TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway

Currently, there is no effective molecular-based therapy for triple-negative breast cancer (TNBC). Canonical transient receptor potential isoform 3 (TRPC3) was previously shown to be upregulated in breast cancer biopsy tissues when compared to normal breast tissues. However, the biological role of TRPC3 in breast cancer still remains to be elucidated. In this study, subcellular fractionation followed by Western blot and immunocytochemistry showed that TRPC3 was over-expressed on the plasma membrane of TNBC line MDA-MB-231 when compared to an estrogen receptor-positive cell line MCF-7. TRPC3 blocker Pyr3 and dominant negative of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231 as measured by proliferation assays. Interestingly, Ras GTPase-activating protein 4 (RASA4), a Ca2+-promoted Ras-MAPK pathway suppressor, was found to be located on the plasma membrane of MDA-MB-231. Blocking TRPC3 decreased the amount of RASA4 located on the plasma membrane, with concomitant activation of MAPK pathways. Our results suggest that, in TNBC MDA-MB-231 cells, Ca2+ influx through TRPC3 channel sustains the presence of RASA4 on the plasma membrane where it inhibits the Ras-MAPK pathway, leading to proliferation and apoptosis resistance. Our study reveals the novel TRPC3-RASA4-MAPK signaling cascade in TNBC cells and suggests that TRPC3 may be exploited as a potential therapeutic target for TNBC

Factors impacting gas content measurements using gas desorption by drilling underground boreholes

Accurate determination of the gas content in coalbeds is important for safe mining. Currently, gas desorption by drilling underground boreholes is the most commonly used gas determination method. However, this method is not very accurate and needs to be improved. In this study, we established a laboratory protocol based on coal adsorption studies to analyse factors affecting the measurement accuracy. The results showed that exposure time, sampling method, sample weight, particle size and gas loss estimate significantly affected the gas content measurement using gas desorption by drilling underground boreholes. Longer exposure time and increased particle size resulted in higher relative errors. Sampling by coring is more accurate than sampling by drilling. The higher the sample weight is for samples weighing less than 240 g, the larger the error of the in situ measurements of desorbed gas, residual gas and lost gas is. The error tends to stabilize for heavier samples. The gas losses at different exposure times calculated using the commonly used Barrer model, power function method and negative exponent method were compared. The gas loss error within 0–12 min, computed with the Barrer model, and after 12 min, computed with the power function method, is minimal. The modified formula of gas loss was obtained using the combination of a fitting analysis and the relationship between gas loss and exposure time. Subsequently, the optimal procedure for in situ gas content measurements using gas desorption by drilling underground boreholes was determined. The gas content errors for anthracite, gas coal, lean coal and long flame coal, which were measured using gas desorption by drilling underground boreholes and corrected using the gas loss formula, decreased significantly to less than 10%, thus, meeting the engineering accuracy norm

Organophosphate flame retardants, tetrabromobisphenol A, and their transformation products in sediment of e-waste dismantling areas and the flame-retardant production base

Due to the prohibition of polybrominated diphenyl ethers, organophosphate flame retardants (OPFRs) and tetrabromobisphenol A (TBBPA) have become emerging flame retardants. However, knowledge about their occurrence, especially their transformation products, is still limited. This study collected sediment samples from two rivers, i.e., Lianjiang River (located at an e-waste dismantling area) and Xiaoqing River (situated at a flame retardant production base), to investigate the occurrence, composition, and spatiality distribution of OPFRs, TBBPA, and their transformation products. Both targets were detected in the Lianjiang River in the range of 220-1.4 x 10(4) and 108-3.1 x 10(3) ng/g dw (dry weight) for OPFRs and TBBPA, and 0.11-2.35 and 4.8-414 ng/g dw for their respective transformation products, respectively. The concentrations of OPFRs and TBBPA in the Xiaoqing River ranged from 4.15 to 31.5 and 0.76-2.51 ng/g dw, respectively, and no transformation products were detected. Different compositional characteristics of OPFRs and distinct spatial distribution from mainstream and tributary observed between the two rivers are attributed to the difference in the local industries. Spatial distribution and principal component analysis indicated that e-waste dismantling activities could be a vital source of local pollution. Besides, the confluence of tributaries seemed to determine the contaminant levels in the Xiaoqing River. Also, concentration ratios and Spearman's correlation between metabolites and parent chemicals were analyzed. Low concentration ratios (3.6 x 10(-4) to 0.16) indicated a low transformation degree, and Spearman's correlation analysis suggested transformation products were partly stemming from commercial products. Considering the limited study of these transformation products, more studies on their sources, transform mechanism, and toxicity are required

Maternal transfer of resorcinol-bis(diphenyl)-phosphate perturbs gut microbiota development and gut metabolism of offspring in rats

