Serum exosomal microRNA-146a as a novel diagnostic biomarker for acute coronary syndrome.

BACKGROUND: Circulating microRNAs (miRNAs) have emerged as potential biomarkers for cardiovascular diseases. However, few studies have focused on the role of exosomal miRNAs in acute coronary syndrome (ACS). The purpose of this study was to explore weather serum exosomal microRNA-146a (exo-miR-146a) could be used as a novel diagnostic biomarker for ACS and to investigate its relationship with inflammatory response. METHODS: A total of 63 ACS patients and 25 patients with normal coronary arteries (Control) were enrolled respectively. The serum exosomes were isolated and then identified by transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). The expression levels of exo-miR-146a in serum were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and the expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in serum were assessed by enzyme-linked immunosorbent assay (ELISA). Spearman's correlation analysis was used to appraise the potential factors related to serum exo-miR-146a and receiver operating characteristic (ROC) curve analysis was applied for predicting the accuracy of ACS via the area under curve (AUC). RESULTS: Exosomes isolated from serum were of typical cup-like shape, with 50-150 nm diameter, and expressed CD9, CD63, CD81, and HSP70. The expression levels of serum exo-miR-146a, IL-1ß, IL-6, and TNF-α were significantly increased in ACS patients compared with the control group, Spearman's correlation analysis indicated that exo-miR-146a expression was markedly positively correlated with IL-1ß, IL-6, and TNF-α. The ROC curve analyses revealed that exo-miR-146a could distinguish ACS patients from their normal controls. CONCLUSIONS: The serum exo-miR-146a may be used as a novel diagnostic biomarker for ACS patients, and it is also associated with inflammatory response.

Anti-HIV drug repurposing against SARS-CoV-2.

A novel severe acute respiratory syndrome human coronavirus (SARS HCoV) was identified from respiratory illness patients (named SARS-CoV-2 by ICTV) in December 2019 and has recently emerged as a serious threat to world public health. However, no approved drugs have been found to effectively inhibit the virus. Since it has been reported that HIV protease inhibitors can be used as anti-SARS drugs by targeting SARS-CoV-1 3CLpro, we chose six approved anti-HIV drugs and investigated their binding interactions with 3CLpro to evaluate their potential to become clinical drugs for the new coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection. The molecular docking results indicate that the 3CLpro of SARS-CoV-2 has a higher binding affinity for all the studied inhibitors than does SARS-CoV-1. Two docking complexes (indinavir and darunavir) with high docking scores were further subjected to MM-PBSA binding free energy calculations to detail the molecular interactions between these two protease inhibitors and SARS HCoV 3CLpro. Our results show that, among the inhibitors tested, darunavir has the highest binding affinity with SARS-CoV-2 and SARS-CoV-1 3CLpro, indicating that it may have the potential to be used as an anti-COVID-19 clinical drug. The mechanism behind the increased binding affinity of HIV protease inhibitors toward SARS-CoV-2 3CLpro (as compared to SARS-CoV-1) was investigated by MD simulations. Our study provides insight into the possible role of structural flexibility during interactions between SARS HCoV 3CLpro and inhibitors and sheds light on structure-based design of anti-COVID-19 drugs targeting SARS-CoV-2 3CLpro.

The Hybrid Strategy of Thermoactinospora rubra YIM 77501T for Utilizing Cellulose as a Carbon Source at Different Temperatures.

