Src-Dependent NM2A Tyrosine Phosphorylation Regulates Actomyosin Remodeling.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr158) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr158 defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr158 as a novel layer of regulation of actomyosin cytoskeleton organization.

The Yin and Yang of Pneumolysin During Pneumococcal Infection.

Pneumolysin (PLY) is a pore-forming toxin produced by the human pathobiont Streptococcus pneumoniae, the major cause of pneumonia worldwide. PLY, a key pneumococcal virulence factor, can form transmembrane pores in host cells, disrupting plasma membrane integrity and deregulating cellular homeostasis. At lytic concentrations, PLY causes cell death. At sub-lytic concentrations, PLY triggers host cell survival pathways that cooperate to reseal the damaged plasma membrane and restore cell homeostasis. While PLY is generally considered a pivotal factor promoting S. pneumoniae colonization and survival, it is also a powerful trigger of the innate and adaptive host immune response against bacterial infection. The dichotomy of PLY as both a key bacterial virulence factor and a trigger for host immune modulation allows the toxin to display both "Yin" and "Yang" properties during infection, promoting disease by membrane perforation and activating inflammatory pathways, while also mitigating damage by triggering host cell repair and initiating anti-inflammatory responses. Due to its cytolytic activity and diverse immunomodulatory properties, PLY is integral to every stage of S. pneumoniae pathogenesis and may tip the balance towards either the pathogen or the host depending on the context of infection.

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Lenstar LS 900 versus Pentacam-AXL: analysis of refractive outcomes and predicted refraction.

Analysis of refractive outcomes, using biometry data collected with a new biometer (Pentacam-AXL, OCULUS, Germany) and a reference biometer (Lenstar LS 900, HAAG-STREIT AG, Switzerland), in order to assess differences in the predicted and actual refraction using different formulas. Prospective, institutional study, in which intraocular lens (IOL) calculation was performed using the Haigis, SRK/T and Hoffer Q formulas with the two systems in patients undergoing cataract surgery between November 2016 and August 2017. Four to 6 weeks after surgery, the spherical equivalent (SE) was derived from objective refraction. Mean prediction error (PE), mean absolute error (MAE) and the median absolute error (MedAE) were calculated. The percentage of eyes within ± 0.25, ± 0.50, ± 1.00, and ± 2.00 D of MAE was determined. 104 eyes from 76 patients, 35 males (46.1%), underwent uneventful phacoemulsification with IOL implantation. Mean SE after surgery was - 0.29 ± 0.46 D. Mean prediction error (PE) using the SRK/T, Haigis and Hoffer Q formulas with the Lenstar was significantly different (p > 0.0001) from PE calculated with the Pentacam in all three formulas. Percentage of eyes within ± 0.25 D MAE were larger with the Lenstar device, using all three formulas. The difference between the actual refractive error and the predicted refractive error is consistently lower when using Lenstar. The Pentacam-AXL user should be alert to the critical necessity of constant optimization in order to obtain optimal refractive results.

Achillea millefolium L. hydroethanolic extract inhibits growth of human tumor cell lines by interfering with cell cycle and inducing apoptosis.

The cell growth inhibitory activity of the hydroethanolic extract of Achillea millefolium was studied in human tumor cell lines (NCI-H460 and HCT-15) and its mechanism of action was investigated. The GI50 concentration was determined with the sulforhodamine B assay and cell cycle and apoptosis were analyzed by flow cytometry following incubation with PI or Annexin V FITC/PI, respectively. The expression levels of proteins involved in cell cycle and apoptosis were analyzed by Western blot. The extracts were characterized regarding their phenolic composition by LC-DAD-ESI/MS. 3,5-O-Dicaffeoylquinic acid, followed by 5-O-caffeoylquinic acid, were the main phenolic acids, while, luteolin-O-acetylhexoside and apigenin-O-acetylhexoside were the main flavonoids. This extract decreased the growth of the tested cell lines, being more potent in HCT-15 and then in NCI-H460 cells. Two different concentrations of the extract (75 and 100 µg/mL) caused alterations in cell cycle profile and increased apoptosis levels in HCT-15 and NCI-H460 cells. Moreover, the extract caused an increase in p53 and p21 expression in NCI-H460 cells (which have wt p53), and reduced XIAP levels in HCT-15 cells (with mutant p53). This work enhances the importance of A. millefolium as source of bioactive phenolic compounds, particularly of XIAP inhibitors.

An Aqueous Extract of Tuberaria lignosa Inhibits Cell Growth, Alters the Cell Cycle Profile, and Induces Apoptosis of NCI-H460 Tumor Cells.

Tuberaria lignosa (Sweet) Samp. is found in European regions, and has antioxidant properties due to its composition in ascorbic acid and phenolic compounds. Given its traditional use and antioxidant properties, the tumor cell growth inhibitory potential of aqueous extracts from T. lignosa (prepared by infusion and decoction) was investigated in three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and HCT-15 (human colorectal adenocarcinoma). Both extracts inhibited the growth of these cell lines; the most potent one being the T. lignosa extract obtained by infusion in the NCI-H460 cells (GI50 of approximately 50 µg/mL). Further assays were carried out with this extract in NCI-H460 cells. At 100 µg/mL or 150 µg/mL it caused an increase in the percentage of cells in the G0/G1 phase and a decrease of cells in S phase of the cell cycle. Additionally, these concentrations caused an increase in the percentage of apoptotic cells. In agreement, a decrease in total poly (ADP-ribose) polymerase (PARP) and pro-caspase 3 levels was found. In conclusion, the T. lignosa extract obtained by infusion was more potent in NCI-H460 cells, altering the cell cycle progression and inducing apoptosis. This work highlights the importance of T. lignosa as a source of bioactive compounds with tumor cell growth inhibitory potential.