Sevoflurane enhances autophagy via Rac1 to attenuate lung ischaemia‒reperfusion injury.

Sevoflurane can attenuate lung ischaemia‒reperfusion injury (LIRI). However, the protective mechanism is unclear. In this study, we developed a LIRI model in vivo that animals (SD, n = 15) were subjected to the administration of 2.2 % sevoflurane 30 min before the onset of left pulmonary artery clamping for 45 min, which was then followed by 60 min of reperfusion treatment. Then, transcriptome sequencing was used to analyse lung tissues. Autophagy inhibition (3-MA) and Rac1-overexpression transfection plasmids were used in BEAS-2B cells, and BEAS-2B cells were subjected to hypoxia reoxygenation (H/R) and sevoflurane treatment. In both animal tissue and cells, inflammatory cytokines and apoptotic and autophagy molecules were measured by quantitative real-time PCR, western blotting and immunostaining. As a result, decreased arterial partial oxygen and damage to the histological structure of lung tissues were observed in LIRI model rats, and these effects were reversed by sevoflurane treatment. Activation of inflammation (elevated IL-1ß, IL-6, and TNF-α) and apoptosis (elevated cleaved caspase3/caspase3 and Bax, degraded expression of Bcl2) and inhibition of autophagy (elevated P62, degraded expression of Beclin1 and LC3-II/LC3I) in the model group were ameliorated by sevoflurane. Transcriptome sequencing indicated that the PI3K/Akt pathway regulated by Rac1 plays an important role in LIRI. Furthermore, overexpression of Rac1 in a cell line inhibited the protective effect of sevoflurane in LIRI. Autophagy inhibition (3-MA) also prevented the protective effect of sevoflurane on inflammation and apoptosis. As shown in the present study, sevoflurane enhances autophagy via Rac1/PI3K/AKT signalling to attenuate lung ischaemia‒reperfusion injury.

Dexmedetomidine promotes colorectal cancer progression via Piwil2 signaling.

PURPOSE: α2-adrenoceptor agonist dexmedetomidine (DEX) has been reported to promote tumorigenesis. Stem-cell protein Piwil2 is associated with cancer progression. Whether Piwil2 plays a role in tumor-promoting effects of DEX is unknown. METHODS: We examined the expression of Piwil2 in human colorectal cancer (CRC) cell lines with/without DEX treatment. We also studied the roles of Piwil2 in proliferation, invasion, migration, as well as expressions of epithelial-mesenchymal transition (EMT)-related proteins in DEX-treated in vitro and in vivo CRC models. And the experiments with genetic and pharmacological treatments were conducted to investigate the underlying molecular mechanism. RESULTS: RNA-sequencing (RNA-seq) analysis found Piwil2 is one of most upregulated genes upon DEX treatment in CRC cells. Furthermore, Piwil2 protein levels significantly increased in DEX-treated CRC cancer cells, which promoted proliferation, invasion, and migration in both CRC cell lines and human tumor xenografts model. Mechanistically, DEX increased nuclear factor E2-related factor 2 (Nrf2) expression, which enhanced Piwil2 transcription via binding to its promoter. Furthermore, in vitro experiments with Piwil2 knockdown or Siah2 inhibition indicated that DEX promoted EMT process and tumorigenesis through Siah2/PHD3/HIF1α pathway. The experiments with another α2-adrenoceptor agonist Brimonidine and antagonists yohimbine and atipamezole also suggested the role of Piwil2 signaling in tumor-promoting effects via an α2 adrenoceptor-dependent manner. CONCLUSION: DEX promotes CRC progression may via activating α2 adrenoceptor-dependent Nrf2/Piwil2/Siah2 pathway and thus EMT process. Our work provides a novel insight into the mechanism underlying tumor-promoting effects of α2-adrenoceptor agonists.

Parecoxib sodium attenuates acute lung injury following burns by regulating M1/M2 macrophage polarization through the TLR4/NF-κB pathway.

