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Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia., Competing Interests: Declaration of competing interest Informed consent statement not applicable., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cimicifugae rhizoma is a traditional Chinese herbal medicine in China, and modern pharmacological research showed that it has obvious antiviral activity. Many polysaccharides have been proved to have immune enhancement and antiviral activity, but there are few studies on the biological activity of Cimicifuga rhizoma polysaccharide (CRP). The aim was to explore the character of CRP and its effects on improving immune activity and inhibiting transmissible gastroenteritis virus (TGEV). The monosaccharide composition, molecular weight, fourier transform infrared spectra and electron microscopy analysis of CRP was measured. The effect of CRP on immune activity in lymphocytes and RAW264.7 cells were studied by colorimetry, FITC-OVA fluorescent staining and ELISA. The effect of CRP on TGEV-infected PK-15 cells was determined using Real-time PCR, Hoechst fluorescence staining, trypan blue staining, acridine orange staining, Annexin V-FITC/PI fluorescent staining, DCFH-DA loading probe, and JC-1 staining. Network pharmacology was used to predict the targets of CRP in enhancing immunity and anti-TGEV, and molecular docking was used to further analyze the binding mode between CPR and core targets. The results showed that CRP was mainly composed of glucose and galactose, and its molecular weight was 64.28 kDa. The content of iNOS and NO in CRP group were significantly higher than the control group. CRP (125 and 62.5 μg/mL) could significantly enhance the phagocytic capacity of RAW264.7 cells, and imprive the content of IL-1β content compared with control group. 250 μg/mL of CRP possessed the significant inhibitory effect on TGEV, which could significantly reduce the apoptosis compared to TGVE group and inhibit the decrease in mitochondrial membrane potential compared to TGVE group. The mRNA expression of TGEV N gene in CRP groups was significantly lower than TGEV group. PPI showed that the core targets of immune-enhancing were AKT1, MMP9, HSP90AA1, etc., and the core targets of TGE were CASP3, MMP9, EGFR, etc. Molecular docking show that CRP has binding potential with target. These results indicated that CRP possessed the better immune enhancement effect and anti-TGEV activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Applying organic fertilizer is the main way to enhance soil fertility through the interfacial reaction between mineral and dissolved organic matter (DOM). However, the interfacial reaction between minerals and DOM may influence antimony(V) (Sb(V)) mobility in agricultural soils around antimony mines. In our study the ferrihydrite (Fh) was chosen as a representative mineral, to reveal the effect of its interaction with chicken manure organic fertilizer (CM-DOM) with Fh on Sb(V) migration. In this study, we investigated different organic matter molecular weights and C/Fe molar ratios. Our findings indicated that the addition of CM-DOM decreased the adsorption of Sb(V) by Fh and promoted the re-release of Sb(V) adsorbed on Fh. This effect was enhanced by increasing the C/Fe molar ratio. Fh mainly affects its interaction with Sb(V) through electrostatic gravitational interaction and ligand exchange, but the presence of CM-DOM weakens the electrostatic interaction between Fh and Sb(V) as well as competes with Sb(V) for the hydroxyl reactive site on Fh surface. In addition, the smaller molecular weight fraction (<10 kDa) of CM-DOM has higher aromaticity and hydrophobicity, which potentially leads to more intense competition with Sb(V) for the reaction sites on Fh. Therefore, the application of organic fertilizer may promote Sb(V) migration, posing significant risks to soil ecosystems and human health, which should be a concern in field soil cultivation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, a low molecular weight poly-d-mannose (LMWM) was separated from a mixed polysaccharide synthesized previously. Monosaccharide composition, Fourier-Transform infrared spectroscopy (FT-IR), periodate oxidation and smith degradation were determined. After safety evaluation, the inhibition of LMWM on the different biofilm formation stages of Salmonella enterica serovar Typhimurium (S. Typhimurium) was tested in vitro. Furthermore, the effect of LMWM on the adhesion of S. Typhimurium to Caco-2 cells and cell surface hydrophobicity (CSH) were observed. Results indicated that LMWM was a homopolysaccharide without cytotoxicity and hemolysis, containing both α-mannose and β-mannose. It showed obvious anti-biofilm activity on S. Typhimurium and mainly activated on the initial adhesion and formation stage, even better than the commercial S. cerevisiae mannan (CM). LMWM inhibited the adhesion of S. Typhimurium on Caco-2 cells with the inhibition rate of 61.04 % at 2 mg/ml. Meanwhile, LMWM decreased the hydrophobicity of S. Typhimurium cell surface. In conclusion, the inhibitory effect on S. Typhimurium biofilm was not caused by bacteriostatic or bactericidal activity of LMWM. The specific anti-adhesion and the decrease of bacterial CSH by LMWM may closely relate to anti-biofilm mechanism. This study provides some supports for the application of LMWM as antibiotics alternative on S. Typhimurium in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Bacterial fibrinolytic enzymes have an important role in thrombolytic therapy due to their ability to dissolve fibrin. Therefore, purification, characterization and activity determination are of prime importance for bacterial fibrinolytic enzymes. In the current study, marine Bacillus amyloliquefaciens was first screened from Sipunculus nudus living in the Beibu Gulf of China and denoted as Guangxi University-1 (GXU-1). Then, an extracellular fibrinolytic enzyme (FEB-1) was purified from GXU-1 using ammonium sulfate precipitation, hydrophobic chromatography and gel filtration chromatography. The specific activity of FEB-1 was determined to be as high as 6789.74 U/mg. The relative molecular weight of FEB-1 was measured as 30 kDa through SDS‒PAGE. The optimum