Stimulation Of Cells Research Articles

Large-scale phosphoproteomics reveals activation of the MAPK/GADD45β/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells

BackgroundBMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.MethodsTo get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry.ResultsLarge-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1.ConclusionsOverall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

Adjuvant Activity and Toxicological Risks of Lipid Nanoparticles Contained in the COVID‑19 “mRNA Vaccines”

The LNPs reportedly used as the platform by Pfizer/BioNTech for its SARS-CoV-2 “mRNA vaccines” allegedly consist of a mixture of phospholipids, cholesterol, PEGylated lipids, and an ionizable cationic lipid. This study reviews some of the main toxicological risks and immunostimulatory properties of such nanomaterials, with particular attention to the ionizable LNPs and their adjuvant properties, inflammatory responses, stimulation of immune cells, and formation of ROS inside transfected cells. The decision not to carry out safety pharmacology, carcinogenicity, and genotoxicity tests on these nanomaterials appears unjustifiable and in contradiction with the international policies provided for novel adjuvants. Important gaps are highlighted on the activities by the relevant regulatory bodies, related to the scientific evaluation, risk management, and pharmacovigilance for new medicinal products in the EU. Given the findings discussed here, it is strongly urged that the mRNA-LNP-based “vaccines” and their boosters should be removed from the worldwide market because of unacceptable and potentially fatal safety risks.

Defective Mitophagy Impairs Response to Inflammatory Activation of Macrophage-Like Cells.

The role of mitophagy in atherosclerosis has been extensively studied during the last few years. It was shown that mitophagy is involved in the regulation of macrophages, which are important players as immune cells in atherosclerosis development. In this study, we investigated the relationship between mitophagy and response to inflammatory stimulation of macrophage-like cells. Six cybrid cell lines with normal mitophagy, that is, increasing in response to stimulation, and 7 lines with defective mitophagy not responding to stimulation were obtained. The objective of the study was to compare the nature of the inflammatory response in normal and defective mitophagy in order to elucidate the role of mitophagy defects in inflammation. We used cytoplasmic hybrids (cybrids) as cellular models, created using mitochondrial DNA from different atherosclerosis patients. Mitophagy was stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and assessed as the degree of colocalization of mitochondria with lysosomes using confocal microscopy. Western blotting methods were used for the determination of proteins involved in the exact mechanism of mitophagy. Experiments with stimulation of mitophagy show a high correlation between these two approaches (microscopy and blotting). The pro-inflammatory response of cybrids was stimulated with bacterial lipopolysaccharide (LPS). The extent of the inflammatory response was assessed by the secretion of cytokines CCL2, IL8, IL6, IL1β, and TNF measured by ELISA. Basal level of secretion of cytokines CCL2, IL8 and TNF was 1.5-2 times higher in cultures of cybrids with defective mitophagy compared to cells with normal mitophagy. This suggests a persistently elevated inflammatory response in cells with defective mitophagy, even in the absence of an inflammatory stimulus. Such cells in the tissue will constantly recruit other immune cells, which is characteristic of macrophages derived from monocytes circulating in the blood of patients with atherosclerosis. We observed significant differences in the degree and type of response to inflammatory activation in cybrids with defective mitophagy. These differences were not so much quantitative as they were dramatically qualitative. Compared with cells with normal mitophagy, in cells with defective mitophagy, the relative (to basal) secretion of IL8, IL6 and IL1b increased after the second LPS activation. This indicates a possible lack of tolerance to inflammatory activation in cells with defective mitophagy, since typically, re-activation reveals a smaller pro-inflammatory cytokine response, allowing the inflammatory process to resolve. In cells with normal mitophagy, exactly this normal (tolerant) inflammatory reaction was observed. Data on the involvement of mitophagy, including defective mitophagy, in disturbances of the inflammatory response in sepsis, viral infections, autoimmune diseases and other pathologies have previously been reported. In this work, we studied the role of defective mitophagy in non-infectious chronic inflammatory diseases using the example of atherosclerosis. We showed a dramatic disruption of the inflammatory response associated with defective mitophagy. Compared with cybrids with normal mitophagy, in cybrids with defective mitophagy, the secretion of all studied cytokines changed significantly both quantitatively and qualitatively. In particular, the secretion of 3 of 5 cytokines demonstrated an intolerant inflammatory response manifested by increased secretion after repeated inflammatory stimulation. Such an intolerant reaction likely indicates a significant disruption of the pro-inflammatory response of macrophages, which can contribute to the chronification of inflammation. Elucidating the mechanisms of chronification of inflammation is extremely important for the search for fundamentally new pharmacological targets and the development of drugs for the prevention and treatment of chronic inflammatory diseases, including atherosclerosis and diseases characteristic of inflammation. Such diseases account for up to 80% of morbidity and mortality.

