Regulation of CCL5 Expression in Smooth Muscle Cells Following Arterial Injury

Chemokines play a crucial role in inflammation and in the pathophysiology of atherosclerosis by recruiting inflammatory immune cells to the endothelium. Chemokine CCL5 has been shown to be involved in atherosclerosis progression. However, little is known about how CCL5 is regulated in vascular smooth muscle cells. In this study we report that CCL5 mRNA expression was induced and peaked in aorta at day 7 and then declined after balloon artery injury, whereas IP-10 and MCP-1 mRNA expression were induced and peaked at day 3 and then rapidly declined

Volume-regulated Cl- current: contributions of distinct Cl- channel and localized Ca2+ signals.

The swelling-activated chloride current (ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume regulated anion channel (VRAC). LRRC8A (SWELL1) was recently identified as an essential component of VRAC but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl- channels, such as anoctamin 1 (ANO1) were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients and these Ca2+ signals were required to activate both, the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the GPCR-independent PLC isoforms

Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca2+ channels

T-type Ca2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions

Microwave breast cancer detection using time-frequency representations

Microwave-based breast cancer detection has been proposed as a complementary approach to compensate for some drawbacks of existing breast cancer detection techniques. Among the existing microwave breast cancer detection methods, machine learning-type algorithms have recently become more popular. These focus on detecting the existence of breast tumours rather than performing imaging to identify the exact tumour position. A key component of the machine learning approaches is feature extraction. One of the most widely used feature extraction method is principle component analysis (PCA). However, it can be sensitive to signal misalignment. This paper proposes feature extraction methods based on time-frequency representations of microwave data, including the wavelet transform and the empirical mode decomposition. Time-invariant statistics can be generated to provide features more robust to data misalignment. We validate results using clinical data sets combined with numerically simulated tumour responses. Experimental results show that features extracted from decomposition results of the wavelet transform and EMD improve the detection performance when combined with an ensemble selection-based classifier

A Hybrid Semi-Supervised Anomaly Detection Model for High-Dimensional Data

Anomaly detection, which aims to identify observations that deviate from a nominal sample, is a challenging task for high-dimensional data. Traditional distance-based anomaly detection methods compute the neighborhood distance between each observation and suffer from the curse of dimensionality in high-dimensional space; for example, the distances between any pair of samples are similar and each sample may perform like an outlier. In this paper, we propose a hybrid semi-supervised anomaly detection model for high-dimensional data that consists of two parts: a deep autoencoder (DAE) and an ensemble k-nearest neighbor graphs- (K-NNG-) based anomaly detector. Benefiting from the ability of nonlinear mapping, the DAE is first trained to learn the intrinsic features of a high-dimensional dataset to represent the high-dimensional data in a more compact subspace. Several nonparametric KNN-based anomaly detectors are then built from different subsets that are randomly sampled from the whole dataset. The final prediction is made by all the anomaly detectors. The performance of the proposed method is evaluated on several real-life datasets, and the results confirm that the proposed hybrid model improves the detection accuracy and reduces the computational complexity

The role of p38 MAPK in IP-10 expression.

<p>(A) 1×10⁶ A7r5 cells were treated with different amounts of a p38 MAPK antagonist SB203580 for 1 h prior to addition of various stimuli as indicated. An identical amount of dissolvent DMSO was used for negative controls. 6 hrs later, total RNA was extracted for measurement of IP-10 mRNA expression by qRT-PCR. 5 µg of different expression plasmids encoding different molecules involved in the p38 MAPK signaling pathway including p38α (B), and DN-MK2 (C), as well as MLK3, MKK3, and MKK6 (D) or empty vector were transiently transfected with Lipofectamine. 48 h after transfection, the cells were treated with LPS (1 µg/ml) plus IFN-γ (10 ng/ml) for 6 h, followed by measurement of IP-10 mRNA expression by qRT-PCR. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1. Results shown are mean plus SD of three independent experiments.</p

Kinetic expression of chemokines in rat vascular smooth muscle cells.

<p>Total RNA was extracted from rat smooth vascular muscle cells treated with IFN-γ, LPS, or IFN-γ plus LPS for different times as indicated and reverse transcribed into cDNA for measurement of CCL5 (A), IP-10 (B) and MCP-1 (C) expression by qRT-PCR. Data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1.</p

Kinetic expression of chemokines in aorta after balloon injury.

<p>Total RNA was extracted from aorta and used for real time quantitative PCR (qRT-PCR) measurement of CCL5 (A), IP-1 (B) and MCP-1 (C) mRNA expression as well as CCR1 (D), CCR3 (E) and CCR5 (F) mRNA expression. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated aorta, which was set as 1. Results shown are mean plus SD of three to four independent experiments.</p

The role of p38 MAPK in CCL5 expression.

<p>(A) 1×10⁶ A7r5 cells were treated with different amounts of a p38 MAPK antagonist SB203580 for 1 h prior to addition of various stimuli as indicated. An identical amount of dissolvent DMSO was used for negative controls. 6 hrs later, total RNA was extracted for measurement of CCL5 mRNA expression by qRT-PCR. 5 µg of different expression plasmids encoding different molecules involved in the p38 MAPK signaling pathway including p38α (B), and DN-MK2 (C), as well as MLK3, MKK3, and MKK6 (D) or empty vector were transiently transfected with Lipofectamine2000. 48 h after transfection, the cells were treated with LPS (1 µg/ml) plus IFN-γ (10 ng/ml) for 6 h, followed by measurement of CCL5 mRNA expression by qRT-PCR. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1. Results shown are mean plus SD of three independent experiments.</p