Synergistic efficacy of ultrasound and ammonium persulfate in inactivating Escherichia coli O157:H7 in buffered peptone water and orange juice.

This study investigated the synergistic effects of ammonium persulfate (PS) and ultrasound (US) on the inactivation of Escherichia coli O157:H7 in buffered peptone water (BPW) and orange juice products. A comprehensive assessment of PS concentrations ranging from 1 to 300 mM, considering not only the statistical significance but also the reliability and stability of the experimental outcomes, showed that 150 mM was the optimal PS concentration for the inactivation of E. coli O157:H7. Additionally, US output intensities varying from 30 % to 60 % of the maximum US intensity were evaluated, and 50 % US amplitude was found to be the optimal US condition. A 50 % amplitude setting on the sonicator corresponds to half of its maximum displacement, approximately 60 µm, based on a maximum amplitude of 120 µm. The inactivation level of E. coli O157:H7 was significantly enhanced by the combined treatment of PS and US, compared to each treatment of PS and US alone. In the BPW, a 10-min treatment with the combination of PS and US resulted in a significant synergistic inactivation, achieving up to a log reduction of 3.86 log CFU/mL. Similarly, in orange juice products, a 5-min treatment with the combination of PS and US yielded a significant synergistic inactivation, with a reduction reaching 5.90 log CFU/mL. Although the treatment caused a significant color change in the sample, the visual differences between the treated and non-treated groups were not pronounced. Furthermore, the combined treatment in orange juice demonstrated significantly enhanced antimicrobial efficacy relative to BPW. Despite identical 5-min treatment periods, the application in orange juice resulted in a substantially higher log reduction of E. coli O157:H7, achieving 7.16 log CFU/mL at a reduced PS concentration of 30 mM, whereas the same treatment in BPW yielded only a 2.89 log CFU/mL reduction at a PS concentration of 150 mM, thereby highlighting its significantly superior antimicrobial performance in orange juice. The mechanism underlying microbial inactivation, induced by the combined treatment of PS and US, was identified as significant cell membrane damage. This damage is mediated by sulfate radicals, generated through the sono-activation of persulfate. In addition, the low pH of orange juice, measured at 3.7, is likely to have further deteriorated the E. coli O157:H7 cells compared to BPW (pH 7.2), by disrupting their cell membranes, proton gradients, and energy metabolism. These findings underscore the effectiveness of PS and US integration as a promising approach for non-thermal pasteurization in the food industry. Further research is needed to optimize treatment parameters and fully explore the practical application of this technique in large-scale food processing operations. Sensory evaluation and nutritional assessment are also necessary to address the limitations of PS.

Role of WSOCs and pH on Ammonium Nitrate Aerosol Efflorescence: Insights into Secondary Aerosol Formation.

Efflorescence of ammonium nitrate (AN) aerosols significantly impacts atmospheric secondary aerosol formation, climate, and human health. We investigated the effect of representative water-soluble organic compounds (WSOCs) (sucralose (SUC), glycerol (GLY), and citric acid (CA) on AN:WSOC aerosol efflorescence using vacuum Fourier transform infrared spectroscopy. Combining efflorescence relative humidity (ERH) measurements, heterogeneous nucleation rates, and model predictions, we found that aerosol viscosity, correlating with molecular diffusion, effectively predicted ERH variations among the AN:WSOC aerosols. WSOCs with higher viscosity (SUC and CA) hindered efflorescence, while GLY with a lower viscosity showed a minor effect. At a low AN:CA molar ratio (10:1), CA promoted ERH, likely due to CA crystallization. Increasing the droplet pH inhibited AN:CA aerosol efflorescence. In contrast, for AN:SUC and AN:GLY aerosols, efflorescence is pH-insensitive. With the addition of trivial sulfate, AN:SUC droplets exhibited two-stage efflorescence, coinciding with ammonium sulfate and AN efflorescence. Given the atmospheric abundance, the morphology, phase, and mixing state of nitrate aerosols are significant for atmospheric chemistry and physics. Our results suggest that AN:WSOCs aerosols can exist in the amorphous phase in the atmosphere, with efflorescence behavior depending on the aerosol composition, viscosity, pH, and the cation and anion interactions in a complex manner.