MAIT cells are associated with responsiveness to neoadjuvant immunotherapy in COPD-associated NSCLC.

Patients with non-small cell lung cancer (NSCLC) and chronic obstructive pulmonary disease (COPD) experience worse clinical outcomes but respond better to immunotherapy than patients with NSCLC without COPD. Mucosal-associated invariant T (MAIT) cells, a versatile population of innate immune T lymphocytes, have a crucial function in the response to infection and tumors. This study investigated the distribution of MAIT cells in COPD-associated NSCLC and their involvement in the immune response. Flow cytometry, immunohistochemistry, and immunofluorescence were performed on tissue samples of patients with NSCLC, with or without COPD, treated with or without anti-programmed death 1 (PD1) immunotherapy. MAIT cells were stimulated with 5-OP-RU using a mouse subcutaneous tumor model. Tumors contained significantly more MAIT cells than paraneoplastic tissues, and CD8+ MAIT cells accounted for more than 90% of these cells. Patients with NSCLC and COPD had higher CD8+ MAIT cell counts than those with NSCLC without COPD. Additionally, patients with NSCLC and COPD displayed reduced expression of the activation marker, CD69, and functional markers, granzyme B (GZMB) and interferon γ (IFNγ), and higher expression of the immune exhaustion marker, PD1. Among patients who received immunotherapy, the proportion with a complete or partial response was higher in those with COPD than in those without COPD. In patients with NSCLC and COPD, the major pathologic response (MPR) group had higher MAIT levels than those in the no major pathologic response (NPR) group. In the mouse subcutaneous tumor model stimulation of MAIT cells using 5-OP-RU enhanced the antitumor effects of anti-PD1. In patients with NSCLC and COPD, response to immunotherapy is associated with accumulation of CD8+ MAIT cells showing immune exhaustion. These findings may contribute to innovative approaches for immunotherapy targeting CD8+ MAIT cells.

Mechanoregulation of MSC spheroid immunomodulation.

Mesenchymal stromal cells (MSCs) are widely used in cell-based therapies and tissue regeneration for their potent secretome, which promotes host cell recruitment and modulates inflammation. Compared to monodisperse cells, MSC spheroids exhibit improved viability and increased secretion of immunomodulatory cytokines. While mechanical stimulation of monodisperse cells can increase cytokine production, the influence of mechanical loading on MSC spheroids is unknown. Here, we evaluated the effect of controlled, uniaxial cyclic compression on the secretion of immunomodulatory cytokines by human MSC spheroids and tested the influence of load-induced gene expression on MSC mechanoresponsiveness. We exposed MSC spheroids, entrapped in alginate hydrogels, to three cyclic compressive regimes with varying stress (L) magnitudes (i.e., 5 and 10 kPa) and hold (H) durations (i.e., 30 and 250 s) L5H30, L10H30, and L10H250. We observed changes in cytokine and chemokine expression dependent on the loading regime, where higher stress regimes tended to result in more exaggerated changes. However, only MSC spheroids exposed to L10H30 induced human THP-1 macrophage polarization toward an M2 phenotype compared to static conditions. Static and L10H30 loading facilitated a strong, interlinked F-actin arrangement, while L5H30 and L10H250 disrupted the structure of actin filaments. This was further examined when the actin cytoskeleton was disrupted via Y-27632. We observed downregulation of YAP-related genes, and the levels of secreted inflammatory cytokines were globally decreased. These findings emphasize the essential role of mechanosignaling in mediating the immunomodulatory potential of MSC spheroids.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Golgi apparatus-targeting fluorescent probe for the imaging of superoxide anion (O2•−) in living cells during ferroptosis

