Peptidoglycan biosynthesis-associated enzymatic kinetic characteristics and ß-lactam antibiotic inhibitory effects of different Streptococcus pneumoniae penicillin-binding proteins.

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different ß-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56-9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s-1) than the other rPBPs (Kcat = 3.20-7.49 × 10-2 s-1). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s-1). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (KD = 3.71-9.35 × 10-4 M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

Study on the mechanism and kinetics of manganese release from waste manganese ore waste rock under rainfall leaching.

Manganese released from the piled manganese ore wastes is a great threat to the local ecosystem and human health. The mechanism and dynamic characteristics of manganese release from the manganese ore wastes were studied based on the static and dynamic experiments. The concentration of manganese in the leaching solution under the intensive state is twice that resulted from the static state; the manganese release from the waste rock increased with the increase of the solid-liquid ratio and reached 922.3 mg/L when the solid-liquid ratio was 1:5. When the particle size of waste rock was less than 180 µm, the release amount of manganese was the largest and reached 491.3 mg/L. When the pH was 7 and the rainfall intensity was 80 mL/h, the increase of leaching time contributed to the rapidly decreased amount of manganese released, and the leaching process reached equilibrium gradually. The cumulative release of manganese increased with the increase of rainfall duration. In the dynamic leaching process, the change of pH and EC of the leachate had nothing to do with the initial pH of leaching agent but has a close relationship with the hydrolysis of minerals in waste. According to the experimental results, it was found that the double constant equation model fitted the kinetic process of release process better. The purpose of this study was to provide a scientific basis for the assessment and control of manganese pollution in soil and groundwater in manganese mining area.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases-bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2-8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Osmotic tolerance of rabbit spermatozoa is affected by extender composition and temperature.

Sperm osmotic adaptability to anisosmotic conditions is important for sperm epididymal maturation, motility activation at ejaculation, and female tract colonization, or for conducting technological procedures such as cryopreservation. Several factors affect this adaptability, including the fluid composition that contributes to water flow dynamics, and the temperature at which osmotic stress is initiated. This study was designed to investigate the effect of medium composition (electrolyte- or sugar-based extender) and temperature (25 and 5 °C) on rabbit sperm adaptability to anisosmotic conditions. Rabbit spermatozoa, therefore, were diluted at both temperatures (25 and 5 °C) in electrolyte- or sugar-based media at increasing osmotic conditions (100 to 1,000 mOsm/kg), and values for sperm variables (sperm kinetics, membrane integrity, mitochondrial membrane potential) were estimated as endpoints. Sperm kinetics seemed to be more sensitive to osmotic stress than membrane integrity or mitochondrial function. The effect of moderate hypoosmotic stress did not differ when there was use of sugar- and electrolyte-based extenders at 25 °C (P > 0.05). In hyper-tonic conditions at 25 °C, the sugar-based extender was more effective in protecting sperm membrane integrity and mitochondrial function (P < 0.05). The lesser temperature made the differences more relevant because of the detrimental effect of hyperosmotic stress was more evident in the electrolyte-based extender at 5 °C (P < 0.05). The results from this study indicated rabbit spermatozoa have different adaptability to anisosmotic conditions induced by sugar- and electrolyte-based media and that the temperature at which the osmotic stress is initiated affects the cellular response.

Effect of potassium on the pyrolysis of biomass components: Pyrolysis behaviors, product distribution and kinetic characteristics.

Potassium is an inorganic mineral element in biomass and has a significant catalytic effect on biomass pyrolysis. In this work, the effect of potassium on the pyrolysis of biomass components (cellulose, xylan and lignin) was investigated with the help of thermogravimetric analyzer coupled to fourier transform infrared spectrometer (TG-FTIR) and pyrolysis-gas chromatography coupled to mass spectrometry (Py-GC/MS). The results showed that potassium accelerated the start of the main pyrolysis stage of the biomass components, reduced the weight loss rate for cellulose and lignin, and increased the weight loss rate for xylan. On the other hand, potassium presented a promotion effect on the formation of char for cellulose but a suppression effect for lignin. In addition, an increasing potassium content promoted the release of volatile products for xylan. Product distribution analysis found that potassium promoted the scission of glycosidic bonds and the decomposition of glucose units, resulting in a sharp yield decrease of carbohydrates and a yield increase of furans, aldehydes and ketones. In addition, an increased production of CO2 was obtained, indicating that potassium favors the cleavage and reforming of carboxyl (COOH) and carbonyl (CO) groups. Furthermore, the effect of potassium on the pyrolysis of cellulose and xylan was stronger than that on lignin pyrolysis. The effect on the pyrolysis reaction also resulted in a higher activation energy for the decomposition of biomass components, especially at high temperature intervals. Moreover, the higher the content of potassium added, the greater the increase was in the activation energy.