Influence of mixed salts on retention behavior of model proteins in cation exchange chromatography.

Mobile phase composition is an important factor for a further improvement of ion exchange chromatography steps of proteins. In this work, the effects of mixed salts on the retention factors of the two model proteins lysozyme (LYZ) and bovine serum albumin (BSA) in cation exchange chromatography (CEC) were investigated and compared to effects previously observed in hydrophobic interaction chromatography (HIC). The model equation describing the effects in HIC was adjusted for linear gradient elution experiments in CEC. The investigated salts were sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. By varying binary salt mixtures as well as using pure salts, model parameters were determined. The normalized root mean square error (NRMSE) of the predicted retention factors for the calibration runs was 4.1% for BSA and 3.1% for LYZ. Additional validation experiments proved the ability of the model to describe and predict retention behavior of the proteins for further salt compositions. Hereby, the NRMSE values for BSA and LYZ were 2.0% and 1.5%, respectively. While the retention factors of LYZ changed linearly with the salt composition, non-linearities in the impact of the anion composition were found for BSA. This was contributed to an overlay of a synergetic salt effect on a protein-specific effect by sulfate on BSA with non-specific effects of the ions for CEC. However, the impact of the synergetic effects on protein separation is lower for CEC than for HIC, as mixed salts do not increase the separation of these proteins. The best salt composition for separating BSA and LYZ is pure ammonium sulfate. Thus, synergetic salt effects can also occur in CEC, but they have a lower impact than in HIC.

Phase State and Relative Humidity Regulate the Heterogeneous Oxidation Kinetics and Pathways of Organic-Inorganic Mixed Aerosols.

Inorganic species always coexist with organic materials in atmospheric particles and may influence the heterogeneous oxidation of organic aerosols. However, very limited studies have explored the role of the inorganics in the chemical evolution of organic species in mixed aerosols. This study examines the heterogeneous oxidation of glutaric acid-ammonium sulfate and 1,2,6-hexanetriol-ammonium sulfate aerosols by hydroxyl radicals (OH) under varied organic mass fractions (forg) and relative humidity in a flow tube reactor. Coupling the oxidation kinetics and product measurements with kinetic model simulations, we found that under both low relative humidity (RH, 30-35%) and high RH conditions (85%), the decreased forg from 0.7 to 0.2 accelerates the oxidation of the organic materials by a factor of up to 11. We suggest that the faster oxidation kinetics under low-RH conditions is due to full or partial phase separation, with the organics greatly enriched at the particle outer region, while enhanced "salting-out" of the organics and OH adsorption caused by higher inorganics could explain the observations under high-RH conditions. Analysis of the oxidation products reveals that the dilution of organics by the inorganic salts and corresponding water uptake under high-RH conditions will favor alkoxy radical fragmentation by a factor of 3-4 and inhibit its secondary chain propagation chemistry. Our results suggest that atmospheric organic aerosol oxidation lifetime and composition are strongly impacted by the coexistent inorganic salts.

Kinetics, Products, and Brown Carbon Formation by Aqueous-Phase Reactions of Glycolaldehyde with Atmospheric Amines and Ammonium Sulfate.

