Downregulation of TRPC4 and TRPC5 Inhibits Smooth Muscle Cell Proliferation without Affecting Endothelial Cell Proliferation

Aims. The main treatment for coronary heart disease is percutaneous coronary intervention (PCI), and drug-eluting stents are designed to inhibit vascular smooth muscle cell (VSMCs) proliferation and migration causing restenosis by releasing pharmacological agents into the vessel wall. Once drug-eluting stents are deployed, these pharmacological agents exert many biological effects in the coronary circulation, not only inhibition of VSMCs but also extension to vascular endothelial cells (VECs). The purpose of this study was to explore target molecules that inhibit VSMCs proliferation without affecting VECs. Methods. mRNA and protein expressions of transient receptor potential channels (TRPCs) in cultured VSMCs and VECs were determined by western blotting and RT-qPCR. VSMCs and VECs proliferation was evaluated using CCK-8 assays and western blotting of proliferating cell nuclear antigen (PCNA). Calcium backfilling assays were performed to detect intracellular calcium ion concentration in cultured VSMCs and VECs. Results. The TRPC6 expression was more abundant in VECs than VSMCs, while TRPC4 and TRPC5 expressions were more abundant in VSMCs than VECs. Knockdown of TRPC4 or TRPC5 alone had no remarkable inhibitory effect on VSMC proliferation. Synergistic knockdown of TRPC4 and TRPC5 inhibited the proliferation of VSMCs, declined the expression of the PCNA, and reduced the intracellular calcium ion concentration but not VECs. Conclusion. These data suggest that concurrent inhibition of TRPC4 and TRPC5 inhibits VSMCs proliferation without affecting VECs, thus providing novel targets for developing pharmacological agents for drug-eluting stents.

Spices and Hypertension: An Insight for Researchers

: Hypertension is a global public health concern since it can lead to complications like stroke, heart disease and kidney failure. These complications can add to disability, increase healthcare costs and can even result in mortality. In spite of the availability of a large number of antihypertensive drugs, the control of blood pressure is suboptimal in many patients. Spices have been used as flavouring agent and to treat diseases in folk medicine since they are considered to be rich sources of phytochemicals especially polyphenols. Hence, during recent years there is renewed interest among researchers to explore natural sources, especially spices in an attempt to find cheaper alternatives with fewer side effects. Our aim is to review the relevant preclinical and clinical studies focused towards the potential use of spices in the management of hypertension. Studies conducted on the most common spices such as celery, cinnamon, cardamom, garlic, ginger, saffron, and turmeric, have been elaborated in this review. These spices may be lowering blood pressure via several possible mechanisms including antioxidant effect, increase in nitric oxide production, reduction in calcium ion concentration, modulation of renin angiotensin pathway etc.

Extracellular Calcium Ion Concentration Regulates Chondrocyte Elastic Modulus and Adhesion Behavior

Extracellular calcium ion concentration levels increase in human osteoarthritic (OA) joints and contribute to OA pathogenesis. Given the fact that OA is a mechanical problem, the effect of the extracellular calcium level ([Ca2+]) on the mechanical behavior of primary human OA chondrocytes remains to be elucidated. Here, we measured the elastic modulus and cell–ECM adhesion forces of human primary chondrocytes with atomic force microscopy (AFM) at different extracellular calcium ion concentration ([Ca2+]) levels. With the [Ca2+] level increasing from the normal baseline level, the elastic modulus of chondrocytes showed a trend of an increase and a subsequent decrease at the level of [Ca2+], reaching 2.75 mM. The maximum increment of the elastic modulus of chondrocytes is a 37% increase at the peak point. The maximum unbinding force of cell-ECM adhesion increased by up to 72% at the peak point relative to the baseline level. qPCR and immunofluorescence also indicated that dose-dependent changes in the expression of myosin and integrin β1 due to the elevated [Ca2+] may be responsible for the variations in cell stiffness and cell-ECM adhesion. Scratch assay showed that the chondrocyte migration ability was modulated by cell stiffness and cell-ECM adhesion: as chondrocyte’s elastic modulus and cell-ECM adhesion force increased, the migration speed of chondrocytes decreased. Taken together, our results showed that [Ca2+] could regulate chondrocytes stiffness and cell-ECM adhesion, and consequently, influence cell migration, which is critical in cartilage repair.

Comprehensive Profiling of Microbiologically Induced CaCO3 Precipitation by Ureolytic Bacillus Isolates from Alkaline Soils

Microbiologically induced CaCO3 precipitation (MICP) is a well-known bio-based solution with application in environmental, geotechnical, and civil engineering. The significance of the MICP has increased explorations of process efficiency and specificity via natural bacterial isolates. In this study, comprehensive profiling of five soil ureolytic Bacillus strains was performed through a newly formed procedure that involved six steps from selection and identification, through kinetic study, to the characterization of the obtained precipitates, for the first time. To shorten the whole selection procedure of 43 bioagents with the MICP potential, Standard Score Analysis was performed and five selected bacteria were identified as Bacillus muralis, B. lentus, B. simplex, B. firmus, and B. licheniformis by the MALDI-TOF mass spectrometry. Despite following the targeted activity, kinetic studies were included important aspects of ureolysis and the MICP such as cell concentration, pH profiling, and reduction in calcium ion concentration. At the final step, characterization of the obtained precipitates was performed using FTIR, XRD, Raman, DTA/TGA, and SEM analysis. Although all tested strains showed significant potential in terms of precipitation of calcite or calcite and vaterite phase, the main differences in the MICP behavior can be observed at the bacterial strain level. B. licheniformis showed favorable behavior compared to the reference Sporosarcina pasteurii DSM 33.