in vitro fibrinolytic activity of FEB-1 was identified at 37 °C and pH = 8. Furthermore, the activity of FEB-1 can be well preserved at 20-45 °C and pH = 6.0 to 9.0. The combination analysis of SDS‒PAGE and the molecular docking calculation revealed that FEB-1 can cleave more Aα- and Bβ-chains of fibrinogen than γ-chain. It is noteworthy that FEB-1 is comparatively stable under human-body environmental conditions, indicating its potential application in thrombosis therapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dissolved organic matter (DOM) is ubiquitous and widespread in natural water and influences the transformation and removal of antibiotics. Nevertheless, the influence of DOM molecular weight (MW) on the indirect photodegradation of antibiotics has rarely been reported. This study attempted to explore the influence of the molecular weight of DOM on the indirect photodegradation of two fluoroquinolone antibiotics (FQs), ofloxacin (OFL) and norfloxacin (NOR), by using UV-vis absorption and fluorescence spectroscopy. The results showed that indirect photodegradation was considered the main photodegradation pathway of FQs in DOM fractions. Triplet-state excited organic matter ( 3 DOM*) and singlet oxygen ( 1 O 2 ) were the main reactive intermediates (RIs) that affected the indirect photodegradation of FQs. The indirect photodegradation rate of FQs was significantly promoted in DOM fractions, especially in the low molecular weight DOM fractions (L-MW DOM, MW < 10 kDa). The results of excitation-emission matrix spectroscopy combined with parallel factor analysis (EEM-PARAFAC) showed that terrestrial humic-like substances had a higher humification degree and fluorophore content in L- MW DOM fractions, which could produce more 3 DOM* and 1 O 2 to promote the indirect photodegradation of FQs. This study provided new insight into the effects of DOM at the molecular weight level on the indirect photodegradation of antibiotics in natural water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hypothesis: We propose that during the evaporation of sessile droplets, the evaporative flux profile is primarily influenced by droplet geometry and composition under diffusion-limited conditions. Most studies have focused solely on the evaporation feature from the liquid to the gas phase, neglecting the extent to which the evaporated vapors affect the evaporation process. We hypothesize that if the molecular weight of the evaporated vapors is significantly high or low compared to the ambient gas, it could alter the evaporative flux., Experiments: We employed a direct optical measurement technique, specifically Mach-Zehnder interferometry. This demonstrated that the distribution of evaporated vapor molecules can substantially modify the evaporative flux profile., Findings: Our study discovered that substantial density gradients between vapor and air could either suppress or enhance the evaporative flux, depending on the droplet's orientation. This research offers fresh insights into evaporative fluxes by taking into account the relative vapor concentration and gravitational effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dissolved organic matter (DOM) is a kind of substance with complex compositions and wide molecular weight distribution, which can strongly combine with various pollutants. Therefore, the binding characteristics of DOM and heavy metal pollutants can be studied specifically according to the binding characteristics of DOM and pollutants. In this study, DOM in surface water bodies was divided into three levels (MW < 1 kDa, 1 kDa < MW < 5 kDa, MW > 5 kDa) according to different molecular weights (MW). The binding properties were investigated by fluorescence spectrum analysis and complex model. Four components (C1-C4) were identified by PARAFAC. Among them, the contribution rate of protein-like components C1, C2 and C4 to the total fluorescence intensity reached more than 78%, and the log K a values of low molecular weight components were the highest, which were 3.28, 3.14 and 3.47, respectively, indicating higher binding ability with Cu 2+ .With the decrease of molecular weight, the log K b value increases, indicating that the complexation is more stable. The humic component C3 in high molecular weight has stronger binding stability with Cu 2+ , but the number of binding sites for C3 is 0.36, while that for C2 is 1.51, indicating that its binding sites and binding ability are relatively low. The results showed that the DOM ligand of Cu 2+ in surface water showed a certain molecular weight dependence. In addition, different MW DOM lead to different pollution forms. Different properties of DOM ligand combined with Cu 2+ were studied in order to control the migration, transformation, bioavailability, morphology and stability of heavy metal pollutants, and to provide theoretical support for the practical application management of surface water pollution control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lignin is an abundant phenolic polymer produced vastly in pulping processes that could be further valorized. In this work, anionic (AKLs) and cationic (CKLs) lignin-based polymers were made by polymerizing kraft lignin (KL) with acrylic acid (AA) or [2-(methacryloyloxy) ethyl] trimethyl-ammonium chloride (METAC), respectively. In the polymerization reactions, various molar ratios of AA or METAC to KL were applied to produce AKLs and CKLs with different characteristics. The produced AKLs and CKLs were used in single and dual systems to flocculate aluminum oxide in suspension. To assess the interaction of these lignin-based polymers with the aluminum oxide particles; the zeta potential, adsorption, and flocculation of the colloidal systems were evaluated comprehensively. The flocculation performance of the lignin-derived polymers was compared with that of the homopolymers of AA and METAC (PAA and PMETAC) and commercially used flocculants. In single polymer systems, among the anionic synthesized polymers and homopolymers, KL-A4 (an AKL) was the best flocculant for the aluminum oxide suspensions owing to its largest molecular weight (330 × 10 3  g/mol) and highest charge density (-4.2 mmol/g). Remarkably, when KL-A4 and KL-C4 (the