Distinct Dynamic and Conformational Features of Human STING in Response to 2′3′-cGAMP and c-di-GMP

The human stimulator of interferon genes protein (hSTING) can bind cyclic dinucleotides (CDNs) to activate the production of type I interferons and inflammatory cytokines. These CDNs can be either bacterial secondary messengers, 3′3′-CDNs, or endogenous 2′3′-cGAMP. cGAMP, with a unique 2′–5′ bond, is the most potent activator of hSTING among all CDNs. However, current understanding of the molecular principles underlying the unique ability of 2′3′-cGAMP to potently activate hSTINGs other than 3′3′-CDNs remains incomplete. In this work, molecular dynamics simulations were used to provide an atomistic picture of the binding of 2′3′-cGAMP and one 3′3′-CDN (c-di-GMP) to hSTING. The results suggest that hSTING binds more strongly to 2′3′-cGAMP than to c-di-GMP, which prefers to bind with a more open and flexible state of hSTING. Finally, a potential “dock–lock–anchor” mechanism is proposed for the activation of hSTING upon the binding of a potent ligand. It is believed that deep insights into understanding the binding of hSTING with 3′3′-CDNs and the endogenous 2′3′-cGAMP would help to establish the principles underlying powerful 2′3′-cGAMP signaling and the nature of hSTING activation, as well as related drug design.     

Bacterial Over-Expression and Purification of the 3′phosphoadenosine 5′phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ_1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ_1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ_1-328-hFADS enzyme shows a slight, but not significant, change of K_m values (0.24 and 6.23 μM for FMN and ATP, respectively) and of k_cat (4.2 × 10⁻² s⁻¹) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ_1-328-hFADS versus wild-type protein.     

Evolutionary Adaptation and Amyloid Formation: Does the Reduced Amyloidogenicity and Cytotoxicity of Ursine Amylin Contribute to the Metabolic Adaption of Bears and Polar Bears?

Much of our knowledge of diabetes is derived from studies of rodent models. An alternative approach explores evolutionary solutions to physiological stress by studying organisms that face challenging metabolic environments. Polar bears eat an enormously lipid-rich diet without deleterious metabolic consequences. In contrast, transgenic rodents expressing the human neuropancreatic polypeptide hormone amylin develop hyperglycemia and extensive pancreatic islet amyloid when fed a high-fat diet. The process of islet amyloid formation by human amylin contributes to β-cell dysfunction and loss of β-cell mass in type 2 diabetes. We show that ursine amylin is considerably less amyloidogenic and less toxic to β-cells than human amylin, consistent with the hypothesis that part of the adaptation of bears to metabolic challenges might include protection from islet amyloidosis-induced β-cell toxicity. Ursine and human amylin differ at four locations: H18R, S20G, F23L, and S29P. These are interesting from a biophysical perspective, since the S20G mutation accelerates amyloid formation, but the H18R slows it. An H18RS20G double mutant of human amylin behaves similarly to the H18R mutant, indicating that the substitution at position 18 dominates the S20G replacement. These data suggest one possible mechanism underpinning the protection of bears against metabolic challenges and provide insight into the design of soluble analogs of human amylin.     

Development and Validation of an HPLC–DAD Method for Analysis of the Six Major Isoflavones in Extracts from Soybean Processing

In this study, a new HPLC method was developed and validated for the determination of daidzin, genistin, glycitin, and their respective aglycones daidzein, genistein and glycitein in extracts from different stages of soy processing. The chromatographic separation was achieved using a Bonus RP 100 × 3.0 mm (3.5-μm particle size) column maintained at 35 °C. The mobile phase consisted of a gradient of water and acetonitrile both containing 0.1% formic acid. Analyses were conducted at a flow rate of 1.0 mL min⁻¹ and the detector was set at 254 nm. Validation parameters indicated that the method has high selectivity and is linear over a range of 1.5–150 μg mL⁻¹ for all compounds. The range of recovery was 95–102% with a RSD% <5% for intra-day and inter-day precision. The robustness study indicated that the flow rate was the only critical factor. The new HPLC–DAD method was successfully applied to real samples and excellent separation was achieved without the need of any sample pretreatment.     

Does Genetic Predisposition Contribute to the Exacerbation of COVID-19 Symptoms in Individuals with Comorbidities and Explain the Huge Mortality Disparity between the East and the West?