Resorcinol-bis(diphenyl)-phosphate (RDP), an emerging organophosphate flame retardant, is increasingly used as a primary alternative for decabromodiphenyl ether and is frequently detected in global environmental matrices. However, the long-term effects of its exposure to humans remain largely unknown. To investigate its intergenerational transfer capacity and health risks, female Sprague Dawley rats were orally exposed to RDP from the beginning of pregnancy to the end of the lactation period. The RDP content, gut microbiota homeostasis, and metabolic levels were determined. RDP accumulation occurred in the livers of maternal rats and offspring and increased with exposure time. 16S rRNA gene sequencing showed that exposure to RDP during pregnancy and/or lactation significantly disrupted gut microbiota homeostasis, as evidenced by decreased abundance and diversity. In particular, the abundance of Turicibacter, Adlercreutzia, and YRC22 decreased, correlating significantly with glycollipic metabolism. This finding was consistent with the reduced levels of short-chain fatty acids, the crucial gut microbial metabolites. Meanwhile, RDP exposure resulted in changes in gut microbiome-related metabolism. Nine critical overlapping KEGG metabolic pathways were identified, and the levels of related differential metabolites decreased. Our results suggest that the significant adverse impacts of RDP on gut microbiota homeostasis and metabolic function may increase the long-term risks related to inflammation, obesity, and metabolic diseases

Role of TRPV1 in the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes.

Cytosolic Ca2+ ([Ca2+]i) is an important signal that regulates cardiomyocyte differentiation during cardiogenesis. TRPV1 is a Ca2+-permeable channel that is expressed in cardiomyocytes. In the present study, we utilized mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) as a model to investigate the functional role of TRPV1 in cardiomyocyte differentiation. Induction of embryonic stem cells into cardiomyocytes was achieved using embryoid body (EB)-based differentiation method. Quantitative PCRs showed an increased TRPV1 expression during the differentiation process. In [Ca2+]i measurement study, application of TRPV1 agonists, capsaicin and camphor, elicited a [Ca2+]i rise in mESC-CMs, the effect of which was abolished by TRPV1-shRNA. In functional study, treatment of EBs with TRPV1 antagonists (capsazepine and SB366791) and TRPV1-shRNA reduced the size of the EBs and decreased the percentage of spontaneously beating EBs. TRPV1 antagonists and TRPV1-shRNA also suppressed the expression of cardiomyocyte marker genes, including cardiac actin, c-TnT, c-TnI, and α-MHC. Taken together, this study demonstrated an important functional role of TRPV1 channels in the differentiation of mESCs into cardiomyocytes

Capsaicin and camphor-induced rise in basal [Ca²⁺]i level in mESC-CMs.

<p>(A and B) Representative traces showing the [Ca²⁺]i responses to 1 μM capsaicin (A) and 2.5 mM camphor (B). (C and D) summary of data comparing the maximal [Ca²⁺]i, while rise in response to 1 μM capsaicin (C) and 2.5 mM camphor (D). Shown were effects of TRPV1-shRNA and SB366791 (10 μM). (E) Effectiveness of TRPV1-shRNA in suppressing TRPV1 mRNA by qPCR. ctl-shRNA stands for control scrambled shRNA. Values were Mean ± SEM. n = 3–5 experiments as labeled. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of TRPV1-shRNA on mESC differentiation to cardiomyocytes.

<p>(A) Summary of FACS data showing that TRPV1-shRNA reduced the c-TnT-positive cardiomyocytes on the differentiation day 12. (B) Data summary showing the effect of TRPV1-shRNA on EB sizes on the differentiation day 7. Each group contained more than 80 EBs from 3 independent experiments. (C) The effect of TRPV1-shRNA on the percentage of beating EBs. The beating was recorded from differentiation day 8 to day 17. (D) qPCR data on the differentiation day 12, showing the expressional levels of cardiac actin (car. actin), cardiac troponin T (c-TnT), cardiac troponin I (c-TnI) and alpha-myosin heavy chain (α-MHC). ctl-shRNA stands for control scrambled shRNA. Values were Mean ± SEM. n = 3 experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Expression pattern of TRPV1 mRNA during the differentiation of mESCs to cardiomyocytes.

<p>Shown was the relative expression of TRPV1 mRNA at different differentiation days. Values were Mean ± SEM. n = 3.</p

Effect of TRPV1 agonist or antagonists on EB sizes and EB beating curves.

<p>(A) (A) Summary of data illustrating EB size distribution on the differentiation day 7 after treatment with 0.1% DMSO (vehicle control), 1 μM capsaicin, 10 μM capsazepine, or 10 μM SB366791. Each group contained more than 200 EBs from 3 independent experiments, with red bar representing the average size. (B) Data summary of EB sizes under different treatments. (C) The percentage of beating EBs after different treatments. The beating was recorded from differentiation day 8 to day 17. Results were Mean ± SEM. n = 3 experiments. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 compared to DMSO control.</p