Ferroptosis is an emerging iron-dependent oxidative cell death type, and recently has been demonstrated to show close relation with Golgi apparatus (GA). Exploring the fluctuation of superoxide anion (O2•−) level in GA during ferroptosis is of great significance to profoundly study the biological functions of GA in ferroptosis. Here, we present a GA-targeting probe (N-GA) to monitor cellular O2•− during ferroptosis. N-GA employed a triflate group and a tetradecanoic amide unit as the recognition site for O2•− and GA-targeting unit, respectively. After the response of N-GA to O2•−, the triflate unit of N-GA converted into hydroxyl group with strong electron-donating ability, generating bright green fluorescence under UV light. N-GA exhibited excellent sensitivity and selectivity towards O2•−. Fluorescence imaging results showed that N-GA could be applied as a GA-targeting probe to monitor cellular O2•−. The stimulation of cells with PMA and rotenone could result in the massive generation of endogenous O2•− in GA. Erastin-induced ferroptosis can markedly induce the increase of O2•− level in GA. Similar to Fer-1 and DFO, dihydrolipoic acid (DHLA) and rutin were demonstrated to inhibit the enormous production of O2•− in GA of the living cells during ferroptosis.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-β2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-β2 (TGF-β2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-β2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-β2. The treatment of RPE cells with TGF-β2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-β2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-β2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-β2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

Daturataturin A Ameliorates Psoriasis by Regulating PPAR Pathway.

Psoriasis is a kind of severe immune-mediated systemic skin disorder, becoming a worldwide public health concern. Daturataturin A (DTA), a withanolide compound, exerts excellent anti-inflammatory and anti-proliferative properties. The objective of this study is to elucidate the effect of DTA on psoriasis and its potential mechanism. We established psoriasis-like keratinocytes model by stimulating HaCaT cells with M5 cocktail cytokines including Interleukin (IL)-17A, IL-22, oncostatin M, IL-1α, and tumor necrosis factor-α (TNF-α), followed by intervention with DTA. The potential effects and mechanisms of DTA on psoriasis were evaluated in vitro. DTA was found to be able to inhibit hyperproliferation, promote apoptosis, decrease the release of pro-inflammatory cytokines, downregulate keratin expression, and improve lipid metabolism via regulating the peroxisome proliferator-activated receptor (PPAR) signaling pathway by M5 cocktail cytokines stimulation in HaCaT cells. DTA ameliorated lipid metabolism of psoriasis and exerted the potential anti-psoriasis effects by regulating PPAR pathway in vitro, suggesting that DTA may act as a new therapeutic agent for psoriasis.

Icariin Attenuates Human Renal Tubular Epithelial Cell Senescence by Targeting PAK2 via miR-23b-3p.

Renal tubular epithelial cells (RTECs) senescence is crucial in kidney diseases. Icariin is shown to have protective effects against renal fibrosis, acute kidney injury, and proteinuria. We aimed to explore the role of icariin in protecting RTECs from senescence and the underlying mechanism involved. An in vitro model of RTEC senescence was established by incubating HK-2 cells with urine exosomes from patients with diabetic kidney disease. Stimulated cells were treated with icariin at various doses to evaluate the compound's therapeutic effects. After RNA transfection, cell cycle arrest and senescence, flow cytometry, and SA-β-Gal staining were analyzed. At the same time, quantitative real-time PCR examined microRNA expression. Biochemical assays. Urine exosomes induced senescence and cell cycle arrest in the G1 stage in HK-2 cells, which were inhibited by icariin. Urine exosome stimulation up-regulated miR-23b-3p expression, which in turn suppressed PAK2 expression. Significantly, the induced and inhibited miR- 23b-3p expressions weakened and augmented the resistance of cells against urine exosome stimulation, respectively, while PAK2 overexpression provided additional protection. Icariin suppressed miR-23b-3p expression, and miR-23b-3p induction blocked the effects of icariin and promoted RTEC senescence. miR-23b-3p and PAK2 form a signaling axis that regulates RTEC senescence upon urine exosome stimulation. Icariin can increase the resistance of RTECs against senescence via miR-23b-3p/PAK2. Our findings shed light on the mechanism of the clinical effects of icariin on renal diseases, which can be exploited to develop effective drugs targeting RTEC senescence in the future.