The elderly and patients with several comorbidities experience more severe cases of coronavirus disease 2019 (COVID-19) than healthy patients without underlying medical conditions. However, it is unclear why these people are prone to developing alveolar pneumonia, rapid exacerbations, and death. Therefore, we hypothesized that people with comorbidities may have a genetic predisposition that makes them more vulnerable to various factors; for example, they are likely to become more severely ill when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To test this hypothesis, we searched the literature extensively. Polymorphisms of genes, such as those that encode angiotensin-converting enzyme 1 (ACE1), have been associated with numerous comorbidities, such as cardiovascular disease, hypertension, diabetes, chronic kidney disease, and obesity, and there are potential mechanisms to explain these associations (e.g., DD-type carriers have greater ACE1 activity, and patients with a genetic alpha-1 anti-trypsin (AAT) deficiency lack control over inflammatory mediators). Since comorbidities are associated with chronic inflammation and are closely related to the renin–angiotensin–aldosterone system (RAAS), these individuals may already have a mild ACE1/ACE2 imbalance before viral infection, which increases their risk for developing severe cases of COVID-19. However, there is still much debate about the association between ACE1 D/I polymorphism and comorbidities. The best explanation for this discrepancy could be that the D allele and DD subtypes are associated with comorbidities, but the DD genotype alone does not have an exceptionally large effect. This is also expected since the ACE1 D/I polymorphism is only an intron marker. We also discuss how polymorphisms of AAT and other genes are involved in comorbidities and the severity of SARS-CoV-2 infection. Presumably, a combination of multiple genes and non-genetic factors is involved in the establishment of comorbidities and aggravation of COVID-19.     

Development and characterization of a “store and create” microfluidic device to determine the heterogeneous freezing properties of ice nucleating particles

Abstract

Understanding heterogeneous ice nucleation induced by ice nucleating particles (INPs) is hindered by analytical challenges in accurately determining the freezing temperature spectrum, abundance, and physicochemical properties of INPs. Here we evaluate the performance of a microfluidic device that employs a “store and create” approach to measure the ice nucleation properties of approximately 600 uniformly sized nanoliter water droplets. These droplets are immersed in surfactant-free environmentally sustainable squalene oil and do not contact the polymer walls of the microfluidic device. The device interfaced with a cold plate temperature controller has a greatly reduced background freezing temperature spectrum for filtered water droplets compared to conventional microliter droplet-on-substrate freezing methods. Droplets containing particles of interest are readily generated on-chip from a suspension of particles in water. Background freezing for 6 nL water droplets exhibits a median freezing temperature of ?33.7?±?0.4?°C, close to the theoretical freezing temperature of ?34.5?°C. The immersion freezing temperature spectra obtained from Snomax bacterial and illite mineral particles compares well with literature data, and the freezing contribution from either type of particle can be separated from a mixed suspension. Our approach generates a highly uniform droplet size distribution, causes no clogging of the microfluidic device, and is capable of reproducible droplet refreezes. The high-resolution freezing spectra obtained from large droplet number arrays enables the use of the derivative INP temperature spectrum analysis to quantitatively distinguish between different classes of INPs. The lower and consistent filtered water background freezing temperature enables measurements of almost the entire immersion freezing temperature regime from ?33 to 0?°C, and quantification of weaker but often abundant INPs such as those found in biomass-burning smoke aerosol.

Copyright © 2019 American Association for Aerosol Research     

Development and Continuous Operation of a Bench-Scale System for the Production of O3 + O2 + CO2 Hydrates

Ozone (O₃) has many industrial applications such as in sterilization. One of the long-term O₃ preservation methods is molecular storage in clathrate hydrate. In this study, an experimental system was developed for continuously forming O₃ + O₂ + CO₂ hydrates. The parameters that affect the continuous operation of the system and that lead to increases in the concentration of O₃ in the hydrates were also experimentally evaluated, implementing the method of quality engineering. After optimizing these operating parameters, the O₃ storage capacity in the hydrates was measured to be 0.26 wt % at 2 h of total operation time. By X-ray diffraction, it was found that the produced sample contained hydrates, and long-term preservation for 6 months was possible at the temperature of general freezing warehouses.     

Development of Self—flowing Al2O3—SiC—C Castable and Its Application to Troughs of Large Blast Furnaces

The developed self-flowing Al2O3-SiC-C castable for troughs of blast furnace(BF) can self-flow under its own gravity,leveling,degassing,compacting and setting without vibration by aplication of external energy,The self-flwoing castable with high strength,improved thermal shock resistance and good resistance to molten slag and hot metal,has been used to troughs of large blast furnace,resulting in molten iron throughput of more than 100000 tons.     