Glycolaldehyde (GAld) is a C2 water-soluble aldehyde produced during the atmospheric oxidation of isoprene and many other species and is commonly found in cloudwater. Previous work has established that glycolaldehyde evaporates more readily from drying aerosol droplets containing ammonium sulfate (AS) than does glyoxal, methylglyoxal, or hydroxyacetone, which implies that it does not oligomerize as quickly as these other species. Here, we report NMR measurements of glycolaldehyde's aqueous-phase reactions with AS, methylamine, and glycine. Reaction rate constants are smaller than those of respective glyoxal and methylglyoxal reactions in the pH range of 3-6. In follow-up cloud chamber experiments, deliquesced glycine and AS seed particles were found to take up glycolaldehyde and methylamine and form brown carbon. At very high relative humidity, these changes were more than 2 orders of magnitude faster than predicted by our bulk liquid NMR kinetics measurements, suggesting that reactions involving surface-active species at crowded air-water interfaces may play an important role. The high-resolution liquid chromatography-electrospray ionization-mass spectrometric analysis of filter extracts of unprocessed AS + GAld seed particles identified sugar-like C6 and C12 GAld oligomers, including proposed product 3-deoxyglucosone, with and without modification by reactions with ammonia to diimine and imidazole forms. Chamber exposure to methylamine gas, cloud processing, and simulated sunlight increased the incorporation of both ammonia and methylamine into oligomers. Many C4-C16 imidazole derivatives were detected in an extract of chamber-exposed aerosol along with a predominance of N-derivatized C6 and C12 glycolaldehyde oligomers, suggesting that GAld is capable of forming brown carbon SOA.

Hygroscopic Growth, Phase Morphology, and Optical Properties of Model Aqueous Brown Carbon Aerosol.

Brown carbon aerosol in the atmosphere contain light-absorbing chromophores that influence the optical scattering properties of the particles. These chromophores may be hydrophobic, such as PAHs, or water soluble, such as nitroaromatics, imidazoles, and other conjugated oxygen-rich molecules. Water-soluble chromophores are expected to exist in aqueous solution in the presence of sufficient water and will exhibit physical properties (e.g., size, refractive index, and phase morphology) that depend on the environmental relative humidity (RH). In this work, we characterize the RH-dependent properties of 4-nitrocatechol (4-NC) and its mixtures with ammonium sulfate, utilizing a single-particle levitation platform coupled with Mie resonance spectroscopy to probe the size, real part of the complex refractive index (RI), and phase morphology of individual micron-sized particles. We measure the hygroscopic growth properties of pure 4-NC and apply mixing rules to characterize the growth of mixtures with ammonium sulfate. We report the RI at 589 nm for these samples as a function of RH and explore the wavelength dependence of the RI at non-absorbing wavelengths. The real part of the RI at 589 nm was found to vary in the range 1.54-1.59 for pure 4-NC from 92.5 to 75% RH, with an estimated pure component RI of 1.70. The real part of the RI was also measured for mixtures of AS and 4-NC and ranged from 1.39 to 1.51 depending on the component ratio and RH. We went on to characterize phase transitions in mixed particles, identifying the onset RH of liquid-liquid phase separation (LLPS) and efflorescence transitions. Mixtures showed LLPS in the range of 85-76% RH depending on the molar ratio, while efflorescence typically fell between 22 and 42% RH. Finally, we characterized the imaginary part of the complex RI using an effective oscillator model to capture the wavelength-dependent absorption properties of the system.

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size.

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Phycocyanin, a super functional ingredient from algae; properties, purification characterization, and applications.

Phycocyanins (PCYs) are a group of luxuriant bioactive compounds found in blue-green algae with an estimated global market of about US$250 million within this decade. The multifarious markets of PCYs noted by form (e.g. powder or aqueous forms), by grade (e.g. analytical, cosmetic, or food grades), and by application (such as biomedical, diagnostics, beverages, foods, nutraceuticals and pharmaceuticals), show that the importance of PCYs cannot be undermined. In this comprehensive study, an overview on PCY, its structure, and health-promoting features are diligently discussed. Methods of purification including chromatography, ammonium sulfate precipitation and membrane filtration, as well as characterization and measurement of PCYs are described. PCYs could have many applications in food colorants, fluorescent markers, nanotechnology, nutraceutical and pharmaceutical industries. It is concluded that PCYs offer significant potentials, although more investigations regarding its purity and safety are encouraged.

Reduction of lignin heterogeneity using aqueous two-phase system: A facile and universal "one-step-three-fractions" approach.

