Pharmacological Research

raw data of western blot

Supplementary file: search strategies, supplementary analyses

This dataset contains clinical and genetic information on case reported in the related article

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations

Asymmetric cell division (ACD) is the process whereby one dividing cell gives rise to two daughter cells of differing fates. ACD is known to be controlled, at least in part, by protein complexes that reside asymmetrically at the cortex of dividing cells. These asymmetric domains are involved in orienting the division plane, localising proteins important for cell fate, as well as for generating polarity in cytosolic structures important for the dispatch of cell fate determinants. One protein complex of particular interest is the Par complex, which is known to asymmetrically localise to the plasma membrane in many systems. It is known that an asymmetric Par complex is required (in many systems), for orienting the division plane, and for generating asymmetry in the density of microtubules in the anaphase-B central spindle (at least in Drosophila). However, whether the Par complex is sufficient to generate these features, or whether they rely on other polarity pathways, is unknown. In this work, I sought to address this question, by developing two techniques for reimparting cortical polarity back into unpolarised cells (cells that have lost polarity). The first of these techniques relies on protein micropatterning, which is the process by which proteins can be ‘printed’ onto a surface. For successful establishment of this assay, a new system of micropatterning, based on ‘anchoring’ proteins to Fibrinogen (a large glycoprotein complex), was developed. Fibrinogen-anchoring remarkably improves the micropatterning of previously difficult-to-pattern proteins, and gives a universal patterning platform upon which complex, multi-protein patterning experiments can be performed. For the first time, protein micropatterns were used to control the subcellular localisation of cell receptors. Further, with this technology, intracellular plasma membrane asymmetries, akin to those that occur in multicellular organisms, could be reconstituted. The second technology for reconstituting plasma membrane asymmetry was based on novel de novo designed proteins, which, upon mixing, spontaneously assemble into large, two-dimensional lattices. Using quantitative fluorescence microscopy and Atomic Force Microscopy, we demonstrated that these lattices assemble in a near-crystalline fashion on cells. With these proteins, robust asymmetric ‘caps’ of plasma membrane complexes could be generated. Using this technique, I showed that an asymmetric Par complex is indeed sufficient to orient division, and to generate asymmetry in the central spindle. Finally, I showed that these two pathways, while both downstream of an asymmetric Par complex, can be untangled. This represents the most complete reconstitution of asymmetric cell division in symmetrically dividing, unpolarised cells to date. This is the first demonstration that an asymmetric Par complex is sufficient to reinstate polarised signalling and important aspects of ACD in unpolarised cells, and is also the first example of central spindle asymmetry in mammalian cells, revealing this to be a conserved feature of ACD. Further, the technology developed in this study will be of broad use to the field, and will no doubt be used to shed light onto more of the mechanisms controlling cortical symmetry breaking and polarised signalling.

This record contains raw data related to the article"Cyclophilin A Inhibition as Potential Treatment of Human Aortic Valve Calcification". Abstract Calcific aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification of aortic valve in AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that i) CyPA is up-regulated in stenotic valves of AS patients, ii) pro-calcifying medium promotes CyPA secretion by VICs, iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

This record contains raw data related to the article “12(S)-Hydroxyeicosatetraenoic acid downregulates monocyte-derived macrophage efferocytosis: new insights in atherosclerosis" Abstract:The involvement of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipooxygenase product of arachidonic acid, has been suggested in atherosclerosis. However,its effecton macrophage functions is not completely understood, so far. The uptake of apoptotic cells (efferocytosis) by macrophages is an anti-inflammatory process, impaired in advanced atherosclerotic lesions. This process induces the release of the anti-inflammatory cytokine interleukin-10 (IL-10), and it is regulated by Rho-GTPases, whose activation involves the isoprenylation, a modification inhibited by statins. We assessed 12-HETE levels in serum of coronary artery disease (CAD) patients, and explored12(S)-HETE invitro effect on monocyte-derived macrophage (MDM) efferocytosis.Sixty-four CAD patients and 24 healthy subjects (HS) were enrolled. Serum12-HETE levels were measured using a tandem mass spectrometry method. MDMs, obtained from a spontaneous differentiation of adherent monocytes, were treated with12(S)-HETE (10–50 ng/mL). Efferocytosis and RhoA activation were evaluated by flow cytometry. IL-10 was measured by ELISA. CAD patients showed increased 12-HETE serum levels compared to HS (665.2 [438.1–896.2] ng/mL and 525.1 [380.1–750.1] ng/mL, respectively, p < 0.05) and reduced levels of IL-10. MDMs expressed the12(S)-HETE cognate receptor GPR31. CAD-derived MDMs displayed defective efferocytosis vs HS-MDMs (9.4 [7.7–11.3]% and 11.1 [9.6–14.1]% of MDMs that have engulfed apoptotic cells, respectively, p < 0.01). This reduction is marked in MDMs obtained from patients not treated with statin (9.3 [7.4–10.6]% statin-free CAD vs HS, p=0.01; and 9.9 [8.6–11.6]% statin-treated CAD vs HS, p=0.07). The in vitro treatment of MDMs with12 (S)-HETE (20ng/mL) induced 20% decrease of efferocytosis (p < 0.01) and 71% increase of RhoA activated form(p < 0.05). Atorvastatin (0.1μM) counteracted these 12(S)-HETE-mediated effects.These results show a12(S)-HETE pro-inflammatory effect and suggest a new potential contribution of this mediator in the development of atherosclerosis.

Related Article: Yi-Han Zuo, Yong-Bei Liu, Chun-Song Cheng, Yu-Pei Yang, Ying Xie, Pei Luo, Wei Wang, Hua Zhou |2020|Pharmacological Research|158|104897|doi:10.1016/j.phrs.2020.104897

Related Article: Qianqian Di, Xibao Zhao, Ruihan Zhang, Xingyu Ma, Xinxin Liang, Xiaoli Li, Junbo Gao, Haimei Tang, Weilin Chen, Weilie Xiao|2020|Pharmacological Research|164|105386|doi:10.1016/j.phrs.2020.105386