High temperature-induced burn injury often leads to an excessive inflammatory cascade resulting in multiple organ dysfunction syndrome, such as acute lung injury (ALI), in addition to skin tissue damage. As a specific COX2 inhibitor, parecoxib sodium suppresses the inflammatory response during burn injury. The effect of parecoxib sodium on ALI induced by burn injury and the associated molecular mechanism still need to be investigated. The role of parecoxib sodium in burn injury-induced ALI through the TLR4/NF-κB pathway was explored in the present study. A burn-induced ALI mouse model was constructed, and M1/M2 macrophages in lung tissue and markers involved in the TLR4/NF-κB signalling pathway were evaluated in bronchoalveolar lavage fluid (BALF) and MH-S mouse alveolar macrophages in vitro. The results indicated that parecoxib sodium attenuated lung injury after burn injury, decreased iNOS and TNF-α expression, increased IL-10 expression in BALF, and regulated the CD86-and CD206-mediated polarization of M1/M2 macrophages in lung tissue along with MH-S mouse alveolar macrophages. The effect of parecoxib sodium might be reversed by a TLR4 agonist. Overall, the results suggested that parecoxib sodium can regulate the polarization of M1/M2 macrophages through the TLR4/NF-κB pathway to attenuate ALI induced by skin burns.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Preoperative Acute Pain Is Associated with Postoperative Delirium.

BACKGROUND: Studies have provided some evidence that pain is a risk factor for postoperative delirium (POD). Therefore, we investigated the relationship between preoperative pain and POD after noncardiac surgery. METHODS: POD was assessed with the Montreal Cognitive Assessment, and preoperative cognition was assessed with the Mini-Mental State Examination. Plasma C-reactive protein (CRP) was detected by enzyme-linked immunosorbent assay before surgery. Preoperative pain was classified by its duration before surgery as chronic pain (lasting more than 1 month), acute pain (lasting less than 1 month), or no pain (no obvious pain). Multiple linear regression was used to adjust for confounding. RESULTS: From October 15, 2018, through August 12, 2019, a total of 67 patients were randomized; 7 were excluded because they were discharged before the seventh postoperative day. The prevalence of POD was significantly higher in the acute pain group (13 of 20; 65%) than in the chronic pain group (5 of 20; 25%) or the no pain group (6 of 20; 30%) (P = 0.019), indicating that delirium is associated with preoperative acute pain. The plasma level of preoperative CRP was also higher in the acute pain group than in the other two groups (mean [interquartile range]: 10.7 [3.3, 29.3] vs 1 [0.5, 3.8]mg/l; P < 0.001), suggesting that elevated preoperative plasma levels of CRP were associated with delirium. CONCLUSIONS: Preoperative acute pain was associated with POD, and increased plasma levels of CRP provide a marker. In addition, we found that illiteracy and advanced age were risk factors for POD.

Preoperative Chronic and Acute Pain Affects Postoperative Cognitive Function Mediated by Neurotransmitters.

The effective prevention of postoperative cognitive dysfunction (POCD) needs to be explored, and the effect of preoperative pain on POCD remains unclear. We established a chronic pain model induced by chronic constriction injury (CCI) and models of acute pain and anxiety without pain in mice that were subsequently subjected to partial hepatectomy surgery. Morris water maze (MWM) tests were performed to evaluate the learning and memory abilities of the mice. ELISA was used to measure IL-1ß, IL-6, and TNF-α in serum, and HPLC-MS was used to detect neurotransmitters in the prefrontal cortices and hippocampi of the mice. The results indicated that chronic pain induced by CCI might have significantly impaired the learning and memory abilities of mice, while acute pain and anxiety without pain only affected the memory abilities of mice. Perioperative acute pain increased the level of IL-1ß in serum, and CCI might have increased the level of IL-6. CCI and acute pain increased dopamine (DA) levels in the cortex, similar to anxiety. Like anxiety, CCI increased 5-hydroxytryptamine (5-HT) levels in the prefrontal cortex and hippocampus. Acute pain led to a decrease in the acetylcholine (ACH) level in the hippocampus. Our results suggest that acute pain and CCI-induced chronic pain might aggravate postoperative cognitive dysfunction via neurotransmitters and by changing the levels of inflammatory factors such as IL-1ß and IL-6.