As the most abundant aromatic biopolymer, lignin presents great potential to produce valuable materials and chemicals. However, its large-scale value-added application is still facing many practical challenges and one of them is the unstable properties caused by lignin heterogeneity. Herein, we developed a novel "one-step-three-fractions" fractionation strategy to reduce lignin heterogeneity using aqueous two-phase system (ATPS) composed of (NH4)2SO4 and ethanol. In contrast to conventional step-wise fractionation processes, the proposed process subdivided heterogeneous lignin into three homogeneous fractions in only one step: the first fraction (F1) dissolved in the ethanol-rich top layer; the second fraction (F2) dissolved in the salt-rich bottom layer and the last fraction (F3) insoluble in both two layers. F2 presented the lowest molecular weight followed by F1 while F3 showed the highest molecular weight. With the increase of molecular weight, the contents of guaiacyl unit and ß-O-4 linkage increased while the content of hydrophilic groups (carboxyl and aromatic hydroxyl) decreased significantly. Moreover, the ATPS exhibited satisfactory recyclability and the fractionation approach could be applied to different types/sources of lignin. Consequently, the work indicates that ATPS is a novel and effective way to fractionate lignin and reduce its molecular weight polydispersity and structural heterogeneity in one step.

CBP22, a Novel Bacteriocin Isolated from Clostridium butyricum ZJU-F1, Protects against LPS-Induced Intestinal Injury through Maintaining the Tight Junction Complex.

A novel bacteriocin secreted by Clostridium butyricum ZJU-F1 was isolated using ammonium sulfate fractionation, cation exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The bacteriocin, named CBP22, contained 22 amino acids with the sequence PSAWQITKCAGSIAWALGSGIF. Analysis of its structure and physicochemical properties indicated that CBP22 had a molecular weight of 2264.63 Da and a +1 net charge. CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923. The effects and potential mechanisms of bacteriocin CBP22 on the innate immune response were investigated with a lipopolysaccharide- (LPS-) induced mouse model. The results showed that pretreatment with CBP22 prevented LPS-induced impairment in epithelial tissues and significantly reduced serum levels of IgG, IgA, IgM, TNF-α, and sIgA. Moreover, CBP22 treatment increased the expression of the zonula occludens and reduced permeability as well as apoptosis in the jejunum in LPS-treated mice. In summary, CBP22 inhibits the intestinal injury and prevents the gut barrier dysfunction induced by LPS, suggesting the potential use of CBP22 for treating intestinal damage.

Synthesis of a novel superabsorbent with slow-release urea fertilizer using modified cellulose as a grafting agent and flexible copolymer.

In this work, a number of glucose unites in polymeric structure of cellulose was converted to 2,4-dihydroxy-3-(1-hydroxy-2-oxoethoxy)butanal (cellulose containing di aldehyde units (CCDAUs)) by oxidation with sodium periodate, followed by condensation with acetone to produce 5,7-dihydroxy-6-((1-hydroxy-4-oxopent-2-en-1-yl)oxy)hept-3-en-2-one unites (cellulose containing di ene units (CCDEUs)). This modified cellulose was characterized by different methods and applied as a copolymer and grafting agent to synthesize an eco-friendly (CCDEUs-g-poly(AA)/urea) superabsorbent with slow-release urea fertilizer. The created double bonds in C2 and C3 positions of ß-d-glucose units increased the linkage between cellulose and acrylic acid, leading to the formation of a strong network for slow-release urea fertilizer. Also, this modification created an expanded network for storage a high amount of water by increasing the cellulose flexibility. The reaction conditions for modification and synthesis of the superabsorbent, the oxidation degree value of glucose units, kinetics models, the effect of different saline solutions, various pH and reswelling time on the water absorbency, water retention capacity, reusability, biodegradability, and slow-release property were investigated. Also, the effect of synthesized CCDEUs-g-poly(AA)/urea on plant growth was tested and excellent results were obtained.

Fusarium solani G6, a novel vitexin-producing endophytic fungus: characterization, yield improvement and osteoblastic proliferation activity.