Thermoactinospora rubra YIM 77501T is an aerobic, Gram-positive, spore-forming and cellulose degrading thermophilic actinomycete isolated from a sandy soil sample of a volcano. Its growth temperature range is 28-60°C. The genomic sequence of this strain revealed that there are 27 cellulase genes belonging to six glycoside hydrolase families. To understand the strategy that this strain uses to utilize carbon sources such as cellulose at different temperatures, comparative transcriptomics analysis of T. rubra YIM 77501T was performed by growing it with cellulose (CMC) and without cellulose (replaced with glucose) at 30, 40, and 50°C, respectively. Transcriptomic analyses showed four cellulase genes (TrBG2, TrBG3, TrBG4, and ThrCel6B) were up-regulated at 30, 40, and 50°C. The rate of gene expression of TrBG2, TrBG3, TrBG4, and ThrCel6B were 50°C > 30°C > 40°C. One cellulase gene (TrBG1) and two cellulase genes (TrBG5 and ThrCel6A) were up-regulated only at 30 and 50°C, respectively. These up-regulated cellulase genes were cloned and expressed in Escherichia coli. The enzymatic properties of up-regulated cellulases showed a variety of responses to temperature. Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation for each growth condition. These expression patterns revealed that a hybrid strategy was used by T. rubra to utilize carbon sources at different temperatures. This study provides genomic, transcriptomics, and experimental data useful for understanding how microorganisms respond to environmental changes and their application in enhancing cellulose hydrolysis for animal feed and bioenergy production.

Effect of the R119G mutation on human P5CR structure and its interactions with NAD: Insights derived from molecular dynamics simulation and free energy analysis.

Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex. These MD simulations and ED analyses suggest that the R119G mutation decreases the flexibility of P5CR, specifically in the substrate binding pocket, which could decrease the kinetics of the cofactor entrance and egress. Furthermore, the MM-PBSA calculations suggest the R119G mutant has a lower cofactor binding affinity for NAD than WT. Our study provides insight into the possible role of the R119G mutation during interactions between P5CR and NAD, thus bettering our understanding of how the mutation promotes cutis laxa.

In silico screening, molecular docking, and molecular dynamics studies of SNP-derived human P5CR mutants.

Pyrroline-5-carboxylate reductase (P5CR) encoded by PYCR1 gene is a housekeeping enzyme that catalyzes the reduction of P5C to proline using NAD(P)H as the cofactor. In this study, we used in silico approaches to examine the role of nonsynonymous single-nucleotide polymorphisms in the PYCR1 gene and their putative functions in the pathogenesis of Cutis Laxa. Among the 348 identified SNPs, 15 were predicted to be potentially damaging by both SIFT and PolyPhen tools; of them two SNP-derived mutations, R119G and G206W, have been previously reported to correlate with Cutis Laxa. These two mutations were therefore selected to be mapped to the wild-type (WT) P5CR structure for further structural and functional analyses. The results of comparative computational analyses using I-Mutant and Autodock reveal reductions in both stability and cofactor binding affinity of these two mutants. Comparative molecular dynamics (MD) simulations were performed to evaluate the changes in dynamic properties of P5CR upon mutations. The results reveal that the two mutations enhance the rigidity of P5CR structure, especially that of cofactor binding site, which could result in decreased kinetics of cofactor entrance and egress. Comparison between the structural properties of the WT and mutants during MD simulations shows that the enhanced rigidity of mutants results most likely from the increased number of inter-atomic interactions and the decreased number of dynamic hydrogen bonds. Our study provides novel insight into the deleterious effects of the R119G and G206W mutations on P5CR, and sheds light on the mechanisms by which these mutations mediate Cutis Laxa.

Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

Characterizing the fluorescent products of waste activated sludge in dissolved organic matter following ultrasound assisted ozone pretreatments.

This study investigated the effects of ozone and ultrasound (US) pretreatments, both individually and combined, on waste activated sludge reduction. Batch tests were conducted first to optimize the individual ozone and US pretreatments. Maximum sludge reduction ratios of 10.89% and 23% were obtained at 0.15g O3/g total solids ozone dose and 1.5W/mL US energy density, respectively. The combined ozone and US pretreatments were studied using response surface methodology. A maximum sludge reduction ratio of 40.14% was achieved by the combined ozone/US pretreatment with an ozone dose of 0.154g O3/g total solids and an US energy density of 1.445W/mL. The analysis of the dissolved organic matter by three-dimensional excitation-emission matrix fluorescence spectroscopy showed that the combined pretreatment was superior to the individual ozone and US pretreatments, and also demonstrated the synergetic effect of these two combined pretreatments.