A53 MOLECULAR MECHANISMS FOR A ROLE OF SHP-2 IN TLR4 SIGNALING IN INTESTINAL EPITHELIAL CELLS

Abstract Background Shp-2 gene polymorphisms are associated with greater susceptibility to ulcerative colitis. How Shp-2 contributes to disease susceptibility is however unclear. Intestinal epithelial cell (IEC)-specific deletion of Shp-2 results in severe colitis in mice (Shp-2IEC-KO). We found important alterations in microbiota composition, namely increased Enterobacteria of the Proteobacteria phylum and decreased Firmicutes levels, that preceded clinical signs of inflammation. Antibiotherapy or epithelial KO of Myd88 inhibits colitis in Shp-2IEC-KO mice. Notably, feces from Shp-2IEC-iKO mice display much more LPS-like bioactivity than do feces from controls (unpublished data). These observations support a role for TLR4/Myd88-bacterial recognition in initiation of colitis in Shp-2IEC-KO mice. Aims The aim of the present study was to identify the molecular mechanisms by which Shp-2 may regulate TLR4 signaling in IEC. Methods 1) The regulation of Shp-2 phosphorylation was analysed in IEC-6 cells stimulated or not with 10 ug/ml of LPS, a TLR4 ligand. 2) We mapped the Shp-2-dependent interactome in LPS-stimulated IEC-6 cells by an APEX2 proximity assay used in combination with mass spectrometry (MS) analysis. Cells stably expressing APEX2-tagged Shp-2 were generated. Addition of doxycycline results in expression of “physiological” levels of Shp-2;APEX2 construct. Proximity biotinylation labeling of Shp-2 by APEX2 was performed after 10 min stimulation with LPS and labeled candidates were analyzed by MS. Results 1) Upon stimulation of IEC-6 cells with LPS, Shp-2 protein is rapidly phosphorylated (Y542, Y580) and potentially activated. This increased phosphorylation is abolished by pretreatment with N-acetyl cystein, a commonly used antioxydant. 2) Using Prostar R package, the three replicates of MS data were filtered for the minimum quantification of 66,66% of each protein in at least one condition. They were normalized within conditions using VSN normalization, then the Partially Observed Values (POV) and the Missing in Entire Column (MEC) values were imputed respectively with SLSA and DetQuantile (Quantile 1%, Factor 0,2). A differential analysis between the LPS and non-treated conditions identified 334 proteins enriched with a FC of ± ≥2,5 with a p-value inferior then 0,05. Among the top Shp-2 interactors, we found Tak1 (ampersand:003E3,5X change) and Tab1 (ampersand:003E3,0X change), two proteins involved in NF-kB signaling. Decreased interaction with the TLR effectors Myd88 (ampersand:003C2,5X change) and Irak1 (ampersand:003C3,2X change) was also noted after LPS treatment. Conclusions These data suggest for the first time that Shp-2 may regulate NF-kB signaling downstream of TLR4 in IEC. Thus, SHP-2 may contribute to the maintenance of mucosal tolerance to microbiota in the colon. Funding Agencies CIHR

Stimulation of skeletal stem cells in the growth plate promotes linear bone growth.

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.

Efficacy of biologically-directed daylight therapy on sleep and circadian rhythm in Parkinson's disease: a randomised, double-blind, parallel-group, active-controlled, phase 2 clinical trial