Syringol Isolated From Eleusine Coracana (L.) Gaertn Bran Suppresses Inflammatory Response Through the Down-Regulation of cPLA2, COX-2, IκBα, p38 and MPO Signaling in sPLA2 Induced Mice Paw Edema.

Eleusine coracana (L.) Gaertn (E. coracana) is one of the highest consuming food crops in Asia and Africa. E. coracana is a plant with several medicinal values including anti-ulcerative, anti-diabetic, anti-viral and anti-cancer properties. However, the anti-inflammatory property of E. coracana remains to be elucidated. Therefore, the objective of present study was to investigate the potential in isolated molecule from E. coracana via a combination of in vitro, in vivo and in silico methods. In this study we have isolated, purified and characterized an anti-inflammatory molecule from E coracana bran extract known as syringol. Purification of syringol was accomplished by combination of GC-MS and RP-HPLC techniques. Syringol significantly inhibited the enzyme activity of sPLA2 (IC50 = 3 µg) and 5-LOX (IC50 = 0.325 µg) in vitro. The inhibition is independent of substrate concentration, calcium ion concentration and was irreversible. Syringol interacts with purified sPLA2 enzymes as evidenced by fluorescence and molecular docking studies. Further, the syringol molecule dose dependently inhibited the development of sPLA2 and carrageenan induced edema. Furthermore, syringol decreases the expression of cPLA2, COX-2, IκBα, p38 and MPO in edematous tissues as demonstrated by western blots. These studies revealed that syringol isolated from E coracana bran may develop as a potent anti-inflammatory molecule.

Regulation of Dendrobium Polysaccharides on Proliferation and Oxidative Stress of Human Umbilical Vein Endothelial Cells in the High Glucose Environment

Backgrounds. Polysaccharides of Dendrobium candidum (PDC) showed a strong antioxidant effect on islet cells while the effects of PDC on human umbilical vein endothelial cells (HUVECs) under the high glucose condition remain unclear. Material and Method. HUVECs were incubated with high glucose (33.3 mmol/L) for 48 hours to induce injury, and cells were treated with PDC (100, 200, and 400 μg/mL) for 48 hours. The tetrazolium blue colorimetric (MTT) assay was used to detect cell proliferation, superoxide dismutase (SOD), and nitric oxide (NO) content in cell supernatants. Flow cytometry was used to detect reactive oxygen species (ROS) and calcium levels. Results. (1) Compared with the control group, the proliferation of HUVECs cells in the high glucose (33.3 mmol/L) group decreased ( P < 0.05 ). The intracellular calcium ion concentration and the intracellular ROS level increased ( P < 0.01 and P < 0.05 ). SOD activity and the level of NO in the culture medium were reduced (P <0.05). (2) Compared with the control group, PDC (50, 100, 200, 400, and 800 μg/mL) did not significantly affect the cell proliferation of HUVECs ( P > 0.05 ). (3) Compared with the high glucose group, the HUVEC cell viability of the high glucose + PDC (100, 200, and 400 μg/mL) group increased while the intracellular calcium ion concentration decreased in a concentration-dependent manner ( P < 0.05 ). Intracellular ROS levels were reduced, while SOD activity and the level of NO in culture fluids increased ( P < 0.05 ). Conclusion. PDC can promote the proliferation of HUVECs in the high glucose environment by reducing oxidative stress injury of HUVECs induced by high glucose.

A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling

Background Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs of uterine diseases. RNA was extracted from the cytobrush samples and samples from 11 selected subfertile and 11 fertile mares were used for Illumina RNA-sequencing. Results The cytobrush sampling was a suitable technique to isolate enough RNA of high quality for transcriptome analysis. Comparing subfertile and fertile mares, 114 differentially expressed genes (FDR = 10%) were identified. Metascape enrichment analysis revealed that genes with lower mRNA levels in subfertile mares were related to ‘extracellular matrix (ECM)’, ‘ECM-receptor interaction’, ‘focal adhesion’, ‘immune response’ and ‘cytosolic calcium ion concentration’, while DEGs with higher levels in subfertile mares were enriched for ‘monocarboxyl acid transmembrane transport activity’ and ‘protein targeting’. Conclusion Our study revealed significant differences in the uterine transcriptome between fertile and subfertile mares and provides leads for potential uterine molecular biomarkers of subfertility in the mare.

StalGrowth—A Program to Estimate Speleothem Growth Rates and Seasonal Growth Variations

Speleothems are one of the few archives which allow us to reconstruct the terrestrial paleoclimate and help us to understand the important climate dynamics in inhabited regions of our planet. Their time of growth can be precisely dated by radiometric techniques, but unfortunately seasonal radiometric dating resolution is so far not feasible. Numerous cave environmental monitoring studies show evidence for significant seasonal variations in parameters influencing carbonate deposition (calcium-ion concentration, cave air pCO2, drip rate and temperature). Variations in speleothem deposition rates need to be known in order to correctly decipher the climate signal stored in the speleothem archive. StalGrowth is the first software to quantify growth rates based on cave monitoring results, detect growth seasonality and estimate the seasonal growth bias. It quickly plots the predicted speleothem growth rate together with the influencing cave environmental parameters to identify which parameter(s) cause changes in speleothem growth rate, and it can also identify periods of no growth. This new program has been applied to multiannual cave monitoring studies in Austria, Gibraltar, Puerto Rico and Texas, and it has identified two cases of seasonal varying speleothem growth.

A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca2+-signalling

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-d-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.