The effect of sulfate on nitrite-denitrifying anaerobic methane oxidation (nitrite-DAMO) process.

Sulfate is generally found in natural water and wastewater. Nitrite-DAMO bacteria live in natural water or wastewater containing different sulfates. To determine the effect of sulfate on the nitrite-DAMO process, we conducted batch tests and continuous tests to investigate the performance and microbial structure of the nitrite-DAMO system at different sulfate concentrations. The results indicated that the nitrogen removal performance of the nitrite-DAMO system was initially promoted and then inhibited at 0-200 mg SO42-/L, and the denitrification rate was highest at 80 mg SO42-/L which was 1.26 mgN/(L·d). When stimulated by sulfate, protein stabilized nitrite-DAMO bacteria. The denitrification kinetics conformed to the Edward equation, and the initial inhibitory concentration of the nitrite-DAMO system was 189.70 mg SO42-/L. Changes in the proportion of unclassfied_c_ABY1 of the phylum Patescibacteria and norank_f_LD-RB-34 of the phylum Bacteroidetes were the main factors influencing how the nitrogen removal rate of the nitrite-DAMO system responded to sulfate.

Effect of extracellular polymeric substances on arsenic accumulation in Chlorella pyrenoidosa.

Inorganic arsenic (iAs) in its dominant dissolved phase in the environment is known to pose major threats to ecological and human health. While the biological effects in many arsenic-bearing freshwaters have been extensively studied, the behavior and bioaccumulation of dissolved iAS in the presence of extracellular polymeric substances (EPS) still remains to be a critical knowledge gap. In this study, the uptakes and kinetic characteristics of iAs were studied using Chlorella pyrenoidosa (a typical freshwater green algae) by addressing the different effects of EPS on arsenite (AsШ) and arsenate (AsV). The arsenic uptake capacity increased as the exposure concentration increased from 0 to 300 µmol L-1, and the uptake rate constants (Ku) in the Bio-dynamic model were greater for AsV than AsШ (0.63-11.57 L g-1 h-1 vs. 0.44-5.43 L g-1 h-1). The toxic effects as mitigated by EPS were observed through the morphological changes of algal cells by TEM and SEM. When compared with the EPS-free algal cells (EPS-F), EPS-covered cells (EPS-C) had a higher arsenic adsorption capacity through EPS-enhanced surface adsorption and reduced intracellular uptake. The overall decrease (35% and 23.3% for AsШ and AsV, respectively) in the maximum uptake capacity in intact algae cells favors cell's tolerance to the toxic effects of iAs. These observed discrepancies between AsШ and AsV and between EPS-C and EPS-F were further elucidated through morphological images (TEM and SEM) and molecular/atomic spectroscopic data that combine three-dimensional excitation-emission matrix fluorescence spectroscopy (3D-EEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Altogether, the spectroscopic evidence revealed the interactions of iAs with C-O-C, C-O-H and -NH2 functional groups in EPS' tyrosine- and tryptophan-like proteins as the binding sites. Overall, this study for the first time provides comprehensive evidence on the iAs-EPS interactions. Such insights will benefit our understanding of the biogeochemical processes of iAs and the strategic development of bioremediation techniques involving microalgae in the natural and engineered systems.

Investigation of spectral and kinetic properties of polymer films based on some analogs of bacteriorhodopsin.

We investigated the characteristics of modified forms of bacteriorhodopsin in which the native retinal chromophore is replaced by a chemical analog ("bacteriorhodopsin analogs"), embedded in a polymer film. We found they displayed differential absorption spectra and kinetic curves for the most long-lived intermediates of the BR photocycle. We also studied the influence of chemical reagents on the functioning of bacteriorhodopsin analogs in polymeric films. We found that the immobilization of BR analogs in polymer leads, as in the case of native BR, to a slowing down of their photocycles. Kinetic analysis showed that M-like state intermediates of all the BR analogs have a longer dark relaxation time than native BR. The retention and retardation of the photocycle in these films suggest that films based on BR analogs can be used as photochromic materials. Moreover, 4-keto BR seems to be more promising for this application as compared not only with native BR, but also with other analogs of BR studied in this work.

Migration and transformation of nitrogen in bioretention system during rainfall runoff.