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to produce vitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72 mg/L) for the strain was observed in PDB liquid medium containing 20.54 g/L of glucose and 8.90 g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28 °C for 6 days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

Colorimetric determination of acid phosphatase activity and inhibitor screening based on in situ polymerization of aniline catalyzed by gold nanoparticles.

A colorimetric assay for acid phosphatase (ACP) was constructed that is based on in situ polymerization of aniline catalyzed by gold nanoparticles (AuNPs). Aniline can be polymerized by ammonium persulfate (APS) in acidic condition and form gold-polyaniline core-shell nanoparticles (Au@PANI NPs) in the presence of AuNPs with the assistance of sodium dodecyl sulfate (SDS). AuNPs were also found to accelerate the polymerization process of aniline and thus shorten the reaction time. Upon the introduction of ascorbic acid (AA), the oxidant APS was consumed via the redox reaction. That led to the suppression of the formation of PANI. Consequently, ACP activity can be supervised on the basis of hydrolysis of 2-phospho-L-ascorbic acid trisodium salt (AAP) catalyzed by ACP to release AA. With the increase of ACP activity, the intensity ratio of the absorbance at λ705 nm (A705) and the absorbance at λ530 nm (A530) gradually decreased and the color gradually changed from dark-green to light-green to blue-gray to purple and eventually to pink. This method for ACP determination worked in the range 0.40 to 2.00 U·L-1. The detection limit is 0.043 U·L-1. The assay was applied to determine ACP in human serum. The recovery ranged from 81.0 to 104.6%. Relative standard deviation was less than 5%. This suits the request for biological sample analysis. Graphical abstract Schematic presentation of the colorimetric determination of acid phosphatase activity and inhibitor screening based on in situ polymerization of aniline catalyzed by gold nanoparticles. : acid phosphatase (ACP); : gold nanoparticles (AuNPs); : gold-polyaniline core-shell nanoparticles (Au@PANI NPs); ascorbic acid (AA); 2-phospho-L-ascorbic acid trisodium salt (AAP).

A buffered media system for yeast batch culture growth.

BACKGROUND: Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system. RESULTS: We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations. CONCLUSION: Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

Swellable catheters based on a dynamic expanding inner diameter.

Intravenous (IV) fluid administration is critical for all patients undergoing care in a hospital setting. In-patient hospital practice, surgeries, and emergency care require functional IVs for fluid replacement and medication administration. Proper placement of IVs is vital to providing medical services. The ease of placement of an IV catheter, however, depends not only on the size of the catheter but also on provider experience and patient demographics such as age, body mass index, hydration status, and medical comorbidities present challenges to successful IV placement. Smaller diameter IV placement can improve success and there are instances where multiple small diameter catheters are placed for patient care when larger bore access is unattainable. Smaller inner-diameter catheters for anesthesia have functional constraints. Ideally, there would be a smaller catheter for placement that could function as a larger catheter for patient care. One solution is the idea of functionally responsive catheters. Here, we evaluated tubular-shaped hydrogels as potential functional catheters that can increase in inner diameter through fluid swelling using cross-linked homopolymers of polyacrylamide, PAM (10-40% w/w), and their copolymers with 0-8% w/w Poly-(Ethylene Glycol)-Diacrylate, PEGDA. For the PAM gels, the water transport mechanism was shown to be concentration-dependent Fickian diffusion, with the less concentrated gels exhibiting increasingly anomalous modes. Increasing the PEGDA content in the network yielded an initial high rate of water uptake, characterized by Case II transport. The swelling kinetics depended strongly on the sample geometry and boundary conditions. Initially, in a submerged swelling, the annulus expands symmetrically in both outward and inward directions (it thickens), reducing the internal diameter by up to 70%. After 1 h, however, the inner diameter increases steadily so that at equilibrium, there is a net (>100%) increase in all the dimensions of the tube. The amount of linear swelling at equilibrium depended only on the polymer volume fraction as made, while the rate of inner diameter expansion depended on the hydrophilicity of the matrix and the kinetics of sorption. This study serves as proof of concept to identify key parameters for the successful design of hydrogel-based catheter devices with expanding inner-diameters for applications in medical care.