Aqua-chlorido(3,5-dinitro-2-oxidobenzo-ato-κ(2)O(1),O(2))(1,10-phenanthroline-κ(2)N,N')chromium(III).

In the title compound, [Cr(C(7)H(2)N(2)O(7))Cl(C(12)H(8)N(2))(H(2)O)], the Cr(III) atom displays a distorted octa-hedral coordination geometry, with the chelating phenantroline and 3,5-dinitro-salicylate ligands in trans positions. In the crystal, mol-ecules are connected via O-H⋯O hydrogen bonds into a two-dimensional framework parallel to (100). In addition, there are π-π stacking inter-actions between phenanthroline ligands along the c axis, with a mean inter-planar distance of 3.456 (4) Å.

5,6-Dihydro-1,10-phenanthroline-1,10-diium µ-oxido-bis-[penta-fluoridotantalate(V)].

In the title compound, (C(12)H(12)N(2))[Ta(2)F(10)O], the doubly protonated 5,6-dihydro-1,10-phenantroline-1,10-diium cation is located on a twofold rotation axis, whereas the isolated [Ta(2)OF(10)](2-) dianion has -1 symmetry. In the so far unknown dianion, the symmetry-related Ta(V) atoms are octa-hedrally coordinated by five F atoms and a bridging O atom, the latter being located on an inversion centre. The two pyridine rings in the cation make a dihedral angle of 22.8 (4)°. The cations and dianions are arranged in layers parallel to (100) and are connected through N-H⋯F and C-H⋯F hydrogen-bonding inter-actions into a three-dimensional structure.

Poly[[bis-(nitrato-κO)bis-(µ(4)-pyridine-4-carboxyl-ato)tetra-kis-(µ(3)-pyridine-4-carboxyl-ato)octa-silver(I)] hemihydrate].

In the title coordination polymer, {[Ag(8)(C(6)H(4)NO(2))(6)(NO(3))(2)]·0.5H(2)O}(n), two Ag(I) ions are two-coordinate within an AgN(2) set and six are three-coordinate within AgN(2)O and AgO(3) sets. The Ag-N and Ag-O distances are in the ranges 2.150 (5)-2.198 (5) and 2.142 (4)-2.702 (5) Å, respectively. A two-dimensional coordination network is formed parallel to (100). The O atom of the disordered solvent water mol-ecule is located on an inversion center.

2-(2-Pyrid-yl)pyridinium (2,2'-bipyridine-κN,N')tetra-kis-(nitrato-κO,O')bis-muthate(III).

The structure of the title compound, (C(10)H(9)N(2))[Bi(NO(3))(4)(C(10)H(8)N(2))], consists of 2-(2-pyrid-yl)pyridinium cations and anions [Bi(NO(3))(4)(C(10)H(8)N(2))](-). The Bi(3+) ion lies on the twofold axis. It is coordinated by two nitro-gen atoms from one 2,2'-bipyridine ligand and eight oxygen atoms from four NO(3) (-) anions. The disordered cation is positioned at the inversion centre. The [Bi(NO(3))(4)(C(10)H(8)N(2))](-) anions and 2-(2-pyrid-yl)pyridinium cations are connected via N-H⋯O hydrogen bonds into chains. Moreover, these chains are further linked into a two-dimensional layered structure through π-π stacking inter-actions between bipyridine ligands along the c axis [centroid-centroid distance = 2.868 (4) Å].

Optimization of operating parameters for sludge process reduction under alternating aerobic/oxygen-limited conditions by response surface methodology.

Batch tests were employed to estimate the optimal conditions for excess sludge reduction under an alternating aerobic/oxygen-limited environment using response surface methodology. Three key operating parameters, initial mixed liquor suspended solids (initial MLSS), HRT (hydraulic retention time) and reaction temperature (T), were selected, and their interrelationships studied by the Box-Behnken design. The experimental data and ANOVA analysis showed that the coefficient of determination (R(2)) was 0.9956 and the adjR(2) was 0.9912, which demonstrates that the modified model was significant. The optimum conditions were predicted to give a maximal ΔMLSS yield of 226 mg/L at an initial MLSS of 10,021 ± 50 mg/L, an HRT of 9.1h and a reaction temperature of 29°C. The prediction was tested by triplicate experiments, where a ΔMLSS yield of 233 mg/L was achieved under the chosen optimal conditions. This excellent correlation between the predicted and measured values provides confidence in the model.