Bioretention systems have been extensively studied as a highly efficient technical measure to tackle the global threat of nitrogen pollution during global rainfall runoff. However, the migration and transformation of various forms nitrogen in bioretention system is unclear. So, in this paper, the bioretention systems with different flow regimes and planted configurations were designed to study the nitrogen removal performance and migration and transformation mechanism. The dynamic changes of NH4+-N and NO3--N were continuously monitored within 60 h after rainfall, and the abundance of 15N isotopes in soil layer NH4+-N was simultaneously measured. The results indicated that NH4+-N was mainly intercepted in soil layer in four constructed bioretention systems with similar removal efficiencies (95.42-97.69%). However, NO3--N was retained in submerged layer with significant different removal efficiencies (43.03-83.00%). After fitting calculation, the nitrification rate of NH4+-N (0.0626 mg kg-1 h-1) in soil was 5.31 times higher than that of the accumulation rate of NO3--N (0.0118 mg kg-1 h-1). During the elimination process of residual NH4+-N in soil, 41.46% removed by denitrification and plant absorption assimilation, another 57.28% stored in the form of organic nitrogen or inorganic nitrogen, only 1.26% leaked out. Based on this, the content variation of TN, NH4+-N and NO3--N could be analyzed by a system-wide and established the nitrogen balance model, which provides a new insight into the enhancement of nitrogen removal in the bioretention system.

Physiological-phased kinetic characteristics of microalgae Chlorella vulgaris growth and lipid synthesis considering synergistic effects of light, carbon and nutrients.

To comprehensively understand kinetic characteristics of microalgae growth and lipid synthesis in different phases, a phase-feeding strategy was proposed to simultaneously regulate light, carbon and nutrients in adaption, growth and stationary phases of microalgae cultivation. Physiological-phased kinetic characteristics of microalgae Chlorella vulgaris growth and lipid synthesis under synergistic effects of light, carbon and nutrients were investigated, and supply-demand relationships of electrons and energy between light and dark reactions of photosynthesis process were discussed. Finally, the optimized cultivation strategy for microalgae in various phases were obtained, under which the lipid productivity was significantly improved from 130.11 mg/L/d to 163.42 mg/L/d. The study provided some important guidance for the large-scale production of biofuels from microalgae.

Thermal decomposition kinetics of sorghum straw via thermogravimetric analysis.

The thermal decomposition of sorghum straw was investigated by non-isothermal thermogravimetric analysis, where the determination of kinetic triplet (activation energy, pre-exponential factor, and reaction model), was the key objective. The activation energy was determined using different isoconversional methods: Friedman, Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS), Starink, Iterative method of Chai & Chen, Vyazovkin AIC method, and Li & Tang equation. The pre-exponential factor was calculated using Kissinger's equation; while the reaction model was predicted by comparison of z-master plot obtained from experimental values with the theoretical plots. The values of activation energy obtained from isoconversional methods were further used for evaluation of thermodynamic parameters, enthalpy, entropy and Gibbs free energy. Results showed three zones of pyrolysis having average activation energy values of 151.21kJ/mol, 116.15kJ/mol, and 136.65kJ/mol respectively. The data was well fitting with two-dimension 'Valensi' model for conversion values from 0 to 0.4 with a coefficient of determination (R2) value of 0.988, and with third order reaction model for values from 0.4 to 0.9 with an R2 value of 0.843.

Inhibition factors and Kinetic model for ammonium inhibition on the anammox process of the SNAD biofilm.

The aim of the present work was to evaluate the anaerobic ammonium oxidation (anammox) activity of simultaneous partial nitrification, anammox and denitrification (SNAD) biofilm with different substrate concentrations and pH values. Kaldnes rings taken from the SNAD biofilm reactor were incubated in batch tests to determine the anammox activity. Haldane model was applied to investigate the ammonium inhibition on anammox process. As for nitrite inhibition, the NH4+-N removal rate of anammox process remained 87.4% of the maximum rate with the NO2--N concentration of 100mg/L. Based on the results of Haldane model, no obvious difference in kinetic coefficients was observed under high or low free ammonia (FA) conditions, indicating that ammonium rather than FA was the true inhibitor for anammox process of SNAD biofilm. With the pH value of 7.0, the rmax, Ks and KI of ammonium were 0.209kg NO2--N/kg VSS/day, 9.5mg/L and 422mg/L, respectively. The suitable pH ranges for anammox process were 5.0 to 9.0. These results indicate that the SNAD biofilm performs excellent tolerance to adverse conditions.