CKL with the highest molecular weight and charge density) were used subsequently in a dual polymer system, a larger adsorbed mass and a more viscous adlayer were formed than those of single polymer systems on the surface of aluminum oxide particles. The synergy between KL-A4 and KL-C4 was even stronger than that between homopolymers, which led to more significant adsorption on the aluminum oxide surface and, consequently, more efficient flocculation, producing larger (22 μm) and stronger flocs, regardless of the agitation intensity used in the systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Low-molecular-weight dissolved organic nitrogen (LMW-DON) is an emerging issue in concentrated leachate (CL). Ozonation is crucial to remove LMW-DON, but selectivity mechanisms of different reactive oxygen species were unknown. Here, reactions of O 3 and • OH with LMW-DON at different dosages were determined from composition, unsaturation/redox potential, and precursor-product relationship. The molecular weight of LMW-DON in CL presented a normal distribution and 76.5% was below 450 Da. LMW-DON with 400-1000 Da increased to 55.6%-66.7% and O/C wa increased by over 40.0% due to electrophilic substitution of O 3 . LMW-DON with <400 Da and 550-1000 Da were preferentially degraded by • OH at the low and high O 3 dosage, respectively. O 3 preferred to remove lipid-like (69.1%), protein-like (58.2%), and amino sugars-like (72.8%) LMW-DON, whereas • OH preferred to the refractory LMW-DON, such as carbohydrates-like (71.1%), lignin-like (49.6%), and tannins-like (72.5%) LMW-DON. Forty-three transformation reactions were quantified using mass difference analysis, and O 3 preferred to oxygen addition (e.g., +2O) and conversed amino to nitro groups, and saturated LMW-DON increased via unsaturated bonds rupture. • OH attacked the carbon groups (e.g., -CH 2 ) and nitrogen groups (e.g., -NH 3 +O, -NO 2 +H). These findings provide molecular evidence for the selectivity of oxidants with LMW-DON and improve the ozonation application in wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hypothesis: We have shown earlier that low molecular weight gels based on N-heptyl-d-galactonamide hydrogels can be 3D printed by solvent exchange, but they tend to dissolve in the printing bath. We wanted to explore the printing of less soluble N-alkyl-d-galactonamides with longer alkyl chains. Less soluble hydrogels could be good candidates as cell culture scaffolds., Experiments: N-hexyl, N-octyl and N-nonyl-d-galactonamide solutions in dimethylsulfoxide are injected in a bath of water following patterns driven by a 2D drawing robot coupled to a z-platform. Solubilization of the gels with time has been determined and solubility of the gelators has been measured by NMR. Imbricated structures have been built with N-nonyl-d-galactonamide as a persistent ink and N-hexyl or N-heptyl-d-galactonamide as sacrificial inks. Human mesenchymal stem cells have been cultured on N-nonyl-d-galactonamide hydrogels prepared by cooling or by 3D printing., Findings: The conditions for printing well-resolved 3D patterns have been determined for the three gelators. In imbricated structures, the solubilization of N-hexyl or N-heptyl-d-galactonamide occurred after a few hours or days and gave channels. Human mesenchymal stem cells grown on N-nonyl-d-galactonamide hydrogels prepared by heating-cooling, which are stable and have a fibrillar microstructure, developed properly. 3D printed hydrogels, which microstructure is made of micrometric flakes, appeared too fragile to withstand cell growth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hypothesis: The emulsifying ability of starch is influenced by its molecular weight. Reducing the molecular weight of starch is expected to influence interfacial adsorption and membrane elasticities, thereby affecting its emulsifying ability through Pickering effects. Hence, it should be possible to tailor the emulsifying ability of starch by adjusting its molecular weight., Experiments: Waxy corn starch (CS) and rice starch (RS) were hydrolyzed with pullulanase to obtain high (HM) and low molecular weight (LM) fractions. After the molecular weight was determined by size exclusion chromatography, the fractions were used to prepare model oil-in-water emulsions. The stability, microscopy, and particle size of the emulsions were characterized, and the underlying emulsification mechanism was subsequently studied through dynamic laser scattering, surface tension analysis, interfacial rheology, and Pearson's correlation calculations., Findings: In the molecular weight range obtained in this study, the smaller the molecular weight of starch, the stronger its emulsifying ability. The decrease in molecular weight resulted in considerable different adsorption and interfacial elasticities with smaller fractions occupying less area on the interface and forming interfaces with higher elasticities, resulting in higher stabilities through Pickering effects. Results thus suggest that the emulsifying ability of starch may be tailored by adjusting its molecular weight., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Spider dragline silk is a remarkable fiber made of unique proteins-spidroins-secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L -1 ) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m -3 . The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Shear thickening fluids (STFs) have been the research focus for decades because of the prospect as a damping ingredient. However, their inherent liquid character confines their practical applications. In this work, inspired by the assembly engineering, novel gelatinous shear thickening fluids (GSTFs) are fabricated by integrating low molecular weight gelators (LMWGs) into STFs and investigated by rheological experiments. The results show that the apparent performances of GSTFs are determined by the LMWGs content. LMWGs inside GSTFs can assemble into three-dimensional network that can constraint the flowability of liquid molecular and their content dominate the density and strength of assembly network. At a moderate content, GSTFs exhibit desired properties with restricted quasi-static flowability and almost undamaged