New non-pharmacological treatments for improving non-motor symptoms in Parkinson's disease (PD) are urgently needed. Previous light therapies for modifying sleep behaviour lacked standardised protocols and were not personalised for an individual patient chronotype. We aimed to assess the efficacy of a biologically-directed light therapy in PD that targets retinal inputs to the circadian system on sleep, as well as other non-motor and motor functions. In this randomised, double-blind, parallel-group, active-controlled trial at the Queensland University of Technology, Australia, participants with mild to moderate PD were computer randomised (1:1) to receive one of two light therapies that had the same photometric luminance and visual appearance to allow blinding of investigators and participants to the intervention. One of these biologically-directed lights matched natural daylight (Day Mel), which is known to stimulate melanopsin cells. The light therapy of the other treatment arm of the study, specifically supplemented the stimulation of retinal melanopsin cells (Enhanced Mel), targeting deficits to the circadian system. Both lights were administered 30min per day over 4-weeks and personalised to an individual patient's chronotype, while monitoring environmental light exposure with actigraphy. Co-primary endpoints were a change from baseline in mean sleep macrostructure (polysomnography, PSG) and an endocrine biomarker of circadian phase (dim light melatonin secretion onset, DLMO) at weeks 4 and 6. Participants data were analysed using an intention to treat principle. All endpoints were evaluated by applying a mixed model analysis. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12621000077864. Between February 4, 2021 and August 8, 2022, 144 participants with PD were consecutively screened, 60 enrolled and randomly assigned to a light intervention. There was no significant difference in co-primary outcomes between randomised groups overall or at any individual timepoint during follow-up. The mean (95% CI) for PSG, N3% was 24.15 (19.82-28.48) for Day Mel (n=23) and 19.34 (15.20-23.47) for the Enhanced Mel group (n=25) in week 4 (p=0.12); and 21.13 (16.99-25.28) for Day Mel (n=26) and 18.48 (14.34-22.62) for the Enhanced Mel group (n=25) in week 6, (p=0.37). The mean (95% CI) DLMO (decimal time) was 19.82 (19.20-20.44) for Day Mel (n=22) and 19.44 (18.85-20.04) for the Enhanced Mel group (n=24) in week 4 (p=0.38); and 19.90 (19.27-20.53) for Day Mel (n=23) and 19.04 (18.44-19.64) for the Enhanced Mel group (n=25) in week 6 (p=0.05). However, both the controlled daylight (Day Mel) and the enhanced melanopsin (Enhanced Mel) interventions demonstrated significant improvement in primary PSG sleep macrostructure. The restorative deep sleep phase (PSG, N3) significantly improved at week 6 in both groups [model-based mean difference to baseline (95% CI):-3.87 (-6.91 to-0.83), p=0.04]. There was a phase-advance in DLMO in both groups which did not reach statistical significance between groups at any time-point. There were no safety concerns or severe adverse events related to the intervention. Both the controlled daylight and melanopsin booster light showed efficacy in improving measures of restorative deep sleep in people with mild to moderate PD. That there was no significant difference between the two intervention groups may be due to the early disease stage. The findings suggest that controlled indoor daylight that is personalised to the individuals' chronotype could be effective for improving sleep in early to moderate PD, and further studies evaluating controlled daylight interventions are now required utilising this standardised approach, including in advanced PD. The Michael J Fox Foundation for Parkinson's Research, Shake IT Up Australia, National Health and Medical Research Council, and Australian Research Council.

PRL-mediated STAT5B/ARRB2 pathway promotes the progression of prostate cancer through the activation of MAPK signaling

Previous study showed that higher expression of prolactin (PRL) was found in CRPC samples compared with hormone-naive prostate cancer (HNPC) and benign prostatic hyperplasia (BPH) samples. We further investigate the function of PRL in prostate cancer (PCa) and explored its downstream effects. We found heterogeneous expression of the PRLR in clinical prostate samples. The VCaP and 22Rv1 cells exhibited PRLR expression. Among the downstream proteins, STAT5B was the dominant subtype in clinical samples and cell lines. Human recombinant PRL stimulation of PCa cells with PRLR expression resulted in increased phosphorylation of STAT5B(pSTAT5B) and progression of PCa in vitro and in vivo, and STAT5B knockdown can suppress the malignant behavior of PCa. To understand the mechanism further, we performed Bioinformatic analysis, ChIP qPCR, and luciferase reporter gene assay. The results revealed that ARRB2 was the transcription target gene of STAT5B, and higher expression of ARRB2 was related to higher aggression and poorer prognosis of PCa. Additionally, Gene set enrichment analysis indicated that higher expression of ARRB2 was significantly enriched in the MAPK signaling pathway. Immunohistochemistry (IHC) demonstrated elevated pSTAT5B, ARRB2, and pERK1/2 expression levels in CRPC tissues compared to HNPC and BPH. Mechanically, ARRB2 enhanced the activation of the MAPK pathway by binding to ERK1/2, thereby promoting the phosphorylation of ERK1/2 (pERK1/2). In conclusion, our study demonstrated that PRL stimulation can promote the progression of PCa through STAT5B/ARRB2 pathway and activation of MAPK signaling, which can be suppressed by intervention targeting STAT5B. Blockade of the STAT5B can be a potential therapeutic target for PCa.

