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Abstract Background Bed‐rest (BR) of only a few days duration reduces muscle protein synthesis and induces skeletal muscle atrophy and insulin resistance, but the scale and juxtaposition of these events have not been investigated concurrently in the same individuals. Moreover, the impact of short‐term exercise‐supplemented remobilization (ESR) on muscle volume, protein turnover and leg glucose uptake (LGU) in humans is unknown. Methods Ten healthy males (24 ± 1 years, body mass index 22.7 ± 0.6 kg/m2) underwent 3 days of BR, followed immediately by 3 days of ESR consisting of 5 × 30 maximal voluntary single‐leg isokinetic knee extensions at 90°/s each day. An isoenergetic diet was maintained throughout the study (30% fat, 15% protein and 55% carbohydrate). Resting LGU was calculated from arterialized‐venous versus venous difference across the leg and leg blood flow during the steady‐state of a 3‐h hyperinsulinaemic–euglycaemic clamp (60 mU/m2/min) measured before BR, after BR and after remobilization. Glycogen content was measured in vastus lateralis muscle biopsy samples obtained before and after each clamp. Leg muscle volume (LMV) was measured using magnetic resonance imaging before BR, after BR and after remobilization. Cumulative myofibrillar protein fractional synthetic rate (FSR) and whole‐body muscle protein breakdown (MPB) were measured over the course of BR and remobilization using deuterium oxide and 3‐methylhistidine stable isotope tracers that were administered orally. Results Compared with before BR, there was a 45% decline in insulin‐stimulated LGU (P

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Abstract Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.

S6 Kinase 2 (S6K2) is a key regulator of cellular signaling and is crucial for cell growth, proliferation, and survival. This review is divided into two parts: the first focuses on the complex network of upstream effectors, downstream modulators, and post-translational modifications (PTMs) that regulate S6K2 activity. We emphasize the dynamic nature of S6K2 regulation, highlighting its critical role in cellular homeostasis and its potential as a therapeutic target in diseases like cancer. The second part utilizes in silico analyses, employing computational tools to model S6K2's three-dimensional structure and predict its interaction networks. Molecular dynamics simulations and docking studies reveal potential binding sites and interactions with novel known inhibitors. We also examine the effects of environmental contaminants that potentially disrupt S6K2 function and provide insights into the role of external factors that could impact its regulatory mechanisms. These computational findings provide a deeper understanding of the conformational dynamics of S6K2 and its interactions with its inhibitors. Together, this integrated biochemical and computational approach enhances our understanding of S6K2 regulation and identifies potential new therapeutic strategies targeting S6K2 in the oncology setting. [ABSTRACT FROM AUTHOR]

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors. [ABSTRACT FROM AUTHOR]

Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy. Recently, T2A has been demonstrated to function through sestrin 2 (SESN) to inhibit mTORC1 activity, but its possible impact on autophagy through this pathway has not been investigated. Here, the model system Dictyostelium discoideum and GBM cell lines were employed to investigate the cellular role of T2A in regulating SESN to inhibit mTORC1 and activate autophagy through a GATOR2 component MIOS. In D. discoideum, T2A treatment induced autophagy and inhibited mTORC1 activity, with both effects lost upon the ablation of SESN (sesn-) or MIOS (mios-). We further investigated the targeting of MIOS to reproduce this effect of T2A, where computational analysis identified 25 novel compounds predicted to strongly bind the human MIOS protein, with one compound (MIOS inhibitor 3; Mi3) reducing cell proliferation in two GBM cells. Furthermore, Mi3 specificity was demonstrated through the loss of potency in the D. discoideum mios- cells regarding cell proliferation and the induction of autophagy. In GBM cells, Mi3 treatment also reduced mTORC1 activity and induced autophagy. Thus, a potential T2A mimetic showing the inhibition of mTORC1 and induction of autophagy in GBM cells was identified. [ABSTRACT FROM AUTHOR]