dynamic shear thickening character. While a higher content will disappear shear thickening and a lower content cannot gelate STFs. Besides, three different LMWGs are employed to gelate STFs and all they can gelate STFs in spite of the distinct minimum gelation concentration, indicating the universality for GSTFs preparation and the superiority of a reasonable molecular structure of LMWGs. Further, the temperature sweep experiments suggest that GSTFs can endure higher temperature without flowing due to its higher gel-sol transition temperature. Basing on these advanced mechanical properties, we believe that the GSTFs with more expected characters have significance for the study of non-Newtonian fluids and will broaden the special application field of STFs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Polymer flooding is an enhanced oil recovery technique to extract the large portion of leftover subsurface oil following conventional extraction methods. In the flooding process, a long-chain polymer, such as partially hydrolyzed polyacrylamide (HPAM), is added to the displacing fluid to increase the mobility and extraction of the oil phase. Nevertheless, the challenge of managing produced water from polymer flooding operations is high because residual HPAM results in significantly high viscosity and organic content in the stream. Commonly used methods for produced water treatment, such as gravity settling and flotation, cannot be applied to obtain a purified stream efficiently, while innovative techniques are not yet feasible in practical operations. In this work, a simple method of polymer precipitation prompted by divalent ions is evaluated, optimized, and compared to membrane ultrafiltration. The physico-chemical properties of the HPAM are investigated and polymer precipitation tests are conducted by varying the main operational parameters, including pH, salinity, temperature, calcium and/or magnesium concentration, and polymer concentration. Response surface developed by central composite design method is used to optimize the process and identify the correct dosage of divalent cations coagulants and pH, the two main factors promoting HPAM separation. The removal of HPAM is well-described and maximized (>85%) by the model, which is also validated on three synthetic samples representing real wastewaters from polymer flooding applications. Optimized ultrafiltration, using ceramic membranes with surface pore size of 15 kDa, also shows the ability to remove HPAM effectively from water, but the precipitation method seems to be more versatile and easier to apply. The two processes, precipitation and ultrafiltration, may potentially be used in sequence as they complement each other in several ways., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Membrane structuration of Large Hybrid Unilamellar Polymer/Lipid Vesicle (LHUV) is an important parameter on the optimization of their properties and thus their valuation in various fields. However, this kind of information is hardly accessible. In this work, we will focus on the development of LHUV obtained from the self-assembly of diblock poly(dimethylsiloxane)-b-poly(ethylene oxide) (PDMS-b-PEO) of different molar masses combined with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 15% and 25% w/w content. The hybrid character of the resulting vesicles as well as their membrane structure are characterized by the mean of different techniques such as small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM). We show that hybrid vesicles with homogeneous membrane structure are obtained whatever the molar mass of the block copolymer (from 2500 to 4000 g/mol), with of a small number of tubular structures observed with the higher molar mass. We also demonstrate that the permeability of the LHUV, evaluated through controlled release experiments of fluorescein loaded in LHUV, is essentially controlled by the lipid/polymer composition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, K m and V max were 11.2 μM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iβ DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3-7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are K m PEP 1100 ± 100 μM, K m E4P 530 ± 100 μM, and k cat 10.3 ± 1.2 s -1 . The kinetic parameters for AroG2 are K m PEP 90 ± 20 μM, K m E4P 130 ± 40 μM, and k cat 2.0 ± 0.2 s -1 . At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Several studies have demonstrated that low-molecular weight heparin (LMWH) is superior to unfractionated heparin (UFH) in trauma patients. The superiority of either one has not been established for the elderly. In this study, we compared LMWH to UFH in elderly trauma patients., Methods: A retrospective analysis of the American College of Surgeons' Trauma Quality Improvement Program database was performed for patients aged ≥65 y. Propensity score matching was performed to minimize confounders between the two groups. Outcomes included venous thromboembolic (VTE) and bleeding events., Results: Overall, 93,987 patients were identified (mean age 77.1 ± 7.3 y, females 55,035 [58.6%]), of which 67,738 (72.1%) patients received LMWH and 26,249 (27.9%) received UFH. After Propensity score matching, LMWH was associated with a lower incidence of deep venous thrombosis (1.7% versus 2.1%, P = 0.007) and pulmonary embolisms (0.6% versus 1%, P< 0.001). LMWH was also associated with fewer bleeding complications (transfusions: 2.8% versus 3.5%, P< 0.001, procedures: 0.7% versus 0.9%, P = 0.007). Sub-analyses showed that differences in VTE rates were identified in patients with mild injuries (Injury Severity Score [ISS] <16, 0.6% versus 1.9%, P< 0.001). Differences in bleeding complications were identified in patients with injuries of mild (ISS <16, transfusions: 3% versus 3.8%, P< 0.001, surgeries: 0.3% versus 0.4%, P= 0.015) and moderate severity (ISS 16-24, transfusions: 1.9% versus 2.7%, P= 0.038, surgeries: 1% versus 1.7%, P= 0.013)., Conclusion: LMWH prophylaxis is superior to UFH for VTE prevention among elderly trauma patients. LMWH prophylaxis is associated with fewer bleeding complications compared to UFH in patients with injuries of mild or moderate severity., (Copyright © 2021. Published by Elsevier Inc.)