Tetra-aqua-(nitrato-κO,O')bis-(pyridinium-4-carboxyl-ate-κO)europium(III) dinitrate.

The asymmetric unit of the title compound, [Eu(NO(3))(C(6)H(5)NO(2))(2)(H(2)O)(4)](NO(3))(2), consists of one-half of the C(2) symmetric coordination cation and one nitrate anion. The eight-coordinated Eu(III) atom is in a distorted dodeca-hedral coordination environment. The coordination cations and nitrate anions are connected via O-H⋯O and N-H⋯O hydrogen bonds into a three-dimensional network.

The effect of calciums on molecular motions of proteinase K.

The native serine protease proteinase K binds two calcium cations. It has been reported that Ca(2+) removal decreased the enzyme's thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca(2+)-bound and Ca(2+)-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca(2+) sites. Although Ca(2+) removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca(2+), the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca(2+) removal, but also complement the experimentally determined structural and biochemical data.

Effect of the chloro-substitution on lowering diabetic hyperglycemia of vanadium complexes with their permeability and cytotoxicity.

The effect of the chloro-substitution of dinuclear vanadium (V) complexes on lowering diabetic hyperglycemia was evaluated. The in vivo tests for hypoglycemic activity show that complex 2 at the dose of 10.0 and 20.0 mg V kg(-1), could significantly decrease the blood glucose level. Importantly, our results the chloro substituent increased the insulin-enhancing properties of the complex 2. The two vanadium compounds had permeability above 10(-5) cm/s. It suggested that two complexes permeate via a passive diffusion mechanism. It was also suggested that two complexes has better good lipophilic properties. The cytotoxicity of two complexes on Caco-2 cells suggested the chlorine atom at C4 of complex 2 increased cytotoxicity for vanadium complexes.

Insulin-enhancing activity of a dinuclear vanadium complex: 5-chloro-salicylaldhyde ethylenediamine oxovanadium(V) and its permeability and cytotoxicity.

A new insulin-enhancing oxovanadium complex 5-chloro-salicylaldhyde ethylenediamine oxovanadium (V) ([V(2)O(2)(mu-O)(2)L(2)]) has been synthesized. The complex was characterized by a variety of physical methods, including X-ray crystallography. The X-ray diffraction analysis show a dinuclear complex of two six-coordinate vanadium centers doubly bridged by the oxygen atoms of the Schiff base ligand with a V(2)O(2) diamond core. The complex was administered intragastrically to STZ-diabetic rats for 2 weeks. The biological activity results show that the complex at the dose of 10.0 and 20.0 mg V kg(-1), could significantly decrease the blood glucose level and ameliorate impaired glucose tolerance in STZ-diabetic rats. That results suggested that the complex exerts an antidiabetic effect in STZ-diabetic rats. Furthermore, the complex ([V(2)O(2)(mu-O)(2)L(2)]) had permeability above 10(-5)cm/s. The experimental results suggested that the vanadium complex permeates via a passive diffusion mechanism. It was also suggested the complex with salen-type ligands has good lipophilic properties and better oral administration. The cytotoxicity of the complex ([V(2)O(2)(mu-O)(2)L(2)]) on Caco-2 cells was measured by a decrease of cell viability using the MTT assay suggesting that the chlorine atom at C4 of complex [V(2)O(2)(mu-O)(2)L(2)] increased cytotoxicity for vanadium complexes.

A new insulin-enhancing agent: [N,N'-bis(4-hydroxysalicylidene)-o-phenylene-diamine]oxovanadium(IV) and its permeability and cytotoxicity.