An Efficient and Green Synthesis of 1′Hspiro[isoindoline-1,2′-quinazoline]-3,4′(3′H)-dione Derivatives in the Presence of Nano Fe3O4–GO–SO3H

ABSTRACT

In this article a simple, rapid, green and efficient method for the synthesis of various 1′Hspiro[isoindoline-1,2′-quinazoline]-3,4′(3′H)-diones in the presence of acidic magnetic graphene oxide nanosheets (Fe₃O₄–GO–SO₃H) as catalyst is reported. Different substituted spirooxindoles have been synthesized from one-pot three component reaction of isatoic anhydride, aliphatic or aromatic primary amines and isatin in the presence of Fe₃O₄–GO–SO₃H. This reaction allowed us to prepare some new and known derivatives of substituted spirooxindoles. The worthwhile advantages of this method are high product yields, short reaction times, the possibility of using of a magnetic, reusable and non-toxic catalyst and solvent-free conditions.     

BMP3 Alone and Together with TGF-β Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-β on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-β, and compared with cultures in basal and TGF-β media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-β on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-β, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-β, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-β induced positive effects. These actions are likely related to the activation of TGF-β signaling pathway.     

Studies Related to the Processing of U–Zr and U–Pu–Zr Metallic Fuels Using Tri-iso-amyl Phosphate (TiAP) as Extractant

Metallic fuel is reprocessed by a compact nonaqueous pyrochemical process. The present study explores the possible uses of an aqueous reprocessing-based method as an alternative to the pyro-processing of metallic fuels. Solvent extraction studies in the batch mode were carried out for both U–Zr- and U–Pu–Zr-based metal alloy systems. Earlier studies carried out in our laboratory have established that Tri-iso-amyl Phosphate (TiAP) is a promising extractant for the reprocessing of spent fuels. In the present study, the distribution data has been generated for the extraction of uranium and zirconium as a function of equilibrium aqueous phase metal ion and nitric acid concentration with TiAP and Tri-n-butyl Phosphate (TBP)-based solvents. These studies indicate the formation of a third phase with zirconium in the presence of uranium with TBP under certain experimental conditions whereas it was not encountered with the TiAP system. Flow sheet for the co-extraction and co-stripping of heavy metal ions by 1.1M TiAP and 1.1M TBP in n-dodecane from U–Zr as well as U–Pu–Zr feed solutions in stage-wise mode has been evaluated. Percentage extraction and stripping of metal ions were calculated stage-wise and the results are discussed.     

Reconstruction of Grenfell Tower fire. Part 3—Numerical simulation of the Grenfell Tower disaster: Contribution to the understanding of the fire propagation and behaviour during the vertical fire spread

The dramatic event of the Grenfell Tower (June 2017), involving a combustible façade system, has raised concerns regarding the fire risk that these systems address. Indeed, as façades are complex systems, it is not straightforward to assess which part of the system is involved in the global fire behaviour. Understanding such façade fires is thus very complex as it depends on a combination of various products and system characteristics, including window frames or air gap or cavity barriers. Fire development inside the initial apartment was investigated using an appropriate CFD model with different scenarios for the fire source and ventilation conditions in a previous study. Fire propagation through the window to the external façade and to higher apartments was modelled and validated against visual observations. This paper describes CFD modelling of the complete Grenfell tower facade, and investigates vertical fire spread behaviour over the full height façade from the initial apartment. Contributions from the combustion of all the apartments' furniture, depending on window failure, and architectural details of the refurbished façade are considered in the numerical model. The modelling results are validated by comparison with photographic and video observations of the real fire.     

Microstructures and Corrosion Resistance of Al2O3—C Based Refractories to the Melts Containing Titania

The corrosion resisance of the Al2O3-C based refrac-tories in melts containing titania has been studied by quasi-station immersion and rotary immersionThe corrosion rate is decreased with the addition of graphite carbon and ZrO2 in the refractories.The corrosion mehanism of Al2O3-C refractories is the oxidization of graphite carbon by the oxides of the melts and the formation of deteriorate layer,For the Al2O3-C-ZrO2 refractories,the corrosion behavior is due to the interaction between melts and refrac-tories,The new compounds of FeO.SiO2,SiZrO4,Feo.3CaO,2CaO.SiO2 and CaO.SiO2 are formed in the deteriorate layer.     

Cerebral Hyperperfusion after Revascularization Inhibits Development of Cerebral Ischemic Lesions Due to Artery-to-Artery Emboli during Carotid Exposure in Endarterectomy for Patients with Preoperative Cerebral Hemodynamic Insufficiency: Revisiting the “Impaired Clearance of Emboli” Concept