Hypothesis: Development of highly efficient low-molecular weight gelators (LMWGs) for safe energy storage materials is of great demand. Energy storage materials as fuel gels are often achieved by construction of hybrid organic frameworks capable of multiple noncovalent interactions in self-assembly, which allow tuning required properties at the molecular level by altering individual building blocks of the LMWG. However, LMWGs have limited rechargeable capability due to their chemical instability., Experiments: We designed, synthesized and characterized a novel, bio-inspired chiral gemini amphiphile derivative 1 containing N-hexadecyl aliphatic tails from quaternized nicotinamide-based segment and bromide anion showing supergelation ability in water, alcohols, aprotic polar and aromatic solvents, with critical gel concentrations as low as 0.1 and 0.035 wt% in isopropanol and water, respectively., Findings: Nanostructural architecture of the network depended on the solvent used and showed variations in size and shape of 1D nanofibers. Supergelation is attributed to a unique asymmetric NH⋯OC, H⋯Br - hydrogen bonding pattern between H-2 hydrogens from nicotinamide-based segment, amide functional groups from chiral trans-cyclohexane-1,2-diamide-based segment and bromide ions, supporting the intermolecular amide-amide interactions appearing across one strand of the self-assembly. Gels formed from 1 exhibit high stiffness, self-healing, moldable and colorable properties. In addition, isopropanol gels of 1 are attractive as reusable, shape-persistent non-toxic fuels maintaining the chemical structure with gelation efficiency for at least five consecutive burning cycles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Polychlorinated biphenyls (PCBs) are compounds of significant interest due to high toxicity, persistence, long-range atmospheric transport, and bioaccumulation. These compounds can interact with components present in the environment, including dissolved organic matter (DOM) in soils and waters, thereby modifying its availability and movement. In this study, DOM was fractionated by ultrafiltration and characterized according to its hydrophobicity and hydrophilicity, then the interaction of a series of PCBs and different DOM fractions was evaluated. The DOM was collected from the surface waters of three sectors located along a river in the southern part of America. These sectors are subject to different anthropic activities, thus the DOM of sector 1, with the least anthropic influence, was mainly hydrophobic and with a high content of aromatic structures. In contrast, the DOM collected from sectors 2 and 3, where anthropic activity is highest, was slightly hydrophobic and hydrophilic, respectively. The DOM of these two sectors was mainly composed of low molecular weight macromolecules. These results revealed that more hydrophobic PCBs (i.e., 101, 118, 138, and 180) have a greater affinity to DOM with a higher molecular weight (i.e., >1 kDa). In turn, PCBs with lesser chlorination and hydrophobicity presented a greater affinity to DOM with a lower molecular weight. In conclusion, our study shows that the high molecular weight DOM is responsible for mobilizing PCBs with a high degree of chlorination., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hypothesis: Recently, a low molecular weight hydrogel based on a carbohydrate alkyl amide has been successfully used as biomaterial for neuron cell culture and for 3D printing. Varying the molecular structure should make it possible to extend the library of carbohydrate low molecular weight hydrogels available for these applications and to improve their performances., Experiments: Thirteen molecules easy to synthetize and designed to be potentially biocompatible were prepared. They are based on gluconamide, glucoheptonamide, galactonamide, glucamide, aliphatic chains and glycine. Their gelation in water was investigated in thermal conditions and wet spinning conditions, namely by dimethylsulfoxide-water exchange under injection., Findings: Nine molecules give hydrogels in thermal conditions. By wet spinning, six molecules self-assemble fast enough, within few seconds, to form continous hydrogel filaments. Therefore, the method enables to shape by injection these mechanically fragile hydrogels, notably in the perspective of 3D printing. Depending on the molecular structure, persistent or soluble gel filaments are obtained. The microstructures are varied, featuring entangled ribbons, platelets or particles. In thermal gelation, molecules with a symmetrical polar head (galacto, glucoheptono) give flat ribbons and molecules with an asymmetrical polar head (gluco) give helical ribbons. The introduction of an extra glycine linker disturbs this trend., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate β-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD + over NADP + , in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD + -dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aquaculture by-products have been of great interest for producing protein hydrolysates with multiple biological activities. The present experiment was carried out to evaluate dietary effects of a low-molecular fraction (<10 kDa) from shrimp waste hydrolysate in forms of unprotected and nanocapsulated on growth and immunity of rainbow trout. Therefore, six diets were designed including a control diet (no supplementation), D1 (1 g kg -1 of unprotected fraction), D2 (1 g kg -1 chitosan nanocapsules), D3 (1 g kg -1 liposome nanocapsules), D4 (1 g kg -1 of fraction-loaded chitosan nanocapsules), D5 (1 g kg -1 of fraction-loaded liposome nanocapsules). Fish (0.91 ± 0.15 g) were fed with experimental diets until apparent satiation for six weeks followed by a 5-day experimental challenge with Streptococcus iniae. Results revealed that growth is strongly affected in fish receiving the fraction with D4 treatment showing the highest weight gain, SGR, final weight and the lowest FCR (p < 0.05). Nanocapsules without fraction did not show remarkable effects when compared to control group. In terms of serum and mucus immune parameters of lysozyme, complement activity, myeloperoxidase activity, and total protease, fish from D4 group showed the highest measured values followed by D5 (p < 0.05). Key immune related genes of IL-6 and TNF-α were noticeably up-regulated in fish from D1, D4, and D5 groups, which were consistent with survival rate after 5 days challenge with Streptococcus iniae. All together, the present findings highlighted the application of chitosan and liposome nanocarriers in aquaculture and potential of low-molecular weight fraction (<10 kDa) from shrimp wastes hydrolysate to improve growth performance and immune status of rainbow trout., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