A new insulin-enhancing agent: [N,N'-bis(4-hydroxysalicylidene)-o-phenylene-diamine] oxovanadium(IV) (BPOV) was synthesized and characterized by X-ray crystallography. BPOV was administered intragastrically to STZ-diabetic rats for 4 weeks. The results showed that BPOV could significantly decrease the blood glucose level and ameliorated impaired glucose tolerance in STZ-diabetic rats. BPOV has been further tested on insulin, glycogen and serum lipid studies. The results suggested BPOV has glucose-lowering activity in diabetic rats, as well as improved the disorder of lipid metabolism in diabetes. BPOV had permeability above 10(-5) cm s(-1). It was suggested good lipophilic properties. The cytotoxicity of BPOV on Caco-2 cells was measured by MTT assay which suggested BPOV have higher effect on impairment of cellular associated with lower level capacity of cellular accumulation.

Study on the heterogeneous degradation of chitosan with H(2)O(2) catalyzed by a new supermolecular assembly crystal: [C(6)H(8)N(2)](6)H(3)[PW(12)O(40)].2H(2)O.

A new supermolecular assembly crystal, [C(6)H(8)N(2)](6)H(3)[PW(12)O(40)].2H(2)O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H(2)O(2) in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H(2)O(2), 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 degrees C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M(v)) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.

Insights derived from molecular dynamics simulation into the molecular motions of serine protease proteinase K.

Serine protease proteinase K, a member of the subtilisin family of enzymes, is of significant industrial, agricultural and biotechnological importance. Despite the wealth of structural information about proteinase K provided by static X-ray structures, a full understanding of the enzymatic mechanism requires further insight into the dynamic properties of this enzyme. Molecular dynamics simulations and essential dynamics (ED) analysis were performed to investigate the molecular motions in proteinase K. The results indicate that the internal core of proteinase K is relatively rigid, whereas the surface-exposed loops, most notably the substrate-binding regions, exhibit considerable conformational fluctuations. Further ED analysis reveals that the large concerted motions in the substrate-binding regions cause opening/closing of the substrate-binding pockets, thus supporting the proposed induced-fit mechanism of substrate binding. The distinct electrostatic/hydrogen-bonding interactions between Asp39 and His69 and between His69 and Ser224 within the catalytic triad lead to different thermal motions and orientations of these three catalytic residues, which can be related to their different functional roles in the catalytic process. Statistical analyses of the geometrical/functional properties as well as evolutionary conservation of the glycines in proteinase K-like proteins reveal that glycines may play an important role in determining the folding architecture and structural flexibility of this class of enzymes. Our simulation study complements the biochemical and structural studies and provides new insights into the dynamic structural basis of the functional properties of this class of enzymes.

Characterizing structural features of cuticle-degrading proteases from fungi by molecular modeling.

BACKGROUND: Serine proteases secreted by nematode and insect pathogenic fungi are bio-control agents which have commercial potential for developing into effective bio-pesticides. A thorough understanding of the structural and functional features of these proteases would significantly assist with targeting the design of efficient bio-control agents. RESULTS: Structural models of serine proteases PR1 from entomophagous fungus, Ver112 and VCP1 from nematophagous fungi, have been modeled using the homology modeling technique based on the crystal coordinate of the proteinase K. In combination with multiple sequence alignment, these models suggest one similar calcium-binding site and two common disulfide bridges in the three cuticle-degrading enzymes. In addition, the predicted models of the three cuticle-degrading enzymes present an essentially identical backbone topology and similar geometric properties with the exception of a limited number of sites exhibiting relatively large local conformational differences only in some surface loops and the N-, C termini. However, they differ from each other in the electrostatic surface potential, in hydrophobicity and size of the S4 substrate-binding pocket, and in the number and distribution of hydrogen bonds and salt bridges within regions that are part of or in close proximity to the S2-loop. CONCLUSION: These differences likely lead to variations in substrate specificity and catalytic efficiency among the three enzymes. Amino acid polymorphisms in cuticle-degrading enzymes were discussed with respect to functional effects and host preference. It is hoped that these structural models would provide a further basis for exploitation of these serine proteases from pathogenic fungi as effective bio-control agents.