radiation-induced rescue effect (RIRE) in cells refers to the phenomenon where irradiated cells (IRCs) receive help from feedback signals produced by partnered bystander unirradiated cells (UIRCs) or from the conditioned medium (CM) that has previously conditioned the UIRCs. In the present work, we explored the role of poly (ADP-ribose) polymerase 1 (PARP1) regulation in RIRE and the positive feedback loop between PARP1 and nuclear factor-kappa-light-chain-enhancer of activated B cell (NF-κB) in RIRE using various cell lines, including HeLa, MCF7, CNE-2 and HCT116 cells. We first found that when the IRCs (irradiated with 2 Gy X-ray) were treated with CM, the relative mRNA expression levels of both tumor suppressor p53-binding protein 1 (53BP1) and PARP1, the co-localization factor between 53BP1 and γH2AX as well as the fluorescent intensity of PARP1 were reduced. We also found that IRCs treated with the PARP1 inhibitor, Olaparib (AZD2281) had a higher 53BP1 expression. These results illustrated that PARP1 was involved in RIRE transcriptionally and translationally. We further revealed that treatment of IRCs with CM together with Olaparib led to significantly lower mRNA expression levels and fluorescent intensities of NF-κB, while treatment of IRCs with CM together the NF-κB inhibitor BAY-11-7082 led to significantly lower mRNA expression levels as well as fluorescent intensities of PARP1. These results illustrated that PARP1 and NF-κB were involved in the positive feedback loop transcriptionally and translationally. Thus, the results supported the occurrence of a PARP1-NF-κB positive feedback loop in RIRE. The present work provided insights into potential exploitation of inhibition of PARP1 and/or the PARP1-NF-κB positive feedback loop in designing adjuncts to cancer radiotherapeutics.

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Estrogen accelerates heart regeneration by promoting the inflammatory response in zebrafish.

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.