DNase is a powerful tool for a series of molecular biology applications. Developing a strategy for large-scale production of DNase with high purity and activity is critical for scientific research. In this study, a previously uncharacterized gene with nuclease activity was found in Trichogramma pretiosum genome. Pichia pastoris GS115 was preferred as the host to overcome the issues related to prokaryotic expression. Under the optimal conditions, the activity of T. pretiosum DNase (Tp-DNase) reached 1940 U/mL of culture supernatant in fed-batch fermentation. Using ion-exchange chromatography and adsorption chromatography, Tp-DNase was produced with a purity of >99% and molecular weight of 45 kDa. In vitro DNA degradation experiments showed that Tp-DNase could effectively degrade dsDNA, and its activity was slightly higher than that of bovine pancreas DNase I under the same conditions. Moreover, Tp-DNase can be used to eliminate nucleic acid contamination and improve the accuracy of nucleic acid detection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Algal organic matter (AOM) and natural organic matter (NOM) from a typical eutrophic lake were comprehensively investigated in terms of their physico-chemical property, components and disinfection byproduct formation potentials (DBPFPs). The relationships between specific chemical properties of AOM and NOM with their corresponding DBPFPs were further evaluated during chlorination. Results indicated that AOM had lower specific UV absorbance (SUVA) but richer organic nitrogen contents than NOM. Fluorescence excitation emission matrix spectroscopy further demonstrated that AOM were chiefly composed of aromatic protein-like and soluble microbial byproduct-like matters, while NOM were mainly contributed from humic acid-like and soluble microbial byproduct-like substances. Although the molecular weight (MW) distribution of AOM and NOM showed no significant difference, size-exclusion chromatography with organic carbon as well as organic nitrogen detection (LC-OCD-OND) revealed that AOM were concentrated with the fraction of building blocks and NOM had higher concentrations of biopolymers and humics (HS). Moreover, AOM displayed higher DBPFPs than NOM, especially for nitrogenous DBPFP (N-DBPFP). MW < 1 kDa fractions both in AOM and NOM contributed the largest proportion to the formation of carbonaceous disinfection byproducts (C-DBPs). In addition, Pearson correlation analysis showed that bulk parameter SUVA was significantly relevant to the formation potentials of trihalomethane both in AOM and NOM, but was ineffective for carbonaceous DBPFP (C-DBPFP) prediction. Dissolved organic nitrogen contents in biopolymer and HS characterized by LC-OCD-OND had strong correlations with N-DBPFPs from AOM and NOM, indicating that LC-OCD-OND quantitative analysis could improve the prediction accuracy of the DBP formation than bulk parameters during NOM and AOM chlorination., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The impact of ferric iron stimulation on the evolution of microbial structure in marine sediment microbial fuel cells (SMFCs), operated for the bioremediation of a complex mixture of low and high molecular weight PAHs (naphthalene, fluorene, pyrene and benzo(a)pyrene), was assessed. Microbial evolution profiles showed high relative abundances of exoelectrogenic iron-reducing bacteria throughout the biodegradation, namely Geoalkalibacter, under ferric iron stimulation and anode reducing conditions, irrespective of sulfate reducing bacteria (SRB) inhibition. Highest PAHs removal was measured in the absence of anode reduction, under Fe stimulation and SRB inhibition, reaching 40.85% for benzo(a)pyrene, the most persistent PAH used in this study. Results suggest that amendment of contaminated sediment with ferric iron could constitute a better bioremediation strategy than using SMFCs. This becomes significant when considering the well-established and dominant indigenous SRB population in marine sediments that usually limits the performance of the anode as a terminal electron acceptor in marine SMFCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC 50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC 50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK., Objective: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated., Methods and Results: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05)., Conclusion: The findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Developing an effective fibrinolytic drug for treating thrombolysis with minimal undesirable side effects is of great importance. In the current study, an optimum solvent was selected for the extraction of fibrinolytic active components. Furthermore, a strong fibrinolytic enzyme named WPI01 was purified from Whitmania pigra Whitman through various chromatographic steps. WPI01 has a molecular mass of 27044.297 Da, and the N-terminal 8 amino acid sequence was determined as VVGGVEAR. WPI01 was stable within the pH range of 6.0-10.0 and with maximum fibrinolytic activity at 40 °C and a pH of 8.0. At 500 U/mL, WPI01 induced 50.59% blood clot reduction in vitro within 6 h, which was higher than that induced by urokinase at 1000 U/mL. In an analysis of the plasminogen activator activity, WPI01 produced obvious halos on heated and unheated fibrin plates, suggesting that WPI01 may not only act as a plasminogen activator but also degrade fibrin clots directly, and more study is needed to support this. In conclusion, WPI01 is obviously different from known fibrinolytic enzymes in terms of substrate specificity and fibrinolytic mode of action, suggesting that it is a novel fibrinolytic enzyme with potential applications in the treatment and prevention of thrombosis., (Copyright © 2020. Published by Elsevier Inc.)

The system performance, sludge property and microbial community shift were evaluated in a nitrifying sludge (NS) bioreactor for simultaneous treating p-Nitrophenol (PNP) and high ammonia wastewater. After long-term acclimation for 80 days, the removal efficiencies of PNP and NH 4 + -N reached to 99.9% and 99.5%, respectively. Meanwhile, the effluent PNP gradually decreased from 7.9 to 0.1 mg/L by acclimation of sludge. The particle size of NS increased from 115.2 μm to 226.3 μm accompanied by the decreased zeta potential as a self-protection strategy. The presence of PNP exposure altered the effluent soluble microbial products (SMP) fluorescent components and molecular composition. The increase in the relative abundance of Thauera, Nitrospiraceae and Nitrosomonas indicated the nitrification and denitrification capacities of NS increased, which maybe the PNP cometabolic biodegradation effect. Moreover, Ignavibacteria and Aeromonas were responsible as the dominant bacteria for degrading PNP in the nitrifying system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We previously explored citrus oil emulsion stabilized by citrus pectin. In this report, we characterized key parameters of the citrus pectin mesoscopic structure and their effect on emulsifying capacity, and explored the underlying mechanism by determining the interfacial properties, emulsifying ability, and micromorphology. To generate different mesoscopic structure, citrus pectins were hydrolyzed or regulated by pH and