Multiple Anesthesia/Surgery Cannot Impair Reference Memory in Adult Mice.

Postoperative cognitive dysfunction increases mortality and morbidity in perioperative patients. Numerous studies have demonstrated that multiple surgery/anesthesia during the neurodevelopmental period affects cognitive function, whereas a single anesthesia/surgery rarely causes cognitive dysfunction in adults. However, whether adults who undergo multiple anesthesia/surgery over a short period will experience cognitive dysfunction remains unclear. In this study, central nervous system inflammation and changes in cholinergic markers were investigated in adult mice subjected to multiple laparotomy procedures over a short period of time. The results showed that despite the increased expression of IL-6 and TNF-α in the hippocampus after multiple operations and the activation of microglia, multiple anesthesia/surgery did not cause a decline in cognitive function in adult mice. There were no changes in the cholinergic markers after multiple anesthesia/surgery.

Central cholinergic neuronal degeneration promotes the development of postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is consistently associated with increased morbidity and mortality. However, its mechanism remains poorly understood. We hypothesized that central cholinergic neuronal degeneration facilitates the development of POCD. The impact of anesthesia/surgery (appendectomy) on learning and memory and the levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and choline transporter (CHT) in adult and aged mice were measured. Separate cohorts were analyzed after pretreatment with donepezil, an AChE inhibitor, in aged mice or with murine-p75-saporin (mu-p75-sap), a cholinergic-specific immunotoxin, in adult mice. Morris Water Maze was used to measure the learning and memory changes after anesthesia/surgery. Western blot was used to measure the changes in the protein levels of the biomarkers of the central cholinergic system. We found that anesthesia/surgery-induced memory decline and attenuation of central cholinergic biomarkers (ChAT and VAChT) in aged mice but not in adult mice. Donepezil pretreatment reduced central cholinergic impairment in the aged mice and prevented learning and memory declines after anesthesia/surgery. In contrast, when central cholinergic neurons were pre-injured with mu-p75-sap, cognitive dysfunction developed in the adult mice after anesthesia/surgery. These data suggest that central cholinergic neuronal degeneration facilitates the development of POCD.

Central cholinergic system mediates working memory deficit induced by anesthesia/surgery in adult mice.

Background: Postoperative cognitive dysfunction (POCD) is consistently associated with increased morbidity and mortality, which has become a major concern of patients and caregivers. Although POCD occurs mainly in aged patients, it happens at any age. Previous studies demonstrated that anesthesia/surgery had no effects on reference memory of adult mice. However, whether it impairs working memory remains unclear. Working memory deficit would result in many deficits of executive function. We hypothesized that anesthesia/surgery impaired the working memory of adult mice and the central cholinergic system was involved. Method: Tibial fracture internal fixation under the anesthesia of isoflurane was performed in two-month-old C57BL/6 mice. Two days later, the spatial reference memory and working memory were measured by a Morris Water Maze (MWM). Donepezil, an inhibitor of acetylcholinesterase (AChE), was administered in another cohort mice for 4 weeks. Then, the working memory was measured by MWM 2 days after anesthesia/surgery. Western blot was used to detect the protein levels of acetylcholine transferase (ChAT), AChE, vesicular acetylcholine transporter (VAChT), and choline transporter (ChT) in the prefrontal cortex (PFC). Results: We found that anesthesia/surgery had no effects on the reference memory, but it impaired the working memory in adult mice. Meanwhile, we also found that the protein level of ChAT in PFC decreased significantly compared with that in control group. Donepezil pretreatment prevented working memory impairment and the decrease of the protein levels of ChAT induced by anesthesia/surgery. Conclusion: These results suggest that anesthesia/surgery leads to working memory deficits in adult mice and central cholinergic system impairment is involved.

A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors.

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Epitope characterization of an anti-PD-L1 antibody using orthogonal approaches.

The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques.

Evidence for Follicle-stimulating Hormone Receptor as a Functional Trimer.

Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor, is an important drug target in the development of novel therapeutics for reproductive indications. The FSHR extracellular domains were observed in the crystal structure as a trimer, which enabled us to propose a novel model for the receptor activation mechanism. The model predicts that FSHR binds Asnα(52)-deglycosylated FSH at a 3-fold higher capacity than fully glycosylated FSH. It also predicts that, upon dissociation of the FSHR trimer into monomers, the binding of glycosylated FSH, but not deglycosylated FSH, would increase 3-fold, and that the dissociated monomers would in turn enhance FSHR binding and signaling activities by 3-fold. This study presents evidence confirming these predictions and provides crystallographic and mutagenesis data supporting the proposed model. The model also provides a mechanistic explanation to the agonist and antagonist activities of thyroid-stimulating hormone receptor autoantibodies. We conclude that FSHR exists as a functional trimer.

Structural biology of glycoprotein hormones and their receptors: insights to signaling.

This article reviews the progress made in the field of glycoprotein hormones (GPH) and their receptors (GPHR) by several groups of structural biologists including ourselves aiming to gain insight into GPH signaling mechanisms. The GPH family consists of four members, with follicle-stimulating hormone (FSH) being the prototypic member. GPH members belong to the cystine-knot growth factor superfamily, and their receptors (GPHR), possessing unusually large N-terminal ectodomains, belong to the G-protein coupled receptor Family A. GPHR ectodomains can be divided into two subdomains: a high-affinity hormone binding subdomain primarily centered on the N-terminus, and a second subdomain that is located on the C-terminal region of the ectodomain that is involved in signal specificity. The two subdomains unexpectedly form an integral structure comprised of leucine-rich repeats (LRRs). Following the structure determination of hCG in 1994, the field of FSH structural biology has progressively advanced. Initially, the FSH structure was determined in partially glycosylated free form in 2001, followed by a structure of FSH bound to a truncated FSHR ectodomain in 2005, and the structure of FSH bound to the entire ectodomain in 2012. Comparisons of the structures in three forms led a proposal of a two-step monomeric receptor activation mechanism. First, binding of FSH to the FSHR high-affinity hormone-binding subdomain induces a conformational change in the hormone to form a binding pocket that is specific for a sulfated-tyrosine found as sTyr 335 in FSHR. Subsequently, the sTyr is drawn into the newly formed binding pocket, producing a lever effect on a helical pivot whereby the docking sTyr provides as the 'pull & lift' force. The pivot helix is flanked by rigid LRRs and locked by two disulfide bonds on both sides: the hormone-binding subdomain on one side and the last short loop before the first transmembrane helix on the other side. The lift of the sTyr loop frees the tethered extracellular loops of the 7TM domain, thereby releasing a putative inhibitory influence of the ectodomain, ultimately leading to the activating conformation of the 7TM domain. Moreover, the data lead us to propose that FSHR exists as a trimer and to present an FSHR activation mechanism consistent with the observed trimeric crystal form. A trimeric receptor provides resolution of the enigmatic, but important, biological roles played by GPH residues that are removed from the primary FSH-binding site, as well as several important GPCR phenomena, including negative cooperativity and asymmetric activation. Further reflection pursuant to this review process revealed additional novel structural characteristics such as the identification of a 'seat' sequence in GPH. Together with the 'seatbelt', the 'seat' enables a common heteodimeric mode of association of the common α subunit non-covalently and non-specifically with each of the three different ß subunits. Moreover, it was possible to establish a dimensional order that can be used to estimate LRR curvatures. A potential binding pocket for small molecular allosteric modulators in the FSHR 7TM domain has also been identified.

Synthesis, antitumor evaluation and docking study of novel 4-anilinoquinazoline derivatives as potential epidermal growth factor receptor (EGFR) inhibitors.

Strike a pose! A series of 4-anilinoquinazolines were designed, synthesized and evaluated in vitro against lung and breast cancer cell lines. Several compounds were found to be endowed with cytotoxicity in the low micromolar range. Molecular docking suggests that these compounds bind to EGFR in a similar manner to known EGFR inhibitors.

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors.

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.

Tumor necrosis factor (TNF)-soluble high-affinity receptor complex as a TNF antagonist.

A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the alpha and beta chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-alpha bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.

Structure-expression relationship of tumor necrosis factor receptor mutants that increase expression.

The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.