The purpose of the present study was to determine whether cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during exposure of the carotid arteries in carotid endarterectomy (CEA). In patients undergoing CEA for internal carotid artery stenosis (≥70%), cerebral blood flow (CBF) was measured using single-photon emission computed tomography (SPECT) before and immediately after CEA. Microembolic signals (MES) were identified using transcranial Doppler during carotid exposure. Diffusion-weighted magnetic resonance imaging (DWI) was performed within 24 h after surgery. Of 32 patients with a combination of reduced cerebrovascular reactivity to acetazolamide on preoperative brain perfusion SPECT and MES during carotid exposure, 14 (44%) showed cerebral hyperperfusion (defined as postoperative CBF increase ≥100% compared with preoperative values), and 16 (50%) developed DWI-characterized postoperative cerebral ischemic lesions. Postoperative cerebral hyperperfusion was significantly associated with the absence of DWI-characterized postoperative cerebral ischemic lesions (95% confidence interval, 0.001–0.179; p = 0.0009). These data suggest that cerebral hyperperfusion after revascularization inhibits development of cerebral ischemic lesions due to artery-to-artery emboli during carotid exposure in CEA, supporting the “impaired clearance of emboli” concept. Blood pressure elevation following carotid declamping would be effective when embolism not accompanied by cerebral hyperperfusion occurs during CEA.     

Combined 3D-QSAR, Molecular Docking and Molecular Dynamics Study on Derivatives of Peptide Epoxyketone and Tyropeptin-Boronic Acid as Inhibitors Against the β5 Subunit of Human 20S Proteasome

An abnormal ubiquitin-proteasome is found in many human diseases, especially in cancer, and has received extensive attention as a promising therapeutic target in recent years. In this work, several in silico models have been built with two classes of proteasome inhibitors (PIs) by using 3D-QSAR, homology modeling, molecular docking and molecular dynamics (MD) simulations. The study resulted in two types of satisfactory 3D-QSAR models, i.e., the CoMFA model (Q(2) = 0.462, R(2) (pred) = 0.820) for epoxyketone inhibitors (EPK) and the CoMSIA model (Q(2) = 0.622, R(2) (pred) = 0.821) for tyropeptin-boronic acid derivatives (TBA). From the contour maps, some key structural factors responsible for the activity of these two series of PIs are revealed. For EPK inhibitors, the N-cap part should have higher electropositivity; a large substituent such as a benzene ring is favored at the C6-position. In terms of TBA inhibitors, hydrophobic substituents with a larger size anisole group are preferential at the C8-position; higher electropositive substituents like a naphthalene group at the C3-position can enhance the activity of the drug by providing hydrogen bond interaction with the protein target. Molecular docking disclosed that residues Thr60, Thr80, Gly106 and Ser189 play a pivotal role in maintaining the drug-target interactions, which are consistent with the contour maps. MD simulations further indicated that the binding modes of each conformation derived from docking is stable and in accord with the corresponding structure extracted from MD simulation overall. These results can offer useful theoretical references for designing more potent PIs.     

Formation of O‘—Sialon Matrix and Microstructure Development in the Refractory of Si3N4—SiO2—ZrO2—Al2O3 System

An O‘-Sialon-Al2O3 composite refractory was prepared by in situ reaction of Si3N4-ZrSiO4-Al2O3 mixture with fused alumina aggregates.Densification,in situreaction procedure and microstructure of the material were investigated by means of X-ray diffraction,optical and scanning electronic microscopes and EDAX.     

Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine

Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).     

Unraveling the cooperative effects of acid sites and kinetics for pyrolysis of CHF3 to C2F4 and C3F6 on SO42−/ZrO2-SiO2

The treatment or upgrading of waste trifluoromethane (CHF₃, R23), which has a significant greenhouse effect, is of great importance in industry. Herein, series of SO₄²⁻/ZrO₂-SiO₂ catalysts with different Brønsted and Lewis acid site densities and ratios were prepared for pyrolysis of R23 to tetrafluoroethylene (C₂F₄, TFE) and hexafluoropropylene (C₃F₆, HFP). The effects of impregnation concentration of (NH₄)₂SO₄ on specific surface area, crystal phase, and Brønsted and Lewis acid site densities and ratios were respectively demonstrated. The Brønsted and Lewis acid sites were observed to have cooperative effects on R23 transformation and up to 94.6% selectivity of (TFE + HFP) could be achieved at 750°C. The kinetic studies revealed the decomposition of R23 into CF₂ carbene and HF was the rate-determining step, and a deactivation behavior was found due to the site coverage and pore blockage by the oligomers of TFE and HFP.     

The system Clinker–MgO–CaZrO3 and its application to the corrosion behavior of CaZrO3/MgO refractory matrix by clinker

Phase relations in the clinker-rich region and the limits between the different stability fields of the isothermal section Clinker (CK)–MgO–CaZrO₃ at 1500 °C were experimentally established. The corrosion behavior by clinker of Portland cement of a CaZrO₃-rich/MgO refractory matrix obtained from both natural and synthetic raw materials were also studied. The attack mechanism to CaZrO₃-rich substrates was discussed in terms of microstructural features of the refractory matrix and the information supplied by the isothermal section Clinker–MgO–CaZrO₃ at 1500 °C.