NaCl. Hydrolysis decreased the size of citrus pectin mesoscopic structure with constant compactness, leading to superior interfacial properties but inferior emulsifying ability. In contrast, pH and NaCl regulation decreased the mesoscopic structure size and increased the compactness, and pH- and NaCl-regulated citrus pectin formed a compact absorbed layer at the interface to resist droplet coalescence/flocculation during homogenization. Our results support the importance of compactness of the citrus pectin mesoscopic structure on emulsifying capacity. This study increased our understanding on the relationship between the mesoscopic structures of polysaccharide emulsifier and emulsifying ability., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-1343 bp). The molecular weight of AACT protein is 43.40 kDa, it is hydrophilic with a theoretical isoelectric point of 8.96. Furthermore, The region of the transcription start site is 1997-2047 bp of AACT promoter, and it contained promoter elements (TATA Boxs, CAAT Boxs, CAAT-box, ABRE, G-Boxs, Sp1, MSA-like, LTR). AACT recombinant protein (43.40 KDa + Tag protein 22.68 KDa) was subjected in SDS-PAGE. AACT the transcription levels of in different development stages were investigated. The expression of AACT in primordia (2.4-fold) and 15 d mycelia (2.3- fold) were significantly higher than 9 d mycelia (contral). The expression level of the AACT downstream genes and triterpenoids content were determined at different developmental stages. Triterpenoid content reached its peak on day 15(7.21 mg/g)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation., Competing Interests: Declaration of competing interest Philip A. Pemberton is affiliated with and has ownership interests in Serplus Technology LLC., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t 1/2 ) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The K m values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 μM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu 2+ and Zn 2+ . Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the present investigation, cloning and overexpression of xylanase (XynF1), the main xylanase of A. oryzae LC1, was performed in prokaryotic system E. coli BL21(DE3) to produce recombinant xylanase with high titer of specific activity (1037.3 U/mg), which was 9.3-fold higher than the native strain. Further, the recombinant XynF1 of size 37 kDa was purified using Ni 2+ -NTA resins followed by cation exchange chromatography, which showed an 1.8-fold increase in purity with 71.4% yield. The r-XynF1 exhibited a wide range of activity at different pH (3.0-10.0) range and temperature (30-70 °C) with an optimum pH at 5.0 and temperature at 30 °C. The results from the current study, clearly demonstrate that this is an effective method to generate a recombinant enzyme with improved activity, making it useful for possible industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recently, microbial exopolysaccharides (EPSs) have offered very large field for medical applications owing to their bioactive characteristics. This study aimed to obtain antitumor EPS and to optimize its production using different optimization approaches. Eighty EPSs-producing bacteria were obtained from mud samples. Isolate BS4 was selected as the most potent antitumor EPS-producer and identified as Bacillus mycoides BS4 using 16S rRNA gene sequence analysis. Cell viability and antitumor activity of produced EPS were investigated using microscopic examination and MTT assay. Interestingly, the produced EPS exhibited low cytotoxicity against normal cell baby hamster kidney (BHK) with IC 50 at 254 μgml -1 while it exhibited an inhibitory effect against cancer cells of human hepatocellular carcinoma (HepG2) and Colorectal adenocarcinoma cells (Caco-2) with IC 50 of 138 μgml -1 and 159 μgml -1 , respectively. The purified EPS was characterized using Fourier transform infrared, gel permeation chromatography and gas chromatography-mass spectroscopy. It showed molecular weight of 1.90 × 10 4  Da and consists of galactose, mannose, glucose and glucuronic acid. The factors affecting EPS production were optimized using one-factor-at-a time and statistical optimization methods. The Placket-Burman (PB) design results indicated that sugarcane molasses, peptone and shaking conditions were the most significant variables, which were further optimized by Response Surface Methodology (RSM). The optimum conditions for EPS production were 8.0% (w/v) sugarcane molasses, 6 gL ‒1 peptone and 300 rpm that produce 8.02gL ‒1 of EPS. This indicates the potentiality of Bacillus mycoides BS4 for production of EPS with biomedical applications., Competing Interests: Declaration of competing interest The Authors declares that they have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Angiogenesis is a fundamental process of wound healing, embryogenesis etc. but occurs in cancer and chronic inflammation pathologically. HET-CAM assay is a useful, well established and animal alternative test to screen anti-inflammatory potentials of pharmaceutical products as well as nano-formulations. Dexketoprofen trometamol (DT) belongs to the nonsteroidal anti-inflammatory drug (NSAID) group which is a rapidly acting analgesic ingredient. Because DT has a short half-life, high and frequent dosing is used in treatment. The need of design and producing a new oral prolonged-release dosage form containing DT is the major aim of the study with low dose and low side effects. Chitosan (CS) has been widely used in the pharmaceutical area because of its favorable biological properties. In this study, DT loaded CS nanoparticles (CS-NPs) were produced by spray drying method for oral drug delivery. Structures of CS-NPs were elucidated by particle size, zeta potential, SEM, DSC, FT-IR and 1 H NMR. High encapsulation efficiency was obtained (73-84%) for the prepared formulations. In vitro release was examined in pH 1.2 buffer and pH 6.8 buffer. DT-loaded CS-NPs showed prolonged release, particularly at pH 6.8. Weibull kinetic model was found to fit best to DT release from CS-NPs in both release medium. The anti-inflammatory activity of optimum formulation (M-DT) was examined using the in vivo HET-CAM assay. The anti-inflammatory activity results indicated that M-DT coded NPs formulation showed significantly good anti-inflammatory potential with closer inhibition value to the standard anti-inflammatory DT at one fifth lower dosage. According to the proposed method and results it can be successfully applicable to the NP preparation containing DT and it could be concluded that DT loaded CS-NPs seem to be a promising prolonged release drug delivery system for oral administration with low dose and high efficiency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 10 6 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG) 40 with X = V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1 H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELP S /nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH 2 ), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CPFX is a highly effective antibiotic, but it has been reported to significantly impair both testicular function and structure in rats. In this study, we assessed reversal of CPFX-induced variation in mice testicular structure and testosterone synthesis by probiotic microbes in the infected model and normal model. We detected testicular weight, testicular structure and Leydig cell variables in numbers. We detected the levels of serum testosterone and steroidogenic enzymes, as well as DBC1, Sirt1, NF-κB, and related redox state and inflammatory response in the testes. The results showed that probiotic microbes had significantly elevated serum testosterone levels and steroidogenic enzymes, higher Sirt1, anti-oxidative enzymes and anti-inflammatory cytokine expression, and lower NF-κB, DBC1, oxidative damage, pro-inflammatory cytokine expression. The results suggest that the testis-protective, antiinflammatory and antioxidation effects of probiotics largely resulted from its ability to decrease oxidative stress and preserve antioxidant activity by stabilizing antioxidant defense systems, reducing oxidative damage and inflammatory response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Magnetic powdered activated carbon (Mag-PAC) was successfully developed and applied as an adsorbent for dissolved organic matter (DOM) removal from the effluent of a membrane bioreactor (MBR) using batch experiments. The results show that a coating of iron oxide particles is consistently distributed on the surface of powdered activated carbon (PAC), resulting in a decrease in the specific surface area and in the pH at the point of zero charge, even though the particle sizes of Mag-PAC and PAC were similar. A Mag-PAC dosage of 4 g/L exhibited efficient and fast DOM adsorption with a relatively short contact time of 5 min. The iron oxide coating on the surface of PAC may play an important role in the DOM removal efficiency. Temkin isotherm and pseudo-second order kinetic models well described the DOM adsorption, suggesting that the adsorption of DOM onto Mag-PAC could be mainly governed by a chemisorption mechanism. Humic acid- and fulvic acid-like compounds and aromatic DOM with molecular weights (MWs) between 2610 Da and 3030 Da were efficiently removed by Mag-PAC, whereas aromatic DOM with an MW of 1760 Da was poorly removed. Our results demonstrate that the application of Mag-PAC for DOM adsorption is attractive and yields benefits as a posttreatment system for MBR effluent due to its efficient and fast DOM adsorption., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A new alginate lyase gene of algl17 was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene contained 2220 bp and encoded a 739-amino acid protein classified into the PL-17 family. The recombinant alginate lyase AlgL17 was overexpressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 84.9 kDa. This enzyme showed activities towards sodium alginate, polyM and alginate oligosaccharide, but very low activity towards polyG. These results indicate that AlgL17 is a polyM-specific oligoalginate lyase. When sodium alginate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 35 °C and pH 8.0, respectively. Recombinant AlgL17 was stable at 25 °C, but not stable at 30 °C and 35 °C. It showed good stability over a pH range of 5.0-8.0. The enzyme activity was increased to 1.7 times by adding NaCl to a final concentration of 0.7 M. The ability of the recombinant AlgL17 producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from sodium alginate and polyM block indicates that AlgL17 is an exo-type alginate lyase., (Copyright © 2019. Published by Elsevier Inc.)

Humic substances are important redox-active organic compounds with various kinds of functional groups in different molecular sizes and connections. They can be formed during the degradation of biomass and biowaste in the environment. In soil environments, insoluble heavy metals and large size organic pollutants trapped in soil micropores (under 2.5 nm) are hard to be removed by bulk HS or microorganisms poorly accessible to pollutants. Recently, low molecular weight fractions (LMWF) of humic acids (HA) obtained from dialysis demonstrate a stronger reducing capacity than bulk HA, and the reducing capacity is found out to be related to the relative fluorescence intensity of LMWF HA by a fluorescence spectra method. The continuous disaggregation of LMWF HA from bulk HA molecules in rapid groundwater flow area provides a possibility by LMWF HA to influence the transformation and transport of the pollutants trapped in soil micropores. Therefore, the objective of this study is to explore the mathematical relationship between reducing capacity of LMWF HA and relative fluorescence intensity of quinone moieties in LMWF HA. We found that Leonardite humic acids (LHA) 3500-LMWF (under 1.25 nm) only account for 0.40%-1.53% of the total organic carbon of bulk HA. However, the reducing capacity of LMWF HA per gram carbon were up to 206 times greater than bulk LHA. Similar trends were found for other two standard LMWF HA samples. Fluorescence spectroscopy was used to characterize the changes in fluorophores of LMWF HA stripped in process. We found that an excitation/emission (E x /E m ) position representing a quinone-like fluorophore in all native LMWF HA state samples was located at 260-270/455-495 nm. The quinone-like fluorophore in native LMWF HA can be further reduced into a fluorophore A and a fluorophore B (E x /E m at 300-305/420-430 nm and 215-225/415-430 nm) related to the quinonoid π-π* transition and benzenoid π-π* transition, respectively. Moreover, the relative fluorescence intensities of these two fluorophores (A and B) have strong correlations with reducing capacities of LMWF HA, confirming a significant role of quinone-like fluorophores in redox properties of HA. LMWF HA can enter micropores to react with pollutants, the application of biomass will have a constant benefit in remediation of contaminated soil site especially in a long time scale., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Xanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt medium (MSM) of pH 7.0 supplemented with 0.8% yeast extract, 8.5 mM xanthine and incubation at 30 °C for 24 h. Under these culture conditions 11.57 fold increase in the production of this enzyme was achieved. The enzyme was purified from A. calcoaceticus RL2-M4 using anion exchange chromatography to 8.18 fold with 31% yield and specific activity of 4.58 U/mg protein. SDS-PAGE analysis of the purified enzyme revealed that it was homodimer of 95 kDa and its native molecular mass was estimated to be 190 kDa. This enzyme was found to be stable at 35 °C for 5 h. The purified xanthine oxidase of A. calcoaceticus RL2-M4 had K m 0.3 mM and V max 5.8 U/mg protein using xanthine as substrate. The activity and stability characteristic of xanthine oxidase of A. calcoaceticus RL2-M4 makes it a potentially good enzyme for industrial applications., (Copyright © 2019. Published by Elsevier Inc.)