Choriocarcinoma (CC) is a malignant trophoblastic tumor that occurs in women of childbearing age. Its cure rate could be over 90% following single drug or multidrug chemotherapy with methotrexate, 5-fluorouracil and etc. Chemoresistance is the main cause of treatment failure in CC, and the mechanisms have yet to be elucidated. Most of the previous works on the chemoresistance of CC focused on the transcript and protein levels, and much less attention was put on the metabolic level. In this study, the metabolomic phenotypes of two lines of CC cell, methotrexate-resistant cell (JEG3R) and methotrexate-sensitive cell (JEG3), as well as the medium extracts, were investigated using an NMR-based metabonomics method. The results showed that the chemoresistance of CC was likely related to disturbed glucose metabolism. Glucitol accumulation was observed only in the JEG3R cell line, suggesting the involvement of aldose reductase in the development of chemoresistance. In addition, methotrexate chemoresistance was also associated with lactate accumulation and stimulated alanine synthesis to fuel the Krebs cycle. The levels of amino acids, nucleotides and phosphocholine in the resistant line showed significant differences from the non-resistant line, and positive correlations with the proliferation rate of CC. This study provided insights into the chemoresistance of CC from the metabolism aspect.

# Background After the introduction of the maternal near-miss (MNM) criteria by the World Health Organization (WHO), an adapted version for low-income countries was defined but has never been validated in a rural hospital in this setting. Aim of this study was to identify the occurrence of MNM by both the use of the WHO and the adapted sub-Saharan Africa (SSA) MNM tool and to compare the applicability of both versions. # Methods This cross-sectional study was done between November 2019 and July 2020 in Ndala Hospital, Tanzania. All pregnant women and women within 42 days after giving birth or termination of pregnancy were included when fulfilling at least one criterion according to either the WHO or the SSA MNM tool. # Results The SSA MNM criteria identified 47 near-miss cases and all seven maternal deaths. The WHO criteria identified ten near-miss cases and five maternal deaths. There were 948 livebirths, consequently leading to maternal near-miss ratio (MNMR) of 50 (95% CI 34-60) and 11 (95% CI 4 – 16) per 1,000 livebirths for the SSA criteria and respectively the WHO MNM criteria. The difference in these numbers seems to be primarily attributed to the addition of defined severe complications in the clinical criteria and the adapted threshold for blood transfusions. Eclampsia and severe malaria form roughly half of these complications. # Conclusions SSA MNM criteria are more suitable than the WHO criteria to identify patients with potentially life-threatening conditions (PLTC) in this rural hospital. The addition of clinical criteria and rejection of several unapplicable laboratory and management-based parameters in the SSA MNM tool seem appropriate adaptations for low-resource settings. However, some non-acute cases are discussable near-misses and the criterion “severe pre-eclampsia with ICU admission” seems not feasible in similar settings. Implementation of the SSA MNM criteria forms a strong basis for auditing. Better monitoring and documentation of patients will improve the use of the criteria and potentially the quality of audit sessions.

Abstract Enhancing endogenous cannabinoid (eCB) signaling has been considered as a potential strategy for the treatment of stress-related conditions. Fatty acid amide hydrolase (FAAH) represents the primary degradation enzyme of the eCB anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). This study describes a potent reversible FAAH inhibitor, SSR411298. The drug acts as a selective inhibitor of FAAH, which potently increases hippocampal levels of AEA, OEA and PEA in mice. Despite elevating eCB levels, SSR411298 did not mimic the interoceptive state or produce the behavioral side-effects (memory deficit and motor impairment) evoked by direct-acting cannabinoids. When SSR411298 was tested in models of anxiety, it only exerted clear anxiolytic-like effects under highly aversive conditions following exposure to a traumatic event, such as in the mouse defense test battery and social defeat procedure. Results from experiments in models of depression showed that SSR411298 produced robust antidepressant-like activity in the rat forced-swimming test and in the mouse chronic mild stress model, restoring notably the development of inadequate coping responses to chronic stress. This preclinical profile positions SSR411298 as a promising drug candidate to treat diseases such as post-traumatic stress disorder, which involves the development of maladaptive behaviors.

Cancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.