Establishment of an immunological detection method of fleroxacin by fluorescence-linked immunosorbent assay.

The direct and indirect competitive fluorescence-linked immunosorbent assay (FLISA and icFLISA) incorporating quantum dots (QDs) for the detection of fleroxacin (FLE) was established for the first time in this study. The monoclonal antibody specific for FLE was successfully conjugated with QDs after purification by the caprylic acid-ammonium sulphate method. The limits of detection of FLISA and icFLISA were 0.012 ng/mL and 0.006 ng/mL, respectively; IC50 were 0.32 ng/mL and 0.19 ng/mL; and the detection ranges were 0.012-24.490 ng/mL and 0.006-16.210 ng/mL. The recovery was 93.8%-112.4% and the coefficient of variation was below 11.75%. The fabricated FLISA and icFLISA are cost-effective, high sensitive and can be an alternative method in the detection of FLE residues.

Effect of Hofmeister Ions on Transport Properties of Aqueous Solutions of Sodium Hyaluronate.

Tracer diffusion coefficients obtained from the Taylor dispersion technique at 25.0 °C were measured to study the influence of sodium, ammonium and magnesium salts at 0.01 and 0.1 mol dm-3 on the transport behavior of sodium hyaluronate (NaHy, 0.1%). The selection of these salts was based on their position in Hofmeister series, which describe the specific influence of different ions (cations and anions) on some physicochemical properties of a system that can be interpreted as a salting-in or salting-out effect. In our case, in general, an increase in the ionic strength (i.e., concentrations at 0.01 mol dm-3) led to a significant decrease in the limiting diffusion coefficient of the NaHy 0.1%, indicating, in those circumstances, the presence of salting-in effects. However, the opposite effect (salting-out) was verified with the increase in concentration of some salts, mainly for NH4SCN at 0.1 mol dm-3. In this particular salt, the cation is weakly hydrated and, consequently, its presence does not favor interactions between NaHy and water molecules, promoting, in those circumstances, less resistance to the movement of NaHy and thus to the increase of its diffusion (19%). These data, complemented by viscosity measurements, permit us to have a better understanding about the effect of these salts on the transport behaviour of NaHy.

Homogeneous batch micro-crystallization of proteins from ammonium sulfate.

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate α-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.

Hydroxyapatite sonosensitization of ultrasound-triggered, thermally responsive hydrogels: An on-demand delivery system for bone repair applications.

While bones have the innate capability to physiologically regenerate, in certain cases regeneration is suboptimal, too slow, or does not occur. Biomaterials-based growth factor delivery systems have shown potential for the treatment of challenging bone defects, however, achieving controlled growth factor release remains a challenge. The objective of this study was to develop a thermally responsive hydrogel for bone regeneration capable of ultrasound-triggered on-demand delivery of therapeutic agents. Furthermore, it was hypothesized that incorporation of hydroxyapatite (HA) into the hydrogel could increase sonosensitization, augmenting ultrasound sensitivity to enable controlled therapeutic release to the target tissue. Alginate thermally responsive P(Alg-g-NIPAAm) hydrogels were fabricated and varying quantities of HA (1, 3, 5, and 7% wt./vol.) incorporated. All hydrogels were highly injectable (maximum injection force below 6.5 N) and rheological characterization demonstrated their ability to gel at body temperature. The study demonstrated the ultrasound-triggered release of sodium fluorescein (NaF), bovine serum albumin (BSA), and bone morphogenetic protein 2 (BMP-2) from the hydrogels. Release rates of BSA and BMP-2 were significantly enhanced in the HA containing hydrogels, confirming for the first time the role of HA as a son sensitizer. Together these results demonstrate the potential of these ultrasound-triggered thermally responsive hydrogels for on-demand delivery of therapeutic agents for bone regeneration.

Effect of the ammonium salt anion on the structure of doxorubicin complex and PEGylated liposomal doxorubicin nanodrugs.

BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.