Pickering emulsions stabilized by starch nanocrystals prepared from various crystalline starches by ultrasonic assisted acetic acid: Stability and delivery of curcumin.

Ultrasonic assisted acetic acid hydrolysis was applied to prepare starch nanocrystals (SNCs) from native starches with different crystalline structures (A, B, and C types). The structure properties, morphology, Pickering emulsion stability and curcumin deliver capacity of both SNCs and native starches were investigated and compared. Compared with native starches, SNCs showed smaller size and higher crystallinity. The size of SNCs varied with different crystalline types, with C-type starch exhibiting the smallest SNCs (107.4 nm), followed by A-type (113.8 nm), and B-type displaying the largest particle size (149.0 nm). SNCs-Pickering emulsion showed enhanced stability with smaller emulsion droplets, higher static stability, and denser oil/water interface. SNCs-Pickering emulsions displayed higher curcumin loading efficiency (53.53 %-61.41 %) compared with native starch-Pickering emulsions (13.93 %-19.73 %). During in vitro digestion, SNCs-Pickering emulsions proved to be more proficient in protecting and prolonging the biological activity of curcumin due to their smaller size and better interfacial properties. These findings demonstrated the potential of SNCs for application in Pickering emulsion and delivery of bioactive components.

Comparison of properties and application of starch nanoparticles optimized prepared from different crystalline starches.

Starch nanoparticles (SNPs) were produced by nanoprecipitation combined with ultrasonication with the use of different starches (corn, potato and sago starch) and used to stabilize Pickering emulsions. The orthogonal experiment was used to optimize preparation conditions of gelatinization pretreatment duration of 30 min, ultrasonic power of 600 W, and ultrasonic time of 40 min. Compared with native starch, the SNPs were spherical in shape and displayed a V-type crystalline structure with low relative crystallinity and higher degree of double-helix. Compared with native starch-Pickering emulsion, the SNP-Pickering emulsion had a smaller droplet size, more uniform distribution, clearer oil/water interface, and higher static stability of droplets. The sago SNP-Pickering emulsion had the great gelatinous structure and emulsion stability. In addition, the SNP-Pickering emulsion had the better loading efficiency and controlled release performance of curcumin. Meanwhile, the bioavailability of curcumin in sago SNP-Pickering emulsion was highest.

Optimization of Corn Resistant Starch Preparation by Dual Enzymatic Modification Using Response Surface Methodology and Its Physicochemical Characterization.

Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents.

Preparation and characterization of quinoa starch nanoparticles as quercetin carriers.

Quinoa starch nanoparticles (QSNPs) prepared by nanoprecipitation method under the optimal condition was developed as a carrier for quercetin. The QSNPs prepared under the optimal condition (90 DMSO/H2O ratio, 10 ethanol/solvent ratio, and ultrasonic oscillation dispersion mode) had the smallest particle size and polymer dispersity index through full factorial design. Compared with maize starch nanoparticles (MSNPs), QSNPs exhibited a smaller particle size of 166.25 nm and a higher loading capacity of 26.62%. Starch nanoparticles (SNPs) interacted with quercetin through hydrogen bonding. V-type crystal structures of SNPs were disappeared and their crystallinity increased after loading with quercetin. QSNPs was more effective in protecting and prolonging quercetin bioactivity because of their small particle sizes and high loading capacities. This study will be useful for preparing starch-based carrier used to load sensitive bioactive compounds.

Physicochemical and structural properties of sago starch.

A comprehensive study was conducted to elucidate physicochemical and structural properties of sago starches. Two sago starch granules were oval in shape with an average diameter of 34.41 µm and had C-type polymorph with a crystallinity of about 28.13%. The amylose and resistant starch (RS) contents of two sago starches were higher than those of corn and potato starches. The two sago starches had a large amount of A and B1 chains (DP 6-24) which could form double helix structures. FTIR exhibited that the structure of two sago starches had a lower degree of order. The peak viscosity and breakdown of sago starch 2 were lower than corn starch, and the setback was higher than potato starch. Additionally, sago starches had lower gelatinization enthalpy and higher regeneration tendency. According to rheological results, sago starches showed lower shear thinning degree and thixotropy compared to corn and potato starches. Sago starch 1 gels represented the highest hardness, adhesiveness, springiness and cohesiveness, which could be used as potential food stabilizer. This study revealed the characteristics of two sago starches compared with other starches. The results indicated that the amylose content and amylopectin structures had significant influence on the physicochemical properties of sago starch.

Dynamic human MutSα-MutLα complexes compact mismatched DNA.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

The preparation, formation, fermentability, and applications of resistant starch.

Resistant starch (RS) cannot be digested in the small intestine but can be fermented by microflora in the colon. To meet the demand for RS, effective methods and advanced equipment for preparing RS have emerged, but further development is needed. RS contents are affected by different prepared methods, starch source and certain nutrients such as protein, phenols, and hydrocolloids interacted with RS. As a beneficial fermentation substrate, RS modifies and stabilizes the intestinal flora to balance the intestinal environment and improve intestinal tract health and function. RS is also a kind of ingredient with potential physiological function, even better than that dietary fiber, but also in terms of providing various health benefits. RS has good food-processing characteristics as well and can thus be widely used in the food industry.

[In vitro effects of tacrolimus on platelet function].

OBJECTIVE: To investigate the in vitro effects of immune inhibitor tacrolimus on platelet function. METHODS: Fresh venous blood was collected from healthy volunteers at ages of 18-25 years old, who are not taking antiplatelet drugs within two weeks. The platelets were isolated from the blood and incubated with different concentrations of tacrolimus (0.06, 0.6, 6, 60, 120, 240 µmol/L) at 37 °C for 2 hours, and then the changes of mitochondrial membrane potential and P-selection of platelets were detected by flow cytometry, the expression of apoptosis related protein by Western Blot, and the change of the platelet aggregation function by platelet aggregation analyzer. RESULTS: Tacrolimus at concentration of 0.06 µmol/L could promote collagen induced platelet aggregation, inhibit thrombin induced platelet aggregation, have no effect on ristocetin and vWF induced platelet aggregation function. Tacrolimus at concentration of 120 µmol/L and 240 µmol/L could reduce the platelet mitochondrial membrane potential and induce the expression of apoptosis protein caspase-3. CONCLUSION: In vitro experimental results showed that high concentration of tacrolimus could lead to platelet apoptosis. But the current therapeutic dose of tacrolimus at 0.06 µmol/L (which is equivalent to 50 ng/ml blood concentration) could have different effects on platelet aggregation function according to different stimulating agents.

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D).

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

In vitro studies of DNA mismatch repair proteins.

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O(6)meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein-DNA complexes and determining the stoichiometries and affinities of protein-protein complexes. We extend the capabilities of AFM to the determination of protein-DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein-DNA complexes.

Formation of a DNA mismatch repair complex mediated by ATP.

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.

DNA bending and unbending by MutS govern mismatch recognition and specificity.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.