Finite element model and modal analysis of CT shelter based on ANSYS / 医疗卫生装备

To perform modal analysis of CT shelter by applying computer simulation technology so as to pro-vide theoretical guidance for CT shelter structure optimization. Based on CAD model, the finite element model of a CT shelter was established with ANSYS simulation platform. Through modal analysis, different-order modal frequency and modal shape of the shelter were computed and the kinetic characteristics were evaluated. Low order modal frequency was kept away from the natural frequency range of chassis system resonance to avoid the overall structure reso-nance; the 3rd and the 4th modal frequency and engine idle speed frequency were very close so that local resonance might occur; road roughness excitation frequency covered the first 6 order modal frequencies and the further vibration-re-ducing measures of CT equipment were suggested. Based on the theories of finite element method and current software platform, modal analysis of shelter structure can be simulated and the results can provide valuable data for the improvement of kinetic characteristics and structure design.

The characteristics and two-step reaction model of p-nitroacetophenone biodegradation mediated by Shewanella decolorationis S12 and electron shuttle in the presence/absence of goethite.

The current study mainly focused on the biodegradation process of p-nitroacetophenone (NP) in the presence and absence of goethite mediated by iron-reducing microbe (Shewanella decolorationis S12) and electron shuttle. The results showed that introduction of electron shuttle could obviously lead to an accumulation of biodegradation intermediate, especially in reaction systems containing high content of electron shuttle in the absence of goethite. Goethite could enhance the degree and rate of NP biodegradation. The microbial reductively generated Fe(II) played an active role in the biodegradation process. The relationship between the concentrations of biodegradation end product and the reaction times could be fitted by a consecutive reaction model with correlation coefficients (adjusted R(2)) in the range from 0.9241 to 0.9831 during the biodegradation stage from the beginning to about 250 h of incubation. However, during the subsequent biodegradation stages, in the presence and absence of goethite, transitions from the consecutive reaction model to zero-order reaction model and from the consecutive reaction model to exponential growth reaction model were observed, respectively. The newly proposed two-step reaction model will help understand the mechanism of the biodegradation process of nitroaromatic compounds and related pollutants.

Kinetics of substrate degradation and electricity generation in anodic denitrification microbial fuel cell (AD-MFC).

Effect of substrate concentration on substrate degradation and electricity generation in anodic denitrification microbial fuel cell (AD-MFC) was investigated over a broad range of substrate concentrations. Substrate degradation rates and power generation could be promoted with increasing substrate concentration in a certain range, but both of them would be inhibited at high substrate concentrations. Maximum denitrification rate of 1.26 ± 0.01 kg NO(-)-N/m(3)d and maximum output voltage of 1016.75 ± 4.74 mV could be achieved when initial NO3(-)-N concentration was 1999.95 ± 2.86 mg/L. Based on Han-Levenspiel model, kinetics of substrate degradation and power generation in the AD-MFC were established. According to the kinetic model, the half-saturation coefficient and the critical inhibitory concentration for nitrate were more than 200 and 4300 mg/L, respectively. The results demonstrated that AD-MFC was capable of treating nitrate-containing wastewater and generating electricity simultaneously, and tolerant to high strength nitrate-containing wastewater.

Contribution of kinetic characteristics of axillary lymph nodes to the diagnosis in breast magnetic resonance imaging.

OBJECTIVE: To assess the contribution of kinetic characteristics in the discrimination of malignant-benign axillary lymph nodes. MATERIAL AND METHODS: One hundred fifty-five female patients were included in the study. Following magnetic resonance imaging (MRI) examinations postprocessing applications were applied, dynamic curves were obtained from subtracted images. Types of dynamic curves were correlated with histopathological results in malignant cases or final clinical results in patients with no evidence of malignancy. Sensitivity, specificity, positive likehood ratio (+LHR), negative (-LHR) of dynamic curves characterizing the axillary lymph nodes were calculated. RESULTS: A total of 178 lymph nodes greater than 8 mm were evaluated in 113 patients. Forty-six lymph nodes in 24 cases had malignant axillary involvement. 132 lymph nodes in 89 patients with benign diagnosis were included in the study. The sensitivity of type 3 curve as an indicator of malignancy was calculated as 89%. However the specificity, +LHR, -LHR were calculated as 14%, 1.04, 0.76 respectively. CONCLUSION: Since kinetic analysis of both benign and malignant axillary lymph nodes, rapid enhancement and washout (type 3) they cannot be used as a discriminator, unlike breast lesions. MRI, depending on the kinetic features of the axillary lymph nodes, is not high enough to be used in the clinical management of breast cancer patients.