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

The role of extracellular vesicle fusion with target cells in triggering systemic inflammation

Extracellular vesicles (EVs) play a crucial role in intercellular communication by transferring bioactive molecules from donor to recipient cells. As a result, EV fusion leads to the modulation of cellular functions and has an impact on both physiological and pathological processes in the recipient cell. This study explores the impact of EV fusion on cellular responses to inflammatory signaling. Our findings reveal that fusion renders non-responsive cells susceptible to inflammatory signaling, as evidenced by increased NF-κB activation and the release of inflammatory mediators. Syntaxin-binding protein 1 is essential for the merge and activation of intracellular signaling. Subsequent analysis show that EVs transfer their functionally active receptors to target cells, making them prone to an otherwise unresponsive state. EVs in complex with their agonist, require no further stimulation of the target cells to trigger mobilization of NF-κB. While receptor antagonists were unable to inhibit NF-κB activation, blocking of the fusion between EVs and their target cells with heparin mitigated inflammation in mice challenged with EVs.

The Effect of Pulsed Electromagnetic Field Stimulation of Live Cells on Intracellular Ca2+ Dynamics Changes Notably Involving Ion Channels

The Effect of Pulsed Electromagnetic Field Stimulation of Live Cells on Intracellular Ca2+ Dynamics Changes Notably Involving Ion Channels

The Effect of Pulsed Electromagnetic Field Stimulation of Live Cells on Intracellular Ca2+ Dynamics Changes Notably Involving Ion Channels

The Effect of Pulsed Electromagnetic Field Stimulation of Live Cells on Intracellular Ca2+ Dynamics Changes Notably Involving Ion Channels

Magnetically Stimulated Integrin Binding Alters Cell Polarity and Affects Epithelial-Mesenchymal Plasticity in Metastatic Cancer Cells.

Inorganic nanoparticles (NPs) have been widely recognized for their stability and biocompatibility, leading to their widespread use in biomedical applications. Our study introduces a novel approach that harnesses inorganic magnetic nanoparticles (MNPs) to stimulate apical-basal polarity and induce epithelial traits in cancer cells, targeting the hybrid epithelial/mesenchymal (E/M) state often linked to metastasis. We employed mesocrystalline iron oxide MNPs to apply an external magnetic field, disrupting normal cell polarity and simulating an artificial cellular environment. These led to noticeable changes in the cell shape and function, signaling a shift toward the hybrid E/M state. Our research suggests that apical-basal stimulation in cells through MNPs can effectively modulate key cellular markers associated with both epithelial and mesenchymal states without compromising the structural properties typical of mesenchymal cells. These insights advance our understanding of how cells respond to physical cues and pave the way for novel cancer treatment strategies. We anticipate that further research and validation will be instrumental in exploring the full potential of these findings in clinical applications, ensuring their safety and efficacy.

Роль К+-провідного каналу TREK-1 у механочутливості гладеньком’язових клітин детрузора сечового міхура щура

Currently, TREK-1 is considered to be the main mechanosensitive channel in detrusor smooth muscle (DSM) cells. The aim of our study was to detect the functioning of the K+-conducting mechanosensitive TREK-1 channel in rat DSM cells using the patch-clamp technique in response to hydrodynamic stimulation (shear stress) and to determine the effects of a TREK-1 agonist – arachidonic acid (AA) and an antagonist – L-methionine. Mechanical stimulation of DSM cells using hydrodynamic stress led to the appearance of a membrane current with signs of pronounced outward rectification at positive membrane potentials, which is typical of TREK-1 activation. The application of AA (50 mcmol/l) activated a current with similar characteristics of the outward rectification to the shear stress-activated one. L-methionine (10 mcmol/l) almost completely prevented the generation of an outwardly rectifying current in response to shear stress stimulation. DSM cells also retained the ability to generate a mechanoactivated current with a more pronounced inward component when extracellular and intracellular K+ were replaced by Cs+. It was concluded that the dominant mechanoactivated current in rat DSM cells is carried by K+-selective TREK-1 channels, but a small portion of this current can also be carried by other nonselective mechanosensitive cation channels.